IDENTIFICATION OF COMMON AND UNCOMMON GENE VARIANTS IN PBC

描述（由申请人提供）：我们将使用第二代和第三代深度测序选择候选区域，全外显子组和mrna的组合，以确定易患PBC的常见和不常见变异。对150例患者和150例对照患者选定的染色体区域进行测序，目的是鉴定出在PBC GWAS中鉴定出的IL12A、SPIB和17号染色体位点（IKZF3/ORMDL3）之间关联的变异。这些区域的配对测序将提供确定编码和非编码变异的机会，包括拷贝数变异。400例病例和400例对照的外显子组测序将筛选不适合GWAS检测的不常见遗传变异，并提供测试不依赖于共同变异假设的替代范例的机会。重要的是，涉及PBC发病机制的两个细胞群（CD8+和CD4+ T细胞）的mRNA测序将补充染色体区域和外显子组的结果。为此，我们将对75例病例和75例对照进行mRNA测序。这种mRNA测序以及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)替代外显子使用，4)RNA编辑和5)优先等位基因表达相关的能力。使用组合数据的各种信息学方法将建立snp的优先级，以便使用Golden Gate 1536 SNPlex在大量1100例PBC病例和2200例对照（不包括发现主题集）中进行验证和测试。测序和复制研究将在意大利同质人群中进行。总之，这种设计应该最大限度地提高我们识别在这种自身免疫性疾病的发病机制中重要的不常见和更常见变异的能力。

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.