Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measuredgenotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL ؊1 (P ‫؍‬ .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3 splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant. (Blood. 2007;109:3713-3724)

Summary. Background: High-normal and elevated plasma FIX activity (FIX:C) levels are associated with increased risk for venous-and possibly arterial-thrombosis. Objective: Because the broad normal range for FIX:C involves a substantial unknown genetic component, we sought to identify quantitative-trait loci (QTLs) for this medically important hemostasis trait. Methods: We performed a genome-wide screen and a resequencing-based variation scan of the known functional regions of every distinct FIX gene (F9) in the genetic analysis of idiopathic thrombophilia project (GAIT), a collection of 398 Spanish-Caucasians from 21 pedigrees. Results: We found no evidence for linkage (LOD scores <1.5) despite genotyping more than 540 uniformly-spaced microsatellites. We identified 27 candidate F9 polymorphisms, including three in cis-elements responsible for the increase in FIX:C that occurs with aging, but found no significant genotype-specific differences in mean FIX:C levels (P-values ‡ 0.11) despite evaluating every polymorphism in GAIT by marginal multicovariate measured-genotype association analysis. Conclusions: The heritable component of interindividual FIX:C variability likely involves a collection of QTLs with modest effects that may reside in genes other than F9. Nevertheless, because the alleles of these 27 polymorphisms exhibited a low overall degree of linkage disequilibrium, we are currently defining their haplotypes to interrogate several highly-conserved non-exonic sequences and other F9 segments not examined here.

Thrombosis is a genetically complex disorder that plays a prominent pathogenic role in myocardial infarction, stroke and pulmonary embolism. Because these distinct endpoints of cardiovascular disease are the most common cause of death worldwide, the goal of investigators in several countries is to comprehensively catalogue human DNA polymorphisms influencing thrombosis susceptibility. It is now generally accepted that accomplishing this goal will require the use of linkage analysis and individuals from extended-pedigrees, even though previous investigations -- which predominantly were based on association analyses and case/control candidate gene studies -- resulted in several notable discoveries including the factor V Leiden mutation. In this regard, we recently completed the first genome linkage screen for DNA regions affecting normal variance of factor VII activity (fVII:C) levels in the 21 families from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. Only one quantitative trait locus (QTL) (LOD score=3.2; p-value=0.022) was identified and localized to the region of chr13q containing the fVII gene, F7. We performed the first comprehensive variation scan of this locus by resequencing the F7 genes in 40 unrelated GAIT subjects. The 49 polymorphisms identified were then genotyped in the full GAIT Project. Using a newly developed BQTN (Bayesian Quantitative Trait Nucleotide) method to estimate the posterior probability of a given variant being functional, we identified strong evidence for the functionality of 2 rare variants, including a novel SNP in intron 7 (C9830T) and a previously known SNP in the coding region of exon 8 (A294V). We also found 2 clusters of promoter (P) and intron 1 (I1) polymorphisms, designated C1: {A-630C, G-402A, T700C, G707A} and C2 {C-2989A, G-401T, -323ins0/10, T-122C, G73A, T835C}, within which all variants exhibit strong linkage disequilibrium (LD) and posterior probabilities of >90% for affecting fVII:C levels. In this study, we will use in vitro transcription to test the statistical findings of the BQTN analysis and, if confirmed, to specifically delineate the functional variant(s) in each cluster. We are creating G-free cassette (GfC) reporter constructs representing the natural P/I1 haplotypes statistically predicted as functional and 10 pairs of constructs where each pair represents the 2 alleles of each of the 10 variants in C1 and C2. All constructs have the same size P (0.9kb) and I1 (1kb) fragments ligated 5′ and 3′ of the GfC, respectively. Because C1 and C2 contain P and I1 variants, a new 120bp GfC was made to match the size of exon 1a and to approximate the native spacing of these polymorphisms in vivo. Once these constructs are sequenced, we will measure and compare their rate of transcriptional initiation using mouse liver nuclear extracts. Because of the strong LD among these P and I1 polymorphisms, population studies alone essentially only analyze the natural haplotypes that are present. The in vitro assays developed in this study will allow the specific influence of each polymorphism to be investigated in the context of a near native configuration of these known regulatory regions.

Regardless of advances in prenatal diagnosis, carrier detection and gene therapy for hemophilia-A, new patients with bleeding diatheses due to inadequate plasma FVIII activity (FVIII:C) levels will still require specialized management at treatment centers. In the ‘post-genome era’ the possibility exists for personalized medicine, in which an individual’s genetic information will be used to tailor prophylactic and/or treatment regimens that will optimize patient outcomes. As listed in the HAMSTeRS database, ~1,000 distinct loss-of-function F8 variants, representing all mutation types including inversions, insertion/deletions and single nucleotide substitutions (SNSs), have been associated with deficiencies of FVIII. To estimate how soon a complete catalogue of every possible mutation affecting FVIII:C levels may become available, we first determined the theoretical number of potential missense and nonsense F8 alleles, whether loss-of-function or not, based on each possible SNS in the coding region as compared to the reference sequence. While the impact of a missense change on FVIII:C, if any, is not always obvious, in contrast to premature-termination codons (PTCs), which are almost always deleterious, findings from a recent resequencing study raises the possibility that non-hemophilic structural differences between a patient’s endogenous FVIII protein and the infused “wildtype” molecule may increase risk of alloimmunization during replacement therapy. Wildtype FVIII contains 2,351 amino acid (aa) residues: 2,332 in the mature protein and 19 in the signal peptide (SP). Appropriate SNSs within the codons for 793 of these residues would create a PTC (UAA, UAG, UGA). Since three distinct base substitutions are possible at each of the three codon positions, 996 nonsense alleles could theoretically arise naturally. As only 123 distinct nonsense mutations are listed in HAMSTeRS, <15% of the theoretical number, many more likely await discovery. Since suitable SNSs within codons for every residue allow for as many as 15,631 naturally-occurring missense variations, of which 462 are in HAMSTeRS, only 2% of all possible alleles, even more mutations of this type likely remain to be identified. The Table presents the number of possible nonsense and missense F8 alleles and the FVIII protein domain/region affected. Although substantial time and diligent surveillance will be required to document the complete allelic architecture of hemophilia-A, since SNS-mutations can occur at every F8 nucleotide, not just those already identified, doing so could potentially have far reaching implications with respect to personalizing both the current strategy of replacement therapy, based on intravenous infusions and future gene-based methods. Table. Potential nonsense and missense F8 alleles based on the FVIII domain/region and position in codon Nucleotide Position in Codon Domain/Region Amino Acids 1st Codon 2nd Codon 3rd Codon Nonsense (Missense) Nonsense (Missense) Nonsense (Missense) SP 0001–0019 3 (51) 1 (56) 3 (23) A1 0001–0336 61 (911) 27 (981) 38 (317) a1 0337–0372 6 (101) 0 (108) 2 (50) A2 0373–0719 70 (935) 41 (1000) 55 (353) a2 0720–0740 5 (55) 3 (60) 4 (26) B 0741–1648 221 (2400) 139 (2585) 35 (1032) a3 1649–1689 12 (111) 4 (119) 4 (53) A3 1690–2019 71 (887) 15 (975) 45 (364) C1 2020–2172 30 (417) 17 (442) 20 (154) C2 2173–2332 35 (432) 14 (466) 15 (167)

SummaryWe identified a previously unknown mutation by sequencing the factor (F)X gene in a severely haemorrhagic 14-year-old maleAfrican-American individual with undetectable plasma FX-activity and -antigen levels. This mutation, called F10-Augusta, was homozygote and is a combination of an 8bp insertion in flanking 3’-genomic-DNA and a 5bp terminal exon-8 deletion involving codons 437 and 438. Sequencing of RT-PCR and 3’-RACE products showed that the F10-Augusta transcript is normally processed but lacks an in-frame stop codon. An allele specific 3’-RACE-based RFLP assay demonstrated that the steady-state concentration of the mutant transcript was markedly lower than that of the wild-type message in total-RNA samples from the patient’s unaffected heterozygous parents. The recently discovered nonstop decay mechanism, a component pathway of the mRNA surveillance system, is a possible explanation for the reduced concentration of the mutant FX transcript. This is the first report implying such a mechanism in the pathogenesis of inherited bleeding disorders.