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: 9100744
• 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/9100744
• 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-in; Knock-out; Knockout Mice; Maps; Mediating; Mining; Mutation; PTPN22 gene; Pathogenesis; Patients; Peripheral Blood Mononuclear Cell; Phase; Phenotype; Population; Preventive therapy; 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; predictive tools; risk variant; success

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.

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，致病的遗传变异及其作用基因尚未确定。作为生殖系因素，遗传风险变异是存在的，而且随时都可以研究--在糖尿病发生之前、期间和之后。因此，遗传信息可以作为一种潜在的预测工具，并提供对疾病临床前阶段可能应用预防性治疗期间发生的发病机制的洞察。在这项建议中，我们描述了一种识别T1D风险变异并描述其功能的方法，该方法侧重于有3个或更多受影响的兄弟姐妹的T1D家庭，其特征是发病年龄较早。在这些不同寻常的“高危”T1D家系中，我们使用测序来识别罕见的、明显有害的变异，这些变异影响位于先前GWAS研究中定义的T1D风险区域的基因。我们的前提是，任何导致疾病风险的基因不太可能只有一个共同的风险变量，而应该包含具有一定频率范围的多个风险等位基因。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更容易识别的影响，既可以从与T1D相关的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中鉴定了一个新的与T1D相关的有害变体，它影响基因的剪接，导致产生一种独特的蛋白质亚型，其功能与野生型等位基因的产物不同。OU的初步数据显示，UBASH3A在人类T细胞激活中发挥了重要作用，这是以前的小鼠基因敲除研究没有预测到的。拟议的研究以两种方式建立在这些发现的基础上。首先，我们进行UBASH3A的机制研究，以确定其在人类T细胞中的功能，了解其在核中先前未被认识的角色，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，允许引入特定的突变构建物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，将整个外显子组测序扩展到更多的高危T1D家族，改进我们的致病变异筛选方法，并识别新的导致T1D发病机制的基因，这些基因可以作为功能研究的靶点。