A multistep approach for the analysis of Hereditary Hearing Loss genes in deaf patients: looking for a needle in a haystack.

DALAMÓN, VIVIANA KARINA; BUONFIGLIO PAULA; LOTERSZTEIN VANESA; GOLDSCHMIDT E; ANA BELEN ELGOYHEN

Mar del Plata

Congreso; LXI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA; 2016

Sociedad Argentina de Investigacion Clinica

Introduction: Hereditary Hearing Loss (HHL) is a common trait affecting 1 in 2000 children. It is characterized by the presence of a large genetic heterogeneity, and to date, over 100 different genes have been identified worldwide. In order to overcome this problem, we designed a multistep strategy: Step 1) screening of frequent mutations in 11 HHL genes by direct sequencing; Step 2) screening of 120 HHL genes by targeted -sequencing (TS); Step 3) validation of identified mutations by in-silico studies and design of functional in-vivo analysis. Methods: A total of 1150 samples were analyzed; 600 from non-syndromic unrelated Argentinean deaf patients (135 familial and 465 sporadic cases) and 550 from relatives and siblings. The first step of the study consisted in investigating and reporting the spectrum and frequency of reported mutations in GJB2, GJB6, OTOF, MT-RNR1, PJVK,TECTA, EYA4, EYA1, SIX1, TMC1, COL4A5 and COCH genes in deaf patients from Argentina. 10 samples that resulted negative for searched mutations in step 1, were candidates for following analysis in step 2 through a commercial optimized platform of massive sequencing for deafness diagnostic. Targeted genomic enrichment was performed to isolate and enrich all exons of 120 genes known to cause non-syndromic hearing loss and some forms of syndromic hearing loss. After genome variants annotation, data were filtered according to quality value, allele frequency, pathogenicity prediction and conservation score. Results: Algorithm performed by direct sequencing in Step 1 allowed us to detect 44 different sequence variations in 252 of the 600 patients (42%) located in genes GJB2, GJB6, OTOF and EYA1. Target genomic enrichment and massive sequencing of 120 HHL genes were performed in 7 samples so far, in the remaining 3 samples data are under the final step of analysis. Overall, 22 different genes presented 37 variations. All of the patients were positively characterized leading to the identification of pathogenic variants. Nevertheless, as many genes were analyzed, in some patients we detected 2, 4 or 8 mutated genes, making genotype/phenotype relationship difficult to assess. We show in the present study some clearcut results, others that are uncertain, and also a third set requiring further analysis or subsequent functional studies to establish which mutations underlay the pathology. Functional studies of some of the identified mutations using Zebra fish models followed by an accurate phenotypic characterization are under way. Conclusions: These findings clearly highlight the importance of genetic studies followed by animal model validation to better understand the genetic basis of HHL.