Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

The extreme genetic heterogeneity of nonsyndromic hearing loss (NSHL) makes genetic diagnosis expensive and time consuming using available methods. To assess the feasibility of target-enrichment and massively parallel sequencing technologies to interrogate all exons of all genes implicated in NSHL, we tested nine patients diagnosed with hearing loss. Solid-phase (NimbleGen) or solution-based (SureSelect) sequence capture, followed by 454 or Illumina sequencing, respectively, were compared. Sequencing reads were mapped using GSMAPPER, BFAST, and BOWTIE, and pathogenic variants were identified using a custom-variant calling and annotation pipeline (ASAP) that incorporates publicly available in silico pathogenicity prediction tools (SIFT, BLOSUM, Polyphen2, and Align-GVGD). Samples included one negative control, three positive controls (one biological replicate), and six unknowns (10 samples total), in which we genotyped 605 single nucleotide polymorphisms (SNPs) by Sanger sequencing to measure sensitivity and specificity for SureSelect-Illumina and NimbleGen-454 methods at saturating sequence coverage. Causative mutations were identified in the positive controls but not in the negative control. In five of six idiopathic hearing loss patients we identified the pathogenic mutation. Massively parallel sequencing technologies provide sensitivity, specificity, and reproducibility at levels sufficient to perform genetic diagnosis of hearing loss.

[1]  I. Schrijver,et al.  Genotyping with a 198 Mutation Arrayed Primer Extension Array for Hereditary Hearing Loss: Assessment of Its Diagnostic Value for Medical Practice , 2010, PloS one.

[2]  Emily H Turner,et al.  Target-enrichment strategies for next-generation sequencing , 2010, Nature Methods.

[3]  P. Stankiewicz,et al.  Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy. , 2010, The New England journal of medicine.

[4]  Eamonn Sheridan,et al.  Genetic diagnosis of familial breast cancer using clonal sequencing , 2010, Human mutation.

[5]  H. Rehm,et al.  High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays , 2010, BMC biotechnology.

[6]  P. Shannon,et al.  Exome sequencing identifies the cause of a Mendelian disorder , 2009, Nature Genetics.

[7]  M. Metzker.  Sequencing technologies — the next generation , 2010, Nature Reviews Genetics.

[8]  S. Nelson,et al.  Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing , 2009, BMC Genomics.

[9]  I. Tikhonova,et al.  Genetic diagnosis by whole exome capture and massively parallel DNA sequencing , 2009, Proceedings of the National Academy of Sciences.

[10]  K. Frazer,et al.  Enrichment of sequencing targets from the human genome by solution hybridization , 2009, Genome Biology.

[11]  Richard J. H. Smith,et al.  Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? , 2009, Mutation research.

[12]  Adam P. DeLuca,et al.  Mutation in the COCH gene is associated with superior semicircular canal dehiscence , 2009, American journal of medical genetics. Part A.

[13]  J. Maguire,et al.  Solution Hybrid Selection with Ultra-long Oligonucleotides for Massively Parallel Targeted Sequencing , 2009, Nature Biotechnology.

[14]  Emily H Turner,et al.  Targeted Capture and Massively Parallel Sequencing of Twelve Human Exomes , 2009, Nature.

[15]  Timothy B. Stockwell,et al.  Evaluation of next generation sequencing platforms for population targeted sequencing studies , 2009, Genome Biology.

[16]  Adam P. DeLuca,et al.  Audioprofile-directed screening identifies novel mutations in KCNQ4 causing hearing loss at the DFNA2 locus , 2008, Genetics in Medicine.

[17]  E. Aller,et al.  Mutation profile of the CDH23 gene in 56 probands with Usher syndrome type I , 2008, Human mutation.

[18]  M. Behlke,et al.  Advances in Molecular and Cellular Therapies for Hearing Loss. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[19]  P. Heyning,et al.  A splice-site mutation and overexpression of MYO6 cause a similar phenotype in two families with autosomal dominant hearing loss , 2008, European Journal of Human Genetics.

[20]  Andrew J. Grimm,et al.  Interpreting missense variants: comparing computational methods in human disease genes CDKN2A, MLH1, MSH2, MECP2, and tyrosinase (TYR) , 2007, Human mutation.

[21]  K. Kahrizi,et al.  Sensorineural deafness and male infertility: a contiguous gene deletion syndrome , 2006, Journal of Medical Genetics.

[22]  J. Bale,et al.  Sensorineural hearing loss in children , 2005, The Lancet.

[23]  W. Kimberling,et al.  Prevalent connexin 26 gene (GJB2) mutations in Japanese , 2000, Journal of medical genetics.

[24]  J. W. Askew,et al.  Novel mutation in the KCNQ4 gene in a large kindred with dominant progressive hearing loss , 1999, Human mutation.

[25]  V. Sheffield,et al.  Carrier rates in the midwestern United States for GJB2 mutations causing inherited deafness. , 1999, JAMA.

[26]  P. Willems,et al.  Nonsyndromic hearing impairment: unparalleled heterogeneity. , 1997, American journal of human genetics.

[27]  G. Camp,et al.  The hereditary hearing loss homepage , 1997 .

[28]  N. E. MORTON,et al.  Genetic Epidemiology of Hearing Impairment , 1991, Annals of the New York Academy of Sciences.

[29]  A. Eisenberg,et al.  A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. , 1989, Nucleic acids research.

[30]  S. Liebowitz.  Retinitis pigmentosa. , 1979, Journal - American Intra-Ocular Implant Society.