Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 107; no. 49; pp. 21104 - 21109
• Main Authors: Shearer, A. Eliot, DeLuca, Adam P., Hildebrand, Michael S., Taylor, Kyle R., Gurrola, José, Scherer, Steve, Scheetz, Todd E., Smith, Richard J. H., King, Mary-Claire
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 07.12.2010; National Acad Sciences
• Subjects: Biological Sciences; Deafness; DNA; DNA Mutational Analysis; Exons; Genetic disorders; genetic heterogeneity; Genetic mutation; Genetic screening; Genetic testing; Genetic Testing - methods; Genomics; Genotype; Genotype & phenotype; hearing disorders; Hearing loss; Hearing Loss - genetics; Heterogeneity; high-throughput nucleotide sequencing; Human genetics; Humans; Medical diagnosis; Medical genetics; Mutation; Pathogenicity; Pathogens; patients; Polymorphism; Polymorphism, Single Nucleotide; prediction; Sequence Analysis, DNA - methods; Sequencing; Single-nucleotide polymorphism; Software; Usher syndromes
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1012989107

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 (Nimble-Gen) 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.