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，涉及PBC发病机制的两个细胞群体(CD8和CD4T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。

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.