Effect of F7 Gene Promoter Polymorphisms on Factor VII Antigenic Levels.

Abstract

Genetic variation in the F7 gene has been shown in several studies to influence quantitative variation in Factor VII antigenic levels. Previously in the GAIT Study, we comprehensively resequenced the F7 gene and obtained strong evidence of multiple functional variants. Two independent sets of highly correlated promoter polymorphisms appeared to contain at least two of these functional variants. However, the high degree of linkage disequilibrium within these two clusters precluded statistical identification of the most likely functional polymorphisms. In an attempt to further disentangle these highly correlated single nucleotide polymorphisms (SNPs), we performed an independent association study on 706 unrelated Spanish subjects enriched for arterial disease. Inclusion criteria included subjects with ischemic stroke or acute coronary artery disease and subjects without a personal history of arterial disease and from the same geographical area. Of these, 179 had acute coronary artery disease, 231 had ischemic stroke and 296 were subjects without personal history of arterial thromboembolic disease. For each individual, we measured Factor VII antigenic levels (FVIIag) using a standard ELISA method. Using an ABI 3100 automated sequencing platform, we typed each individuals for six promoter polymorphisms. These polymorphisms included SNPs observed in the 5′ proximal promoter region at positions −670, −630, −402, −401, and −323. As in our original observations, we observed two tight clusters of SNPs, {−670, −630, −402} and {−401, −323}, that exhibited strong intraset linkage disequilibrium. Robust measured genotype analysis, as implemented in the computer program, SOLAR, was used to assess the association between the F7 SNPs and FVIIag levels. Bayesian quantitative trait nucleotide analysis was used to calculate the Bayes Factors (which represent the relative evidence for one SNP over another)for each possible SNP within a linkage disequilibrium cluster. All analyses allowed for covariate effects such as sex and age. Each of the six promoter polymorphisms showed strong statistical association with levels of FVIIag. The observed associations were stable when analysis was limited to only normal subjects or to only subjects showing prior arterial disease. In Table 1, the variants and local sequence of each polymorphism is detailed as is the p-value showing the statistical evidence of association for each polymorphism with FVIIAg levels. Bayesian analysis suggested that the −402 and −323 polymorphisms best predicted the FVIIag phenotype (p value:3.2×10−18). Our results confirm in an Spanish population-based association study that these F7 promoter polymorphisms are in substantial linkage disequilibrium and show a strong association with levels of FVIIag, While the linkage disequilibrium within each of these SNP sets is very high and precludes unequivocal statistical identification of the true functional variants, our method has provided additional support for the −402 and −323 polymorphisms as being the most likely functional sites. This additional evidence now allows us to prioritize these polymorphisms for more expensive gold-standard molecular functional analyses. Table 1 Nucleotide site Variant Sequence P-value −670 A/C ccA/Cgcc 4.8×10–8 −630 A/G aaA/Gca 4.4× 10–7 −401 G/A cgG/Agt 2.8×10–8 −402 G/T cgG/Tct 7.7×10–14 −323 Ins 0/10 agCCTATATCCTg 4.6×10–14