Deep Re-Sequencing of Rheumatoid Arthritis Risk Loci

描述(由申请人提供)：类风湿性关节炎(RA)的全基因组关联研究(GWAS)已导致确定了基因组中25个以上与疾病风险有关的区域(RA风险基因)。尽管在基因定位方面取得了令人兴奋的进展，但这些等位基因解释了类风湿性关节炎风险总体变异的20%，这表明仍有更多的风险等位基因有待识别。在目前的应用中，我们建议对25个RA风险基因的候选基因的蛋白质编码部分进行重新排序，以寻找罕见的功能突变。如果发现了一种罕见的疾病相关突变，那么几乎可以肯定这个基因就是致病基因。这些罕见的功能突变将为研究RA发病的基本机制提供机会。在这个应用中，我们假设罕见的功能等位基因对类风湿性关节炎的风险有贡献，这些基因也包含GWAS确定的独立的共同等位基因。为了验证这一假设，我们将实施下一代测序技术，该技术提供了一种具有成本效益的方法来对候选基因进行重新排序。目的1：利用下一代测序技术对750例类风湿关节炎患者和750例匹配对照的27个生物候选基因的编码外显子进行重新测序。我们将使用我们在布罗德研究所的同事最近开发的强大的测序管道来识别所有常见和罕见的等位基因。目的：在750例患者和750例对照中，对所有功能等位基因进行基因分型，并进行与疾病风险的关联研究。我们将在同一样本中对所有假定的功能突变进行基因分型，以确认序列调用并确定每个受试者的基因类型。我们将通过两种方法测试与RA风险的关联：基于等位基因的测试和基于基因的测试。目标3：我们将在更多的样本中复制我们的发现。如果我们的基于等位基因的测试确定了低频率但分离在祖先单倍型上的疾病相关功能等位基因，那么我们将通过在10,000个额外的病例对照样本中对罕见的等位基因进行基因分型来重复这一发现。如果我们的基于基因的测试确定了一组与疾病相关的罕见等位基因的集合，这些等位基因是个体特有的(即来自最近的祖先起源，不分离于祖先的单倍型)，那么我们将在另外1,500个病例和1,500个对照中对该基因的编码外显子进行重新排序。 我们已经组建了一支独特的科学家团队，能够领导该项目的各个方面。我们的研究将在欣赏下一代测序能力的科学环境中进行，而不会低估它的技术和分析挑战。 公共卫生相关性：了解类风湿性关节炎的遗传基础的一个长期目标是改善对这种常见且使人衰弱的疾病患者的护理。从理论上讲，识别特定的DNA片段(“等位基因”)应该有助于在症状出现之前或在骨骼破坏发生之前的疾病过程中早期诊断可治疗的疾病。此外，遗传学应该提供对疾病途径的重要步骤的洞察，允许开发针对高危个体这些途径的新疗法。

DESCRIPTION (provided by applicant): Genome-wide association studies (GWAS) in rheumatoid arthritis (RA) have led to the identification of more than 25 regions of the genome that contribute to risk of disease (RA risk loci). Despite this exciting progress at gene mapping, these alleles explain <20% of the overall variation in RA risk, indicating that many more risk alleles remain to be identified. In the current application, we propose to re-sequence the protein-coding portion of candidate genes from 25 RA risk loci to search for rare functional mutations. If a rare disease-associated mutation is identified, then this gene is almost certainly the causal gene. These rare functional mutations will provide an opportunity to study fundamental mechanisms of RA pathogenesis. In this application, we hypothesize that rare functional alleles contribute to risk of RA in genes that also harbor independent common alleles identified by GWAS. To test this hypothesis, we will implement next-generation sequencing technology, which provides a cost-effective approach to re- sequence candidate genes. Aim 1: Re-sequence the coding exons of 27 biological candidate genes in 750 RA cases and 750 matched controls using next-generation sequencing technology. We will identify all common and rare alleles using a robust sequencing pipeline recently developed by our colleagues at the Broad Institute. Aim 2: Genotype all functional alleles in the same 750 cases and 750 controls, and conduct an association study with disease risk. We will genotype all putative functional mutations in the same samples to confirm the sequence calls and to determine the genotype of each subject. We will test for association with RA risk in two ways: an allele-based test and a gene-base test. Aim 3: We will replicate our findings in additional samples. If our allele-based test identifies disease-associated functional alleles that are low frequency yet segregate on an ancestral haplotype, then we will replicate the finding by genotyping the rare allele in >10,000 additional case-control samples. If our gene-based test identifies a collection of disease-associated rare alleles that are private to individuals (i.e., are of recent ancestral origin, do not segregate on ancestral haplotypes), then we will re-sequence the coding exons of that gene in an additional 1,500 cases and 1,500 controls. We have assembled a team of scientists uniquely positioned to conduct all aspects of the project. Our studies will be conducted within a scientific environment that appreciates the power of next-generation sequencing, without underestimating it's technical and analytical challenges. PUBLIC HEALTH RELEVANCE: A long-term goal of understanding the genetic basis of rheumatoid arthritis is to improve care of patients with this common and debilitating disease. In theory, identifying specific pieces of DNA ("alleles") should aid in diagnosing a treatable condition either prior to onset of symptoms, or early in the course of disease before bone destruction occurs. In addition, genetics should provide insight into important steps of the disease pathway, allowing for the development of new therapies that target these pathways in at-risk individuals.