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

BUONFIGLIO, PAULA; DALAMÓN, VIVIANA KARINA; LOTERSZTEIN, VANESA; GOLDSCHMIDT, ERNESTO; ELGOYHEN, ANA BELÉN

Caixas do Sul, Rio Grande do Sul

Workshop; XIII Curso Escola Latino-Americana de Genética Humana e Médica; 2017

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 GJB2 and GJB6 which are the most common disease causing genes, by direct sequencing; Step 2) screening of frequent mutation in another 10 HHL genes by direct sequencing according to patient`s phenotype; Step 3) Screening of 155 HHL genes by Whole Exome-sequencing (WES); Step 4) Analysis of Potential Disease-Causing Mutations by in-silico studies and pending functional validation by in-vivo analysis. There are some models which can be used in our laboratory (Xenopus laevis Ooocytes and zebra- fish models); however, the large amount of genes involved in the pathology make this step still challenging.A total of 1181 samples were analyzed so far; 631from non-syndromic unrelated Argentinean deaf patients 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 in deaf patients from Argentina. In step 2, we expand the screening in OTOF, MT-RNR1, PJVK, TECTA, EYA4, EYA1, SIX1, TMC1, COL4A5 and COCH genes. In step 3, 5 samples were candidates for Whole- Exome Sequencing. After genome variants annotation, data were filtered according to specific hearing loss genes (155), mode of inheritance, variations?s localization, allele frequency, pathogenicity prediction and conservation score, and other evidences. Direct sequencing allowed us to detect 47 different sequence variations in 245 of the 631 patients (39%) located in genes GJB2 and GJB6. Then, we study MT-RNR1, OTOF, EYA4, TECTA, PJVK, TMC1, COCH, COL4A5 and SLC26A4 in 200 of the patients who were normal for GJB2 and GJB6 obtaining a low raise in the percentage of diagnosed patients. Whole-Exome Sequencing in 5 samples showed, after filtering process, 11 variations in 9 different genes. Most of the patients were positively characterized leading to the identification of potential disease causing mutations. Nevertheless, as many genes were analyzed, in some patients we detected 3 or 4 suspicious variations, making genotype/phenotype relationship difficult to assess. In one of the analyzed patient no mutation was detected in targeted genes; therefore, familial analysis is in progress. 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. These findings clearly highlight the importance of genetic studies followed by in sílico and in-vivo analysis to better understand the genetic basis of HHL.