The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results.

Background: Each of the cardiomyopathies, classically categorized as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy, has a signature genetic theme. Hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning >10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. Methods: An international panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The panel used the Clinical Genome Resource semiquantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories on the basis of the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. Results: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from 8 gene ontologies were classified as having definitive ( BAG3 , DES , FLNC , LMNA , MYH7 , PLN , RBM20 , SCN5A , TNNC1 , TNNT2 , TTN ) or strong ( DSP ) evidence. Seven genes (14%; ACTC1 , ACTN2 , JPH2 , NEXN , TNNI3 , TPM1 , VCL ) including 2 additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, 6 were similarly classified for hypertrophic cardiomyopathy and 3 for arrhythmogenic right ventricular cardiomyopathy. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of the 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. Conclusions: In the curation of 51 genes, 19 had high evidence (12 definitive/strong, 7 moderate). It is notable that these 19 genes explain only a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high-evidence genes; however, genes lacking robust evidence are also commonly included. We recommend that high-evidence DCM genes be used for clinical practice and that caution be exercised in the interpretation of variants in variable-evidence DCM genes.

Numerous genes are known to cause dilated cardiomyopathy (DCM). However, until now technological limitations have hindered elucidation of the contribution of all clinically relevant disease genes to DCM phenotypes in larger cohorts. We now utilized next-generation sequencing to overcome these limitations and screened all DCM disease genes in a large cohort. In this multi-centre, multi-national study, we have enrolled 639 patients with sporadic or familial DCM. To all samples, we applied a standardized protocol for ultra-high coverage next-generation sequencing of 84 genes, leading to 99.1% coverage of the target region with at least 50-fold and a mean read depth of 2415. In this well characterized cohort, we find the highest number of known cardiomyopathy mutations in plakophilin-2, myosin-binding protein C-3, and desmoplakin. When we include yet unknown but predicted disease variants, we find titin, plakophilin-2, myosin-binding protein-C 3, desmoplakin, ryanodine receptor 2, desmocollin-2, desmoglein-2, and SCN5A variants among the most commonly mutated genes. The overlap between DCM, hypertrophic cardiomyopathy (HCM), and channelopathy causing mutations is considerably high. Of note, we find that >38% of patients have compound or combined mutations and 12.8% have three or even more mutations. When comparing patients recruited in the eight participating European countries we find remarkably little differences in mutation frequencies and affected genes. This is to our knowledge, the first study that comprehensively investigated the genetics of DCM in a large-scale cohort and across a broad gene panel of the known DCM genes. Our results underline the high analytical quality and feasibility of Next-Generation Sequencing in clinical genetic diagnostics and provide a sound database of the genetic causes of DCM.

Background— According to clinical-pathological correlation studies, the natural history of arrhythmogenic right ventricular dysplasia/cardiomyopathy is purported to progress from localized to global right ventricular dysfunction, followed by left ventricular (LV) involvement and biventricular pump failure. The inevitable focus on sudden death victims and transplant recipients may, however, have created a skewed perspective of a genetic disease. We hypothesized that unbiased representation of the spectrum of disease expression in arrhythmogenic right ventricular dysplasia/cardiomyopathy would require in vivo assessment of families in a genetically heterogeneous population. Methods and Results— A cohort of 200 probands and relatives satisfying task force or modified diagnostic criteria for arrhythmogenic right ventricular dysplasia/cardiomyopathy underwent comprehensive clinical evaluation. Desmosomal mutations were identified in 39 individuals from 20 different families. Indices of structural severity correlated with advancing age and were increased in long-term endurance athletes. Fulfillment of modified criteria indicated phenotypically mild disease, whereas asymptomatic status did not. In >80%, ECG, rhythm monitoring, and/or gadolinium-enhanced cardiovascular magnetic resonance were suggestive of LV involvement, the extent of which often was marked among individuals with chain-termination mutations and/or desmoplakin disease. Three patterns of disease expression were identified: (1) classic , with isolated right ventricular disease or LV involvement in association with significant right ventricular impairment; (2) left dominant , with early and prominent LV manifestations and relatively mild right-sided disease; and (3) biventricular , characterized by parallel involvement of both ventricles. Conclusions— LV involvement in arrhythmogenic right ventricular dysplasia/cardiomyopathy may precede the onset of significant right ventricular dysfunction. Recognition of disease variants with early and/or predominant LV involvement supports adoption of the broader term arrhythmogenic cardiomyopathy.

At least 4% of sudden deaths are unexplained at autopsy [sudden arrhythmic death syndrome (SADS)] and a quarter may be due to inherited cardiac disease. We hypothesized that comprehensive clinical investigation of SADS families would identify more susceptible individuals and causes of death. Fifty seven consecutively referred families with SADS death underwent evaluation including resting 12 lead, 24 h and exercise ECG and 2D echocardiography. Other investigations included signal averaged ECG, ajmaline testing, cardiac magnetic resonance imaging, and mutation analysis. First-degree relatives [184/262 (70%)] underwent evaluation, 13 (7%) reporting unexplained syncope. Seventeen (30%) families had a history of additional unexplained premature sudden death(s). Thirty families (53%) were diagnosed with inheritable heart disease: 13 definite long QT syndrome (LQTS), three possible/probable LQTS, five Brugada syndrome, five arrhythmogenic right ventricular cardiomyopathy (ARVC), and four other cardiomyopathies. One SCN5A and four KCNH2 mutations (38%) were identified in 13 definite LQTS families, one SCN5A mutation (20%) in five Brugada syndrome families and one (25%) PKP2 (plakophyllin2) mutation in four ARVC families. Over half of SADS deaths were likely to be due to inherited heart disease; accurate identification is vital for appropriate prophylaxis amongst relatives who should undergo comprehensive cardiological evaluation, guided and confirmed by mutation analysis.