Abstract

BackgroundHereditary 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.ResultsIn 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.ConclusionsWe 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.

Highlights

  • Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing

  • Five patients with familial congenital deafness were screened for 15 deafness genes on the 454 Genome Sequencer in order to evaluate if generation sequencing enables the detection of mutations

  • We have developed an assay to improve the molecular diagnosis of autosomal recessive nonsyndromic hearing loss (ARNSHL) by simultaneous sequencing of the Prediction (PolyPhen-2)

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Summary

Introduction

Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). GJB2 mutations are the most frequent cause of autosomal recessive non-syndromic hearing loss (ARNSHL) and account for about 20 % of the cases [3]. Newborns that are diagnosed with severe-to-profound HL in the absence of other abnormal findings on physical examination are analyzed for mutations in the GJB2 gene. In some cases, when imaging of the inner ear shows abnormalities such as an enlarged vestibular aquaduct, the SLC26A4 gene is analyzed. Besides these genes there is hardly any other gene that is routinely analyzed in DNA diagnostics. Sequencing of all genes by traditional DNA sequencing technology is labor intensive and not cost effective [5]

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