One approach to the identification of genetic loci that influence complex diseases is through the study of quantitative risk factors correlated with disease susceptibility. Factor XII (FXII) plasma levels, a related phenotype correlated with thrombosis, is such a risk factor. We conducted the first genomewide linkage screen to localize genes that influence variation in FXII levels. Two loci were detected: one on chromosome 5 and another on chromosome 10 (LOD scores 4.73 and 3.53, respectively). On chromosome 5, the peak LOD score occurred in the 5q33-5ter region, near the FXII gene. Addition of a 46C/T mutation in the FXII gene increased the multipoint LOD score to 10.21 (P=3.6 x 10(-12)). A bivariate linkage analysis of FXII activity and thrombosis further improved the linkage signal (LOD = 11.73) and provided strong evidence that this quantitative-trait locus (QTL) has a pleiotropic effect on the risk of thrombosis (P=.004). Linkage analysis conditional on 46C/T indicated that this mutation alone cannot explain the chromosome 5 signal, implying that other functional sites must exist. These results represent the first direct genetic evidence that a QTL in or near the FXII gene influences both FXII activity and susceptibility to thrombosis and suggest the presence of one or more still unknown functional variants in FXII.

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As part of the GAIT (genetic analysis of idiopathic thrombophilia) project, we analyzed polymorphisms in the factor V (FV) gene to assess their role as genetic determinants of normal phenotypic variation of hemostasis-related traits in a Spanish population. During the analysis of exon 13 polymorphisms, we detected an abnormal PCR-amplified fragment in some members of the GAIT19 family. Direct sequence analysis revealed a deletion of 108 bp in eight out of 20 individuals in this family. This deletion removes exactly 36 amino acids from the B domain of FV; thus it does not alter the reading frame of the sequence. Among the deleted amino acids there is the 4070A>G polymorphism (H1299R), which could affect the level or function of FV. In addition, in the same family we identified three novel DNA variants (L1257I, Q1317Q and T1327T) in exon 13 of the F5 gene. Despite these variants, we did not detect any differences either in the coagulant or anticoagulant traits, or in the plasma protein levels involved in the blood coagulation cascade, between the carriers compared with their non-carrier relatives. From these results, we can conclude that the mutant allele is expressed and the resultant protein is functional. Moreover, it is unlikely that the 4070A>G polymorphism, within the deletion, and the novel DNA variants alter the functional properties of the mature FV protein. Further analyses of this naturally occurring mutation and the novel DNA variants should yield useful information for the understanding of the function of the B domain of FV.

Delineating the genetic variability of loci coding for complex diseases helps to understand the individual variation in disease susceptibility and drug response. We present the allelic architecture of the F7 gene. This gene is the major determinant of FVII plasma levels, and these plasma levels constitute an important intermediate risk factor for cardiovascular disease. As part of the Genetic Analysis of Idiopathic Thrombophila Project, we completely re-sequenced the F7 locus (promoter, exons, introns, and 3'-untranslated region) in 40 unrelated individuals. We found 49 polymorphisms with only two amino acid changes suggesting that regulatory non-coding and intronic variants are responsible for the FVII variability. These results are important for mapping susceptibility alleles of complex diseases, because differences in pair-wise linkage disequilibrium patterns between DNA variants and haplotype frequency distributions may help to detect disease-associated alleles. In addition, we present the results of an in silico search that established genomic comparisons among different species. In conclusion, our study of the F7 DNA sequence variations is an example of a strategy for analyzing the genetic architecture of a quantitative trait locus. Furthermore, it provides a model for future analyses of genetic factors that contribute to the susceptibility of complex diseases in humans.

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