SNP Analysis Of The VWF GENE Identifies Multiple Common Variants THAT Affect VWF Levels and OCCUR At Different Frequencies In Patients With TYPE 1 VWD

von Willebrand disease (VWD) type 1 is characterized by incomplete penetrance and variable expressivity. Individuals with very low levels of VWF (< 20 IU/dl) typically have mutations in the VWF gene. However, no such mutations can be identified in a significant subset of type 1 VWD patients, particularly those with VWF levels > 30 IU/dL. ABO is the best-characterized genetic modifier of VWF levels, accounting for up to 30% of the genetic variability and blood type O is overrepresented in patients with VWD type 1. VWF is highly polymorphic with over 1,100 reported single nucleotide variants (SNVs). The impact of many of these changes remains unknown. We hypothesize that common variants in VWF affect VWF levels and may play a role in the diagnosis of VWD type 1. We genotyped 94 tagging SNPs through VWF in a large Amish family (n=445) and study their association with VWF levels. We performed single SNP association tests using age, gender and ABO status as covariates and multiple testing corrections were implemented. We observed significant associations (p value ≤ 0.0005) between VWF levels and twelve tagging SNPs: rs2362479 (telomeric to VWF), rs10849363 and rs7964554 (intron 4); rs216308 (intron 24); rs216310 and rs216311 (exon 28); rs11612370 and rs11612384 (intron 33); rs216330, rs11064003, and rs2239162 (intron 37); and rs216293 (intron 38). To replicate the association of these SNPs with VWF levels in a larger Caucasian population, we conducted association analyses in three population-based cohorts of healthy individuals: 1) the Zimmerman Program for Molecular and Clinical Biology of VWD (ZPMCB-VWD) control cohort (n=150), 2) the Genes and Blood-Clotting Study (GABC) cohort (n=934) and 3) the Trinity Student Study (TSS) cohort (n=2,145). All association analyses were modeled including age, gender, rs687289 (a proxy marker of blood group O) and adjusted for multiple testing. Three SNP associations were replicated in the ZPMCB-VWD control cohort. An additional four SNP associations were replicated in the ZPMCB-VWD control and TSS cohorts. Associations of the intron 37 variants rs11064003 and rs2239162 with VWF levels were replicated in all cohorts and reached genome wide significance with p values of 1.34 x 10 ^-11 and 7.35 x 10 ^-15 respectively in the TSS cohort. We then genotyped the 12 candidate SNPs that showed significance in the original family in a cohort of 310 Caucasian VWD type 1 index cases (ZPMCB-VWD). We compared the minor allele frequency (MAF) of the tagging SNPs between the ZPMCB-VWD control cohort (n=150) and the ZPMCB-VWD type 1 index case cohort (n=310).

The minor allele of rs7964554 (associated with lower VWF levels) was more common (46.2%) in patients with VWD type 1 than in controls (34.0%), and that difference was statistically significant (p=0.0005). The minor alleles of rs11612370, rs11612384, rs11064003, and rs2239162 (all associated with higher VWF levels) were less common (p=0.018, 0.009, 0.012, and 0.0004, respectively) in VWD type 1 index cases compared to controls. In this population if the subject has VWD type 1, then the odds of being heterozygous for the minor allele of rs7964554 were 1.47 (95% CI: 1.08, 2.00; p=0.014). Similarly, the odds of being heterozygous for the minor allele of rs2239162 were 0.64 (95% CI: 0.45, 0.91; p=0.014). In summary we have identified several common variants in VWF that have a strong effect on VWF levels and more importantly are either under or over represented in patients with VWD type 1 indicating that may influence its diagnosis.