Fine Mapping and Functional Evaluation of Selected Type 1 Diabetes Loci

选定 1 型糖尿病基因座的精细绘图和功能评估

• 批准号: 7798886
• 负责人: Hakon Hakonarson
• 金额: $ 477.39万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-20 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7798886
• 关键词: Accounting; African American; Alleles; Animal Model; Autoimmune Process; Blood Cells; Cells; Childhood; Chronic; Code; Collection; Correlation Studies; DNA; Diabetes Mellitus; Disease; Epigenetic Process; Evaluation; Frequencies; Future; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genome; Genotype; Human; Immune; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Knock-in Mouse; Life Expectancy; Linkage Disequilibrium; Maps; Medicine; Methylation; Mus; Nucleic Acid Regulatory Sequences; PTPN22 gene; Parents; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Predisposition; Production; Quality of life; RNA Splicing; Research; Risk; Sampling; Sensitivity and Specificity; Source; Stratification; Structure of beta Cell of islet; Testing; Therapeutic; Translations; Variant; clinically relevant; cohort; diabetes risk; genome wide association study; public health relevance; research study

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).

描述（由申请人提供）：拟议研究的总体目标是精细定位包含常见风险变体的选定T1 D基因座，并确定其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）块内的所有变体，其频率足够高，以解释在这些位点观察到的关联。我们选择了八个基因座，所有这些基因座都涉及免疫机制，所有这些基因座都已得到很好的建立和复制。将对300个病例的所有基因座进行重新测序，选择携带尽可能多的易感等位基因的病例;对照组将来自1000个基因组计划。在目标1b中，我们建议评估LD区组内的所有变体对T1 D风险的独立影响。选择的标签标记将在我们收集的12，000份DNA样本（4，000例病例及其父母）以及非洲裔美国人血统的T1 D患者（200例）和对照组（400例）的较小队列中进行基因分型。将通过条件回归分析结果，以确定与T1 D相关的多态性不能通过LD解释为区组内的另一种效应。在目标2a中，我们提出了一个大规模的，高通量的等位基因效应，顺式，T1 D相关的多态性对转录，剪接和翻译的评价。在目标2b中，我们将使用来自病例和健康对照的外周血细胞确定T1 D相关变体编码的细胞表型。对于具有已知功能的基因，例如IFIH 1、PTPN 22、RASGRP 1、BACH 2、UBASH 3，将在来自不同基因型个体的细胞之间离体比较该功能。对于功能未知的基因，如CLEC 16 A，第一步将是通过过表达和SiRNA敲低实验来确定其功能;在Aim 2c中，我们将使用人类基因座的动物模型作为功能线索的重要来源。我们还建议创建携带人类多态性的两个等位基因的敲入（KI）小鼠，从涉及PTPN 22和IFIH 1基因中的编码变体的良好定位的基因座开始，并继续到CLEC 16 A基因。将小鼠与NOD背景杂交，并研究人类多态性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T1 D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机制。在Aim 3a中，我们将对重新测序的所有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基因型效应。在目标3b中，我们将使用与T1 D相关性最强的SNP标记建立预测性测试，其灵敏度和特异性在预测T1 D风险方面具有临床相关性。我们预计这些研究将显着提高我们对T1 D发病机制的理解。 公共卫生相关性： 1型糖尿病（T1 D）是一种多因素的儿童疾病，具有很强的遗传成分。它是最常见的严重慢性儿童疾病之一，预期寿命严重缩短，生活质量受损。它是由胰腺β细胞的自身免疫性破坏引起的，导致缺乏胰岛素的产生。虽然我们和其他研究小组通过最近的全基因组关联研究已经确定了几个T1 D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）发现基因座内可能解释观察到的关联的所有变体，并确定它们对T1 D风险的独立影响; 2）功能性表征变体;以及3）通过综合的基因型/表型相关性进一步表征病因学途径，并建立在风险分层和未来治疗指导中使用这些变体的概念证明（即个性化医疗）。

项目成果

Yeast one-hybrid screen of a thymus epithelial library identifies ZBTB7A as a regulator of thymic insulin expression.

胸腺上皮文库的酵母单杂交筛选将 ZBTB7A 鉴定为胸腺胰岛素表达的调节剂。

• DOI: 10.1016/j.molimm.2013.05.238
• 发表时间: 2013
• 期刊: Molecular immunology
• 影响因子: 3.6
• 作者: St-Jean,JulienR;Ounissi-Benkalha,Houria;Polychronakos,Constantin
• 通讯作者: Polychronakos,Constantin

Expression profile of a clonal insulin-expressing epithelial cell in the thymus.

胸腺中表达胰岛素的克隆上皮细胞的表达谱。

• DOI: 10.1016/j.molimm.2013.07.015
• 发表时间: 2013
• 期刊: Molecular immunology
• 影响因子: 3.6
• 作者: Levi,Dina;Polychronakos,Constantin
• 通讯作者: Polychronakos,Constantin

Genes involved in type 1 diabetes: an update.

• DOI: 10.3390/genes4030499
• 发表时间: 2013-09-16
• 期刊: Genes
• 影响因子: 3.5
• 作者: Bakay M;Pandey R;Hakonarson H
• 通讯作者: Hakonarson H

CLEC16A regulates splenocyte and NK cell function in part through MEK signaling.

CLEC16A 部分通过 MEK 信号传导调节脾细胞和 NK 细胞功能。

• DOI: 10.1371/journal.pone.0203952
• 发表时间: 2018
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Pandey,Rahul;Bakay,Marina;Hain,HeatherS;Strenkowski,Bryan;Elsaqa,BarakatZB;Roizen,JeffreyD;Kushner,JakeA;Orange,JordanS;Hakonarson,Hakon
• 通讯作者: Hakonarson,Hakon