Fine Mapping and Functional Evaluation of Selected Type 1 Diabetes Loci

描述(由申请人提供)：拟议研究的总体目标是精细定位含有常见风险变异的选定T1D基因座，并确定它们的作用机制和对细胞破坏的贡献。在目标1a中，我们建议发现相关连锁不平衡(LD)块中的所有变异，其频率足够高，以解释在这些基因座观察到的关联。我们选择了8个基因座，它们都与免疫机制有关，都得到了很好的建立和复制。将对300例病例进行所有基因座的重新测序，选择携带尽可能多的易感等位基因；对照将来自1000基因组计划。在目标1b中，我们建议评估LD区块内的所有变异对T1D风险的独立影响。选定的标签标记将在我们收集的12,000份DNA样本(4,000名患者及其父母)以及一小部分T1D患者(200名)和非裔美国人对照(400名)中进行基因分型。结果将通过条件回归分析，以确定其与T1D的关联不能被LD解释为区块内的另一效应的多态。在目标2a中，我们建议对T1D相关多态对转录、剪接和翻译的等位基因效应进行大规模、高通量的顺式评估。在目标2b中，我们将使用病例和健康对照的外周血细胞来确定T1D相关变体编码的细胞表型。对于已知功能的基因，如IFIH1、PTPN22、RASGRP1、BACH2、UBASH3，这种功能将在体外在不同基因类型的个体细胞之间进行比较。对于功能未知的基因，如CLEC16A，第一步将通过过度表达和siRNA敲除实验来确定其功能；在目标2c中，我们将使用人类基因座的动物模型作为发挥功能的重要线索来源。我们还建议创建携带人类多态的两个等位基因的敲入(Ki)小鼠，从涉及PTPN22和IFIH1基因编码变体的良好映射的基因座开始，直到CLEC16A MOSE。这些小鼠将与NOD背景杂交，并将研究人类基因多态对糖尿病易感性和免疫失调的特定方面的影响。在目标2d中，我们将通过分析与T1D风险相关的表达模式的基因调控区域内的甲基化模式来评估表观遗传学机制。在Aim3a中，我们将对所有重新测序的基因座进行详细的表型-基因型相关性研究，以寻找等位基因特异性的基因型效应。在目标3b中，我们将使用与T1D关联最强的SNP标志物建立一种预测试验，其敏感性和特异性在预测T1D风险方面具有临床意义。我们预计这些研究将显著提高我们对T1D发病机制的理解。 公共卫生相关性： 项目简介1型糖尿病(T1D)是一种具有强烈遗传成分的多因素儿童疾病。它是最常见的严重儿童慢性疾病之一，预期寿命严重缩短，生活质量受到影响。这是由于自身免疫性破坏胰岛β细胞，导致胰岛素分泌不足所致。虽然我们和其他研究小组已经通过最近的全基因组关联研究确定了几个T1D基因座，但这些基因座赋予风险的实际因果机制仍然未知。该项目建议1)发现基因座内所有可能解释观察到的关联的变异，并确定它们对T1D风险的独立影响；2)从功能上表征这些变异；以及3)通过全面的基因/表型相关性进一步表征病因路径，并为在风险分层和未来的治疗指导(即个性化药物)中使用这些变异建立概念证明。

DESCRIPTION (provided by applicant): The general objective of the proposed research is to fine-map selected T1D loci harboring common risk variants and to determine their mechanism of action and contribution to ¿-cell destruction. In Aim 1a, we propose to discover all variants within the associated linkage disequilibrium (LD) blocks of a frequency high enough to account for the observed association at these loci. We have selected eight loci all of which involve immune mechanisms, all of which have been well established and replicated. Three hundred cases will be re- sequenced for all the loci, selected for carrying the susceptibility alleles in as many as possible; the controls will come from the 1000 genome project. In Aim 1b, we propose to evaluate all variants within the LD blocks for independent effects on T1D risk. Selected tag markers will be genotyped on our collection of 12,000 DNA samples (4,000 cases and both their parents) as well as in a smaller cohort of T1D patients (200) and controls (400) of African-American ancestry. The results will be analyzed by conditional regression to identify polymorphisms whose association with T1D cannot be accounted for by LD to another effect within the block. In Aim 2a, we propose a large-scale, high throughput evaluation of allelic effects, in cis, of the T1D-associated polymorphisms on transcription, splicing and translation. In Aim 2b, we will determine the cellular phenotypes encoded by the T1D-associated variants using peripheral blood cells from cases and healthy controls. For genes with known function, such as IFIH1, PTPN22, RASGRP1, BACH2, UBASH3, this function will be compared, ex vivo, between cells from individuals of different genotypes. For genes whose function is not known, such as CLEC16A, the first step will be to define its function by over-expression and SiRNA knockdown experiments; In Aim 2c, we will use animal models of human loci as an important source of clues to function. We also propose to create knock-in (KI) mice carrying the two alleles of the human polymorphism, starting with the well-mapped loci involving coding variants in the PTPN22 and IFIH1 genes and proceeding to the CLEC16A mose. The mice will be crossed to the NOD background and the effect of the human polymorphism on diabetes susceptibility and on specific aspects of immune dysregulation will be studied. In Aim 2d, we will evaluate epigenetic mechanisms through the analysis of methylation patterns within regulatory regions of genes with expression patterns that correlate with T1D risk. In Aim3a, we will perform detailed phenotype- genotype correlations studies of all loci re-sequenced in search for allele-specific genotype effects. In Aim 3b, we will establish a predictive test using SNP markers that associate most strongly with T1D with sensitivity and specificity that is of clinical relevance in predicting T1D risk. We anticipate these studies will markedly enhance our understanding of the pathogenesis of T1D. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Type 1 Diabetes (T1D) is a multifactorial childhood disease with a strong genetic component. It is one of the most common severe chronic childhood diseases, with life expectancy severely shortened and quality of life compromised. It is caused by autoimmune destruction of the pancreatic beta cells with resulting lack of production of insulin. Although we and other groups have identified several T1D loci through recent genome- wide association studies, the actual causal mechanisms by which these loci confer risk remain unknown. This project proposes to 1) discover all variants within the loci that may account for the observed association and determine their independent effects on T1D risk; 2) functionally characterize the variants; and 3) further characterize the etiological pathways through comprehensive genotype/phenotype correlations and establish a proof of concept for use of these variants in risk stratification and future therapeutic guidance (ie, personalized medicine).