title: Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome.
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.
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Krawitz
authors.affiliation: Department of Audiology and Phoniatrics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Schiska
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Krüger
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Appelt
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Heinrich
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Parkhomchuk
authors.affiliation: Max Planck Institute for Molecular Genetics Berlin, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Timmermann
authors.affiliation: Unidad de Genetica, Hospital Universitario La Fe and CIBERER Valencia, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Millan
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Peter N
authors.initials: PN
authors.lastname: Robinson
authors.affiliation: Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mundlos
authors.affiliation: Berlin Brandenburg Center for Regenerative Therapies BCRT Berlin, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Hecht
authors.affiliation: Department of Audiology and Phoniatrics, Charité Universitätsmedizin Berlin Berlin, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Gross
keywords: Gene panel diagnostics; Usher syndrome; next-generation sequencing