Replication of findings on the association of genetic variation in 24 hemostasis genes and risk of incident venous thrombosis

A major obstacle to advancing knowledge of the genetic predictors of disease has been the failure to replicate findings when tested in new populations. In this letter, we present findings that attempted to replicate statistical significant associations from our previously published investigation of variation in 24 hemostasis genes and the risk of incident venous thrombosis (VT) in post-menopausal women.1 In the original work, we identified 21 single nucleotide polymorphisms (SNPs) and 6 haplotypes across 24 hemostasis genes that were associated with a nominal p-value of less than 0.05. Among these, 5 SNPs remained significant after adjusting for multiple testing using a 0.2 threshold of the false-discovery rate q value.2 Three of the 5 were “new” findings not previously reported. In this report, we used data from 2 studies to replicate the 3 findings and to further explore the other 24 associations in addition to the 2 well-characterized variants, factor V Leiden (rs6024) and prothrombin 20210A (rs1799963). Using meta-analytic methods, we combined data from an on-going, population-based, case-control study in women 18–89 years of age and data from the Cardiovascular Health Study (CHS), a prospective cohort study of men and women aged 65 years and older. None of the subjects in this meta-analysis was included in the original report. The case-control data included only newly recruited subjects to the on-going study that produced the original report. Detailed methods were previously published.1 Briefly, new cases included 91 incident VT events in premenopausal women that occurred between years 1995 and 2004 and 120 incident VT events in postmenopausal women that occurred in years 2003 and 2004. Also included were 187 and 435 matched controls, respectively. The median age at the time of the VT event was 54 years (interquartile range 41–72 years), 100% were women, and 89% were white. Genotyping was performed on genomic DNA using an Illumina GoldenGate custom panel. The cohort data were incident VT events in CHS identified by the Longitudinal Investigation of Thromboembolism Etiology (LITE). Methods for CHS and LITE were previously published.3, 4 Briefly, CHS recruited 5,888 participants in 2 waves: 1989–90 (original cohort) and 1992–93 (minority cohort). Among CHS participants without a history of VT at baseline and for whom we had DNA and consent to use it for research, we identified 150 incident VT events through 2002, the last year of VT event identification in CHS-LITE. The median age at the time of the VT event was 79 years (interquartile range 74–84 years), 55% were women, and 79% were white. Genotyping was performed on genomic DNA using an Illumina GoldenGate custom panel. Each study independently conducted association analyses of the 23 SNPs (including factor V Leiden and prothrombin 20210A variants) and 6 haplotypes with the risk of incident VT. Logistic regression was used to estimate relative risk (RR) of VT in the case-control study and Cox proportional hazards regression was used to estimate risk of VT in the cohort study. For both analyses, age- and race (white versus non-white) adjusted additive genetic models were used for SNP and haplotype associations. The case-control study also adjusted for case-control matching variables, which included index year, menopausal status, and hypertension status. The cohort study also adjusted for sex and clinic site as well as sampling variables, which included hypertension status and duration of follow-up. We used a fixed-effects, inverse-variance-weighted meta-analysis that combined risk-estimate coefficients and standard errors across the studies to produce a summary RR with alpha-based confidence intervals and p-value for each test. For the 3 SNPs that were “new” findings in the original publication and declared significant after adjustment for multiple testing, the 1-sided p-value threshold for replication was 0.0167 (0.05/3 with Bonferroni correction). For the 15 SNPs and haplotypes that were “new” associations in the original publication but not declared significant after adjusting for multiple testing, the 2-sided p-value threshold for significance was 0.0033 (0.05/15 with Bonferroni correction). For the remaining 11 SNPs for which there were previous reports in the literature of an association at the time of the original study, we attempted confirmation in the meta-analyzed data and used a 1-sided p-value threshold of 0.05. Furthermore, we tested all comparisons across the original and meta-analysis data to see if risk estimates were not statistically different. Statistical power was calculated based on the minor allele frequency (MAF) and relative risk (RR) from the original study. The results of the meta-analysis that included 361 incident VT events are listed in the Table along with the original findings. For each SNP and haplotype, the MAF and RR with 95% confidence interval and p-value are provided. Also listed are the status of the finding at the time of the original publication (Original Status) and the current status (Updated Status) based on the results of the meta-analysis. Table 1 Replication of associations between selected variants and haplotypes across hemostasis genes and the risk of incident venous thrombosis.