Open Access Research article

Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform

Sarah De Keulenaer1, Jan Hellemans12, Steve Lefever2, Jean-Pierre Renard3, Joachim De Schrijver3, Hendrik Van de Voorde2, Mohammad Amin Tabatabaiefar56, Filip Van Nieuwerburgh14, Daisy Flamez13, Filip Pattyn2, Bieke Scharlaken1, Dieter Deforce14, Sofie Bekaert1, Wim Van Criekinge13, Jo Vandesompele12, Guy Van Camp5 and Paul Coucke12*

Author Affiliations

1 NXTGNT, Ghent University, Ghent, Belgium

2 Center for Medical Genetics, Ghent University, Ghent, Belgium

3 Biobix, Laboratory for Bioinformatics and Computational Genomics, Ghent University, Ghent, Belgium

4 Laboratory for Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium

5 Department of Medical Genetics, University of Antwerp, Antwerp, Belgium

6 Department of Medical Genetics, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

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BMC Medical Genomics 2012, 5:17  doi:10.1186/1755-8794-5-17

Published: 18 May 2012

Abstract

Background

Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.

Results

In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.

Conclusions

We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.

Keywords:
Deafness; Next generation sequencing; PCR based enrichment; Genetic diagnostics