Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies Tobias Eisenberger 1 , Christine Neuhaus 1 , Arif O. Khan 2 , Christian Decker 1 , Markus N. Preising 3 , Christoph Friedburg 3 , Anika Bieg 1 , Martin Gliem 4 , Peter Charbel Issa 4 , Frank G. Holz 4 , Shahid M. Baig 5 , Yorck Hellenbroich 6 , Alberto Galvez 2 , Konrad Platzer 6 , Bernd Wollnik 7,8,9 , Nadja Laddach 10 , Saeed Reza Ghaffari 11 , Maryam Rafati 12 , Elke Botzenhart 13 , Sigrid Tinschert 14,15 , Doris Bo ¨ rger 16 , Axel Bohring 17 , Julia Schreml 7,8 , Stefani Ko ¨ rtge-Jung 18 , Chayim Schell-Apacik 19 , Khadijah Bakur 20 , Jumana Y. Al-Aama 20 , Teresa Neuhann 21 , Peter Herkenrath 22 , Gudrun Nu ¨ rnberg 8,23 , Peter Nu ¨ rnberg 8,23 , John S. Davis 24 , Andreas Gal 25 , Carsten Bergmann 1,26 , Birgit Lorenz 3 , Hanno J. Bolz 1,7 * 1 Bioscientia Center for Human Genetics, Ingelheim, Germany, 2 Division of Pediatric Ophthalmology, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia, 3 Department of Ophthalmology, Justus-Liebig-University Giessen, University Hospital Giessen and Marburg GmbH, Giessen Campus, Giessen, Germany, 4 Department of Ophthalmology, University of Bonn, Bonn, Germany, 5 Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, 6 Institute of Human Genetics, University of Lu ¨ beck, Lu ¨ beck, Germany, 7 Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany, 8 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany, 9 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, 10 MRC Holland, Amsterdam, The Netherlands, 11 Comprehensive Genetic Center, Tehran University of Medical Sciences, Tehran, Iran, 12 Avicenna Biotechnology Research Institute, Tehran, Iran, 13 Pra ¨natalzentrum Hamburg und Humangenetik, Hamburg, Germany, 14 Institute of Clinical Genetics, Technical University Dresden, Dresden, Germany, 15 Division of Human Genetics, Medical University Innsbruck, Innsbruck, Austria, 16 Humangenetik, Bremen, Germany, 17 Institute of Human Genetics, Westfa ¨lische Wilhelms-University, Mu ¨ nster, Germany, 18 Praenatal-Medizin und Genetik Du ¨ sseldorf, Du ¨ sseldorf, Germany, 19 Praxis fu ¨ r Humangenetik am DRK-Klinikum Westend, Berlin, Germany, 20 Princess Al Jawhara Albrahim Center of Excellence in Research of Hereditary Disorders, King Abdulaziz University, Jeddah, Saudi Arabia, 21 Medizinisch Genetisches Zentrum, Munich, Germany, 22 Department of Pediatrics, University Hospital of Cologne, Cologne, Germany, 23 Cologne Center for Genomics and Center for Molecular Medicine, University of Cologne, Cologne, Germany, 24 Department of Ophthalmology, Zayed Military Hospital, Abu Dhabi, United Arab Emirates, 25 Institute of Human Genetics, University Medical Center Hamburg- Eppendorf, Hamburg, Germany, 26 Center for Clinical Research, University Hospital of Freiburg, Freiburg, Germany Abstract Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover ‘‘hidden mutations’’ such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 59 exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 59-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading. Citation: Eisenberger T, Neuhaus C, Khan AO, Decker C, Preising MN, et al. (2013) Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies. PLoS ONE 8(11): e78496. doi:10.1371/journal.pone.0078496 Editor: Tiansen Li, National Eye Institute, United States of America Received July 15, 2013; Accepted September 12, 2013; Published November 12, 2013 Copyright: ß 2013 Eisenberger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this study. Competing Interests: TE, CN, CD, AB, CB and HJB are employees of Bioscientia, which is part of a publicly traded diagnostic company. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: hanno.bolz@bioscientia.de PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e78496