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Mol Genet Genomic Med. 2014 Sep;2(5):393-401. doi: 10.1002/mgg3.92. Epub 2014 Jun 15.

Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome.

Author information

  • 1Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
  • 2Department of Audiology and Phoniatrics, Charité Universitätsmedizin Berlin Berlin, Germany.
  • 3Max Planck Institute for Molecular Genetics Berlin, Germany.
  • 4Unidad de Genetica, Hospital Universitario La Fe and CIBERER Valencia, Spain.
  • 5Berlin Brandenburg Center for Regenerative Therapies BCRT Berlin, Germany.

Abstract

Usher syndrome is an autosomal recessive disorder characterized both by deafness and blindness. For the three clinical subtypes of Usher syndrome causal mutations in altogether 12 genes and a modifier gene have been identified. Due to the genetic heterogeneity of Usher syndrome, the molecular analysis is predestined for a comprehensive and parallelized analysis of all known genes by next-generation sequencing (NGS) approaches. We describe here the targeted enrichment and deep sequencing for exons of Usher genes and compare the costs and workload of this approach compared to Sanger sequencing. We also present a bioinformatics analysis pipeline that allows us to detect single-nucleotide variants, short insertions and deletions, as well as copy number variations of one or more exons on the same sequence data. Additionally, we present a flexible in silico gene panel for the analysis of sequence variants, in which newly identified genes can easily be included. We applied this approach to a cohort of 44 Usher patients and detected biallelic pathogenic mutations in 35 individuals and monoallelic mutations in eight individuals of our cohort. Thirty-nine of the sequence variants, including two heterozygous deletions comprising several exons of USH2A, have not been reported so far. Our NGS-based approach allowed us to assess single-nucleotide variants, small indels, and whole exon deletions in a single test. The described diagnostic approach is fast and cost-effective with a high molecular diagnostic yield.

KEYWORDS:

Gene panel diagnostics; Usher syndrome; next-generation sequencing

PMID:
25333064
PMCID:
PMC4190874
DOI:
10.1002/mgg3.92
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