Comprehensive studies of novel SNPs affecting warfarin dose in African Americans

描述（由申请人提供）：华法林（Coumadin（R））一直是一个长期的研究目标，因为它既难以确定正确的剂量，又有严重的不良反应。目前，已经开发了使用CYP 2C 9和VKORC 1基因多态性的算法来预测高加索人和亚洲人华法林的正确维持剂量。然而，这些算法，其中包括已知的非遗传变量，如年龄，体重和合并用药，是在非洲裔美国人的预测少得多。发现哪些SNPs影响非裔美国人的剂量及其作用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SNP。该提案的目的是为影响非裔美国人华法林剂量的新型遗传变异提供有效证据，研究这些SNP对基因调控的影响，并显示这些SNP对华法林药代动力学及其药物靶点维生素K环氧化物还原酶（VKOR）的影响。在追求这些目标，我们已经确定和基因分型的相关单倍型标记SNP（htSNPs）在CYP 2C 9和VKORC 1在非洲裔美国人，使用比较基因组学和推定的转录结合位点预测。通过观察htSNPs，我们可以对单倍型中的一个SNP进行基因分型，并捕获与该SNP的连锁不平衡（LD）的变化。在发现队列中进行的关联测试揭示了两个基因中有趣的新变异。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）来鉴定表达定量性状基因座（eQTL），这是一种从未用于鉴定与华法林给药相关的新型SNP的方法。为此，我们计划在两个独立的非裔美国人抗凝队列中对最高度相关的SNP进行基因分型。然后将在一系列体外试验中测定复制的那些细胞的功能。然后，我们将通过开展一项评价CYP 2C 9 SNP对S-与R-华法林血浆浓度比影响的药代动力学研究和一项评价VKORC 1 SNP对VKOR蛋白功能替代标志物维生素K缺乏或拮抗作用II（PIVKA-II）诱导的凝血酶原影响的药效学研究，研究这些SNP对药物代谢和靶蛋白的直接临床影响。这项研究的提出是及时和必要的，以填补目前的知识空白，并影响真实的翻译药物遗传学到临床实践。 公共卫生相关性：拟议的研究将有助于确定影响非裔美国人华法林剂量的基因突变，这是一个研究不足的人群。这些新突变的发现可能会导致这种药物的更好剂量和更少的副作用。这些研究还可能揭示对华法林给药重要的基因可能被调节的新方法（即，产生或多或少的蛋白质）。

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. PUBLIC HEALTH RELEVANCE: The proposed studies will help identify genetic mutations that affect warfarin dose in African Americans, an under-studied population. The discovery of these novel mutations may lead to better dosing of this medication and less side-effects. These studies may also uncover new ways in which genes important to warfarin dosing may be regulated (i.e., produce more or less protein).