Novel T1D risk variants from genomic analyses in high risk families

高风险家庭基因组分析中发现的新型 T1D 风险变异

• 批准号: 8955744
• 负责人: Patrick Concannon
• 金额: $ 33.75万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8955744
• 关键词: Address; Affect; Age; Alleles; Biological Assay; Cell Nucleus; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cytoplasm; Data; Development; Diabetes Mellitus; Disease; Environmental Risk Factor; Family; Frequencies; Genes; Genetic; Genetic Risk; Genomic approach; Genomics; Genotype; Health; Human; Insulin-Dependent Diabetes Mellitus; Jurkat Cells; Knock-out; Knockout Mice; Maps; Mediating; Mining; Mutation; PTPN22 gene; Pathogenesis; Patients; Peripheral Blood Mononuclear Cell; Phase; Phenotype; Population; Preventive; Process; Production; Protein Isoforms; Proteins; Proteomics; Risk; Role; Siblings; Signal Transduction; Site; Site-Directed Mutagenesis; Spliced Genes; T-Cell Activation; T-Lymphocyte; TCR Activation; Testing; Time; Variant; base; disorder risk; exome sequencing; gene function; genetic information; genetic risk factor; genetic variant; genome wide association study; heterokaryon; high risk; improved; insight; mutant; novel; pre-clinical; risk variant; success; tool

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. Our 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.

描述（由申请人提供）：通过全基因组关联研究（GWAS），已经绘制了超过40个含有T1D风险基因座的染色体区域。然而，对于这些区域中的大多数，致病的遗传变异以及它们作用的基因尚未被确定。作为生殖系因素，遗传风险变异是存在的，并且在糖尿病发展之前，期间和之后的任何时候都可以进行研究。因此，遗传信息可以作为一个潜在的预测工具，以及提供洞察发病机制发生在临床前阶段的疾病，其中预防性治疗可能适用。在这项提案中，我们描述了一种用于识别T1D风险变体并表征其功能的方法，该方法侧重于具有3个或更多受影响兄弟姐妹的T1D家族，其特征在于发病年龄较早。在这些不寻常的“高风险”T1D家族中，我们使用测序来鉴定罕见的，明显有害的变异，这些变异影响了先前GWAS研究中定义的T1D风险区域中的基因。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 常见的风险变异，而是应该包含多个风险等位基因的频率范围。识别风险等位基因，虽然频率较低，但具有更明显和更容易识别的影响 对基因功能的研究既可以从T1D相关染色体区域的许多基因中精确定位相关基因，又可以深入了解其在T1D发病机制中的作用模式。在初步研究中，我们已经使用这种方法来鉴定UBASH3A中一种新的T1D相关有害变体，该变体影响基因的剪接，导致产生一种独特的蛋白质同种型，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细胞活化中的重要作用，而不是从先前的小鼠敲除研究中预测的。 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进行了机制研究，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活化的影响。与此同时，我们建议扩展我们的基因组方法，导致这些发现，将整个外显子组测序扩展到其他高风险T1D家族，改进我们的致病变体过滤方法，并确定有助于T1D发病机制的新基因，这些基因可以作为功能研究的目标。