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.

Development and validation of a whole-exome sequencing test for simultaneous detection of point mutations, indels and copy-number alterations for precision cancer care

“…Green et al ., 19 have recently showed that patients’ DNA samples were successfully sequenced after HaloPlex capture, with >99% of targeted nucleotides in the panel covered by >20×. 19 Similar results were obtained by Berglund et al ., 16 demonstrating that virtually all SNVs in their study (>99%) were covered by at least one sequence read and >96% of the variants were covered at a sequence depth of at least 30×. However, both of these studies have been using HMW DNA and targeted HaloPlex panels.…”

Development of a Comprehensive Sequencing Assay for Inherited Cardiac Condition Genes

“…Targeted sequencing of gene panels is an alternative to WES and has been widely used in research and is increasingly applied in clinical settings [ 13 ]. In the ICC setting, small gene panels have been used for specific ICCs, including long QT syndrome (LQTS), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) [ 14 – 16 ]. Multiple workflows and bioinformatics pipelines are needed to run these various ICC gene panels, and gene coverage is such that Sanger sequencing ‘fill in’ is needed, which has very major manpower implications.…”

Malignant Arrhythmia with Variants of Desmocollin-2 and Desmoplakin Genes

“…So far, 84 site mutations of DSC2 and 148 site mutations of DSP have been reported in ARVC. 16) DSP p. R1207K was the first reported and was detected in the present case of exercise-induced malignant arrhythmia in a teenager. At the same time, we confirmed that the heterozygous missense variants carried by this patient were inherited from his father.…”