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      Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations

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          Abstract

          Purpose:

          Targeted next-generation sequencing provides a remarkable opportunity to identify variants in known disease genes, particularly in extremely heterogeneous disorders such as nonsyndromic hearing loss. The present study attempts to shed light on the complexity of hearing impairment.

          Methods:

          Using one of two next-generation sequencing panels containing either 80 or 129 deafness genes, we screened 30 individuals with nonsyndromic hearing loss (from 23 unrelated families) and analyzed 9 normal-hearing controls.

          Results:

          Overall, we found an average of 3.7 variants (in 80 genes) with deleterious prediction outcome, including a number of novel variants, in individuals with nonsyndromic hearing loss and 1.4 in controls. By next-generation sequencing alone, 12 of 23 (52%) probands were diagnosed with monogenic forms of nonsyndromic hearing loss; one individual displayed a DNA sequence mutation together with a microdeletion. Two (9%) probands have Usher syndrome. In the undiagnosed individuals (10/23; 43%) we detected a significant enrichment of potentially pathogenic variants as compared to controls.

          Conclusion:

          Next-generation sequencing combined with microarrays provides the diagnosis for approximately half of the GJB2 mutation–negative individuals. Usher syndrome was found to be more frequent in the study cohort than anticipated. The conditions in a proportion of individuals with nonsyndromic hearing loss, particularly in the undiagnosed group, may have been caused or modified by an accumulation of unfavorable variants across multiple genes.

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          Most cited references21

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          QuantiSNP: an Objective Bayes Hidden-Markov Model to detect and accurately map copy number variation using SNP genotyping data

          Array-based technologies have been used to detect chromosomal copy number changes (aneuploidies) in the human genome. Recent studies identified numerous copy number variants (CNV) and some are common polymorphisms that may contribute to disease susceptibility. We developed, and experimentally validated, a novel computational framework (QuantiSNP) for detecting regions of copy number variation from BeadArray™ SNP genotyping data using an Objective Bayes Hidden-Markov Model (OB-HMM). Objective Bayes measures are used to set certain hyperparameters in the priors using a novel re-sampling framework to calibrate the model to a fixed Type I (false positive) error rate. Other parameters are set via maximum marginal likelihood to prior training data of known structure. QuantiSNP provides probabilistic quantification of state classifications and significantly improves the accuracy of segmental aneuploidy identification and mapping, relative to existing analytical tools (Beadstudio, Illumina), as demonstrated by validation of breakpoint boundaries. QuantiSNP identified both novel and validated CNVs. QuantiSNP was developed using BeadArray™ SNP data but it can be adapted to other platforms and we believe that the OB-HMM framework has widespread applicability in genomic research. In conclusion, QuantiSNP is a novel algorithm for high-resolution CNV/aneuploidy detection with application to clinical genetics, cancer and disease association studies.
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            Testing for an Unusual Distribution of Rare Variants

            Introduction High throughput sequencing of the human genome is now a reality: recent advances in sequencing technology now permit near complete ascertainment of genetic variation, including rare variants ( p0), some neutral, and some protective (pi
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              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.
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                Author and article information

                Journal
                Genet Med
                Genet. Med
                Genetics in Medicine
                Nature Publishing Group
                1098-3600
                1530-0366
                December 2014
                29 May 2014
                : 16
                : 12
                : 945-953
                Affiliations
                [1 ]Institute of Human Genetics, Julius-Maximilians-Universität Würzburg , Würzburg, Germany
                [2 ]Department of Bioinformatics, Julius-Maximilians-Universität Würzburg , Würzburg, Germany
                [3 ]Institute of Human Genetics, University Medical Centre, Johannes Gutenberg University , Mainz, Germany
                [4 ]Division of Communication Disorders, Department of Otorhinolaryngology, University Medical Centre, Johannes Gutenberg University , Mainz, Germany
                [5 ]Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, Comprehensive Hearing Center, Julius-Maximilians-Universität Würzburg , Würzburg, Germany.
                Author notes
                Article
                gim201465
                10.1038/gim.2014.65
                4262760
                24875298
                31c9f489-cdf6-4254-bda1-1b076f12bea0
                Copyright © 2014 American College of Medical Genetics and Genomics

                This work is licensed under a Creative Commons Attribution 3.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/

                History
                : 24 January 2014
                : 06 May 2014
                Categories
                Original Research Article

                Genetics
                deafness gene panel,mutational load,nonsyndromic hearing loss,sensorineural hearing loss,targeted next-generation sequencing

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