Discovery of GJB2 cis-regulatory elements and missing DFNB1 mutation

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GJB 2突变，但尚未发现突变负荷的重要部分。诊断和研究实验室已经观察到过多的听力损失个体仅在一条染色体上被鉴定为GJB 2突变，因此无法对遗传病因进行明确诊断。我们的实验室鉴定并表征了一个DFNB 1等位基因，该等位基因包含一个131 kb缺失，其近端断点远离GJB 2和GJB 6的转录起始位点;这种缺失与GJB 2和GJB 6 mRNA的表达减少或丧失分离。这个和其他重要的DFNB 1缺失等位基因的证据表明，GJB 2和GJB 6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，单核苷酸变异和小的缺失影响远程增强子元件的功能，构成了“失踪”的突变个体与听力损失谁是一个单一的DFNB 1突变杂合子。我们建议通过双管齐下的方法来提高缺失DFNB 1突变的识别。首先，我们建议将下一代测序靶向DFNB 1位点，包括已记录GJB 2调节功能的131 kb间隔。我们将比较95名听力损失和GJB 2单等位基因突变患者的序列与其他三个序列数据集：96名人群匹配的对照，1名听力正常的人，我们记录了GJB 2和GJB 6 mRNA的表达，以及任何/所有公开可用的基因组（例如1000个基因组计划）。这些比较将产生一组来自DFNB 1基因座的候选调控突变，我们假设这些突变将包含一个或几个在我们的患者中统计学上过度代表的变体，或者一个集合 一个或多个簇的新变体。第二，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DFNB 1基因座内鉴定出与GJB 2启动子处或附近的DNA特异性相互作用的区域。我们将交叉这些目标中的每一个的研究结果，考虑生物信息学预测和实验确定的调控功能的公共数据集，以优先考虑候选调控区域和相关的候选变体，用于通过报告基因测定进行功能分析。通过这几种策略对DFNB 1基因座的询问将揭示先前未鉴定的DFNB 1突变。 公共卫生相关性：GJB 2突变导致DFNB 1听力损失;已知存在GJB 2调节突变，但尚未确定。通过对选定患者集进行靶向下一代测序并与适当的对照进行比较，我们将编制一组优先的候选GJB 2调节突变。我们还将通过相关细胞系中的染色体构象捕获来评估该位点的调控功能，并通过报告基因测定来测试候选突变对增强子功能的影响。

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.