Discovery of GJB2 cis-regulatory elements and missing DFNB1 mutation

描述(申请人提供)：虽然最常见的先天性听力障碍的遗传原因是GJB2的突变，但很大一部分突变负载尚未被发现。诊断和研究实验室观察到，有太多的听力损失患者只在一条染色体上发现了GJB2突变，因此无法对他们做出明确的遗传病因诊断。我们的实验室发现并鉴定了一个DFNB1等位基因，该等位基因包含一个131 kb的缺失，其近端的断裂点远离GJB2和GJB6的转录起始点；这种缺失分离出GJB2和GJB6的mRNA表达降低或丢失。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，在单个DFNB1突变杂合子的听力损失患者中，影响远距离增强子元件功能的单核苷酸变异和小的缺失构成了“缺失”突变。我们建议通过双管齐下的方法来鉴定缺失的DFNB1突变。首先，我们建议下一代测序的目标是DFNB1基因座，包括已经记录的GJB2调节功能的131kb区间。我们将把95名听力损失和GJB2单等位基因突变的患者的序列与其他三个序列数据集进行比较：96名人群匹配的对照组，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基因座的候选调控突变，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高的变异，或者一个集合 映射为一个或多个簇的新变体。其次，通过在一些代理细胞系中的染色体构象捕获碳拷贝(5C)，我们将从DFNB1基因座内识别与GJB2启动子或附近的DNA特异性相互作用的区域。我们将把来自每个目标的发现与考虑生物信息预测和实验确定的调控功能的公共数据集相结合，以确定候选调控区域和相关候选变异体的优先顺序，以便通过报告分析进行功能分析。通过这几种策略对DFNB1基因座的询问将发现以前未识别的DFNB1突变。

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.