Identification of New Hereditary Hearing Loss Genes Using High-Throughput Sequencing Technologies.

Hearing loss (HL) is the most frequent birth defect in developed societies affecting approximately 1 to 3 in every 1000 live births. HL is a remarkably complex and heterogeneous disease presenting with various phenotypes as a result of both genetic and environmental factors. Within genetic or hereditary hearing loss (HHL) about 70% of cases can be classified as non-syndromic hearing loss (NSHL), i.e. with the absence of abnormalities in other organs, and to date 158 NSHL loci and 95 genes have been reported as causative. Considering that the achievement of a correct molecular diagnosis is essential for uncovering the molecular mechanisms of hearing loss, in order to provide patients with prognostic information and personalized risk assessments and reduce public health costs, this study aims to define the genetic cause of hearing loss in a subset of NSHL familial cases coming from both Italy and Qatar. In order to overcome the high genetic heterogeneity of this disease and the fact that different major players seem to be involved in the Italian and Qatari populations, next generation sequencing techniques have been employed in this study. In particular, in the case of the Qatari population, this study represents the first high-throughput screening for the molecular diagnosis of hearing loss, being thus extremely valuable from an epidemiological point of view. As a first step, patients have been screened for 96 deafness-genes using a custom targeted re-sequencing (TRS) panel. Data analysis led to the identification of the molecular cause in 50% of all families, highlighting TECTA and MYO7A as major players in the Italian population, and CDH23 and TMC1 in the Qatari one. . Families negative to TRS have been selected for whole exome sequencing (WES) analysis, with the purpose of discovering new disease-related genes. So far two new candidates, SPATC1L and PLS1, in two Italian families have been identified. SPATC1L encodes the speriolin-like protein, whose function is still unknown. A novel stop variant has been identified in an Italian family affected by autosomal dominant NSHL (ADNSHL) and some functional studies (i.e. expression analysis in mouse whole cochleae, in vitro molecular cloning) together with statistical analysis (i.e. a candidate-gene population-based statistical study in cohorts from Caucasus and Central Asia) supported the role of this gene in hearing function and loss. In the case of PLS1, a new missense variant has been identified in an Italian ADNSHL family. The gene encodes the plastin-1 protein, which has already been associated to hearing loss in mice. The generation of a knock-in in the Zebrafish model (in collaboration with ZeClinics, a Biotech Contract Research Organization (CRO) and early-phase biopharmaceutical (PHARMA) company using Zebrafish for the study of human diseases, located in Barcelona, Spain) is now in progress and its gene expression in Zebrafish larvae inner ear has been preliminary confirmed. Altogether these results clearly proved that TRS followed by WES and functional studies are powerful tools for both the molecular diagnosis of NSHL, and the identification of new disease-related genes