Genetic and epigenomic determinants of hearing loss in Hispanic populations

摘要感音神经性听力损失(SNHL)是致残的主要原因，影响约14亿人 全球各地的人，包括不同的年龄段和种族。尽管已经确定了大约150个基因 对于SNHL，西班牙裔人口对SNHL的研究仍然不足，大多数西班牙裔研究集中在 单基因GJB2。一些国家被研究--智利、墨西哥/拉美裔美国人、尼加拉瓜、菲律宾-- GJB2变异的患病率很低，这表明来自这些国家的SNHL队列具有新的基因 或用于发现的变种。此外，西班牙裔儿童在环境中暴露于化学品的风险很大。 这可能会导致表观遗传修饰并导致SNHL。我们的主要假设是SNHL有一个 西班牙裔人群中独特的、特定于群体的等位基因和表观遗传谱。我们 聚集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这个项目将是 工作效率高。在我们之前的研究中，我们在西班牙裔美国人和菲律宾人患者中发现了新的变异 SNHL，包括与颞骨异常相关的基因变异，并预测 人工耳蜗术后的结果。尼加拉瓜家庭被提交进行外显子组测序，结果为阴性 变异；这些家族可能含有非编码变异或具有SNHL的表观遗传机制。我们已经进入了 在家系和差异甲基化区域建立新的SNHL基因的高效鉴定管道 (DMRS)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 目的：确定西班牙裔儿童SNHL的遗传和表观遗传危险因素。对于目标1，我们将确定 SNHL变体来自使用分层方法的下一代序列数据，其包括Sanger测序， 筛选、纯合性作图、连锁分析和传递不平衡测试。我们将招募500人 西班牙裔家庭，包括足以在全基因组范围内显著连锁的大家庭，并提交DNA 对样品进行测序和分析。在这些家族中发现的新的SNHL基因和变种将被跟踪 在小鼠和斑马鱼模型中的蛋白质定位和听力研究以及 上皮细胞。对于目标2，我们将进行表观基因组范围的关联研究，方法是分析一个 根据年龄、性别和人口匹配的500名SNHL先证者和500名听力正常儿童组成的强大队列 以识别与SNHL相关联的DMR。对于排名靠前的DMR，我们将使用CRISPR-dCas9 对上皮细胞的技术，以确定靶向甲基化位置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的甲基化图谱和遗传数据将有助于 了解遗传和环境对SNHL的贡献。总体而言，该项目将对基因筛查产生影响 方案和遗传咨询，特别是在西班牙裔人群中，以及提高对 为SNHL的治疗开辟了新的靶点。

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.