Fibrinogen Gene Research Articles

Genetically Determined Hemocoagulatory Abnormalities as a Cause of Ischemic Strokes in Children

We present here results from studies of the histories of thrombosis and polymorphisms of the hemocoagulation system genes in 27 children with ischemic stroke. The structures of allelic variants are compared in the Russian and non-Russian populations. All patients had more than four procoagulatory mutations. The most frequent were polymorphisms of the FGB fibrinogen gene (G-455A), the ITGA2 thrombocyte receptor gene (C807T), and the PAI-1 fibrinolysin gene (675 4G4G). The family histories of 81.5% of the children included thrombotic episodes and vascular events in relatives aged less than 50 years. Current data on the frequencies of mutations in populations of sick and healthy children are analyzed, along with their phenotypic characteristics.

SiRNA down-regulation of FGA mRNA in HepG2 cells demonstrated that heterozygous abnormality of the Aα-chain gene does not affect the plasma fibrinogen level

IntroductionWe encountered two afibrinogenemia patients with homozygous and compound heterozygous FGA mutation. Of interest, the patients' parents, who are heterozygous, had normal levels of plasma fibrinogen; thus, we hypothesized that liver FGA mRNA levels were higher than those of FGB and/or FGG mRNA. Materials and MethodsTo test the hypothesis, we quantitated mRNA levels of a normal liver and a human hepatocyte cell line, HepG2 cells, and performed siRNA-mediated down-regulation of the fibrinogen gene in HepG2 cells. mRNA levels were determined using real-time quantitative RT- PCR for three normal livers and HepG2 cells. Down-regulation of FGA, FGB, or FGG in HepG2 cells was performed by the addition of siRNA corresponding to each of the three genes, and the mRNA levels determined in the cells and the secreted fibrinogen concentration in media. ResultsThe mRNA level of normal human liver was FGA=FGB>FGG and the FGG mRNA level was about 2-fold lower than the others, that of HepG2 cells was FGA>FGG>FGB and FGA mRNA was approximately 2- or 4-fold higher than FGG mRNA and FGB mRNA. When FGA, FGB, or FGG mRNA expression levels were down-regulated by nearby 50%, fibrinogen concentrations in media were 78%, 49%, or 57% of the control, respectively. ConclusionsOur results suggest that FGG mRNA levels limit fibrinogen expression in normal liver and HepG2 cells and that 50% reduction of FGA mRNA levels would not limit fibrinogen expression in normal liver and HepG2 cells.

A novel fibrinogen B beta chain frameshift mutation causes congenital afibrinogenaemia

Congenital afibrinogenaemia is a rare autosomal recessive disorder caused by various mutations within the fibrinogen genes FGA, FGB and FGG. Ins/del mutations in FGB are extremely rare. We report a patient with afibrinogenaemia who suffered from umbilical cord bleeding and repeated bleeding episodes. His plasma fibrinogen levels could not be detected using the Clauss method and immunological methods. Molecular analyses revealed homozygosity in a novel four bases insertion in codon 40 of FGB exon 2 (g. 2833_2834 ins GTTT), which resulted in a truncated 50-residue polypeptide that contained 11 exceptional abnormal residues. In the transient expression experiments, mutant fibrinogen could be detected at higher level than wild-type fibrinogen in COS-7 cell lysates but not in culture media. These results suggest that the homozygous mutation in FGB could be responsible for congenital afibrinogenaemia in this patient. This frameshift mutation could impair fibrinogen assembly and secretion without influencing the protein synthesis.

A novel mutation in the FGB: c.1105C>T turns the codon for amino acid Bβ Q339 into a stop codon causing hypofibrinogenemia

Routine coagulation tests on a 14year-old male with frequent epistaxis showed a prolonged thrombin time together with diminished functional (162mg/dl) and gravimetric (122mg/dl) fibrinogen concentrations. His father showed similar aberrant results and sequencing of the three fibrinogen genes revealed a novel heterozygous nonsense mutation in the FGB gene c.1105C>T, which converts the codon for residue Bβ 339Q to stop, causing deletion of Bβ chain residues 339-461. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and RP-HPLC (reverse-phase high-pressure liquid chromatography) of purified fibrinogen showed only normal Aα, Bβ, and γ chains, indicating that molecules with the truncated 37,990Da β chain were not secreted into plasma. Functional analysis showed impaired fibrin polymerization, fibrin porosity, and elasticity compared to controls. By laser scanning confocal microscopy the patient's fibers were slightly thinner than normal. Electrospray ionization mass spectrometry (ESI MS) presented normal sialylation of the oligosaccharide chains, and liver function tests showed no evidence of liver dysfunction that might explain the functional abnormalities.

The dilemma of inherited dysfibrinogenemia during pregnancy

Inherited dysfibrinogenemia is a rare disorder caused by mutations in the fibrinogen gene, described in approximately 400 families to date. We present the case of a 20-year-old woman at 7 weeks of pregnancy with a history of two first-trimester spontaneous abortions and a family history of thrombotic events. Her testing revealed evidence of dysfibrinogenemia, necessitating multidisciplinary management planning including Hematology, OB-GYN, Maternal-Fetal Medicine, Blood Bank Services and Anesthesia. Antenatal care included a combination of intravenous fibrinogen infusions to maintain fibrinogen levels above 100 mg/dl and anticoagulation with low molecular weight heparin. She had an uneventful full-term delivery and continued fibrinogen infusions and thromboprophylaxis for 6 weeks postpartum. The combination of fibrinogen infusions and anticoagulation maintained the balance between bleeding and clotting in our patient during pregnancy. We recommend a multidisciplinary team approach for the management of dysfibrinogenemia during pregnancy to provide successful pregnancy outcomes.

Clinical phenotype, laboratory features and genotype of 35 patients with heritable dysfibrinogenaemia.

Heritable dysfibrinogenaemia (HD) is a rare qualitative disorder of fibrinogen (FGN). To better describe the clinical, laboratory and genotypic spectrum of HD, we evaluated 35 subjects identified at two UK centres using laboratory criteria. 12/35(34%) subjects with HD experienced bleeding (bleeding score >1 at any site), 3/35(9%) thrombosis and 20/35(57%) were asymptomatic. Amongst subjects with bleeding, symptoms were typically mild, at one anatomical site and seldom occurred after invasive procedures. All subject showed dry clot weight within or above laboratory reference interval (median 3·2 g/l; range 1·9-5·1), reduced Clauss fibrinogen (median 0·52 g/l; range 0·21-1·3), and prolonged thrombin (median 30·7 s; range 21·3-45·7) and reptilase (median 42·0 s; range 20·0-68·0) times. In all subjects, the prothrombin time ratio (PTR), determined by Sysmex CA-1500 coagulometer and Innovin activator, was abnormal (median 1·42; range 1·22-1·61). The activated partial thromboplastin time ratio and PTR with other coagulometers and activators were comparatively insensitive to HD. All subjects with HD harboured heterozygous candidate nucleotide variations within known hotspots in the FGN genes. The HD variants identified in this cross-sectional study seldom have significant clinical manifestations and show similar laboratory features irrespective of genotype. Selection of coagulometer and PT activator may markedly affect the detection of new HD cases using coagulation screening tests.

C0096 Assessing the combined effects two single nucleotide genetic polymorphisms of fibrinogen genes on the coagulation cascade in patients with stable angina

Studies have shown that genetic polymorphisms such as the G58A and the G455A polymorphisms on alpha and beta chain genes of fibrinogen respectively are associated with fibrinogen plasma levels in healthy individuals, but their effects on thrombotic process in patients with coronary artery disease (CAD) are unclear. In the present study we examined the combined effect of these polymorphisms on prothrombotic profile of patients with CAD.

Molecular analysis of afibrinogenemic mutations caused by a homozygous FGA1238 bp deletion, and a compound heterozygous FGA1238 bp deletion and novel FGA c.54+3A>C substitution

We identified two afibrinogenemic girls in two Japanese families and performed molecular analysis to clarify the mechanisms of fibrinogen defects. Genetic analyses were performed by PCR amplification of the fibrinogen gene and DNA sequence analysis. To analyze the mechanisms of mature fibrinogen defects in plasma, we cloned minigenes from the proposita’s PCR-amplified DNA, transfected them into CHO cells, and sequenced the cDNA amplified with the RT reaction followed by PCR. Sequence analyses indicated that one was caused by a homozygous 1238 bp deletion of the fibrinogen Aα-chain gene (FGAΔ1238) and the other was a compound heterozygous FGAΔ1238 and novel FGA c.54+3A>C substitution. The minigene corresponding to FGAΔ1238 generates two aberrant mRNAs, both of which may induce a frameshift and terminate prematurely. In contrast, the minigene corresponding to FGA c.54+3A>C generates two aberrant mRNAs, one of which may induce a frameshift and terminate prematurely, and the other uses a cryptic 5′ splice site in exon 1, resulting in the deletion of six amino acids in signal peptides. Molecular analyses of both genetic variants suggest that the lack of a mature Aα-chain, impaired assembly, and/or secretion of the fibrinogen molecule may lead to afibrinogenemia.

Multiple gene polymorphisms predisposing to the prothrombotic state in an adolescent with acute myocardial infarction

Acute myocardial infarction with ST-segment elevation (STEMI) is rare in adolescents and its pathogenesis is unclear. Growing evidence shows an association between the prothrombotic state and acute STEMI. Prothrombotic genetic factors may be involved in the pathogenesis of STEMI. We present a case of an adolescent with acute STEMI who had multiple prothrombotic gene polymorphisms: in the beta fibrinogen, methylenetetrahydrofolate reductase and cholesteryl ester transfer protein genes, as well as genotypes in plasminogen activator inhibitor-1 and human platelet antigen type-1. He had normal coronary arteries with catheterinduced spasm and was treated with a calcium antagonist and aspirin.

Compound heterozygous mutations of a family with inherited hypofibrinogenemia

Objective To analyze the phenotype and genotype of a Chinese family with inherited hypofibrinogenemia, and to investigate its molecular mechanism. Methods Peripheral blood was collected from seven people of this family and then plasma was separated.Activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), the activities of antithrombin (AT∶A), protein C (PC∶A) and protein S (PS∶A) were tested.The activity and antigen of plasma fibrinogen were analyzed by Clauss method and immunoturbidimetry method, respectively.The fibrinogen peptide chain of the proband was semiquantitatively assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Thrombin generation test was performed by calibrated automated thrombogram.The dynamic process of blood coagulation was evaluated by the thrombelastography (TEG).Genomic DNA was extracted from the peripheral blood.The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes FGA, FGB and FGG were amplified by polymerase chain reaction（PCR） and analyzed by direct sequencing. Results The activity and the antigen levels of the proband′s plasma fibrinogen were reduced to 0.48 g/L and 0.68 g/L, respectively.TT prolonged to 29.2 s and RT prolonged to 75.8 s.The assays of SDS-PAGE showed no abnormal molecular weight of fibrinogen.Peak height of thrombin generation was reduced to 249.93 nmol/L and endogenous thrombin potential was reduced to 1007.0 nmol·L-1·min.Hypocoagulability state of the whole blood was found by TEG test.The coagulation index was -8.6.The proband was diagnosed as inherited hypofibrinogenemia by phenotype analysis.Two mutations (Gln143Pro and g.4642delC) were found in the proband′s fibrinogen Aa-chain gene, Gln143Pro came from her mother and g.4642delC came form her father. Conclusion Compound Heterozygous Mutations (Gln143Pro and g.4642delC) of fibrinogen Aa-chain causes the proband congenital hypofibrinogenemia.（Chin J Lab Med,2012,35:322-327） Key words: Afibrinogenemia; Fibrinogen; Pedigree; Heterozygote; Mutation

A genetic instrument for Mendelian randomization of fibrinogen

Mendelian randomization studies on fibrinogen commonly use a single genetic variant as an instrument, but this may explain only a small proportion of the total phenotypic variance. We examined the contribution of multiple common single nucleotide polymorphisms (SNPs) and haplotypes in the entire fibrinogen gene cluster to plasma fibrinogen levels in two prospective cohorts, for use as instruments in future Mendelian randomization studies. Genotypes for 20 SNPs were determined in 2,778 middle-age (49-64 years) men from the Second-Northwick-Park-Heart Study (NPHS-II). These were replicated in 3,705 men from the Whitehall-II study (WH-II). Plasma fibrinogen levels were determined six times in NPHS-II and three times in WH-II. The minor alleles of four SNPs from the FGB gene, two from the FGA gene, and one from the FGG gene were associated with higher plasma fibrinogen levels. SNP rs1800790 (-455G>A) commonly used in Mendelian randomization studies was associated with R2=1.22% of the covariate adjusted residual variance in fibrinogen level. A variable selection procedure identified one additional SNP: rs2070011 (FGA) altogether explaining R2=1.45% of the residual variance in fibrinogen level. Using these SNPs no evidence for causality between the fibrinogen levels and coronary heart diseases was found in instrumental variables analysis. In the replication cohort, WH-II, the effects of the two SNPs on fibrinogen levels were consistent with the NPHS-II results. There is statistical evidence for several functional sites in the fibrinogen gene cluster that determine an individual's plasma fibrinogen levels. Thus, a combination of several SNPs will provide a stronger instrument for fibrinogen Mendelian randomization studies.

Fibrinogen polymorphisms associated with sporadic cerebral hemorrhage in a Chinese population

Fibrinogen plays an important role in the intrinsic and extrinsic pathways of blood coagulation. This study investigated the association between common variants in the fibrinogen gene and the risk of developing sporadic cerebral hemorrhage (CH). We performed genotyping analyses for three single nucleotide polymorphisms (SNP) in the fibrinogen gene in a case-controlled study involving 195 patients with CH and 116 control participants; both groups were of southern Han-Chinese origin. Logistic regression analysis indicated that haplotypes ATA (rs1800790+rs1800787+rs6050), AA (rs1800790+rs6050) and TA (rs1800787+rs6050) could nearly double the risk of sporadic CH (odds ratio [OR]=1.738, 95% confidence interval [CI]: 1.103–2.740, p=0.017; adjusted OR=1.762, 95% CI: 1.042–2.982, p=0.035), although the three SNP were not associated with sporadic CH when analyzed separately. These findings indicate that rs1800790, rs1800787 and rs6050 polymorphisms may contribute to the etiology of sporadic CH in the Chinese population.

Fibrinogen gene regulation

The Aα, Bβ and γ polypeptide chains of fibrinogen are encoded by a three gene cluster on human chromosome four. The fibrinogen genes (FGB-FGA-FGG) are expressed almost exclusively in hepatocytes where their output is coordinated to ensure a sufficient mRNA pool for each chain and maintain an abundant plasma fibrinogen protein level. Fibrinogen gene expression is controlled by the activity of proximal promoters which contain binding sites for hepatocyte transcription factors, including proteins which influence fibrinogen transcription in response to acute-phase inflammatory stimuli. The fibrinogen gene cluster also contains cis regulatory elements; enhancer sequences with liver activities identified by sequence conservation and functional genomics. While the transcriptional control of this gene cluster is fascinating biology, the medical impetus to understand fibrinogen gene regulation stems from the association of cardiovascular disease risk with high level circulating fibrinogen. In the general population this level varies from about 1.5 to 3.5 g/l. This variation between individuals is influenced by genotype, suggesting there are genetic variants contributing to fibrinogen levels which reside in fibrinogen regulatory loci. A complete picture of how fibrinogen genes are regulated will therefore point towards novel sources of regulatory variants. In this review we discuss regulation of the fibrinogen genes from proximal promoters and enhancers, the influence of acute-phase stimulation, post-transcriptional regulation by miRNAs and functional regulatory variants identified in genetic studies. Finally, we discuss the fibrinogen locus in light of recent advances in understanding chromosomal architecture and suggest future directions for researching the mechanisms that control fibrinogen expression.

A novel regulatory element between the human FGA and FGG genes

High circulating fibrinogen levels correlate with cardiovascular disease (CVD) risk. Fibrinogen levels vary between people and also change in response to physiological and environmental stimuli. A modest proportion of the variation in fibrinogen levels can be explained by genotype, inferring that variation in genomic sequences that regulate the fibrinogen genes ( FGA , FGB and FGG ) may affect hepatic fibrinogen production and perhaps CVD risk. We previously identified a conserved liver enhancer in the fibrinogen gene cluster (CNC12), between FGB and FGA . Genome-wide Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that transcription factors which bind fibrinogen gene promoters also interact with CNC12, as well as two potential fibrinogen enhancers (PFE), between FGA and FGG . Here we show that one of the PFE sequences has potent hepatocyte enhancer activity. Using a luciferase reporter gene system, we found that PFE2 enhances minimal promoter- and FGA promoter-driven gene expression in hepatoma cells, regardless of its orientation with respect to the promoters. A region within PFE2 bears a short series of conserved nucleotides which maintain enhancer activity without flanking sequence. We also demonstrate that PFE2 is a liver enhancer in vivo, driving enhanced green fluorescent protein expression in transgenic zebrafish larval livers. Our study shows that combining public domain ChIP-seq data with in vitro and in vivo functional tests can identify novel fibrinogen gene cluster regulatory sequences. Variation in such elements could affect fibrinogen production and influence CVD risk.

Developmental expression and organisation of fibrinogen genes in the zebrafish

The zebrafish is a model organism for studying vertebrate development and many human diseases. Orthologues of the majority of human coagulation factors are present in zebrafish, including fibrinogen. As a first step towards using zebrafish to model human fibrinogen disorders, we cloned the zebrafish fibrinogen cDNAs and made in situ hybridisations and quantitative reverse transcription-polymerase chain reactions (qRT-PCR) to detect zebrafish fibrinogen mRNAs. Prior to liver development or blood flow we detected zebrafish fibrinogen expression in the embryonic yolk syncytial layer and then in the early cells of the developing liver. While human fibrinogen is encoded by a three-gene, 50 kilobase (kb) cluster on chromosome 4 ( FGB-FGA-FGG ), recent genome assemblies showed that the zebrafish fgg gene appears distanced from fga and fgb , which we confirmed by in situ hybridisation. The zebrafish fibrinogen Bβ and γ protein chains are conserved at over 50% of amino acid positions, compared to the human polypeptides. The zebrafish Aα chain is less conserved and its C-terminal region is nearly 200 amino acids shorter than human Aα. We generated transgenic zebrafish which express a green fluorescent protein reporter gene under the control of a 1.6 kb regulatory region from zebrafish fgg . Transgenic embryos showed strong fluorescence in the developing liver, mimicking endogenous fibrinogen expression. This regulatory sequence can now be used for overexpression of transgenes in zebrafish hepatocytes. Our study is a proof-of-concept step towards using zebrafish to model human disease linked to fibrinogen gene mutations.

Replication and characterisation of genetic variants in the fibrinogen gene cluster with plasma fibrinogen levels and haematological traits in the Third National Health and Nutrition Examination Survey.

Previous genetic association studies of the fibrinogen gene cluster have identified associations with plasma fibrinogen levels. These studies are typically limited to plasma fibrinogen measured among European-descent populations. We sought to replicate previous well-known associations with fibrinogen variants and plasma fibrinogen. We then sought to identify and characterise novel associations with fibrinogen variants with plasma fibrinogen and several haematological traits in three racial/ethnic populations. We genotyped 25 single nucleotide polymorphisms (SNPs) in the fibrinogen gene cluster in 2,631 non-Hispanic whites, 2,108 non-Hispanic blacks, and 2,073 Mexican-Americans from the Third National Health and Nutrition Examination Survey (NHANES). We performed single SNP tests of association for plasma fibrinogen, mean platelet volume, platelet distribution width, platelet count, white blood cell count, and serum triglycerides. Five previously identified associations with plasma fibrinogen replicated in our study in non-Hispanic whites and blacks. We identified two novel associations between genetic variants and decreased plasma fibrinogen: rs2227395 (p=0.0007; non-Hispanic whites) and rs2070022 (p=0.001; Mexican-Americans). Several fibrinogen SNPs were also associated with haematological traits: rs6050 with decreased platelet distribution width in non-Hispanic whites; rs6050 and rs2066879 with decreased and increased platelet distribution width, respectively, in non-Hispanic whites;rs2227409 with increased mean platelet volume, rs2070017 with decreased platelet count, and rs6063 with increased platelet distribution width in non-Hispanic blacks; and rs4220 and rs2227395 with decreased white blood cell count, rs2227409 with increased platelet distribution width, rs2066860 and rs1800792 with increased and decreased triclyceride levels, respectively, and rs1800792 with decreased platelet counts in Mexican-Americans. We successfully replicated and identified novel associations with fibrinogen variants and plasma fibrinogen. These data confirm the importance of the fibrinogen gene cluster for plasma fibrinogen levels as well as suggest this gene cluster may have pleiotropic effects on haematological traits.

Replication of Candidate Gene Single Nucleotide Polymorphisms (SNPs) Previously Reported As Associated with Venous Thromboembolism (VTE)

Abstract 1238 Background:SNPs within genes encoding factor XI (F11), fibrinogen genes (FGA, FGG) and other candidate genes within the procoagulant, anticoagulant, fibrinolytic, innate immunity and endocrine pathways have been reported as associated with VTE. However, the independent risk of VTE associated with many of these SNPs after controlling for factor V Leiden, Prothrombin G20210A and ABO blood group non-O carrier status is uncertain. Objective:To replicate candidate gene SNPs previously reported as associated with VTE. Methods:As part of a large replication study, we included 17 SNPs previously reported as associated with VTE in a custom Illumina Golden gate (total n=1093 SNPs) genotyping array. We genotyped 1270 non-Hispanic adults of European ancestry with objectively-diagnosed VTE (cases; no cancer, venous catheter or antiphospholipid antibodies) and 1302 controls (frequency-matched on case age, gender, race, MI/stroke status). Genotyping results from high-quality control DNA (SNP call rate ≥ 95%) was used to generate a cluster algorithm. The primary outcome was VTE status, a binary measure. The covariates were age at interview or blood sample collection, sex, stroke and/or MI status, and state of residence. To adjust for population stratification, we performed the multidimensional scaling (MDS) analysis option in PLINK v 1.07 to identify outliers in our population using the ancestry informative markers. We tested for an association between each SNP and VTE using unconditional logistic regression, adjusting for age, sex, stroke/MI status, state of residence and ABO rs514659 (in high linkage disequilibrium with non-O blood type). The analyses were corrected for multiple comparisons using an extension of false discovery rates. The false discovery rate (reported as a Q-value) is an analogue measure of the p-value that takes into account the number of statistical tests and estimates the expected proportion of false positive tests incurred when a particular SNP is significant. All analyses were performed using PLINK v 1.07. Results:MDS gave no evidence of population stratification. Genotyping was unsuccessful for two of the 17 SNPs. We found significant associations between VTE and SNPs in F11, FGG, TC2D and FGA (Table). However, the false discovery rates for all significant SNPs except F11 rs3756008 were >0.05, suggesting that the observed associations were likely falsely positive due to multiple comparisons. Even at a false discovery rate of Q-value=0.0099, one would expect ∼13 SNPs (0.0099 × 1302 SNPs) to be falsely associated with VTE due to multiple comparisons. Consequently, even our observed association between F11 rs3756008 and VTE remains tentative. Conclusions:We were unable to replicate reported associations between 15 SNPs and VTE. Our results emphasize the necessity of replication studies in different populations to confirm reported associations of SNPs with VTE.TableAssociation of candidate gene SNPs with venous thromboembolismSNPGene†Odds ratio (95% CI)P-valueQ-valuers3756008FXI1.25 (1.11, 1.40)0.00020.0099rs2036914FXI0.84 (0.75, 0.94)0.00270.094rs2066854FGG1.17 (1.03, 1.32)0.01710.33rs10133762TC2D1.13 (1.01, 1.26)0.03510.42rs6050FGA1.14 (1.00, 1.29)0.04880.47rs2726953SCARA51.07 (0.95, 1.21)0.26530.77rs2234693ESR10.95 (0.84, 1.06)0.33830.80rs6585234HABP20.94 (0.81, 1.09)0.40230.83rs10895068PGR1.10 (0.86, 1.41)0.44180.83rs1799863CCR50.87 (0.60, 1.26)0.47110.83rs33989577APOA41.07 (0.87, 1.30)0.53120.85rs253061F2R0.97 (0.85, 1.10)0.59620.87rs1800872IL101.02 (0.90, 1.17)0.72820.88rs344782PLAUR0.99 (0.89, 1.11)0.8910.89rs2227589SERPINC11.00 (0.82, 1.21)0.99010.90TC2D, tandem C2 domains, nuclearCCR5, chemokine receptor 5FGA, fibrinogen alpha chainAPOA4, apolipoprotein A 4SCARA5, scavenger receptor class A, member 5 F2R, thrombin receptorESR1, estrogen receptor 1PLAUR, uPA receptorHABP2, hyaluran binding protein 2SERPINC1, antithrombin†FGG, fibrinogen gamma chainPGR, progesterone receptor Disclosures:Heit:Daiichi Sankyo: Consultancy, Honoraria.

A Novel Missense Mutation in FGG (c.944C>A) Encodes for An Amino Acid Change (p.Ala315Asp) in the Gamma Chain of Fibrinogen Causing Hypofibrinogenemia and a Thrombotic Phenotype

Abstract 856Fibrinogen plays a central role in maintaining hemostasis. Disruption of its normal function may lead to hemorrhagic or thrombotic events. Fibrinogen is encoded by three genes, FGA, FGB, and FGG, clustered on chromosome 4q28-q31. Hereditary defects of fibrinogen, although uncommon, can affect the quantity (hypofibrinogenemia and afibrinogenemia) or the quality (dysfibrinogenemia) of the circulating protein. We report a family with two affected individuals (father and daughter) that presented with a mild bleeding predisposition, but also exhibited thrombotic events, i.e. unprovoked deep vein thrombosis in a father and an in utero middle cerebral artery stroke in a daughter. The Clauss fibrinogen levels for the father and daughter were 105 and 98 mg/dL (normal 150–400 mg/dL) and fibrinogen antigen levels were 138 and 152 mg/dL (normal 170–400 mg/dL), respectively. Thrombin times were prolonged in both patients. All other coagulation factors were within normal range and the patients did not carry either the prothrombin G20210A or factor V Leiden mutation.All exons and intron-exon junctions of the three fibrinogen genes were sequenced. The father and daughter were found to be heterozygous for a novel missense mutation in the gamma chain (Ala315Asp) while this mutation was not observed in an unaffected family member (mother of the daughter).Transient transfection experiments using CHO-K1 cells showed that gamma chain with p.Ala315Asp mutation was detected in both cell lysates and supernatant, although in reduced amount as compared with cells transfected with wild-type (WT) FGG cDNA.Structurally, there was no significant difference in fibrin fiber diameter, as measured manually by scanning electron microscopy, between the affected (father and daughter), and the daughter's unaffected mother. Similarly, there was no difference in thrombus height between the three individuals. As expected, hydraulic permeability of platelet rich plasma clots correlated with the fibrinogen levels in each person. Clotting kinetics in plasma of affected family members was consistent with hypofibrinogenemia, but did not suggest abnormal fibrin polymerization. Clot structure and fibrin deposition was further characterized with a microfluidics flow assay, an experimental technique in which platelet and fibrin accumulation is observed in real-time using fluorescent probes in whole blood under shear stress on a collagen/tissue factor micropatterned surface. Interestingly, fibrin density and platelet accumulation, as measured by platelet and fibrin surface area coverage, was highest in the father and daughter, the two individuals with hypofibrinogenemia and thrombotic phenotype.In order to evaluate the possible changes resulting from the Ala315Asp mutation we examined predicted alterations in protein structure, energy, and protein-protein interaction between two fibrinogen molecules via in silico molecular modeling using published protein crystal structure. The molecular modeling showed that alanine for aspartic acid substitution predicted to result in substantial changes in overall conformation of the interaction interface (distal D-domain) between the two fibrinogen molecules due to introduction of a bulkier side chain. Potential energy (energy of minimization) for Ala315Asp mutant homodimer, and for WT-mutant heterodimer were lower than the WT homodimer, predicting a more stable structure from an energetic standpoint. Overall interaction energy (measurement of the energy of association between the two molecules) was lower for Ala315Asp mutant homodimer as compared with WT homodimer or WT-mutant heterodimer, suggesting more favorable interaction for Ala315Asp mutant dimers. Overall, the in silico modeling studies predicted that Ala315Asp mutation may lead to more rapid or stronger interaction between fibrinogen molecules.These data suggest that a novel FGG mutation, c.944C>A, that predicts pAla315Asp change, if expressed and incorporated into fibrinogen molecule in vivo, may result in a more stable or more resistant to lysis clot. Characterizing the association between a specific genetic defect and its phenotypic expression will advance the current understanding of the molecular and biochemical mechanisms of the inherited disorders of fibrinogen, and may contribute to the development of safer therapeutic interventions for the patients with fibrinogen disorders. Disclosures:No relevant conflicts of interest to declare.

Phenotype and Genotype of 22 Subjects with Heritable Dysfibrinogenaemia

Abstract 1212▪▪This icon denotes a clinically relevant abstractThe heritable dysfibrinogenaemias (HD) are a heterogenous group of qualitative disorders of fibrinogen in which there is abnormal fibrin clot formation. Affected individuals usually display prolonged thrombin (TCT) and reptilase clotting times (RCT) and reduced plasma fibrinogen activity. The plasma fibrinogen concentration is normal or is reduced less than the fibrinogen activity. HD is associated with pathogenic mutations in FGA, FGB or FGG genes that encode the fibrinogen Aα, Bβ and g chains respectively. However, most clinical descriptions of HD comprise single case reports and the range of phenotype and genotype in the wider population is difficult to estimate. In order to improve understanding of this disorder, we now report a cross-sectional survey of 22 subjects with HD identified at 2 UK regional Haemostasis centres from 2001–11.We recruited index cases from our Haemostasis laboratory referral practice who satisfied all the following criteria- 1) prolonged TCT; 2) prolonged TCT with protamine (TCTP) and/or prolonged RCT; 3) reduced Clauss fibrinogen and 4) normal fibrinogen dry clot weight. Demographic and clinical characteristics were recorded using a standardised case report form. Genetic analysis was performed by Sanger sequencing of FGA exon 2, FGB exon 2 and FGG exon 8 which we identified as mutation hotspots from the fibrinogen mutation database (http://www.geht.org/databaseang/fibrinogen/). Phenotype and genotype was also determined in available first degree family members.We identified 16 index cases and 6 family members with HD (median age 41, range 12–78; 9 males). HD was identified in plasma submitted to our Haemostasis laboratories for the investigation of bleeding in 8/16 (50%) index cases, for stroke in 1/16 (6%) and for ‘routine coagulation screening' in 7/16 (44%). Mild mucocutaneous bleeding or menorrhagia was documented in the clinical records of 13/22 (59%) subjects and thrombotic stroke in 1/22 (5%). 8/22 (36%) subjects were asymptomatic.The prothrombin time (PT) was prolonged in 21/22 (95%) subjects (median 15.3 s; range 11.4–16.7; reference interval (RI) 9.6–11.6) but the activated partial thromboplastin time (aPTT) was prolonged in only 1/22 (5%; median 23.8 s; range 22.8–37.0, RI 24.0–32.0). The median TCT was 28.6 s (range 20.6–45.7; RI 13–19) and TCTP 31.9 s (n=11; range 20.0–35.5; RI 13–19). The median RCT was 37s (n=12; range 19–68; RI 15–19), Clauss fibrinogen 0.49 g/dL (range 0.29–1.3; RI 1.8–3.6) and dry clot weight 2.3 g/dL (range 1.9–4.2; RI 1.8–3.6). Heterozygous missense nucleotide variations were identified in the candidate exons of the fibrinogen genes in all 16/16 (100%) index cases and segregated with the HD phenotype in all available family members. These variations predicted aminoacid substitutions at the fibrinopeptide A cleavage site in the fibrinogen Aa chain in 10/16 (63%) index cases (FGA p.R35H (n=4); p.R35S (n=3); p.R35P (n=2) and p.G36S (n=1); numbered from RefSeq NP_000499.1) or at nearby residues (FGA p.V39D (n=1) and p.F27C (n=1)). FGG variations were identified in 3 index cases that predicted aminoacid substitutions in the fibrinogen g chain region necessary for D:D domain interactions during fibrin monomer assembly (FGG p.R301H (n=1), p.D342G (n=1) and p.A367T (n=1); RefSeq NP_000499.1). One subject harboured a FGB variation that predicted a p.R44C substitution (Refseq NP_005132.2) at the fibrinopeptide B cleavage site in the fibrinogen Bb chain. The FGA p.R35S, p.R35H, FGB p.R44C and FGG p.R301H and p.A367T substitutions have been associated previously with HD.We show that the HD laboratory phenotype is rare and was frequently identified in subjects with mild bleeding. However, HD was also commonly asymptomatic and did not segregate with bleeding symptoms within affected families suggesting that the HD variants identified in this study do not confer significant bleeding risk. The prevalence of mild bleeding in some study subjects may reflect recruitment bias. We also show that HD is strongly associated with nucleotide variations in the FGA, FGB or FGG genes. All the observed nucleotide variations have been previously associated with HD and/or predict aminoacid substitutions in critical regions for normal fibrin polymerization. Defective fibrin polymerization may affect endpoint determination in some automated clotting assays and is likely to account for the consistent finding of prolonged PT in the study subjects. Disclosures:No relevant conflicts of interest to declare.

Genetic population structure in the boky-boky (Carnivora: Eupleridae), a conservation flagship species in the dry deciduous forests of central western Madagascar

The boky-boky, Mungotictis decemlineata, is an endemic and presumed forest-dependent carnivoran species restricted to lowland central western Madagascar. It inhabits dry deciduous forests, which have been severely reduced in surface area with ∼60% destroyed or degraded by humans during the past 60 years. M. decemlineata is limited to the remnant forests of the central and southern Menabe, and using samples collected from sites across this zone, a phylogeographic study was conducted based on two mitochondrial (1140 base pairs [bp] of cytochrome b and 563 bp of the control region) and one nuclear fragment (591 bp of the seventh intron of the fibrinogen gene). Forty-seven individuals were included from the central Menabe from four principal localities and two animals from the southern Menabe from a single locality. Low sequence divergence (1.65% for the combined fragment, 4.26% for the control region and 0.78% for cytochrome b) characterized specimens across a zone of 130 km delimited by the Tsiribihina River to the north and the Mangoky River to the south; this area includes most of the geographical range of M. decemlineata. Phylogenetic trees, haplotype networks and exact test of population differentiation did not reveal any meaningful geographic partitioning of genetic variation. However, shallow yet significant genetic structure was revealed by ΦST calculations for the combined as well as separate DNA fragments, which we ascribe to isolation-by-distance. We proposed two different scenarios to explain the lack of meaningful phylogeographical structure in Mungotictis: (1) for this forest-dependent species, dispersal during periods of more continuous forest cover gave rise to a genetic meta-population or (2) that it is able to cross non-forested zones and broadly disperses, leading to high levels of genetic homogeneity. Current inferences favour the first hypothesis. The short- and medium-term future of this taxon is in jeopardy associated with habitat destruction across its geographical range.