title: Assessment of HaloPlex amplification for sequence capture and massively parallel sequencing of arrhythmogenic right ventricular cardiomyopathy-associated genes.
abstract: The genetic basis of arrhythmogenic right ventricular cardiomyopathy (ARVC) is complex. Mutations in genes encoding components of the cardiac desmosomes have been implicated as being causally related to ARVC. Next-generation sequencing allows parallel sequencing and duplication/deletion analysis of many genes simultaneously, which is appropriate for screening of mutations in disorders with heterogeneous genetic backgrounds. We designed and validated a next-generation sequencing test panel for ARVC using HaloPlex. We used SureDesign to prepare a HaloPlex enrichment system for sequencing of DES, DSC2, DSG2, DSP, JUP, PKP2, RYR2, TGFB3, TMEM43, and TTN from patients with ARVC using a MiSeq instrument. Performance characteristics were determined by comparison with Sanger, as the gold standard, and TruSeq Custom Amplicon sequencing of DSC2, DSG2, DSP, JUP, and PKP2. All the samples were successfully sequenced after HaloPlex capture, with >99% of targeted nucleotides covered by >20×. The sequences were of high quality, although one problematic area due to a presumptive context-specific sequencing error-causing motif located in exon 1 of the DSP gene was detected. The mutations found by Sanger sequencing were also found using the HaloPlex technique. Depending on the bioinformatics pipeline, sensitivity varied from 99.3% to 100%, and specificity varied from 99.9% to 100%. Three variant positions found by Sanger and HaloPlex sequencing were missed by TruSeq Custom Amplicon owing to loss of coverage.
authors.affiliation: Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Department of Clinical Genetics, County Council of Östergötland, Linköping, Sweden. Electronic address: anna.k.green@lio.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Gréen
authors.affiliation: Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden; Division of Drug Research, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden; Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Gréen
authors.affiliation: Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Department of Clinical Genetics, County Council of Östergötland, Linköping, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Rehnberg
authors.affiliation: Department of Medicine and Health Science, Linköping University, Linköping, Sweden; Department of Cardiology, County Council of Östergötland, Linköping, Sweden.
authors.forename: Anneli
authors.initials: A
authors.lastname: Svensson
authors.affiliation: Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Department of Clinical Genetics, County Council of Östergötland, Linköping, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Gunnarsson
authors.affiliation: Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; Department of Clinical Genetics, County Council of Östergötland, Linköping, Sweden.
authors.forename: Jon
authors.initials: J
authors.lastname: Jonasson
mesh_terms: D019571:Arrhythmogenic Right Ventricular Dysplasia / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D051187:Desmocollins / Q000235:genetics / Q000378:metabolism; D051182:Desmogleins / Q000235:genetics / Q000378:metabolism; D051180:Desmoplakins / Q000235:genetics / Q000378:metabolism; D003896:Desmosomes / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000592:standards*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000592:standards*; D051191:Plakophilins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D051187:Desmocollins; D051182:Desmogleins; D051180:Desmoplakins; D051191:Plakophilins