Comprehensive studies of novel SNPs affecting warfarin dose in African Americans

说明(申请人提供)：华法林(Coumadin(R))长期以来一直是研究的目标，因为它既难以确定正确的剂量，又有严重的不良反应。目前，已经开发出利用CYP2C9和VKORC1基因的多态来预测高加索人和亚洲人华法林的正确维持剂量的算法。然而，这些算法，包括已知的非遗传变量，如年龄、体重和伴随的药物，在非裔美国人中预测能力要差得多。发现哪些SNP会影响非裔美国人的剂量，以及它们的影响机制仍然是目前知识的空白。我们假设这些研究将确定影响非裔美国人华法林剂量的临床相关SNPs。这项建议的目的是为影响非裔美国人华法林剂量的新基因变异提供有效证据，调查这些SNPs对基因调控的影响，并显示这些SNPs对华法林及其药物靶点维生素K环氧化物还原酶(VKOR)的药代动力学的影响。为了实现这些目标，我们利用比较基因组学和推测的转录结合位点预测方法，在非裔美国人中确定了相关的单倍型标签SNPs(HtSNPs)，并对其进行了基因分型。通过观察htSNP，我们可以在单倍型中只对一个SNP进行基因分型，并捕捉到与该SNP的连锁不平衡(LD)的变异。发现队列中的关联测试显示，这两个基因都有有趣的新奇变异。然而，需要对这些发现进行验证。为了进一步评估调控，我们使用全基因组生物信息学工具(SCAN)来识别表达数量性状基因座(EQTL)，这种方法从未被用于识别与华法林剂量相关的新SNP。为此，我们计划对两个独立的非裔美国人抗凝队列中关联度最高的SNPs进行基因分型。然后，将在一系列体外测试中对那些复制的细胞进行功能测试。然后，我们将通过药代动力学研究和VKORC1SNPs对VKOR蛋白功能替代标记物-维生素K缺乏或拮抗II诱导的凝血酶原(PIVKA-II)的影响来研究这些SNPs对药物代谢和靶蛋白的直接临床作用。这项拟议的研究是及时和必要的，以填补当前知识的空白，并影响药物遗传学真正转化为临床实践。

DESCRIPTION (provided by applicant): Warfarin (Coumadin (R)) has been a long-standing target of research because it is both difficult to determine the correct dose and has serious adverse effects. Currently, algorithms using polymorphisms in the CYP2C9 and VKORC1 genes have been developed to predict the correct maintenance dose of warfarin in Caucasians and Asians. However these algorithms, which include known non-genetic variables such as age, weight and concomitant medications, are much less predictive in African Americans. Discovery of which SNPs affect dose in African Americans and the mechanism underlying their effect remain a gap in the current knowledge. We hypothesize that these studies will identify clinically relevant SNPs that affect warfarin dose in African Americans. The aims of this proposal are to provide validated evidence for novel genetic variation that affects warfarin dose in African Americans, to investigate the effect these SNPs have on the gene regulation, and to show the effect of these SNPs on both the pharmacokinetics of warfarin and its drug target Vitamin K epoxide reductase (VKOR). In pursuit of these goals, we have determined and genotyped the relevant haplotype tagging SNPs (htSNPs) in CYP2C9 and VKORC1 in African Americans, using comparative genomics and putative transcriptional binding sites prediction. By looking at htSNPs we can genotype just one SNP in the haplotype and capture the variation in linkage disequilibrium (LD) with that SNP. Tests for association in the discovery cohort have revealed interesting novel variation in both genes. However, validation of these findings is needed. To further evaluate regulation, we used a genome-wide bioinformatics tool (SCAN) to identify expression Quantitative Trait Loci (eQTLs), a method that has never been used to identify novel SNPs associated with warfarin dosing. To this end, we plan on genotyping the most highly associated SNPs in two independent African American anticoagulation cohorts. Those that replicate will then be assayed for function in a series of in vitro assays. We will then investigate the direct clinical effect of these SNPs on drug metabolism and the target protein by conducting a pharmacokinetic study evaluating the effect of CYP2C9 SNPs on S- to R-warfarin plasma concentration ratio and a pharmacodynamic study evaluating the effect of VKORC1 SNPs on a surrogate marker of VKOR protein function, Prothrombin induced by vitamin K absence or antagonism II (PIVKA-II). This proposed research is both timely and necessary to fill gaps in the current knowledge and to affect real translation of pharmacogenetics into clinical practice.