Genetic investigation

We have previously described the methods for the genetic investigations in Hertz et al.¹⁵ In brief, DNA was extracted from whole blood in 57 of the individuals using the QIAamp DNA Mini Kit (Qiagen, Stockach, Germany). DNA was extracted from fresh frozen muscle from one individual and from fresh frozen spleen tissue from three individuals, using the EZ1 DNA Investigator Kit (Qiagen). The extracted DNA was quantified with the Quantifiler Human DNA Quantification Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol. DNA from two samples was whole-genome amplified using the REPLI-g Midi kit (Qiagen) following the manufacturer's protocol due to low DNA concentration.

NGS was used to investigate 100 genes previously reported to be associated with inherited cardiac diseases and SCD (Table 1). The genes were selected from the available literature in PubMed and Online Mendelian Inheritance in Man (http://omim.org/).

Abbreviations: ARVC, arrhythmogenic right ventricular cardiomyopathy; BrS, Brugada syndrome; CCD, cardiac conduction disease; CPVT, catecholaminergic polymorphic ventricular tachycardia; CTD, carnitine transporter deficiency; DCM, dilated cardiomyopathy; ERS, early repolarization syndrome; FAF, familial atrial fibrillation, FD, Fabry disease; HCM, hypertrophic cardiomyopathy; LL, Lenegre-Lev syndrome; LQTS, long QT syndrome; LVNC, left ventricular non-compaction; PFVF, paroxysmal familial ventricular fibrillation; RBBB, right bundle branch block; SIDS, sudden infant death syndrome; SSS, sick sinus syndrome; SQTS, short QT syndrome; WPW, Wolff–Parkinson–White Syndrome.

SureDesign (Agilent Technologies, Santa Clara, CA, USA) was used to design capture probes for the target regions of the exons and UTRs of the 100 genes for the Haloplex Target Enrichment system (Agilent Technologies) with 150 bp read lengths. The design included all coding exons, 25 bp of the adjacent introns and 5′- and 3′-UTR regions of the 100 genes of interest to a total of 2076 regions with a total size of 787 943 bp. Overall, 99.6% of the target was covered >50 ×. Two exons were not covered sufficiently by the sequencing and seven exons were not sequenced due to missing probe designs. Details of the genomic regions not covered sufficiently is found in Supplementary Table S1. Median coverage for amplicons was 950 (range: 206–3065).

The Haloplex Target Enrichment protocol version D.5 was used according to the manufacturer's instruction. The protocol included the following steps: (1) digestion of 200 ng of genomic DNA with different restriction enzymes in eight tubes and analysis of the fragments using a 2100 Bioanalyzer (Agilent Technologies); (2) hybridization of the digested DNA to Haloplex probes with ends of the probes that were complementary to the fragments of the targets. During hybridization, the fragments were circularized and indexes, sequencing motifs and biotin were incorporated; (3) the target DNA was captured with Haloplex magnetic beads; (4) the nicks in the circularized fragments were closed by ligation; and (5) elution with NaOH, PCR amplification, purification and final elution with Tris-HCl buffer of the captured target DNA was performed. After library build, the amount of DNA was measured using a Qubit Fluorometer 2.0 with the dsDNA HS assay (Invitrogen, Life Technologies Europe BV, Nærum, Denmark). The size distribution of the DNA was analyzed using a 2100 Bioanalyzer and the High Sensitivity DNA kit (Agilent Technologies). All samples were sequenced on a MiSeq (Illumina, San Diego, CA, USA) according to the manufacturer's recommendations with 150 bp paired-end sequencing using the MiSeq Reagent Kit V2 (300 cycles).