IDENTIFICATION OF COMMON AND UNCOMMON GENE VARIANTS IN PBC

描述(申请人提供)：我们将使用第二代和第三代深度测序相结合的选定候选区域，整个外显子和mRNAs，以确定常见和不常见的变异，易于PBC的敏感性。对150例患者和150名对照的选定染色体区域的测序将针对在我们的PBC GWAS中发现的IL12A、SPIB和17号染色体基因座(IKZF3/ORMDL3)之间的关联的变异的识别。这些区域的配对测序将提供机会来确定编码和非编码变异，包括拷贝数变体。400个病例和400个对照的外显子组测序将筛选不适用于GWAS检测的罕见遗传变异，并提供机会测试不依赖于共同变异假设的替代范式。重要的是，与PBC发病有关的两个细胞群(CD8+和CD4+T细胞)的mRNA测序将补充染色体区域和外显子组结果。在这方面，将对75例病例和75例对照进行mRNA测序。这种mRNA测序与目标染色体区域测序和外显子组测序将提供将序列变异与1)基因表达、2)eQTN数据、3)可选外显子使用、4)RNA编辑和5)优先等位基因表达相关联的能力。使用组合数据的各种信息学方法将使用Golden Gate 1536 SNPlex建立SNPs的优先顺序，以用于大量1100个PBC病例和2200个对照(不包括发现主题集)的验证和测试。测序和复制研究都将使用同质的意大利种群进行。总之，这项设计应该最大限度地提高我们识别不常见和更常见的变异的能力，这些变异在这种自身免疫性疾病的病因中很重要。 公共卫生相关性：这项建议的目标是确定导致原发性胆汁性肝硬变风险的原因基因变异。原发性胆汁性肝硬变被认为是一种典型的自身免疫性疾病，明确免疫病理学的遗传基础不仅有助于这种疾病的患者，而且对自身免疫也具有普遍意义。这项研究将利用最新的技术进步来提供DNA序列差异，并提供将遗传变异与功能变化联系起来的机会，这种疾病的易感性具有很高的发病率和死亡率。这些研究将对PBC的发病机制提供有价值的见解，从而可能导致治疗的发展。

DESCRIPTION (provided by applicant): We will use a combination of second and third generation deep sequencing of selected candidate regions, whole exomes and mRNAs to identify both common and uncommon variants that predispose to PBC susceptibility. The sequencing of selected chromosome regions in 150 cases and 150 controls will target identification of variants that underlie the association of IL12A, SPIB, and a chromosome 17 locus (IKZF3/ORMDL3) that are identified in our PBC GWAS. The paired sequencing of these regions will provide the opportunity to ascertain coding and non-coding variation including copy number variants. The exome sequencing of 400 cases and 400 controls will screen for uncommon genetic variants that are not amenable to GWAS detection and provide the opportunity to test an alternate paradigm that does not depend on the common variant hypothesis. Importantly, mRNA sequencing of two cell populations implicated in PBC pathogenesis (CD8+ and CD4+ T cells) will complement both the chromosome region and exome results. For this aspect, mRNA will be sequenced in 75 cases and 75 controls. This mRNA sequencing together with the targeted chromosomal region sequencing and exome sequencing will provide the ability to correlate sequence variation with 1) gene expression, 2) eQTN data, 3) alternative exon usage, 4) RNA editing and 5) preferential allelic expression. A variety of informatics approaches using the combined data will establish a prioritization of SNPs for validation and testing in large numbers 1100 PBC cases and 2200 controls (not including discovery subject set) using a Golden Gate 1536 SNPlex. Both the sequencing and replication studies will be performed using a homogeneous Italian population. Together this design should maximize our ability to identify uncommon as well as more common variants that are important in the etiopathogenesis of this autoimmune disease. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to identify causal genetic variants underlying the risk for primary biliary cirrhosis. Primary biliary cirrhosis is considered a model autoimmune disease and defining the genetic basis of immunopathology will not only help patients with this disease, but will also be generically important for autoimmunity. The study will utilize the recent advances in technology to provide DNA sequence differences and an opportunity to link genetic variation to functional changes import in the susceptibility of this disease with high morbidity and mortality. These studies will provide valuable insight into the pathogenesis of PBC that may lead to therapeutic development.