G2Cdb::Gene report

Gene id
G00001303
Gene symbol
FGB (HGNC)
Species
Homo sapiens
Description
fibrinogen beta chain
Orthologue
G00000054 (Mus musculus)

Databases (7)

Gene
ENSG00000171564 (Ensembl human gene)
2244 (Entrez Gene)
FGB (GeneCards)
Literature
134830 (OMIM)
Marker Symbol
HGNC:3662 (HGNC)
Protein Expression
1900 (human protein atlas)
Protein Sequence
P02675 (UniProt)

Literature (259)

Pubmed - other

  • Abnormal fibrinogens IJmuiden (B beta Arg14----Cys) and Nijmegen (B beta Arg44----Cys) form disulfide-linked fibrinogen-albumin complexes.

    Koopman J, Haverkate F, Grimbergen J, Engesser L, Nováková I, Kerst AF and Lord ST

    IVVO-TNO, Gaubius Laboratory, Leiden, The Netherlands.

    The molecular defects in two congenital abnormal fibrinogens, IJmuiden and Nijmegen, were determined by sequence analysis of genomic DNA amplified by the polymerase chain reaction. Both fibrinogens were heterozygous, IJmuiden having a B beta Arg14----Cys substitution and Nijmegen having a B beta Arg44----Cys substitution. Clotting induced by thrombin or Reptilase was impaired in both fibrinogens, indicating defective fibrin polymerization. Immunoblot analysis of both purified fibrinogens demonstrated that some of the abnormal molecules were linked by disulfide bonds to albumin. In addition, abnormal high molecular weight fibrinogen complexes with Mrs between 600,000 and 700,000 were present. Fibrinogen-albumin and high molecular weight complexes were also detected in the patients' plasmas. Quantitative analysis demonstrated that of the total plasma fibrinogen in the IJmuiden patient, 20% was linked to albumin and 10% was present as high molecular weight complexes. In plasma Nijmegen, 13% was linked to albumin and 15% was present as high molecular weight complexes. These results demonstrate that the additional abnormal cysteine in fibrinogens IJmuiden and Nijmegen resulted in the formation of disulfide-linked complexes with other proteins, predominantly albumin. We also found that a significant fraction of the abnormal fibrinogen molecules contained free sulfhydryl groups. These findings complicate interpretation of functional studies of these altered fibrinogens.

    Funded by: NHLBI NIH HHS: HL31048

    Proceedings of the National Academy of Sciences of the United States of America 

  • Epistatic and pleiotropic effects of polymorphisms in the fibrinogen and coagulation factor XIII genes on plasma fibrinogen concentration, fibrin gel structure and risk of myocardial infarction.

    Mannila MN, Eriksson P, Ericsson CG, Hamsten A and Silveira A

    King Gustaf V Research Institute, Karolinska University Hospital Solna, S-171 76 Stockholm, Sweden, Maria.Nastase.Mannila@ki.se

    An intricate interplay between the genes encoding fibrinogen gamma (FGG), alpha (FGA) and beta (FGB), coagulation factor XIII (F13A1) and interleukin 6 (IL6) and environmental factors is likely to influence plasma fibrinogen concentration, fibrin clot structure and risk of myocardial infarction (MI). In the present study, the potential contribution of SNPs harboured in the fibrinogen, IL6 and F13A1 genes to these biochemical and clinical phenotypes was examined. A database and biobank based on 387 survivors of a first MI and population-based controls were used. Sixty controls were selected according to FGG 9340T > C [rs1049636] genotype for studies on fibrin clot structure using the liquid permeation method. The multifactor dimensionality reduction method was used for interaction analyses. We here report that the FGA 2224G > A [rs2070011] SNP (9.2%), plasma fibrinogen concentration (13.1%) and age (8.1%) appeared as independent determinants of fibrin gel porosity. The FGA 2224G > A SN 17c2 P modulated the relation between plasma fibrinogen concentration and fibrin clot porosity. The FGG-FGA*4 haplotype, composed of the minor FGG 9340C and FGA 2224A alleles, had similar effects, supporting its reported protective role in relation to MI. Significant epistasis on plasma fibrinogen concentration was detected between the FGA 2224G > A and F13A1 Val34Leu [rs5985] SNPs (p < 0.001). The FGG 9340T > C and FGB 1038G > A [rs1800791] SNPs appeared to interact on MI risk, explaining the association of FGG-FGB haplotypes with MI in the absence of effects of individual SNPs. Thus, epistatic and pleiotropic effects of polymorphisms contribute to the variation in plasma fibrinogen concentration, fibrin clot structure and risk of MI.

  • Mutations and polymorphisms in genes affecting haemostasis components in children with thromboembolic events.

    Komitopoulou A, Platokouki H, Kapsimali Z, Moschovi M, Kattamis A, Pergantou H and Aronis S

    Haemostasis/Haemophilia Unit, University of Athens, Aghia Sophia Children's Hospital, Athens, Greece. theokom@otenet.gr

    The distribution of mutations/polymorphisms in genes affecting haemostasis [factor V Leiden (FVL), FV H1298R (FVR(2)), FII 20210A, b-Fib 455G-->A, FXIII V34L, PAI-1 4G, HPA-1b] among 141 children with thrombosis at various sites and 103 controls was compared. Additionally, the carriage of these mutations/polymorphisms was associated with the levels of their corresponding proteins in thrombosed children. Thrombosis was more frequent in boys (p = 0.021). No studied mutation/polymorphism was found to be a risk factor for thrombosis, except for FVL (odds ratio 3.8, 95% CI 1.4-10.6). The risk of thrombosis for FVL carriers was twice as high in children with an idiopathic thrombosis (odds ratio 5.4) than in thrombosed children with an underlying disease or a triggering event (odds ratio 2.7). FVL carriers had an odds ratio of 5.9 (95% CI 1.8-19.6) when FVR(2) was absent. In thrombosed children, the activated protein C resistance ratio was significantly lower in the presence of FVL (p < 0.001). Prothrombin and fibrinogen levels, although higher in FII 20210A and b-Fib 455G-->A carriers, did not reach statistical significance.

    Pathophysiology of haemostasis and thrombosis 

  • Genetic cardiovascular risk factors and age-related macular degeneration.

    Haas P, Aggermann T, Steindl K, Krugluger W, Pühringer H, Oberkanins C, Frantal S and Binder S

    Ludwig Boltzmann Institute for Retinology and Biomicroscopic Lasersurgery, Rudolf Foundation Clinic, Vienna, Austria. paulina.haas@wienkav.at

    Purpose: To investigate the association between genetic cardiovascular risk factors and exudative age-related macular degeneration (AMD) in a White Austrian population.

    Methods: Seventy-five unrelated AMD patients and 75 unrelated healthy, sex- and age-matched control patients were genotyped for the following 19 single nucleotide polymorphisms (SNPs) in 14 different genes: blood coagulation factor V (FV) R506Q, factor II (prothrombin) G20210A and factor XIII (FXIII) V34L; 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; plasminogen activator inhibitor 1 (PAI-1) 4G/5G; endothelial protein C receptor (EPCR) 4600 A>G (A3 haplotype), 4678 G>C (A1 haplotype); apolipoprotein B (ApoB) R3500Q; apolipoprotein E (ApoE) E2/E3/E4; β-fibrinogen -455 G>A; human platelet antigen 1 (HPA1) a/b; angiotensin-converting enzyme (ACE) I/D; endothelial nitric oxide synthase (eNOS) 786 T>C, 894 G>T; lymphotoxin alpha (LTA) 804 C>A and 9p21 rs10757278. Genotyping was carried out by polymerase chain reaction (PCR) followed by reverse hybridization (CVD StripAssays; ViennaLab Diagnostics, Vienna, Austria).

    Results: No statistically significant association could be observed between AMD and the investigated genetic risk factors for cardiovascular disease (CVD). All factors seem to be uniformly distributed in the two groups of AMD patients and healthy controls. Two variables -β-fibrinogen: -455 G>A (p = 0.0786) and apolipoprotein E4 (p = 0.0636) - were not as far from association as the others.

    Conclusion: Our data show that the 19 tested CVD risk markers do not play a significant role in AMD. β-Fibrinogen and apolipoprotein E4 should be examined in a larger cohort.

    Acta ophthalmologica 2011;89;4;335-8

  • The effect of four hemostatic gene polymorphisms on the outcome of septic critically ill patients.

    Tsantes AE, Tsangaris I, Bonovas S, Kopterides P, Rapti E, Dimopoulou I, Markatos C, Orfanos S, Armaganidis A and Travlou A

    Laboratory of Haematology & Blood Bank Unit, Attikon University General Hospital, Medical School, University of Athens, 1 Rimini Str., Athens, Greece. atsantes@yahoo.com

    Genetic variants of hemostatic factors leading to prothrombotic phenotypes of hypercoagulability and hypofibrinolysis might affect prognosis of septic critically ill patients. Our aim was to evaluate the effect of four hemostatic genetic variants, namely fibrinogen-beta-455G/A, factor XIII (FXIII) V34L, plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphisms and factor V Leiden (FVL) mutation on survival of critically ill patients with severe sepsis or septic shock. A prospective, observational study in an 18-bed general ICU included 73 patients with severe sepsis or septic shock. Epidemiological, laboratory data and comorbidities along with severity scores were recorded. Genotyping for fibrinogen-beta-455G/A, FXIII V34L and PAI-1 4G/5G polymorphism and FVL mutation was carried out in all patients. The primary outcomes were the 28-day and the 90-day survival. Age, septic shock, severity indexes, prior steroid use and arterial pH were identified as predictors of the 28-day and 90-day survival in both the univariate and the multivariate models. On the contrary, none of the examined polymorphisms was found to significantly affect either the 28-day or the 90-day survival. Our data suggest that the importance of these hemostatic polymorphisms as predictors of the prognosis of sepsis in critically ill patients is probably very small.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2010;21;2;175-81

  • Influence of Amerindian mit 1ad ochondrial DNA haplogroups on thrombosis susceptibility and frequency of four genetic prothrombotic variants in Southern Chilean subjects.

    Guzmán N, Lanas F and Salazar LA

    Laboratorio de Biología Molecular & Farmacogenética, Departamento de Ciencias Básicas, Facultad de Medicina, Francisco Salazar 01145, Casilla 54-D, Temuco, Chile.

    Background: Recent evidence suggests that the ethnic background may determine the susceptibility to cardiovascular diseases. Considering the genetic composition of Chilean population, the aim of the present study was to evaluate the possible association between Amerindian mitochondrial DNA (mtDNA) haplogroups and venous thrombosis susceptibility and the influence on frequency of factor V 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 677C>T and beta-fibrinogen -148C>T polymorphisms in Southern Chilean population.

    Methods: A total of 172 individuals, 60 patients with diagnosis of deep venous thrombosis (DVT) confirmed by Doppler ultrasonography and 112 controls were included in this study. The single nucleotide polymorphisms (SNP) and the Amerindian mtDNA haplogroups were detected by PCR and PCR-RFLP techniques, respectively.

    Results: The presence of mtDNA haplogroups did not modify the thrombosis susceptibility. Of 4 SNPs genotyped, only the MTHFR 677C>T variant was more frequent in DVT patients when compared to controls (OR 3.46; 95%CI, 1.50-8.00). A lower frequency of 1691G>A (factor V) and a total absence of prothrombin 20210G>A variants were observed in subjects with Amerindian background.

    Conclusions: The presence of Amerindian haplogroups did not modify the susceptibility to DVT in the studied subjects. Only MTHFR 677C>T polymorphism was associated to venous thrombosis in Chilean subjects. The lower frequency of factor V 1691G>A and the absence of prothrombin 20210G>A polymorphism confirm the poor utility of the molecular detection of these variants in the thrombophilia study in populations with Amerindian background.

    Clinica chimica acta; international journal of clinical chemistry 2010;411;5-6;444-7

  • Identification of human plasma proteins as major clients for the extracellular chaperone clusterin.

    Wyatt AR and Wilson MR

    School of Biological Sciences, University of Wollongong, Wollongong, New South Wales 2522, Australia.

    Clusterin (CLU) is an extracellular chaperone that is likely to play an important role in protein folding quality control. This study identified three deposition disease-associated proteins as major plasma clients for clusterin by studying CLU-client complexes formed in response to physiologically relevant stress (shear stress, approximately 36 dynes/cm(2) at 37 degrees C). Analysis of plasma samples by size exclusion chromatography indicated that (i) relative to control plasma, stressed plasma contained proportionally more soluble protein species of high molecular weight, and (ii) high molecular weight species were far more abundant when proteins purified by anti-CLU immunoaffinity chromatography from stressed plasma were compared with those purified from control plasma. SDS-PAGE and Western blot analyses indicated that a variety of proteins co-purified with CLU from both stressed and control plasma; however, several proteins were uniquely present or much more abundant when plasma was stressed. These proteins were identified by mass spectrometry as ceruloplasmin, fibrinogen, and albumin. Immunodot blot analysis of size exclusion chromatography fractionated plasma suggested that CLU-client complexes generated in situ are very large and may reach >or=4 x 10(7) Da. Lastly, sandwich enzyme-linked immunosorbent assay detected complexes containing CLU and ceruloplasmin, fibrinogen, or albumin in stressed but not control plasma. We have previously proposed that CLU-client complexes serve as vehicles to dispose of damaged misfolded extracellular proteins in vivo via receptor-mediated endocytosis. A better understanding of these mechanisms is likely to ultimately lead to the identification of new therapies for extracellular protein deposition disorders.

    The Journal of biological chemistry 2010;285;6;3532-9

  • A prospective case-control study analyzes 12 thrombophilic gene mutations in Turkish couples with recurrent pregnancy loss.

    Yenicesu GI, Cetin M, Ozdemir O, Cetin A, Ozen F, Yenicesu C, Yildiz C and Kocak N

    Department of Obstetrics and Gynecology, Cumhuriyet University School of Medicine, Sivas, Turkey. imirgonca@yahoo.com

    Problem: Recurrent pregnancy loss (RPL) is a heterogeneous disorder. The contribution of specific thrombophilic genes to the pathophysiology of RPL has remained controversial. We evaluated the prevalences of 12 thrombophilic gene mutations among homogenous Caucasian couples with RPL and fertiles.

    Method: of study This was a prospective case-control study evaluating 272 women with RPL and 152 of their male partners, and a control group of 56 fertile couples. We investigated mutations including FV Leiden, factor V H1299R, factor II prothrombin G20210A, F XIII V34L, beta-fibrinogen -455G>A, plasminogen activator inhibitor-1, GPIIIa L33P (HPA-1 a/b L33P), MTHFR C677T, MTHFR A1298C, ACE I/D, Apo B R3500Q, and Apo E.

    Results: Overall, heterozygous mutations of FV Leiden, FXIII V34L, GPIIIa L33P, Apo E4, and prothrombin G20210A and homozygous mutations of PAI-1and MTHFR C677T were associated with RPL. There was no meaningful association between RPL and other studied genes.

    Conclusion: In contrast to the other mutations and polymorphisms, FV Leiden, FXIII V34L, GPIIIa L33P, Apo E, prothrombin G20210A, PAI-1 and MTHFR C677T gene mutations may help to identify the couples at risk for recurrent pregnancy loss.

    American journal of reproductive immunology (New York, N.Y. : 1989) 2010;63;2;126-36

  • Hemostatic gene polymorphisms in young Sardinian with non-fatal acute myocardial infarction.

    Musino L, Rossi R, Partenza A, Mureddu G, Zinellu A, Pilo P, Maoddi I, Spazzafumo L, Franconi F, Deiana L and Carru C

    Department of Biomedical Sciences, Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, Sassari, Italy.

    Although the role of environmental factors in the development of acute myocardial infarction (AMI) has been clearly established, the role of genetic factors is still undefined. The aim of this study was to investigate the association between various gene polymorphisms in the haemostatic system and the risk of myocardial infarction in a very genetic restricted area population of Sardinian young adults with AMI. The study case-control involved 71 patients who had survived a first MI at a mean age of 47,2 years and 150 healthy subjects. No differences in the allele or genotype frequencies were seen between the study groups for the fibrinogen, prothrombin, factor V, factor VII, vWF, TM, PAI-1, TPO gene, and PLA and HPA-2 genes polymorphisms. Indeed differences statistically significant were detected for A5709G in the TPO gene (P= 0,041), and I/D dimorphism in the eNOS gene (P= 0,016). We therefore conclude that among all the investigated polymorphisms only the 5709G and eNOS4a alleles seem to confer protection against MI in the young age of Sardinian people.

    Frontiers in bioscience (Elite edition) 2010;2;559-65

  • G-455A polymorphism of beta-fibrinogen gene and the risk of premature myocardial infarction in Greece.

    Rallidis LS, Gialeraki A, Fountoulaki K, Politou M, Sourides V, Travlou A, Lekakis I and Kremastinos DT

    Second Department of Cardiology, Attikon Hospital, School of Medicine, University of Athens, Greece. rallidis@ath.forthnet.gr

    Introduction: There are limited and controversial data regarding the impact of G-455A polymorphism of beta-fibrinogen gene in the pathogenesis of premature myocardial infarction (MI). We examined whether the G-455A polymorphism of beta-fibrinogen gene is associated with the development of MI< or =35 years of age.

    Methods: We recruited 181 consecutive patients who had survived their first acute MI< or =35 years of age (mean age=32.2+/-3.4 years). The control group consisted of 129 healthy individuals matched with cases for age and sex, without a family history of premature coronary heart disease. G-455A polymorphism of beta-fibrinogen was tested with polymerase chain reaction and reverse hybridization.

    Results: There was a higher prevalence of carriers of the A allele (GA+AA genotype) in controls than in patients (odds ratio [OR] 0.57, 95% confidence interval [CI] 0.36 to 0.91, p=0.02). G-455A polymorphism of beta-fibrinogen gene was associated with lower risk for acute MI (OR 0.46, 95% CI 0.25 to 0.83, p=0.01) after adjusting for major cardiovascular risk factors. Fibrinogen levels were higher in patients compared to controls [332 (292-385) vs. 311 (262-373) mg/dL, p=0.01], but the adjusted for classical risk factors fibrinogen levels did not differ (OR 1.003, 95% CI 0.99 to 1.01, p=0.37). Patients possessing the A allele did not differ in their fibrinogen and lipid levels compared to patients with the -455GG genotype.

    Conclusions: Our data indicate that the presence of the G-455A polymorphism of beta-fibrinogen gene has a "protective effect" against the development of non-fatal acute MI< or =35 years of age in Greece.

    Thrombosis research 2010;125;1;34-7

  • Impact of genetic polymorphisms on platelet function and aspirin resistance.

    Pamukcu B, Oflaz H, Onur I, Hancer V, Yavuz S and Nisanci Y

    Istanbul University, Istanbul Faculty of Medicine, Department of Cardiology, Turkey. bpamukcu@istanbul.edu.tr

    Genetic polymorphisms may affect platelets' responses to the antiplatelet therapy. Our aim was to determine the role of genetic polymorphisms on aspirin resistance in patients with coronary heart disease (CHD). A total of 126 consecutive patients (35-85 years old, 32% women) with chronic stable CHD was enrolled in the study. Platelet function assays were realized by the platelet function analyzer (PFA)-100 with collagen and epinephrine (Col/Epi) and collagen and adenosine diphosphate (Col/ADP) cartridges. Aspirin resistance was defined as having a closure time of less than 186 s with Col/Epi cartridges despite regular aspirin therapy. Factor V, prothrombin, factor XIII, beta-fibrinogen, plasminogen activator inhibitor I (PAI-1), glycoprotein IIIa, methylene tetrahydrofolate reductase, ACE and ApoB gene polymorphisms were determined by three consecutive steps: isolation and amplification of DNA and reverse hybridization. We determined that 30 patients (23.8%) had aspirin resistance by the PFA-100. Mean closure time measured with the Col/ADP cartridges was 74 +/- 12 s (51-104 s). Ten of the 30 patients with aspirin resistance were women (33.3%). Genetic polymorphisms were determined in 30 aspirin-resistant and 17 aspirin-sensitive patients. No statistically significant relationship was determined between aspirin resistance and the genetic panel. In our study we did not determine a significant relationship between the aspirin resistance and factor V, prothrombin, factor XIII, beta-fibrinogen, PAI-1, glycoprotein IIIa, methylene tetrahydrofolate reductase, ACE and ApoB gene polymorphisms.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2010;21;1;53-6

  • Interaction between fibrinogen and IL-6 genetic variants and associations with cardiovascular disease risk in the Cardiovascular Health Study.

    Carty CL, Heagerty P, Heckbert SR, Jarvik GP, Lange LA, Cushman M, Tracy RP and Reiner AP

    Department of Epidemiology, University of Washington, Seattle, USA. calyca@u.washington.edu

    The inflammatory cytokine interleukin-6 (IL-6) is a main regulator of fibrinogen synthesis, though its interaction with fibrinogen genes (FGA, FGB, FGG) and subsequent im 1d pact on cardiovascular diseas 1f1b e (CVD) risk is not well-studied. We investigated joint associations of fibrinogen and IL6 tagSNPs with fibrinogen concentrations, carotid intima-media thickness, and myocardial infarction or ischemic stroke in 3900 European-American Cardiovascular Health Study participants. To identify combinations of genetic main effects and interactions associated with outcomes, we used logic regression. We also evaluated whether the relationship between fibrinogen SNPs and fibrinogen level varied by IL-6 level using linear regression models with multiplicative interaction terms. Combinations of fibrinogen and IL6 SNPs were significantly associated with fibrinogen level (p < 0.005), but not with other outcomes. Fibrinogen levels were higher in individuals having FGB1437 (rs1800790) and lacking FGA6534 (rs6050) minor alleles; these SNPs interacted with IL6 rs1800796 to influence fibrinogen level. Marginally significant (p= 0.03) interactions between IL-6 level and FGA and FGG promoter SNPs associated with fibrinogen levels were detected. We identified potential gene-gene interactions influencing fibrinogen levels. Although IL-6 responsive binding sites are present in fibrinogen gene promoter regions, we did not find strong evidence of interaction between fibrinogen SNPs and IL6 SNPs or levels influencing CVD.

    Funded by: NHLBI NIH HH 1f40 S: N01-HC-85079; NHLBI NIH HHS: N01 HC-55222, N01 HC015103, N01 HC035129, N01 HC045133, N01-HC-75150, N01-HC-85080, N01-HC-85081, N01-HC-85082, N01-HC-85083, N01-HC-85084, N01-HC-85085, N01-HC-85086, N01HC55222, N01HC75150, N01HC85079, N01HC85086, R01 HL071862, R01 HL071862-05A1, T32 HL007902, T32 HL007902-10, U01 HL080295, U01 HL080295-01

    Annals of human genetics 2010;74;1;1-10

  • Fulfilling the promise of personalized medicine? Systematic review and field synopsis of pharmacogenetic studies.

    Holmes MV, Shah T, Vickery C, Smeeth L, Hingorani AD and Casas JP

    Centre for Clinical Pharmacology, University College London, London, United Kingdom.

    Background: Studies of the genetic basis of drug response could help clarify mechanisms of drug action/metabolism, and facilitate development of genotype-based predictive tests of efficacy or toxicity (pharmacogenetics).

    Objectives: We conducted a systematic review and field synopsis of pharmacogenetic studies to quantify the scope and quality of available evidence in this field in order to inform future research.

    Original research articles were identified in Medline, reference lists from 24 meta-analyses/systematic reviews/review articles and U.S. Food and Drug Administration website of approved pharmacogenetic tests. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS, AND INTERVENTION CRITERIA: We included any study in which either intended or adverse response to drug therapy was examined in relation to genetic variation in the germline or cancer cells in humans.

    Study characteristics and data reported in abstracts were recorded. We further analysed full text from a random 10% subset of articles spanning the different subclasses of study.

    Results: From 102,264 Medline hits and 1,641 articles from other sources, we identified 1,668 primary research articles (1987 to 2007, inclusive). A high proportion of remaining articles were reviews/commentaries (ratio of reviews to primary research approximately 25 ratio 1). The majority of studies (81.8%) were set in Europe and North America focussing on cancer, cardiovascular disease and neurology/psychiatry. There was predominantly a candidate gene approach using common alleles, which despite small sample sizes (median 93 [IQR 40-222]) with no trend to an increase over time, generated a high proportion (74.5%) of nominally significant (p<0.05) reported associations suggesting the possibility of significance-chasing bias. Despite 136 examples of gene/drug interventions being the subject of >or=4 studies, only 31 meta-analyses were identified. The majority (69.4%) of end-points were continuous and likely surrogate rather than hard (binary) clinical end-points.

    The high expectation but limited translation of pharmacogenetic research thus far may be explained by the preponderance of reviews over primary research, small sample sizes, a mainly candidate gene approach, surrogate markers, an excess of nominally positive to truly positive associations and paucity of meta-analyses. Recommendations based on these findings should inform future study design to help realise the goal of personalised medicines. SYSTEMATIC REVIEW REGISTRATION NUMBER: Not Registered.

    Funded by: British Heart Foundation: FS 05/125; Medical Research Council: G0802432; Wellcome Trust: 082178

    PloS one 2009;4;12;e7960

  • A candidate gene approach to genetic prognostic factors of IgA nephropathy--a result of Polymorphism REsearch to DIstinguish genetic factors Contributing To progression of IgA Nephropathy (PREDICT-IgAN).

    Yamamoto R, Nagasawa Y, Shoji T, Inoue K, Uehata T, Kaneko T, Okada T, Yamauchi A, Tsubakihara Y, Imai E, Isaka Y and Rakugi H

    Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Osaka, Japan.

    Background: Renal prognosis of IgA nephropathy (IgAN) is affected by environmental and genetic factors. Other studies demonstrated that some atherosclerotic disease-related genes were significantly associated with renal prognosis.

    Methods: The Polymorphism REsearch to DIstinguish genetic factors Contributing To progression of IgAN (PREDICT-IgAN) was a multicentre retrospective observational study to investigate associations between progression of IgAN (a 50% increase of serum creatinine level and slope of eGFR) and a hundred atherosclerotic disease-related gene polymorphisms, mainly single nucleotide polymorphisms (SNPs) in 320 IgAN patients who had more than a normal range of urinary protein (> or =0.25 g/day) at diagnosis.

    Results: During 8.3 +/- 4.2 years of a follow-up period, 83 patients (25.9%) developed progression. In log-rank tests, glycoprotein Ia GPIa C807T and G873A and intercellular adhesion molecule-1 ICAM-1 A1548G (K469E) were found to be significantly associated with progression even after adjustment for multiple comparisons by the method of Bonferroni (adjusted P = 0.0174, 0.0176 and 0.0430, respectively). In a multivariate Cox proportional-hazards model, GPIa 807TT (873CC) [versus 807TT, adjusted hazard ratio 2.05 (95% confidence interval 1.13-3.71)] and ICAM-1 1548GG [versus 1548AA, 2.55 (1.40-4.65)] were identified as independent genetic predictors of progression, along with conventional clinical prognostic factors such as eGFR, urinary protein and use of antihypertensives at diagnosis.

    Conclusions: PREDICT-IgAN distinguished GPIa C807T/ G873A and ICAM-1 A1548G from multiple athero- sclerotic disease-related gene polymorphisms by their predictive indicator for progression of IgAN.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2009;24;12;3686-94

  • Genetic risk factors for periodontitis in a Japanese population.

    Kobayashi T, Nagata T, Murakami S, Takashiba S, Kurihara H, Izumi Y, Numabe Y, Watanabe H, Kataoka M, Nagai A, Hayashi J, Ohyama H, Okamatsu Y, Inagaki Y, Tai H and Yoshie H

    General Dentistry and Clinical Education Unit, Niigata University Medical and Dental Hospital, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan. kotetsuo@dent.niigata-u.ac.jp

    Genetic variants at multiple loci have been shown to be associated with susceptibility to periodontitis. To better assess the genetic risk factors for periodontitis, we performed a case-control study in 319 Japanese individuals with periodontitis (172 aggressive and 147 chronic disease) and 303 race-matched healthy control individuals. Thirty-five functional gene polymorphisms that had been previously associated with immune responses were genotyped. For all gene polymorphisms tested, no significant differences were observed in the allele frequencies of persons with aggressive, chronic, and combined (aggressive and chronic) periodontitis, compared with control individuals. Multiple logistic regression analysis revealed a significant association of the vitamin D receptor +1056 T/C polymorphism with susceptibility to chronic periodontitis, after adjustment for age, gender, and smoking status (P = 0.002). These results suggest that none of the polymorphisms tested was strongly associated with periodontitis in a Japanese population. However, the vitamin D receptor +1056 polymorphism may be related to chronic periodontitis.

    Journal of dental research 2009;88;12;1137-41

  • Early pregnancy loss in celiac women: The role of genetic markers of thrombophilia.

    Ciacci C, Tortora R, Scudiero O, Di Fiore R, Salvatore F and Castaldo G

    Gastroenterologia, Dipartimento di Medicina Clinica e Sperimentale, University of Naples Federico II, Naples, Italy. ciacci@unina.it

    Background: Adverse pregnancy outcomes are more frequent in celiac than in non-celiac women.

    Aims: To investigate a possible role of genetic prothrombotic variants in early pregnancy loss of celiac women.

    Methods: Thirty-nine celiac women who had experienced early pregnancy losses (at least two losses within the first 3 months of pregnancy), and 72 celiac women with a history of one or more normal pregnancies and no pregnancy loss (controls) entered the study, at the moment of diagnosis for celiac disease. A clinical history was obtained from each woman. DNA from leukocytes was tested for: factor V Leiden (mutation G1691A), factor V R2 (H1299R), factor II (G20210A), methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), beta-fibrinogen (-455 G>A), PAI-1 alleles 4G/5G, factor XIII (V34L), and HPA-1 (L33P).

    Results: Age at diagnosis was significantly higher (p=0.002) in the celiac women with pregnancy losses than in controls. Of the gene variants studied, the allelic frequency of 4G variant of PAI-1, and the frequency of mutant genotypes were significantly more frequent in the group of celiac women with early pregnancy loss (p=0.00003 and 0.028, respectively). Surprisingly, the beta-fibrinogen -455 G>A genotype distribution (but not the allelic frequency of the variant allele) significantly differed between the two groups, since variant genotypes were more frequent in the control group (p=0.009).

    Conclusion: The 4G variant of the PAI-I gene may predispose to miscarriage a subset of celiac women; these data should be verified on larger populations.

    Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver 2009;41;10;717-20

  • [Complex analysis of genetic predisposition to ischemic stroke in Russians].

    Parfenov MG, Titov BV, Sudomoina MA, Martynov MIu, Favorov AV, Ochs MF, Gusev EI and Favorova OO

    Carriage frequencies of alleles and genotypes of 10 functionally important single nucleotide polymorphisms that are located in genes FGA, FGB, APOE, LPL, ACE and CMA1 were analyzed in the ischemic stroke (IS) patients of Russian ethnic descent and in the control group of the same ethnic descent and of similar gender and age. Comparison between patients and control group revealed no significant differences in frequencies of individual alleles and genotypes for all the polymorphic loci studied. However, complex analysis of genetic predisposition using APSampler algorithm revealed carriage of allele (-491A) APOE as a predisposing factor for IS (p = 0.044, OR 3.8, 95% CI 1.0-15.1). Accordingly, carriage of genotype (-491T/T) APOE was associated with resistance to IS (p = 0.044, OR 0.26, 95% CI 0.07-1.0). The allele -249C FGB carriage addition to this genotype enhances its protective properties, p-value of the combination is 2-fold lower than that of the genotype (-491T/T) APOE (OR 0.17, 95% CI 0.04-0.8). Two more protective combinations were identified: biallelic (-427C) APOE + (1595G) LPL and triallelic (-491C) APOE + (1595G) LPL + (-1903G) CMAI (in both cases p = 0.0052, OR 0.18, 95% CI 0.05-0.66). Overall, involvement in formation of the risk of IS development in Russians was evidenced for alleles of four genes: APOE, FGB, LPL and CMA1, where APOE gene involvement was evidenced for alleles of two polymorphic loci, -491T and -427C. Linkage analysis suggested that involvement of these loci in insusceptibility to IS is mutually independent.

    Funded by: NLM NIH HHS: LM008932

    Molekuliarnaia biologiia 2009;43;5;937-45

  • Modification of the interleukin-6 response to air pollution by interleukin-6 and fibrinogen polymorphisms.

    Ljungman P, Bellander T, Schneider, Breitner S, Forastiere F, Hampel R, Illig T, Jacquemin B, Katsouyanni K, von Klot S, Koenig W, Lanki T, Nyberg F, Pekkanen J, Pistelli R, Pitsavos C, Rosenqvist M, Sunyer J and Peters A

    Department of Cardiology, Karolinska Institutet, Stockholm South Hospital, Stockholm, Sweden. petter.ljungman@ki.se

    Background: Evidence suggests that cardiovascular effects of air pollution are mediated by inflammation and that air pollution can induce genetic expression of the interleukin-6 gene (IL6).

    Objectives: We investigated whether IL6 and fibrinogen gene variants can affect plasma IL-6 responses to air pollution in patients with cardiovascular disease.

    Methods: We repeatedly determined plasma IL-6 in 955 myocardial infarction survivors from six European cities (n = 5,539). We conducted city-specific analyses using additive mixed models adjusting for patient characteristics, time trend, and weather to assess the impact of air pollutants on plasma IL-6. We pooled city-specific estimates using meta-analysis methodology. We selected three IL6 single-nucleotide polymorphisms (SNPs) and one SNP each from the fibrinogen alpha-chain gene (FGA) and beta-chain gene (FGB) for gene-environment analyses.

    Results: We found the most consist 76b ent modifications for variants in IL6 rs2069832 and FBG rs1800790 after exposure to carbon monoxide (CO; 24-hr average; p-values for interaction, 0.034 and 0.019, respectively). Nitrogen dioxide effects were consistently modified, but p-values for interaction were larger (0.09 and 0.19, respectively). The strongest effects were seen 6-11 hr after exposure, when, for example, the overall effect of a 2.2% increase in IL-6 per 0.64 mg/m(3) CO was modified to a 10% (95% confidence interval, 4.6-16%) increase in IL-6 (p-value for interaction = 0.002) for minor homozygotes of FGB rs1800790.

    Conclusions: The effect of gaseous traffic-related air pollution on inflammation may be stronger in genetic subpopulations with ischemic heart disease. This information could offer an opportunity to identify postinfarction patients who would benefit more than others from a cleaner environment and antiinflammatory treatment.

    Environmental health perspectives 2009;117;9;1373-9

  • [Relationship between multi-locus fibrinogen polymorphisms and fibrinogen concentration, molecular reactivity and cerebral infarction].

    Yuan XD, Wang SJ, Xu YR, Gao J, Yang N, Li J and Li HF

    Department of Neurology, Kailuan Hospital, North China Coal Medical College, Tangshan 063000, China.

    Objective: To study the distribution characteristics of Beta-fibrinogen (Fg)B gene-854G/A, -455G/A, -249C/T, -148C/T, 448G/A and Bcl-1 G/A polymorphism in North China Han population, and the influence on plasma Fg concentration and molecular reactivity. Further more, to explore the role of Fg gene polymorphisms combining with multi-physiological and environmental factors in the development of cerebral infarction.

    Methods: Cluster sampling, health examination and questionnaires surveys of 1652 subjects from Tangshan Kailuan Group Corporation were conducted. Blood biochemistry, Fg concentration, fibrin monomer polymerized velocity (FMPV), absorbance maximum (Amax) and FMPV/Amax were measured. The six polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism.

    Results: In the population, the proportion of the FgB beta-249 T variation allele was 65.49%, while the proportion of the rest loci was predominantly wild type. The significant differences in Fg concentration and FMPV/Amax were found in -854 genotype groups. The Fg concentration in -854GA group was higher than those in GG and AA group. Only the distribution frequencies of FgB beta Bcl-1 A variation allele, GA and AA genotype in the cerebral infarction group were higher than those in non-infarction group, and the prevalence of cerebral infarction in AA genotype group was higher than other groups (the probability value of above-mentioned results were all P<0.01).

    Conclusions: FgB beta Bcl-1A allele and variation genotype were susceptible to cerebral infarction. FgB beta-455GA/448G linkage genotype may contribute to the increased plasma Fg concentration. FgB beta-854 was one of the main controlling gene loci for plasma Fg concentration and molecular reactivity.

    Zhonghua xue ye xue za zhi = Zhonghua 1f40 xueyexue zazhi 2009;30;9;582-7

  • High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension.

    Morris TA, Marsh JJ, Chiles PG, Magaña MM, Liang NC, Soler X, Desantis DJ, Ngo D and Woods VL

    Division of Pulmonary and Critical Care Medicine, University of California, San Diego, CA 92103-8378, USA. t1morris@ucsd.edu

    The mechanism by which chronic thromboembolic pulmonary hypertension (CTEPH) develops after acute pulmonary thromboembolism is unknown. We previously reported that fibrin from CTEPH patients is relatively resistant to fibrinolysis in vitro. In the present study, we performed proteomic, genomic, and functional studies on fibrin(ogen) to investigate whether abnormal fibrin(ogen) might contribute to the pathogenesis of CTEPH. Reduced and denatured fibrinogen from 33 CTEPH patients was subjected to liquid chromatography-mass spectrometry analysis. Fibrinogen from 21 healthy controls was used to distinguish atypical from commonly occurring mass peaks. Atypical peaks were further investigated by targeted genomic DNA sequencing. Five fibrinogen variants with corresponding heterozygous gene mutations (dysfibrinogenemias) were observed in 5 of 33 CTEPH patients: Bbeta P235L/gamma R375W, Bbeta P235L/gamma Y114H, Bbeta P235L, Aalpha L69H, and Aalpha R554H (fibrinogens(San Diego I-V)). Bbeta P235L was found in 3 unrelated CTEPH patients. Functional analysis disclosed abnormalities in fibrin polymer structure and/or lysis with all CTEPH-associated mutations. These results suggest that, in some patients, differences in the molecular structure of fibrin may be implicated in the development of CTEPH after acute thromboembolism.

    Funded by: NCI NIH HHS: 2 P30CA023100-23, CA099835, CA118595, P30 CA023100, R21 CA118595, R33 CA099835; NHLBI NIH HHS: HL-080302, HL-095089, R01 HL095089, R21 HL080302; NIAID NIH HHS: AI076961, R21 AI076961

    Blood 2009;114;9;1929-36

  • Clinical factors such as B-type natriuretic peptide link to factor VII, endothelial NO synthase and estrogen receptor alpha polymorphism in elderly women.

    Funami J, Hayashi T, Nomura H, Ding QF, Ishitsuka-Watanabe A, Matsui-Hirai H, Ina K, Zhang J, Yu ZY and Iguchi A

    Department of Geriatrics, Nagoya University Graduate School of Medicine, Tsuruma-cho 65, Showa-ku, Nagoya, Aichi 466-8550, Japan.

    Aims: This study evaluated the presence of genetic mutations in relation to thrombosis or atherosclerosis in elderl 2ec y women.

    This is an observational study of 93 Japanese women with a mean age of 80.9 years recruited from outpatient clinics of Nagoya University and its related hospitals. Ten single nucleotide polymorphisms (SNPs) were studied. Each gene studied acts in or is related to either blood coagulation (factor V Leiden, prothrombin G20210A, factor XIII Val34Leu, factor VII Arg353Gln, MTHFR C677T, beta-fibrinogen G-455A, PAI-1 4G/5G), metabolic syndrome-related pathways (PPARalpha Leu162Val), or endothelium/estrogen system (eNOS Glu298Asp, ERalpha IVS1-401). SNPs were analyzed for their relation to clinical values including lipids, B-type natriuretic peptide 1f40 (BNP), fasting plasma glucose, tumor necrosis factor-alpha, interleukin-6, cyclic GMP, and nitric oxide metabolites.

    Comparisons between the distributions of different genotypes and clinical values showed three relationships. First, factor VII Arg353Gln and HDL-cholesterol (HDL-C) were linked to Arg/Arg carriers at higher levels (P=.049). The HDL-C to LDL-cholesterol ratio supported this link (P=.027). Second, eNOS Glu298Asp and triglycerides were linked to Glu/Glu carriers at higher levels (P=.031). Third, ERalpha IVS1-401 and BNP were related to CC genotype at lower levels (P=.031). Additionally, the last two relations showed that genotype does not influence the demarcation line of biomarkers, but the plasma/serum levels of biomarkers instead.

    Significance: Correlations of factor VII Arg353Gln with HDL-C and eNOS Glu298Asp with triglycerides are new findings. Polymorphisms in the endothelium/estrogen system and the heart failure marker BNP are also correlated, with ERalpha IVS1-401 being the first identified marker. SNPs may be helpful for understanding the pathophysiology of atherosclerotic diseases in elderly women.

    Life sciences 2009;85;7-8;316-21

  • Association of genetic variants with chronic kidney disease in individuals with different lipid profiles.

    Yoshida T, Kato K, Yokoi K, Oguri M, Watanabe S, Metoki N, Yoshida H, Satoh K, Aoyagi Y, Nishigaki Y, Nozawa Y and Yamada Y

    Department of Cardiovascular Medicine, Inabe General Hospital, Inabe, Japan.

    The purpose of the present study was to identify genetic variants that confer susceptibility to chronic kidney disease (CKD) in individuals with low or high serum concentrations of triglycerides (TG), high-density lipoprotein (HDL)-cholesterol, or low-density lipoprotein (LDL)-cholesterol, thereby contributing to the personalized prevention of CKD in such individuals. The study population comprised 5944 Japanese individuals, including 1706 subjects with CKD [estimated glomerular filtration rate (eGFR)<60 ml/min/1.73 m2] and 4238 controls (eGFR>or=60 ml/min/1.73 m2). The genotypes for 296 polymorphisms of 202 candidate genes were determined. The Chi-square test, multivariable logistic regression analysis with adjustment for covariates, and a stepwise forward selection procedure revealed that seven different polymorphisms were significantly (P<0.005) associated with the prevalence of CKD in individuals with low or high serum concentrations of TG or HDL- or LDL-cholesterol: the Aright curved arrow G (Glu23Lys) polymorphism of KCNJ11 and the 125592Cright curved arrow A (Thr431Asn) polymorphism of ROCK2 in individuals with low serum TG; the 734Cright curved arrow T (Thr254Ile) polymorphism of ACAT2 and the Cright curved arrow G (Gln27Glu) polymorphism of ADRB2 in individuals with high serum TG; the -1607/1Gright curved arrow 2G polymorphism of MMP1 in individuals with low serum HDL-cholesterol; the Gright curved arrow A (Val158Met) polymorphism of COMT in individuals with low serum LDL-cholesterol; the 584Gright curved arrow A (Gln192Arg) polymorphism of PON1 in individuals with high serum LDL-cholesterol. No polymorphism was associated with CKD in individuals with high serum HDL-cholesterol. These results suggest that polymorphisms associated with CKD may differ among individuals with different lipid profiles. Stratification of subjects according to lipid profiles may thus be important for personalized prevention of CKD based on genetic information.

    International journal of molecular medicine 2009;24;2;233-46

  • Phenotype-genotype interactions on renal function in type 2 diabetes: an analysis using structural equation modelling.

    Song XY, Lee SY, Ma RC, So WY, Cai JH, Tam C, Lam V, Ying W, Ng MC and Chan JC

    Department of Statistics, The Chinese University of Hong Kong, Shatin, Hong Kong, SAR, People's Republic of China.

    Cardiovascular and renal diseases share common risk factors. We used structural equation modelling (SEM) to evaluate the independent and combined effects of phenotypes and genotypes implicated in cardiovascular diseases on renal function in type 2 diabetes.

    Methods: 1,188 type 2 diabetic patients were stratified into high-risk and low-risk groups according to bimodal distributions of the logarithmically transformed (log(e)) urinary albumin:creatinine ratio and plasma creatinine levels. Models for these groups, comprising continuous and non-ranking categorical data, were developed separately to evaluate the inter-relationships among measured variables and latent factors using non-linear SEMs, Bayesian estimation and model selection as assessed by a goodness-of-fit statistic.

    Results: Inter-correlated measured variables (obesity, glycaemia, lipid, blood pressure) and variants of the genes encoding endothelial nitric oxide synthase (NOS), beta-adrenergic receptor (ADRB), components of the renin-angiotensin system (RAS) and lipid metabolism were loaded onto their respective latent factors of phenotypes and genotypes. In addition to direct and indirect effects, latent factors of obesity, lipid and BP interacted with latent factors of ADRB and RAS genotypes to influence renal function. Together with variants of the genes encoding peroxisome proliferator-activated receptor gamma, atrial natriuretic peptide, adducin, G protein beta(3) subunit, epithelial sodium channel alpha subunit and matrix metallopeptidase 3, these parameters explained 39-80% of the variance in renal function in the high-risk and low-risk models.

    SEM is a useful tool for confirming and quantifying multiple interactions of biological pathways with genetic determinants. The combined and interactive effects of blood pressure, lipid and obesity on renal function may have therapeutic implications, especially in type 2 diabetic individuals with genetic risk factors.

    Diabetologia 2009;52;8;1543-53

  • A candidate gene association study of 77 polymorphisms in migraine.

    Schürks M, Kurth T, Buring JE and Zee RY

    Division of Preventive Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 900 CommonwealthAvenue East, 3rd Floor, Boston, MA 02215-1204, SUA. mschuerks@rics.bwh.harvard.edu

    Unlabelled: Population-based studies have established an association between migraine and cardiovascular disease (CVD). We sought to investigate whether genetic variants implicated in CVD are associated with migraine. We performed an association study among 25,713 women participating in the Women's Health Study, with information on 77 previously characterized polymorphisms. Migraine and migraine aura status were self-reported. We used logistic regression to investigate the genotype-migraine association. At baseline, 4705 (18.3%) women reported history of migraine; 39.6% of the 3306 women with active migraine indicated aura. Regarding any history of migraine, the multivariable-adjusted odds ratios (95% confidence intervals) for TNF rs673 were 0.52 (0.30 to 0.89), for TGFB1 rs1800469 0.93 (0.89 to 0.98), and for CCR2 rs1799864 1.12 (1.03 to 1.21). Among active migraine with aura, the odds ratios (95% confidence intervals) were 1.35 (1.0 to 1.81) for TNF rs1800750, 1.13 (1.02 to 1.26) for TNF rs1800629, and 1.22 (1.07 to 1.40) for CCR2 rs1799864; among active migraine without aura, 0.9 (0.84 to 0.97) for TGFB1 rs1800469, 1.13 (1.01 to 1.27) for NOS3 rs3918226, and 1.12 (1.02 to 1.24) for IL9 rs2069885. After correction for multiple testing using the false discovery rate, none of the results remained significant. Our data suggest an association of polymorphisms implicated in inflammatory pathways and migraine in women. TNF, CCR2, TGFB1, NOS3, and IL9 warrant further investigation.

    Perspective: This article presents results from an association study of 77 polymorphisms, implicated in CVD, 85a and migraine. Variants in TNF, CCR2, TGFB1, NOS3, and IL9 were found to be associated with migraine but did not remain significant after adjustment for multiple testing. Variations in these genes warrant further investigation.

    Funded by: NCI NIH HHS: CA-47988, R01 CA047988, R01 CA047988-09, R01 CA047988-10, R01 CA047988-11, R01 CA047988-12, R01 CA047988-13, R01 CA047988-14, R01 CA047988-15, R01 CA047988-16, R01 CA047988-17, R01 CA047988-18; NHLBI NIH HHS: R01 HL043851, R01 HL043851-09, R01 HL043851-10

    The journal of pain : official journal of the American Pain Society 2009;10;7;759-66

  • The genomic basis of cerebral palsy: a HuGE systematic literature review.

    O'Callaghan ME, MacLennan AH, Haan EA, Dekker G and South Australian Cerebral Palsy Research Group

    Discipline of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, SA, Australia, michael.ocallaghan@student.adelaide.edu.au

    Cerebral palsy has been associated with a number of candidate genes. To date, no systematic review has been conducted to synthesise genetic polymorphism associations with cerebral palsy. We apply the HuGE NET guidelines to search PubMed and EMBASE databases for publications investigating single nucleotide polymorphisms (SNPs) and cerebral palsy outcome. 22 papers were identified and are discussed in this review. Candidate genes were grouped as (1) thrombophilic, (2) cytokine, (3) apolipoprotein E or (4) other SNPs, largely related to cardiovascular physiology/pathophysiology and the functioning of the immune system. Of the studies identified, cohorts were usually small, without adequate control and ethnically diverse, making direct comparison between studies difficult. The most promising candidate genes include factor V Leiden, methylenetetrahydrofolate reductase, lymphotoxin-alpha, tumour necrosis factor-alpha, eNOS and mannose binding lectin. Large case-control studies are needed to confirm these candidates with attention given to cohort ethnicity, cerebral palsy subtype analysis and possible multiple gene and gene-environment interactions.

    Human genetics 2009;126;1;149-72

  • Analysis of inherited thrombophilic mutations and natural anticoagulant deficiency in patients with idiopathic portal hypertension.

    Bayan K, Tüzün Y, Yilmaz S, Canoruc N and Dursun M

    Department of Gastroenterology, Dicle University Faculty of Medicine, Diyarbakir, Turkey. drserif@dicle.edu.tr

    Idiopathic portal hypertension (IPH) is characterized by non-cirrhotic presinusoidal intrahepatic portal hypertension. The etiopathogenesis of the disease is poorly understood. Obliteration with microthrombosis of the small portal vein branches may lead to lesions underlying portal hypertension. We aimed to put forward a comprehensive thrombophilic mutation profile in IPH and its probable contribution to pathogenesis. Eleven patients and 12 controls were included. We used the CVD-StripAssay which is based on the reverse-hybridization principle to identify a total of 12 thrombophilic gene mutations: Factor V R506Q, Factor V H1299R, prothrombin G20210A, Factor XIII V34L, beta-Fibrinogen -455 G-A, PAI-1 4G/5G, platelet GPIIIa L33P, MTHFR C677T, MTHFR A1298C, ACE I/D, Apo B R3500Q and Apo E2/E3/E4, respectively. We also evaluated some blood parameters and protein C, protein S, AT-III levels using commercially available assays. IPH patients and controls were similar in respect to gender distribution (P = 1.000). Mean age was 31.2 in patients and 29.1 in controls (P = 0.622). Pica history was present in 54.5% of the patients. Mean protein C and AT-III levels were lower in patients than that of controls (P = 0.002 and 0.001, respectively). Factor XIII V34L, PAI-1, GPIIIa L33P, MTHFR C677T and MTHFR A1298C frequencies of genetic polymorphisms were found to be significantly higher among patients than that of controls. Apolipoprotein E2/E3/E4 analysis showed an inverse relationship with IPH when E2 plus E4 compared with E3. A higher frequency of Beta-Fibrinogen -455G-A mutation was observed in patients, but this difference did not reach a statistical significance. Our data represent the most comprehensive study to date with respect to thrombophilic gene polymorphisms in IPH. The data support a possible pathogenetic role in IPH, at least by some of the prothrombotic mutations. In order to confirm or refuse this proposal, a larger cohort of patients is needed.

    Journal of thrombosis and thrombolysis 2009;28;1;57-62

  • Antihypertensive pharmacogenetic effect of fibrinogen-beta variant -455G>A on cardiovascular disease, end-stage renal disease, and mortality: the GenHAT study.

    Lynch AI, Boerwinkle E, Davis BR, Ford CE, Eckfeldt JH, Leiendecker-Foster C and Arnett DK

    Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA.

    Objective: The FGB gene codes for fibrinogen-beta, a polypeptide of the coagulation factor fibrinogen, which is positively associated with cardiovascular diseases. Studies show that angiotensin-converting enzyme (ACE) inhibitors lower plasma fibrinogen concentrations, whereas diuretics and calcium-channel blockers do not. As carriers of the FGB-455 minor 'A' allele have higher levels of fibrinogen while ACE inhibitors lower it, we hypothesize that 'A' allele carriers benefit more from antihypertensive treatment with ACE inhibitors than calcium-channel blockers or diuretics, relative to 'GG' genotype individuals.

    Methods: The Genetics of Hypertension Associated Treatment (GenHAT) study [ancillary to Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT)] genotyped hypertensive participants for several hypertension-related candidate genes, making this a post-hoc analysis of a randomized trial. In total, 90.1% of the ALLHAT population was successfully genotyped for FGB-455. We included participants (n=30 076) randomized to one of three antihypertensive medications (lisinopril, amlodipine, chlorthalidone), with two treatment comparisons: lisinopril versus chlorthalidone and lisinopril versus amlodipine. The primary outcome of ALLHAT/GenHAT was coronary heart disease, defined as fatal coronary heart disease or non-fatal myocardial infarction, and secondary outcomes included stroke, heart failure, all-cause mortality, and end-stage renal disease (ESRD) with mean follow-up time of 4.9 years. Genotype-by-treatment interactions (pharmacogenetic effects) were tested with the Cox regression.

    Results: Stroke: common 'GG' homozygotes had higher risk on lisinopril versus amlodipine [hazard ratio (HR)=1.38, P<0.001], whereas minor 'A' allele carriers had slightly lower risk (HR=0.96, P=0.76; P value for interaction=0.03). Mortality: 'GG' homozygotes had higher risk on lisinopril versus amlodipine (HR=1.12, P=0.02) or chlorthalidone (1.05, P=0.23), whereas 'A' allele carriers had slightly lower risk (HR=0.92, P=0.33 for lisinopril versus amlodipine; HR=0.88, P=0.08 for lisinopril versus chlorthalidone; P value for interactions 0.04 and 0.03, respectively). ESRD: 'GG' homozygotes had higher risk on lisinopril versus chlorthalidone (HR=1.27, P=0.08), whereas 'A' allele carriers had lower risk (HR=0.64, P=0.12; P value for interaction=0.03).

    Conclusion: There was evidence of pharmacogenetic effects of FGB-455 on stroke, ESRD, and mortality, suggesting that relative to those homozygous for the common allele, variant allele carriers of the FGB gene at position -455 have a better outcome if randomized to lisinopril than chlorthalidone (for mortality and ESRD) or amlodipine (for mortality and stroke). For the models in which a pharmacogenetic effect was observed, the outcome rates among 'GG' homozygotes were higher in those randomized to lisinopril versus amlodipine or chlorthalidone, whereas minor 'A' allele carriers had lower event rates when randomized to lisinopril versus the other medications.

    Funded by: NHLBI NIH HHS: HL63082, N01-HC-35130, N01HC35130, R01 HL063082, R01 HL063082-02, R01 HL083498

    Pharmacogenetics and genomics 2009;19;6;415-21

  • Association between -455G/A and fibrinogen in a Chinese population.

    Sun A, Ma H, Xu D, Wang Y, Liu M, Xu L, Chen J, Hu T, Wang Z, Wang K, Zou Y, Huang W and Ge J

    Shanghai Institute of Cardiovascular Diseases, Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, China.

    Objective: The aim of the study was to evaluate the association between beta-fibrinogen gene -455G/A polymorphism and the plasma fibrinogen level in Chinese patients with different subtypes of coronary heart disease (CHD).

    We investigated beta-fibrinogen gene -455G/A polymorphism and plasma fibrinogen level in non-CHD control subjects (n = 466) and CHD patients (n = 1,019) including patients with stable angina pectoris (SAP) (n = 674), and acute coronary syndrome (ACS) (n = 345). Increased plasma fibrinogen levels were observed in the CHD groups compared with the control subjects (ACS: 380.92 +/- 92.35 mg/dl, SAP: 352.49 +/- 94.89 mg/dl, control: 311.72 +/- 87.09* mg/dl, *P < 0.001 vs. ACS and SAP). Individuals with the -455A/A genotype were associated with the highest plasma fibrinogen in the control subjects (P < 0.001) and patients with SAP (P < 0.001) but not in patients with ACS (P > 0.05). Allele frequency and genotype distribution were similar among the three groups (P = 0.314).

    Conclusions: This study demonstrated that elevated plasma fibrinogen level is related to increased CHD risk. The presence of -455A allele is significantly associated with higher fibrinogen in non-CHD control subjects and SAP patients but not in ACS patients while -455G/A polymorphism is not a risk factor for CHD in the Chinese population.

    Acta cardiologica 2009;64;3;357-61

  • ORF3 protein of hepatitis E virus interacts with the Bbeta chain of fibrinogen resulting in decreased fibrinogen secretion from HuH-7 cells.

    Ratra R, Kar-Roy A and Lal SK

    Virology Group, International Centre for Genetic Engineering and Biotechnology, PO Box 10504, Aruna Asaf Ali Road, New Delhi 110067, India.

    The ORF3 protein of hepatitis E virus (HEV), the precise cellular functions of which remain obscure, was used in a yeast two-hybrid screen to identify its cellular binding partners. One of the identified interacting partners was fibrinogen Bbeta protein. The ORF3-fibrinogen Bbeta interaction was verified by co-immunoprecipitation and fluorescence resonance energy transfer in mammalian cells. Fibrinogen is a hepatic acute-phase protein and serves as a central molecule that maintains host homeostasis and haemostasis during an acute-phase response. Metabolic labelling of ORF3-transfected HuH-7 cells showed that secreted as well as intracellular levels of fibrinogen were decreased in these cells compared with vector-transfected controls. Northern hybridization and RT-PCR analyses revealed that the mRNA levels of all three chains of fibrinogen, Aalpha, Bbeta and gamma, were transcriptionally downregulated in ORF3-transfected cells. The constitutive expression of fibrinogen genes can be significantly upregulated by interleukin (IL)-6, an important mediator of liver-specific gene expression during an acute-phase response. Transcription of fibrinogen genes after IL-6 stimulation was less in ORF3-expressing cells compared with controls. This report adds one more biological function to, and advances our understanding of, the cellular role of the ORF3 protein of HEV. The possible implications of these findings in the virus life cycle are discussed.

    The Journal of general virology 2009;90;Pt 6;1359-70

  • Prothrombotic genetic variants and atherosclerosis in patients with cerebral ischemia of arterial origin.

    Pruissen DM, Kappelle LJ, Rosendaal FR, Algra A and SMART Study Group

    Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, The Netherlands.

    Objective: Prothrombotic genetic variants associated with arterial disease probably affect arterial thrombus formation but may also promote atherosclerosis. We hypothesized that specific prothrombotic variants lead to advanced atherosclerosis in patients with cerebral ischemia of arterial origin.

    We included 689 patients with nondisabling cerebral ischemia of arterial origin. Twenty-two variants in 14 genes were genotyped. None of the variants was associated with carotid intima-media thickness or younger age at the occurrence of cerebral ischemia. Factor V Leiden (mean prevalence difference 25%; 95% CI 11-40) and the glycoprotein 1b-alpha Thr145Met variant (mean prevalence difference 12%; 95% CI 2.3-22) were associated with symptomatic carotid stenosis. After accounting for multiple testing by determination of the false discovery rate, only the association between factor V Leiden and symptomatic carotid stenosis remained present.

    Conclusions: Prothrombotic genetic variants showed no consistent association with three markers of advanced atherosclerosis in patients with cerebral ischemia of arterial origin. This study does not support the hypothesis that prothrombotic genetic variants have a direct role in the pathogenesis of atherosclerosis.

    Atherosclerosis 2009;204;1;191-5

  • Prothrombotic polymorphisms, mutations, and their association with pediatric non-cardioembolic stroke in Asian-Indian patients.

    Biswas A, Tiwari AK, Ranjan R, Meena A, Akhter MS, Yadav BK, Behari M and Saxena R

    Department of Hematoloy, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.

    Genes involved in the hemostatic mechanism are logical candidate genes for association studies in prothrombotic conditions such as stroke. Since the underlying etiology in pediatric strokes is different than adults, looking for genetic causes would be the logical thing to do in the pediatric stroke population. Fifty-eight Asian-Indian stroke patients below 15 years of age and equal number of age- and sex-matched healthy controls were the subjects for the study. The subjects were screened for 13 polymorphisms and three mutations spread across seven different candidate genes involved in the hemostatic system. Of the 13 polymorphisms and three mutations studied, four polymorphisms, HPA-I, TAFI 147Ala>Thr, methylene tetrahydrofolate reductase (MTHFR) 677 C>T, and MTHFR 1298 A>C, showed significant association with the disease phenotype. MTHFR 677 C>T showed the strongest association and therefore may have a strong predisposing role for pediatric strokes. Gene-gene interaction studies showed a strong interaction between HPA-I and MTHFR 677 C>T polymorphism. The wild type of both these polymorphisms synergistically showed a strong protective effect [p < 0.0001, O.R: 10.06(4.26-23.71)]. Polymorphisms in HPA-I and MTHFR may have important predisposing roles in the development of pediatric stroke.

    Annals of hematology 2009;88;5;473-8

  • Two novel mutations at contiguous codons in the fibrinogen Bbeta chain as 1765 sociated with hypofibrinogenaemia.

    Davis RL, May S, Chunilal S and Brennan SO

    Thrombosis and haemostasis 2009;101;5;980-2

  • Fibrinogen gene polymorphism (Bbeta-148C/T) in Uygur patients with cerebral infarction.

    Guo X, Zhang D and Zhang X

    Department of Neurology, First Affiliated Hospital of Xinjiang Medical University, Urumqi, China.

    Objective: To explore the frequency distribution of the fibrinogen gene polymorphism (Bbeta-148C/T) and its relationship with plasma fibrinogen (Fg) level in Uygur patients with cerebral infarction in Xinjiang.

    Methods: The frequency distribution of FgBbeta-148C/T was analysed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in the Uygur cerebral infarction group (n=62) and the Uygur control group without cerebral infarction (n=64). Plasma fibrinogen levels were measured by the automatic coagulation analyser.

    Results: The frequency of the T allele in the cerebral infarction group was significantly higher than that of the control group (0.31 versus 0.19, p<0.05). The plasma fibrinogen level of the cerebral infarction group was also significantly higher than that of the control group (4.19 +/- 1.29 g/l versus 3.08 +/- 0.56 g/l, p<0.01). The plasma fibrinogen levels in both CT and TT genotype subgroups in patients with cerebral infarction were higher than those in the CC genotype subgroups (p<0.01). They were also higher than those in the CT and TT genotype subgroups within the control group (p<0.05).

    Conclusion: The fibrinogen gene polymorphism (Bbeta-148C/T) affects plasma fibrinogen concentration. T allele, alone or cooperating with other risk factors, increases the plasma fibrinogen level. Thus, the fibrinogen gene polymorphism (Bbeta-148C/T) may be related with cerebral infarction. T allele may be a risk factor for cerebral infarction by increasing the plasma fibrinogen level. The above conclusion is in accordance with similar studies on the Han nationality in China.

    Neurological research 2009;31;4;381-4

  • Association of novel genetic Loci with circulating fibrinogen levels: a genome-wide association study in 6 population-based cohorts.

    Dehghan A, Yang Q, Peters A, Basu S, Bis JC, Rudnicka AR, Kavousi M, Chen MH, Baumert J, Lowe GD, McKnight B, Tang W, de Maat M, Larson MG, Eyhermendy S, McArdle WL, Lumley T, Pankow JS, Hofman A, Massaro JM, Rivadeneira F, Kolz M, Taylor KD, van Duijn CM, Kathiresan S, Illig T, Aulchenko YS, Volcik KA, Johnson AD, Uitterlinden AG, Tofler GH, Gieger C, Wellcome Trust Case Control Consortium, Psaty BM, Couper DJ, Boerwinkle E, Koenig W, O'Donnell CJ, Witteman JC, Strachan DP, Smith NL and Folsom AR

    Department of Epidemiology, Erasmus Medical Center, Rotterdam, The Netherlands.

    Background: Fibrinogen is both central to blood coagulation and an acute-phase reactant. We aimed to identify common variants influencing circulation fibrinogen levels.

    We conducted a genome-wide association analysis on 6 population-based studies, the Rotterdam Study, the Framingham Heart Study, the Cardiovascular Health Study, the Atherosclerosis Risk in Communities Study, the Monitoring of Trends and Determinants in Cardiovascular Disease/KORA Augsburg Study, and the British 1958 Birth Cohort Study, including 22 096 participants of European ancestry. Four loci were marked by 1 or more single-nucleotide polymorphisms that demonstrated genome-wide significance (P<5.0 x 10(-8)). These included a single-nucleotide polymorphism located in the fibrinogen beta chain (FGB) gene and 3 single-nucleotide polymorphisms representing newly identified loci. The high-signal single-nucleotide polymorphisms were rs1800789 in exon 7 of FGB (P=1.8 x 10(-30)), rs2522056 downstream from the interferon regulatory factor 1 (IRF1) gene (P=1.3 x 10(-15)), rs511154 within intron 1 of the propionyl coenzyme A carboxylase (PCCB) gene (P=5.9 x 10(-10)), and rs1539019 on the NLR family pyrin domain containing 3 isoforms (NLRP3) gene (P=1.04 x 10(-8)).

    Conclusions: Our findings highlight biological pathways that may be important in regulation of inflammation underlying cardiovascular disease.

    Funded by: Chief Scientist Office: CZB/4/540; Medical Research Council: G0000934, G0600329, G0701003, G0800759, MC_QA137934; NCRR NIH HHS: M01RR00069, UL1 RR025005, UL1-RR-025005; NHGRI NIH HHS: U01 HG004402, U01 HG004402-02, U01-HG-004402; NHLBI NIH HHS: HL073410, N01 HC-55222, N01 HC015103, N01 HC025195, N01 HC035129, N01 HC045133, N01 HC055015, N01 HC055016, N01 HC055018, N01 HC055019, N01 HC055020, N01 HC055021, N01 HC055022, N01 HC055222, N01 HC075150, N01 HC085079, N01 HC085086, N01-HC-25195, N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, N01-HC-55022, N01-HC-75150, N01-HC-85079, N01-HC-85080, N01-HC-85081, N01-HC-85082, N01-HC-85083, N01-HC-85084, N01-HC-85085, N01-HC-85086, N01HC25195, N01HC55015, N01HC55016, N01HC55018, N01HC55019, N01HC55020, N01HC55021, N01HC55022, N01HC55222, N01HC75150, N01HC85079, N01HC85086, N02 HL64278, N02-HL-64278, R01 HL 087652, R01 HL059367, R01 HL059367-09, R01 HL073410, R01 HL073410-04, R01 HL086694, R01 HL086694-02, R01 HL087641, R01 HL087641-03, R01 HL087652, R01 HL087652-02, R01 HL087652-03, R01-HL-086694, R01-HL-087641, R01-HL-59367, U01 HL080295, U01 HL080295-05; NIDDK NIH HHS: DK063491, P30 DK063491, P30 DK063491-019004, P30 DK063491-029004, P30 DK063491-039004, P30 DK063491-049004, P30 DK063491-05; PHS HHS: HHS N268200625226C; Wellcome Trust: 061858, 068545/Z/02, 090532

    Circulation. Cardiovascular genetics 2009;2;2;125-33

  • Candidate genetic variants in the fibrinogen, methylenetetrahydrofolate reductase, and intercellular adhesion molecule-1 genes and plasma levels of fibrinogen, homocysteine, and intercellular adhesion molecule-1 among various race/ethnic groups: data from the Women's Genome Health Study.

    Albert MA, Pare G, Morris A, Rose L, Buri 1efe ng J, Ridker PM and Zee RY

    Center for Cardiovascular Disease Prevention, Donald W. Reynolds Center for Cardiovascular Disease Research, Brigham and Women's Hospital, Harvard Medical School, Boston MA, USA. maalbert@partners.org

    Background: Although inflammation is a core element of atherogenesis and plasma levels of fibrinogen (FGB), homocysteine, and intercellular adhesion molecule-1 (ICAM-1) differ by race/ethnicity, little is known about the role of genetic polymorphisms in the FGB, methylenetetrahydrofolate reductase (MTHFR), and ICAM-1 genes in determining plasma levels of these biomarkers. We examined the relationship between specific polymorphisms in the FGB, homocysteine, and ICAM-1 genes and their respective inflammatory biomarker concentrations at baseline in women from different race/ethnic groups.

    Methods: We genotyped specific polymorphisms in FGB (-455G>A/rs1800790), MTHFR (677C>T/rs1801133), and ICAM-1 (Lys56Met/rs5491 and Gly241Arg/rs1799969) at baseline and evaluated their relationship with respective inflammatory biomarker levels in 25,565 white, 476 African-American (black), 277 Hispanic, and 370 Asian women participating in the Women's Genome Health Study.

    Results: Overall, the minor allele frequencies for -455G>A were similar among white, Hispanic, and Asian women (17.2%-21.9%) but significantly lower in black women (6.6%, P < .001). The minor allele was associated with elevated FGB levels only in whites and Asians. After adjustment for age, body mass index, smoking, postmenopausal status, diabetes, hormone replacement therapy use, hypertension, and education, black women had the highest FGB levels compared to other race/ethnic groups. The minor allele frequency of the MTHFR 677C>T polymorphism was lowest in blacks (blacks 12.1%, whites 33.1%, Hispanics 39.0%, Asians 24.0%), and the T allele was only significantly associated with homocysteine levels in white women. Among whites, Hispanics, and Asians, the Lys56Met polymorphism was rare compared to the frequency in blacks (P < .001). Neither the Lys56Met nor Gly241Arg polymorphisms were common in Asians. Nonetheless, both polymorphisms were generally associated with lower ICAM-1 levels; the lowest levels were observed in black women.

    Conclusion: We found significant associations between certain candidate genetic polymorphisms and baseline plasma levels of FGB, homocysteine, and ICAM-1 in women from various race/ethnic groups. The present investigation is hypothesis generating and suggests genetic determination of differential concentrations of these atherosclerosis-related inflammatory biomarkers differ among various race/ethnic groups.

    Funded by: NCI NIH HHS: R01 CA047988, R01 CA047988-09, R01 CA047988-10, R01 CA047988-11, R01 CA047988-12, R01 CA047988-13, R01 CA047988-14, R01 CA047988-15, R01 CA047988-16, R01 CA047988-17, R01 CA047988-18; NHLBI NIH HHS: R01 HL043851, R01 HL043851-09, R01 HL043851-10

    American heart journal 2009;157;4;777-83.e1

  • Deletion of five residues from the coiled coil of fibrinogen (Bbeta Asn167_Glu171del) associated with bleeding and hypodysfibrinogenemia.

    Brennan SO, Davis RL, Lowen R and Ruskova A

    Molecular Pathology Laboratory, Canterbury Health Laboratories, PO Box 151, Christchurch, New Zealand. steve.brennan@otago.ac.nz

    Routine pre-surgical coagulation investigations led to the detection of a novel type of hypodysfibrinogenemia whose functional defect appears to result from an alteration in the spacing between the functional domains of the fibrinogen molecule. The detection, by reverse phase HPLC, of a minor isoform of Bbeta chain with a 554 Da decrease in mass led to the identification of a deletion of five amino acids (NVVNE) from the center of the coiled coil. The variant chain contributed only 10% of the total Bbeta material and the mutation (BbetaAsn167_Glu171del) was associated with both increased clotting times and low functional and physical fibrinogen concentrations in 3 family members. There was a significant history of pregnancy-associated bleeding and miscarriage within the first trimester. Mechanistically ad4 the 15-nucleotide deletion appears to arise from replication advancement during DNA synthesis caused by a flanking pentanucleotide repeat of AATGA.

    Haematologica 2009;94;4;585-8

  • Novel loci, including those related to Crohn disease, psoriasis, and inflammation, identified in a genome-wide association study of fibrinogen in 17 686 women: the Women's Genome Health Study.

    Danik JS, Paré G, Chasman DI, Zee RY, Kwiatkowski DJ, Parker A, Miletich JP and Ridker PM

    Center for Cardiovascular Disease Prevention, Donald W. Reynolds Center for Cardiovascular Research, and Translational Medicine Division, Brigham and Women's Hospital, 900 Commonwealth Ave. East, Boston, MA 02215, USA.

    Background: Fibrinogen is a multifunctional circulating glycoprotein involved in wound healing, thrombosis, platelet aggregation, and inflammation, and elevated levels predict vascular disease. Despite evidence of crucial biological function and moderate heritability, comprehensive analysis of the influence of genetic variation on fibrinogen is not available.

    To address this issue, we undertook a genome-wide association study evaluating the potential relationships between 337 343 single-nucleotide polymorphisms (SNPs) and plasma fibrinogen levels among 17 686 apparently healthy women participating in the Women's Genome Health Study. As C-reactive protein is also an inflammatory marker known to predict cardiovascular diseases, we compared the determinants of fibrinogen levels with those of C-reactive protein. Four novel loci were identified, in addition to the fibrinogen gene cluster, which were associated with fibrinogen levels at genome-wide levels of significance (range of probability values from 8.82 x 10(-09) to 8.04 x 10(-39)). Two of the loci are related to common chronic inflammatory diseases: the first, at locus 5q31.1 (SLC22A5, SLC22A4, IRF1), lies immediately adjacent to a locus linked to Crohn disease (P value for lead SNP, 1.24 x 10(-12)) and the second, at locus 17q25.1 (CD300LF, SLC9A3R1, NAT9), has been associated with psoriasis (P value for lead SNP, 7.72 x 10(-11)). A third locus at 1q21.3 (IL6R) lies within the interleukin 6 receptor gene, a critical component of the inflammatory cascade (P value for lead SNP, 1.80 x 10(-11)). A novel locus at 2q34 (CPSI) participates in the urea cycle (P=8.82 x 10(-09)). The majority of implicated SNPs showed little evidence of dual association with C-reactive protein levels.

    Conclusions: A genome-wide survey of the human genome identifies novel loci related to common chronic inflammatory diseases as genetic determinants of fibrinogen levels, in addition to loci that relate to the inflammatory cascade, the urea cycle, and the fibrinogen gene cluster.

    Funded by: NCI NIH HHS: R01 CA047988, R01 CA047988-09, R01 CA047988-10, R01 CA047988-11, R01 CA047988-12, R01 CA047988-13, R01 CA047988-14, R01 CA047988-15, R01 CA047988-16, R01 CA047988-17, R01 CA047988-18; NHLBI NIH HHS: K08 HL076443-01, K08 HL076443-02, K08 HL076443-03, K08 HL076443-04, K08 HL076443-05, R01 HL043851, R01 HL043851-09, R01 HL043851-10, R01 HL080467, R01 HL080467-01, R01 HL080467-02, R01 HL080467-03

    Circulation. Cardiovascular genetics 2009;2;2;134-41

  • Thrombophilic gene polymorphisms are risk factors for unexplained infertility.

    Coulam CB and Jeyendran RS

    Pregnancy Success Center and Rinehart Center for Reproductive Medicine, Chicago, Illinois, USA. cbcoulam@aol.com

    Earlier studies have shown inherited thrombophilia to be a risk factor for recurrent implantation failure, raising the question of whether this risk is related to the underlying cause of unexplained infertility or to the mechanisms involved in the implantation process. When nine thrombophilic gene polymorphisms were compared among 92 women with the diagnosis of unexplained infertility and 60 fertile control women, women with a history of unexplained infertility displayed a higher prevalence of MTHFR C677T polymorphisms than control women.

    Fertility and sterility 2009;91;4 Suppl;1516-7

  • Fibrinogen genes modify the fibrinogen response to ambient particulate matter.

    Peters A, Greven S, Heid IM, Baldari F, Breitner S, Bellander T, Chrysohoou C, Illig T, Jacquemin B, Koenig W, Lanki T, Nyberg F, Pekkanen J, Pistelli R, R� 93f �ckerl R, Stefanadis C, Schneider A, Sunyer J, Wichmann HE and AIRGENE Study Group

    Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Epidemiology, Ingolstaedter Landstrasse, Neuherberg 85764, Germany. peters@helmholtz-muenchen.de

    Rationale: Ambient particulate matter has been associated with systemic inflammation indicated by blood markers such as fibrinogen, implicated in promoting atherothrombosis.

    Objectives: This study evaluated whether single-nucleotide polymorphisms (SNPs) within the fibrinogen genes modified the relationship between ambient particles and plasma fibrinogen.

    Methods: In 854 myocardial infarction survivors from five European cities plasma fibrinogen levels were determined repeatedly (n = 5,082). City-specific analyses were conducted to assess the impact of particulate matter on fibrinogen levels, applying additive mixed models adjusting for patient characteristics, time trend, and weather. City-specific estimates were pooled by meta-analysis methodology.

    Seven SNPs in the FGA and FGB genes shown to be associated with differences in fibrinogen levels were selected. Promoter SNPs within FGA and FGB were associated with modifications of the relationship between 5-day averages of particulate matter with an aerodynamic diameter below 10 microm (PM(10)) and plasma fibrinogen levels. The PM(10)-fibrinogen relationship for subjects with the homozygous minor allele genotype of FGB rs1800790 compared with subjects homozygous for the major allele was eightfold higher (P value for the interaction, 0.037).

    Conclusions: The data suggest that susceptibility to ambient particulate matter may be partly genetically determined by polymorphisms that alter early physiological responses such as transcription of fibrinogen. Subjects with variants of these frequent SNPs may have increased risks not only due to constitutionally higher fibrinogen concentrations, but also due to an augmented response to environmental inflammatory stimuli such as ambient particulate matter.

    American journal of respiratory and critical care medicine 2009;179;6;484-91

  • IL6 and CRP haplotypes are associated with COPD risk and systemic inflammation: a case-control study.

    Yanbaeva DG, Dentener MA, Spruit MA, Houwing-Duistermaat JJ, Kotz D, Passos VL and Wouters EF

    Department of Respiratory Medicine, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University Medical Centre, Maastricht, The Netherlands. d.yanbaeva@pul.unimaas.nl

    Background: Elevated circulating levels of C-reactive protein (CRP), interleukin (IL)-6 and fibrinogen (FG) have been repeatedly associated with many adverse outcomes in patients with chronic obstructive pulmonary disease (COPD). To date, it remains unclear whether and to what extent systemic inflammation is primary or secondary in the pathogenesis of COPD. The aim of this study was to examine the association between haplotypes of CRP, IL6 and FGB genes, systemic inflammation, COPD risk and COPD-related phenotypes (respiratory impairment, exercise capacity and body composition).

    Methods: Eighteen SNPs in three genes, representing optimal haplotype-tagging sets, were genotyped in 355 COPD patients and 195 healthy smokers. Plasma levels of CRP, IL-6 and FG were measured in the total study group. Differences in haplotype distributions were tested using the global and haplotype-specific statistics.

    Results: Raised plasma levels of CRP, IL-6 and fibrinogen were demonstrated in COPD patients. However, COPD population was very heterogeneous: about 40% of patients had no evidence of systemic inflammation (CRP < 3 mg/uL or no inflammatory markers in their top quartile). Global test for haplotype effect indicated association of CRP gene and CRP plasma levels (P = 0.0004) and IL6 gene and COPD (P = 0.003). Subsequent analysis has shown that IL6 haplotype H2, associated with an increased COPD risk (p = 0.004, OR = 4.82; 1.64 to 4.18), was also associated with very low CRP levels (p = 0.0005). None of the genes were associated with COPD-related phenotypes.

    Conclusion: Our findings suggest that common genetic variation in CRP and IL6 genes may contribute to heterogeneity of COPD population associated with systemic inflammation.

    BMC medical genetics 2009;10;23

  • A meta-analysis of candidate gene polymorphisms and ischemic stroke in 6 study populations: association of lymphotoxin-alpha in nonhypertensive patients.

    Wang X, Cheng S, Brophy VH, Erlich HA, Mannhalter C, Berger K, Lalouschek W, Browner WS, Shi Y, Ringelstein EB, Kessler C, Luedemann J, Lindpaintner K, Liu L, Ridker PM, Zee RY, Cook NR and RMS Stroke SNP Consortium

    Laboratory of Human Genetics, Beijing Hypertension League Institute, Beijing, China.

    Ischemic stroke is a multifactorial disease with a strong genetic component. Pathways, including lipid metabolism, systemic chronic inflammation, coagulation, blood pressure regulation, and cellular adhesion, have been implicated in stroke pathophysiology, and candidate gene polymorphisms in these pathways have been proposed as genetic risk factors.

    Methods: We genotyped 105 simple deletions and single nucleotide polymorphisms from 64 candidate genes in 3550 patients and 6560 control subjects from 6 case-control association studies conducted in the United States, Europe, and China. Genotyping was performed using the same immobilized probe typing system and meta-analyses were based on summary logistic regressions for each study. The primary analyses were fixed-effects meta-analyses adjusting for age and sex with additive, dominant, and recessive models of inheritance.

    Results: Although 7 polymorphisms showed a nominal additive association, none remained statistically significant after adjustment for multiple comparisons. In contrast, after stratification for hypertension, 2 lymphotoxin-alpha polymorphisms, which are in strong linkage disequilibrium, were significantly associated among nonhypertensive individuals: LTA 252A>G (additive model; OR, 1.41 with 95% CI, 1.20 to 1.65; P=0.00002) and LTA 26Thr>Asn (OR, 1.19 with 95% CI, 1.06 to 1.34; P=0.003). LTA 252A>G remained significant after adjustment for multiple testing using either the false discovery rate or by permutation testing. The 2 single nucleotide polymorphisms showed no association in hypertensive subjects (eg, LTA 252A>G, OR, 0.93; 95% CI, 0.84 to 1.03; P=0.17).

    Conclusions: These observations may indicate an important role of LTA-mediated inflammatory processes in the pathogenesis of ischemic stroke.

    Funded by: NHLBI NIH HHS: HL-58755, HL-63293, R01 HL058755-02, R01 HL058755-03, R01 HL063293, R01 HL063293-01, R01 HL063293-02, R01 HL063293-03, R01 HL063293-04; NIA NIH HHS: 2 R01 AG005394-22A1, 2 R01 AG027574-22A1, AG05394, AG05407, R01 AG005394, R01 AG005407, R01 AG005407-14, R01 AG005407-15, R01 AG005407-16, R01 AG005407-17, R01 AG005407-18, R01 AG005407-19, R01 AG005407-20, R01 AG005407-21A1, R01 AG005407-22, R01 AG005407-23, R01 AG027574, R01 AG027576, R01 AG027576-22; NIAMS NIH HHS: AR35582, AR35583, AR35584, R01 AR035582, R01 AR035583, R01 AR035584

    Stroke 2009;40;3;683-95

  • Association of a polymorphism of the apolipoprotein E gene with chronic kidney disease in Japanese individuals with metabolic syndrome.

    Yoshida T, Kato K, Fujimaki T, Yokoi K, Oguri M, Watanabe S, Metoki N, Yoshida H, Satoh K, Aoyagi Y, Nishigaki Y, Tanaka M, Nozawa Y and Yamada Y

    Department of Cardiovascular Medicine, Inabe General Hospital, Inabe, Japan.

    The purpose of the present study was to identify genetic variants that confer susceptibility to chronic kidney disease (CKD) in Japanese individuals with metabolic syndrome. The study population comprised 2150 Japanese individuals with metabolic syndrome, including 411 subjects with CKD [estimated glomerular filtration rate (eGFR) <50 mL/min/1.73m(2)] and 1739 controls (eGFR >/=60 mL/min/1.73m(2)). The genotypes for 100 polymorphisms of 80 candidate genes were determined. The chi-square test, multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that nine polymorphisms of APOE, ABCA1, PTGS1, TNF, CPB2, AGTR1, OR13G1, and GNB3 were associated (P<0.05) with the prevalence of CKD. Among these polymorphisms, the -219G-->T polymorphism of APOE (rs405509) was most significantly associated with CKD in Japanese individuals with metabolic syndrome.

    Genomics 2009;93;3;221-6

  • Genetic variation in fibrinogen; its relationship to fibrinogen levels and the risk of myocardial infarction and ischemic stroke.

    Siegerink B, Rosendaal FR and Algra A

    Department of Clinical Epidemiology, Leiden University Medical Centre, Leiden, the Netherlands.

    Background: Confounding by common causes and reverse causation have been proposed as explanations for the association between high fibrinogen levels and cardiovascular disease. Genetic variants can alter fibrinogen characteristics and are not subject to these problems.

    Objectives: To determine the fibrinogen plasma levels for genotypic variants in fibrinogen-A alpha (FGA Thr312Ala) and fibrinogen-B beta (FGB - 455G/A), and whether these variants are associated with arterial thrombosis.

    Methods: Fibrinogen genotypes were determined in a population-based case-control study including women aged 18-50 years; 218 cases with myocardial infarction, 192 cases with ischemic stroke, and 769 healthy controls. Fibrinogen levels were determined in the control population.

    Results: The FGB - 455G/A variant increased plasma fibrinogen levels, whereas the FGA Thr312Ala variant lowered plasma fibrinogen levels, albeit to a modest extent. The risk of ischemic stroke was altered when the homozygote minor allele was compared with the homozygote major allele. The FGA Thr312Ala single-nucleotide polymorphism (SNP) was associated with a decrease in risk [odds ratio (OR) 0.43; 95% confidence interval (CI) 0.21-0.87], whereas the FGB - 455G/A SNP might have increased the risk (OR 1.76; 95% CI 0.7-4.03). The risk of myocardial infarction was not altered for either SNP (FGA Thr312Ala, OR 0.98, 95% CI 0.40-2.40; FGB - 455G/A, OR 0.98, 95% CI 0.40-2.40).

    Conclusions: With the genetic variations as markers of plasma fibrinogen levels alterations, thereby ruling out confounding and reverse causation, our results suggest that plasma fibrinogen levels could play a more pronounced role as risk factors for ischemic stroke than for myocardial infarction.

    Journal of thrombosis and haemostasis : JTH 2009;7;3;385-90

  • A deep intronic mutation in FGB creates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment.

    Davis RL, Homer VM, George PM and Brennan SO

    Molecular Pathology Laboratory, Department of Pathology, Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, New Zealand. ryan.davis@otago.ac.nz

    We previously described a novel homozygous point mutation (FGB c.115-600A>G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50-bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre-mRNA splicing factor (SF2)/arginine/serine-rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bbeta chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re-established normal pre-mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein-RNA interaction and successfully restored normal splicing ( approximately 50% at 2 microM and approximately 90% at 10 microM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation.

    Human mutation 2009;30;2;221-7

  • Association study between variants in the fibrinogen gene cluster, fibrinogen levels and hypertension: results from the MONICA/KORA study.

    Kolz M, Baumert J, Gohlke H, Grallert H, Döring A, Peters A, Wichmann HE, Koenig W and Illig T

    Department of Internal Medicine II - Cardiology, University of Ulm Medical Center, Robert-Koch Str. 8, D-89081 Ulm, Germany.

    Previous studies reported a gender-specific association between plasma fibrinogen concentrations and incident hypertension. We systematically analysed polymorphisms and haplotypes across the fibrinogen gene cluster with fibrinogen levels and assessed their contribution to prevalent hypertension in 2,200 men and 2,159 women from the population-based MONICA/KORA Augsburg study. Eleven tagging single nucleotide polymorphisms (SNPs) were systematically selected in the three fibrinogen genes and haplotypes were reconstructed. The minor alleles of two SNPs, rs2227401 (FGB) and rs2070016 (FGA) and the haplotypes tagged by those variants, were significantly associated with higher fibrinogen concentrations in both, men and women, explaining 1% of the total variance of fibrinogen concentrations. In addition, a FGG haplotype, tagged by rs1049636, was associated with lower concentrations of fibrinogen in women, but not in men. Regarding hypertension, we detected a significant association with a FGA promoter variant (rs2070008) in women only, whereas fibrinogen haplotypes were not associated with hypertension after correction for multiple comparisons in either men or women. In conclusion, our results suggest that variants in all three fibrinogen genes are significantly associated with differences in fibrinogen concentrations with modest contribution to phenotypic variance. It is likely that other genetic variants outside the fibrinogen gene loci are involved in the regulation of fibrinogen concentrations. In addition, one FGA promoter variant was significantly associated with hypertension in women. Confirmation of these findings by future studies is warranted.

    Thrombosis and haemostasis 2009;101;2;317-24

  • Prevalence in the United States of selected candidate gene variants: Third National Health and Nutrition Examination Survey, 1991-1994.

    Chang MH, Lindegren ML, Butler MA, Chanock SJ, Dowling NF, Gallagher M, Moonesinghe R, Moore CA, Ned RM, Reichler MR, Sanders CL, Welch R, Yesupriya A, Khoury MJ and CDC/NCI NHANES III Genomics Working Group

    National Office of Public Health Genomics, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA. mdc9@cdc.gov

    Population-based allele frequencies and genotype prevalence are important for measuring the contribution of genetic variation to human disease susceptibility, progression, and outcomes. Population-based prevalence estimates also provide the basis for epidemiologic studies of gene-disease associations, for estimating population attributable risk, and for informing health policy and clinical and public health practice. However, such prevalence estimates for genotypes important to public health remain undetermined for the major racial and ethnic groups in the US population. DNA was collected from 7,159 participants aged 12 years or older in Phase 2 (1991-1994) of the Third National Health and Nutrition Examination Survey (NHANES III). Certain age and minority groups were oversampled in this weighted, population-based US survey. Estimates of allele frequency and genotype prevalence for 90 variants in 50 genes chosen for their potential public health significance were calculated by age, sex, and race/ethnicity among non-Hispanic whites, non-Hispanic blacks, and Mexican Americans. These nationally representative data on allele frequency and genotype prevalence provide a valuable resource for future epidemiologic studies in public health in the United States.

    American journal of epidemiology 2009;169;1;54-66

  • The functional effects of the -455G/A polymorphism on the IL-6-induced expression of the beta-fibrinogen gene may be due to linkage disequilibrium with other functional polymorphisms.

    Morozumi T, Sharma A and De Nardin E

    Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY 14214, USA.

    Elevated plasma fibrinogen levels have been identified as an independent risk factor for coronary heart diseases, stroke and peripheral artery disease. The -455G/A polymorphism in the promoter region of the beta-fibrinogen gene has been associated with increased plasma fibrinogen levels. However, the functional effect of this polymorphism has been controversial and other polymorphisms in the fibrinogen gene have also been implicated in higher fibrinogen levels. In this study, we evaluated the transcriptional activity of 4 natural haplotypes and 6 artificial haplotypes in the promoter region of the beta-fibrinogen gene. Significantly lower IL-6-induced activity was observed in the -1420A and -148T alleles. In contrast, the -854A allele had significantly higher activity. Artificial haplotypes containing the -1420A, -854A and -148T alleles were also analyzed to confirm individual functional effects. The -1420A and -148T alleles significantly lowered the activities, while the -854A allele significantly raised the activity. From this study we conclude that the -1420G/A, -854G/A and -148C/T polymorphisms in the beta-fibrinogen promoter region are functional polymorphisms while the -455G/A polymorphism may not be a functional one, and that the association of the -455G/A polymorphism with higher fibrinogen levels may actually be due to linkage disequilibrium between the -455G/A polymorphism and other truly functional polymorphisms.

    Immunological investigations 2009;38;3-4;311-23

  • Fibrinogen beta-chain tyrosine nitration is a prothrombotic risk factor.

    Parastatidis I, Thomson L, Burke A, Chernysh I, Nagaswami C, Visser J, Stamer S, Liebler DC, Koliakos G, Heijnen HF, Fitzgerald GA, Weisel JW and Ischiropoulos H

    Stokes Research Institute and Departments of Pediatrics and Pharmacology, Children's Hospital of Philadelphia and the University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Elevated levels of circulating fibrinogen are associated with an increased risk of atherothrombotic diseases although a causative correlation between high levels of fibrinogen and cardiovascular complications has not been established. We hypothesized that a potential mechanism for an increased prothrombotic state is the post-translational modification of fibrinogen by tyrosine nitration. Mass spectrometry identified tyrosine residues 292 and 422 at the carboxyl terminus of the beta-chain as the principal sites of fibrinogen nitration in vivo. Immunoelectron microscopy confirmed the incorporation of nitrated fibrinogen molecules in fibrin fibers. The nitration of fibrinogen in vivo resulted in four distinct functional consequences: increased initial velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness, and reduced rate of clot lysis. The rate of fibrin clot formation and clot architecture was restored upon depletion of the tyrosine-nitrated fibrinogen molecules. An enhanced response to the knob "B" mimetic peptides Gly-His-Arg-Pro(am) and Ala-His-Arg-Pro(am) suggests that incorporation of nitrated fibrinogen molecules accelerates fibrin lateral aggregation. The data provide a novel biochemical risk factor that could explain epidemiological associations of oxidative stress and inflammation with thrombotic complications.

    Funded by: NHLBI NIH HHS: P50 HL70128, R01 HL54926; NIEHS NIH HHS: ES013508

    The Journal of biological chemistry 2008;283;49;33846-53

  • Lp(a) and risk of recurrent cardiac events in obese postinfarction patients.

    Corsetti JP, Ryan D, Rainwater DL, Moss AJ, Zareba W, Block RC and Sparks CE

    Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. James_Corsetti@urmc.rochester.edu

    Studies of recurrent coronary events in obese postinfarction patients show mixed results despite potential importance of obesity-related pathophysiologic processes and associated markers in establishing and predicting risk. The study aim was to determine specific markers of recurrent risk in obese postinfarction patients. Nondiabetic patients of the Thrombogenic Factors and Recurrent Coronary Events (THROMBO) postinfarction study were classified according to BMI as normal weight (<25 kg/m(2)), overweight (25.0-29.9 kg/m(2)), and obese (> or = 30 kg/m(2)). Cox multivariable regression with adjustment for significant clinical covariates was performed in each group monitoring outcome (cardiac death, myocardial infarction (MI), or unstable angina with 26 months follow-up) as a function of 17 thrombogenic, inflammatory, and metabolic blood markers and 17 cardiovascular disease-associated genetic polymorphisms. Results revealed no statistically significant genetic or blood marker variables in normal or overweight patients. For obese postinfarction patients, elevated lipoprotein(a) (Lp(a))was found to be a highly significant risk marker with hazard ratio and 95% confidence interval of 3.94 (2.11-7.35), P = 0.000017 (upper tertile vs. lower two tertiles). Additionally, elevated Lp(a) was found to interact with the -75G>A polymorphism of the apolipoprotein A-I gene and the -250G>A polymorphism of the hepatic lipase gene in establishing risk. We conclude that interactions of elevated Lp(a) with polymorphisms of the apolipoprotein A-I and hepatic lipase genes, primarily reflective of altered lipoprotein metabolism, play an important role in the establishment of recurrent coronary event risk in obese, nondiabetic postinfarction patients. These findings suggest close monitoring and consideration of weight reduction for obese postinfarction patients with elevated Lp(a) levels.

    Funded by: NCRR NIH HHS: KL2 RR 024136, KL2 RR024136; NHLBI NIH HHS: HL-48259

    Obesity (Silver Spring, Md.) 2008;16;12;2717-22

  • Prevalence of prothrombotic polymorphisms in Greece.

    Gialeraki A, Politou M, Rallidis L, Merkouri E, Markatos C, Kremastinos D and Travlou A

    Haematology Laboratory, Attikon Hospital, School of Medicine, University of Athens, Athens, Greece. agialer@med.uoa.gr

    The aim of this study was to assess the prevalence of several polymorphisms in genes that are involved in several pathways such as hemostasis, fibrinolysis, platelet membrane receptor activity, endothelial integrity and function, lipid metabolism, and regulation of blood pressure in healthy subjects of Greek origin. Most of these polymorphisms are mainly associated with conditions such as venous thromboembolism and atherothrombosis, and their prevalence has not been studied yet in Greece. We tested 140 healthy individuals for factor V (FV)1691G/A, FV4070G/A, FII 20210G/A, factor XIII (FXIII) exon 2G/T, fibrinogen beta-455G/A, plasminogen activator inhibitor-1 (PAI-1)-675 4G/5G, human platelet antigens 1 (HPA1) a/b, apolipoprotein B (ApoB) 10708 G/A, apolipoprotein E (ApoE) E2, E3, and E4, angiotensin-converting enzyme (ACE) D/I, 5,10 methylenetetrahydrofolate reductase (MTHFR) 677C/T, and MTHFR 1298A/C polymorphisms using a PCR and reverse hybridization technique that detects all of them simultaneously. The allele frequencies observed are in accordance with those reported in other Caucasian populations and almost identical to those of East Mediterranean populations. This first report from Greece may serve as a baseline for planning further investigations of these polymorphisms in association with several clinical entities and for launching guidelines for patient testing of various disease settings in this population.

    Genetic testing 2008;12;4;541-7

  • Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study.

    Ennis S, Jomary C, Mullins R, Cree A, Chen X, Macleod A, Jones S, Collins A, Stone E and Lotery A

    Genetic Epidemiology and Bioinformatics Group, University of Southampton, Human Genetics Division (Mp 808), Southampton General Hospital, Southampton, UK.

    Background: Age-related macular degeneration is the most prevalent form of visual impairment and blindness in developed countries. Genetic studies have made advancements in establishing the molecular cause of this disease, identifying mutations in the complement factor H (CFH) gene and a locus on chromosome 10 encompassing the HTRA1/LOC387715/ARMS2 genes. Variants in complement 3 (C3) and an HLA locus containing both factor B and C2 genes have also been implicated. We aimed to identify further genetic risk factors for this disease.

    Methods: We used a case-control study design in a UK sample of patients with age-related macular degeneration (n=479) and controls (n=479) and undertook a low-density screen of 32 genes using 93 single nucleotide polymorphisms (SNPs). Genes were selected as candidates on the basis of potential functional relevance to age-related macular degeneration. Significant initial findings were confirmed by replication in an independent US cohort of 248 unrelated patients with disease and 252 controls, and by high-density genotyping around association signals.

    Findings: The SNP variant rs2511989, located within intron six of the SERPING1 gene, showed highly significant genotypic association with age-related macular degeneration (uncorrected p=4.0x10(-5), corrected p=0.00372). We detected no evidence for association between disease and the other 31 candidate genes. The odds ratio for age-related macular degeneration in rs2511989 G/A heterozygotes compared with wild type G/G homozygotes was 0.63 (95% CI 0.47-0.84). A similar comparison of the A/A homozygotes with the wild type yielded an odds ratio of 0.44 (0.31-0.64). We replicated the observed genotypic association in a US cohort (p=0.008). Furthermore, a secondary high-density genotyping study across the SERPING1 gene region identified five additional SNP variants similarly associated with age-related macular degeneration (rs2244169, rs2511990, rs2509897, rs1005510, and rs2511988).

    Interpretation: Genetic variation in SERPING1 significantly alters susceptibility to age-related macular degeneration. SERPING1 encodes the C1 inhibitor, which has a crucial role in inhibition of complement component 1 (C1) and might implicate the classic pathway of complement activation in this disease.

    Funded by: Howard Hughes Medical Institute; NEI NIH HHS: EY-016822, EY-017451; Wellcome Trust: 076169

    Lancet (London, England) 2008;372;9652;1828-34

  • Candidate genes and cerebral palsy: a population-based study.

    Gibson CS, Maclennan AH, Dekker GA, Goldwater PN, Sullivan TR, Munroe DJ, Tsang S, Stewart C and Nelson KB

    Schools of aPaediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia.

    Objective: The objective of this study was to examine whether selected genetic polymorphisms in the infant are associated with later-diagnosed cerebral palsy.

    Methods: A population-based case-control study was conducted of 28 single-nucleotide polymorphisms measured in newborn screening blood spots. A total of 413 children with later-diagnosed cerebral palsy were born to white women in South Australia in 1986-1999, and there were 856 control children. Distributions of genotypic frequencies were examined in total cerebral palsy, in gestational age groups, and by types of cerebral palsy and gender. Genotyping was performed by using a TaqMan assay.

    Results: For inducible nitric-oxide synthase, possession of the T allele was more common in all children with cerebral palsy and for heterozygotes who were born at term. For lymphotoxin alpha, homozygous variant status was associated with risk for cerebral palsy and with spastic hemiplegic or quadriplegic cerebral palsy. Among term infants, heterozygosity for the endothelial protein C receptor single-nucleotide polymorphism was more frequent in children with cerebral palsy. In preterm infants, the variant A allele of interleukin 8 and heterozygosity for the beta-2 adrenergic receptor were associated with cerebral palsy risk. Interleukin 8 heterozygote status was associated with spastic diplegia. Variants of several genes were associated with cerebral palsy in girls but not in boys.

    Conclusions: Two of the 28 single-nucleotide polymorphisms examined were associated with all types of spastic cerebral palsy in both gestational age groups and others with cerebral palsy in gestational age or cerebral palsy subgroups. Some of these associations support previous findings. There may be a genetic contribution to cerebral palsy risk, and additional investigation is warranted of genes and gene-environment interactions in cerebral palsy.

    Funded by: NCI NIH HHS: N01-CO-12400

    Pediatrics 2008;122;5;1079-85

  • Comparison of thrombophilic gene mutations among patients experiencing recurrent miscarriage and deep vein thrombosis.

    Coulam CB, Wallis D, Weinstein J, DasGupta DS and Jeyendran RS

    Rinehart Center for Reproductive Medicine, Evanston, IL, USA. cbcoulam@aol.com

    Problem: Inherited thrombophilia has been shown to be a risk factor for cardiovascular disease including deep venous thrombosis as well as reproductive disorders including recurrent pregnancy loss. We have previously reported three out of the 10 thrombophilic mutations studied, plasminogen activator inhibitor-1 (PAI-1) 4G/5G, factor XIII V34L, and homozygous MTHFR C667T, correlated significantly with recurrent pregnancy loss compared with controls. This study was undertaken to compare the frequencies of nine inherited thrombophilias among women with a history of recurrent pregnancy loss with individuals experiencing deep venous thrombosis and fertile controls.

    Six hundred thirty-four participants including 550 women with a history of recurrent pregnancy loss, 43 individuals with deep vein thrombosis and 41 fertile women without a history of recurrent miscarriage. All participants had buccal swabs taken for DNA analyses of nine gene polymorphisms including factor V G1691A, factor V H1299R (R2), factor II Prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, human platelet antigen 1 a/b (L33P), MTHFR C677T, MTHFR A1298C. Frequencies of thrombophilic gene polymorphisms were compared among the three populations studied.

    Results: Individuals with a history of DVT had a significantly higher frequency of all of the polymorphisms studied compared with women experiencing a history of recurrent pregnancy loss and the fertile controls. The frequencies of mutations for V34L and PAI-1 4G/5G were significantly increased among women experiencing recurrent pregnancy loss compared with controls. The most prevalent polymorphisms were factor XIII V34L and PAI-1 4G/4G for both individuals with a history of deep vein thrombosis and recurrent pregnancy loss compared with controls.

    Conclusion: Screening for risk factors for inherited thrombophilia with only polymorphisms for factor V von Leiden, factor II prothrombin and MTHFR may be missing the more prevalent identifiers of jeopardy.

    American journal of reproductive immunology (New York, N.Y. : 1989) 2008;60;5;426-31

  • Fibrinogen Foxton: a novel BbetaA277V mutation causing low normal plasma fibrinogen concentration.

    Davis RL, Baker B and Brennan SO

    Molecular Pathology Laboratory, Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, Christchurch, New Zealand. ryan.davis@otago.ac.nz

    Thrombosis and haemostasis 2008;100;4;708-10

  • Common genetic polymorphisms and haplotypes of fibrinogen alpha, beta, and gamma chains affect fibrinogen levels and the response to proinflammatory stimulation in myocardial infarction survivors: the AIRGENE study.

    Jacquemin B, Antoniades C, Nyberg F, Plana E, Müller M, Greven S, Salomaa V, Sunyer J, Bellander T, Chalamandaris AG, Pistelli R, Koenig W and Peters A

    Centre for Environmental Research, Municipal Institute of Medical Research, Barcelona, Spain.

    Objectives: This study was designed to investigate whether single nucleotide polymorphisms (SNPs) and haplotypes of the fibrinogen gene-cluster (fibrinogen chains alpha [FGA], beta [FGB], and gamma [FGG]) could explain the inter- and intraindividual variability of fibrinogen levels in patients with atherosclerosis. We also searched for genetic determinants affecting the responses of fibrinogen genes to proinflammatory stimulation.

    Background: The mechanisms regulating fibrinogen levels are not fully understood, and they are likely to be regulated by complex gene-environment interactions.

    Methods: In the AIRGENE study, 895 survivors of myocardial infarction from 5 European cities were followed prospectively for 6 to 8 months, and plasma fibrinogen, interleukin (IL)-6, and C-reactive protein levels were determined monthly. We analyzed 21 SNPs and the corresponding haplotypes in the 3 fibrinogen genes.

    Results: Eight SNPs in FGA and FGB were significantly associated with fibrinogen levels. Similarly, 2 different haplotypes in FGA and 3 in FGB were also associated with mean fibrinogen levels. The IL-6 levels had a significant impact on the associations between SNPs/haplotypes in FGA/FGB and fibrinogen levels. We also identified SNPs and haplotypes in FGA and FGB with strong impact on the intraindividual variability of fibrinogen during the follow-up period.

    Conclusions: We identified common SNPs and haplotypes on FGA/FGB genes, explaining the interindividual and intraindividual variability of fibrinogen levels, in patients with a history of myocardial infarction. We have also identified for the first time, SNPs/haplotypes on FGA/FGB whose effects on fibrinogen expression are modified by the underlying IL-6 levels. These findings may have an impact on risk stratification and the design of genetically guided therapeutic approaches in patients with advanced atherosclerosis.

    Journal of the American College of Cardiology 2008;52;11;941-52

  • Association of beta-fibrinogen gene -148C/T and -455G/A polymorphisms and coronary artery disease in Chinese population: a meta analysis.

    Chen X, Xu M, Jin L, Chen J and Chen W

    Department of Cardiology, Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, 519000, China.

    The purpose of our study was to evaluate the correlation between the beta-fibrinogen gene -148C/T and -455G/A polymorphisms and susceptibility to coronary artery disease in the Chinese population using a meta-analytic approach. Eligible studies about this correlation were identified by searching the PubMed, EMBASE, and CNKI databases. Of the 13 identified, 7 (with 1488 cases and 1234 controls) involved the -148C/T polymorphism and 9 (with 1023 cases and 1081 controls) involved the -455G/A polymorphism. No publication bias was detectable and heterogeneity testing found significant differences between the ORs for both groups of studies. The combined OR for the 7 studies on susceptibility to coronary artery disease in -148T allele carriers compared to the -148C/C wild-type homozygotes was 1.31 (95%CI: 0.94-1.84, P = 0.11). The combined OR for the 9 studies on susceptibility to coronary artery disease in -455A allele carriers compared to the -455G/G wild-type homozygotes was 1.75 (95%CI: 1.24-2.46, P = 0.001). Our results suggest the absence of an association between the beta-fibrinogen gene -148C/T polymorphism and susceptibility to coronary artery disease and the possibility that -455G/A polymorphism (in particular, allele A) increases susceptibility to this disease in the Chinese population.

    Science in China. Series C, Life sciences 2008;51;9;814-20

  • Frequency distribution of the G/A alleles of the beta-fibrinogen gene in the Lebanese population.

    Shammaa DM, Sabbagh AS, Taher AT, Zaatari GS and Mahfouz RA

    Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Riad El Solh, Beirut, Lebanon.

    Fibrinogen is a plasma protein that has been reported to be associated with an increased risk of atherothrombotic diseases and venous thrombosis. The most common polymorphism that has been studied so far in different populations is the G-455-->A polymorphism in the promoter region of the beta-fibrinogen gene. We studied 160 healthy unrelated Lebanese individuals for the prevalence of -455G/G, -455G/A and -455A/A genotypes of the beta-fibrinogen gene and the frequency of G and A alleles using a reverse hybridization PCR assay. The prevalence of the G/G, G/A, and A/A genotypes were found to be 60.6, 31.9 and 7.5%, respectively. The frequency of the G and A alleles were found to be 0.77 and 0.23, respectively. As compared to other ethnic groups, the Lebanese individuals were found to have a relatively high prevalence of the A allele which may predispose them to develop cardiovascular diseases as well as thrombotic events. This study provides additional unique genetic information pertaining to the Lebanese population.

    Molecular biology reports 2008;35;3;307-11

  • Influence of fibrinogen beta-chain gene variations on risk of myocardial infarction in a Chinese Han population.

    Lu XF, Yu HJ, Zhou XY, Wang LY, Huang JF and Gu DF

    Department of Evidence Based Medicine and Division of Population Genetics, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China.

    Background: Although the role of fibrinogen as a predictor of acute myocardial infarction (MI) has been well-established, the association of genetic polymorphisms in the fibrinogen gene with MI is still controversial. This study was conducted to elucidate the association between the genetic polymorphisms of the fibrinogen beta-chain (FGB) gene and MI in Chinese Han population.

    Methods: The occurrence of 3 common polymorphisms (i.e., -455G/A, R448K and 8558C/G) in a case-control study including 508 patients with MI and 503 healthy controls was investigated. Results Analyses of single polymorphisms showed that individuals carrying the rare alleles for the 3 polymorphisms were significantly associated with a decreased risk of MI. Logistic regression analysis indicated that R448K remained independently associated with MI after adjustment for environmental risk factors (adjusted odds ratio(OR) = 0.71 for KK/RK versus RR, P = 0.023). The three polymorphisms were found to be in strong linkage disequilibrium. Haplotype analyses showed that the A-K-G haplotype (-455A, 448K, 8558G) was associated with a protective effect against MI. Compared with the common haplotype G-R-C, the adjusted OR for A-K-G was 0.68 (95% CI, 0.51-0.90; P = 0.006).

    Conclusion: These data indicate that individuals carrying the FGB 448K allele may be protective against having MI in this population.

    Chinese medical journal 2008;121;16;1549-53

  • Haplotypes of the fibrinogen gene and cerebral small vessel disease: the Rotterdam scan study.

    van Oijen M, Cheung EY, Geluk CE, Hofman A, Koudstaal PJ, Breteler MM and de Maat MP

    Department of Epidemiology and Biostatistics, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands.

    Objective: Fibrinogen levels and fibrinogen clot structure have been implicated in the pathogenesis of vascular disease. We examined fibrinogen levels and variation in fibrinogen genes (fibrinogen gamma (FGG), alpha (FGA) and beta (FGB)), which have been associated with fibrin clot structure and fibrinogen levels, in relation to cerebral small vessel disease (SVD).

    This study was performed as part of the Rotterdam Scan Study, a population based study in 1077 elderly patients undergoing cerebral MRI. Plasma fibrinogen levels and haplotypes were determined. We examined the association between fibrinogen levels and haplotype with silent brain infarcts and white matter lesions using logistic regression models. We constructed seven haplotypes (frequency >0.01) that describe the total common variation in the FGG and FGA genes. Haplotype 2 (GATAGTG) was associated with the presence of silent brain infarcts compared with the most frequent haplotype (GGTGGTA) (OR 1.41, 95% CI 1.03 to 1.94). Haplotype 3 (GGCGATA) was associated with periventricular white matter lesions in the highest tertile of the distribution (OR 1.40, 95% CI 1.01 to 1.92). No association was found between plasma fibrinogen levels and SVD.

    Conclusions: Our study provides evidence for an association of common variation in the FGG and FGA genes with cerebral SVD. It is possible that the structure of the fibrin clot rather than plasma fibrinogen levels plays a role in the pathogenesis of cerebral SVD.

    Journal of neurology, neurosurgery, and psychiatry 2008;79;7;799-803

  • Fibrinogen gene variation and ischemic stroke.

    Jood K, Danielson J, Ladenvall C, Blomstrand C and Jern C

    Institute of Neuroscience and Physiology, the Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden.

    Background: Plasma fibrinogen level and fibrin clot structure are heritable traits that may be of importance in the pathogenesis of ischemic stroke.

    Objectives: To investigate associations between variation in the fibrinogen gamma (FGG), alpha (FGA) and beta (FGB) genes, fibrinogen level, and ischemic stroke.

    Methods: The Sahlgrenska Academy Study on Ischemic Stroke comprises 600 cases and 600 matched population controls. Stroke subtypes were defined according to TOAST criteria. Plasma fibrinogen level was measured by an automated clot-rate assay. Eight tagging single nucleotide polymorphisms (SNPs) were selected to capture genetic variation in the FGA, FGG, and FGB genes.

    Results: Plasma fibrinogen was independently associated with overall ischemic stroke and all subtypes, both in the acute stage (P < 0.001) and at three-month follow-up (P < 0.05). SNPs belonged to two haplotype blocks, one containing the FGB gene and the other the FGG and FGA genes. FGB haplotypes were associated with fibrinogen level (P < 0.01), but not with ischemic stroke. In contrast, FGG/FGA haplotypes showed independent association to ischemic stroke but not to fibrinogen level. In an additive model with the most common FGG/FGA haplotype (A1) as reference, the adjusted odds ratios of ischemic stroke were 1.4 [95% confidence interval (95% CI) 1.1-1.8], P < 0.01, 1.4 (95% CI 1.0-1.8), P < 0.05, and 1.5 (95% CI 1.0-2.1), P < 0.05 for the A2, A3, and A4 FGG/FGA haplotypes, respectively.

    Conclusion: FGG/FGA haplotypes show association to ischemic stroke. This association is independent of fibrinogen level, thus suggesting that the association between ischemic stroke and variation at the FGG/FGA genes is mediated by qualitative rather than quantitative effects on fibrin(ogen).

    Journal of thrombosis and haemostasis : JTH 2008;6;6;897-904

  • New application of intelligent agents in sporadic amyotrophic lateral sclerosis identifies unexpected specific genetic background.

    Penco S, Buscema M, Patrosso MC, Marocchi A and Grossi E

    Medical Genetics, Clinical Chemistry and Clinical Pathology Laboratory, Niguarda Ca' Granda Hospital P.za Ospedale Maggiore 3, 20100 Milan, Italy. silvana.penco@ospedaleniguarda.it

    Background: Few genetic factors predisposing to the sporadic form of amyotrophic lateral sclerosis (ALS) have been identified, but the pathology itself seems to be a true multifactorial disease in which complex interactions between environmental and genetic susceptibility factors take place. The purpose of this study was to approach genetic data with an innovative statistical method such as artificial neural networks to identify a possible genetic background predisposing to the disease. A DNA multiarray panel was applied to genotype more than 60 polymorphisms within 35 genes selected from pathways of lipid and homocysteine metabolism, regulation of blood pressure, coagulation, inflammation, cellular adhesion and matrix integrity, in 54 sporadic ALS patients and 208 controls. Advanced intelligent systems based on novel coupling of artificial neural networks and evolutionary algorithms have been applied. The results obtained have been compared with those derived from the use of standard neural networks and classical statistical analysis

    Results: Advanced intelligent systems based on novel coupling of artificial neural networks and evolutionary algorithms have been applied. The results obtained have been compared with those derived from the use of standard neural networks and classical statistical analysis. An unexpected discovery of a strong genetic background in sporadic ALS using a DNA multiarray panel and analytical processing of the data with advanced artificial neural networks was found. The predictive accuracy obtained with Linear Discriminant Analysis and Standard Artificial Neural Networks ranged from 70% to 79% (average 75.31%) and from 69.1 to 86.2% (average 76.6%) respectively. The corresponding value obtained with Advanced Intelligent Systems reached an average of 96.0% (range 94.4 to 97.6%). This latter approach allowed the identification of seven genetic variants essential to differentiate cases from controls: apolipoprotein E arg158cys; hepatic lipase -480 C/T; endothelial nitric oxide synthase 690 C/T and glu298asp; vitamin K-dependent coagulation factor seven arg353glu, glycoprotein Ia/IIa 873 G/A and E-selectin ser128arg.

    Conclusion: This study provides an alternative and reliable method to approach complex diseases. Indeed, the application of a novel artificial intelligence-based method offers a new insight into genetic markers of sporadic ALS pointing out the existence of a strong genetic background.

    BMC bioinformatics 2008;9;254

  • Polymorphisms in coagulation factors and the risk of recurrent cardiovascular events in men after a first myocardial infarction.

    van der Krabben MD, Rosendaal FR, van der Bom JG and Doggen CJ

    Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.

    Background: The aim of this study was to examine whether genetic predisposition to high levels of coagulation factors influences the risk of developing fatal and non-fatal arterial cardiovascular events in men with a first myocardial infarction (MI).

    Methods: We performed a cohort study among 542 MI patients with a mean age of 56 years (range 32-70 years) at the time of the event. All of the men had a first MI between 1990 and 1996 and were followed until 1 September 2004. DNA was analyzed for polymorphisms of fibrinogen, prothrombin (factor II), factor V, factor VII and plasminogen activator inhibitor type 1, all of which are associated with gain of function of the protein. We collected information from hospital files and general practitioners on the occurrence of major arterial events.

    Results: In total, 254 major arterial cardiovascular events occurred during a median follow-up period of 11 years (range 0.2-15 years). The point estimates of the relative rates (RRs) of these events for the variant genotypes were all between 0.7 and 1.1 except for the prothrombin 20210A mutation: RR 1.8 (95% confidence interval 0.8-4.1).

    Conclusion: These findings suggest that there is no association between coagulation factor polymorphisms, previously associated with plasma levels, and the risk of recurrent cardiovascular events.

    Journal of thrombosis and haemostasis : JTH 2008;6;5;720-5

  • Fibrinogen Aalpha Thr312Ala polymorphism is associated with chronic thromboembolic pulmonary hypertension.

    Suntharalingam J, Goldsmith K, van Marion V, Long L, Treacy CM, Dudbridge F, Toshner MR, Pepke-Zaba J, Eikenboom JC and Morrell NW

    Pulmonary Vascular Diseases Unit, Papworth Hospital, Papworth Everard.

    Although chronic thromboembolic pulmonary hypertension (CTEPH) is characterised by the persistence of organised thrombus, few pro-thrombotic risk factors have been identified in subjects with the disease. The aim of the present study was to compare the prevalence of eight functionally relevant haemostatic polymorphisms between CTEPH subjects and healthy controls. Genomic DNA was isolated from 214 CTEPH subjects and 200 healthy controls, and analysed for Factor V Leiden, prothrombin guanine (G) to adenine (A) substitution at nucleotide 20210 (20210G>A), plasminogen activator inhibitor-1 4G/5G, tissue plasminogen activator 7351 cytosine (C)>thymidine (T), Factor XIII 100G>T, fibrinogen Aalpha substitution of threonine with alanine at position 312 (Thr312Ala), fibrinogen Bbeta substitution of arginine with lysine at position 448 (Arg448Lys) and fibrinogen Bbeta 455G>A polymorphisms. A significant difference was demonstrated in fibrinogen Aalpha Thr312Ala genotype and allele frequencies between CTEPH subjects and controls. The presence of the alanine allele significantly increased the risk of CTEPH. The fibrinogen Aalpha alanine 312 allele alters fibrinogen alpha-alpha chain cross-linkage and has previously been associated with both increased risk of embolisation and increased resistance to thrombolysis. An association between this polymorphism and chronic thromboembolic pulmonary hypertension, therefore, supports an embolic aetiology for this disease, and may provide a mechanism by which thrombus persists following an acute event.

    Funded by: Medical Research Council: MC_U105260799

    The European respiratory journal 2008;31;4;736-41

  • Fibrinogen genes and myocardial infarction: a haplotype analysis.

    Koch W, Hoppmann P, Biele J, Mueller JC, Schömig A and Kastrati A

    Deutsches Herzzentrum München and 1. Medizinische Klinik, Klinikum rechts der Isar, Munich, Germany. wkoch@dhm.mhn.de

    Objective: Fibrinogen has a role in inflammatory processes and participates in atherosclerotic plaque formation. Despite intensive investigation, there is no clear evidence for a role of variations in the genes coding for the fibrinogen-alpha, fibrinogen-beta, and fibrinogen-gamma polypeptide chains in myocardial infarction. We examined the association of haplotypes in the 50-kb fibrinogen gene region with myocardial infarction in 2 large case-control samples.

    Study sample 1 consisted of 3657 patients with myocardial infarction and 1211 control individuals and sample 2 comprised 1392 patients and 1392 controls. Haplotypes were inferred from genotype analyses of tagging single nucleotide polymorphisms dispersed among the fibrinogen genes. The frequencies of these haplotypes were not significantly different between the case and control groups in either sample (P > or = 0.07). In addition, haplotypes specific for individual fibrinogen genes were analyzed. No substantial differences in the frequencies of these haplotypes were observed between the groups (P > or = 0.13). Finally, haplotypes composed of SNPs that exhibited relatively low pairwise allelic associations among each other were examined. The proportions of the haplotypes were not significantly different between cases and controls (P > or = 0.12).

    Conclusions: A haplotype analysis did not reveal a link between genetic variations in the fibrinogen gene region and myocardial infarction.

    Arteriosclerosis, thrombosis, and vascular biology 2008;28;4;758-63

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Combined effect of hemostatic gene polymorphisms and the risk of myocardial infarction in patients with advanced coronary atherosclerosis.

    Martinelli N, Trabetti E, Pinotti M, Olivieri O, Sandri M, Friso S, Pizzolo F, Bozzini C, Caruso PP, Cavallari U, Cheng S, Pignatti PF, Bernardi F, Corrocher R and Girelli D

    Department of Clinical and Experimental Medicine, University of Verona, Verona, Italy.

    Background: Relative little attention has been devoted until now to the combined effects of gene polymorphisms of the hemostatic pathway as risk factors for Myocardial Infarction (MI), the main thrombotic complication of Coronary Artery Disease (CAD). The aim of this study was to evaluate the combined effect of ten common prothrombotic polymorphisms as a determinant of MI.

    We studied a total of 804 subjects, 489 of whom with angiographically proven severe CAD, with or without MI (n = 307; n = 182; respectively). An additive model considering ten common polymorphisms [Prothrombin 20210G>A, PAI-1 4G/5G, Fibrinogen beta -455G>A, FV Leiden and "R2", FVII -402G>A and -323 del/ins, Platelet ADP Receptor P2Y12 -744T>C, Platelet Glycoproteins Ia (873G>A), and IIIa (1565T>C)] was tested. The prevalence of MI increased linearly with an increasing number of unfavorable alleles (chi(2) for trend = 10.68; P = 0.001). In a multiple logistic regression model, the number of unfavorable alleles remained significantly associated with MI after adjustment for classical risk factors. As compared to subjects with 3-7 alleles, those with few (</=2) alleles had a decreased MI risk (OR 0.34, 95%CIs 0.13-0.93), while those with more (>/=8) alleles had an increased MI risk (OR 2.49, 95%CIs 1.03-6.01). The number of procoagulant alleles correlated directly (r = 0.49, P = 0.006) with endogenous thrombin potential.

    Conclusions: The combination of prothrombotic polymorphisms may help to predict MI in patients with advanced CAD.

    Plo 1f40 S one 2008;3;2;e1523

  • Associations between common fibrinogen gene polymorphisms and cardiovascular disease in older adults. The Cardiovascular Health Study.

    Carty CL, Cushman M, Jones D, Lange LA, Hindorff LA, Rice K, Jenny NS, Durda JP, Walston J, Carlson CS, Nickerson D, Tracy RP and Reiner AP

    Cardiovascular Health Research Unit, Department of Epidemiology, University of Washington, 1760 Minor Avenue, Seattle, WA 98101, USA. calyca@u.washington.edu

    Elevated plasma fibrinogen is a risk factor for cardiovascular disease (CVD), but associations between fibrinogen single nucleotide polymorphisms (SNPs) and disease risk are inconsistent. We investigated whether common (> or = 5% minor allele frequency) variation in the fibrinogen genes (FGA, FGB, FGG) is associated with fibrinogen concentration, carotid artery intima-medial thickness (IMT) and risk of incident myocardial infarction (MI), ischemic stroke and CVD mortality in European- (EA) and African-descent (AA) adults (> or = 65 years) from the Cardiovascular Health Study. TagSNPs were genotyped in 3,969 EA and 719 AA free of MI or stroke at baseline. Race-specific models included multiple testing correction and adjustment for sex, age and site. Among EA, minor alleles of FGA3807, FGB1437 and FGG902 were associated with higher fibrinogen levels; whereas FGA251, FGA2224, FGA6534 and FGG10034 were associated with lower levels, p<0.004 for each. Strongest associations were seen for FGB1437; each additional copy of the minor allele was associated with 13 mg/dl (95%CI: 9-16) higher fibrinogen level. Similar trends in AA were not significant. Fibrinogen haplotypes were not significantly associated with internal or common carotid IMT. No associations with MI or CVD mortality were seen in EA, though FGB1038 and FGG902 were significantly associated with increased and decreased risk of stroke in men, respectively, as were related haplotypes. FGB1038 was also associated with CVD mortality in AA, HR = 1.9 (95%CI: 1.3-2.7). In conclusion, while fibrinogen genetic variation was strongly associated with fibrinogen levels, there was less evidence of association with the more complex outcomes of IMT and CVD events.

    Funded by: NHLBI NIH HHS: HL 071862, N01 HC 15103, N01 HC 35129, N01 HC 55222, N01 HC 85079, N01 HC 85080, N01 HC 85081, N01 HC 85082, N01 HC 85083, N01 HC 85084, N01 HC 85085, N01 HC 85086, U01 HL 080295

    Thrombosis and haemostasis 2008;99;2;388-95

  • [Correlation between levels of fibrinogen, beta455 g/A fibrinogen gene polymorphism and chronic periodontitis].

    Ge S, Wu YF, Liu TJ, He QM, Zhao L and Meng S

    Department of Periodontology, West China School of Stomatology, Sichuan University, Chengdu 610041, China.

    Objective: To investigate the relationship between plasma levels of fibrinogen, the-beta455 G/A fibrinogen gene polymorphism and the severity of periodontal inflammation and to explore the possible role of fibrinogen in the association of periodontitis with coronary heart disease (CHD).

    Methods: A total of 121 patients with moderate to severe periodontitis and periodontally healthy and gingivitis controls were enrolled in the study. Peripheral blood samples were collected and the plasma fibrinogen levels were determined by the clotting method of Clauss. Polymerase chain reaction and restriction fragment length polymorphism analysis with Hae III were used to examine the -beta455 G/A fibrinogen gene polymorphism.

    Results: Fibrinogen levels were significantly higher in moderately or severely chronic periodontitis patients [(3.45 +/- 0.68) g/L] than periodontally healthy and gingivitis controls [(2.47 +/- 0.42) g/L, P < 0.001]. The carrier status of the A allele at position -455 in the beta fibrinogen gene was associated with elevated fibrinogen levels and the frequency of the-A455 allele in the beta fibrinogen gene in the patient group was significantly higher than in the control group (P = 0.032). Carriers of the -A455 allele were about 3-fold more likely to have moderate or severe periodontitis as compare to individuals without the -A455 allele( OR = 3. =135, P= 0.008).

    Conclusions: Fg-beta455 G/A polymorphism may contribute to the elevated plasma fibrinogen levels and put individuals at higher risk of having severe periodontitis. As the independent risk factor of CHD, fibrinogen levels and Fg-beta455 G/A polymorphism may play a role in the pathogenesis of periodontitis.

    Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 2008;43;2;87-91

  • Common variation in the C-terminal region of the fibrinogen beta-chain: effects on fibrin structure, fibrinolysis and clot rigidity.

    Ajjan R, Lim BC, Standeven KF, Harrand R, Dolling S, Phoenix F, Greaves R, Abou-Saleh RH, Connell S, Smith DA, Weisel JW, Grant PJ and Ariëns RA

    Academic Unit of Molecular Vascular Medicine, Leeds Institute for Genetics, Health and Therapeutics, University of Leeds, UK.

    Fibrinogen BbetaArg448Lys is a common polymorphism, positioned within the carboxyl terminus of the Bbeta-chain of the molecule. Studies suggest that it is associated with severity of coronary artery disease and development of stroke. The effects of the amino acid substitution on clot structure remains controversial, and the aim of this study was to investigate the effect(s) of this polymorphism on fibrin clot structure using recombinant techniques. Permeation, turbidity, and scanning electron microscopy showed that recombinant Lys448 fibrin had a significantly more compact structure, with thin fibers and small pores, compared with Arg448. Clot stiffness, measured by means of a novel method using magnetic tweezers, was significantly higher for the Lys448 compared with the Arg448 variant. Clots made from recombinant protein variants had similar lysis rates outside the plasma environment, but when added to fibrinogen-depleted plasma, the fibrinolysis rates for Lys448 were significantly slower compared with Arg448. This study demonstrates for the first time that clots made from recombinant BbetaLys448 fibrinogen are characterized by thin fibers and small pores, show increased stiffness, and appear more resistant to fibrinolysis. Fibrinogen BbetaArg448Lys is a primary example of common genetic variation with a significant phenotypic effect at the molecular level.

    Funded by: NHLBI NIH HHS: HL30954

    Blood 2008;111;2;643-50

  • [The beta-fibrinogen gene polymorphism, fibrinogen level and platelet aggregation in patients with ischemic stroke].

    Favorova OO, Sudomoina MA, Martynov MIu, Serdiuk IE, Parfenov MG, Nikonova AA, Kolesnikova TI, Iasamanova AN and Gusev EI

    We examined an association of the C-148T beta-fibrinogen gene polymorphism with fibrinogen level and platelet aggregation in patients with ischemic stroke and in people with vascular risk factors but no ischemic stroke. Among stroke patients, carriers of an -148T allele had significantly higher plasma fibrinogen level and platelet aggregation to norepinephrin compared to the C/C homozygotes. No significant differences in frequencies of a C-148 allele between stroke patients and controls were observed.

    Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova 2008;Suppl 23;10-4

  • Association of fibrinogen and fibrinogen gene beta148 and beta854 polymorphisms with coronary heart disease.

    Sun AJ, Ma HL, Chen XY, Wang Y, Fang YM, Ma YL, Wang KQ, Zou YZ, Huang W and Ge JB

    Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai Institute of Cardiovascular Disease, Shanghai, China.

    Background: The aim of this study was to observe the association between fibrinogen C148T, G854A polymorphisms and plasma fibrinogen levels in a large cohort of Chinese patients with coronary heart disease (CHD).

    Methods: Fibrinogen gene beta148/beta854 polymorphisms were screened and plasma fibrinogen levels and lipids were measured in patients with angiographically confirmed CHD (n = 836) and in controls without CHD (n = 418).

    Results: Age, sex, smoking, hypertension, diabetes mellitus, blood lipids and fibrinogen levels were related to CHD (all p < 0. 05) while the frequencies of fibrinogen beta148 and beta854 alleles, gene and genotypes between the two groups were similar (all p > 0.05).

    Conclusion: This study demonstrates that the plasma fibrinogen level, but not fibrinogen beta148 and beta854 genotypes, was associated with CHD in the Chinese population.

    Cardiology 2008;111;3;167-70

  • Connection between genetically determined blood coagulation factors and haemorheology.

    Pongrácz E, Andrikovics H, Bernát IS and Nagy Z

    Department of Neurology-Stroke, State Health Centre, Budapest, Hungary. pangracze@gmail.com

    Background: The phenotype caracteristic of blood clotting factors are well known, however few data has been documented about effects on haemorheology. The connection among genetic polymorphisms, haemorheological factors and vascular mortality is also studied poorly.

    Purpose: Our aim was: to study six genetic polymorphisms of blood clotting factors, which presents the role of platelet-plasmaprotein-endothel system in thrombotic course in controls and ischaemic stroke cohort. Second, to study the connection of genotypes and haemorheologic factors and both with five years vascular mortality in patients.

    It was studied the genetic polymorhisms of GP IIb/IIIa Leu33Pro, prothrombin gene G20210A, ACE I/D, fibrinogen gene-455 G/A, Leiden mutation and MTHFR C677T alleles or genotypes in blood samples of 433 ischaemic stroke patients and 173 controls by PCR. Haematocrit values, plasma fibrinogen (FIB) concentration, whole blood viscosity (WBV) at 90 s(-1) and also the plasma viscosity (PV) were measured. Vascular mortality of patients were followed during five years and studied by curves Kaplan-Meier.

    Results: A higher plasma FIB concentration in non smoker patients, carrying A alleles of FIB gene could be observed as compared to wild types (p<0.05). Also a moderate WBV increasing in smoker patients with A alleles was found against wild types (p=0.11), at the same time we observed a significant WBV increasing in non smoker patients (p<0.05). The highest quartile of PV showed a connection with Leiden mutation in whole group of patients (p=0.01), in subgroup of young patients (<50 years) (p=0.03) and also in non smoker groups (p<0.05) as compared to patients having wild types. No association could be detected between different genetic polymorphisms and vascular mortality, however it was observed significant mortality increasing in patients having PV above 1.51 mPa s (p=0.03).

    Conclusion: Certain genetic polymorphisms of coagulation system could result unfavorable haemorheological changes, however non of them increases the mortality. The connection between higher mortality and PV focuses the attention for the necessity of PV measuring and correction in stroke patients.

    Clinical hemorheology and microcirculation 2008;39;1-4;333-41

  • Gender differences in genetic risk profiles for cardiovascular disease.

    Silander K, Alanne M, Kristiansson K, Saarela O, Ripatti S, Auro K, Karvanen J, Kulathinal S, Niemelä M, Ellonen P, Vartiainen E, Jousilahti P, Saarela J, Kuulasmaa K, Evans A, Perola M, Salomaa V and Peltonen L

    Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland. kaisa.silander@ktl.fi

    Background: Cardiovascular disease (CVD) incidence, complications and burden differ markedly between women and men. Although there is variation in the distribution of lifestyle factors between the genders, they do not fully explain the differences in CVD incidence and suggest the existence of gender-specific genetic risk factors. We aimed to estimate whether the genetic risk profiles of coronary heart disease (CHD), ischemic stroke and the composite end-point of CVD differ between the genders.

    We studied in two Finnish population cohorts, using the case-cohort design the association between common variation in 46 candidate genes and CHD, ischemic stroke, CVD, and CVD-related quantitative risk factors. We analyzed men and women jointly and also conducted genotype-gender interaction analysis. Several allelic variants conferred disease risk for men and women jointly, including rs1801020 in coagulation factor XII (HR = 1.31 (1.08-1.60) for CVD, uncorrected p = 0.006 multiplicative model). Variant rs11673407 in the fucosyltransferase 3 gene was strongly associated with waist/hip ratio (uncorrected p = 0.00005) in joint analysis. In interaction analysis we found statistical evidence of variant-gender interaction conferring risk of CHD and CVD: rs3742264 in the carboxypeptidase B2 gene, p(interaction) = 0.009 for CHD, and rs2774279 in the upstream stimulatory factor 1 gene, p(interaction) = 0.007 for CHD and CVD, showed strong association in women but not in men, while rs2069840 in interleukin 6 gene, p(interaction) = 0.004 for CVD, showed strong association in men but not in women (uncorrected p-values). Also, two variants in the selenoprotein S gene conferred risk for ischemic stroke in women, p(interaction) = 0.003 and 0.007. Importantly, we identified a larger number of gender-specific effects for women than for men.

    A false discovery rate analysis suggests that we may expect half of the reported findings for combined gender analysis to be true positives, while at least third of the reported genotype-gender interaction results are true positives. The asymmetry in positive findings between the genders could imply that genetic risk loci for CVD are more readily detectable in women, while for men they are more confounded by environmental/lifestyle risk factors. The possible differences in genetic risk profiles between the genders should be addressed in more detail in genetic studies of CVD, and more focus on female CVD risk is also warranted in genome-wide association studies.

    PloS one 2008;3;10;e3615

  • [Genetic polymorphism and risk of arterial thrombosis in patients with atrial fibrillation].

    Serdechnaia EV, Tatarskiĭ BA and Kazakevich EV

    This study revealed the importance of 6 genetic determinants (accounting for predisposition to thrombophilia) as risk factors of thromboembolic stroke in patients with atrial fibrillation (AF). The occurrence of A-allele of FGB gene of patients with complicated form of AF was shown to be significantly higher compared with controls. Carriage of FGB-455A, grade II mitral regurgitation, and AF longer than 48 hours are assumed to be independent predictors of thromboembolic stroke.

    Klinicheskaia meditsina 2008;86;12;30-3

  • Meta-analysis of genetic studies from journals published in China of ischemic stroke in the Han Chinese population.

    Xu X, Li J, Sheng W and Liu L

    Department of Neurology, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China.

    Background: The aim of this study was to confirm the nature and number of genes contributing to stroke risk and qualify the genetic risk of each susceptibility gene in the Han Chinese population.

    Methods: After collecting all case-control studies related to DNA polymorphism of any candidate gene for ischemic stroke in Han Chinese, strict selection criteria and exclusion criteria were determined and different effect models were used according to the difference in heterogeneity. Meta-analyses were carried out by Revman 4.0 software and the publication bias was further evaluated through calculation of fail-safe numbers in the included gene polymorphisms.

    Results: Seventy-six studies were included in the meta-analyses which were all published in mainland China and referred to 6 candidate genes and 7 polymorphisms. Among the gene polymorphisms tested in the study, association of gene polymorphisms with increasing risk of ischemic stroke was confirmed in 6 polymorphisms including angiotensin-converting enzyme insertion/deletion (ACE I/D; OR = 1.87, 95% CI = 1.45-2.42), methylenetetrahydrofolate reductase (MTHFR) C677T (OR = 1.55, 95% CI = 1.26-1.90), plasminogen activator inhibitor 1 (PAI-1) 4G/5G (OR = 1.79, 95% CI = 1.20-2.67), beta-fibrinogen (beta-Fg) -455A/G (OR = 1.48, 95% CI = 1.14-1.92), beta-Fg -148T/C (OR = 1.72, 95% CI = 1.42-2.07), apolipoprotein E (ApoE) epsilon2-4 (OR = 2.39, 95% CI = 1.94-2.95). Because of the obvious publication bias, the association between paraoxonase 1 (PON-1) polymorphisms and stroke risk was not established although the OR of the meta-analysis suggested a positive result (OR = 1.14, 95% CI = 1.01-1.35).

    Conclusions: ACE D/I, MTHFR C677T, beta-Fg -455A/G, beta-Fg -148T/C, PAI-1 4G/5G, and ApoE epsilon2-4 were associated with risk of ischemic stroke in Han Chinese.

    Cerebrovascular diseases (Basel, Switzerland) 2008;26;1;48-62

  • Polymorphisms of the fibrinogen-beta gene are related to 2-hour glucose level after oral glucose tolerance test in Hong Kong Chinese.

    Wong LY, Ong KL, Cheung BM, Leung RY, Man YB and Lam TH

    Department of Medicine, University of Hong Kong, Hong Kong, China.

    Fibrinogen, an acute phase protein, is an important inflammatory marker that is associated with cardiovascular diseases. We studied the association of three common human fibrinogen-beta gene (FGB) variants, -455G>A, -249C>T, and -148C>T with glycemic parameters in 265 non-diabetic Hong Kong Chinese subjects. Both FGB variants, -455G>A and -148C>T were in comple 1512 te linkage disequilibrium and were associated with higher levels of plasma fibrinogen and 2-h glucose after a 75-g oral glucose load (p<0.01). Carriers of FGB AC-haplotype, comprising the two nucleotide variants at positions -455 and -249, had higher fibrinogen level (2.64 +/- 0.65 vs 2.42 +/- 0.52 g/L, p=0.002) and 2-h glucose after a 75-g oral glucose load (5.87 +/- 1.14 vs 5.47 +/- 1.22 g/L, p=0.006). The associations were significant in men, but not women. In stepwise multiple regression analysis, AC-haplotype was independently associated with plasma fibrinogen level and 2-h glucose (p=0.002 and 0.010 respectively). This suggests that fibrinogen may play a role in the development of impaired glucose tolerance.

    Disease markers 2008;24;3;167-73

  • Haemostatic genetic variants, ABO blood group and bleeding risk during oral anticoagulant treatment after cerebral ischaemia of arterial origin.

    Pruissen DM, Rosendaal FR, Gorter JW, Garcia AA, Kappelle LJ, Algra A and SPIRIT Study Group

    Dept. of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, The Netherlands.

    Oral anticoagulant treatment for secondary prevention after cerebral ischaemia of presumed arterial origin is associated with a higher bleeding rate than cardioembolic stroke. This discrepancy is only partly explained by known bleeding risk factors. Haemostatic genetic variants and AB0 blood group may be involved. We performed a nested casecontrol study in patients with cerebral ischaemia of presumed arterial origin on anticoagulant treatment (International Normalized Ratio between 3.0-4.5). All 34 cases with non-fatal haemorrhage (10 intracranial and 24 extracranial) and 68 control patients on anticoagulant treatment without such a bleeding were selected from the SPIRIT study. AB0 blood group and 11 haemostatic genetic variants were investigated. The Thr312Ala variant of the alpha fibrinogen gene was associated with a decreased bleeding risk (odds ratio (OR) 0.3 for Ala/Ala and Thr/Ala versus Thr/Thr genotype; 95% CI 0.1-0.8). Factor V Leiden was associated with an increased bleeding risk (OR 11.6; 95% CI 1.3-103). The APOE2 allele (OR 0.5; 95% CI 0.2-1.7) and the Tyr204Phe variant in the factor XIII subunit A (OR 2.1; 0.9-5) had nonsignificant relationships with bleeding risk. AB0 blood group and 7 other genetic variants in coagulation factors II and XIII, vitamin K epoxide reductase complex, beta fibrinogen and apolipoprotein E were not related with the risk of haemorrhage. The Ala312Thr variant in the alpha fibrinogen gene is associated with a decreased and factor V Leiden with an increased bleeding risk in patients on anticoagulant treatment after cerebral ischaemia of presumed arterial origin.

    Journal of neurology 2007;254;12;1660-5

  • Fibrinogen Tolaga Bay: a novel gammaAla341Val mutation causing hypofibrinogenaemia.

    Davis RL and Brennan SO

    Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, New Zealand. ryan.davis@chmeds.ac.nz

    Thrombosis and haemostasis 2007;98;5;1136-8

  • Polymorphism of genes related to cardiovascular disease in patients with rheumatoid arthritis.

    Arlestig L, Wållberg Jonsson S, Stegmayr B and Rantapää-Dahlqvist S

    Department of Public Health and Clinical Medicine/Rheumatology and Medicine, University Hospital, Umeå, Sweden.

    Objective: To analyze candidate genes, related to cardiovascular disease (CVD) in general, and potentially involved in the inflammatory process, in RA patients from northern Sweden.

    Methods: Four hundred and sixty-seven individuals (345 females; 122 males) with RA (ACR criteria), having a mean age of 61.8 +/- 13.0 years and mean disease duration of 16.2 +/- 12.1 years, were consecutively recruited and followed-up for 3 years. The prevalence of CVD, [(ischemic heart disease (IHD), deep vein thromboses/pulmonary embolism (DVT/PE) and/or stroke/TIA] and hypertension was registered. Candidate genes encoding for Beta-fibrinogen (G-455A), Factor XIIIA (Val34Leu), plasminogen activator inhibitor type-1 (PAI-1 4G/5G), and tumor necrosis factor receptor (TNFR)II (M196R) were analysed. Controls (n = 672) were randomly selected according to age and gender from the Medical Biobank of Northern Sweden. Polymorphisms were genotyped using a TaqMan 9700HT and the 5'nuclease allelic discrimination assay.

    Results: The genotypes, carriers and alleles did not differ in distribution between patients and controls. Carriage of the TNFRII R variant was more frequent among patients with hypertension (p = 0.018). The genotype distribution of PAI-1 in patients with IHD differed significantly (p = 0.002) because carriage of 4G was more frequent (p = 0.024). Combined carriage of TNFRII 196R variant and Beta-fibrinogen-455A was a stronger predictor for hypertension than each genotype separately. The distribution of FXIIIA genotypes deviated significantly in RA patients with DVT/PE (p = 0.028) with an increased frequency of the 8d4 Leu34 variant.

    Conclusion: The unusual alleles of TNFRII, PAI-1 and FXIIIA were associated with CVD in RA patients. The combination of several of the rare types further increased the predictive values for CVD.

    Clinical and experimental rheumatology 2007;25;6;866-71

  • A meta-analysis of beta-fibrinogen gene-455G/A polymorphism and plasma fibrinogen level in Chinese cerebral infarction patients.

    Chen XC, Xu MT, Zhou W, Han CL and Chen WQ

    Department of Cardiology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, Guangdong, China. cxcoffice@21cn.com

    Objective: To evaluate the correlation between the beta-fibrinogen gene-455G/A polymorphism and cerebral infarction in Chinese population by means of meta-analysis.

    Methods: Genetic association studies on evaluating the beta-fibrinogen gene -455G/A polymorphism and cerebral infarction involving Chinese population published before December 2005 were collected from database of PubMed, EMBASE, and CNKI. All the data in literature were abstracted based on the defined selection criteria by two independent investigators. Publication bias was tested by funnel plot and the odd ratios of all studies were combined dependent on the result of heterogeneity test among the individual studies. The software Review Manager (Version 4.2) was used for meta-analysis.

    Results: Eleven studies including 1405 patients and 1600 controls met the selection criteria. There was no publication bias in 11 reviewed studies. Heterogeneity test of reviewed studies showed statistically significant differences (chi2=24.58, P=0.006) among the ORs of individual studies. The combined OR of 11 studies of susceptibility to cerebral infarction in -455A allele carriers compared with the -455G/G wild homozygotes was 1.33 (95%CI 1.04-1.71, P=0.02). In the patients with cerebral infarction in 6 studies, the summarized average plasma fibrinogen level of allele A carrier was 0.29 g/L (95%CI 0.14-0.44, P=0.0002) higher than that of -455G/G homozygous ones.

    Conclusions: beta-fibrinogen gene -455G/A polymorphism might contribute to susceptibility of cerebral infarction in Chinese population; allele A increases the individual susceptibility to the disease.

    Biomedical and environmental sciences : BES 2007;20;5;366-72

  • Probing the beta-chain hole of fibrinogen with synthetic peptides that differ at their amino termini.

    Doolittle RF and Pandi L

    Department of Chemistry and Biochemistry and Division of Biology, University of California, San Diego, La Jolla, California 92093-0314, USA. rdoolittle@ucsd.edu

    In a recent report, we showed that alanine can replace glycine at the amino terminus of synthetic B-knobs that bind to human fibrin(ogen). We now report a survey of 13 synthetic peptides with the general sequence XHRPYam, all tested with regard to their ability to delay fibrinolysis in an in vitro system activated by t-PA, the results being used as measures of binding affinity to the betaC hole. Unexpectedly, some large and bulky amino acids, including methionine and arginine, are effective binders. Amino acids that branch at the beta carbon (valine, isoleucine, and threonine) do not bind effectively. Crystal structures were determined for two of the peptides (GHRPYam and MHRPYam) complexed with fibrin fragment D-dimer; the modeling of various other side chains showed clashing in the cases of beta-carbon substituents. The two crystal structures also showed that the enhanced binding observed with pentapeptides with carboxyl-terminal tyrosine, compared with that of their tetrapeptide equivalents, is attributable to an interaction between the tyrosine side chain and a guanidino group of a nearby arginine (beta406). The equivalent position in gamma-chains of human fibrin(ogen) is occupied by a lysine (gamma338), but in chicken and lamprey fibrin(ogen), it is an arginine, just as occurs in beta chains. Accordingly, the peptides GPRPam and GPRPYam, which are surrogate A-knobs, were tested for their influence on fibrin polymerization with fibrinogen from lamprey and humans. In lampreys, GPRPYam is a significantly better inhibitor, but in humans, it is less effective than GPRPam, indicating that in the lamprey system the same tyrosine-arginine interaction can also occur in the gamma-chain setting.

    Funded by: NHLBI NIH HHS: HL-81553

    Biochemistry 2007;46;35;10033-8

  • The chymotrypsin-like protease complex of Treponema denticola ATCC 35405 mediates fibrinogen adherence and degradation.

    Bamford CV, Fenno JC, Jenkinson HF and Dymock D

    Department of Oral and Dental Science, University of Bristol, Lower Maudlin St., Bristol BS1 2LY, United Kingdom.

    Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The Aalpha and Bbeta chains of fibrinogen, but not the gamma chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, approximately 100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100 degrees C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP(-) mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP(-)) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

    Infection and immunity 2007;75;9;4364-72

  • Clinical and biological features of 3 cases of hypofibrinogenemia associated with three different mutations (gamma Ala341Thr, Bbeta Tyr326Cys and Aalpha Asp496Asn).

    Hanss M, Chevreaud C, French P, Négrier C and de Mazancourt P

    Thrombosis and haemostasis 2007;98;3;689-91

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • A meta-analysis of relationship between beta-fibrinogen gene -148C/T polymorphism and susceptibility to cerebral infarction in Han Chinese.

    Chen XC, Xu MT, Zhou W, Han CL and Chen WQ

    Department of Cardiology, Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, Guangdong Province, China. cxcoffice@21cn.com

    Objective: The results of studies on association between -148C/T polymorphism in promoter region of beta-fibrinogen gene and susceptibility to cerebral infarction in Chinese population are controversial. In this study, we summarize the results of published works in this field by a meta-analysis. Data sources Genetic association studies evaluating the beta-fibrinogen gene -148C/T polymorphisms and cerebral infarction involving Chinese population published before December 2005 were collected from PubMed, EMBASE and CNKI. Study selection Case control studies involving unrelated, Han subjects aged from 18 to 80 years, and the internationally recognized diagnostic standard of cerebral infarction and genotype frequencies in control group consistent with Hardy-Weinberg equilibrium were used. Publication bias was tested by funnel plot and the odds ratios of all studies were combined dependent on the result of heterogeneity test among the individual studies. The software Review Manager (Version 4.2) was used for meta-analysis.

    Results: Eleven studies including 1223 patients and 1433 controls met the selection criteria. There was no heterogeneity among the odds ratios (ORs) of individual studies (chi(2) = 17.82, P = 0.06). The combined OR of susceptibility to cerebral infarction in -148T allele carriers compared to the wild homozygote was 1.32 (95% CI 1.12 to 1.55, P = 0.0008). In the patients with cerebral infarction, the average plasma fibrinogen level of allele T carrier was 0.42 g/L (95% CI 0.29 to 0.54, P < 0.001), higher than that of -148C/C homozygous ones.

    Conclusions: beta-fibrinogen gene -148C/T polymorphism might contribute to susceptibility to cerebral infarction in Han Chinese. To reach a definitive conclusion, further gene to gene and gene to environment interactions studies on beta-fibrinogen polymorphisms and cerebral infarction with large sample size are required.

    Chinese medical journal 2007;120;13;1198-202

  • Fibrinogen-beta gene haplotype is associated with mortality in sepsis.

    Manocha S, Russell JA, Sutherland AM, Wattanathum A and Walley KR

    Critical Care Research Laboratories, James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, St. Paul's Hospital, 1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6.

    Objectives: Fibrinogen plays a key role in coagulation and inflammation. Transcription of the fibrinogen-beta gene (FGB) is the rate-limiting step in fibrinogen production. Our aim was to determine whether haplotypes of FGB are associated with mortality and organ dysfunction in a cohort of patients with sepsis.

    Methods: A prospective cohort of 631 consecutive Caucasian patients with sepsis from a tertiary care medical-surgical ICU were enrolled in a gene association study. Patients were genotyped for three polymorphisms in FGB: -854 G/A, -455 G/A, and +9006 G/A. Haplotypes were inferred using PHASE. The primary outcome was mortality. Secondary outcomes were severity of organ dysfunction as measured by days alive and free (DAF) of organ dysfunction.

    Results: Haplotype GAA was associated with a significantly lower 28-day mortality (28.9% vs. 36.9% for all other haplotypes, p=0.03). Carriers of two copies of haplotype GAA (vs. one and zero copies) had more DAF of organ dysfunction. In a multivariate analysis, haplotype GAA was an independent predictor for lower mortality (OR=0.66, 95% CI=0.46-0.94, p=0.02).

    Conclusions: Haplotype GAA in FGB is associated with lower mortality and lower severity of organ dysfunction. Haplotype GAA encompasses a previously described haplotype -1420A/-854G/-455A/-249C/-148T/+1690G that is associated with higher fibrinogen levels.

    The Journal of infection 2007;54;6;572-7

  • Epistatic effect of plasminogen activator inhibitor 1 and beta-fibrinogen genes on risk of glomerular microthrombosis in lupus nephritis: interaction with environmental/clinical factors.

    Gong R, Liu Z and Li L

    Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China.

    Objective: Glomerular microthrombosis (GMT) is not uncommon in lupus nephritis and has been associated with active renal injury and progressive kidney destruction. We undertook this study to determine whether genetic variations of hemostasis factors, such as plasminogen activator inhibitor 1 (PAI-1) and fibrinogen, affect the risk of GMT.

    Methods: A cross-sectional cohort of 101 lupus nephritis patients with or without GMT was genotyped for PAI-1 -675 4G/5G and beta-fibrinogen (FGB) -455 G/A gene polymorphisms and analyzed.

    Results: PAI-1 4G/4G homozygotes and FGB A allele carriers were both at increased risk for GMT. When the data were stratified for both gene polymorphisms, an epistatic effect was detected. The PAI-1 4G/4G genotype was found to predispose to GMT not equally in all lupus nephritis patients, but only in FGB A allele carriers. Likewise, the association between the FGB A allele and GMT was restricted to lupus nephritis patients homozygous for the PAI-1 4G allele. This epistatic effect was revalidated by the multifactor dimensionality reduction (MDR) analysis and further assessed by incorporating a variety of environmental and clinical factors into the MDR analysis. The most parsimonious model that had a cross-validation consistency of 100% included joint effects of PAI-1 and FGB gene polymorphisms and anticardiolipin antibody (aCL) status and yielded the best prediction of GMT, with 66.6% accuracy.

    Conclusion: Our findings suggest that risk of GMT in lupus nephritis is attributable, at least in part, to an epistatic effect of PAI-1 and FGB genes, likely via an interaction with environmental/clinical factors, such as aCL.

    Arthritis and rheumatism 2007;56;5;1608-17

  • Fibrinogen and fibrin: scaffold proteins in hemostasis.

    Lord ST

    Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. stl@med.unc.edu

    Elevated fibrinogen is a cardiovascular risk factor. Recent work provides a rationale for this risk, as abnormal fibrin clot structure, strength and stability correlates with coronary artery disease. This review describes in-vitro experiments whose intent is to define the molecular mechanisms that control clot architecture and function in vivo.

    Biochemical and structural data continue to define the interactions between monomer units that assemble into a fibrin clot. In particular, 'A: a' interactions dominate the first step in fiber formation, while the analogous 'B: b' interactions have a minor role. Studies show the N-terminus of Bbeta, the C-terminus of Aalpha, and the splice variant gamma' modulate fibrin clot structure. Measurement of the mechanical properties of fibrinogen and fibrin show fibrin fibers are among the strongest in nature. Studies have identified fibrinogen-binding proteins that influence clot structure and function.

    Summary: These findings defined mechanisms that control fibrin clot structure, strength and stability. This basic information provides direction for clinical studies to examine clot properties in pathologic thrombosis and pharmaceutical studies to develop therapeutic interventions to prevent or control cardiovascular disease. These studies also establish novel techniques to examine individual bonds, molecules and fibers.

    Funded by: NHLBI NIH HHS: HL31048

    Current opinion in hematology 2007;14;3;236-41

  • Analysis of the effect of multiple genetic variants of cardiovascular disease risk on insulin concentration variability in healthy adults of the STANISLAS cohort. The role of FGB-455 G/A polymorphism.

    Maumus S, Marie B, Vincent-Viry M, Siest G and Visvikis-Siest S

    Institut National de la Santé et de la Recherche Médicale (INSERM) U525, 30 rue Lionnois, F-54000 Nancy, France.

    Introduction: Given the hypothesis of a common soil for atherosclerosis, type 2 diabetes and metabolic syndrome, we tested the contribution of gene polymorphisms involved in cardiovascular diseases on fasting insulin concentration (FIC).

    Methods: The polymorphisms were investigated by a multiplex assay in 308 apparently healthy French middle-aged men and women, taken from the STANISLAS cohort. FIC was measured by a microparticular enzymatic immunoassay.

    Results: After a series of regression analyses involving 34 polymorphisms, FGB -455G/A was the only polymorphism that remained significantly associated with FIC when adjusting the analyses for multiple testing. Stepwise models showed that FGB polymorphism accounted for 4.39% of FIC variability in men. Additionally, interactions between FGB and with environmental factors (alcohol and smoking in men, and BMI in women) were found.

    Discussion: To our knowledge, this is the first study reporting an influence of FGB polymorphism on FIC in a healthy population. Our results concord with the already shown link between fibrinogen concentration and FIC, and support the hypothesis of a relationship between fibrinogen and endothelium in FIC homeostasis whose alteration may induce several metabolic disorders. The contribution of this gene, although modest, is consistent with the polygenic nature of insulin levels.

    Atherosclerosis 2007;191;2;369-76

  • Elevated plasma fibrinogen gamma' concentration is associated with myocardial infarction: effects of variation in fibrinogen genes and environmental factors.

    Mannila MN, Lovely RS, Kazmierczak SC, Eriksson P, Samnegård A, Farrell DH, Hamsten A and Silveira A

    Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, Karolinska Institutet, Solna, Sweden. maria.nastase.mannila@ki.se

    Background: Fibrinogen gamma', a fibrinogen gamma-chain variant generated via alternative mRNA processing, has been associated with susceptibility to thrombotic disease.

    Objective: The present case-control study searched for potential determinants of the plasma fibrinogen gamma' concentration and examined the relationship between this variant and risk of myocardial infarction (MI).

    The Stockholm Coronary Artery Risk Factor study, comprising 387 postinfarction patients and 387 healthy individuals, was employed. The fibrinogen gamma (FGG) 9340T > C [rs1049636], fibrinogen alpha (FGA) 2224G > A [rs2070011] and fibrinogen beta (FGB) 1038G > A [rs1800791] polymorphisms were determined. The plasma fibrinogen gamma' concentration was measured by enzyme-linked immunosorbent assay. The multifactor dimensionality reduction method was used for interaction analyses on risk of MI.

    Results: The FGG 9340T > C and FGA 2224G > A polymorphisms, total plasma concentrations of fibrinogen, insulin and high-density lipoprotein, and gender appeared to be independent determinants of plasma fibrinogen gamma' concentration in patients, and the corresponding determinants in controls included FGG 9340T > C and FGA 2224G > A polymorphisms and plasma fibrinogen concentration. An elevated plasma fibrinogen gamma' concentration proved to be an independent predictor of MI [adjusted odds ratio (OR) (95% CI): 1.24 (1.01, 1.52)]. The plasma fibrinogen gamma' concentration was involved in a high-order interaction with total plasma fibrinogen and the FGG 9340T > C and FGA 2224G > A polymorphisms, associated with a further increased risk of MI [OR (95% CI): 3.22 (2.35, 4.39)].

    Conclusions: Plasma fibrinogen gamma' concentration influences the risk of MI, and this relationship seems to be strengthened by the presence of an elevated total plasma fibrinogen concentration and the FGG 9340T and FGA 2224G alleles.

    Funded by: NHLBI NIH HHS: 1 R21 HL75006

    Journal of thrombosis and haemostasis : JTH 2007;5;4;766-73

  • TagSNP evaluation for the association of 42 inflammation loci and vascular disease: evidence of IL6, FGB, ALOX5, NFKBIA, and IL4R loci effects.

    Carlson CS, Heagerty PJ, Nord AS, Pritchard DK, Ranchalis J, Boguch JM, Duan H, Hatsukami TS, Schwartz SM, Rieder MJ, Nickerson DA and Jarvik GP

    Division of Public Health Sciences, The Fred Hutchinson Cancer Research Center, The University of Washington, Seattle, WA, USA.

    Inflammatory markers have consistently been associated with vascular disease. Evidence of genetic polymorphisms in inflammatory loci that predict severe carotid artery disease (CAAD) would suggest that this relationship is not secondary to other correlated factors, but related to inflammation itself. We examined the full common genetic variation in 42 inflammatory loci for prediction of severe CAAD versus ultrasound proven controls using a tagSNP approach. For selected loci, monocyte RNA levels were contrasted in subjects with and without CAAD. We confirm the association of IL6(-174), FGB (-455), and ALOX5 with CAAD and show that multiple ALOX5 SNPs independently predict CAAD. We provide evidence for previously unreported associations of SNPs in IL4R, NFKBIA, and PLG with CAAD, and weaker evidence for associations with CSF3, IL10RA, and VCAM1. The NFKBIA and IL10RA expression levels significantly differed between subjects with CAAD and controls. These results support a role for genetic variation related to inflammation in CAAD and a causal role for specific gene products.

    Funded by: NHLBI NIH HHS: P01 HL072262, R01 HL073401, R01 HL074366, R01HL67406, U01 HL66682

    Human genetics 2007;121;1;65-75

  • AMI is associated with polymorphisms in the NOS3 and FGB but not in PAI-1 genes in young adults.

    Sampaio MF, Hirata MH, Hirata RD, Santos FC, Picciotti R, Luchessi AD, de Quateli Doi S, Armaganijan D and Batlouni M

    Instituto Dante Pazzanese de Cardiologia, Sao Paulo, SP, Brazil.

    Background: We investigated the relationship between NOS3, FGB and PAI-1 polymorphisms and endothelial dysfunction and risk factors for acute myocardial infarction (AMI) in young adults.

    Methods: Endothelial function was measured by response to flow mediated vasodilation (FMV) and induced by nitrate (FMN). Biochemical parameters were measured by standard enzymatic methods and plasma total nitrate was analyzed by the NOA system. NOS3 (T-786C, G894T and intron 4A/B STR), FGB (C-148T and G-455A) and PAI-1 (4G/5G) polymorphisms were determined by PCR-RFLP.

    Results: Concentrations of total and LDL cholesterol, apo B, triglycerides, nitrate, PAI-1 and fibrinogen were higher and apo AI, HDL cholesterol and FMV were lower in AMI patients than in controls (p<0.001). PAI-1 (p<0.001) but not nitrate was higher in AMI patients with low response to FMV. NOS3 T-786C and FGB C-148T polymorphisms were associated with AMI (p<0.050). NOS3 T-786C was also related to hypertension (p=0.049). NOS3 intron 4A/B STR was associated with increased concentrations of total cholesterol and apo B. NOS3-786TT/894GT haplotype was associated with increased FMV (p=0.018) than the other haplotypes.

    Conclusions: Our data suggest NOS3 and FGB polymorphisms are associated with AMI. NOS3 is also related to hypertension, endothelial dysfunction and variation on serum cholesterol in young adults with AMI.

    Clinica chimica acta; international journal of clinical chemistry 2007;377;1-2

  • Genetic variation in estrogen receptor, C-reactive protein and fibrinogen does not predict the plasma levels of inflammation markers after longterm hormone replacement therapy.

    de Maat MP, Madsen JS, Langdahl B, Bladbjerg EM, Tofteng CL, Abrahamsen B, Rejnmark L, Brixen K, Christensen K, Jespersen J and Kristensen SR

    Department for Thrombosis research, Institute of Public Health, University of Southern Denmark, Denmark. m.demaat@erasmusmc.nl

    Markers of inflammation, such as C-reactive protein (CRP) and fibrinogen, are associated with the risk of atherothrombosis. Plasma levels of these markers of inflammation are affected by hormone replacement therapy (HRT) and modulated by smoking. We studied whether genetic variation in the estrogen receptor- 1 (ESR1), CRP and fibrinogen-beta genes influences the plasma levels of inflammation markers after HRT. Plasma CRP and fibrinogen were measured after five years follow-up in healthy postmenopausal women (per-protocol group) who were randomised to hormone therapy (n=187) or no treatment (n=249). The effect of HRT, smoking and genetic variations in ESR1 (PvuII and XbaI), CRP (1444C/T) and fibrinogen-beta (FGB, -455G/A) were determined. The plasma concentration of CRP was higher in the HRT group than in the control group (2.03 mg/l and 1.41 mg/l, respectively; p < 0.001), while the concentration of fibrinogen was lower in the HRT group than in the control group (3.02 g/l and 3.20 g/l, respectively; p < 0.001), indicating that it is unlikely that inflammation is the common underlying pathway. There was a significant interaction between smoking and HRT on the fibrinogen (p=0.02), but not on the CRP concentration (n.s.). Genetic polymorphisms in ESR1, CRP and fibrinogen were not associated with an effect of HRT on the CRP and fibrinogen plasma levels, and no significant interaction with smoking was observed. In conclusion, higher plasma levels of CRP and lower plasma levels of fibrinogen were observed in women using HRT; however, genetic polymorphisms in ESR1, CRP and FGB were not associated with these effects of HRT.

    Thrombosis and haemostasis 2007;97;2;234-9

  • Polymerization of fibrin: Direct observation and quantification of individual B:b knob-hole interactions.

    Litvinov RI, Gorkun OV, Galanakis DK, Yakovlev S, Medved L, Shuman H and Weisel JW

    Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 1040 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104-6058, USA. litvinov@mail.med.upenn.edu

    The polymerization of fibrin occurs primarily through interactions between N-terminal A- and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a- and b-holes in the gamma- and beta-modules. Of the potential knob-hole interactions--A:a, B:b, A:b, and B:a--the first has been shown to be critical for fibrin formation, but the roles of the others have remained elusive. Using laser tweezers-based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the gammaD364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mimetic peptide, the (beta15-66)2 fragment containing 2 B-knobs, and a monoclonal antibody against the beta15-21 sequence. The interaction of desB-fibrin with fragment D containing a- and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer.

    Funded by: NHLBI NIH HHS: HL-30954, HL-31048, HL-56051, R01 HL030954, R01 HL031048, R01 HL056051

    Blood 2007;109;1;130-8

  • Associations of the beta-fibrinogen Hae III and factor XIII Val34Leu gene variants with venous thrombosis.

    Cushman M, Cornell A, Folsom AR, Wang L, Tsai MY, Polak J and Tang Z

    Department of Medicine, University of Vermont College of Medicine, Burlington, VT 05446, USA. mary.cushman@uvm.edu

    Introduction: The factor XIII Val34Leu (100 G-->T) and beta-fibrinogen Hae III (-455 G-->A) gene variants have been associated with reduced risk of venous thrombosis, but not in all studies.

    Methods: We investigated the associations of these polymorphisms with risk of venous thrombosis in a prospective, population-based study of 21,680 men and women aged 45-100 years at enrollment. Factor XIII 100 G/T and beta-fibrinogen -455 G/A were analyzed on stored DNA from 511 thrombosis cases and 1028 control subjects without thrombosis during follow up.

    Results: The beta-fibrinogen A allele was present in 24.4% of cases and 32.3% of controls. Compared to GG subjects, the age, race, and sex adjusted odds ratio (OR) of venous thrombosis was 0.77 (95% CI 0.59-0.99) for GA subjects, and 0.60 (95% CI 0.31-1.16) for AA subjects. The adjusted OR of thrombosis associated with factor XIII 100 G/T was 1.01 (95% CI 0.81-1.26) for GT subjects and 0.45 (95% CI 0.44-1.19) for TT subjects, compared to GG. For both genotypes, ORs of thrombosis were similar in whites and non-whites, although there were no non-white fibrinogen AA cases. beta-fibrinogen -455 GA or AA attenuated the thrombosis risk associated with obesity (from 2.14 to 1.25) and factor V Leiden (from 3.89 to 2.36).

    Conclusions: beta-fibrinogen -455 G/A, but not factor XIII 100 G/T, was associated with a lower risk of venous thrombosis in this general population sample. beta-fibrinogen -455 A may attenuate the increased thrombosis risk associated with obesity or factor V Leiden.

    Funded by: NHLBI NIH HHS: N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, N01-HC-55022, N01-HC-85079, N01-HC-85086, N01HC55015, N01HC55016, N01HC55018, N01HC55019, N01HC55020, N01HC55021, N01HC55022, N01HC85079, N01HC85086, R01 HL059367, R01 HL059367-07, R01 HL59367

    Thrombosis research 2007;121;3;339-45

  • A comprehensive analysis of 12 thrombophilic mutations and related parameters in patients with inflammatory bowel disease: data from Turkey.

    Yilmaz S, Bayan K, Tüzün Y, Batun S and Altintaş A

    Department of Gastroenterology, Dicle University Faculty of Medicine, Diyarbakir, 21280, Turkey. drserif@dicle.edu.tr

    Background: Possible association of inflammatory bowel disease (IBD) with the most common inherited prothrombotic conditions has been the focus of many investigations. Advance in modern molecular biology is expanding the thrombophilia evaluation steadily. We tried to put forward a comprehensive thrombophilic profile in IBD and to see the probable role of this profile in pathogenesis.

    Methods: A total of 60 adults (33 patients and 27 healthy controls) were included. We used the CVD-StripAssay which is based on the reverse-hybridization principle to identify a total of 12 thrombophilic gene mutations: Factor V R506Q, Factor V H1299R, prothrombin G20210A, Factor XIII V34L, beta-Fibrinogen-455 G-A, PAI-1 4G/5G, platelet GPIIIa L33P, MTHFR C677T, MTHFR A1298C, ACE I/D, Apo B R3500Q and Apo E2/E3/E4, respectively. Besides, we evaluated many related blood parameters such as protein C, protein S, AT-III, IL-6, TNF-alpha, Apo-A1, Apo-B100, homocysteine (tHcy) etc. using commercially available assays.

    Results: The frequencies of genetic polymorphisms were found to be statistically insignificant among patients and controls, except for three: Beta-Fibrinogen-455G-A, MTHFR A1298C and ACE-I/D. Two patients with a history of deep venous thrombosis had more than one polymorphism. Patients with MTHFR C677T and MTHFR A1298C gene mutations had a similar mean tHcy levels with controls. Patients with Apolipoprotein B R3500Q and Apolipoprotein E4 gene mutations had similar mean LDL-cholesterol levels. Mean total cholesterol and triglyceride levels were similar in patients and controls of Apo E2, E3, E4 alleles.

    Conclusion: Predominantly, the presence of genetic mutations that predispose to hypercoagulable states does not appear to be in correlation with IBD. There was a statistical difference between the proportions of the mutated allele frequencies of Beta-Fibrinogen-455G-A, MTHFR A1298C and ACE-I/D in IBD.

    Journal of thrombosis and thrombolysis 2006;22;3;205-12

  • [Association of fibrinogen B beta -1420G/A, -993C/T and -854G/A gene polymorphism with coronary heart disease].

    Xing HY, Cai WW, Li XM, Fu Q and Liu GX

    Department of Hematology, Affiliated Hospital, Luzhou Medical College, Luzhou, Sichuan, 646000, P. R. China. xinghy52003@126.com

    Objective: To analyze the frequency of FGB gene -1420G/A, -993C/T and -854G/A polymorphisms, and their association with plasma fibrinogen levels in patients with coronary heart disease and in health adults.

    Methods: The FGB gene -1420G/A, -993C/T and -854G/A polymorphisms were analyzed with restriction fragment length polymorphisms, polymerase chain reaction with allele-specific primer and nucleotide sequencing methods. Plasma fibrinogen levels were determined by turbidimetry.

    Results: The frequencies of -1420A were 0.33 in patients with coronary heart disease and 0.26 in health adults. The frequencies of -1420A in coronary heart disease were apparently higher than that in health adults. There were no difference in frequencies of other two alleles. The logistic study suggested -1420G/A polymorphism was associated with coronary heart disease. There are significantly difference in plasma fibrinogen levels in two groups. Plasma fibrinogen levels were significantly increased in patients with coronary heart disease.

    Conclusion: This study suggests -1420G/A polymorphism may be associated with occurrence of coronary heart disease.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2006;23;6;622-6

  • Matrix-specific suppression of integrin activation in shear stress signaling.

    Orr AW, Ginsberg MH, Shattil SJ, Deckmyn H and Schwartz MA

    Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

    Atherosclerotic plaque develops at sites of disturbed flow. We previously showed that flow activates endothelial cell integrins, which then bind to the subendothelial extracellular matrix (ECM), and, in cells b6e on fibronectin or fibrinogen, trigger nuclear factor-kappaB activation. Additionally, fibronectin and fibrinogen are deposited into the subendothelial ECM at atherosclerosis-prone sites at early times. We now show that flow activates ECM-specific signals that establish patterns of integrin dominance. Flow induced alpha2beta1 activation in cells on collagen, but not on fibronectin or fibrinogen. Conversely, alpha5beta1 and alphavbeta3 are activated on fibronectin and fibrinogen, but not collagen. Failure of these integrins to be activated on nonpermissive ECM is because of active suppression by the integrins that are ligated. Protein kinase A is activated specifically on collagen and suppresses flow-induced alphavbeta3 activation. Alternatively, protein kinase Calpha is activated on fibronectin and mediates alpha2beta1 suppression. Thus, integrins actively cross-inhibit through specific kinase pathways. These mechanisms may determine cellular responses to complex extracellular matrices.

    Funded by: NHLBI NIH HHS: R01 HL056595, R01 HL075092, R01 HL75092

    Molecular biology of the cell 2006;17;11;4686-97

  • Gene polymorphisms implicated in influencing susceptibility to venous and arterial thromboembolism: frequency distribution in a healthy German population.

    Hoppe B, Tolou F, Dörner T, Kiesewetter H and Salama A

    Institute of Transfusion Medicine, Campus Benjamin Franklin, Charité - Universitätsklinikum Berlin, Berlin, Germany. berthold.hoppe@charite.de

    Evolvement and progression of cardiovascular diseases affecting the venous and arterial system are influenced by a multitude of environmental and hereditary factors. Many of these hereditary factors consist of defined gene polymorphisms, such as single nucleotide polymorphisms (SNPs) or insertion-deletion polymorphisms, which directly or indirectly affect the hemostatic system. The frequencies of individual hemostatic gene polymorphisms in different normal populations are well defined. However, descriptions of patterns of genetic variability of a larger extent of different factors of hereditary hypercoagulability in single populations are scarce. The aim of this study was i) to give a detailed description of the frequencies of factors of hereditary thrombophilia and their combinations in a German population (n = 282) and ii) to compare their distributions with those reported for other regions. Variants of coagulation factors [factor V 1691G>A (factor V Leiden), factor V 4070A>G (factor V HR2 haplotype), factor VII Arg353Gln, factor XIII Val34Leu, beta-fibrinogen -455G>A, prothrombin 20210G>A], coagulation inhibitors [tissue factor pathway inhibitor 536C>T, thrombomodulin 127G>A], fibrinolytic factors [angiotensin converting enzyme intron 16 insertion/deletion, factor VII-activating protease 1601G>A (FSAP Marburg I), plasminogen activator inhibitor 1-675 insertion/deletion (5G/4G), tissue plasminogen activator intron h deletion/insertion], and other factors implicated in influencing susceptibility to thromboembolic diseases [apolipoprotein E2/E3/E4, glycoprotein Ia 807C>T, methylenetetrahydrofolate reductase 677C>T] were included. The distribution of glycoprotein Ia 807C>T deviated significantly from the Hardy-Weinberg equilibrium, and a comparison with previously published data indicates marked region and ethnicity dependent differences in the genotype distributions of some other factors.

    Thrombosis and haemostasis 2006;96;4;465-70

  • Which thrombophilic gene mutations are risk factors for recurrent pregnancy loss?

    Goodman CS, Coulam CB, Jeyendran RS, Acosta VA and Roussev R

    Millenova Immunology Laboratories, 233 East Erie Street, Chicago, IL 60611, USA.

    Problem: Thrombophilia has been associated with poor obstetrical outcomes. To determine the association of specific inherited thrombophilias and recurrent pregnancy loss, 10 thrombophilic genes were investigated.

    A total of 550 women with a history of recurrent pregnancy loss had buccal swabs taken for DNA analyses of the following gene mutations: factor V G1691A, factor V H1299R (R2), factor V Y1702C, factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b(L33P), methylenetetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C. The frequencies of these mutations were compared with controls published in the literature.

    Results: When examined individually, PAI-1 4G/5G (P = 0.009), factor XIII V34L (P < 0.0001), and homozygous MTHFR C667T (P < 0.0001) correlated significantly with recurrent pregnancy loss compared with controls. The frequency of the factor V Y1702C mutation was extremely low in patients and controls; thus, this gene was removed from further calculations. The remaining six mutated genes, when analyzed cumulatively, also corresponded with recurrent pregnancy loss (P < 0.0001).

    Conclusion: A panel of thrombogenic gene mutations consisting of factor V G1691A, factor V H1299R (R2), factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b(L33P), MTHFR C677T, and MTHFR A1298C can identify individuals at risk for recurrent pregnancy loss.

    American journal of reproductive immunology (New York, N.Y. : 1989) 2006;56;4;230-6

  • Fibrinogen Nottingham II: a novel Bbeta Arg264gly substitution causing hypofibrinogenaemia.

    Hill MB, Brennan SO, Dear A, Strong J, Nejim T and Dolan G

    Department of Clinical Chemistry, Nottingham University Hospitals, Nottingham, UK. marian.hill@nottingham.ac.uk

    Thrombosis and haemostasis 2006;96;3;378-80

  • [A related analysis for alpha, beta fibrinogen gene haplotypes and nucleotide polymorphisms associated with the ischemic stroke in Hainan Han population].

    Liang L, Sun C, Liao XP, Xiao F, Chen XD, Huang SX, Tang XL, Wen GQ, Long ZG, Wang XY, Liu GX, Cheng S and Cai WW

    Department of Biochemistry, Hainan Medical College, Haikou, Hainan, 571101 PR China.

    Objective: To analyze the association of that the polymorphisms and haplotypes of Taq I site in beta fibrinogen gene and the single nucleotide sites -455 G/A, -249 C/T, -148 C/T, +1689T/G, Bsm A I G/C, 448 G/A, Bcl I G/A, Hinf I A/C in beta-fibrinogen gene are linked up with the ischemic stroke(IS).

    Methods: Turbidmetric assay was used to measure the plasma fibrinogen level of one hundred and sixty cases with ischemic stroke and one hundred and thirty healthy individuals from Hainanese Han population. The polymorphisms and genotypes were characterized by PCR-RFLP. Hardy-Weinberg equilibrium and statistical differences of allelic, genotype and haplotype frequencies were obtained by Chi-square test. Pairwise linkage disequilibrium was calculated and haplotypes of nine or four polymorphisms were estimated by the EH + program.

    Results: There were highly significant differences in genotype frequencies and allelic frequencies of the polymorphisms -455 G/A, -148 C/T, 448 G/A, which happened between the IS group and control subjects (P< 0.01). However, the significant differences of the allelic frequencies in the other six polymorphisms were not found between the IS group and the control (P> 0.05). The odds ratio(OR) with the rare alleles of A -455, T -148 and A 448 is 2.46, 2.30 and 2.08 (95% confidence interval 1.153%-3.924%, 1.429%-3.694% and 1.298%-3.329%) respectively. No definite haplotype block was found by linkage disequilibrium analysis in the control group and the IS group. Association of haplotypes constructed from the nine polymorphisms with IS was not found. Among the haplotypes constructed from four polymorphisms including -455 G/A, -148 a67 C/T, 448 G/A alleles, haplotype differences were found between the control group and the IS group. Haplotypes with G -455, C -148, G448 alleles appeared more frequently in control group(P< or = 0.01), whereas haplotypes with A -455, T -148, A 448 occurred more frequently in the IS group(P< 0.01).

    Conclusion: The results of multi-allele and haplotype analysis indicated that the polymorphisms -455 G/A, -148 C/T, 448 G/A in beta fibrinogen gene were the possible risk factors associated with the occurrence of ischemic stroke in Hainan Han population.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2006;23;3;316-9

  • Association between patterns of nucleotide variation across the three fibrinogen genes and plasma fibrinogen levels: the Coronary Artery Risk Development in Young Adults (CARDIA) study.

    Reiner AP, Carty CL, Carlson CS, Wan JY, Rieder MJ, Smith JD, Rice K, Fornage M, Jaquish CE, Williams OD, Tracy RP, Lewis CE, Siscovick DS, Boerwinkle E and Nickerson DA

    Departments of Epidemiology and Genome Sciences, University of Washington, Seattle, WA 98101-1448, USA. apreiner@u.washington.edu

    Background: Previous genotype-phenotype association studies of fibrinogen have been limited by incomplete knowledge of genomic sequence variation within and between major ethnic groups in FGB, FGA, and FGG.

    Methods: We characterized the linkage disequilibrium patterns and haplotype structure across the human fibrinogen gene locus in European- and African-American populations. We analyzed the association between common polymorphisms in the fibrinogen genes and circulating levels of both 'functional' fibrinogen (measured by the Clauss clotting rate method) and total fibrinogen (measured by immunonephelometry) in a large, multi-center, bi-racial cohort of young US adults.

    Results: A common haplotype tagged by the A minor allele of the well-studied FGB-455 G/A promoter polymorphism (FGB 1437) was confirmed to be strongly associated with increased plasma fibrinogen levels. Two non-coding variants specific to African-American chromosomes, FGA 3845 A and FGG 5729 G, were each associated with lower plasma fibrinogen levels. In European-Americans, a common haplotype tagged by FGA Thr312Ala and several other variant alleles across the fibrinogen gene locus was strongly associated with decreased fibrinogen levels as measured by functional assay, but not by immunoassay. Overall, common polymorphisms within the three fibrinogen genes explain < 2% of the variability in plasma fibrinogen concentration.

    Conclusions: In young adults, fibrinogen multi-locus genotypes are associated with plasma fibrinogen levels. The specific single nucleotide polymorphism and haplotype patterns for these associations differ according to population and also according to phenotypic assay. It is likely that a substantial proportion of the heritable component of plasma fibrinogen concentration is due to genetic variation outside the three fibrinogen genes.

    Funded by: NHLBI NIH HHS: HL66642, HL66682, HL71017, N01-HC-05187, N01-HC-45134, N01-HC-48047, N01-HC-48048, N01-HC-48049, N01-HC-48050, N01-HC-95095

    Journal of thrombosis and haemostasis : JTH 2006;4;6;1279-87

  • Common genetic variation in five thrombosis genes and relations to plasma hemostatic protein level and cardiovascular disease risk.

    Kathiresan S, Yang Q, Larson MG, Camargo AL, Tofler GH, Hirschhorn JN, Gabriel SB and O'Donnell CJ

    National Heart, Lung, and Blood Institute Framingham Heart Study, Framingham, MA 01702-5827, USA.

    Objective: We undertook a linkage disequilibrium (LD)-based genetic approach to investigate the hypothesis that common sequence variants in 5 thrombosis genes influence plasma hemostatic protein levels or risk of cardiovascular disease (CVD).

    In a reference panel, we characterized LD structure at the fibrinogen gene cluster (fibrinogen-beta[FGB], FGA, and FGG), factor VII (F7), and tissue plasminogen activator (PLAT) loci. Forty-one tag single nucleotide polymorphisms (SNPs) were genotyped in 1811 unrelated Framingham Heart Study participants. There were significant associations of 9 FGB SNPs with fibrinogen level (minimum P=0.002) and of 7 F7 SNPs and factor VII level (minimum P<0.0001). SNPs at the PLAT locus were not associated with PLAT level. In stepwise analysis, a single FGB variant explained 1% of the residual variance in fibrinogen level, and 2 F7 SNPs together explained 10% of the residual variance in factor VII level. Two PLAT haplotypes were associated with CVD (multivariable-adjusted global P=0.0004).

    Conclusions: A comprehensive survey of common sequence variation demonstrates that cis-regulatory SNPs explain a modest proportion of the residual variance in circulating fibrinogen and factor VII level and PLAT haplotypes increase the risk of CVD. Additional studies are warranted to confirm the association of PLAT sequence variation and risk of CVD.

    Funded by: NHLBI NIH HHS: HL66582, N01-HC-25195

    Arteriosclerosis, thrombosis, and vascular biology 2006;26;6;1405-12

  • Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon.

    Spena S, Tenchini ML and Buratti E

    Department of Biology, University of Milan, Italy.

    In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.

    Funded by: Telethon: GGP02453, GGP030261

    RNA (New York, N.Y.) 2006;12;6;948-58

  • Association studies of genetic polymorphism, environmental factors and their interaction in ischemic stroke.

    Gao X, Yang H and ZhiPing T

    Department of Neurology, Peking University People's Hospital, 11 Xizhimen South St, Beijing, 100044, China. gxg56@tom.com

    Genetic background plays an important role in susceptibility to ischemic stroke. Our aim was to investigate the association of genetic polymorphisms and ischemic stroke and to evaluate their interaction with environmental risk factors in the Chinese population. Angiotensin-converting enzyme (ACE) gene I/D polymorphism, apolipoprotein E (ApoE) gene polymorphism, methylenetetrahydrofolate reductase (MTHFR) gene 677C/T polymorphism, and beta fibrinogen (Fgbeta) gene 148C/T polymorphism were analyzed in 100 patients and 100 matched controls. The subjects were genotyped by polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC) analysis. Persons with the Fgbeta CT/TT, MTHFR CT/TT, and ACE ID/DD genotypes had an elevated incidence of ischemic stroke (OR 3.907, 95% CI, 1.160-13.162, P=0.028). Smokers with the Fgbeta CT/TT or APOEepsilon4epsilon3 genotype, as well as individuals with the Fgbeta CT/TT genotype who consumed alcohol were more likely to develop a stroke. The data indicate that certain unfavorable genotypic combinations act synergistically in the development of ischemic stroke in the Chinese population. Synergism was also observed between genotype and environmental risk factors. This study may facilitate the development of a strategy to effectively prevent ischemic strokes.

    Neuroscience letters 2006;398;3;172-7

  • Multiple thrombophilic gene mutations rather than specific gene mutations are risk factors for recurrent miscarriage.

    Coulam CB, Jeyendran RS, Fishel LA and Roussev R

    Pregnancy Success Center or the Rinehart Center for Reproductive Medicine, Chicago, IL, USA. cbcoulam@aol.com

    Problem: Recurrent miscarriage is a heterogeneous condition. While the role of acquired thrombophilia has been accepted as an etiology of recurrent miscarriage, the contribution of specific inherited thrombophilic genes to this disorder has remained controversial. We compared the prevalence of 10 thrombophilic gene mutations among women with a history of recurrent miscarriages and fertile control women.

    A total of 150 women with a history of two or more recurrent pregnancy losses and 20 fertile control women with no history of pregnancy losses had buccal swabs taken for DNA analyses of 10 gene mutations [factor V G1691A, factor V H1299R (R2), factor V Y1702C, factor II prothrombin G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, HPA1 a/b (L33P), MTHFR C677T, MTHFR A1298C]. The prevalence of these mutations was compared between women experiencing recurrent miscarriage and controls.

    Results: No differences in the frequency of specific gene mutations were detected when women with recurrent miscarriage were compared with control women. However, the prevalence of homozygous mutations and total gene mutations among patients with recurrent miscarriage was significantly higher than among controls. Homozygous mutations were found in 59% of women with a history of recurrent pregnancy loss contrasted to 10% of control women. More than three gene mutations among the 10 genes studied were observed in 68% of women with recurrent miscarriage and 21% of controls.

    Conclusion: Inherited thrombophilias are associated with recurrent miscarriage. This association is manifest by total number of mutations rather than specific genes involved.

    American journal of reproductive immunology (New York, N.Y. : 1989) 2006;55;5;360-8

  • p130Cas: a versatile scaffold in signaling networks.

    Defilippi P, Di Stefano P and Cabodi S

    Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena 5 bis 10126 Turin, Italy. paola.defilippi@unito.it

    The Cas family of multiadaptor and scaffold molecules has an essential role in intracellular signaling events. Although these proteins do not have enzymatic or transcriptional activity, they spatially and temporally control signaling events through their ability to undergo changes in phosphorylation and to associate with effectors proteins in multimolecular complexes. The involvement of p130Cas in cell motility as a component of the integrin signaling machinery is well established. Here, we discuss recent developments that highlight a fundamental role in cell transformation and microbial pathogenesis and the implications of these developments on p130Cas function under normal and pathological conditions.

    Trends in cell biology 2006;16;5;257-63

  • [A linkage between beta-fibrinogen gene -148C/T polymorphism and cerebral infarction].

    Ma AJ, Pan XD, Zhang CS, Xing Y and Zhang YN

    Department of Neurology, General Hospital of Tianjin Medical University, Tianjin, 300070 P.R.China.

    Objective: To study the linkage between -148C/T polymorphism of beta-fibrinogen gene and plasma fibrinogen levels in patients with acute cerebral infarction.

    Methods: One hundred and fifty-one patients with cerebral infarction and 101 healthy individuals were enrolled in this trial. The beta-fibrinogen gene -148C/T polymorphism was analyzed by PCR-restriction fragment length polymorphism, and plasma fibrinogen levels were obtained from prothrombin time assay.

    Results: Plasma fibrinogen levels of patients were significantly higher than those of controls (P<0.01). In both groups, T allele carriers had higher plasma fibrinogen levels than other those did (P<0.01); and the fibrinogen level difference was still significant if both groups was based on their sex (P<0.05). Divided by age, each group of the study cases has significant difference between two genotypes (P<0.05). T -148 allele frequency of the middle age case in study group was higher than that in control group (P<0.05).

    Conclusion: High plasma fibrinogen level is a risk factor to cerebral infarction. Plasma fibrinogen level is affected by -148C/T polymorphism of beta-fibrinogen gene. With or without other risk factors and environmental factors affecting, T allele increases plasma fibrinogen level and may be a heritable risk factor to cerebral infarction.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2006;23;2;202-4

  • Hypofibrinogenaemia resulting from novel single nucleotide deletion at codon Bbeta58 (3404del A) associated with thrombotic stroke in infancy.

    Brennan SO, Mosesson MW, Lowen R, Siebenlist KR and Matsunaga A

    Molecular Pathology Laboratory, Canterbury Health Laboratories, P.O. Box 151, Christchurch, New Zealand. steve.brennan@chmeds.ac.nz

    Thrombosis and haemostasis 2006;95;4;738-9

  • Gender differences in the expression of erythrocyte aggregation in relation to B beta-fibrinogen gene polymorphisms in apparently healthy individuals.

    Ben Assayag E, Bova I, Berliner S, Peretz H, Usher S, Shapira I and Bornstein NM

    Department of Neurology, Tel-Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel.

    An increased erythrocyte aggregation (EA) is associated with capillary slow flow, tissue hypoxemia and endothelial dysfunction. Fibrinogen is a major determinant in the formation of aggregated red blood cells. It has been suggested that the B beta-fibrinogen -455 G/A polymorphism is associated with erythrocyte hyperaggregability in men with coronary artery disease. The purpose of this study was to investigate the influence of the beta-fibrinogen -455 G/A polymorphism on erythrocyte aggregation in apparently healthy individuals. Plasma fibrinogen, red blood cell count, serum lipids, erythrocyte sedimentation rate, and the genotype of the B beta-fibrinogen -455 G/A polymorphism were examined in a cohort of 545 apparently healthy individuals and those with atherothrombotic risk factors. A whole blood erythrocyte aggregation test was performed by using a simple slide test and image analysis. In men, EA levels and plasma fibrinogen levels were significantly higher in subjects carrying the -455 A allele compared to subjects with the -455 GG genotype. This association did not exist in women carrying the fibrinogen -455 A allele. The -455 GA/AA men presented significantly higher correlation between the plasma fibrinogen concentrations and EA. This observation raises the prospect of possible change in the functional properties of the -455 GA/AA fibrinogen, enhancing its ability to induce EH. This study suggests that the B beta-fibrinogen -455 A allele is related to EH in men only. Putative mechanism could be hyperfibrinogenemia and a functional change in the fibrinogen molecule that alters its ability to interact with red blood cells and supports the aggregability of these cells.

    Thrombosis and haemostasis 2006;95;3;428-33

  • Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia.

    Sauls DL, Lockhart E, Warren ME, Lenkowski A, Wilhelm SE and Hoffman M

    Research Service, Durham Veteran's Affairs Medical Center, 508 Fulton Street, Durham, North Carolina 27705, USA.

    We have previously shown functional differences in fibrinogen from hyperhomocysteinemic rabbits compared to that in control rabbits. This acquired dysfibrinogenemia is characterized by fibrin clots that are composed of abnormally thin, tightly packed fibers with increased resistance to fibrinolysis. Homocysteine thiolactone is a metabolite of homocysteine (Hcys) that can react with primary amines. Recent evidence suggests that Hcys thiolactone-lysine adducts form in vivo. We now demonstrate that the reaction of Hcys thiolactone with purified fibrinogen in vitro produces fibrinogen (Hcys fibrinogen) with functional properties that are strikingly similar to those we have observed in homocysteinemic rabbits. Fibrinogen purified from homocysteinemic rabbits and Hcys fibrinogen are similar in that (1) they both form clots composed of thinner, more tightly packed fibers than their respective control rabbit and human fibrinogens; (2) the clot structure could be made to be more like the control fibrinogens by increased calcium; and (3) they both form clots that are more resistant to fibrinolysis than those formed by the control fibrinogens. Further characterization of human fibrinogens showed that Hcys fibrin had similar plasminogen binding to that of the control and an increased capacity for binding tPA. However, tPA activation of plasminogen on Hcys fibrin was slower than that of the control. Mass spectrometric analysis of Hcys fibrinogen revealed twelve lysines that were homocysteinylated. Several of these are close to tPA and plasminogen binding sites. Lysines are major binding sites for fibrinolytic enzymes and are also sites of plasmin cleavage. Thus, modification of lysines in fibrinogen could plausibly lead to impaired fibrinolysis. We hypothesize that the modification of lysine by Hcys thiolactone might occur in vivo, lead to abnormal resistance of clots to lysis, and thereby contribute to the prothrombotic state associated with homocysteinemia.

    Funded by: NHLBI NIH HHS: HL48320

    Biochemistry 2006;45;8;2480-7

  • Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.

    Lewandrowski U, Moebius J, Walter U and Sickmann A

    Protein Mass Spectrometry and Functional Proteomics Group, Rudolf Virchow Center for Experimental Biomedicine, Versbacher Strasse 9, 97078 Wuerzburg, Germany.

    Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

    Molecular & cellular proteomics : MCP 2006;5;2;226-33

  • Multi-locus candidate gene polymorphisms and risk of myocardial infarction: a population-based, prospective genetic analysis.

    Zee RY, Cook NR, Cheng S, Erlich HA, Lindpaintner K and Ridker PM

    Center for Cardiovascular Disease Prevention, Harvard Medical School, Boston, MA 02215, USA. rzee@rics.bwh.harvard.edu

    Background: Polymorphisms in candidate genes related to lipid metabolism, thrombosis, hemostasis, cell-matrix adhesion, and inflammation have been suggested clinically useful in risk assessment of cardiovascular disease.

    Methods: We evaluated a panel of 92 candidate gene polymorphisms, using a multiplex polymerase chain reaction-immobilized probe assay amongst 523 individuals who subsequently deve 1f40 loped myocardial infarction (MI), and amongst 2092 individuals who remained free of reported cardiovascular disease over a mean follow-up period of 13.2 years.

    Results: Of the 92 polymorphisms tested, three that we previously reported on were associated with risk of MI, [pro12ala in the peroxisome proliferator activated-receptor gamma gene (odds ratio, OR = 0.75, P = 0.02); thr164ile in the beta-2 adrenergic receptor gene (OR = 0.14, P = 0.007); and ala23thr polymorphism in the eotaxin gene (OR = 1.87, P = 0.01)]. However, when adjusted for the other 89 polymorphisms evaluated, these findings were no longer statistically significant. Further, in contrast to reports from other investigators, we found little evidence for association of a C677T polymorphism in the 5,10-methylenetetrahydrofolate reductase gene, the angiotensin-I-converting enzyme 1 insertion/deletion polymorphism, a 4G/5G polymorphism in the serine/cysteine proteinase inhibitor-clade E-member 1 gene, the factor V Leiden mutation, the G20210A factor II mutation, a -455G>A polymorphism in the beta-fibrinogen gene, the cys112arg/arg158cys apolipoprotein E gene polymorphism, a gly460trp polymorphism in the alpha-adducin gene, and a -629C>A polymorphism in the cholesteryl ester transfer protein gene with risk of MI.

    Conclusions: After correction for multiple comparisons, the addition of genetic information observed in the present study had little impact on risk prediction models for MI. The present investigation highlights the importance of replication and validation of findings from genetic association studies.

    Funded by: NHLBI NIH HHS: HL-58755, HL-63293

    Journal of thrombosis and haemostasis : JTH 2006;4;2;341-8

  • [Study on the relationship between polymorphisms of susceptible genes in coagulation pathway related to pulmonary thromboembolism in Chinese Han population].

    Zhai ZG, Wang C, Yang YH, Pang BS, Xiao B and Mao YL

    Division of Pulmonary Disease, Beijing Institute of Respiratory Medicine, Beijing Chaoyang Hospital, Capital University of Medical Sciences, Beijing 100020, China.

    Objective: To determine the prevalence of beta-fibrinogen gene -455G/A, -148C/T polymorphisms in Chinese Han population and to investigate whether they were associated with pulmonary thromboembolism (PTE).

    Methods: The subjects consisted of 101 patients with PTE and 101 healthy controls matched with age and sex, from the same geographic area. All patients were diagnosed by high probability of lung ventilation/perfusion scan and/or multi-slice CT pulmonary angiography as well as medical history and clinical manifestations. Genome DNA was extracted from whole blood using KI-phenol-chloroform. Genotypes and allele frequencies of fibrinogen beta g b94 ene -455G/A, -148C/T polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Restriction enzyme HaeIII and HindIII digestion were used for detecting -455G/A, -148C/T polymorphisms respectively.

    Results: Regarding fibrinogen beta gene -455G/A and -148C/, the allele frequencies G and A of fibrinogen beta -455 in the controls were 0.931, 0.069 while C and T of -148 were 0.777, 0.223 respectively, which were in good agreement with Hardy-Weinberg equilibrium. There was significant difference of -455G/A genotype frequencies distribution of AA, GA, GG between cases and in controls respectively, but no significant difference was found in the -148C/T polymorphisms. The frequencies of mutation allele -455A were 0.193, 0.169 in cases and in controls with P < 0.05 but there was no statistically significant difference of -148T allele. The presence of A allele of fibrinogen beta -455 was found to be a greater risk factor in cases than in controls. The odds ratio (OR) of GA and GA + AA were 3.723 (1.786 - 7.759), 3.749 (1.842 - 7.630), respectively. When compared with GG genotype, the P value was 0.0001.

    Conclusion: There was a complete linkage disequilibrium between fibrinogen beta -148C/T and -455G/A found. The frequencies of -455A, alleles in PTE disease were apparently higher than that of healthy adults but there was no difference in -148T alleles.

    Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 2006;27;2;165-9

  • Contribution of the -455G/A polymorphism at beta-fibrinogen gene and of the Leiden mutation to hemorheological parameters in ischemic stroke patients.

    Pongrácz E, Andrikovics H, Csornai M, Bernát IS and Nagy Z

    Central Hospital of Home Office, Neurological Department, Budapest, Hungary. epongracz@bm.gov.hu

    Unlabelled: The concentration of plasma fibrinogen (FIB) is an important factor in the coagulation cascade and also in the determination of blood and plasma viscosity depending on both genetic and acquired factors. The -455G/A polymorphism of the beta-FIB gene is connected to the plasma concentration of FIB but the effect of Leiden mutation on hemorheological parameters is unclear. The two genetic polymorphisms were studied by polymerase chain reaction in healthy subjects and ischemic stroke cohort and the effects on the concentration of plasma FIB, whole blood and plasma viscosity of patients as well. A total of 278 ischemic stroke patients and 173 control subjects were enrolled. Marcro-rheological parameters as plasma FIB concentration, whole blood viscosity (90 sec(-1) shear rate) and plasma viscosity have been measured also in the subgroup of young (age < 50 years) and in a subgroup of non-smoker patients.

    Results: No significant difference was found in the prevalency of H1/H2 genotype between controls and cases in pooled stroke group OR 0.95 (95% CI: 0.47-1.27), however H2/H2 genotype frequency was increased in young subgroup of patients (OR: 1.66 95% CI: 0.52-5.25). Plasma FIB concentration was increased both in the total cohort (p < 0.05) and in the non-smoker subgroup (p < 0.03) of patients carried H2/H2 as compared to H1/H1 genotype and the prevalence was increased in the group of patients having plasma FIB concentration > 4 g/l (p < 0.05). The whole blood viscosity was elevated in the H2/H2 group as compared to the group carrying wild type (p < 0.03). A tendency of increased plasma viscosity in the group of patients with H2/H2 genotype as compared to wild type was found (p = 0.07). Leiden mutation prevalence showed an increased risk OR: 1.67 (95% CI: 0.75-3.70) in the young patients group as compared to controls. In patients who have had the highest plasma viscosity, higher frequency of Leiden mutation was detected as compared to wild type, in total group (p = 0.01), in young patients (p = 0.03) and in subgroup of non-smoker patients (p = 0.05).

    Conclusions: Our findings support the notion that the homozigous variant of beta-FIB gene can raise both plasma FIB concentration and whole blood viscosity. Leiden mutation connected to the elevation of plasma viscosity could demonstrate a new pathway of increased thrombophylic potential in ischemic stroke patients.

    Clinical hemorheology and microcirculation 2006;35;1-2;75-82

  • Mutations and polymorphisms in genes affecting hemostasis proteins and homocysteine metabolism in children with arterial ischemic stroke.

    Komitopoulou A, Platokouki H, Kapsimali Z, Pergantou H, Adamtziki E and Aronis S

    Hemostasis and Hemophilia Unit, Aghia Sophia Children's Hospital, Athens, Greece.

    Background: The pathogenesis of thrombosis in childhood seems to be multifactorial implicating genetic and environmental factors.

    Aim: To compare the distributions of mutations/polymorphisms in genes affecting hemostasis (factor V Leiden - FVL, FV H1298R-FVR2, FII 20210A, b-Fib 455G>A, FXIII V34L, PAI-1 4G, HPA-1b) or homocysteine metabolism (MTHFR C677T, MTHFR A1298C) among 90 children with arterial ischemic stroke (AIS) and 103 controls, and to associate the carriage of these mutations/polymorphisms with their corresponding proteins in children with AIS.

    Results: AIS was more frequent in boys (p < 0.01). No studied mutation/polymorphism was found to be a risk factor for AIS, except for FVL [odds ratio 4.2 (95% CI 1.5-12.1)], the presence of which was even higher in 31 children with congenital AIS [odds ratio 6.82 (95% CI 2.0-22.8)]. FVL carriers had an odds ratio of 5.76 (95% CI 1.6-6.4) when FVR2 was absent. In thrombosed children, activated protein C resistance, prothrombin and fibrinogen levels were higher in the presence of FVL, FII20210A or b-Fib 455G-->A, respectively. Double heterozygotes in both MTHFR C677T and A1298T or homozygotes in one had significantly elevated homocysteine levels.

    Conclusion: Except for FVL, no definite conclusion could be reached regarding the involvement of the studied mutations/polymorphisms in childhood AIS.

    Cerebrovascular diseases (Basel, Switzerland) 2006;22;1;13-20

  • Polymorphisms associated with apolipoprotein B levels in Greek patients with familial hypercholesterolemia.

    Choumerianou DM, Maumus S, Skoumas J, Pitsavos C, Stefanadis C, Visvikis-Siest S and Dedoussis GV

    Laboratory of Molecular Biology, Department of Science of Dietetics-Nutrition, Harokopio University of Athens, Athens, Greece.

    Background: Familial hypercholesterolemia (FH) is a genetic disorder characterized by high low-density lipoprotein-cholesterol (LDL-C) concentrations, which frequently gives rise to premature coronary artery disease. The clinical expression of FH is highly variable, even in patients carrying the same LDL receptor (LDLR) gene mutation. This variability may be due to environmental and other genetic factors.

    Methods: We investigated paraoxonase 2 (PON 2) Ser311Cys, lipoprotein lipase (LPL) Asn291Ser, plasminogen activator inhibitor-1 (PAI-1) T11053G, beta-fibrinogen (FGB) -455 G>A and nitric oxide synthase gene (NOS) -922 A>G polymorphisms in 84 patients with FH. The effect of polymorphisms as independent factors of high lipid values was evaluated.

    Results: The PON 2 Cys311 allele was correlated with high total cholesterol and LDL-C and apolipoprotein B levels, while LPL Asn291, PAI-1 T11053, FGB -455 G and NOS -922 A alleles were correlated with high apolipoprotein B levels.

    Conclusions: These results suggest that apolipoprotein B levels in FH heterozygotes may be affected by several different genetic variants.

    Clinical chemistry and laboratory medicine 2006;44;7;799-806

  • Relationship among urinary albumin excretion rate, lipoprotein lipase PvuII polymorphism and plasma fibrinogen in type 2 diabetic patients.

    Javorský M, Kozárová M, Salagovic J and Tkác I

    Department of Medicine IV, L. Pasteur University Hospital, Rastislavova 43, SK-041 90 Kosice, Slovakia.

    Plasma fibrinogen level represents a strong cardiovascular risk factor and is regulated by an interplay of genetic and environmental factors. Hyperfibrinogenemia frequently occurs in cluster with dyslipidemia w cbd ithin the frame of insulin resistance syndrome (IRS) and type 2 diabetes mellitus. Genetic variants with a pleiotropic effect have been proposed to cause IRS features including hyperfibrinogenemia. We studied the influence of polymorphisms in lipoprotein lipase (LPL) gene, beta-fibrinogen gene (FIBB) and environmental factors on plasma fibrinogen levels in type 2 diabetes patients. 131 type 2 diabetes patients (mean age 62+/-10 years, 33% male) were genotyped for polymorphisms in LPL gene (intron 6 PvuII, intron 8 HindIII) and FIBB gene (-148C/T, -455G/A) by PCR-RFLP method. Fibrinogen was measured by thrombin coagulation method, albuminuria by immunoturbidimetric assay. Polymorphism LPL PvuII showed a gene-dose effect on fibrinogen levels, with the highest fibrinogen in P-P- homozygotes (p = 0.05, analysis of variance). P-carriers (P-P- and P+P- combined) had significantly higher fibrinogen levels compared with P+P+ homozygotes (3.74+/-1.40 g/l vs 3.06+/-1.20 g/l, p=0.03). Other studied polymorphisms were not significantly related to fibrinogen levels. Age- and sex-adjusted fibrinogenemia correlated significantly with albuminuria (r = 0.48, p=0.001), serum uric acid (r = 0.42, p=0.006) and serum creatinine (r = 0.32, p=0.04). Multiple stepwise linear regression identified interaction term of LPL PvuII and albuminuria as an independent predictor of fibrinogen level, explaining 18% of fibrinogen variance. Albuminuria thus appears to be the best predictor of fibrinogen plasma levels in type 2 diabetic patients. Relationship between albuminuria and fibrinogenemia may be modified by the genotype LPL PvuII, which also shows a weak association with plasma fibrinogen level in type 2 diabetes patients.

    Physiological research 2006;55;1;55-62

  • Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma' levels.

    Uitte de Willige S, de Visser MC, Houwing-Duistermaat JJ, Rosendaal FR, Vos HL and Bertina RM

    Hemostasis and Thrombosis Research Center, Department of Hematology (C2-R), Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.

    We investigated the association between haplotypes of fibrinogen alpha (FGA), beta (FGB), and gamma (FGG), total fibrinogen levels, fibrinogen gamma' (gammaA/gamma' plus gamma'/gamma') levels, and risk for deep venous thrombosis. In a population-based case-control study, the Leiden Thrombophilia Study, we typed 15 haplotype-tagging single nucleotide polymorphisms (htSNPs) in this gene cluster. None of these haplotypes was associated with total fibrinogen levels. In each gene, one haplotype increased the thrombosis risk approximately 2-fold. After adjustment for linkage disequilibrium between the genes, only FGG-H2 homozygosity remained associated with risk (odds ratio [OR], 2.4; 95% confidence interval [95% CI], 1.5-3.9). FGG-H2 was also associated with reduced fibrinogen gamma' levels and reduced ratios of fibrinogen gamma' to total fibrinogen. Multivariate analysis showed that reduced fibrinogen gamma' levels and elevated total fibrinogen levels were both associated with an increased risk for thrombosis, even after adjustment for FGG-H2. A reduced fibrinogen gamma' to total fibrinogen ratio (less than 0.69) also increased the risk (OR, 2.4; 95% CI, 1.7-3.5). We propose that FGG-H2 influences thrombosis risk through htSNP 10034C/T [rs2066865] by strengthening the consensus of a CstF site and thus favoring the formation of gammaA chain above that of gamma' chain. Fibrinogen gamma' contains a unique high-affinity, nonsubstrate binding site for thrombin, which seems critical for the expression of the antithrombin activity that develops during fibrin formation (antithrombin 1).

    Blood 2005;106;13;4176-83

  • [The relationship between the five beta-fibrinogen gene polymorphisms and cerebral infarction].

    Fu Y, Wei X, Ni PH, Ying YY, Song YY and Chen SD

    Department of Neurology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.

    Objective: To investigate the relationship among beta-fibrinogen (Fg) concentration, related gene polymorphisms (including -148C/T, -249C/T, -455G/A, 448G/A, 1689T/G) and cerebral infarction.

    Methods: Fg level and its five gene polymorphisms were analyzed with by polymerase chain reaction-restriction fragment length polymorphism in 132 patients with cerebral infarction, 79 patients with other neurological diseases and 92 healthy elders.

    Results: The plasma Fg level in cerebral infarction patients was significantly higher than that in the patients with other neurological diseases or healthy elders (P < 0.05). In the three groups, the plasma Fg levels in individuals with T-148 and A-455 alleles were higher than those in individuals without T-148 and A-455 alleles (P < 0.05). However, there were no statistically significant differences in the genotype and allele frequencies in the five mutation gene polymorphisms among the three groups (P > 0.05).

    Conclusions: Cerebral infarction is a multifactorial disease and an increased Fg level is a risk factor for cerebral infarction. T-148 and A-455 allele can lead to elevated Fg concentration.

    Zhonghua nei ke za zhi 2005;44;12;914-7

  • A role of TNF-alpha gene variant on juvenile ischemic stroke: a case-control study.

    Rubattu S, Speranza R, Ferrari M, Evangelista A, Beccia M, Stanzione R, Assenza GE, Volpe M and Rasura M

    IRCCS Neuromed, Pozzilli Is, Italy.

    The role of genetic factors in the individual predisposition to develop ischemic stroke has been assessed by previous studies performed both in animal models and in humans. The main goal of the current investigation was to determine the possible contribution of genes encoding procoagulant and inflammatory factors on the occurrence of ischemic stroke in a cohort of young cases and corresponding controls. One hundred and fifteen cases of ischemic stroke were recruited for this study. A detailed clinical assessment, a definite etiologic diagnosis, as well as the presence/absence of known risk factors for ischemic stroke were obtained for each patient. As a control group 180 healthy, unrelated subjects were included. The whole population was screened for polymorphisms belonging to genes encoding FII, FV, alpha-fibrinogen, beta-fibrinogen, GP IIb/IIIa, tumor necrosis factor (TNF)-alpha, interleukin 1-beta. Hypertension was the most important risk factor for ischemic stroke in our cohort [OR = 6.9, confidence interval (CI) 2.9-16.7, P < 0.0001]. Among all genes tested, the TNF-alpha gene variant exerted a significant, independent effect on individual predisposition to ischemic stroke occurrence (OR = 1.8, CI = 1.01-3.3, P < 0.05). Our findings, obtained in a cohort of young Italian patients, may support the existence of a direct contributory role of TNF-alpha, a proinflammatory cytokine protein, in the susceptibility to brain damage.

    European journal of neurology 2005;12;12;989-93

  • Quality control of fibrinogen secretion in the molecular pathogenesis of congenital afibrinogenemia.

    Vu D, Di Sanza C, Caille D, de Moerloose P, Scheib H, Meda P and Neerman-Arbez M

    Department of Genetic Medicine and Development, Swiss Institute of Bioinformatics, University Medical Centre, Geneva, Switzerland.

    Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q31. A subset of afibrinogenemia mutations has been shown to specifically impair fibrinogen secretion, but the underlying molecular mechanisms remained to be elucidated. Here, we show that truncation of the seven most C-terminal residues (R455-Q461) of the Bbeta chain specifically inhibits fibrinogen secretion. Expression of additional mutants and structural modelling suggests that neither the last six residues nor R455 is crucial per se for secretion, but prevent protein misfolding by protecting hydrophobic residues in the betaC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bbeta, gamma and angiopoietin-2 chimeric molecules demonstrated that the betaC domain prevents the secretion of single chains and complexes, whereas the gammaC domain allows their secretion. Our data provide new insight into the mechanisms accounting for the quality control of fibrinogen secretion and confirm that mutant fibrinogen retention is one of the pathological mechanisms responsible for congenital afibrinogenemia.

    Human molecular genetics 2005;14;21;3271-80

  • Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

    Liu T, Qian WJ, Gritsenko MA, Camp DG, Monroe ME, Moore RJ and Smith RD

    Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.

    The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

    Funded by: NCRR NIH HHS: P41 RR018522, RR18522; NIGMS NIH HHS: U54 GM-62119-02, U54 GM062119

    Journal of proteome research 2005;4;6;2070-80

  • Role of thrombophilic gene polymorphisms in branch retinal vein occlusion.

    Weger M, Renner W, Steinbrugger I, Cichocki L, Temmel W, Stanger O, El-Shabrawi Y, Lechner H, Schmut O and Haas A

    Department of Ophthalmology, Medical University Graz, Graz, Austria. martin.weger@meduni-graz.at

    Objective: Branch retinal vein occlusion (BRVO) is a common cause of severe visual loss. Numerous risk factors, including arterial hypertension, diabetes mellitus, and arteriosclerosis, have been identified. Gene polymorphisms affecting hemostasis may also play a role in the pathogenesis of BRVO. The present study was therefore done to determine the prevalence of genetic polymorphisms in factors implicated in hypercoagulability among patients with BRVO.

    Design: Retrospective case-control study.

    Participants: The study cohort consisted of 294 patients with BRVO and 294 control subjects, matched for age and gender.

    Methods: Determination of genotypes was done by allele-specific digestion of polymerase chain reaction products, or by 5' exonuclease assay (TaqMan).

    Genotypes of factor V R506Q (factor V Leiden), prothrombin 20210G>A, fibrinogen beta -455G> A, factor XII (FXII) 46C>T, and ITGA2 807C>T (platelet glycoprotein Ia [GPIa] 807C>T) and ITGB3 L59P (platelet GPIIIa PlA1/PlA2) polymorphisms.

    Results: Genotype distributions of the investigated gene polymorphisms did not differ significantly between patients and control subjects. In contrast, significantly increased prevalences of arterial hypertension and hypercholesterolemia were found among patients with BRVO. In a logistic regression analysis, the presence of arterial hypertension was associated with an odds ratio (OR) of 2.32 (95% confidence interval [CI], 1.62-3.32), whereas hypercholesterolemia yielded an OR of 2.54 (95% CI, 1.74-3.70) for BRVO.

    Conclusion: Our data indicate that the prevalences of the investigated gene polymorphisms do not differ significantly in patients with BRVO and control subjects. This suggests that these polymorphisms are not major risk factors for BRVO.

    Ophthalmology 2005;112;11;1910-5

  • [Correlation between fibrinogen polymorphisms and the type of cerebral infarction].

    Wang SJ, Yuan XD, Gao J, Pei HZ and Li HF

    Department of Neurology, Kailuan Hospital, Tangshan, Hebei, PR China.

    Objective: To study the association of beta-fibrinogen(Fg) gene -148 C/T and 448 G/A polymorphisms, plasma Fg concentration, molecular reactivity and the type of cerebral infarction.

    Methods: Gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. The plasma Fg concentration and the molecular reactivity were also determined.

    Results: The Fg concentration in MCI patients with T -148 allele was higher than that in PCI patients and controls. The MCI patients with A448 allele had higher Fg concentration, FMPV and FMPV/Amax when compared with controls, and had higher FMPV/Amax when compared with PCI patients.

    Conclusion: FgB beta -148 and 448 mutational genotypes have impact on Fg concentrationì and therefore increase the risk of MCI.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2005;22;5;572-4

  • Integrins and Src: dynamic duo of adhesion signaling.

    Shattil SJ

    Hematology-Oncology Division, Department of Medicine, University of California-San Diego, La Jolla, CA 92093, USA. sshattil@ucsd.edu

    Src family protein tyrosine kinases (SFKs) play important roles downstream of integrin adhesion receptors, and they are necessary for the generation of "outside-in signals" that regulate cytoskeletal organization, cell motility and gene expression in response to cell adhesion. One relatively under-explored facet of this relationship is the possible physical interaction of integrins with SFKs. Recently, it has been established that beta3 integrins and c-Src can interact directly, and this pool of c-Src is activated by cell adhesion to initiate outside-in signaling in platelets, osteoclasts and cells of the vasculature. Here, the biochemical basis for and biological significance of this integrin-SFK interaction is summarized, and I propose a general mechanism for initiation of outside-in integrin signaling.

    Trends in cell biology 2005;15;8;399-403

  • [Nine polymorphisms of fibrinogen gene and their association with plasma fibrinogen levels in Hainan Han population].

    Liang L, Sun C, Xiao F, Tang XL, Chen XD, Zhou DF, Wang XY, Wang Z, Liu GX, Cheng S, Ling GX, Bao CX and Cai WW

    Department of Biochemistry, Hainan Medical College, Haikou, Hainan, 571101 PR China.

    Objective: To investigate the allelic frequencies of polymorphisms of alpha Taq I and beta Bcl I, Hinf I A/C, 448 G/A, beta BsmA I G/C, +1689T/G, -148C/T, -249C/T, -455G/A in Hainan Han population and their association with plasma fibrinogen level.

    Methods: Turbidmetric assay was used to measure plasma fibrinogen level of two hundred and thirty-eight healthy individuals. The genotypes were characterized by PCR-RFLP and sequence analysis. The relationships between the genotypes and plasma fibrinogen levels were analyzed by t test and ANOVA.

    Results: The frequencies of the rare alleles of alpha Taq I and beta Bcl I, Hinf I A/C, 448 G/A, beta BsmA I G/C, +1689T/G, -148C/T, -249C/T, -455G/A polymorphisms were 0.445, 0.239, 0.134, 0.235, 0.273, 0.241, 0.265, 0.441, 0.254 respectively. In the general population, the plasma fibrinogen level is significantly higher in the groups of genotypes -455GA and AA, -148CT and TT, alpha Taq I T1T1 than in the group of wild types(P=0.004, 0.015 and 0.043 respectively). In the men, plasma fibrinogen level is significantly higher in the groups of genotypes -455GA and AA, -148CT and TT, alpha Taq I T1T1, alpha Taq I T1T2 than in the group of wild types(P=0.001, 0.023, 0.003 and 0.032 respectively). In the women, no significant genotype association with plasma fibrinogen level was detected.

    Conclusion: There was linkage disequilibrium between the fibrinogen gene loci. The beta -455G/A beta 448G/A, alpha Fg Taq I polymorphisms were associated with the difference in plasma fibrinogen in men. A(-455), T(-148) and alpha Taq I T1 alleles were associated with higher fibrinogen levels.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2005;22;4;457-61

  • [Association of matrix metalloproteinase-9 and platelet membrane glycoprotein VI polymorphisms with acute coronary syndrome].

    Qin Q, Zhao BR, Mao YM, Cui RZ, Kou L, Li YL, Zhao FM and Hui RT

    Tianjin Chest Hospital, Tianjin 300051, China.

    Objective: To investigate serum level and gene polymorphisms of matrix metalloproteinase 9 (MMP-9), and platelet glycoprotein VI (GPVI) in patients with acute coronary syndrome (ACS).

    Methods: In a prospective study of 179 patients with documented ACS and 164 controls, we measured baseline serum MMP-9 levels using ELISA and determined the MMP-9/C-1562T and MMP-9/G5564A genotypes using PCR-restriction fragment length polymorphism. Fib serum level was measured by Clauss assay. We also analyzed the Fib/Bbeta-148C/T and GPVI/T13254C polymorphisms.

    Results: Serum levels of MMP-9 and Fib in ACS patients were significantly higher than in controls (P < 0.001), and serum level of Fib in the acute myocardial infarction group was higher than in patients with unstable angina (P < 0.05). No significant difference between ACS patients and controls was found in frequencies of MMP-9/C-1562T, MMP-9/G5564A, Fib/Bbeta-148C/T, and GPVI/T13254C genotypes and alleles (P > 0.05). The T allele of the Fib/Bbeta-148T polymorphism was associated with increased plasma Fib level (P < 0.05). There was a strong positive correlation between serum level of MMP-9 and Fib (r = 0.289, P < 0.01).

    Conclusion: Serum levels of MMP-9 and Fib were independent risk factors of ACS. There was an obvious relationship between the Bbeta-148C/T mutation and high Fib level. No significant difference between controls and ACS patients was found in the frequencies of MMP-9 C-1562T and G5564A, Fib Bbeta-148C/T and GPVI T13254C genotypes and alleles (P > 0.05).

    Zhonghua xin xue guan bing za zhi 2005;33;7;622-6

  • Beta-fibrinogen haplotypes and the risk for cardiovascular disease in a dialysis cohort.

    Liu Y, Berthier-Schaad Y, Fink NE, Fallin MD, Tracy RP, Klag MJ, Smith MW and Coresh J

    Wake Forest University School of Medicine, Winston-Salem, NC, USA. yoliu@wfubmc.edu

    Background: Elevated plasma fibrinogen levels are common in dialysis patients and may be related to an elevated risk for cardiovascular disease (CVD). We tested the hypothesis that genetic variation in the beta-fibrinogen ( FGB ) gene, shown to explain 1% to 5% of fibrinogen level variation in the general population, has an important role in elevated fibrinogen levels and excess CVD risk in dialysis patients.

    Methods: Plasma fibrinogen was measured in 735 dialysis patients a median of 3 months from the start of dialysis therapy by using an automated clot-rate assay. Seven polymorphisms of the FGB gene were determined. Haplotype analysis was conducted using the Phase program to estimate haplotypes, with stratification for race. CVD events were ascertained from medical records.

    Results: During a median follow-up of 2.1 years, 279 CVD events occurred. Genotype frequencies were in Hardy-Weinberg equilibrium. Four common haplotypes identified were not associated with fibrinogen levels or CVD risk in the entire cohort or after stratification by race. The -455A allele, known to increase gene expression in vitro, was marginally associated with fibrinogen levels only in patients without diabetes (regression coefficient [beta], 20 mg/dL [for +1 copy of the A allele; P = 0.06]), adjusted for age, sex, race, smoking, baseline dialysis modality, comorbidity, and history of diabetes and CVD. Post hoc analysis showed that -249C-->T (defining haplotype 3) was associated with greater fibrinogen levels and CVD risk among patients without diabetes and current smokers.

    Conclusion: The FGB gene likely does not have an important role in determining the variation in elevated plasma fibrinogen levels or excess CVD risk in dialysis patients.

    Funded by: AHRQ HHS: R01-HS-08365; NCI NIH HHS: N01-CO-12400; NHLBI NIH HHS: HL 46696, HL 58329, R01-HL-62985; NIDDK NIH HHS: K24-DK-02856, R01-DK-59616

    American journal of kidney diseases : the official journal of the National Kidney Foundation 2005;46;1;78-85

  • Common promoter polymorphisms of inflammation and thrombosis genes and longevity in older adults: the cardiovascular health study.

    Reiner AP, Diehr P, Browner WS, Humphries SE, Jenny NS, Cushman M, Tracy RP, Walston J, Lumley T, Newman AB, Kuller LH and Psaty BM

    Department of Epidemiology, University of Washington, Seattle, WA 98101, USA. apreiner@u.washington.edu

    Inflammatory response genes may influence life span or quality at advanced ages. Using data from the population-based cardiovascular health study (CHS) cohort, we examined the associations between promoter polymorphisms of several inflammation and thrombosis genes with longevity. We ascertained genotypes for interleukin (IL)-6 -174 G/C, beta-fibrinogen -455 G/A, plasminogen activat 1f40 or inhibitor (PAI)-1 -675 4G/5G, and thrombin-activatable fibrinolysis inhibitor (TAFI) -438 G/A in 2224 men and women > or = 65 years old at baseline. During 10 years of follow-up, men with the TAFI -438 A/A genotype had decreased mortality due to all causes, and lived, on average, 0.9 more years of life, or 1.1 more years of healthy life, than men with the -438 G allele. The effects of TAFI -438 G/A in women were smaller and not statistically significant. PAI-1 4G/4G genotype appeared to be associated with lower non-cardiovascular mortality in men, but with greater cardiovascular mortality in women. In exploratory analyses, we observed a possible interaction among anti-inflammatory drugs, interleukin-6 -174 C/C genotype, and longevity. These findings suggest that modulators of fibrinolytic activity may have a generalized influence on aging, and merit further investigation in studies of genetic determinants of human longevity.

    Funded by: NHLBI NIH HHS: N01-HC-15103, N01-HC-35129, N01-HC-85079, N01-HC-85086; NIA NIH HHS: AG 05407

    Atherosclerosis 2005;181;1;175-83

  • [Pharmacogenetics of the local thrombolysis in patients with deep vein thrombosis].

    Falkowski A, Kaczmarczyk M, Goracy I, Górecka-Szyld B, Poncyljusz W, Parczewski M and Ciechanowicz A

    Katedra i Zakład Radiologii Ogólnej i Stomatologicznej Pomorskiej AM w Szczecinie.

    Thrombophilia, the state of increased tendency for blood clotting, is considered the disorder of a complex etiology, caused by both environmental and genetic factors. As gene variants predisposing to thrombophilia and influencing the increased risk of vein thrombosis might influence response to local thrombolysis, the aim of the work was to characterize the pharmacogenetic conditions for local streptokinase treatment in patients with a deep vein thrombosis (DVT) of lower extremities based on the following polymorphism analyses: G1691A polymorphism of factor V (FV), G20210A polymorphism of prothrombin (PT), A4250G (Thr312Ala) polymorphism of fibrinogen-alpha (FGA), G(-455)A polymorphism of fibrinogen-beta (FGB), 4G/5G polymorphism of plasminogen activator inhibitor type 1(PAI-1) and insertion/deletion (I/D) polymorphism of tissue plasminogen activator (t-PA). The study included 40 DVT patients who underwent a local thrombolytic treatment within 14-day period from diagnosis. Full recanalization was achieved in 20 subjects (50%) [group R(+)], whereas incomplete or total lack of recanalization was identified in the remaining 20 patients [group R(-)]. No major complications of thrombolytic treatment occurred in the studied group. In the case of prothrombin gene all individuals carried homozygous wild type genotype (GG). Prevalence of the genotypes and alleles of the remaining five polymorphisms did not differ significantly between the groups R(+) and R(-). Neither sex nor age, smoking or time period from diagnosis to introduction of the thrombolytic treatment significantly influenced treatment efficacy. The results of the study suggest that a local thrombolysis with streptokinase introduced within two week period from the diagnosis is a safe and efficient method of treatment for deep vein thrombosis of lower extremities. However, size of the group is insufficient to clearly determine the association between investigated polymorphisms and efficacy of local treatment with streptokinase.

    Polskie Archiwum Medycyny Wewnetrznej 2005;114;1;644-51

  • Polymorphisms in prothrombotic genes and their impact on ischemic stroke in a Sardinian population.

    Rubattu S, Di Angelantonio E, Nitsch D, Gigante B, Zanda B, Stanzione R, Evangelista A, Pirisi A, Rosati G and Volpe M

    IRCCS Neuromed, Localita' Camerelle, 86077 Pozzilli (Is), Italy. rubattu.speranza@neuromed.it

    Genetic factors are involved in the individual predisposition to develop ischemic stroke (IS). In the present study we tested the role of the Factor VII G10976A and -C122T polymorphisms on the susceptibility to develop IS in a genetically homogenous and clinically well ascertained case-control study including 294 cases (median age 75 years; 176 males/118 females) and 286 controls (median age 73 years; 163 males/123 females) in Sardinia, Italy. In addition, we carried out an exploratory analysis with respect to other frequently studied polymorphisms of haemostatic factor genes:Factor II G20210A, Factor V G1691A,,Fibrinogen alpha-chain Thr312Ala, Fibrinogen beta-chain -C148T, Factor XIII G185T, GPIIb/IIIa T1565C. Among all the genes tested, FVII -C122T showed a significant, independent contribution to IS predisposition both in crude and adjusted analyses (crude OR 1.52, 95% CI 1.09-2.10, P=0.013; adjusted OR 1.48, 95% CI 1.04-2.09, P=0.028, respectively). Haplotype analyses revealed a conserved population structure with high linkage disequilibrium between both FVII mutations tested. Blood levels of FVII had an inverse relationship with the polymorphism involved. Apart from genetic influence, there was a significant role for hypertension (OR=1.7, 95% CI 1.19-2.43, P=0.003), hypercholesterolemia (OR=2.21, 95% CI 1.38-3.54, P=0.001) and atrial fibrillation (OR=1.66, 95% CI 1.06-2.58, P=0.026) on IS occurrence. In summary, we describe evidence for a possible direct association of FVII gene molecular variants with the occurrence of IS in a genetically homogenous human sample.

    Thrombosis and haemostasis 2005;93;6;1095-100

  • Coagulation gene polymorphisms as risk factors for myocardial infarction in young Indian Asians.

    Pegoraro RJ, Ranjith N and Rom L

    Department of Chemical Pathology, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban.

    Background: The relationship between pro-coagulant gene polymorphisms, clinical features and the risk of premature coronary heart disease (CHD) in Indian Asian subjects resident in South Africa has been investigated.

    Methods: The prevalence of the beta-fibrinogen -455G/A and -148C/T, and the factor VII 10 bp 5' promoter insertion/deletion and R353Q polymorphisms were examined in 195 unrelated Indian Asian patients (< or = 45 years) who presented with myocardial infarction (MI). Results were compared with those from 107 unaffected siblings (18-45 years) and 300 healthy age- and race-matched control subjects.

    Results: Overall, none of the polymorphisms examined here showed any association with MI. However, when stratified according to obesity, patients with a BMI > 30 kg/m2 had a significantly higher frequency of the beta-fibrinogen variant alleles, compared with non-obese patients (19% vs 9%; p = 0.025) and controls (19% vs 9%; p = 0.003). Furthermore, the highest frequency of variant alleles occurred in obese smokers (24%), compared with 4% in non-obese non-smokers (p = 0.003) and 9% in control subjects (p < 0.001). The factor VII R353Q and promoter insertion variants, on the other hand, were associated with higher HDL and lower LDL levels (p = 0.034 and 0.04, respectively).

    Conclusion: In young Indian Asians who are both obese and smoke, the beta-fibrinogen genetic polymorphisms -455G-->A and -148C-->T, which are in linkage disequilibrium, are significant risk factors for the development of MI. Factor VII genetic variants, namely the 10 bp promoter insertion/deletion and R353Q polymorphisms, may possibly play a protective role through their association with elevated HDL and low LDL levels, respectively.

    Cardiovascular journal of South Africa : official journal for Southern Africa Cardiac Society [and] South African Society of Cardiac Practitioners 2005;16;3;152-7

  • A hepatocyte nuclear factor-3 site in the fibrinogen beta promoter is important for interleukin 6-induced expression, and its activity is influenced by the adjacent -148C/T polymorphism.

    Verschuur M, de Jong M, Felida L, de Maat MP and Vos HL

    Hemostasis and Thrombosis Research Center, Department of Hematology, Leiden University Medical Center, P. O. Box 9600, 2300 RC Leiden, Gaubius Laboratory, TNO Quality of Life, P. O. Box 2215, 2301 CE Leiden, The Netherlands.

    An elevated plasma fibrinogen level is an independent risk factor for cardiovascular disease. Therefore, an understanding of the regulation of fibrinogen expression is important. Inflammation and genetic variation of the fibrinogen beta gene regulate plasma fibrinogen levels, and there are indications that inflammation and genetic variation interact. The aim of our study was to gain more understanding of the regulation of the inflammatory response of the fibrinogen beta gene and to determine the effects of genetic variation. Luciferase reporter gene assays in hepatoma cells, mutation analysis, and electrophoretic mobility shift assays were used to investigate the transcriptional regulation of the fibrinogen beta promoter. We identified a hepatocyte nuclear factor-3 (HNF-3) site located just upstream of previously identified interleukin-6 (IL6)-responsive sequences. This HNF-3 site is essential for a full response of the promoter to IL6, which is a new function for HNF-3. The activity of the CCAAT box/enhancer-binding protein site (located 18 nucleotides downstream of the HNF-3 site and important to the IL6 response) depends on the integrity of the HNF-3 site and vice versa, explaining the necessity of HNF-3 in the IL6 response of the fibrinogen beta promoter. Furthermore, small interfering RNA to HNF-3 reduces the fibrinogen beta mRNA levels. The rare T allele of the -148C/T polymorphism, which is present between the binding sites of HNF-3 and CCAAT box/enhancer-binding protein, interferes with this mechanism, and this polymorphism is in our assay system the only genetic determinant of IL6-induced promoter activity among six polymorphisms in the fibrinogen beta promoter.

    The Journal of biological chemistry 2005;280;17;16763-71

  • Regulation of gamma-fibrinogen chain expression by heterogeneous nuclear ribonucleoprotein A1.

    Xia H

    Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021, USA. hxia@nybloodcenter.org

    Earlier studies showed that HepG2 cells stably transfected with any one fibrinogen chain cDNA enhanced the expression of the other two fibrinogen chains. In this report, a regulatory element "TGCTCTC" in the gamma-fibrinogen promoter region, -322 to -316, is identified, which is involved in increased expression of gamma chain in HepG2 cells that are transfected with Bbeta fibrinogen cDNA. By electrophoretic mobility shift assay, three DNA-protein complexes were found to form with the regulatory element. The amount of the protein complexes that bind with the regulatory element was much reduced in HepG2 cells transfected with Bbeta cDNA. By DNA-affinity chromatography, mass spectrometry, and supershift assay, human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was identified as a component of the complexes. Overexpression of hnRNP A1 suppressed basal gamma-fibrinogen transcription. These results indicate that the basal expression of gamma-fibrinogen is regulated by a constitutive transcriptional repressor protein, hnRNP A1, and the decreased binding activity of hnRNP A1 leads to the overexpression of gamma chain in HepG2 cells that overexpress the Bbeta chain.

    The Journal of biological chemistry 2005;280;13;13171-8

  • gammaAla82Gly represents a common fibrinogen gamma-chain variant in Caucasians.

    Ivaskevicius V, Jusciute E, Steffens M, Geisen C, Hanfland P, Wienker TF, Seifried E and Oldenburg J

    Institute for Transfusion Medicine and Immune Haematology, DRK Blood Donor Service Baden-Württemberg/Hessen, Sandhofstrasse 1, D-60528 Frankfurt am Main, Germany.

    Screening of 200 blood donors for the presence of polymorphisms in three fibrinogen genes (FGA, FGB, FGG), revealed two individuals with a heterozygous missense mutation (c.323C > G, gammaAla82Gly) in the FGG gene. This mutation has been reported previously to cause mild hypofibrinogenaemia. Analysis of an additional 416 blood donors showed two more heterozygous gammaAla82Gly mutations, resulting in an overall gammaAla82Gly allele frequency of 0.0032. Haplotype analysis demonstrated that the gammaAla82Gly mutation originated from a common founder. From these data we estimated that homozygous individuals for gammaAla82Gly should occur at a frequency of 1: 95 000, suggesting that hypofibrinogenaemia represents a more frequent condition in the population than so far believed.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2005;16;3;205-8

  • [Inherited dysfibrinogenemia caused by Arg275His in the beta chain of fibrinogen].

    Fang Y, Wang X, Qi H, Wu W, Ding Q, Dai J, Zhou R, Wang W, Xie S and Wang H

    Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai, 200025 P. R. China.

    Objective: To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia.

    Methods: Laboratory tests including activated particle thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and the activity of protein C (PC), protein S(PS) and antithrombin (AT) were conducted in the proband and 4 family members. The activity and antigen of fibrinogen in plasma were measured by functional and immunoturbidimetry assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes were analyzed by direct sequencing.

    Results: The proband had normal APTT and PT, but prolonged TT. Her plasma fibrinogen levels were extremely reduced, which was also found in her mother. The sequencing results of the proband revealed heterozygous g.5678 G>A in the exon 8 of FGG gene originating from her mother, which caused Arg275His missense mutation.

    Conclusion: Dysfibrinogenemia in the family is caused by Arg275His in the beta chain of fibrinogen and it is the first report on a Chinese family with inherited dysfibrinogenemia.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2005;22;2;201-3

  • Liver histology of an afibrinogenemic patient with the Bbeta-L353R mutation showing no evidence of hepatic endoplasmic reticulum storage disease (ERSD); comparative study in COS-1 cells of the intracellular processing of the Bbeta-L353R fibrinogen vs. the ERSD-associated gamma-G284R mutant.

    Duga S, Braidotti P, Asselta R, Maggioni M, Santagostino E, Pellegrini C, Coggi G, Malcovati M and Tenchini ML

    Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy.

    Background: Type I fibrinogen deficiencies (hypofibrinogenemia and afibrinogenemia) are rare congenital disorders characterized by low or unmeasurable plasma fibrinogen antigen levels. Their genetic bases are represented by mutations within the three fibrinogen genes. Among the 11 reported missense mutations, a few have been characterized by expression studies and found to have an impaired fibrinogen assembly and/or secretion. Histopathological analyses were previously reported in two hypofibrinogenemic cases with discernible hepatic disease, revealing that both underlying mutations (gamma-Gly284Arg and gamma-Arg375Trp) were associated with hepatic fibrinogen endoplasmic reticulum storage disease (ERSD).

    Objective: The objective of this study was to investigate the liver histology in an afibrinogenemic patient, homozygous for the Bbeta-Leu353Arg mutation, and to study the intracellular processing of the mutant protein.

    Liver histology was evaluated by light microscopy, electron microscopy and immunocytochemistry. Intracellular processing of mutant fibrinogen was analyzed by pulse-chase labeling and immunoprecipitation experiments. Messenger RNA levels were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR).

    Results: The histopathological characterization of the liver showed no signs of fibrinogen accumulation, a difference from the previously reported findings in two hypofibrinogenemic kindreds with ERSD. To evaluate whether the Bbeta-Leu353Arg mutation and the ERSD-associated gamma-Gly284Arg mutation affected intracellular fibrinogen trafficking differently, both mutant proteins were expressed in COS-1 cells. Bbeta-Leu353Arg led to a more severe secretion defect, but no differences that could explain phenotype-genotype correlation were found in the intracellular processing. Endoglycosidase-H analysis demonstrated a secretion block before translocation to the Golgi medial stacks. Real-time RT-PCR studies showed normal levels of the Bbeta mRNA in the patient's liver.

    Conclusions: The results confirm that Bbeta-Leu353Arg is associated with impaired fibrinogen secretion, but not with hepatic ERSD.

    Journal of thrombosis and haemostasis : JTH 2005;3;4;724-32

  • No association between pulmonary embolism or deep vein thrombosis and the -455G/A beta-fibrinogen gene polymorphism.

    Camilleri RS and Cohen H

    Haemostasis Research Unit, Department of Haematology, University College London School of Medicine, 3rd floor Jules Thorn Building, The Middlesex Hospital, 48 Riding House Street, London W1W 7EY, UK. r.camillieri@ucl.ac.uk

    Hyperfibrinogenaemia has been reported to be associated with deep vein thrombosis (DVT). However, whether or not the "fibrinogen-raising"-455G/A polymorphism of the beta-fibrinogen gene is associated with DVT is uncertain and there are no data on whether this polymorphism is associated with pulmonary embolism (PE). We have studied relationships between the -455G/A beta-fibrinogen gene polymorphism and the occurrence of PE and/or DVT (n = 339) (PE only, n = 76; DVT only, n = 216; PE and DVT, n = 47). There was no difference between the -455A allelic frequencies for the control (n = 190) and patient groups - PE, 0.187 and 0.171, respectively [P = 0.6087, chi test; odds ratio (OR), 1.12; 95% confidence interval (CI), 0.72-1.74]; DVT, 0.187 and 0.171, respectively (P = 0.5408, chi test; OR, 1.11; 95% CI, 0.78-1.59). This also applied when only Caucasian individuals were considered - PE allelic frequencies, 0.192 and 0.193, respectively (P = 0.9764, chi test; OR, 0.99; 95% CI, 0.62-1.60); DVT allelic frequencies, 0.192 and 0.186, respectively (P = 0.8404, chi test; OR, 1.04; 95% CI, 0.71-1.51). While the results should be interpreted with caution as the frequency of the -455A allele is rare, the -455A allele of the beta-fibrinogen gene does not appear to be associated with an increased risk of PE or DVT.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2005;16;3;193-8

  • The fibrin-derived peptide Bbeta15-42 protects the myocardium against ischemia-reperfusion injury.

    Petzelbauer P, Zacharowski PA, Miyazaki Y, Friedl P, Wickenhauser G, Castellino FJ, Gröger M, Wolff K and Zacharowski K

    Department of General Dermatology, Medical University of Vienna, 18-20 Waehringer Guertel, Vienna, 1090, Austria.

    In the event of a myocardial infarction, current interventions aim to reopen the occluded vessel to reduce myocardial damage and injury. Although reperfusion is essential for tissue salvage, it can cause further damage and the onset of inflammation. We show a novel anti-inflammatory effect of a fibrin-derived peptide, Bbeta15-42. This peptide competes with the fibrin fragment N-terminal disulfide knot-II (an analog of the fibrin E1 fragment) for binding to vascular endothelial (VE)-cadherin, thereby preventing transmigration of leukocytes across endothelial cell monolayers. In acute or chronic rat models of myocardial ischemia-reperfusion injury, Bbeta15-42 substantially reduces leukocyte infiltration, infarct size and subsequent scar formation. The pathogenic role of fibrinogen products is further confirmed in fibrinogen knockout mice, in which infarct size was substantially smaller than in wild-type animals. Our findings conclude that the interplay of fibrin fragments, leukocytes and VE-cadherin contribute to the pathogenesis of myocardial damage and reperfusion injury. The naturally occurring peptide Bbeta15-42 represents a potential candidate for reperfusion therapy in humans.

    Nature medicine 2005;11;3;298-304

  • -455G/A polymorphism and preprocedural plasma levels of fibrinogen show no association with the risk of clinical restenosis in patients with coronary stent placement.

    Monraats PS, Rana JS, Zwinderman AH, de Maat MP, Kastelein JP, Agema WR, Doevendans PA, de Winter RJ, Tio RA, Waltenberger J, Frants RR, van der Laarse A, van der Wall EE and Jukema JW

    Department of Cardiology, Leiden University Medical Center, The Netherlands. J.W.Jukema@lumc.nl

    The effect of preprocedural fibrinogen levels on in-stent restenosis is largely unknown. The -455G/A polymorphism of the fibrinogen beta-gene is associated with baseline plasma level or acute phase increase of fibrinogen. Therefore, we hypothesized that there is a relationship between this polymorphism and preprocedural fibrinogen level and clinical restenosis at follow-up among patients with coronary stent placement. The GENetic DEterminants of Restenosis (GENDER) project is a multicenter follow-up study that enrolled 3,146 consecutive patients after successful percutaneous coronary intervention. A coronary stent was placed in 2,309 patients. 1f40 Of these, 2,257 (97.7%) patients were successfully genotyped for the -455G/A polymorphism. Plasma fibrinogen levels were measured at baseline in a subpopulation of 623 stented patients with the von Clauss method and patients were grouped into tertiles according to fibrinogen levels. Primary endpoint was target vessel revascularization (TVR); secondary combined endpoint was defined as death presumably from cardiac causes, MI not attributable to another coronary artery than the target vessel, and TVR. No association was observed between the -455G/A polymorphism and TVR or combined endpoint (p=0.99, p=0.97, respectively). Multivariate regression analysis revealed that the risk of TVR and combined endpoint was not higher for patients in the highest tertile for fibrinogen versus the lowest tertile (RR=0.60, 95% CI: 0.26-1.37 for TVR, RR=0.64, 95% CI: 0.29-1.44 for combined endpoint). In conclusion, the presence of -455G/A polymorphism in the fibrinogen beta-gene and preprocedural fibrinogen level is not associated with an increased risk of TVR or combined endpoint in a patient population with coronary stent placement. Therefore, these parameters are not worthwhile for stratifying patients at risk for restenosis pre-stenting.

    Thrombosis and haemostasis 2005;93;3;564-9

  • Contribution of haplotypes across the fibrinogen gene cluster to variation in risk of myocardial infarction.

    Mannila MN, Eriksson P, Lundman P, Samnegård A, Boquist S, Ericsson CG, Tornvall P, Hamsten A and Silveira A

    Atherosclerosis Research Unit, Karolinska Institutet, King Gustaf V Research Institute, Karolinska University Hospital, Solna, Stockholm, Sweden. Maria.Nastase.Mannila@medks.ki.se

    Fibrinogen has consistently been recognized as an independent predictor of myocardial infarction (MI). Multiple mechanisms link fibrinogen to MI; therefore disentangling the factors underlying variation in plasma fibrinogen concentration is essential. Candidate regions in the fibrinogen gamma (FGG), alpha (FGA) and beta (FGB) genes were screened for single nucleotide polymorphisms (SNPs). Several novel SNPs were detected in the FGG and FGA genes in addition to the previously known SNPs in the fibrinogen genes. Tight linkage disequilibrium extending over various physical distances was observed between most SNPs. Consequently, eight SNPs were chosen and determined in 377 postinfarction patients and 387 healthy individuals. None of the SNPs were associated with plasma fibrinogen concentration or MI. Haplotype analyses revealed a consistent pattern of haplotypes associated with variation in risk of MI. Of the four haplotypes inferred using the FGA -58G>A and FGG 1299 +79T>C SNPs, the most frequent haplotype, FGG-FGA*1 (prevalence 46.6%), was associated with increased risk of MI (OR 1.51; 95%CI 1.18, 1.93), whereas the least frequent haplotype, FGG-FGA*4 (11.8%), was associated with lower risk of MI (OR 0.79 95%CI 0.64, 0.98). In conclusion, fibrinogen haplotypes, but not SNPs in isolation, are associated with variation in risk of MI.

    Thrombosis and haemostasis 2005;93;3;570-7

  • The -148 C/T fibrinogen gene polymorphism and fibrinogen levels in ischaemic stroke: a case-control study.

    van Goor MP, EB, Leebeek FW, Brouwers GJ, Koudstaal PJ and Dippel DW

    Erasmus MC Rotterdam, Department of Neurology, PO Box 2040, 3000 CA Rotterdam, Netherlands. m.vangoor@erasmusmc.nl

    Background: To determine whether -148 C/T fibrinogen gene promoter polymorphism increases stroke risk by modifying the fibrinogen level.

    Design: A case-control study of patients with first ever ischaemic stroke, confirmed by computed tomography.

    Methods: Venous blood samples were collected for fibrinogen and routine coagulation tests one week after the stroke, and after three months in about half the patients. Population controls were age and sex matched. -148 C/T fibrinogen polymorphism was determined by polymerase chain reaction followed by digestion with restriction enzymes HindIII/AluI.

    Results: There were 124 patients and 125 controls, mean age 56 years (range 18 to 75); 34 patients (27%) and 41 controls (33%) were heterozygous for -148 C/T fibrinogen polymorphism; six patients (5%) and five controls (4%) had the T/T genotype. The odds ratio of ischaemic stroke associated with CC homozygotes v T carriers was 0.8 (95% confidence interval, 0.5 to 1.4). Relative risk for ischaemic stroke associated with fibrinogen levels in the highest quartile was 3.9 (1.9 to 8.4) at one week, decreasing to 1.4 (0.6 to 3.3) at three months.

    Conclusions: -148 C/T fibrinogen gene polymorphism was not a strong risk factor for ischaemic stroke. High fibrinogen levels early after acute stroke probably represent an acute phase response.

    Journal of neurology, neurosurgery, and psychiatry 2005;76;1;121-3

  • [Associations of hemostasis factors genes with early development of ischemic heart disease and manifestation of myocardial infarction in young age].

    Dankovtseva EN, Zateĭshchikov DA, Chudakova DA, Koroleva OS, Brovkin AN, Nosikov VV, Gaĭdukova NV, Tishchenko VA and Sidorenko BA

    Aim: To study polymorphisms of genes of factors of the system of hemostasis in young patients with ischemic heart disease (IHD).

    Material: Two groups of patients participated in the study: patients with first manifestation of IHD at the age < or = 50 years (men) or < or = 55 years (women) (n=158), and patients with first IHD manifestation at the age > or = 70 years (n=92).

    Methods: We studied polymorphic markers of genes encoding clotting factors V (F5) and VII (F7), subunit IIIa of platelet integrin (ITGB3), beta-chain of fibrinogen (FGB) and tissue plasminogen activator type 1 (PLANH1).

    Results: After separation of a subgroup of patients with MI without preceding angina we revealed significant differences in distribution of frequencies of genotypes of polymorphic marker C(-426)T of factor V gene: genotype TT was significantly more frequent in young (14.9%) than in old (2%) patients (p=0.008). Multifactorial logistic regression revealed independent association of early IHD with smoking (OR 6.112 [2.567-14.552]; p<0.001) and presence of genotype TT of C(-426)T polymorphic marker of F5 gene (OR=9.410 [1.074-82.459]; p=0.043).

    Conclusion: Thus we obtained data on the presence of independent association between IHD risk and manifestation of MI in young age with genotype TT of polymorphic marker C(-426)T of F5 gene as well as with traditional risk factors of IHD.

    Kardiologiia 2005;45;12;17-24

  • Plasma fibrinogen concentration predicts the risk of myocardial infarction differently in various parts of Europe: effects of beta-fibrinogen genotype and environmental factors. The HIFMECH Study.

    Mannila MN, Silveira A, Hawe E, Eriksson P, Aillaud MF, Juhan-Vague I, Yudkin J, Margaglione M, di Minno G, Mussoni L, Tremoli E, Humphries S, Hamsten A and HIFMECH Study Group

    Atherosclerosis Research Unit, King Gustaf V Research Institute, Karolinska University Hospital, S-171 76 Stockholm, Sweden. Maria.Nastase.Mannila@medks.ki.se

    The propensity to atherothrombotic disease differs in Europe, with high-risk regions located in the North of Europe and lowrisk regions in the South of Europe. The HIFMECH study (Hypercoagulability and Impaired Fibrinolytic function MECHanisms predisposing to myocardial infarction (MI) study) was undertaken to elucidate genetic and environmental mechanisms underlying MI based on investigations of postinfarction patients and healthy individuals recruited from Stockholm, Sweden, London, England (North of Europe), Marseille, France and San Giovanni Rotondo, Italy (South of Europe). In the present report, emphasis was placed on fibrinogen, a multifunctional protein, widely recognized as an independent predictor of atherothrombotic disease. The adjusted plasma fibrinogen concentration was an independent discriminator between cases and controls in London (SOR 3.58; 95% CI 1.31; 9.83), but not in the other centres. Genotyping for six beta-fibrinogen promoter single nucleotide polymorphisms was performed of which -249C/T, -455G/A and -854G/A were used in analysis as a consequence of the linkage disequilibrium pattern. Four haplotypes, with similar distribution across Europe, were detected: CGG (46.7%), CAG (20.3%), TGG (18.2%) and CGA (14.8%). A significant haplotype effect on plasma fibrinogen concentration was observed in patients (p < 0.001) but not in controls (p = 0.08). The -455G/A genotype related to plasma fibrinogen concentration amongst patients along with centre and IL-6 concentration (together explaining 11.5% of the variation), whereas predictors amongst controls included centre, body mass index, IL-6 and smoking habit (explaining 15.7%). Thus, plasma fibrinogen concentration contributes differently to MI across Europe, and a disease-related stimulus is required to evoke allele-specific regulation of fibrinogen synthesis.

    Thrombosis and haemostasis 2004;92;6;1240-9

  • [The relationship b 1d44 etween beta fibrinogen gene-455G/A polymorphisms and Budd-Chiari syndrome].

    Wang SY, Wei YF and Li JD

    Department of Hematology, Hebei Provincial People's Hospital, Shijiazhuang 050051, China.

    Objective: To study the effect of the -455G-->A substitution on influencing fibrinogen levels and morbidity in patients with Budd-Chiari syndrome (BCS).

    Methods: 53 patients with BCS diagnosed by color Doppler-ultrasound and venography and 105 healthy persons as control were observed. Assay of plasma fibrinogen was performed by the method of enzymatic reaction. DNA was extracted from white cells using the phenol/chloroform method. beta fibrinogen gene was detected by polymerase chain reaction- restriction fragment length polymorphism techniques using thermostable Taq polymerase under conditions recommended by the manufacturer.

    Results: The frequencies of -455GA+AA genotype were significantly increased in patients with BCS compared with normal controls (P <0.05, OR=2.04, 95% CI: 1.03-4.02). There was a significant difference in plasma fibrinogen levels between BCS subjects and control subjects (P <0.01). Either in patients with BCS or in healthy controls, the plasma fibrinogen levels was significantly increased seen in the subjects with -455A alleles (0.01< P <0.05).

    Conclusions: beta fibrinogen gene -455G/A polymorphism is associated with increased plasma fibrinogen levels and may be an important risk factor in the pathogenesis of BCS.

    Zhonghua nei ke za zhi 2004;43;10;753-5

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • [Association of polymorphic marker G(-455)A of gene FGB with coronary artery disease].

    Chudakova DA, Minushkina LO, Zateĭshchikov DA and Nosik VV

    Patients with coronary artery disease (CAD), including those who have had myocardial infarction (MI), and control subjects have been compared with respect to the distributions of the alleles and genotypes of polymorphic marker G(-455)A of gene FGB encoding the fibrinogen beta-chain. The groups studied do not differ significantly with respect to the distributions of G(-455)A alleles and genotypes. This indicates that this marker is not associated with CAD in the Moscow population. Allele A of the G(-455)A polymorphic marker has been found to be associated with an increased fibrinogen content of blood plasma in women with CAD.

    Genetika 2004;40;10;1406-9

  • Genetic and environmental determinants of fibrin structure and function: relevance to clinical disease.

    Scott EM, Ariëns RA and Grant PJ

    Academic Unit of Molecular Vascular Medicine, Martin Wing, The General Infirmary at Leeds, Leeds, LS1 3EX, UK.

    The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. The structure of the fibrin clot and the genetic and environmental factors that modify it have effects on its biological function. Alterations in fibrin structure and function have implications for the clinical presentation of vascular disease. This review briefly describes the key features involved in the formation of a fibrin clot, its typical structure, and function. This is followed by a review of the current literature on genetic and environmental influences on fibrin structure/function and the relationship to clinical disease. The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. This review discusses how genetic and environmental factors alter fibrin structure and function and the implications this has for the clinical presentation of vascular disease.

    Arteriosclerosis, thrombosis, and vascular biology 2004;24;9;1558-66

  • Association of genetic polymorphisms in the fibrinogen and platelet glycoprotein genes with unstable angina in Chinese patients.

    Ni Y, Hu D, Yu H, Li C, Liu W, Wang H, Li L, Yongbin N, Dayi H, Hong Y, Cuilan L, Wenling L, Hongyu W and Lei L

    Department of Cardiology, Peking University People's Hospital, Beijing, People's Republic of China.

    Background: Inherited predisposition has been associated with coronary artery disease (CAD) in the white population.

    Hypothesis: The objective of this study was to investigate the association between the risk of unstable angina (UA) and genetic factors believed to be associated with an increased tendency toward thrombosis (the variable number of tandem repeats [VNTR] polymorphism of the platelet glycoprotein [GP] Ib alpha gene, Pl(A1/A2) of the platelet GP IIIa gene, 448G/A of the Bbeta fibrinogen gene and Thr312Ala of the Aalpha fibrinogen gene) in Chinese patients with UA.

    Methods: We performed a case/control study evaluating 69 Chinese patients (43 men, 26 women) with UA and 69 control subjects without CAD, individually matched for age and gender. The restriction fragment length polymorphism (RFLP) method was used to determine the genetic polymorphisms.

    Results: The frequencies of GP Ib alpha C/B genotype and Bbeta fibrinogen 448A allele were higher in patients with UA (46.4 vs. 30.4%, odds ratio [OR] 1.977, 95% confidence interval [CI] 0.98-3.97, p = 0.054, and 49.3 vs. 20.3%, OR 3.816, 95% CI 1.797-8.103, p = 0.000, respectively). Only four subjects (two cases, two controls) with GP IIIa Pl(A2) allele were found, and there was no association between Aalpha fibrinogen Thr312Ala polymorphism and UA.

    Conclusions: Chinese patients with UA had increased frequencies of GP Ib alpha C/B genotype and Bbeta fibrinogen 448A allele. These data suggest that some genetic factors may influence the development of UA.

    Clinical cardiology 2004;27;8;455-8

  • [Relationship between fibrinogen B beta gene FGB -455G/A polymorphism and atherosclerotic cerebral infarction].

    Sun H, Lu FH, Tian Q, Wen PE, Wu F and Wang X

    Department of Cardiocerebrolvascular Disease,Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan, Shandong, 250062 P. R. China. sasa303@163.com

    Objective: To explore the distribution of fibrinogen (FGB) B beta polymorphism in Chinese Han population and the association of the polymorphisms with the occurrence of atherosclerotic cerebral infarction (ACI).

    Methods: The B beta gene FGB -455G/A polymorphism was identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) in 132 patients with ACI and 148 healthy controls matching on age and sex. Turbidimetric assays were performed to measure the plasma fibrinogen levels of all cases.

    Results: The plasma fibrinogen level in ACI group (3.42+/-0.52 g/L), was significantly higher than that in the controls (2.96+/-0.42g/L), P<0.001. The A allele was associated with the elevated plasma fibrinogen levels in both patients and controls. Among the A allele carriers, smokers had significantly higher plasma fibrinogen levels than did the non-smokers (P<0.05). The distribution of B beta gene FGB -455G/A polymorphism was in accordance with the Hardy-Weinberg equilibrium (P>0.05). The A allelic frequency in ACI group (0.258) was significantly higher than that in the control group (0.152) (P<0.05). Logistic regression analysis showed that the cases carrying A allele (GA+AA genotype) had 1.653 times the risk of ACI.

    Conclusion: The study demonstrates that A allele of the B beta gene FGB -455G/A polymorphism may be a susceptible predictor of the occurrence of ACI, particularly in smokers.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2004;21;4;382-5

  • Erythrocyte aggregation and -455G/A polymorphism of the beta-fibrinogen gene in survivors of acute myocardial infarction.

    Vayá A, Breña S, Réganon E, Vila V, Martinez-Sales V, Contreras MT, Zorio E, Corella D and Aznar J

    Thrombosis and haemostasis 2004;92;1;223-4

  • [Association between the polymorphism of beta-fibrinogen gene -455G/A and ischemic stroke].

    Dong QL and Zhang C

    Department of Neurology, People's Hospital of Rizhao City, Rizhao, Shandong, 276800 PR China. dqlrz@163.com

    Objective: To study the association between the variation of beta fibrinogen gene promoter -455G/A and ischemic stroke.

    Methods: Eighty-six hypertensive patients with ischemic stroke (stroke group) and 85 hypertensive patients without ischemic stroke (hypertensive group) were randomly selected from the in patients, and 90 healthy persons(control group) were recruited for this study. Polymerase chain reaction with restrictive enzyme Hae III (PCR-restriction fragment length polymorphism method) was employed to analyze the polymorphism of beta-fibrinogen gene promoter -455G/A. Plasma fibrinogen levels were measured with prothrombin time (PT) assay.

    Results: A(-455) allele frequencies of the stroke group (0.22) was significantly higher than that of the control group and the hypertensive group (0.11, 0.13, chi-square=8.3 P<0.05). The genotype exhibited significant difference among the 3 groups; G/A and A/A were more commonly seen in the stroke group(chi-square=10.03,P<0.05). The mean fibrinogen level of the stroke group(4.82+/-0.26 g/L) was significantly higher than that of the control group and the hypertensive group(4.37+/-0.19, 5.50+/-0.20, F=5.98 P<0.01) In both stroke group and hypertensive group, the plasma fibrinogen levels in patients with -455G/G were significantly lower than those in patients with -455G/A, and -455A/A but the difference was not significant among the 3 genotypes in the control group.

    Conclusion: Plasma fibrinogen level could be affected by the beta-fibrinogen gene -455G/A polymorphism. And A(-455) allele may be an independent risk factor for ischemic stroke.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2004;21;3;274-6

  • The human plasma proteome: a nonredundant list developed by combination of four separate sources.

    Anderson NL, Polanski M, Pieper R, Gatlin T, Tirumalai RS, Conrads TP, Veenstra TD, Adkins JN, Pounds JG, Fagan R and Lobley A

    The Plasma Proteome Institute, Washington DC 20009-3450, USA. leighanderson@plasmaproteome.org

    We have merged four different views of the human plasma proteome, based on different methodologies, into a single nonredundant list of 1175 distinct gene products. The methodologies used were 1) literature search for proteins reported to occur in plasma or serum; 2) multidimensional chromatography of proteins followed by two-dimensional electrophoresis and mass spectroscopy (MS) identification of resolved proteins; 3) tryptic digestion and multidimensional chromatography of peptides followed by MS identification; and 4) tryptic digestion and multidimensional chromatography of peptides from low-molecular-mass plasma components followed by MS identification. Of 1,175 nonredundant gene products, 195 were included in more than one of the four input datasets. Only 46 appeared in all four. Predictions of signal sequence and transmembrane domain occurrence, as well as Genome Ontology annotation assignments, allowed characterization of the nonredundant list and comparison of the data sources. The "nonproteomic" literature (468 input proteins) is strongly biased toward signal sequence-containing extracellular proteins, while the three proteomics methods showed a much higher representation of cellular proteins, including nuclear, cytoplasmic, and kinesin complex proteins. Cytokines and protein hormones were almost completely absent from the proteomics data (presumably due to low abundance), while categories like DNA-binding proteins were almost entirely absent from the literature data (perhaps unexpected and therefore not sought). Most major categories of proteins in the human proteome are represented in plasma, with the distribution at successively deeper layers shifting from mostly extracellular to a distribution more like the whole (primarily cellular) proteome. The resulting nonredundant list confirms the presence of a number of interesting candidate marker proteins in plasma and serum.

    Molecular & cellular proteomics : MCP 2004;3;4;311-26

  • -455G/A beta-fibrinogen gene polymorphism, factor V Leiden, prothrombin G20210A mutation and MTHFR C677T, and placental vascular complications.

    Camilleri RS, Peebles D, Portmann C, Everington T and Cohen H

    Haemostasis Research Unit, Department of Haematology, University College London School of Medicine, University College London Hospitals NHS Trust, London, UK. r.camilleri@ucl.ac.uk

    Hyperfibrinogenaemia is associated with systemic arterial and venous thromboembolism and therefore may contribute to placental vascular disease associated with obstetric complications. The fibrinogen-raising -455G/A beta-fibrinogen gene polymorphism may enhance the physiological increase in fibrinogen levels during pregnancy and thereby predispose to obstetric complications. This retrospective case-control study looked at the association between the beta-fibrinogen gene polymorphism -455G/A, the hereditary thrombophilic markers factor V Leiden, prothrombin G20210A mutation (PGM) and C677T methylene tetrahydrofolate reductase (MTHFR), and obstetric complications associated with placental vascular disease. The study group (n = 247) comprised 147 women (90 Caucasian) who met the clinical criteria and a control group of 100 parous women (90 Caucasian) with no history of obstetric or medical complications. No significant differences were observed in the -455A allelic frequencies of the patient and normal control groups, with (allelic frequencies, 0.156 and 0.178, respectively; P = 0.5716, chi2 test, odds ratio = 1.17, 95% confidence interval = 0.65-2.13) or without (allelic frequencies, 0.129 and 0.170, respectively; P = 0.2077, chi2 test, odds ratio = 1.38, 95% confidence interval = 0.81-2.35) the exclusion of non-Caucasian women. There was an increased prevalence of factor V Leiden among Caucasian patients compared with normal controls (allelic frequencies, 0.056 and 0.017, respectively; P = 0.048, chi2 test, odds ratio = 0.29, 95% confidence interval = 0.05-1.15) but there were no differences in the prevalences of PGM or MTHFR. These data suggest that factor V Leiden is associated with an increased risk of obstetric complications, but that the -455A allele of beta-fibrinogen, PGM and MTHFR do not appear to be implicated.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2004;15;2;139-47

  • Absence of association between polymorphisms in the hemostatic factor pathway genes and carotid intimal medial thickness: the Framingham Heart Study.

    Fox CS, Larson MG, Corey D, Feng D, Lindpaintner K, Polak JF, Wolf PA, D'Agostino RB, Tofler GH and O'Donnell CJ

    National Heart, Lung and Blood Institute's Framingham Heart Study, Framingham, Mass, USA.

    Fibrinogen, plasminogen activator inhibitor-1, and other key proteins in the coagulation cascade have been implicated in the origin of cardiovascular disease. Polymorphisms in genes encoding these proteins have been associated with variability in plasma levels of these proteins. Carotid intimal medial thickness (IMT) is a heritable, quantitative measure of atherosclerosis that is predictive of subsequent myocardial infarction and stroke. We sought to test whether carotid IMT is associated with polymorphisms in several well-characterized genes in the hemostatic factor pathways.

    Methods: Here, 867 men and 911 women (mean age, 57 years) in the Framingham offspring cohort underwent B-mode carotid ultrasonography to determine the mean internal (ICA) and common carotid artery (CCA) IMT. Age-, sex-, and multivariable-adjusted linear regression was used to estimate the association of the following variants with log-transformed CCA and ICA IMT: factor V Leiden, factor VII Arg/Gln, fibrinogen HindIII beta-148, plasminogen activator inhibitor-1 4G/5G, and the glycoprotein IIIa Pl(A2) polymorphism.

    Results: Mean ICA IMT was 0.58 mm; mean CCA IMT was 0.60 mm. There were no differences in ICA or CCA IMT by genotype for any of the candidate genes in unadjusted, age- or sex-adjusted, and multivariable-adjusted models.

    Conclusions: There is no evidence for an association between well-studied polymorphisms in the hemostatic factor genes and carotid IMT. Whether other common genetic variants in hemostatic factor genes are associated with subclinical atherosclerosis remains to be determined.

    Funded by: NHLBI NIH HHS: N01-HC-25195; NINDS NIH HHS: 5R01-NS17950-20

    Stroke 2004;35;3;e65-7

  • Beta-fibrinogen promoter -455 G/A (HaeIII) polymorphism prediction of plasma fibrinogen but not of ischemic cerebrovascular disease.

    Bi S, Wang D, Li G, Wen S and Pan S

    Institute of Cerebrovascular Diseases and Related Disorders, Department of Neurology, First Clinical College of Harbin Medical University, Harbin 150001. bisheng98@hotmail.com

    Objective: The -455 G/A (HaeIII) polymorphism of beta-fibrinogen gene influences levels of plasma fibrinogen. We further investigated whether it influences the risk of ischemic cerebrovascular disease.

    Methods: We accumulated 134 acute ischemic cerebrovascular disease (ICVD) cases and compared their -455 G/A status with a control group (n = 166). The beta-fibrinogen gene -455 G/A polymorphism was analyzed for all subjects by PCR-RFLP with the restrictive enzyme HaeIII.

    Results: Plasma fibrinogen was higher in AA homozygous participants (341 mg/dL) than in participants carrying the G allele: GA (290 mg/dL), GG (298 mg/dL) in the control group. Plasma fibrinogen was also higher in AA homozygous patients (353 mg/dL) than in cases carrying the G allele: GA (287 mg/dL), GG (302 mg/dL) in the ICVD group. However, there was no significant association between beta-fibrinogen gene -455 G/A polymorphism and ICVD group.

    Conclusions: Although a small effect cannot be excluded, beta-fibrinogen gene -455 G/A polymorphism is an independent predictor of plasma fibrinogen, but not of ischemic cerebrovascular disease.

    Chinese medical sciences journal = Chung-kuo i hsueh k'o hsueh tsa chih 2004;19;1;1-5

  • Genetic influences on blood pressure within the Stanislas Cohort.

    Sass C, Cheng S, Siest G and Visvikis S

    Unité INSERM U 525, Centre de Médecine Préventive, Vandoeuvre-Lès-Nancy and Université Henri Poincaré, Nancy, France.

    Objective: To investigate the association of 21 polymorphisms within 13 genes, APOE, APOB, APOC3, CETP, LPL, PON1, MTHFR, FGB, F5, GPIIIa, SELE, ACE and AGT, with inter-individual blood pressure (BP) variation.

    Participants: Seven hundred and seventy-six men and 836 women, free of antihypertensive and lipid-lowering medications, were selected from the Stanislas Cohort.

    Results: ANOVA on blood pressure values after adjustment for covariates [age, body mass index (BMI), contraceptive pill, tobacco and alcohol] showed that lipoprotein lipase (LPL) Ser447Ter and glycoprotein IIIA (GpIIIa) Pl polymorphisms were significantly associated with BP in women (0.01 < or = P < or = 0.05), whereas BP levels in men were significantly different according to apolipoprotein CIII (APOC3) 3206T/G and -482C/T polymorphisms (P < or = 0.05). In women, compared to the most common allele, the GpIIIa Pl allele was associated with increased mean arterial pressure (MAP) (P < 0.05) and pulse pressure (PP) (P < 0.001), and the LPL 447Ter allele was associated with decreased systolic blood pressure (SBP) and PP levels (0.001 < or = P < or = 0.05). These two polymorphisms appeared to act independently. In men, the APOC3 3206GG genotype was related to decreased diastolic blood pressure (DBP) and MAP levels (P < or = 0.01), and the APOC3 -482T allele with decreased PP levels (P < or = 0.05). The presence of both the -482C allele and the 3206GG genotype was related to decreased DBP, suggesting that specific haplotypes might be involved.

    Conclusion: The APOC3, LPL and GpIIIa genes were found to be associated with BP levels. The contributions of these genes, although modest, are consistent with the polygenic nature of BP levels.

    Journal of hypertension 2004;22;2;297-304

  • Candidate genetic markers and the risk of restenosis after coronary angioplasty.

    Völzke H, Grimm R, Robinson DM, Wolff B, Schwahn C, Hertwig S, Motz W and Rettig R

    Institute of Epidemiology and Social Medicine, Ernst Moritz Arndt University, Walther Rathenau Strasse 48, D-17487 Greifswald, Germany. voelzke@uni-greifswald.de

    The aim of the present study was to test for possible associations between candidate gene polymorphisms and the risk of restenosis and recurrent restenosis after percutaneous transluminal coronary angioplasty (PTCA) without stenting. We followed up 511 PTCA patients, and restenosis and recurrent restenosis were defined according to angiographical criteria. Genotyping of the beta-fibrinogen -455 G/A, glycoprotein (GP) IIIa PlA1/PlA2, plasminogen activator inhibitor-1 (PAI-1) 4G/5G, factor V Leiden 1691 G/A, tumour necrosis factor alpha (TNFalpha) -238 G/A, TNFalpha -308 G/A, interleukin (IL)-1alpha -889 C/T, IL-1beta -511 C/T, methylenetetrahydrofolate reductase (MTHFR) 677 C/T and endothelial nitric oxide synthase (eNOS) 4 b/a gene polymorphisms was performed by PCR and restriction-fragment-length-polymorphism-based techniques. One hundred and sixty patients (31.3%) developed restenosis and in 130 of these patients, of whom 123 were available for analysis, a second PTCA without stenting was performed. Of these patients, 35 (28.5%) developed recurrent restenosis. None of the investigated genotypes were associated with the risk of restenosis or recurrent restenosis after PTCA. The degree of stenosis before and immediately after PTCA and the severity of the lesion were independent predictors for restenosis after PTCA. In conclusion, there was no association between the beta-fibrinogen -455 G/A, GP IIIa PlA1/A2, PAI-1 4G/5G, factor V Leiden 1691 G/A, TNFalpha -238 G/A, TNFalpha -308 G/A, IL-1alpha -889 C/T, the IL-1beta -511 C/T, MTHFR 677 C/T and eNOS 4 b/a gene polymorphisms and the risk of restenosis after PTCA as well as recurrent restenosis after repeated PTCA.

    Clinical science (London, England : 1979) 2004;106;1;35-42

  • Fibrinogen C-terminal peptidic sequences (Haptides) modulate fibrin polymerization.

    Marx G, Ben-Moshe M, Magdassi S and Gorodetsky R

    HAPTO Biotech (Israel) Ltd. PO Box 12275, Jerusalem 91121, Israel 91121. gmarx@hapto.co.il

    We previously described synthetic peptides of 19-21 amino acid residues, homologous to the C-termini of fibrinogen Fib(340) and Fib(420), from the beta-chain (Cbeta), the extended alphaE chain (CalphaE) and near the end of the gamma-chain (preCgamma) which elicited attachment (haptotactic) responses from mesenchymal cells. We named these haptotactic peptides -Haptides. The effects of Haptides on fibrin clot formation was evaluated and their possible effects on platelet aggregation was examined. The Haptides Cbeta,Ca 80 lphaE and preCgamma, (2-10 micro M) increased fibrin clot turbidity and also decreased thrombin-induced clotting time. Higher co 1eb8 ncentrations (>120 micro M of Cbeta or preCgamma) induced fibrinogen precipitation even without thrombin. These precipi-tates exhibited different ultrastructure from thrombin-induced fibrin by scanning and transmission microscopy. C-terminal peptides of the other fibrinogen chains exerted no such effects. Sepharose beads covalently coated with either whole fibrinogen or Haptides (SB-Fib or SB-Haptide) highly adsorbed free (FITC) Haptides. In aqueous solution, Haptides formed nano-par-ticles with average size of approximately 150 nm in diameter. We suggest that such positively charged aggregates could serve to nucleate and accelerate fibrin gel formation. These results also indicate that Cbeta and preCgamma sequences within fibrin(ogen) participate in the docking and condensation of fibrin(ogen) during its assembly into a fibrin clot. By contrast, Haptides up to 100 micro M did not bind to platelets, and had no effect on platelet aggregation. Our findings highlight the roles of the C-terminal sequences of the beta and gamma chains in fibrin(ogen) polymerization as well as in cell attachment.

    Thrombosis and haemostasis 2004;91;1;43-51

  • Genetic polymorphisms associated with thrombophilia and vascular disease in women with unexplained late intrauterine fetal death: a multicenter study.

    Hefler L, Jirecek S, Heim K, Grimm C, Antensteiner G, Zeillinger R, Husslein P and Tempfer C

    Department of Obstetrics and Gynecology, Vienna University Medical School, Vienna, Austria. lukas.hefler@akh-wien.ac.at

    Objective: We determined whether gene polymorphisms associated with thrombophilia and vascular disease as etiologic factors were involved in the pathogenesis of pregnancy-associated complications.

    Methods: We conducted a multicenter case-control study in which we studied 94 women with late unexplained intrauterine fetal death (IUFD) and 94 healthy women with at least one uncomplicated full-term pregnancy and no history of IUFD. We obtained blood samples from all subjects and analyzed their DNA for 12 common polymorphisms of thrombophilic and vascular genes (factor V Leiden, factor V H1299R, prothrombin G20210A, factor XIII V34L, MTHFR C677T, MTHFR A1298C, beta-fibrinogen-455 G to A, PAI-1 4G/5G, GPIIIa L33P, HFE C282Y, apolipoprotein B R3500Q, and apolipoprotein E2/E3/E4).

    Results: We found no significant association between any of the polymorphisms investigated and IUFD. Subgroup analyses involving various combinations of polymorphisms and in which gestational age and fetal weight were corrected for also showed no significant results.

    Conclusions: Our data represent the largest study to date with respect to thrombophilic and vascular gene polymorphisms in IUFD. In accordance with others, we challenge the importance of thrombophilic and vascular gene polymorphisms in the pathogenesis of this condition.

    Journal of the Society for Gynecologic Investigation 2004;11;1;42-4

  • The -455G/A polymorphism of the beta fibrinogen gene and the Bgl II polymorphism of the alpha2beta1 integrin gene and myocardial infarction in patients with type 2 diabetes.

    Poglajen G, Kirbis J and Milutinović A

    Medical Center Medicor Izola, Slovenia.

    Platelets and fibrinogen might be involved in the pathogenesis of thrombus formation and MI. The Bgl II gene polymorphism of the alpha2beta1 integrin, which is a platelet collagen receptor, and the -455G/A polymorphism in the beta fibrinogen gene have been suggested as genetic risk factors for MI. The aim of this study was to look for a relationship between the -455G/A polymorphism in the beta fibrinogen gene and the development of MI in Caucasians with type 2 diabetes. One hundred and forty-two subjects with type 2 diabetes and MI were compared to 234 diabetic subjects with no history of coronary artery disease. There were no significant differences in the frequency of the Bgl II gene polymorphism or of the -455G/A polymorphism in the beta fibrinogen gene in the patients with MI compared to the patients without MI: Bgl II (+/+) genotype was found in 19.7% of patients with MI and 15.4% of controls and -455GG genotype was found in 58.4% of patients with MI and 57.7% of controls. The present study demonstrates that neither the Bgl II gene polymorphism nor -455G/A polymorphism in the beta fibrinogen gene is a genetic marker for MI in Slovene population (Caucasians) with type 2 diabetes.

    Folia biologica 2004;50;6;203-4

  • Congenital afibrinogenemia: identification and expression of a missense mutation in FGB impairing fibrinogen secretion.

    Vu D, Bolton-Maggs PH, Parr JR, Morris MA, de Moerloose P and Neerman-Arbez M

    Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva, Switzerland.

    Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease: a homozygous deletion of approximately 11 kb of the fibrinogen alpha-chain gene (FGA). Subsequent studies revealed that the great majority of afibrinogenemia mutations are localized in FGA, but mutations were also found in FGG and FGB. Apart from 3 missense mutations identified in the C-terminal portion of FGB, all fibrinogen gene mutations responsible for afibrinogenemia are null. In this study, a young boy with afibrinogenemia was found to be a compound heterozygote for 2 mutations in FGB: an N-terminal nonsense mutation W47X (exon 2) and a missense mutation (G444S, exon 8). Coexpression of the FGB G444S mutant cDNA in combination with wild-type FGA and FGG cDNAs demonstrated that fibrinogen molecules containing the mutant beta chain are able to assemble but are not secreted into the media, confirming the pathogenic nature of the identified mutation.

    Blood 2003;102;13;4413-5

  • Fibrinogen B beta polymorphisms do not directly contribute to an altered in vitro clot structure in humans.

    Maghzal GJ, Brennan SO and George PM

    Molecular Pathology, Canterbury Health Laboratories, PO Box 151, Christchurch, New Zealand. ghassan.maghzal@chmeds.ac.nz

    Fibrinogen B beta polymorphisms, such as the -455 G/A and the Arg448Lys amino acid substitution, have been shown to increase the risk of atherothrombotic disease. Although these polymorphisms are related to fibrinogen concentrations, their effect on fibrin clot structure has not been extensively studied. We examined the frequency of the fibrinogen B beta -455 G/A polymorphism in a group of myocardial infarction (MI) patients. There was no association between this polymorphism and MI. However, we found that patients homozygous for the rare -455 A allele had a higher average age at first MI. A similar result was found for individuals homozygous for the B beta 448 Lys allele who also had a higher age at first MI. We subsequently studied the clotting properties of purified Arg448 and Lys448 fibrinogens in vitro and found that these fibrinogens did not significantly differ in their polymerisation, fibrinolysis kinetics or in their clot permeation properties. Mass spectrometry analysis of endoproteinase Asp-N digests of B beta chains revealed that the Lys(448) and the Arg(448) chains were expressed in approximately equal proportions in a heterozygote Arg448Lys individual. Our results demonstrate that the fibrinogen B beta -455 G/A polymorphism is not associated with myocardial infarction and further-more the closely linked B beta Arg448Lys protein coding variation does not have an influence on the function nor the structure of the protein in a purified system.

    Thrombosis and haemostasis 2003;90;6;1021-8

  • Molecular variation at functional genes and the history of human populations--data on candidate genes for cardiovascular risk in the Mediterranean.

    Moral P, Valveny N, López-Alomar A, Calo C, Kandil M, Harich N, González-Pérez E, Via M, Esteban E, Dugoujon JM and Vona G

    Department of Animal Biology-Anthropology, University of Barcelona, Barcelona, Spain.

    A screening of 22 DNA polymorphisms has been performed in western Mediterranean populations (Iberian Peninsula, Morocco, and Central Mediterranean Islands). The analyzed markers correspond to polymorphic sites in several candidate genes for cardiovascular disease including apolipopoteins and their receptors (APOA1, APOB, APOE, APOC1, APOC2, LPA, and LDLR), genes implied in the hemostasis regulation (Factor VII, alpha and beta-fibrinogen, alpha and beta platelet-integrin, tissue plasminogen activator, and plasminogen activator inhibitor-1), and the angiotensin converting enzyme gene. The results are presented of a partial analysis carried out in following population samples: 6 from the Iberian Peninsula, 2 from Morocco, and 3 from Central Islands. The degree of inter-population diversity was significant and consistent with data from other kind of genetic polymorphisms. The apportionment of the allele frequency variance supported a geographic structure into three main regions: Central Mediterranean Islands, the Iberia Peninsula and North Africa. The genetic distance pattern is compatible with a south-to-north North African influence in the Iberian Peninsula and a remarkable gene flow from sub-Saharan Africa into Morocco. Epidemiologically, North Africa is characterized by high frequencies of LPA PNR alleles with high number of repeats (protective for cardiovascular risk) and high frequencies of the APOE*E4 allele (risk factor) as compared with European populations.

    Collegium antropologicum 2003;27;2;523-36

  • [Preliminary study on single nucleotide polymorphisms and linkage disequilibrium in promoter region of fibrinogen B beta gene].

    Gong WX, Cai YM, Chen H and Ma SG

    The Third Affiliated Hospita, Medical College, Jinan University, Zhuhai, Guangdong, 519000 PR China. gwxzh@163.com

    Objective: To investigate the distribution characters and linkage disequilibrium of single nucleotide polymorphisms (SNPs) -148C/T, -455G/A and -854G/A in the promoter region of fibrinogen B(FGB) beta gene.

    Methods: Genotype and allele frequencies of FGB beta gene were examined by polymerase chain reaction-restriction fragment length polymorphism and nucleotide sequencing methods in 377 Chinese southern Han individuals. Three FGB beta SNPs Hardy-Weinberg equilibrium and linkage disequilibrium were analyzed with population genetics methods.

    Results: The allele frequencies of 3 SNPs are in good agreement with Hardy-Weinberg equilibrium. A total of 9 genotypes among the 377 individuals were identified in 3 SNPs. The genotype frequencies of -148CC, CT and TT were 0.597, 0.358 and 0.045, respectively; the -455G/A genotype frequencies were the same as that of -148C/T SNP; the genotype frequencies of -854GG,GA, AA were 0.820,0.178,0.002, respectively. The frequencies of rare allele -148T, -455A and -854A were 0.224,0.224 and 0.092, respectively, while the common allele frequencies were 0.776 for -148C, 0.776 for -455G, and 0.908 for -854G. There were no statistically significant differences in genotype and allele frequencies between the male and female groups (P>0.05). The relationship between -455G and -148C was completely concordant, but there was a random distribution between -854 and -148 (-445) SNPs.

    Conclusion: The results show there is a complete linkage disequilibrium between -148C/T and -455G/A and a negative linkage disequilibrium between -854G/A and -148C/T, as well as between -854G/A and -455G/A. This study has provided population genetics data on FGB beta gene promoter in Chinese southern Han population.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2003;20;6;512-6

  • The beta fibrinogen gene G-455-A polymorphism is a risk factor for Legg-Perthes disease.

    Dilley A, Hooper WC, Austin H, Jamil M, Miller C, Stokes M, Evatt B and Eldridge J

    Hematologic Diseases Branch, Division of AIDS, STD, and Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, US Department of Health and Human Services, Atlanta, Georgia 30333, USA. adilley@cdc.gov

    Legg-Perthes disease is a pediatric hip disorder characterized by avascular necrosis of the femoral head. The etiology of Legg-Perthes disease may involve repeated interruptions of the blood supply to the proximal femur. Thus, the role of thrombosis in Legg-Perthes disease is of interest. The focus of this analysis is an evaluation of the relationship between Legg-Perthes disease and the beta fibrinogen gene G-455-A polymorphism in 55 cases of Legg-Perthes disease and 56 age, race, and gender-matched healthy controls. Parents of subjects completed a questionnaire about their child's lifestyle and medical history. Blood was obtained for plasma and DNA analysis. Study subjects were predominantly white (93%), male (77%) and under age 16 (70%). Cases were more likely to be exposed to passive smoke than were controls (odds ratio 5.6, 95% confidence interval 2.0-12.0). Assuming a dominant genetic model, individuals who possessed either the G/A or A/A genotype were over three times more likely to have Legg-Perthes disease compared to those without the polymorphism (odds ratio 3.4, 95% confidence interval 1.5-7.8). Separate analyzes by smoke exposure revealed that the excess risk of the G-455-A polymorphism occurred in those exposed (odds ratio 7.0) as opposed to those unexposed to passive smoke (odds ratio 1.9). Although this difference in the odds ratios is not statistically significant (P=0.2), it suggests a possible interactive effect of cigarette smoke and the b fibrinogen gene G-455-A polymorphism in the risk of developing Legg-Perthes disease.

    Journal of thrombosis and haemostasis : JTH 2003;1;11;2317-21

  • Hypofibrinogenemia caused by a nonsense mutation in the fibrinogen Bbeta chain gene.

    Mimuro J, Hamano A, Tanaka T, Madoiwa KS, Sugo T, Matsuda M and Sakata Y

    Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Tichigi-ken, Japan.

    Congenital hypofibrinogenemia, fibrinogen Tottori II, caused by a nonsense mutation in the fibrinogen Bbeta chain gene, was found in a 68-year-old Japanese female. The plasma fibrinogen level was 99.2 mg dL(-1) as determined by the thrombin time method. No overt molecular abnormalities were observed in purified patient fibrinogen by SDS-PAGE analysis. After sequencing all exons and exon-intron boundaries of three fibrinogen genes, we found a heterozygous single point mutation of T-->G at position 3356 of the patient fibrinogen Bbeta chain gene. This nucleotide mutation results in a nonsense mutation (TAT sequence for Bbeta 41Tyr to TAG sequence for a translation termination signal). The mutation was confirmed by polymerase chain reaction-restriction fragment length polymorphism analysis, since this nucleotide mutation results in a new NheI recognition sequence at this position. These data indicated that the nonsense mutation of the fibrinogen Bbeta chain gene caused a truncated fibrinogen Bbeta chain, which may not be assembled in the fibrinogen molecule.

    Journal of thrombosis and haemostasis : JTH 2003;1;11;2356-9

  • Myocardial infarction in patients aged less than 40 years. Frequency of BclI polymorphism in the fibrinogen beta-chain gene and plasma fibrinogen.

    Lewandowski 132b K, Kwaśnikowski P, Elikowski W and Zawilska K

    Department of Haematology of University of Medical Sciences, Poznań, Poland.

    Background: Several polymorphisms in the genes encoding for three separate chains of fibrinogen have been described. Some of them (Hae III and B854) are associated with elevated fibrinogen plasma level.

    Aim: To determine the frequency of BclI polymorphism in the fibrinogen beta-chain gene (BclI betaFb) in young survivors of myocardial infraction (MI) and to assess the relationship between allele status, plasma fibrinogen concentration and the number of affected coronary arteries.

    Methods: The study group consisted of 99 male patients (mean age 43.5, range 29-49 years) with premature coronary artery disease (CAD) diagnosed by coronary angiography who had MI in the mean age of 37.4+/-3.2 years. The control group involved 78 age- and gender-matched healthy volunteers. DNA was extracted from peripheral blood leukocytes using standard methods. Fibrinogen blood concentration was determined using biuretic method. The BclI polymorphism in the fibrinogen beta-chain gene was investigated using polymerase chain reaction (PCR).

    Results: Obesity was found in 15%, smoking - in 89%, hypertension - in 21%, diabetes - in 14% and hyperlipidemia - in 86% of MI patients. A family history of MI was present in 50% of patients. Coronary angiography revealed single-vessel disease in 34%, two-vessel disease in 36%, and three-vessel disease in 16% of patients. In two patients coronary angiography was normal. The frequency of BclI polymorphism of the beta-fibrinogen gene was significantly higher in MI patients than in controls (40.4% vs 29.5%, p<0.01). Moreover, in MI patients carrying the mutant allele a higher concentration of blood fibrinogen was found in comparison to patients without this anomaly (3.87 vs 3.55 g/L, p=0.05). There was no evidence of an association between the number of affected coronary arteries and polymorphism of BclI betaFbg gene status. However, all patients carrying BclI polymorphism of betaFbg gene had abnormal coronary angiography, contrary to patients without any defect.

    Conclusions: 1. Polymorphism of BclI betaFbg gene is associated with an increased fibrinogen plasma level. 2. There is no association between BclI polymorphism of betaFbg gene and the number of affected coronary arteries. This may confirm the hypothesis of multi-factorial aetiology of CAD in young patients suffering MI.

    Kardiologia polska 2003;59;9;205-12

  • Characterization of the enzymatic activity of human kallikrein 6: Autoactivation, substrate specificity, and regulation by inhibitors.

    Magklara A, Mellati AA, Wasney GA, Little SP, Sotiropoulou G, Becker GW and Diamandis EP

    Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ont., Canada M5G 1X5.

    Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.

    Biochemical and biophysical research communications 2003;307;4;948-55

  • Genetic risk factors for cerebrovascular disease in children with sickle cell disease: design of a case-control association study and genomewide screen.

    Adams GT, Snieder H, McKie VC, Clair B, Brambilla D, Adams RJ, Kutlar F and Kutlar A

    Sickle Cell Center, Department of Medicine, Medical College of Georgia, Augusta, GA, USA. gayetadams@hotmail.com

    Background: The phenotypic heterogeneity of sickle cell disease is likely the result of multiple genetic factors and their interaction with the sickle mutation. High transcranial doppler (TCD) velocities define a subgroup of children with sickle cell disease who are at increased risk for developing ischemic stroke. The genetic factors leading to the development of a high TCD velocity (i.e. cerebrovascular disease) and ultimately to stroke are not well characterized.

    Methods: We have designed a case-control association study to elucidate the role of genetic polymorphisms as risk factors for cerebrovascular disease as measured by a high TCD velocity in children with sickle cell disease. The study will consist of two parts: a candidate gene study and a genomewide screen and will be performed in 230 cases and 400 controls. Cases will include 130 patients (TCD > or = 200 cm/s) randomized in the Stroke Prevention Trial in Sickle Cell Anemia (STOP) study as well as 100 other patients found to have high TCD in STOP II screening. Four hundred sickle cell disease patients with a normal TCD velocity (TCD < 170 cm/s) will be controls. The candidate gene study will involve the analysis of 28 genetic polymorphisms in 20 candidate genes. The polymorphisms include mutations in coagulation factor genes (Factor V, Prothrombin, Fibrinogen, Factor VII, Factor XIII, PAI-1), platelet activation/function (GpIIb/IIIa, GpIb IX-V, GpIa/IIa), vascular reactivity (ACE), endothelial cell function (MTHFR, thrombomodulin, VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1), inflammation (TNFalpha), lipid metabolism (Apo A1, Apo E), and cell adhesion (VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1). We will perform a genomewide screen of validated single nucleotide polymorphisms (SNPs) in pooled DNA samples from 230 cases and 400 controls to study the possible association of additional polymorphisms with the high-risk phenotype. High-throughput SNP genotyping will be performed through MALDI-TOF technology using Sequenom's MassARRAY system.

    Discussion: It is expected that this study will yield important information on genetic risk factors for the cerebrovascular disease phenotype in sickle cell disease by clarifying the role of candidate genes in the development of high TCD. The genomewide screen for a large number of SNPs may uncover the association of novel polymorphisms with cerebrovascular disease and stroke in sickle cell disease.

    Funded by: NHLBI NIH HHS: HL67682-01, R01 HL067682

    BMC medical genetics 2003;4;6

  • Prenatal diagnosis for congenital afibrinogenemia caused by a novel nonsense mutation in the FGB gene in a Palestinian family.

    Neerman-Arbez M, Vu D, Abu-Libdeh B, Bouchardy I and Morris MA

    Division of Medical Genetics, University Medical School and University Hospital, Geneva, Switzerland. marguerite.arbez@medecine.unige.ch

    Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of detectable fibrinogen. We previously identified the first causative mutations for this disease, homozygous deletions of approximately 11 kb of the fibrinogen alpha chain gene (FGA). Subsequent analyses revealed that most afibrinogenemia alleles are truncating mutations of FGA, although mutations in all 3 fibrinogen genes, FGG, FGA and FGB have been identified. In this study, we performed the first prenatal diagnosis for afibrinogenemia. The causative mutation in a Palestinian family was a novel nonsense mutation in the FGB gene, Trp467Stop (W467X). Expression of the Trp467Stop mutant FGB cDNA in combination with wild-type FGA and FGG cDNAs showed that fibrinogen molecules containing the mutant beta chain are not secreted into the media. The fetus was found to be heterozygous for the Trp467Stop mutation by direct sequencing and by linkage analysis, a result that was confirmed in the newborn by intermediate fibrinogen levels.

    Blood 2003;101;9;3492-4

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Fibrinogen is a marker for nephropathy and peripheral vascular disease in type 1 diabetes: studies of plasma fibrinogen and fibrinogen gene polymorphism in the DCCT/EDIC cohort.

    Klein RL, Hunter SJ, Jenkins AJ, Zheng D, Semler AJ, Clore J, Garvey WT and DCCT/ECIC STUDY GROUP

    Division of Endocrinology, Metabolism, and Medical Genetics, Department of Medicine, Medical University of South Carolina, Charleston 29425, USA. kleinrl@musc.edu

    We examined whether plasma fibrinogen levels and the beta-fibrinogen gene G(-455)-->A polymorphism were related to microvascular or macrovascular disease in patients (n = 909) with type 1 diabetes enrolled in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/ EDIC). Univariate regression showed that fibrinogen levels were correlated with BMI (r = 0.15; P < 0.0001), HbA(1c) (r = 0.11; P = 0.0014), total cholesterol (r = 0.17; P < 0.0001), and LDL cholesterol (r = 0.16; P < 0.0001) in all patients. In men, but not women, waist-to-hip ratio (r = 0.20; P < 0.0001) and triglycerides (r = 0.13; P = 0.0047) also became powerful predictors of fibrinogen level; in women, but not men, fibrinogen was correlated with both diastolic (r = 0.16; P = 0.0011) and systolic (r = 0.11; P = 0.0241) blood pressure. Fibrinogen was correlated with urinary albumin excretion rates in men (r = 0.13; P = 0.0033), but not in women. In both sexes, however, the development of proteinuria (albumin excretion >300 mg/24 h) was accompanied by 1.5-fold increment in plasma fibrinogen compared with patients with normal excretion or microalbuminuria. In addition, high fibrinogen levels were associated with a lower average ankle-brachial index in women (r = -0.13; P = 0.0075), but not men. Multiple regression analyses demonstrated that plasma fibrinogen was independently correlated with high albumin excretion rate in men, and with low average ankle-brachial index in women. Fibrinogen was not correlated with the severity of retinopathy. Carotid artery intima-medial thickness was not correlated with fibrinogen, and the G(-455)-->A polymorphism in the 5' promoter region of the 1f40 beta-fibrinogen gene did not influence circulating fibrinogen levels. However, the presence of the more common G(-455) allele was associated with greater intima-medial thickness in the internal carotid artery (ANCOVA P = 0.045). Last, hyperfibrinogenemia in type 1 diabetes is associated with components of the insulin resistance syndrome trait cluster, and the association is influenced by sex.

    Funded by: NHLBI NIH HHS: P01-HL55782

    Diabetes care 2003;26;5;1439-48

  • Fibrinogen polymorphisms TaqI, HaeIII and BclI are not associated with a higher risk of deep vein thrombosis.

    Bozic M, Teran N, Peterlin B and Stegnar M

    Department of Vascular Diseases, University Medical Centre, Ljubljana, Slovenia. mojca.bozic@trnovo.kclj.si

    Pathophysiology of haemostasis and thrombosis 2003;33;3;164-9

  • Fibrinogen gene promoter -455 A allele as a risk factor for lacunar stroke.

    Martiskainen M, Pohjasvaara T, Mikkelsson J, Mäntylä R, Kunnas T, Laippala P, Ilveskoski E, Kaste M, Karhunen PJ and Erkinjuntti T

    Department of Forensic Medicine, University of Tampere, Finland.

    Elevated fibrinogen levels are suggested to increase the risk of myocardial infarction and stroke. Carriers of the A allele of the fibrinogen -455G/A polymorphism have increased plasma fibrinogen levels. We studied the association of this polymorphism with stroke subtype in the Stroke Aging Memory (SAM) cohort.

    Methods: The SAM cohort comprises 486 consecutive patients 55 to 85 years of age who, 3 months after ischemic stroke, completed a detailed stroke assessment. Stroke subtypes were examined with MRI. -455G/A genotype was determined by polymerase chain reaction. MRI and genotype data were available for the 299 patients who constitute the present study population.

    Results: Genotype distributions were 64.9% (GG), 31.8% (GA), and 3.3% (AA). In a logistic regression model with age, sex, hypertension, diabetes, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, arrhythmia, atrial fibrillation, peripheral arterial disease, and smoking as possible confounders, there was a significant association between A+ genotype and >or=3 lacunar infarcts (odds ratio [OR], 2.57; 95% CI, 1.23 to 5.36; P=0.01). Hypertensive patients carrying the A allele had increased risk (OR, 4.24; 95% CI, 1.29 to 13.99; P=0.02) for >or=3 lacunar infarcts. A similar increase in risk was observed among smokers with the A+ genotype (OR, 2.67; 95% CI, 0.92 to 7.77; P=0.07).

    Conclusions: Stroke patients carrying the A allele of the Bbeta-fibrinogen -455G/A polymorphism frequently presented with multiple lacunar infarcts. This association was stronger among hypertensives and smokers. These associations suggest that the A allele may predispose to atherothrombotic events in cerebrovascular circulation.

    Stroke 2003;34;4;886-91

  • Genetic variations observed in arterial and venous thromboembolism--relevance for therapy, risk prevention and prognosis.

    Harrington DJ, Malefora A, Schmeleva V, Kapustin S, Papayan L, Blinov M, Harrington P, Mitchell M and Savidge GF

    Centre for Haemostasis and Thrombosis, St. Thomas' Hospital, London, UK.

    We undertook genetic and biochemical assays in patients with arterial (n = 146) and venous (n = 199) thromboembolism and survivors of pulmonary embolism (n = 58) to study causation and gene-life style interactions. In the clinical material from North Western Russia, factor V Leiden was found to be a risk factor in venous thrombosis (OR = 3.6), while the methylenetetrahydrofolate reductase (MTHFR) C677T mutation was a significant variable in both venous (p = 0.03) and arterial thrombosis (p = 0.004). Homocysteine levels were determined (n = 84) and hyperhomocysteinemia correlated with the T allele of the MTHFR gene, and with smoking and coffee consumption. Vitamin supplementation reduced homocysteine levels dependent on MTHFR genotype (36% TT, 25% CT, 22% CC). In pulmonary embolism patients, frequency of the -455G/A beta-fibrinogen dimorphism was studied. Carriers of this allele were significantly underrepresented (p < 0.02) among pulmonary embolism survivors (34.5%) compared to controls (56.7%). Additionally, -455AA homozygotes were found in 11.7% controls but only 1.7% of pulmonary embolism patients (p = 0.006). In venous and arterial thrombosis cases, MTHFR and homocysteine data led to effective dietary supplementation with a reduced risk of disease progression. Results from the pulmonary embolism study may indicate that screening tests for the -455G/A beta-fibrinogen genetic variation could be of prognostic value, and may point the way for novel anticoagulation strategies.

    Clinical chemistry and laboratory medicine 2003;41;4;496-500

  • No evidence of association between prothrombotic gene polymorphisms and the development of acute myocardial infarction at a young age.

    Atherosclerosis, Thrombosis, and Vascular Biology Italian Study Group

    Background: We investigated the association between 9 polymorphisms of genes encoding hemostasis factors and myocardial infarction in a large sample of young patients chosen because they have less coronary atherosclerosis than older patients, and thus their disease is more likely to be related to a genetic predisposition to a prothrombotic state.

    This nationwide case-control study involved 1210 patients who had survived a first myocardial infarction at an age of <45 years who underwent coronary arteriography in 125 coronary care units and 1210 healthy subjects matched for age, sex, and geographical origin. None of the 9 polymorphisms of genes encoding proteins involved in coagulation (G-455A beta-fibrinogen: OR, 1.0; CI, 0.8 to 1.2; G1691A factor V: OR, 1.1; CI, 0.6 to 2.1; G20210A factor II: OR, 1.0; CI, 0.5 to 1.9; and G10976A factor VII: OR, 1.0; CI, 0.8 to 1.3), platelet function (C807T glycoprotein Ia: OR, 1.1; CI, 0.9 to 1.3; and C1565T glycoprotein IIIa: OR, 0.9; CI, 0.8 to 1.2), fibrinolysis (G185T factor XIII: OR, 1.2; CI, 0.9 to 1.6; and 4G/5G plasminogen activator inhibitor type 1: OR, 0.9; CI, 0.7 to 1.2), or homocysteine metabolism (C677T methylenetetrahydrofolate reductase: OR, 0.9; CI, 0.8 to 1.1) were associated with an increased or decreased risk of myocardial infarction.

    Conclusions: This study provides no evidence supporting an association between 9 polymorphisms of genes encoding proteins involved in hemostasis and the occurrence of premature myocardial infarction or protection against it.

    Circulation 2003;107;8;1117-22

  • Association of increased levels of fibrinogen and the -455G/A fibrinogen gene polymorphism with chronic periodontitis.

    Sahingur SE, Sharma A, Genco RJ and De Nardin E

    Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, NY 14214, USA.

    Background: Fibrinogen is one of the acute-phase proteins whose levels are elevated during periodontal disease. Recent studies suggest that excessive fibrinogen production might play a role in upregulating host immune responses. In addition, there is a relationship between the -455G/A polymorphism (HaeIII) in the 5' flanking region of the beta-fibrinogen gene promoter and increased fibrinogen levels. In this study, we investigated the distribution of the -455G/A polymorphism and the relationship of this specific genotype to fibrinogen levels in periodontitis patients.

    Methods: In order to assess the -455G/A polymorphism, restriction fragment length polymorphism (RFLP) analysis with HaeIII enzyme was performed in the promoter region of the beta-fibrinogen gene. This was carried out on 79 chronic periodontitis patients as compared to 75 periodontally healthy subjects, matched to age, gender, and race. Fibrinogen levels were determined by the radial immunodiffusion assay (RID).

    Results: The frequency of homozygocity for the rare allele of the beta-fibrinogen gene (H2H2) was 13% for the periodontitis patients and 3% for the control group (P = 0.01). The distributions of H1H1 and H1H2 genotypes were 48% and 39% in the patient group and 70% and 27% in the control group, respectively. Chi-square analysis indicated that the distribution of these genotypes between the 2 groups was significantly different (P = 0.01). Fibrinogen levels were significantly higher in the patient group (2,496.5 mg/l +/- 105) compared to the control group (2,250.0 mg/l +/- 118.3) after adjusting for age, gender, and smoking status (P = 0.04). Consistent with previous reports, in our study population, those subjects with the H2H2 genotype had significantly higher fibrinogen levels (3,005.7 mg/l +/- 182.5) compared to subjects with the H1H1 genotype (2,325.0 mg/l +/- 91.6) or H1H2 genotype (2,438.0 mg/l +/- 117.4) (P = 0.001). Furthermore, the H1H2 and H2H2 genotypes were found at a higher frequency among periodontitis patients than controls. The odds ratios (OR) for these genotypes were 3.26 (95% confidence interval [CI]: 1.25 to 8.53) for the H1H2 genotype and 6.41 (95% CI: 1.15 to 35.83) for the H2H2 genotype as compared to individuals with the H1H1 genotype, after adjusting for age, gender, and smoking status.

    Conclusions: The results indicate that a higher percentage of chronic periodontitis patients exhibit genotypes associated with higher plasma fibrinogen levels than healthy individuals. Furthermore, periodontitis patients have significantly higher fibrinogen levels compared to healthy individuals. The presence of H1H2 or H2H2 genotypes as well as elevated fibrinogen levels, in conjunction with other factors, may put individuals at higher risk of having periodontal disease, or may result from periodontal infection-genetic interactions.

    Funded by: NIDCR NIH HHS: DE07926, DE12085

    Journal of periodontology 2003;74;3;329-37

  • Potential thrombophilic mutations/polymorphisms in patients with no flow-limiting stenosis after myocardial infarction.

    French JK, Van de Water NS, Sutton TM, Lund M, Gao W, McDowell J, Liu-Stratton Y, Pohorence J, Szymanski D, Goldschmidt-Clermont P, White HD, Browett PJ and Cooke G

    Department of Molecular Medicine, University of Auckland, Auckland, New Zealand. johnf@adhb.govt.nz

    Background: Although inherited thrombophilias are more common in patients with venous thromboembolism, their influence on the development of myocardial infarction (MI) requires clarification.

    To determine whether there are increased frequencies of mutations/polymorphisms in 14 genes potentially causing thrombophilia in patients with no flow-limiting stenoses after MI compared with patients with > or =1 flow-limiting stenosis of >50%, we studied 395 patients (60 with no flow-limiting stenosis) who underwent angiography at approximately 1 month. The mutations/polymorphisms studied included Factor V Leiden, prothrombin variant G20210A, beta-fibrinogen 448 (G/A), endothelial protein C receptor (23-base pair insertion), methyl tetrahydrofolate reductase 677 (C/T), platelet glycoprotein IIIa PlA1/A2, plasminogen activator inhibitor-1 4G/5G, angiotensin II type 1 receptor (A/C), hemochromatosis gene 282 (G/A), nitric oxide synthase (NOS) (3 forms: eNOS, eNOS3, eNOS4), p22 phox of NADPH oxidase C242T, and angiotensin-converting enzyme insertion/deletion polymorphism. The frequencies of Factor V Leiden and the beta-fibrinogen 448 A allele were higher in patients with no flow-limiting stenosis than in patients with > or =1 stenosis (11.7% vs 3.6%, odds ratio [OR] 3.6, 95% CI 1.3-9.4, P =.015; and 42% vs 27%, OR 2.0, 95% CI 1.1-3.5, P =.018, respectively), and there was a trend toward an increased frequency of prothrombin variant G20210A (6.7% vs 2.1%, OR 3.4, 95% CI 0.95-11.8, P =.069). However, in patients with no flow-limiting stenosis after MI the frequencies of the other gene mutations/polymorphisms were not increased. Also, there were no significant interactions between any of these 14 mutation/polymorphisms, major cardiovascular risk factors, and the absence of any flow-limiting stenosis, except for Factor V Leiden and hypertension (OR 6.34, 95% CI 2.67-100, P =.004).

    Conclusions: Patients with no flow-limiting stenosis after MI had increased frequencies of 2 inherited thrombophilias (Factor V Leiden and beta-fibrinogen 448 A allele), and there was a trend toward an increased frequency of prothrombin variant G20210A compared with patients with > or =1 stenosis. These data suggest that polymorphisms/mutations in some gene products influencing coagulation may influence the pathogenesis of MI.

    American heart journal 2003;145;1;118-24

  • Identification of a novel human angiopoietin-like gene expressed mainly in heart.

    Zeng L, Dai J, Ying K, Zhao E, Jin W, Ye Y, Dai J, Xu J, Xie Y and Mao Y

    State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PR China.

    The angiopoietins are an important family of growth factors specific for vascular endothelium. Most of them bind to the TIE2 receptor and are related to regulation of angiogenesis. During large-scale DNA sequencing of the human fetal brain cDNA library, we cloned a novel human angiopoietin-like cDNA and termed it human angiopoietin-like 5 ( ANGPTL5). Like other members of the angiopoietin family, ANGPTL5-deduced protein also has an N-terminal cleavable signal peptide, a predicted coiled-coil domain, and a fibrinogen-like domain. The search against the human genome database indicated that ANGPTL5 maps to 11q22. Expression analysis of ANGPTL5 shows that it is mainly expressed in adult human heart.

    Journal of human genetics 2003;48;3;159-62

  • Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bbeta-chain gene causing activation of cryptic splice sites.

    Spena S, Duga S, Asselta R, Malcovati M, Peyvandi F and Tenchini ML

    Department of Biology and Genetics for Medical Sciences, University of Milan, Italy.

    Congenital afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in afibrinogenemia, 17 in the Aalpha, 6 in the gamma, and only 2 in the Bbeta fibrinogen-chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bbeta-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bbeta-chain gene splicing mutations described in afibrinogenemia. The IVS6 + 13C > T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7 + 1G > T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bbeta-chain minigene constructs, either wild-type or mutant, were transfected in HeLa cells. Assessed by semiquantitative analysis of reverse transcriptase-polymerase chain reaction products, the IVS7 + 1G > T mutation resulted in multiple aberrant splicings, while the IVS6 + 13C > T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bbeta chain for fibrinogen assembly and secretion.

    Blood 2002;100;13;4478-84

  • Spectrum and prevalence of prothrombotic single nucleotide polymorphism profiles in the Greek Cypriot population.

    Xenophontos SL, Hadjivassiliou M, Ayrton N, Karagrigoriou A, Pantzaris M, Nicolaides AN and Cariolou MA

    Molecular Genetics Department-B and Laboratory of Forensic Genetics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.

    Background: This study was performed to establish the allele, genotype and genotype combination/SNP (single nucleotide polymorphism) profile frequencies in the general population of Cyprus for 6 genes implicated in thrombotic disorders. The genes with their respective functional polymorphisms were the following: factor V (G1691A), prothrombin/factor II (G20210A), methylenetetrahydrofolate reductase (C677T), platelet glycoprotein receptor IIIa (P1A1/A2), b-fibrinogen (G/A-455) and plasminogen activator inhibitor-type 1 (4G/5G).

    Methods: DNA samples from 121 unrelated individuals were used for this epidemiological study. The polymerase chain reaction followed by restriction digestion were used to genotype the 6 different polymorphic loci. Allele and genotype frequencies were established and shown to be in Hardy-Weinberg equilibrium.

    Results: Mutant allele frequencies for the 6 genes were as follows: factor V-4%, prothrombin-2%, methylenetetrahydrofolate reductase -39%, platelet glycoprotein receptor IIIa-16%, beta-fibrinogen-17% and plasminogen activator inhibitor - type 1-46%. Combined defects occurred which may increase the risk for vascular events, 33% of individuals (39/118) had 3 or more of the above mutations.

    Conclusions: As in other European populations, prospective case-control studies to estimate the risk for deep vein thrombosis (DVT) and ischemic episodes with respect to genetic and environmental risk factors should be performed. Thrombophilia screening should be applied for primary and secondary prevention of thrombotic episodes in susceptible individuals on the island of Cyprus. Individuals targeted for such screening include those with the following: a positive family history for thrombosis; a previous DVT or other ischemic episode; prior exposure to circumstantial risk factors and in the presence of echolucent plaques.

    International angiology : a journal of the International Union of Angiology 2002;21;4;322-9

  • 2.8 A crystal structures of recombinant fibrinogen fragment D with and without two peptide ligands: GHRP binding to the "b" site disrupts its nearby calcium-binding site.

    Kostelansky MS, Betts L, Gorkun OV and Lord ST

    Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.

    We report two crystal structures, each at a resolution of 2.8 A, of recombinant human fibrinogen fragment D (rfD) in the absence and presence of peptide ligands. The bound ligands, Gly-Pro-Arg-Pro-amide and Gly-His-Arg-Pro-amide, mimic the interactions of the thrombin exposed polymerization sites, "A" and "B", respectively. This report is the first to describe the structure of fragment D in the presence of both peptide ligands. The structures reveal that recombinant fibrinogen is nearly identical to the plasma protein but with minor changes, like the addition of a proximal fucose to the carbohydrate linked to residue betaGln364, and slightly different relative positions of the beta- and gamma-modules. Of major interest in our structures is that a previously identified calcium site in plasma fibrinogen is absent when Gly-His-Arg-Pro-amide is bound. The peptide-dependent loss of this calcium site may have significant biological implications that are further discussed. These structures provide a foundation for the detailed structural analysis of variant recombinant fibrinogens that were used to identify critical functional residues within fragment D.

    Funded by: NHLBI NIH HHS: HL 31048

    Biochemistry 2002;41;40;12124-32

  • G-455A polymorphism of the fibrinogen beta gene and deep vein thrombosis.

    Renner W, Cichocki L, Forjanics A, Köppel H, Gasser R and Pilger E

    Karl-Franzens University, Graz, Austria. wilfried.renner@kfunigraz.ac.at

    Background: Elevated fibrinogen levels have been linked to increased risk for deep venous thrombosis, although it is not clear whether fibrinogen is causal or rather a marker for the presence of other risk factors. A common G/A polymorphism in the gene for the fibrinogen beta-chain (FGB G-455A) is associated with elevated fibrinogen levels. The present study was designed to analyze the role of this genetic marker for deep venous thrombosis.

    We performed a case-control study including 307 patients with documented deep venous thrombosis and 316 control subjects. beta-fibrinogen genotypes were determined by allele-specific polymerase chain reaction.

    Results: GG, GA and AA genotype frequencies were similar among the patients (53.1%, 41.0, 5.9) and controls (51.6%, 42.1, 6.3; P = 0.92). Fibrinogen levels of the patients (median 3.72 g l-1; range 1.93-11.6) did not differ significantly from those of the controls (3.76; 2.17-9.99). Carriers of the homozygous AA genotype had significantly higher fibrinogen levels than noncarriers (patients: 5.32 vs. 3.59; P = 0.024; controls: 6.29 vs. 3.72; P = 0.048).

    Conclusion: Our data suggest that the fibrinogen-elevating FGB G-455A gene polymorphism is not linked to an increased risk for deep venous thrombosis.

    European journal of clinical investigation 2002;32;10;755-8

  • Analysis of Iranian patients allowed the identification of the first truncating mutation in the fibrinogen Bbeta-chain gene causing afibrinogenemia.

    Asselta R, Spena S, Duga S, Peyvandi F, Malcovati M, Mannucci PM and Tenchini ML

    Department of Biology and Genetics for Medical Sciences, University of Milan, via Viotti 3/5, 20133 Milan, Italy.

    Congenital afibrinogenemia is a rare coagulation disorder whose molecular basis is still poorly characterized. Most mutations have been identified in the fibrinogen Aalpha- and gamma-chain genes, whereas only two missense mutations have been reported in the Bbeta-chain gene. The aim of this work was to widen knowledge about the mutational spectrum of this disease by analyzing the molecular bases of congenital afibrinogenemia in three unrelated Iranian patients.

    All patients showed unmeasurable levels of clottable fibrinogen in plasma. Mutational screening was performed by sequencing the whole coding region, including exon-intron boundaries and part of the promoter region of the three fibrinogen genes.

    Results: Sequencing in one patient revealed the presence of a novel nonsense mutation (3282C-->T) in exon 2 of the fibrinogen Bbeta-chain gene, causing a severe truncation of the corresponding polypeptide (R17X). In the remaining probands, two already known small deletions (4209delA and 4220delT), both located in exon 5 of the fibrinogen Aalpha-chain gene, were identified, and their effect at the protein level explored by computer-assisted analysis.

    The identification of the first truncating mutation in the fibrinogen Bbeta-chain gene confirms the involvement of all three fibrinogen genes in the pathogenesis of congenital afibrinogenemia and widens the mutational spectrum of the disease. This knowledge is clinically essential in order to carry out prenatal diagnosis in families at risk.

    Haematologica 2002;87;8;855-9

  • Fibrinogen and factor VII promoter polymorphisms in women with preeclampsia.

    Laasanen J, Hiltunen M, Punnonen K, Mannermaa A and Heinonen S

    Department of Obstetrics and Gynecology, Kuopio University Hospital, Kuopio, Finland.

    Objective: To determine whether genetic variability in the promoter regions of the genes encoding fibrinogen and factor VII contribute to individual differences in susceptibility to the development of preeclampsia.

    Methods: The study involved 133 preeclamptic and 115 healthy control pregnant women who were genotyped for the G-455A polymorphism in the beta-fibrinogen gene promoter and for a decamer insertion or deletion polymorphism at position -323 in the factor VII gene promoter. We used chi(2) analysis to assess genotype frequency differences between preeclamptic women and controls.

    Results: The allelic distribution of the fibrinogen A-455G polymorphism was similar in the two groups, with the frequency of the variant A allele being 18.8% in the preeclampsia group and 20.9% in the control group. We did not find any association between the presence of the factor VII insertion allele and preeclampsia (5.6% versus 6.1%). Accordingly, the genotype distribution of the fibrinogen G-455A and factor VII polymorphisms in the preeclamptic and control groups was similar (P =.852 and P =.308).

    Conclusion: The G-455A polymorphism of the fibrinogen gene promoter and the decamer insertion or deletion polymorphism of the factor VII gene promoter are unlikely to be major genetic predisposing factors for preeclampsia in subjects from eastern Finland.

    Obstetrics and gynecology 2002;100;2;317-20

  • Genetic polymorphisms modify the response of factor VII to oral contraceptive use: an example of gene-environment interaction.

    Bloemenkamp KW, de Maat MP, Dersjant-Roorda MC, Helmerhorst FM and Kluft C

    Department of Obstetrics, Gynaecology and Reproductive Medicine, Leiden University Medical Center, Building 1, H-3-P, P.O. Box 9600, Leiden 2300 RC, The Netherlands.

    Elevated plasma level 1f40 s of factor VII and fibrinogen are risk factors for cardiovascular disease, especially arterial thrombosis. Oral contraceptive use increases factor VII and fibrinogen plasma levels. It has been described that DNA polymorphisms are associated with the plasma levels of hemostatic variables and their regulation. The R/Q353 polymorphism in the factor VII gene and the -455G/A polymorphism in the fibrinogen beta-gene are associated with plasma levels of factor VII and fibrinogen, respectively. We analysed data of a randomised study (n = 95) in which two types of oral contraceptives were compared with regard to their effect on factor VII and fibrinogen, in which we also determined R/Q353 and -455G/A polymorphisms. Women were allocated randomly to either receiving a monophasic oral contraceptive containing 75 micrograms of gestodene and 20 micrograms of ethinyl estradiol, or 150 micrograms of desogestrel and 20 micrograms of ethinyl estradiol. Blood was taken before treatment and after 3 and 6 months of oral contraceptive use. Factor VII and fibrinogen increased significantly after 3 and 6 months of oral contraceptive use; the increase in factor VII was higher in the desogestrel group than in the gestodene group at 3 and 6 months. For fibrinogen, there were no intergroup differences at 3 and 6 months. At baseline, an association between genotype and plasma factor VII and fibrinogen levels was observed. In multivariate analysis, the R/Q353 polymorphism and the type of oral contraceptive were determinants of the effect on the change in factor VII, with the highest increase in women carrying the Q allele and using the desogestrel-containing oral contraceptive, and the lowest increase in women with the RR genotype who use the gestodene-containing oral contraceptive. For fibrinogen, no interaction among type of oral contraceptive, -455G/A polymorphism, and change in plasma levels was observed. We conclude that an individual's genetic variation may contribute to the response of plasma factor VII to oral contraceptive use.

    Vascular pharmacology 2002;39;3;131-6

  • New insights into fibrin (ogen) structure and function.

    Everse SJ

    Dept. of Biochemistry, University of Vermont, Burlinton, VT, USA. stephen.everse@uvm.edu

    Vox sanguinis 2002;83 Suppl 1;375-82

  • Association of beta-fibrinogen and factor VII polymorphism with plasma fibrinogen and factor VII levels, and no association of PAI-1 polymorphism with plasma PAI-1 levels in hemodialysis patients.

    Ando R, Doi M, Yamauchi K, Chida Y, Ida T, Endo K, Yanagi H and S

    Department of Internal Medicine, Nakano General Hospital, Tokyo, Japan. rando@bd.mbn.or.jp

    Aims: Recent studies have stressed the roles of genetic factors on the plasma levels of hemostatic markers and on cardiovascular complications. We investigated the association of DNA polymorphisms for beta-fibrinogen, factor VII, and PAI-1 with plasma levels of these factors and with ischemic heart disease (IHD) and cerebral infarction (CI) in patients undergoing hemodialysis (HD).

    Methods: beta-fibrinogen G/A-455, factor VII R353Q and PAI-1 4G/5G polymorphisms were determined by PCR-RFLP in 149 HD patients and in 100 controls. The plasma levels of fibrinogen, factor VII and PAI-1 were also measured.

    Results: The allele frequencies and the genotype frequencies of these 3 polymorphisms were not different between HD patients and controls. In HD patients, plasma fibrinogen levels were significantly lower in the GG genotype than in the GA genotype, and plasma factor VII activity was significantly higher in the RR genotype than in the RQ genotype. Multiple regression analysis disclosed that CRP and beta-fibrinogen polymorphism were the significant determinants of fibrinogen levels. Plasma PAI-1 levels were not different among the 3 genotypes. The frequency of the A-455 allele was significantly higher in HD patients with CI than in those without CI, and the genotype distribution for beta-fibrinogen differed significantly between the 2 groups. Between the same 2 groups, however, significant differences were found neither in the frequency of the 353Q or 4G allele nor in the genotype distribution for factor VII and PAI-1. No significant differences in the frequency of the G-455, 353Q or 4G alleles, or in the genotype distribution for beta-fibrinogen, factor VII and PAI-1 were observed between patients with IHD and those without IHD. Multiple logistic regression analysis demonstrated that neither polymorphism was associated with CI or IHD.

    Conclusions: In HD patients, beta-fibrinogen and factor VII polymorphisms affected plasma levels of fibrinogen and factor VII, respectively. Beta-fibrinogen polymorphism was not an independent but a possible risk factor for CI in HD patients. Further study will be needed to confirm the precise role of 5-fibrinogen polymorphisms in the pathogenesis of CI in HD patients.

    Clinical nephrology 2002;58;1;25-32

  • Genetic determinants of the response to bezafibrate treatment in the lower extremity arterial disease event reduction (LEADER) trial.

    Jamshidi Y, Flavell DM, Hawe E, MacCallum PK, Meade TW and Humphries SE

    Centre for Cardiovascular Genetics, Department of Medicine, Royal Free and University College London Medical School, The Rayne Institute, London, UK.

    Genetic determinants of baseline levels and the fall in plasma triglyceride and fibrinogen levels in response to bezafibrate treatment were examined in 853 men taking part in the lower extremity arterial disease event reduction (LEADER) trial. Three polymorphisms in the peroxisome proliferator activated receptor alpha (PPARalpha) gene were investiga 1f40 ted (L162V, G>A in intron 2 and G>C in intron 7), two in the apolipoprotein CIII (APOC3) gene (-482C>T and -455T>C) and one in the beta-fibrinogen (FIBB) gene (-455G>A). The presence of diabetes (n=158) was associated with 15% higher triglyceride levels at baseline compared to non-diabetics (n=654) (P<0.05). Among the diabetic group, carriers of the PPARalpha intron 7 C allele had 20% lower triglyceride levels compared to homozygotes for the common G allele (P<0.05), with a similar (non-significant) trend for the L162V polymorphism, which is in linkage disequilibrium with the intron 7 polymorphism. For the APOC3 gene, carriers of the -482T allele had 13% lower baseline triglyceride levels compared to -482C homozygotes (P<0.02), but no effect was observed with the -455T>C substitution. In the non-diabetic patients, the PPARalpha V162 allele was significantly associated with 9% higher baseline triglyceride levels (P<0.03) and a similar, but non-significant trend was seen for the intron 7 polymorphism. Overall, triglyceride levels fell by 26% with 3 months of bezafibrate treatment, and current smokers showed a poorer response compared to ex/non-smokers (23% fall compared to 28% P=0.03), but none of the genotypes examined had a significant influence on the magnitude of response. Carriers of the -455A polymorphism of the FIBB gene had, as expected, marginally higher baseline fibrinogen levels, 3.43 versus 3.36 g/l (P=0.055), but this polymorphism did not affect response to treatment. Overall, fibrinogen levels fell by 12%, with patients with the highest baseline fibrinogen levels showing the greatest decrease in response to bezafibrate. For both the intron 2 and the L162V polymorphisms of the PPARalpha gene there was a significant interaction (both P<0.01) between genotype and baseline levels of fibrinogen on the response of fibrinogen levels to bezafibrate, such that individuals carrying the rare alleles in the lowest tertile showed essentially no overall decrease compared to a 0.18 g/l fall in homozygotes for the common allele. Thus while these genotypes are a minor determinant of baseline triglyceride and fibrinogen levels, there is little evidence from this study that the magnitude of response to bezafibrate treatment in men with peripheral vascular disease is determined by variation at these loci.

    Atherosclerosis 2002;163;1;183-92

  • [Genetics of blood coagulation in young stroke patients].

    Pongrácz E, Tordai A, Csornai M and Nagy Z

    BM Központi Kórház és Intézményei, Neurológiai Osztály, 1071 Budapest, Városligeti fasor 9-13.

    The classical risk factors did not explain all the possible etiology of cerebral stroke. Genetic polymorphisms responsible for thrombophilia were implicated recently as risk factors of stroke. In this genetico-epidemiological study the author's aim was to analyse the tendency of genetic polymorphisms to cluster in a cohort of young and elderly stroke patients and in healthy subjects in Hungary.

    Methods: 253 patients with stroke were compared with 173 healthy blood donors on the basis of genetic polymorphisms of platelet GP IIb/IIIa receptor (33 LeuPro), prothrombin gene G20210A, Factor V Leiden mutation, ACE I/D, methylenetetrahydrofolate reductase (MTHFR) and beta fibrinogen gene G455A. These data were acquired using PCR. Questionnaires were used to investigate the family history and to determine the risk factor profile. The subtypes of stroke were analysed in a stroke cohort grouped according to different polymorphisms.

    Results: An increased frequency of GP IIIa heterozygosity was found as compared to a West-European stroke cohort (31% versus 19%). The prothrombin gene variant (2.9% European and 4.8% in Hungary) was also found to increase in frequency. In young stroke patients (age < 50) compared with control subjects the odds ratios were higher: in prothrombin gene (OR: 4.9), in Leiden mutation (OR: 1.67), in fibrinogen gene (OR: 1.64) and in MTHFR(+/+) (OR: 1.58). Clustering of two polymorphisms could only be detected in young patients. These clustering polymorphisms were GP IIb/IIIa with prothrombin G20210A variant (OR: 6.74, 95% CI 1.1-18.2) and prothrombin gene variant with MTHFR (OR: 5.3, CI95 1.2-8.3).

    Conclusion: Selected and clustered genetic polymorphisms of haemostatic factors could be responsible for the high stroke morbidity in Central Europe. The presence and clustering tendency of these factors have been described in young stroke victims.

    Ideggyogyaszati szemle 2002;55;3-4;111-7

  • beta-fibrinogen gene -455A/G polymorphism and plasma fibrinogen level in Chinese stroke patients.

    Liu Y, Pan J, Wang S, Li X and Huang Y

    Department of Hematology, Peking Union Medical College Hospital, Beijing 100730, China.

    Objectives: To investigate the relationship between the beta-fibrinogen gene -455A/G polymorphism and plasma fibrinogen level and to determine the influence of the mutation on ischemic stroke.

    Methods: Ninety-one patients (63.5 +/- 10.1 years) with ischemic stroke and 74 elderly control subjects (60.6 +/- 10.8 years) without any thromboembolic events and 98 healthy blood donators as young control (37.5 +/- 13.3 years) were enrolled in this trial. The beta-fibrinogen gene -455A/G polymorphism was analyzed for all subjects by PCR-RFLP with the restrictive enzyme Hae III, while plasma fibrinogen levels were obtained from the prothrombin time (PT) assay. For statistical analysis, the parameters were compared between any two different groups by the unpaired Student's t test and the Chi-square test. Before analysis, log transformations for concentrations of fibrinogen were carried out.

    Results: H2 allele frequency was higher in male ischemic stroke patients than in the elderly control (22.7% vs 7.1%, chi(2) = 5.56, P < 0.02). There was no significant difference between the female groups. In those patients without any thromboembolic events (both elderly and young control groups), the frequency of H2 decreased with age (< or = 40, 21.3%; 41 - 59, 15.4%; and > or = 60, 10.2%). In the male elderly and young control groups, the level of plasma fibrinogen was lower in the H1H1 genotype (287 +/- 96 mg/dl and 234 +/- 58 mg/dl) than in H1H2 and H2H2 (331 +/- 44 mg/dl and 307 +/- 55 mg/dl; t = 2.53 and 9.67, P < 0.05). In the female elderly groups, this tendency was not found.

    Conclusion: Plasma fibrinogen expression is affected by the beta-fibrinogen gene -455A/G polymorphism, and the H2 allele may be a risk factor for ischemic stroke in Chinese males.

    Chinese medical journal 2002;115;2;214-6

  • [Genetic variations in plasminogen activator inhibitor-1 gene and beta fibrinogen gene associated with glomerular microthrombosis in lupus nephritis and the gene dosage effect].

    Gong R, Liu Z, Chen Z and Li L

    Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, 210002 P. R. China. zhihong@public1.ppt.js.cn

    Objective: To explore the relationship of plasminogen activator inhibitor-1 (PAI-1) gene -675 4G/5G and beta fibrinogen gene -455 G/A variations to glomerular microthrombosis(T) in lupus nephritis(LN).

    Methods: One hundred and one patients with biopsy proven LN were divided into two groups according to the presence or absence of glomerular microthrombus, i.e. group LN+T(n=46) and group LN-T(n=55). The genotypes of PAI-1 gene and beta fibrinogen gene were profiled by polymerase chain reaction-sequence length polymorphism (PCR-SLP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) respectively. Clinical baseline data at the time of renal biopsy were collected. Normal controls consisted of 128 unrelated healthy adults. The etiologic fractions (EF) were calculated for estimating the contribution of risk genotypes of the two candidate genes to an increase in susceptibility to glomerular microthrombosis in LN patients.

    Results: Both the 4G/4G genotype and the 4G allele of PAI-1 gene occurred more frequently in group LN+T (47.83% and 0.685) than in group LN-T (23.64% and 0.507)(P<0.05) and normal controls (28.13% and 0.570) (P<0.05). The PAI-1 4G/4G genotype was significantly associated with microthrombosis (OR=2.96, 95%CI:1.26-6.92). Besides, the prevalence of the genotypes carrying the A allele of beta fibrinogen gene, i.e. G/A and A/A, as well as the prevalence of the A allele per se, was increased in group LN+T (47.83% and 0.261) versus group LN-T (27.27% and 0.145)(P<0.05). LN patients carrying the A allele had a high risk of glomerular thrombosis(OR=2.44, 95%CI:0.98-5.59). In addition, the presence of the PAI-1 4G/4G genotype together with the A allele of the beta fibrinogen gene was found to be a greater risk factor (OR=4.5, 95%CI: 1.34-15.12) for glomerular thrombosis in LN than the 4G/4G genotype or the A allele alone. The pooled EF (45.98%) for the risk genotypes of both PAI-1 gene and beta fibrinogen gene was also higher than that for the risk genotypes of either gene (31.67% and 28.23%).

    Conclusion: The above findings indicated that genetic variations in PAI-1 and beta fibrinogen loci might represent risk factors for glomerular microthrombosis in LN. They may have synergetic impact and present gene dosage effect on the susceptibility to this pathological subphenotype.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2002;19;1;1-5

  • The effect of fibrinogen genotype on fibrinogen levels after strenuous physical exercise.

    Brull DJ, Dhamrait S, Moulding R, Rumley A, Lowe GD, MJ, Humphries SE and Montgomery HE

    Department of Medicine, Royal Free and UCL Medical School, UK. d.brull@ucl.ac.uk

    We have examined the effect of two beta-fibrinogen gene promoter polymorphisms (-455G>A and -854G>A) on the fibrinogen response to severe exercise in a group of male army recruits undergoing basic training. Fibrinogen was measured pre-training and again serially after severe 48 h final military exercise (FME). Out of 884 subjects, 762 completed training of whom 250 were selected for post-FME study. Fibrinogen levels (g/l) were significantly elevated over baseline levels 2, 48 and 96 h after FME, representing increases of 15.7%, 3.4% and 7.6% (p <0.005; p = 0.05 and p <0.005 respectively), with higher levels in -455A allele carriers than genotype -455GG: 3.17+/-0.05 vs. 2.94+/-0.05 (p <0.001), 2.86+/-0.05 vs. 2.60+/-0.05 (p <0.0005) and 2.98+/-0.06 vs. 2.69+/-0.06 (p <0.0005) at 2, 48 and 96 h respectively. There was no effect of the -854G>A polymorphism on fibrinogen, even after taking in 64c to account beta-fibrinogen -455 genotype. Thus the fibrinogen -455G>A polymorphism influences fibrinogen levels following exercise. The effect of genotype might be clinically relevant at times of hyperfibrinogenaemia such as following an acute inflammatory response.

    Thrombosis and haemostasis 2002;87;1;37-41

  • The contribution of genetic factors to thrombotic and bleeding outcomes in coronary patients randomised to IIb/IIIa antagonists.

    Shields DC, Fitzgerald AP, O'Neill PA, Muckian C, Kenny D, Moran B, Cannon CP, Byrne CE and Fitzgerald DJ

    Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin, Ireland. dshields@rcsi.ie

    Genetic variants are risk factors for coronary disease, but their role in recurrent events and in response to treatment is less clear. We genotyped genetic variants implicated in primary coronary disease in 924 Caucasians with acute coronary syndromes participating in the OPUS-TIMI16 trial of the GPIIb/IIIa antagonist orbofiban. These were the platelet glycoprotein (GP) receptors GPIIIa, GPIa, GPIbalpha; platelet ligands beta-fibrinogen and von Willebrand Factor (vWF); interleukins (IL) IL-1RN, and IL-6; adhesion proteins E-selectin and P-selectin; and metalloproteinase MMP-9. Cox modelling of all genetic variants demonstrated no significant impact on the composite endpoint (P = 0.88), which included myocardial infarction (MI), death, recurrent ischemia, urgent revascularisation and stroke 1f40 , but a significant impact on recurrent myocardial infarction alone (chi(2) = 20.4, 10 df, P = 0.04). There was a significant interaction of the polymorphisms with orbofiban treatment influencing bleeding outcomes (P = 0.004). Thus, genetic polymorphisms may be associated with subsequent myocardial infarction, and may also be associated with treatment-associated bleeding among coronary patients.

    The pharmacogenomics journal 2002;2;3;182-90

  • Multi-locus interactions predict risk for post-PTCA restenosis: an approach to the genetic analysis of common complex disease.

    Zee RY, Hoh J, Cheng S, Reynolds R, Grow MA, Silbergleit A, Walker K, Steiner L, Zangenberg G, Fernandez-Ortiz A, Macaya C, Pintor E, Fernandez-Cruz A, Ott J and Lindpainter K

    Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

    The complexity of recognizing the potential contribution of a number of possible predictors of complex disorders is increasingly challenging with the application of large-scale single nucleotide polymorphism (SNP) typing. In the search for putative genetic factors predisposing to coronary artery restenosis following balloon angioplasty, we determined genotypes for 94 SNPs representing 62 candidate genes, in a prospectively assembled cohort of 342 cases and 437 controls. Using a customized coupled-logistic regression procedure accounting for both additive and interactive effects, we identified seven SNPs in seven genes that, together, showed a statistically significant association with restenosis incidence (P <0.0001), accounting for 11.6% of overall variance observed. Among them are candidate genes for cardiovascular pathophysiology (apolipoprotein-species and NOS), inflammatory response (TNF receptor and CD14), and cell-cycle control (p53 and p53-associated protein). Our results emphasize the need to account for complex multi-gene influences and interactions when assessing the molecular pathology of multifactorial medical entities.

    Funded by: NHGRI NIH HHS: HG00008; NHLBI NIH HHS: K04-HL-03138-01

    The pharmacogenomics journal 2002;2;3;197-201

  • Mutation in the promoter region of the beta-fibrinogen gene and the risk of future myocardial infarction, stroke and venous thrombosis.

    Blake GJ, Schmitz C, Lindpaintner K and Ridker PM

    The Center for Cardiovascular Disease Prevention, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.

    Aim: Polymorphisms in the promoter region of the beta-fibrinogen gene are associated with increased plasma fibrinogen levels. We investigated whether the distribution of the C148T polymorphism is associated with an increase in cardiovascular risk.

    In a nested case-control design, the distribution of the C148T polymorphism was investigated among 751 participants in the Physicians' Health Study who subsequently developed myocardial infarction, stroke or venous thromboembolism (cases) and among 751 age- and smoking-matched controls over follow-up of 8.6 years. Frequency of the T allele was similar among men who had myocardial infarction (22.7%, P=0.5), stroke (18.4%, P=0.2) or venous thromboembolism (17.0%, P=0.1) compared with those with no cardiovascular events (21.5%). The relative risk for any vascular event among men homozygous or heterozygous for the T allele compared with men homozygous for the C allele was 0.94 (95% CI 0.76-1.16). We found no evidence of an association between the T allele and myocardial infarction (relative risk 1.06; 95% CI 0.82-1.36), stroke (0.87, 0.63-1.21) or venous thromboembolism (0.75; 0.51-1.08). Analysis adjusted for aspirin use and traditional cardiovascular risk factors had no significant effect on these findings.

    Conclusion: In a large prospective cohort, carriage of the T allele for the C148T mutation in the beta-fibrinogen promoter gene was not associated with an increased subsequent risk of cardiovascular events.

    Funded by: NHLBI NIH HHS: HL58755

    European heart journal 2001;22;24;2262-6

  • Genetic influences on lipid metabolism trait variability within the Stanislas Cohort.

    Pallaud C, Gueguen R, Sass C, Grow M, Cheng S, Siest G and Visvikis S

    Institut National de la Sante et de la Recherche Médicale U525, 2 Avenue du Doyen Jacques Parisot, 54501 Vandoeuvre-lès-Nancy, France-Université Henri Poincaré, Nancy I, 30 Rue Lionnois, 54000 Nancy, France.

    The contribution of 17 polymorphisms within 13 candidate genes on lipid trait variability was investigated by a multiplex assay in 772 men and 780 women coming for a health checkup examination. The studied genes were APOE, APOB, APOC3, CETP, LPL, PON, MTHFR, FGB, GpIIIa, SELE, ACE, and AGT. We found that APOB-Thr71Ile, APOE-(112/158), APOC3-1100C/T, and SELE-98G/T polymorphisms had a significant effect on lipid traits (P < or = 0.001 to P < or = 0.01). Genetic effects accounted for 3.5-5.7% of variation in apolipoprotein B (apoB)-related traits among men, and for 5.7-9.0% among women. The contribution of APOE polymorphism on apoB-related traits variability was two to three times more important in women than in men. We found suggestive evidence for interactive effects between genetics and age, smoking status, and oral contraceptives. Increase of LDL-cholesterol and apoB concentrations with age was stronger among the epsilon4 carriers in women, and apolipoprotein A-I (apoA-I) concentration decreased with age in epsilon4 male carriers. The effect of epsilon2 allele on LDL-cholesterol was more important in the oral contraceptive users. In nonsmokers only, the APOC3-1100C allele in women was related to lower apoB-related traits concentrations, and in men to higher apoA-I and HDL-cholesterol concentrations. In conclusion, this work, in addition to the reinforcement of the already known associations between APOB, APOE, and APOC3 genes and lipids, leads to new perspectives in the complex relationships among genes and environmental factors. The newly observed relationships between E-selectine gene and lipid concentrations support the hypotheses of multiple metabolic pathways contributing to the complexity of lipids variability.

    Journal of lipid research 2001;42;11;1879-90

  • The alternatively spliced alpha(E)C domain of human fibrinogen-420 is a novel ligand for leukocyte integrins alpha(M)beta(2) and alpha(X)beta(2).

    Lishko VK, Yakubenko VP, Hertzberg KM, Grieninger G and Ugarova TP

    Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.

    The interaction of human plasma fibrinogen with leukocyte integrins alpha(M)beta(2) (CD11b/CD18, Mac-1) and alpha(X)beta(2) (CD11c/CD18, p150,95) is an important component of the inflammatory response. Previously, it was demonstrated that binding of fibrinogen to these integrins is mediated by gammaC, the globular C-terminal domain of the gamma chain. In this study, evidence was found of another fibrinogen domain that can serve as a ligand for the 2 leukocyte integrins: alpha(E)C, a homologous domain that extends the alpha chains in a recently discovered subclass of fibrinogen known as fibrinogen-420. Recombinant alpha(E)C supported strong adhesion and migration of cells expressing alpha(M)beta(2) and alpha(X)beta(2), including nonactivated and activated U937 and THP-1 monocytoid cells, and neutrophils. Cells transfected with complementary DNA for these integrins also bound alpha(E)C. The specificity of interaction was substantiated by inhibition of cell adhesion with antibodies against alpha(M), alpha(X), and beta(2) subunits. Also, neutrophil inhibitory factor, a specific inhibitor of alpha(M)beta(2) and alpha(X)beta(2) function, efficiently blocked cell adhesion to alpha(E)C. In alpha(M)beta(2) and alpha(X)beta(2), the I domain is the binding site for alpha(E)C, since alpha(E)C bound to recombinant alpha(M) I and alpha(X)I domains in a dose-dependent and saturable manner. Synthetic peptides that duplicated sequences gamma190 to 202 and gamma377 to 395, previously considered putative binding sites in gammaC, effectively inhibited alpha(M)beta(2)- and alpha(X)beta(2)-mediated adhesion to alpha(E)C, suggesting that recognition of alpha(E)C by the I domain involves structural features in common with those of gammaC. These findings identify alpha(E)C as a second domain in fibrinogen-420 that binds alpha(M)beta(2) and alpha(X)beta(2) and can mediate leukocyte adhesion and migration.

    Funded by: NHLBI NIH HHS: HL-51050, HL-63199

    Blood 2001;98;8;2448-55

  • Fibrinogen genotypes (alpha and beta) are associated with plasma fibrinogen levels in Chinese.

    Liu Y, Saha N, Heng CK, Hong S and Low PS

    Journal of medical genetics 2001;38;9;E31

  • Variation of plasma D-dimer following surgery: implications for prediction of postoperative venous thromboembolism.

    Lippi G, Veraldi GF, Fraccaroli M, Manzato F, Cordiano C and Guidi G

    Istituto di Chimica e Microscopia Clinica, Dipartimento di Scienze Biomediche e Morfologiche, Università degli Studi di Verona, Ospedale Policlinico, Italy.

    The prognosis of venous thromboembolism is considerably influenced by an accurate and fast diagnosis. Although the role of D-dimer testing in the diagnosis of suspected venous thromboembolism is well established for outpatients, there is controversial evidence on the clinical usefulness of its measurement in surgical patients. In order to recognize patterns of variation of D-dimer following surgery and identify potential pitfalls in prediction of venous thromboembolic complications, plasma D-dimer was assayed in 30 patients undergoing major elective hip surgery and 20 patients undergoing laparoscopic cholecystectomy for acute cholecystitis. The postoperative variation of plasma D-dimer differed widely between the two subgroups. Patients undergoing laparoscopic cholecystectomy showed D-dimer concentrations persistently increased from the baseline to the 15th postoperative day, whereas patients undergoing hip surgery were characterized by a double peak, on the 1st and 7th postoperative days. Mean inter-individual daily coefficient of variations of plasma D-dimer throughout the postoperative period were 49% (range 39%-61%) for laparoscopic cholecystectomy and 101% (range 72%-156%) for orthopedic surgery. The markedly heterogeneous fluctuation of plasma D-dimer suggests that the postoperative activation of the hemostatic system depends on the type and time since surgery, thus limiting the clinical usefulness of D-dimer testing in the diagnostic approach to postoperative venous thromboembolism.

    Clinical and experimental medicine 2001;1;3;161-4

  • The impaired polymerization of fibrinogen Longmont (Bbeta166Arg-->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers.

    Lounes KC, Lefkowitz JB, Henschen-Edman AH, Coates AI, Hantgan RR and Lord ST

    Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7525, USA.

    This study identified a new substitution in the Bbeta chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site "a." Thus, the sites "A" and "a" can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unab 1f40 le to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg166-->Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils. (Blood. 2001;98:661-666)

    Funded by: NHLBI NIH HHS: HL-31048

    Blood 2001;98;3;661-6

  • [Effect of beta-Fibrinogen-455 Gene Polymorphism on Plasma Fibrinogen Levels in Patients with Ischemic Stroke]

    Li FQ, Liu GX, Yang ZG, Cai WW and Ling GX

    Research Laboratory of Blood Disease, The Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China.

    In large prospective studies, plasma fibrinogen levels have been shown to be an independent risk factor of vascular disease, including ischemic stroke. Elevated plasma fibrinogen in an individual could be due to the presence of predisposing genetic and/or environmental factors, such as smoking. Of the polymorphisms studies to date, the beta-fibrinogen-455 (beta-Fg-455) G-->A substitution in the 5' flanking region is associated with the most consistent difference in plasma fibrinogen levels in both case-control studies and in selected groups of healthy individuals. In order to further elucidate the role of the beta-Fg-455 G-->A substitution in determining fibrinogen levels and susceptibility to ischemic stroke in case-control population, including 104 individuals with verified ischemic stroke and 156 healthy individuals. Turbidimetriy assays were used to measure plasma fibrinogen levels of all samples. The beta-Fg-455 G-->A mutation was identified by the polymerase chain reaction followed by restriction enzyme digestion of the amplified DNA with HaeIII. The plasma fibrinogen level in patients with ischemic stroke [(3.51 +/- 1.09) g/L] was significantly higher than that in the control [(3.08 +/- 0.71) g/L] (P < 0.01). The A-allele is associated with elevated fibrinogen levels in both patients and controls. The plasma fibrinogen levels in controls with A-allele in elder people were higher than in younger people (P < 0.05). Those with A allele in males of ischemic stroke had significantly higher plasma fibrinogen levels in smokers than in non-smokers and ex-smokers (P < 0.05), but it was not significantly difference in subjects of GG genotype (P > 0.05). Our data demonstrates an association of the beta-Fg promoter A-455 allele with higher fibrinogen levels in the general population, and suggests that the A-allele may be a susceptible predictor of ischemic stroke, particularly in aging and smoking.

    Zhongguo shi yan xue ye xue za zhi 2001;9;2;165-168

  • Ethnic differences in fibrinogen levels: the role of environmental factors and the beta-fibrinogen gene.

    Cook DG, Cappuccio FP, Atkinson RW, Wicks PD, Chitolie A, Nakandakare ER, Sagnella GA and Humphries SE

    Department of Public Health Sciences, St. George's Hospital Medical School, London. d.cook@sghms.ac.uk

    Fibrinogen is a cardiovascular risk factor, but little is known about levels in ethnic groups that differ in their cardiovascular risk. Fibrinogen was measured in 479 Black individuals, 459 South Asian Indians, and 453 Whites aged 40-59 years living in south London, England, from March 1994 to July 1996. Genotype was determined at two sites in the promoter of the beta-fibrinogen gene (G-455-->A and C-148-->T). Plasma fibrinogen levels were lower in Blacks than in Whites by 0.22 g/liter (95% confidence interval (CI): 0.08, 0.36) in men and 0.11 g/liter (95% CI: -0.01, 0.23) in women. These b6f differences were not explained by measured environmental variables, including smoking, or by genotypes. The fibrinogen levels of South Asians were not consistently different from those of WHITES: The A-455 and T-148 alleles were less common in Blacks than in either Whites or South ASIANS: In Whites and South Asians, but not in Blacks, there was complete allelic association between the two variants. In Blacks, the T allele rather than the A allele was associated with higher fibrinogen levels. The average fibrinogen-raising effect of the T-148 allele across all ethnic groups was 0.14 g/liter (95% CI: 0.02, 0.26 g/liter) in women and 0.15 g/liter (95% CI: 0.03, 0.27 g/liter) in men. Low fibrinogen levels in Blacks may partly explain their lower risk of ischemic heart disease in the United KINGDOM:

    American journal of epidemiology 2001;153;8;799-806

  • Beta-fibrinogen gene -455G/A polymorphism and coronary heart disease incidence: the Atherosclerosis Risk in Communities (ARIC) Study.

    Folsom AR, Aleksic N, Ahn C, Boerwinkle E and Wu KK

    Division of Epidemiology, School of Public Health, University of Minnesota, Minneapolis, MN 55454-1015, USA.

    Purpose: The -455G/A (HaeIII) polymorphism of the beta-fibrinogen gene influences levels of plasma fibrinogen. We determined whether it influences risk of coronary heart disease.

    Methods: We conducted a case-cohort study nested within a prospective investigation, the Atherosclerosis Risk in Communities Study. We accumulated 398 incident coronary heart disease cases over a median of 5.3 years of follow-up and compared their -455G/A status with a random sample of the cohort (n = 498).

    Results: Plasma fibrinogen was higher (p = 0.04) in AA homozygous participants (341 mg/dL) than in persons carrying the G allele: GA (290 mg/dL), GG (298 mg/dL). However, there was no significant association between -455G/A and incident CHD.

    Conclusions: Although a small effect cannot be excluded, -455G/A does not appear to be an important genetic determinant of CHD.

    Funded by: NHLBI NIH HHS: N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, N01-HC-55022

    Annals of epidemiology 2001;11;3;166-70

  • Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion.

    Redman CM and Xia H

    Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67 Street, New York, NY 10021, USA. credman@nybc.org

    Plasma fibrinogen is synthesized primarily in hepatocytes and assembly of the three component chains (A alpha, B beta, and gamma) into its final form as a six-chain dimer (A alpha, B beta, gamma)2 occurs rapidly in the lumen of the endoplasmic reticulum (ER). Assembly takes place in a stepwise manner with single chains interacting with each other to form A alpha-gamma and B beta-gamma complexes. The two-chain complexes then acquire another chain to form half-molecules (A alpha, B beta, gamma)1, which in a final step are linked to form the six-chain (A alpha, B beta, gamma)2 complex. As with other secreted glycoproteins, N-linked glycosylation of B beta and gamma chains commences in the ER and is completed in Golgi organelles. Sulfation and phosphorylation occur at post-ER stages of the secretory process. Since some ER chaperones coisolate with nascent fibrinogen chains they have been implicated in assisting chain assembly. Studies with recombinant systems, using deletion and substitution mutants, indicate that initial chain assembly depends on hydrophobic interactions present in the C-terminal half of the coil-coil domains and that inter- and intra-disulfide bonds that stabilize fibrinogen are needed to complete chain assembly. Not all the chains that are synthesized are assembled into fibrinogen and the unassembled chains are not secreted. HepG2 cells contain surplus A alpha and gamma chains that accumulate as free gamma chains and as an A alpha-gamma complex. A alpha-gamma is degraded by lysosomes whereas the gamma chain is degraded by the proteasome-ubiquitin system. Studies with expression of single chains by COS cells confirm that gamma and B beta are hydrolyzed by proteasomes and indicate that A alpha is degraded partially both by lysosomes and proteasomes. The role of surplus chains in regulating fibrinogen assembly is not understood but overexpression of any one chain, elicited by transfection of HepG2 cells, results in the upregulation of the other two genes, increased fibrinogen synthesis and secretion, and maintenance of surplus intracellular A alpha and gamma chains. HepG2 cells, programmed in this manner to increase basal fibrinogen expression, have higher HMG-CoA reductase mRNA levels, enhanced cholesterol and cholesterol ester synthesis, and increased secretion of apolipoprotein B (apoB). Overexpression of basal levels of fibrinogen does not affect synthesis of other acute phase proteins. Enhanced secretion of apoB is due to diminished degradation of nascent apoB by proteasomes and not to increased expression. Increased secretion of apoB is associated with increased basal expression of fibrinogen and is not affected when fibrinogen expression is stimulated by interleukin-6. In HepG2 cells, a feedback mechanism exists and extracellular sterols specifically downregulate expression of the three fibrinogen genes. These studies link, at the cellular level, basal fibrinogen expression with lipid metabolism.

    Annals of the New York Academy of Sciences 2001;936;480-95

  • Molecular mechanisms of hypo- and afibrinogenemia.

    Brennan SO, Fellowes AP and George PM

    Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch Hospital, P.O. Box 151, Christchurch, New Zealand. steve.brennan@chmeds.ac.nz

    Point mutations responsible for hypo- and afibrinogenemia are yielding new insights into amino acid side chains involved in the molecular processing, assembly, secretion, and domain stability of fibrinogen. Reverse phase chromatography, isoelectric focussing, electrospray mass spectrometry, and tryptic peptide mass mapping have shown that chains with heterozygous mutations of gamma 284 Gly-->Arg, B beta 316 Asp-->Tyr and gamma 371 Thr-->Ile are absent from plasma fibrinogen. The nonexpression of these mutations appears to result from perturbation of the five-stranded beta sheet of the D domain. We propose that this is due to retention of the variant in the endoplasmic reticulum and that in turn this leads to hypofibrinogenemia. Other mutations effect intracellular proteolysis and chain assembly. For example the mutation, A alpha 20 Val-->Asp, makes the protein a substrate for furin, which removes the first 19 residues of the A alpha chain as the mature molecule transits the trans golgi complex. Transient expression of gamma 153 Cys-->Arg chains together with A alpha and B beta chains suggests this mutation might perturb chain assembly, and the incorporation of mutations of B beta 353 Leu-->Arg or B beta 400 Gly-->Asp into intracellular fibrinogen precludes its subsequent export from host cells expressing fibrinogen genes. The graded severity of the hypo- and afibrinogenemias associated with homozygous A alpha chain truncations suggest the absolute minimal requirement for molecular assembly is the formation of the C terminal disulfide ring of the coiled coil.

    Annals of the New York Academy of Sciences 2001;936;91-100

  • Mapping of a minimal apolipoprotein(a) interaction motif conserved in fibrin(ogen) beta - and gamma -chains.

    Klose R, Fresser F, Kochl S, Parson W, Kapetanopoulos A, Fruchart-Najib J, Baier G and Utermann G

    Institute for Medical Biology and Human Genetics, Universität Innsbruck, 6020 Innsbruck, Austria.

    Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated f 15b4 ibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.

    The Journal of biological chemistry 2000;275;49;38206-12

  • Venous thrombosis in relation to fibrinogen and factor VII genes among African-Americans.

    Austin H, Hooper WC, Lally C, Dilley A, Ellingsen D, Wideman C, Wenger NK, Rawlins P, Silva V and Evatt B

    Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA 30333, USA. haustin@sph.emory.edu

    We evaluated the relation between venous thrombosis and plasma fibrinogen levels, the HaeIII and BcI polymorphisms of the beta fibrinogen gene, and the MspI polymorphisms of the factor VII gene in a case-control study of African-Americans. The study included 91 venous thrombosis cases and 185 control subjects obtained from a hospital in Atlanta, Georgia. High plasma fibrinogen was associated with increased risk of venous thrombosis, but the finding was not statistically significant. There was little association between the HaeIII polymorphisms and the BclI polymorphisms and the risk of venous thrombosis. The prevalence of the M2/M2 genotype of the factor VII gene was higher among cases than controls, but the difference was not statistically significant. The prevalence of the HaeIII H2 allele and the BclI B2 allele of the beta fibrinogen gene, both of which have been associated with slightly higher levels of plasma fibrinogen in most studies, is considerably lower among African-Americans in this study than it is among Whites in the United States and among Northern Europeans. The study is limited by its small size. However, despite this limitation, it supports the belief that increased plasma fibrinogen levels are associated with increased venous thrombosis risk. The study also indicated that the HaeIII and the BclI polymorphisms of the beta fibrinogen gene and the MspI polymorphisms of the factor VII gene are not strong determinants of venous thrombosis.

    Journal of clinical epidemiology 2000;53;10;997-1001

  • [The study of beta-fibrinogen gene - 455 G/A, - 148 C/T, 448 G/A polymorphisms and their association with plasma fibrinogen levels].

    Yang Z, Li F, Liu G, Cai W and Ling G

    Objective: To analyze the frequency of beta-fibrinogen (beta-Fg) gene - 455 G/A, - 148 C/T, 448 G/A polymorphisms and their association with plasma fibrinogen levels in Han nationality in Guangdong Chinese.

    Methods: The beta-Fg gene - 455 G/A, - 148 C/T, 448 G/A polymorphisms of 156 individuals were analyzed by restriction fragment length polymorphism (RFLP). Plasma fibrinogen levels were determined by turbidimetry.

    Results: The frequencies of A(- 455), T(- 148) and A(448) allele were 0.276, 0.285 and 0.272, respectively. There were strong linkages of G(- 455), C(- 148) and G(448), and of A(- 455), T(- 148) and A(448), the correspondence was more than 97%. The plasma fibrinogen levels in the presence and absence of A(- 455) allele were (3.13 +/- 0.74) g/L and (2.89 +/- 0.57) g/L (P < 0.05); of T(- 148) allele were (3.12 +/- 0.73) g/L and (2.89 +/- 0.58) g/L (P < 0.05); and of A(448) were (3.13 +/- 0.74) g/L and (2.89 +/- 0.57) g/L (P < 0.05), respectively. The plasma fibrinogen levels of the three polymorphisms with the mutant gene are significantly higher than that in the wild type.

    Conclusion: The three polymorphisms loci are strong linkage disequilibrium. It suggests that beta-Fg gene - 455 G/A, - 148 C/T, 448 G/A polymorphisms are associated with plasma fibrinogen levels.

    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 2000;21;9;463-5

  • Fibrinogen polymorphisms are not associated with the risk of myocardial infarction.

    Doggen CJ, Bertina RM, Cats VM and Rosendaal FR

    Department of Clinical Epidemiology, Leiden University Medical Centre, The Netherlands.

    In the Study of Myocardial Infarctions Leiden, we investigated the prevalence of three polymorphisms in the alpha- and beta-fibrinogen genes among 560 patients with a myocardial infarction and 646 control subjects. Secondly, we studied the relationships between these polymorphisms and fibrinogen activity and antigen levels. The TaqI, HaeIII and BclI polymorphisms in the fibrinogen gene were not associated with myocardial infarction. As we found an association of the rare B2 allele with fibrinogen levels and a similar, but weak, effect for the rare H2 allele, we conclude that a genetic propensity to high fibrinogen levels does not affect the risk of myocardial infarction. This is evidence against a causal role for fibrinogen levels in the aetiology of myocardial infarction.

    British journal of haematology 2000;110;4;935-8

  • [beta-fibrinogen gene -455A/G polymorphism and plasma fibrinogen level in Chinese stroke patients].

    Liu Y, Pan J and Wang S

    Department of Hematology, Peking Union Medical College Hospital. Beijing 100730, China.

    Objective: To explore the relationship between beta-fibrinogen gene -455A/G polymorphism and plasma fibrinogen level and to determine the influence of the mutation on ischaemic stroke.

    Methods: Ninety-one patients (63.5 +/- 10.1 years) with ischaemic stroke, 74 control patients (60.6 +/- 10.8 years) without any thromboembolic events and 98 healthy blood donors (37.5 +/- 13.3 years) were enrolled in this trial. beta-fibrinogen gene -455A/G polymorphism was analyzed from all subjects by polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP) with restrictive enzyme Hae III. Plasma fibrinogen levels were obtained from prothrombin time (PT) assay. The parameters were compared between any two different groups by unpaired t-test and Chi-square test. Before the analysis, log transformation was carried out for concentrations of fibrinogen.

    Results: Frequency of H2 allele was higher in male patients with ischaemic stroke than in control patients (22.7% vs 7.1%, chi(2) = 5.56, P < 0.02) 105b . There was no significant difference in female patients. In the subjects without any thromboembolic events (including control patients and healthy blood donors), frequency of H2 decreased with age (>/= 40, 21.3%; 41 approximately 59, 15.4%; >/= 60, 10.0%). Among male control patients and healthy blood donors, the level of plasma fibrinogen was lower in the H1H1 genotype (287 g/L +/- 96 g/L and 234 g/L +/- 58 g/L) than in H1H2 and H2H2 genotypes (331 g/L +/- 44 g/L and 307 g/L +/- 55 g/L; t = 2.53 and 9.67, P < 0.05); among females, this tendency was not found.

    Conclusion: Plasma fibrinogen expression is affected by the beta-fibrinogen gene - 455A/G polymorphism. H2 allele is a risk factor for ischaemic stroke in Chinese men.

    Zhonghua yi xue za zhi 2000;80;5;336-8

  • Fibrinogen assembly, secretion, and deposition into extracellular matrix by MCF-7 human breast carcinoma cells.

    Rybarczyk BJ and Simpson-Haidaris PJ

    Department of Pathology, University of Rochester School of Medicine and Dentistry, New York 14642, USA.

    A hallmark of breast carcinoma is the deposition of fibrinogen (FBG) without subsequent conversion to fibrin in the tumor stroma. In this study, the ability of the MCF-7 human breast cancer epithelial cell line to synthesize, secrete, and deposit FBG into the extracellular matrix (ECM) was examined. Whereas MCF-7 cells produced low levels of intact FBG, abundant levels of FBG intermediate complexes or degraded Aalpha, Bbeta, and gamma chain polypeptides were observed. Most of the Bbeta chain was degraded and missing an NH2-terminal peptide fragment. Reverse transcription-PCR analysis indicated that only gamma chain mRNA was present in detectable steady-state levels, although Southern hybridization revealed that the FBG Aalpha, Bbeta, and gamma chain genes were intact in MCF-7 cells. Immunostaining showed that extracellular FBG was bound to the surface of MCF-7 cells in a punctate pattern, reminiscent of receptor binding, rather than a fibrillar pattern characteristic of mature ECM. A similar punctate pattern of staining was observed when MCF-7 FBG was added to fibroblasts that normally assemble exogenous FBG into an extensive, fibrillar ECM, suggesting that MCF-7 cells are defective in assembly of a fibrillar ECM. The loss of FBG Bbeta chain NH2-terminal peptides may contribute to the lack of intact FBG assembly in MCF-7 cells, which may further affect its ability to assemble FBG into a fibrillar ECM. Taken together, the data suggest that endogenous synthesis and secretion of FBG is, at least in part, the source of FBG deposition in the ECM of breast cell carcinomas.

    Funded by: NHLBI NIH HHS: HL30616, HL50615

    Cancer research 2000;60;7;2033-9

  • Missense mutations in the human beta fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion.

    Duga S, Asselta R, Santagostino E, Zeinali S, Simonic T, Malcovati M, Mannucci PM and Tenchini ML

    Department of Biology and Genetics for Medical Sciences, the Institute of Veterinary Physiology and Biochemistry, the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, University of Milan, Italy.

    Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen Aalpha-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bbeta-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bbeta fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion. (Blood. 2000;95:1336-1341)

    Blood 2000;95;4;1336-41

  • Signaling through platelet integrin alpha IIb beta 3: inside-out, outside-in, and sideways.

    Shattil SJ

    Department of Vascular Biology, Scripps Research Institute, La Jolla, CA 92037, USA. shattil@scripps.edu

    Thrombosis and haemostasis 1999;82;2;318-25

  • Characterization of single-nucleotide polymorphisms in coding regions of human genes.

    Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, Shaw N, Lane CR, Lim EP, Kalyanaraman N, Nemesh J, Ziaugra L, Friedland L, Rolfe A, Warrington J, Lipshutz R, Daley GQ and Lander ES

    Whitehead Institute/MIT Center for Genome Research, Cambridge, Massachusetts 02139, USA. lander@genome.wi.mit.edu

    A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.

    Nature genetics 1999;22;3;231-8

  • Fibrinogen.

    Herrick S, Blanc-Brude O, Gray A and Laurent G

    PfizerCentral Research, Sandwich, Kent, UK. s.herrick@ucl.ac.uk

    Fibrinogen is a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, fibrinogen is cleaved by thrombin to form fibrin which is the most abundant component of blood clots. As well as controlling blood loss at sites of tissue damage, other properties of fibrinogen have recently been discovered. For example, various cleavage products of fibrinogen and fibrin, released during coagulation and fibrinolysis, respectively, regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types including fibroblasts, endothelial and smooth muscle cells. Current research aims to define the bioactive fibrinogen molecule moieties and cellular receptors involved in these processes. Future studies may provide us with new opportunities to develop agents which are useful in promoting tissue repair or conversely in inhibiting fibrosis in inflammatory and fibroproliferative diseases where endothelial cell damage or chronic leakage of blood proteins is a feature.

    The international journal of biochemistry & cell biology 1999;31;7;741-6

  • Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide.

    Everse SJ, Spraggon G, Veerapandian L and Doolittle RF

    Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0634, USA.

    The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

    Funded by: NHLBI NIH HHS: HL-26873

    Biochemistry 1999;38;10;2941-6

  • Formation of the human fibrinogen subclass fib420: disulfide bonds and glycosylation in its unique (alphaE chain) domains.

    Fu Y, Zhang JZ, Redman CM and Grieninger G

    Lindsley F. Kimball Research Institute of the New York Blood Center, New York, NY, USA.

    COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel alphaE chain and those of the more abundant subclass whose alpha chains lack alphaE's globular C-terminus. That region, referred to as the alphaEC domain, is closely related to the ends of beta and gamma chains of fibrinogen (betaC and gammaC). Transfection of COS cells with alphaE, beta, and gamma cDNAs alone results in secretion of the symmetrical molecule (alphaEbetagamma)2, also known as Fib420. Cotransfection with cDNA for the shorter alpha chain yielded secretion of both (alphabetagamma)2 and (alphaEbetagamma)2 but no mixed molecules of the structure alphaalphaE(betagamma)2. Exploiting the COS cells' fidelity with regard to Fib420 production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the alphaE chain. No evidence from Cys --> Ser replacements was found for interchain disulfide bridges involving the four cysteines of the alphaEC domain. However, for fibrinogen secretion, the alphaE, beta, and gamma subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dissimilar structural roles in the process of fibrinogen assembly.

    Funded by: NHLBI NIH HHS: HL 37457, HL 51050

    Blood 1998;92;9;3302-8

  • Crystal structure of fragment double-D from human fibrin with two different bound ligands.

    Everse SJ, Spraggon G, Veerapandian L, Riley M and Doolittle RF

    Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634, USA.

    Factor XIII-cross-linked fragment D (double-D) from human fibrin was crystallized in the presence of two different peptide ligands and the X-ray structure determined at 2.3 A. The peptide Gly-Pro-Arg-Pro-amide, which is an analogue of the knob exposed by the thrombin-catalyzed removal of fibrinopeptide A, was found to reside in the gamma-chain holes, and the peptide Gly-His-Arg-Pro-amide, which corresponds to the beta-chain knob, was found in the homologous beta-chain holes. The structure shows for the first time that the beta-chain knob does indeed bind to a homologous hole on the beta-chain. The gamma- and beta-chain holes are structurally very similar, and it is remarkable they are able to distinguish between these two peptides that differ by a single amino acid. Additionally, we have found that the beta-chain domain, like its gamma-chain counterpart, binds calcium.

    Funded by: NHLBI NIH HHS: HL-26873

    Biochemistry 1998;37;24

  • Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin.

    Spraggon G, Everse SJ and Doolittle RF

    Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0634, USA.

    In blood coagulation, units of the protein fibrinogen pack together to form a fibrin clot, but a crystal structure for fibrinogen is needed to understand how this is achieved. The structure of a core fragment (fragment D) from human fibrinogen has now been determined to 2.9 A resolution. The 86K three-chained structure consists of a coiled-coil region and two homologous globular entitles oriented at approximately 130 degrees to each other. Additionally, the covalently bound dimer of fragment D, known as 'double-D', was isolated from human fibrin, crystallized in the presence of a Gly-Pro-Arg-Pro-amide peptide ligand, which simulates the donor polymerization site, and its structure solved by molecular replacement with the model of fragment D.

    Nature 1997;389;6650;455-62

  • Polarized secretion of fibrinogen by lung epithelial cells.

    Guadiz G, Sporn LA, Goss RA, Lawrence SO, Marder VJ and Simpson-Haidaris PJ

    Department of Medicine-Vascular Medicine Unit, University of Rochester School of Medicine and Dentistry, New York.

    The lung epithelium has recently been identified as a novel site of fibrinogen (FBG) biosynthesis. A coordinated upregulation of A alpha, B beta, and gamma chain FBG gene transcription occurs upon stimulation of A549 lung epithelial cells with dexamethasone (DEX) and the proinflammatory mediator interleukin-6 (IL-6). Subsequently, the cells synthesize and secrete fully assembled FBG. This study addresses the polarity of such FBG secretion by A549 cells cultured on polycarbonate membrane filters. After induction with IL-6 and DEX, cells were metabolically labeled, and FBG was immunopurified from the apical and 1f40 basolateral chambers. Analysis by gel electrophoresis revealed that A549 cells secreted newly synthesized FBG in a polarized manner, with the majority (80%) of FBG secreted basolaterally. Consistent with this observation, immunoelectron microscopy using Protein A-gold labeling showed FBG within secretory vesicles in close proximity to the basolateral aspect of the A549 cell membrane. Polarized secretion was microtubule-dependent since depolymerization using colchicine significantly reduced the basolateral component of secretion, causing intracellular retention of FBG. These data provide evidence that FBG is secreted by lung alveolar epithelial cells vectorially toward the basement membrane, which may reflect in vivo processes associated with local injury, inflammation, and repair mechanisms.

    Funded by: NHLBI NIH HHS: HL30616, HL50615; NIAID NIH HHS: AI07362

    American journal of respiratory cell and molecular biology 1997;17;1;60-9

  • Induction of fibrinogen biosynthesis and secretion from cultured pulmonary epithelial cells.

    Haidaris PJ

    Department of Medicine/Hematology Unit, University of Rochester School of Medicine and Dentistry, NY 14642, USA.

    Although the liver is the primary site of fibrinogen (FBG) synthesis, epithelial cells from diverse tissues have been shown to express one or more of the FBG A alpha, B beta, and gamma chain genes. In contrast, marrow megakaryocytes, which store FBG in the alpha-granules, are thought not to express the FBG genes. Our earlier studies have shown that epithelial cells in a variety of extrahepatic tissues express the gamma chain gene ubiquitously, while the mRNAs for the A alpha and B beta chain genes are essentially undetectable. During systemic inflammation, the liver secretes increased levels of FBG into the circulation, and lung epithelium responds to local inflammation during pulmonary infection by increased transcription of the gamma-FBG gene. Therefore, to determine whether extrahepatic epithelial cells express the A alpha, B beta, and gamma chain genes in response to proinflammatory mediators, cultured lung epithelial cells were treated with interleukin-6 (IL-6) and dexamethasone (DEX). Northern blot analysis demonstrated that the levels of gamma-FBG mRNA in cultured lung (A549) and liver (HepG2) epithelial cells increased 2- to 10-fold in response to treatment. Reverse-transcriptase-polymerase chain reaction amplification demonstrated increased accumulation of steady state levels of FBG A alpha, B beta, and gamma chain mRNAs in lung epithelial cells after treatment. The basal level of lung cell gamma-FBG gene transcription was not accompanied by appreciable levels of A alpha and B beta chain gene transcription; however, nuclear run-on analysis suggested that the increase in lung cell FBG mRNAs in response to DEX +/- IL-6 was due to new transcription. Furthermore, stimulation of lung epithelial cells with IL-6 + DEX resulted in maximal secretion of intact FBG (340 kD) composed of the characteristic A alpha, B beta, and gamma chain polypeptides. The data suggest that basal expression of the gamma-FBG gene in extrahepatic tissue occurs ubiquitously in the absence of detectable levels of A alpha- and B beta-FBG gene expression, which are then upregulated on induction of an inflammatory response. This would result in local synthesis and secretion of FBG in the injured tissue, supporting the hypothesis that production of FBG by extrahepatic epithelial cells in response to inflammation plays a role in wound repair.

    Funded by: NHLBI NIH HHS: HL 30616, HL 50615

    Blood 1997;89;3;873-82

  • The role of betagamma and alphagamma complexes in the assembly of human fibrinogen.

    Huang S, Cao Z, Chung DW and Davie EW

    Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350, USA.

    The role of alphagamma and betagamma dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing beta and gamma chains and generating betagamma complexes were fused with cells expressing only the alpha chain. Likewise, when cells co-expressing alpha and gamma chains and generating alphagamma complexes were fused with cells expressing only the beta chain, fibrinogen was also formed and secreted. The relative amounts of alphagamma or betagamma intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the beta chain was limiting, the alphagamma dimer was the predominant intermediate; likewise, when the alpha chain was limiting, the betagamma complex was the predominant intermediate. The incorporation of preformed alphagamma and betagamma complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of alphagamma and betagamma complexes as functional intermediates in the assembly of fibrinogen.

    Funded by: NHLBI NIH HHS: HL16919

    The Journal of biological chemistry 1996;271;44;27942-7

  • Regulation of fibrinogen biosynthesis by cytokines, consequences on the vascular risk.

    Vasse M, Paysant J, Soria J, Collet JP, Vannier JP and Soria C

    DIFEMA, Medical University of Rouen., St Etienne du Rouvray, France.

    High level of fibrinogen in plasma is recognised as an important vascular risk factor. However, it is not known if the increase in fibrinogen is directly responsible for the vascular risk or is a marker of vascular inflammation. Our data strengthen the hypothesis that the fibrinogen level is a marker of vascular disease, since a parallel effect of cytokines on fibrinogen biosynthesis and on vascular injury was noted. Among the cytokines which induce the synthesis of fibrinogen, oncostatin M (OSM) is the most potent cytokine synthesised by activated monocytes for inducing fibrinogen synthesis by Hep G2 cells (human hepatoma cell line). Interestingly at the same concentrations needed for fibrinogen biosynthesis, OSM induces smooth muscle cell proliferation. In contrast, the cytokines IL-4, IL-10 and IL-13 which have a protective effect against vascular injury leading to atherosclerosis, dose dependently down regulate the biosynthesis of fibrinogen. This was due to both a decrease of IL-6 induced fibrinogen synthesis by hepatocytes, evidenced by a decrease in fibrinogen secretion in the medium and beta chain mRNA expression and to an inhibition of production of the hepatocyte-stimulating activity for fibrinogen biosynthesis (HSF) by LPS-activated monocytes. Noteworthingly, IL-10 induces a significant decrease of the production of OSM by LPS-activated monocytes. In situ activation of monocytes by cytokines in the vessel wall could also contribute to the deposition of fibrin(ogen) derivatives, identified as pathogenic factor.

    Haemostasis 1996;26 Suppl 4;331-9

  • Generation and analysis of 280,000 human expressed sequence tags.

    Hillier LD, Lennon G, Becker M, Bonaldo MF, Chiapelli B, Chissoe S, Dietrich N, DuBuque T, Favello A, Gish W, Hawkins M, Hultman M, Kucaba T, Lacy M, Le M, Le N, Mardis E, Moore B, Morris M, Parsons J, Prange C, Rifkin L, Rohlfing T, Schellenberg K, Bento Soares M, Tan F, Thierry-Meg J, Trevaskis E, Underwood K, Wohldman P, Waterston R, Wilson R and Marra M

    Genome Sequencing Center, Washington University School of Medicine, St. Louis, Missouri 63108, USA. lhillier@watson.wustl.edu

    We report the generation of 319,311 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' and 3' ends of 194,031 human cDNA clones. Our goal has been to obtain tag sequences from many different genes and to deposit these in the publicly accessible Data Base for Expressed Sequence Tags. Highly efficient automatic screening of the data allows deposition of the annotated sequences without delay. Sequences have been generated from 26 oligo(dT) primed directionally cloned libraries, of which 18 were normalized. The libraries were constructed using mRNA isolated from 17 different tissues representing three developmental states. Comparisons of a subset of our data with nonredundant human mRNA and protein data bases show that the ESTs represent many known sequences and contain many that are novel. Analysis of protein families using Hidden Markov Models confirms this observation and supports the contention that although normalization reduces significantly the relative abundance of redundant cDNA clones, it does not result in the complete removal of members of gene families.

    Genome research 1996;6;9;807-28

  • Glucose-dependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro.

    Morohoshi M, Fujisawa K, Uchimura I and Numano F

    Third Department of Internal Medicine, Tokyo Medical and Dental University, Japan.

    To clarify the mechanisms that cause elevation of plasma fibrinogen levels in diabetes, we first examined the effect of hyperglycemia on the production of interleukin 6 (IL-6) and tumor necrosis factor (TNF) by cultured human peripheral blood monocytes. Monocyte-enriched fractions isolated from 20 healthy volunteers were incubated with 11 mmol/l glucose, 33 mmol/l glucose, or mannitol as an osmolar control for 6 or 24 h. After 6 h of incubation, IL-6 and TNF-alpha mRNA levels were analyzed by reverse transcription and polymerase chain reaction. In addition, after 24 h of incubation, IL-6 and TNF-alpha immunoreactivity in the culture medium was measured by enzyme-linked immunoassay. Both IL-6 and TNF-alpha mRNA levels and immunoreactivity were significantly increased by treatment with 33 mmol/l glucose compared with treatment with 11 mmol/l glucose or 11 mmol/l glucose with 22 mmol/l mannitol. In addition, preincubation of the cells with an anti-TNF monoclonal antibody (mAb) blocked the stimulatory effect of 33 mmol/l glucose on IL-6 synthesis and secretion. Second, we examined the ability of conditioned media from human peripheral blood monocytes to stimulate beta-fibrinogen mRNA synthesis in HepG2 cells. The conditioned medium from monocytes treated with 33 mmol/l glucose increased beta-fibrinogen mRNA levels. The results of this study demonstrate that hyperglycemia stimulated IL-6 and TNF synthesis and secretion by human peripheral monocytes in vitro and that the IL-6 response to hyperglycemia may be mediated by TNF. Furthermore, hyperglycemia may increase fibrinogen levels through stimulation of peripheral monocytes. These results suggest that hyperglycemia may cause hyperfibrinogenemia in diabetic patients through an IL-6-dependent and TNF-dependent mechanism.

    Diabetes 1996;45;7;954-9

  • The mitogenic effects of the B beta chain of fibrinogen are mediated through cell surface calreticulin.

    Gray AJ, Park PW, Broekelmann TJ, Laurent GJ, Reeves JT, Stenmark KR and Mecham RP

    University College London Medical School, Division of Cardiopulmonary Biochemistry, United Kingdom.

    We have previously shown that soluble partially degraded fibrin(ogen) remains in solution after fibrin clot formation and is a potent fibroblast mitogen (Gray, A.J., Bishop, J.E., Reeves J.T., Mecham, R.P., and Laurent, G.J. (1995) Am. J. Cell Mol. Biol. 12, 684-690). Mitogenic sites within the fibrin(ogen) molecule are located on the A alpha and B beta chains of the protein (Gray, A.J., Bishop, J. E., Reeves, J.T., and Laurent, G.J. (1993) J. Cell Sci. 104, 409-413). However, receptor pathways through which mitogenic effects are mediated are unknown. The present study sought to determine the nature of fibrin(ogen) receptors expressed on human fibroblasts which interact with the fibrinogen B beta chain. Receptor complexes were isolated from 125I-surface-labeled fibroblasts and purified on a fibrinogen B beta chain affinity column. Subsequent high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis analysis indicated fibrinogen B beta chain bound specifically to a 60-kDa surface protein. Sequence analysis of the amino terminus of this protein indicated 100% homology to human calreticulin. Immunoprecipitation experiments employing a polyclonal anti-calreticulin antibody provided further evidence that the 60-kDa protein isolated in this study was calreticulin. Further, polyclonal antibodies to human calreticulin significantly inhibited the mitogenic activity of fibrinogen B beta chain on human fibroblasts. The present study has shown that cell surface calreticulin binds to the B beta chain of fibrinogen mediating its mitogenic activity.

    Funded by: Wellcome Trust

    The Journal of biological chemistry 1995;270;44;26602-6

  • Retinoids stimulate fibrinogen production both in vitro (hepatocytes) and in vivo. Induction requires activation of the retinoid X receptor.

    Nicodeme E, Nicaud M and Issandou M

    Laboratoires Glaxo, Centre de Recherches, ZA de Courtaboeuf, Les Ulis, France.

    The in vitro effects of retinoids on fibrinogen synthesis were investigated in HepG2 cells and primary human hepatocytes. In vivo effects were studied in the rat. In HepG2 cells, maximal stimulation (twofold) of fibrinogen secretion was obtained when cells were incubated in the presence of 1 mumol/L all-trans retinoic acid (T-RA) for 24 hours. A comparable increas 17d5 e was observed for both de novo fibrinogen synthesis and fibrinogen beta chain mRNA level. In primary cultures of human hepatocytes, treatment with 1 mumol/L T-RA for 72 hours also gave a twofold increase in fibrinogen production. Furthermore, rats treated for 6 days with 100 mg.kg-1.d-1 T-RA presented increased fibrinogen plasma levels (110%). A selective retinoic X receptor (RXR) agonist, 4-[1-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-ethenyl]benzoi c acid (3-methyl TTNEB), as well as 9-cis retinoic acid, a natural RXR ligand, mimicked the effects of T-RA on fibrinogen synthesis in vitro at lower concentrations. In contrast, a selective retinoic A receptor alpha (RAR alpha) agonist was a poor activator. The ED50 of the different retinoids on fibrinogen secretion by HepG2 cells was 25 nmol/L for T-RA, 4 nmol/L for 9-cis retinoic acid, 11 nmol/L for the synthetic RXR agonist, and > 500 nmol/L for the RAR alpha agonist. However, incubation of HepG2 cells with RXR agonist together with RAR alpha agonist resulted in a further increase in fibrinogen production. The secretion of two other acute-phase proteins, alpha-antichymotrypsin and caeruloplasmin, was also stimulated by retinoids in HepG2 cells but by a different regulatory mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

    Arteriosclerosis, thrombosis, and vascular biology 1995;15;10;1660-7

  • The interaction of fibulin-1 with fibrinogen. A potential role in hemostasis and thrombosis.

    Tran H, Tanaka A, Litvinovich SV, Medved LV, Haudenschild CC and Argraves WS

    Department of Biochemistry, J. H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.

    The fibulins are an emerging family of extracellular matrix and blood proteins presently having two members designated fibulin-1 and -2. Fibulin-1 is the predominant fibulin in blood, present at a concentration of 30-40 micrograms/ml (approximately 1000-fold higher than fibulin-2). During the course of isolating fibulin-1 from plasma by immunoaffinity chromatography, a 340-kDa polypeptide was consistently found to co-purify. This protein was identified as fibrinogen (Fg) based on its electrophoretic behavior and reactivity with Fg monoclonal antibodies. Radioiodinated fibulin-1 was shown to bind to Fg transferred onto nitrocellulose filters after SDS-polyacrylamide gel electrophoresis. In enzyme-linked immunosorbent assay, fibulin-1 bound to Fg (and fibrin) adsorbed onto microtiter well plastic, and conversely, Fg bound to fibulin-1-coated wells. The binding of Fg to fibulin-1 was also observed in surface plasmon resonance assays, and a dissociation constant (Kd) of 2.9 +/- 1.6 microM was derived. In addition, fluorescence anisotropy experiments demonstrated that the interaction was also able to occur in fluid phase, which suggests that complexes of fibulin-1 and Fg could exist in the blood. To localize the portion of Fg that is responsible for interacting with fibulin-1, proteolytic fragments of Fg were evaluated for their ability to promote fibulin-1 binding. Fragments containing the carboxyl-terminal region of the Bbeta chain (residues 216-468) were able to bind to fibulin-1. In addition, it was found that fibulin-1 was able to incorporate into fibrin clots formed in vitro and was immunologically detected within newly formed fibrin-containing thrombi associated with human atherectomy specimens. The interaction between fibulin-1 and Fg highlights potential new roles for fibulin-1 in hemostasis as well as thrombosis.

    Funded by: NIGMS NIH HHS: GM42912

    The Journal of biological chemistry 1995;270;33;19458-64

  • Urinary protein C inhibitor binding region in the B beta-chain of human fibrinogen.

    Hayashi S

    Thrombosis Chemical Institute, Tokyo, Japan.

    The urinary protein C inhibitor (PCI) binding region in the B beta-chain of human fibrinogen was examined by ligand blotting, reverse-phase HPLC and amino acid sequencing. The B beta-chain, isolated from reduced and pyridylethylated fibrinogen, was digested with staphylococcal V8 protease to yield eight peptides consisting of 10, 12, 13, 13.5, 14, 16, 17 and 18 kDa bands and the cleaved peptides were ligand-blotted. The 12 kDa band bound to urinary PCI. Moreover, the digested B beta-chain was isolated by reverse-HPLC and the elution peak showing positive binding to urinary PCI was sequenced. The N-terminal amino acid sequence was A V S Q T, which corresponds to Ala106-Thr110 in the B beta-chain. Judging from the M(r) of this peptide (12 kDa), it comprises the region from Ala106 to Glu192-Glu210 which are partly located in the D-region. It is concluded that the urinary PCI-binding region to the B beta-chain resides in the sequence of Ala110 to Glu192-Glu210.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 1993;4;6;921-6

  • Binding of urinary protein C inhibitor to fibrin(ogen) and its binding mechanism.

    Hayashi S and Yamada K

    Thrombosis Chemical Institute, Tokyo, Japan.

    The region and mechanism of urinary protein C inhibitor (PCI) binding to fibrin(ogen) were examined using fibrin(ogen)-Sepharose and ligand blotting. Urinary PCI bound to fibrin(ogen)-Sepharose in a heparin-dependent manner at a level about 1.6-fold higher to fibrin-Sepharose than to fibrinogen-Sepharose. Scatchard analysis of the binding between urinary PCI and fibrin(ogen)-Sepharose showed that the Kd for fibrin-Sepharose and fibrinogen-Sepharose were 4.0 nM and 5.7 nM respectively. Ligand blotting using urinary PCI and an enzyme-linked immunoassay showed that urinary PCI bound to fibrinogen, fibrinogen degradation products (X, Y, D and E) and the A alpha-, B beta- and gamma-chains of fibrinogen. Binding between urinary PCI and fibrin(ogen)-Sepharose was slightly suppressed (16%) by alpha-methylmannose and largely suppressed (42%) by EACA, which indicates that the carbohydrate chain and the lysine binding site participate in the binding. These findings suggest that urinary PCI binds to fibrin(ogen) via the A alpha-, B beta- and gamma-chains and its binding is partly mediated by carbohydrate and the lysine binding site.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 1993;4;1;153-8

  • Molecular basis of fibrinogen Naples associated with defective thrombin binding and thrombophilia. Homozygous substitution of B beta 68 Ala----Thr.

    Koopman J, Haverkate F, Lord ST, Grimbergen J and Mannucci PM

    University Hospital, Leiden, The Netherlands.

    In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G----A) in the B beta chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the B beta chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human alpha-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound less than 10% of active site inhibited alpha-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound approximately 50% of alpha-thrombin. These results suggest that the mutation of B beta Ala 68----Thr affects the binding of alpha-thrombin to fibrin, and that defective binding results in a decreased release of FpA and FpB in both homozygous and heterozygous abnormal fibrinogens.

    Funded by: NHLBI NIH HHS: HL-31048

    The Journal of clinical investigation 1992;90;1;238-44

  • Potential role of entactin in hemostasis. Specific interaction of entactin with fibrinogen A alpha and B beta chains.

    Wu C and Chung AE

    Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.

    The interactions between the endothelial basement membrane, platelets, and coagulation factors play essential roles in hemostasis. Entactin is an integral and ubiquitous component of the basement membrane. Experiments were designed to study the interactions between entactin and blood coagulation factors. We have demonstrated, for the first time, that entactin interacts with fibrinogen in a specific manner. Entactin binding sites have been localized to the A alpha and B beta chains of fibrinogen. The binding of entactin to either the A alpha chain or the B beta chain was divalent cation-independent. The binding of 35S-labeled entactin to the immobilized fibrinogen A alpha or B beta chain was concentration-dependent and saturable and could be inhibited by unlabeled entactin, soluble fibrinogen, anti-entactin antiserum, or anti-fibrinogen antiserum. In addition, we have provided evidence that entactin can be cross-linked to itself, and probably also to fibrin(ogen), by transglutaminase. These novel properties, together with its cell binding, chemotactic, and phagocytic promoting activities and ubiquitous distribution in basement membranes, suggest that entactin may play important roles in hemostasis and wound healing.

    Funded by: NCI NIH HHS: CA-21246; NIGMS NIH HHS: GM-25690

    The Journal of biological chemistry 1991;266;28;18802-7

  • A new congenital abnormal fibrinogen Ise characterized by the replacement of B beta glycine-15 by cysteine.

    Yoshida N, Wada H, Morita K, Hirata H, Matsuda M, Yamazumi K, Asakura S and Shirakawa S

    Institute of Hematology, Jichi Medical School, Tochigi, Japan.

    A new case of heterozygous dysfibrinogenemia characterized by the replacement of NH2-terminal amino acid of fibrin beta-chain was found in a 50-year-old man. Despite a prolonged thrombin time, the propositus' fibrinogen had a normal reptilase time with the normal release of fibrinopeptide A. Release of fibrinopeptide B by thrombin was strongly affected, but a very high concentration of thrombin almost completely released fibrinopeptide B with a normal elution pattern on reversed-phase high performance liquid chromatography (HPLC). Lysylendopeptidase-cleavage of purified B beta-chains analyzed on HPLC showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide demonstrated that B beta glycine-15, NH2-terminus of the fibrin beta-chain, was replaced by cysteine. These findings will be of particular importance because they strongly support the hypothesis that the NH2-terminal portion of the fibrin beta-chain is involved in the polymerization reaction by thrombin. The propositus' daughter and two sisters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Ise. During these studies, we found that a very high concentration of thrombin cleaves not only the A alpha Arg19-Val20 bond but also the COOH-terminal region of alpha-chains, which results in the generation of further degraded alpha-chains with apparent molecular weights of 44,000 or less.

    Blood 1991;77;9;1958-63

  • Intracellular fate of fibrinogen B beta chain expressed in COS cells.

    Danishefsky K, Hartwig R, Banerjee D and Redman C

    Lindsley F. Kimball Research Institute, New York Blood Center, NY 100221.

    Full-length fibrinogen B beta cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. B beta chain expression was measured by pulse-labelling cells with L-[35S]methionine, immunoprecipitating the B beta chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B beta chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted B beta chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that B beta chain is not transported to the Golgi apparatus. In transfected COS cells, antibody to fibrinogen co-immunoprecipitated B beta chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent B beta chains. Non-secreted B beta chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH4Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that B beta chain by itself does not contain the signal for fibrinogen secretion and that non-secreted B beta chain is associated with BiP and degraded in the rough endoplasmic reticulum.

    Funded by: NHLBI NIH HHS: HL 37457

    Biochimica et biophysica acta 1990;1048;2-3;202-8

  • Nucleotide sequences of the three genes coding for human fibrinogen.

    Chung DW, Harris JE and Davie EW

    Department of Biochemistry, University of Washington, Seattle 98195.

    Funded by: NHLBI NIH HHS: HL 16919

    Advances in experimental medicine and biology 1990;281;39-48

  • DNA polymorphisms at fibrinogen loci and plasma fibrinogen concentration.

    Berg K and Kierulf P

    Institute of Medical Genetics, University of Oslo, Norway.

    Associations have been reported between restriction fragment length polymorphisms (RFLPs) at fibrinogen loci and plasma fibrinogen concentration, in a British study. We have examined a series of unrelated Norwegians. We found no association bet 1f40 ween plasma fibrinogen concentration and any genotype in either of two fibrinogen polymorphisms examined (one at the alpha-fibrinogen locus, the other at the beta-fibrinogen locus). We have also examined monozygotic twins and evaluated heritability of fibrinogen level by the intraclass correlation coefficient. We arrived at an unimpressive estimate of heritability. With such a low level of heritability, it would have been surprising if we had found an association with a single gene marker in a relatively limited series of people. The reason for the discrepancy between the British and the Norwegian study is unknown. Great care has to be exercised in interpreting disease associations, since with DNA variations being examined at an increasing number of "candidate loci", the risk of finding spurious associations increases with the number of analyses conducted.

    Clinical genetics 1989;36;4;229-35

  • High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen.

    Ni F, Konishi Y, Frazier RB, Scheraga HA and Lord ST

    Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.

    The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought 1734 close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.

    Funded by: NHLBI NIH HHS: HL-30616, HL-31048; NIGMS NIH HHS: GM-24893; ...

    Biochemistry 1989;28;7;3082-94

  • [Not Available].

    Corvi A

    Atti e memorie della Accademia italiana di storia della farmacia 1989;6;1;17-27

  • A polymorphism at B beta 448 of fibrinogen identified during structural studies of fibrinogen Baltimore II.

    Schmelzer CH, Ebert RF and Bell WR

    Johns Hopkins University School of Medicine, Baltimore, MD 21205.

    Funded by: NHLBI NIH HHS: HL07525, HL29067, HL36260

    Thrombosis research 1988;52;2;173-7

  • Characterization of the 5'-flanking region for the human fibrinogen beta gene.

    Huber P, Dalmon J, Courtois G, Laurent M, Assouline Z and Marguerie G

    To identify the possible regulatory sequences in the genetic expression of fibrinogen, a human genomic DNA library raised in lambda EMBL 4 phage was screened using cDNA probes coding for the A alpha, B beta and gamma chains of human fibrinogen. The entire fibrinogen locus was characterized and its organization analysed by means of hybridization and restriction mapping. Among the clones identified, a single recombinant lambda phage contained the beta gene and its 5'- and 3'-flanking regions. A 1.5 kb fragment of the immediate 5'-flanking region was sequenced and S1 mapping experiments revealed three transcription start points. Comparison of this sequence with that previously reported for the same region upstream from the human gamma gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different. In contrast, comparison of the 5'-flanking regions of human and rat beta genes revealed a 142 bp sequence of 80% homology situated 16 bp upstream from the human beta gene. This highly conserved region may well represents a potential candidate for a regulatory sequence of the human beta gene.

    Nucleic acids research 1987;15;4;1615-25

  • Characterization of fibrinogen New York 1. A dysfunctional fibrinogen with a deletion of B beta(9-72) corresponding exactly to exon 2 of the gene.

    Liu CY, Koehn JA and Morgan FJ

    Fibrinogen New York 1 (NY-1) was identified in a family with a thrombotic tendency. Studies on fibrinogen NY-1 and the fibrinogen from her brother, designated NY-1a, showed that both have abnormal thrombin-nonclottable fibrinogen (50% of the total fibrinogen in NY-1 and 35-40% in NY-1a) and that the trait is heterozygous and autosomal codominant. The abnormal fibrinogen polymerizes in the presence of calcium and can be further cross-linked by Factor XIIIa. The release rates of fibrinopeptides A and B by thrombin from both (NY-1 and NY-1a) were slower than those from normal fibrinogen. Two mol of fibrinopeptide A but only 0.6-1.0 (NY-1) or 1.0-1.3 (NY-1a) mol of fibrinopeptide B were released per mol of fibrinogen. Additionally, only 1.0 (NY-1) or 1.3 (NY-1a) mol of the B beta(1-42) peptide were released by plasmin/mol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reduced fibrinogen revealed two protein bands in the B beta-chain region (Mr = 54,000 as compared with 57,300 for the normal). When NY-1a fibrinogen was treated with CNBr, two sizes of the NH2-terminal disulfide knot were obtained (Mr = 59,000 and 49,000). The Mr = 49,000 component is consistent with an abnormal NH2-terminal disulfide knot with two defective NH2-terminal B beta-chains. Amino acid sequence analyses demonstrated that the abnormal B beta-chain is the result of a deletion in the sequence from residues 9 to 72. This deletion corresponds exactly to exon 2 of the gene. Since this family has a thrombotic tendency, the defect in the fibrinogen may be important in the pathogenesis of thrombosis in this family.

    The Journal of biological chemistry 1985;260;7;4390-6

  • Fibrinogen and fibrin.

    Doolittle RF

    Funded by: NHLBI NIH HHS: HL 18,576

    Annual review of biochemistry 1984;53;195-229

  • Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.

    Kant JA, Lord ST and Crabtree GR

    Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene.

    Proceedings of the National Academy of Sciences of the United States of America 1983;80;13;3953-7

  • Cloning of fibrinogen genes and their cDNA.

    Chung DW, Rixon MW, Que BG and Davie EW

    Cross-species hybridizations have enabled us to isolate and clone the gene for the beta chain of human fibrinogen. Highlights of the gene for the beta chain revealed by nucleotide sequence analyses, particularly in areas that have a direct bearing on defining the overall organization of the gene, have been presented. Nucleotide sequence determination has confirmed the presence of seven intervening sequences. The positions where several of these intervening sequences interrupt the coding region appear to be related to the functional domains of the polypeptide. A putative signal peptide has been identified. Studies on the cDNA for the human alpha chain indicate that the alpha chain polypeptide may be synthesized in a precursor form with a COOH-terminal extension of 15 amino acids as compared to the alpha chain present in the mature molecule found in plasma. We are in the process of isolating the genes for the alpha and gamma chains by a similar approach. We are hopeful that these studies will provide information as to how they are regulated and how they have undergone changes in the course of evolution.

    Funded by: NHLBI NIH HHS: HL 16919

    Annals of the New York Academy of Sciences 1983;408;449-56

  • Covalent structure of fibrinogen.

    Henschen A, Lottspeich F, Ke 1399 hl M and Southan C

    Annals of the New York Academy of Sciences 1983;408;28-43

  • Characterization of complementary deoxyribonucleic acid and genomic deoxyribonucleic acid for the beta chain of human fibrinogen.

    Chung DW, Que BG, Rixon MW, Mace M and Davie EW

    A total of 148 cDNAs coding for the beta chain of human fibrinogen have been identified from a human liver cDNA library employing a bovine cDNA as a probe. The largest cDNA insert contained 1932 base pairs cloned into the PstI site of plasmid pBR322. This cDNA insert contained 66 base pairs coding for a portion or all of a signal sequence, 1383 base pairs coding for 461 amino acids in the mature protein, a stop codon of TAG, a noncoding region of 431 base pairs, and a poly(A) tail of 19 base pairs. Most of the cDNA inserts coding for the beta chain were found to have a noncoding region of 98 or 167 base pairs rather than 431 base pairs at the 3'-end. The bovine cDNA for the beta chain was also employed as a probe for screening a lambda phage library containing human genomic DNA. Seven positive phage were identified. One of the phage, which contained the entire gene for the beta chain of fibrinogen, was examined by electron microscopy, and portions of its DNA sequence are presented. Seven intervening sequences were identified in the gene for the beta chain of human fibrinogen. The largest intervening sequence (approximately 1.3 kilobases) was found at the 5'-end of the gene and was located between amino acid residues 8 and 9, which are present in fibrinopeptide B. A sequence analysis of the 5'-end of the gene also indicated that the B chain of human fibrinogen contained a signal sequence of either 16, 27, or 30 amino acid residues.

    Funded by: NHLBI NIH HHS: HL 16919, HL 28598

    Biochemistry 1983;22;13;3244-50

  • Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets.

    Phillips DR, Jennings LK and Prasanna HR

    Washed human platelets suspended in buffers containing either 1.8 mM Ca2+ and 0.49 mM Mg2+ or 1 mM EDTA were treated with human alpha-thrombin to induce secretion. Glycoprotein G, a major glycoprotein in alpha-granules, was quantitatively secreted from platelets activated in the EDTA-containing buffer but remained with the platelet in the presence of Ca2+ and Mg2+. Addition of Ca2+ to the platelets that were activated in the presence of EDTA caused glycoprotein G to bind to platelets. To determine if glycoprotein G is expressed on the membrane surface of the activated platelet, platelets were rapidly labeled by a method employing lactoperoxidase-catalyzed iodination. Although glycoprotein G was barely detected on the surface of unstimulated platelets, labveling 1 min after thrombin treatment showed that glycoprotein G rapidly became one of the prominent surface proteins. These findings show that an alpha-granule protein, glycoprotein G, is one of the major glycoproteins on the membrane surface of thrombin-activated platelets and that its binding is dependent on divalent cations.

    Funded by: NHLBI NIH HHS: HL 00080, HL 15616, HL 21487

    The Journal of biological chemistry 1980;255;24;11629-32

  • Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences.

    Laudano AP and Doolittle RF

    Biochemistry 1980;19;5;1013-9

  • Fibrinogen Houston: a dysfibrinogen exhibiting defective fibrin monomer aggregation and alpha-chain cross-linkages.

    Weinger RS, Rudy C, Moake JL, Conlon CL and Cimo PL

    A 38-year-old male patient with a life-long history of easy bruising and mild bleeding had a prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Reptilase (Bathrops atrox) clotting time was normal. His undiluted and diluted plasma prolonged the APTT, PT, and TT of normal plasma. Fibrin produced from patient plasma was insoluble in 5 M urea. Plasma fibrinogen level was increased when measured as clottable protein and by Laurell electroimmunoassay. Specific assays of plasma factors II, V, VII, X, VIII, IX, XI, and XII were normal. A circulating antithrombin in patient plasma was excluded by demonstrating normal thrombin-induced platelet aggregation of gel-separated platelets in the presence of patient plasma. Purified patient fibrinogen reproduced the anticoagulant effect of patient plasma. Patient fibrinogen antigen was similar to normal fibrinogen antigen by immunodiffusion, immunoelectrophoresis (pH 5.2 and 8.6), and crossed immunoelectrophoresis. His unreduced purified fibrinogen had normal migration on polyacrylamide slab gels. Also, the migration in gel slabs of A alpha, B beta and gamma-polypeptide chains, produced by mercaptoethanol reduction of purified patient fibrinogen, was similar to reduced normal fibrinogen. Thrombin-induced total fibrinopeptide release was normal. However, fibrin monomers produced from patient fibrinogen by thrombin (devoid of fibrinopeptides A and B) reaggregated abnormally; fibrin monomers produced by reptilase (devoid of only fibrinopeptides A) reaggregated normally. Fibrin generated from patient plasma in the presence of factor XIII and calcium, was defective in the formation of covalently bonded alpha-alpha polymers and demonstrated an increased susceptibility to the lytic effects of plasmin (generated in vitro by the addition of streptokinase).

    Funded by: PHS HHS: MC-B-480001-01-0

    American journal of hematology 1980;9;3;237-48

  • [Temperature-dependent changes in the profile of the sarcoplasmic reticulum membrane hydrophobic zones].

    Tarakhovskiĭ IuS, Galushchenko IV, Boroviagin VL, Ritov VB and Komarov PG

    The dependence of the state of the hydrophobic zone of rabbit sarcoplasmic reticulum (SR) membranes on temperature of the membrane fragment suspension before rapid freezing was studied by the freeze fracturing technique. It was shown that within the temperature range of--15-- +37 degrees C the amount of intramembrane particles and their distribution in the membrane plane and between their convex and concave surfaces do not practically depend on the temperature of the SR membrane suspension. This is indicative of the lack of correlation between the physical state of the phospholipid matrix (gel -- liquid crystal) before freezing and the nature of the profile of the membrane hydrophobic zone revealed after fracturing. The disturbances in the protein -- lipid interactions in the membrane under the effects of mersalyl or aqueous solutions of diethyl ester followed by complete inactivation of Ca2+-dependent ATPase lead to a decrease in the amount of intramembrane particles, which is especially well-pronounced at 37 degrees and -15 degrees C.

    Biokhimiia (Moscow, Russia) 1979;44;5;897-902

  • Amino acid sequence of the beta chain of human fibrinogen.

    Watt KW, Takagi T and Doolittle RF

    The beta chain of human fibrinogen contains 461 amino acid residues, 15 of which are methionines. The calculated molecular weight, independent of a single carbohydrate cluster, is 52 230. In this regard, we have isolated and characterized all 16 cyanogen bromide fragments. In one case (CNI), we have concentrated on a disputed portion of a previously reported fragment. The arrangement of the cyanogen bromide peptides was deduced by the use of overlap fragments obtained from the tryptic digestion of modified and unmodified beta-chains and from digestions with staphylococcal protease, as well as by considerations involving the plasmic digestion products of fibrinogen. In one case two adjacent fragments were aligned by homology with the corresponding segments of the gamma chain. The homology of the beta chain with the gamma chain is especially strong over the course of the carboxy-terminal two-thirds of the sequence. Neither of these chains appears to be homologous with the alpha chain in these regions. With a few minor exceptions, the sequence reported in this article is in agreement with data reported by other groups in Stockholm and Munich.

    Biochemistry 1979;18;1;68-76

  • Primary structure of human fibrinogen. Characterization of disulfide-containing cyanogen-bromide fragments.

    Gårdlund B, Hessel B, Marguerie G, Murano G and Blombäck B

    European journal of biochemistry 1977;77;3;595-610

  • Disulfide bridges in nh2 -terminal part of human fibrinogen.

    Blombäck B, Hessel B and Hogg D

    Thrombosis research 1976;8;5;639-58

  • Quantitative studies on the release of platelet fibrinogen by thrombin.

    Keenan JP and Solum NO

    British journal of haematology 1972;23;4;461-6

Gene lists (2)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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