Genetic and epigenomic determinants of hearing loss in Hispanic populations

摘要 感音神经性听力损失 (SNHL) 是导致残疾的主要原因，影响约 14 亿人 全球人民，包括不同年龄组和种族。尽管已鉴定出约 150 个基因 对于 SNHL，西班牙裔人群的 SNHL 仍然没有得到充分研究，大多数西班牙裔研究集中在 单基因GJB2。研究了一些国家——智利、墨西哥/西班牙裔美国人、尼加拉瓜、菲律宾—— GJB2 变异的流行率较低，表明来自这些国家的 SNHL 队列具有新基因 或发现的变体。此外，西班牙裔儿童面临环境中化学品暴露的风险 这可能会导致表观遗传修饰并导致 SNHL。我们的首要假设是 SNHL 有一个 西班牙裔人群中独特的、人群特异性的等位基因和表观遗传谱。我们 组建了一个国际研究小组，他们在耳科、遗传学、 表观基因组学和功能基因组学，以及之前的合作经验表明该项目将 高生产力。在我们之前的研究中，我们在西班牙裔美国人和菲律宾患者中发现了新的变异 SNHL，包括与颞骨异常相关并可预测的遗传变异 人工耳蜗植入效果。尼加拉瓜家庭接受了外显子组测序，结果呈阴性 变种；这些家族可能含有非编码变异或具有 SNHL 的表观遗传机制。我们有在 建立有效的管道来鉴定家族和差异甲基化区域中的新型 SNHL 基因 （DMR）在病例对照队列中，以及在动物模型和上皮细胞中的验证方法。我们的目标是 确定患有 SNHL 的西班牙裔儿童的遗传和表观遗传危险因素。对于目标 1，我们将确定 使用分层方法从下一代序列数据中获得 SNHL 变体，其中包括桑格测序、 过滤、纯合性作图、连锁分析和传递不平衡测试。我们将招募500名 西班牙裔家庭，包括足以进行全基因组显着连锁的大家庭，并提交 DNA 样品进行测序和分析。将跟踪在这些家族中发现的新 SNHL 基因和变异 进行小鼠和斑马鱼模型中的蛋白质定位和听力研究以及突变构建体 上皮细胞。对于目标 2，我们将通过分析一个基因的甲基化组来进行全表观基因组关联研究。 500 名 SNHL 先证者和 500 名听力儿童的强大队列，年龄、性别和人口相匹配， 为了识别与 SNHL 相关的 DMR。对于最常见的 DMR，我们将利用 CRISPR-dCas9 上皮细胞技术，以确定靶向甲基化位点是否会影响基因表达。整合 使用甲基化数量性状基因座分析来分析甲基化谱和遗传数据将有助于 了解遗传与环境对 SNHL 的贡献。总体而言，该项目将影响基因筛查 方案和遗传咨询，特别是针对西班牙裔人群，以及提高对遗传的了解 听力机制并导致 SNHL 治疗发展的新目标。

ABSTRACT Sensorineural hearing loss (SNHL) is a leading cause of disability and affects ~1.4 billion people globally, including different age groups and ethnicities. Although around 150 genes have been identified for SNHL, the Hispanic population remains understudied for SNHL, with most Hispanic studies focused on a single gene GJB2. A few countries studied – Chile, Mexico/Hispanic-American, Nicaragua, the Philippines – have a low prevalence of GJB2 variants, suggesting that SNHL cohorts from these countries have novel genes or variants for discovery. Additionally, Hispanic children are at risk for environmental exposures to chemicals that may lead to epigenetic modifications and cause SNHL. Our overarching hypothesis is that SNHL has a unique, population-specific allelic and epigenetic spectrum in Hispanic-descent populations. We assembled an international group of researchers with complementary expertise in otology, genetics, epigenomics and functional genomics, with previous collaborative experience that signals this project will be highly productive. In our previous studies, we identified novel variants in Hispanic-American and Filipino patients with SNHL, including genetic variants that were associated with temporal bone anomalies and predictive of cochlear implant outcomes. Nicaraguan families were submitted for exome sequencing and were negative for variants; these families likely harbor non-coding variants or have epigenetic mechanisms of SNHL. We have in place efficient pipelines for the identification of novel SNHL genes in families and differentially methylated regions (DMRs) in case-control cohorts, as well as validation methods in animal models and epithelial cells. Our goal is to determine genetic and epigenetic risk factors in Hispanic children with SNHL. For Aim 1, we will identify SNHL variants from next-generation sequence data using a tiered approach, which includes Sanger sequencing, filtering, homozygosity mapping, linkage analysis and transmission disequilibrium tests. We will recruit 500 Hispanic families, including large families sufficient for genome-wide significant linkage, and submit DNA samples to sequencing and analyses. Novel SNHL genes and variants identified in these families will be followed up with protein localization and hearing studies in mouse and zebrafish models and mutation constructs in epithelial cells. For Aim 2, we will perform an epigenome-wide association study by profiling the methylome of a well-powered cohort of 500 SNHL probands and 500 hearing children matched by age, sex and population, in order to identify DMRs that are associated with SNHL. For the top identified DMRs, we will utilize CRISPR-dCas9 technology on epithelial cells to determine if targeting the methylation site will affect gene expression. Integration of methylation profiles and genetic data using methylation quantitative trait locus analysis will aid in understanding genetic vs. environmental contributions to SNHL. Overall this project will impact genetic screening protocols and genetic counseling particularly in Hispanic populations, as well as improve understanding of the hearing mechanism and lead to new targets for the development of treatment of SNHL.