Novel T1D risk variants from genomic analyses in high risk families

描述（由申请人提供）：通过全基因组关联研究（GWAS），已经绘制了40多个携带T1D风险位点的染色体区域。然而，对于这些区域中的大多数，致病的遗传变异及其作用的基因尚未被确定。作为种系因素，遗传风险变异在糖尿病发生之前、期间和之后的任何时候都是存在的，并且可以进行研究。因此，遗传信息可以作为一种潜在的预测工具，并提供在疾病的临床前阶段发生的发病机制的见解，预防治疗可能被应用。在本提案中，我们描述了一种识别T1D风险变异并表征其功能的方法，该方法侧重于有3个或更多患病兄弟姐妹的T1D家庭，其特征是发病年龄早。在这些不寻常的“高风险”T1D家族中，我们使用测序来识别影响先前GWAS研究中定义的T1D风险区域基因的罕见的、明显有害的变异。我们的前提是，任何导致疾病风险的基因都不可能只有一个共同的风险变异，而是应该包含多个频率范围内的风险等位基因。识别风险等位基因，虽然不太常见，但对基因功能有更明显和容易识别的影响，既可以从许多T1D相关染色体区域中确定相关基因，也可以深入了解其在T1D发病机制中的作用模式。在初步研究中，我们使用这种方法在UBASH3A中鉴定了一种新的与t1d相关的有害变异，这种变异会影响该基因的剪接，导致产生一种独特的蛋白质异构体，其功能与野生型等位基因的产物不同。我们的初步数据揭示了UBASH3A在人类T细胞激活中的重要作用，这在之前的小鼠敲除研究中没有预测到。拟议的研究以两种方式建立在这些发现的基础上。首先，我们对UBASH3A进行机制研究，以确定其在人类T细胞中的功能，了解其在细胞核中以前未被认识到的作用，并确定其对T1D风险的贡献。为此，我们在Jurkat细胞中产生了基于CRISPR的UBASH3A敲除，允许引入特异性诱变的构建体来测试突变对细胞激活的影响。同时，我们建议扩展导致这些发现的基因组方法，将全外显子组测序扩展到额外的高风险T1D家族，改进我们对致病变异的过滤方法，并确定有助于T1D发病机制的新基因，这些基因可以用于功能研究。

DESCRIPTION (provided by applicant): More than 40 chromosomal regions harboring risk loci for T1D have been mapped through genome-wide association studies (GWAS). However, for most of these regions, the causative genetic variants, and the genes they act upon, have yet to be identified. As germline factors, genetic risk variants are present and amenable to study at all times - before, during and after the development of diabetes. Therefore, genetic information can serve as a potential predictive tool as well as provide insights into pathogenesis occurring during the preclinical phase of the disease where preventive therapies might be applied. In this proposal we describe an approach for identifying T1D risk variants and characterizing their function that focuses on T1D families with 3 or more affected siblings characterized by early ages at onset. In these unusual "high risk" T1D families, we use sequencing to identify rare, overtly deleterious variants affecting genes located in T1D risk regions defined in prior GWAS studies. Our premise is that any gene that contributes to disease risk is unlikely to have just one common risk variant, but rather should contain multiple risk alleles with a range of frequencies. Identifying risk alleles that, while less frequent, have more overt and readily discernible effects on gene function can both pinpoint the relevant gene from among many in a T1D-associated chromosomal region and provide insights into its mode of action in T1D pathogenesis. In preliminary studies we have used this approach to identify a novel T1D-associated deleterious variant in UBASH3A which affects the splicing of the gene, resulting in the production of a unique isoform of the protein that differs in function from the product of the wild type allele. Ou preliminary data reveal a significant role for UBASH3A in human T cell activation not predicted from prior mouse knockout studies. The proposed studies build upon these findings in two ways. First we pursue mechanistic studies of UBASH3A in order to define its function in human T cells, understand its previously unrecognized role in the nucleus and define its contribution to T1D risk. To this end, we have generated CRISPR based knockouts of UBASH3A in Jurkat cells allowing the introduction of specifically mutagenized constructs to test the effects of mutations on cell activation. In parallel, we propose to expand our genomic approach that led to these findings, extending whole exome sequencing to additional high risk T1D families, improving our filtering approaches for causative variants and identifying new genes contributing to T1D pathogenesis which can be targeted for functional studies.