Utility of Post-Mortem Genetic Testing in Cases of Sudden Arrhythmic Death Syndrome

Background Sudden arrhythmic death syndrome (SADS) describes a sudden death with negative autopsy and toxicological analysis. Cardiac genetic disease is a likely etiology. Objectives This study investigated the clinical utility and combined yield of post-mortem genetic testing (molecular autopsy) in cases of SADS and comprehensive clinical evaluation of surviving relatives. Methods We evaluated 302 expertly validated SADS cases with suitable DNA (median age: 24 years; 65% males) who underwent next-generation sequencing using an extended panel of 77 primary electrical disorder and cardiomyopathy genes. Pathogenic and likely pathogenic variants were classified using American College of Medical Genetics (ACMG) consensus guidelines. The yield of combined molecular autopsy and clinical evaluation in 82 surviving families was evaluated. A gene-level rare variant association analysis was conducted in SADS cases versus controls. Results A clinically actionable pathogenic or likely pathogenic variant was identified in 40 of 302 cases (13%). The main etiologies established were catecholaminergic polymorphic ventricular tachycardia and long QT syndrome (17 [6%] and 11 [4%], respectively). Gene-based rare variants association analysis showed enrichment of rare predicted deleterious variants in RYR2 (p = 5 × 10-5). Combining molecular autopsy with clinical evaluation in surviving families increased diagnostic yield from 26% to 39%. Conclusions Molecular autopsy for electrical disorder and cardiomyopathy genes, using ACMG guidelines for variant classification, identified a modest but realistic yield in SADS. Our data highlighted the predominant role of catecholaminergic polymorphic ventricular tachycardia and long QT syndrome, especially the RYR2 gene, as well as the minimal yield from other genes. Furthermore, we showed the enhanced utility of combined clinical and genetic evaluation.

S udden cardiac death (SCD) in the young is a devastating event. The annual incidence in the 1-to 35-year-old age group is estimated at 1.3 to 2.8 per 100,000 (1). Autopsies in younger SCD victims lead to a diagnosis of structural cardiac disease in the majority. Yet, in 30% to 40% of cases, the cause remains elusive despite toxicological and histopathologic analysis (1,2). A proportion is expected to have suffered arrhythmic death and is referred to as succumbing from sudden arrhythmic death syndrome (SADS) (3,4). SADS is caused, in part, by primary electrical disorders such as long QT syndrome (LQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT), which are associated with a structurally normal heart (5,6).
Post-mortem testing of the genes underlying primary electrical disorders in cases of SADS (the "molecular autopsy") allows for ascertainment of the genetic cause (7,8). This may inform clinical and genetic evaluation of surviving relatives for SCD prevention (9). Early case series focused on 4 main genes (KCNQ1, KCNH2, SCN5A, and RYR2). Relatively small studies have interrogated extended gene panels including cardiomyopathy-associated genes by nextgeneration sequencing. The yield varied widely due to variation in variant-calling (2,8,10). More recently,     (18). Libraries were prepared per manufacturer's instructions and sequenced on the Illumina HiSeq (cohort 1) or Next-Seq (cohort 2 and controls). We analyzed 77 genes tested by both systems that have been previously associated with primary electrical diseases or cardiomyopathies (Online Table 1).
All the samples (i.e., cohorts 1, 2, and controls) were processed together. Low quality (Q <20, window size 5) reads/bases were trimmed and read quality assessed. High-quality reads were mapped to UCSC GRCh37/hg19 reference genome. Next-generation sequencing was used to mark duplicate reads, realign locally around indels, and recalibrate base quality scores according to best practices. Alignment summary metrics and coverage and callability metrics were generated. A base was considered "callable" if sequenced with minimum read depth ¼ 10x, base quality $20, and mapping quality $10. Target base callability of >90% was achieved in 98% of samples overall and in 92%, 95%, and 100% of samples from cohort 1, 2, and controls, respectively. Gene level callability is shown in Online  Statistical enrichment of rare variants was analyzed for in SADS cases versus controls using the sequence kernel association test (22) as implemented in RVTESTS (rare variant tests) (23). We restricted the analysis to rare variants (MAF <1 in 10,000 in ExAC) predicted to be deleterious by a Combined Annotation-Dependent Depletion (24) score >25 to decrease genetic background noise and enrich for variants that could potentially be disease causing.

SADS cases and controls of non-European descent
were excluded from the analysis. As a negative control, we performed the gene-based analysis using only rare synonymous variants not predicted to change the protein. Bonferroni correction for multiple testing was used to define statistical significance thresholds. We tested 77 genes and 1 phenotype (SADS): single-gene tests were considered significant if p values were < a ¼ 0.05/77 (p < 6.5 Â 10 -4 ). Additionally, a likely pathogenic variant was identified in KCNQ1, KCNH2, TTN, and MYH7 in this age category. Furthermore, we found a pathogenic  (7) 16/235 (7) 2/33 (6) 17/253 (7) 1/15 (7) 1.0000 Values are median (interquartile range) or n/N (%). *Positive outcome was defined as a pathogenic or likely pathogenic variant per the ACMG guidelines (12). †p values refer to the following comparison: positive genetic testing RYR2 (n ¼ 17) versus no positive RYR2 test (n ¼ 285). ‡Note missing data for these parameters and that percentages are based on nonmissing data. §Includes palpitations, shortness of breath, and chest pain.
ACMG ¼ American College of Medical Genetics; SADS ¼ sudden arrhythmic death syndrome; SCD ¼ sudden cardiac death.  Table 4). Only RYR2 displayed an enrichment of rare variants in cases compared to controls at our pre-specified statistical significance threshold (p ¼ 6.5 Â 10 -4 ). To ensure that this result was not a consequence of systematic sequencing differences between cases and controls, we performed the same analysis using only rare synonymous variants, which did not uncover any differences (p ¼ 0.89).
Predictors of a positive genetic test are listed in

DISCUSSION
We report, to our knowledge, the largest study of molecular autopsy and its clinical utility in a set of   Indeed, the ratio of rare VUS to pathogenic or likely pathogenic cardiomyopathic variants was extremely unfavorable in our cohort ( Figure 2) and illustrated the need for careful and stringent adjudication. A small number of genotype-positive LQTS cases from New Zealand were not included in the study (13,14). Of these, only 3 would have been adjudicated as pathogenic or likely pathogenic by ACMG criteria and would not have affected the yield significantly. In addition, some exons were moderately covered (e.g., exon 1 of KCNQ1 and KCNH2 in cohort 1) (Online Table 3), but this was unlikely to have resulted in significant underestimation of the yield. Interpretation of variants in the small minority of non-Caucasian cases may have been hampered by the absence of large, ethnically specific population databases to which we could refer. Furthermore, we did not sequence genes currently associated with noncardiac disorders that predispose to sudden death.

CONCLUSIONS
Next-generation sequencing based molecular autopsy identified a 13% yield of clinically actionable