title: Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform.
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.
authors.affiliation: NXTGNT, Ghent University, Ghent, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: De Keulenaer
authors.forename: Jan
authors.initials: J
authors.lastname: Hellemans
authors.forename: Steve
authors.initials: S
authors.lastname: Lefever
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Renard
authors.forename: Joachim
authors.initials: J
authors.lastname: De Schrijver
authors.forename: Hendrik
authors.initials: H
authors.lastname: Van de Voorde
authors.forename: Mohammad Amin
authors.initials: MA
authors.lastname: Tabatabaiefar
authors.forename: Filip
authors.initials: F
authors.lastname: Van Nieuwerburgh
authors.forename: Daisy
authors.initials: D
authors.lastname: Flamez
authors.forename: Filip
authors.initials: F
authors.lastname: Pattyn
authors.forename: Bieke
authors.initials: B
authors.lastname: Scharlaken
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
authors.forename: Sofie
authors.initials: S
authors.lastname: Bekaert
authors.forename: Wim
authors.initials: W
authors.lastname: Van Criekinge
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.forename: Guy
authors.initials: G
authors.lastname: Van Camp
authors.forename: Paul
authors.initials: P
authors.lastname: Coucke
mesh_terms: D000072259:Connexin 26; D017630:Connexins; D003638:Deafness / Q000175:diagnosis* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction
chemical_list: D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26