Expression and Replication Studies to Identify New Candidate Genes Involved in Normal Hearing Function

Considerable progress has been made in identifying deafness genes, but still little is known about the genetic basis of normal variation in hearing function. We recently carried out a Genome Wide Association Study (GWAS) of quantitative hearing traits in southern European populations and found several SNPs with suggestive but none with significant association. In the current study, we followed up these SNPs to investigate which of them might show a genuine association with auditory function using alternative approaches. Firstly, we generated a shortlist of 19 genes from the published GWAS results. Secondly, we carried out immunocytochemistry to examine expression of these 19 genes in the mouse inner ear. Twelve of them showed distinctive cochlear expression patterns. Four showed expression restricted to sensory hair cells (Csmd1, Arsg, Slc16a6 and Gabrg3), one only in marginal cells of the stria vascularis (Dclk1) while the others (Ptprd, Grm8, GlyBP, Evi5, Rimbp2, Ank2, Cdh13) in multiple cochlear cell types. In the third step, we tested these 12 genes for replication of association in an independent set of samples from the Caucasus and Central Asia. Nine out of them showed nominally significant association (pCDH13, GRM8, ANK2, SLC16A6, ARSG, RIMBP2 and DCLK1) showed an audiometric pattern with differences between different genotypes further supporting their role in hearing function. These data demonstrate the usefulness of this multistep approach in providing new insights into the molecular basis of hearing and may suggest new targets for treatment and prevention of hearing impairment.在致聋基因的鉴定领域已取得长足进展，但目前学界对听觉功能正常变异的遗传基础仍知之甚少。我们近期在南欧人群中开展了一项针对听觉数量性状的全基因组关联研究（Genome Wide Association Study, GWAS），结果发现了数个具备提示性关联的单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点，但未发现存在显著关联的位点。在本研究中，我们对上述SNPs进行了追踪验证，旨在通过多种替代方法探究其中哪些位点可能与听觉功能存在真实关联。首先，我们从已发表的GWAS结果中筛选出19个候选基因；其次，采用免疫细胞化学技术检测这19个基因在小鼠内耳中的表达情况，结果显示其中12个基因展现出独特的耳蜗表达模式：4个基因的表达仅局限于感觉毛细胞（Csmd1、Arsg、Slc16a6及Gabrg3），1个基因仅在血管纹边缘细胞中表达（Dclk1），其余7个基因则在多种耳蜗细胞类型中均有表达（Ptprd、Grm8、GlyBP、Evi5、Rimbp2、Ank2、Cdh13）；第三步，我们在一组独立的高加索及中亚人群样本中，对这12个基因的关联信号进行了验证，其中9个基因展现出名义显著性关联。其中CDH13、GRM8、ANK2、SLC16A6、ARSG、RIMBP2及DCLK1的听力学表型在不同基因型间存在差异，进一步支持了它们在听觉功能中的作用。本研究数据证实了该多步骤研究策略的实用性，可为听觉功能的分子机制研究提供新视角，并可为听力损伤的治疗与预防提供潜在新靶点。