Discovery of GJB2 cis-regulatory elements and missing DFNB1 mutation

发现 GJB2 顺式调控元件和缺失的 DFNB1 突变

• 批准号: 8292676
• 负责人: Ellen Shields Wilch
• 金额: $ 15.35万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292676
• 关键词: Affect; Alleles; Biological Assay; Carbon; Cell Line; Chromosomes; Code; Collection; DNA; DNA Resequencing; Data Set; Detection; Diagnosis; Diagnostics Research; Distant; Enhancers; Genes; Genetic; Genetic Enhancer Element; Genetic Predisposition to Disease; Genetic Transcription; Genome; Genomics; Hearing; Human Cell Line; Indium; Individual; Luciferases; Maps; Messenger RNA; Methods; Molecular Conformation; Mutation; Nucleic Acid Regulatory Sequences; Nucleotides; Patients; Persons; Population; Proxy; Regulatory Element; Relative (related person); Reporter; Reporter Genes; Sampling; Site; System; Testing; Variant; hearing impairment; histone modification; mutant; next generation; novel; promoter

DESCRIPTION (provided by applicant): Although the most common genetic cause of congenital hearing impairment is mutation in GJB2, a significant portion of the mutation load has not yet been discovered. Diagnostic and research labs have observed an excess of individuals with hearing loss who are identified with a GJB2 mutation on only one chromosome, for whom, consequently, a definitive diagnosis of genetic etiology cannot be made. Our lab identified and characterized a DFNB1 allele that contains a 131-kb deletion with a proximal breakpoint well away from the transcriptional start sites of both GJB2 and GJB6; this deletion segregates with reduced or lost expression of both GJB2 and GJB6 mRNA. The evidence of this and other important DFNB1 deletion alleles suggests that cis-regulatory elements for GJB2 and GJB6 exist at considerable distance from the genes themselves. We hypothesize that single nucleotide variants and small deletions affecting the function of distant enhancer elements constitute the 'missing' mutation among individuals with hearing loss who are heterozygous for a single DFNB1 mutation. We propose to advance the identification of missing DFNB1 mutation by a two-pronged approach. First, we propose to target next-generation sequencing to the DFNB1 locus, including the 131-kb interval in which GJB2-regulatory function has been documented. We will compare the sequences of 95 people with hearing loss and monoallelic mutation of GJB2 to three other sequence data sets: 96 population-matched controls, 1 hearing person in whom we have documented expression of both GJB2 and GJB6 mRNA, and any/all publicly available genomes (e.g. 1000 Genomes Project). These comparisons will yield a set of candidate regulatory mutations from the DFNB1 locus, that we hypothesize will comprise either one or several variants that are statistically over-represented among our patients, or a collection of novel variants that map as one or more clusters. Second, by chromosome conformation capture carbon-copy (5C) in a number of proxy cell lines, we will identify regions from within the DFNB1 locus that interact specifically with DNA at or near the GJB2 promoter. We will intersect the findings from each of these aims with consideration of public datasets of bioinformatically-predicted and experimentally-determined regulatory function to prioritize candidate regulatory regions, and associated candidate variants, for functional analysis by reporter assay. Interrogation of the DFNB1 locus by these several strategies will reveal previously unidentified DFNB1 mutation. PUBLIC HEALTH RELEVANCE: GJB2 mutations cause DFNB1 hearing loss; GJB2-regulatory mutation is known to exist but has not been identified. By targeted next-generation sequencing of a select patient set and comparison to appropriate controls, we will compile a prioritized set o candidate GJB2-regulatory mutations. We will also assess regulatory function in this locus by chromosome conformation capture in relevant cell lines, and test the effect of candidate mutations on enhancer function by reporter assay.

描述（由申请人提供）：虽然先天性听力障碍最常见的遗传原因是GJB2突变，但突变负荷的很大一部分尚未被发现。诊断和研究实验室观察到，有过多的听力损失患者仅在一条染色体上发现GJB2突变，因此无法对其进行明确的遗传病因诊断。我们的实验室鉴定并鉴定了一个DFNB1等位基因，该等位基因包含一个131-kb的缺失，其近端断点远离GJB2和GJB6的转录起始位点；这种缺失与GJB2和GJB6 mRNA的表达减少或缺失相分离。这个和其他重要的DFNB1缺失等位基因的证据表明，GJB2和GJB6的顺式调控元件存在于距离基因本身相当远的地方。我们假设，单核苷酸变异和影响远端增强子元件功能的小缺失构成了听力损失个体中单个DFNB1突变杂合的“缺失”突变。我们建议通过双管齐下的方法来推进缺失DFNB1突变的鉴定。首先，我们建议对DFNB1位点进行下一代测序，包括已记录的gjb2调节功能的131-kb区间。我们将95名听力损失和GJB2单等位基因突变患者的序列与其他三个序列数据集进行比较：96个群体匹配的对照组，1名我们记录了GJB2和GJB6 mRNA表达的听力患者，以及任何/所有公开可用的基因组（例如1000基因组计划）。这些比较将产生一组来自DFNB1位点的候选调节突变，我们假设这些突变将包括一个或几个在我们的患者中统计上过度代表的变异，或者是一个集合