Diagnostic yield of targeted next generation sequencing in 2002 Dutch cardiomyopathy patients.

BACKGROUND: Next-generation sequencing (NGS) is increasingly used for clinical evaluation of cardiomyopathy patients as it allows for simultaneous screening of multiple cardiomyopathy-associated genes. Adding copy number variant (CNV) analysis of NGS data is not routine yet and may contribute to the diagnostic yield. OBJECTIVES: Determine the diagnostic yield of our targeted NGS gene panel in routine clinical diagnostics of Dutch cardiomyopathy patients and explore the impact of exon CNVs on diagnostic yield. METHODS: Patients (N = 2002) referred for clinical genetic analysis underwent diagnostic testing of 55-61 genes associated with cardiomyopathies. Samples were analyzed and evaluated for single nucleotide variants (SNVs), indels and CNVs. CNVs identified in the NGS data and suspected of being pathogenic based on type, size and location were confirmed by additional molecular tests. RESULTS: A (likely) pathogenic (L)P variant was detected in 22.7% of patients, including 3 with CNVs and 25 where a variant was identified in a gene currently not associated with the patient's cardiomyopathy subtype. Only 15 out of 2002 patients (0.8%) were found to carry two (L)P variants. CONCLUSION: The yield of routine clinical diagnostics of cardiomyopathies was relatively low when compared to literature. This is likely due to the fact that our study reports the outcome of patients in daily routine diagnostics, therefore also including patients not fully fulfilling (subtype specific) cardiomyopathy criteria. This may also explain why (L)P variants were identified in genes not associated with the reported subtype. The added value of CNV analysis was shown to be limited but not negligible.

Homozygous damaging SOD2 variant causes lethal neonatal dilated cardiomyopathy.

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM) is recognised to be a heritable disorder, yet clinical genetic testing does not produce a diagnosis in >50% of paediatric patients. Identifying a genetic cause is crucial because this knowledge can affect management options, cardiac surveillance in relatives and reproductive decision-making. In this study, we sought to identify the underlying genetic defect in a patient born to consanguineous parents with rapidly progressive DCM that led to death in early infancy. METHODS AND RESULTS: Exome sequencing revealed a potentially pathogenic, homozygous missense variant, c.542G>T, p.(Gly181Val), in SOD2. This gene encodes superoxide dismutase 2 (SOD2) or manganese-superoxide dismutase, a mitochondrial matrix protein that scavenges oxygen radicals produced by oxidation-reduction and electron transport reactions occurring in mitochondria via conversion of superoxide anion (O2-·) into H2O2. Measurement of hydroethidine oxidation showed a significant increase in O2-· levels in the patient's skin fibroblasts, as compared with controls, and this was paralleled by reduced catalytic activity of SOD2 in patient fibroblasts and muscle. Lentiviral complementation experiments demonstrated that mitochondrial SOD2 activity could be completely restored on transduction with wild type SOD2. CONCLUSION: Our results provide evidence that defective SOD2 may lead to toxic increases in the levels of damaging oxygen radicals in the neonatal heart, which can result in rapidly developing heart failure and death. We propose SOD2 as a novel nuclear-encoded mitochondrial protein involved in severe human neonatal cardiomyopathy, thus expanding the wide range of genetic factors involved in paediatric cardiomyopathies.

No major role for rare plectin variants in arrhythmogenic right ventricular cardiomyopathy.

AIMS: Likely pathogenic/pathogenic variants in genes encoding desmosomal proteins play an important role in the pathophysiology of arrhythmogenic right ventricular cardiomyopathy (ARVC). However, for a substantial proportion of ARVC patients, the genetic substrate remains unknown. We hypothesized that plectin, a cytolinker protein encoded by the PLEC gene, could play a role in ARVC because it has been proposed to link the desmosomal protein desmoplakin to the cytoskeleton and therefore has a potential function in the desmosomal structure. METHODS: We screened PLEC in 359 ARVC patients and compared the frequency of rare coding PLEC variants (minor allele frequency [MAF] <0.001) between patients and controls. To assess the frequency of rare variants in the control population, we evaluated the rare coding variants (MAF <0.001) found in the European cohort of the Exome Aggregation Database. We further evaluated plectin localization by immunofluorescence in a subset of patients with and without a PLEC variant. RESULTS: Forty ARVC patients carried one or more rare PLEC variants (11%, 40/359). However, rare variants also seem to occur frequently in the control population (18%, 4754/26197 individuals). Nor did we find a difference in the prevalence of rare PLEC variants in ARVC patients with or without a desmosomal likely pathogenic/pathogenic variant (14% versus 8%, respectively). However, immunofluorescence analysis did show decreased plectin junctional localization in myocardial tissue from 5 ARVC patients with PLEC variants. CONCLUSIONS: Although PLEC has been hypothesized as a promising candidate gene for ARVC, our current study did not show an enrichment of rare PLEC variants in ARVC patients compared to controls and therefore does not support a major role for PLEC in this disorder. Although rare PLEC variants were associated with abnormal localization in cardiac tissue, the confluence of data does not support a role for plectin abnormalities in ARVC development.

Titin gene mutations are common in families with both peripartum cardiomyopathy and dilated cardiomyopathy.

AIM: Peripartum cardiomyopathy (PPCM) can be an initial manifestation of familial dilated cardiomyopathy (DCM). We aimed to identify mutations in families that could underlie their PPCM and DCM. METHODS AND RESULTS: We collected 18 families with PPCM and DCM cases from various countries. We studied the clinical characteristics of the PPCM patients and affected relatives, and applied a targeted next-generation sequencing (NGS) approach to detect mutations in 48 genes known to be involved in inherited cardiomyopathies. We identified 4 pathogenic mutations in 4 of 18 families (22%): 3 in TTN and 1 in BAG3. In addition, we identified 6 variants of unknown clinical significance that may be pathogenic in 6 other families (33%): 4 in TTN, 1 in TNNC1, and 1 in MYH7. Measurements of passive force in single cardiomyocytes and titin isoform composition potentially support an upgrade of one of the variants of unknown clinical significance in TTN to a pathogenic mutation. Only 2 of 20 PPCM cases in these families showed the recovery of left ventricular function. CONCLUSION: Targeted NGS shows that potentially causal mutations in cardiomyopathy-related genes are common in families with both PPCM and DCM. This supports the earlier finding that PPCM can be part of familial DCM. Our cohort is particularly characterized by a high proportion of TTN mutations and a low recovery rate in PPCM cases.

New clinical molecular diagnostic methods for congenital and inherited heart disease.

IMPORTANCE OF THE FIELD: For patients with congenital and inherited heart disorders, causative mutations are often not identified owing to limitations of current screening techniques. Identifying the mutation is of major importance for genetic counseling of patients and families, facilitating the diagnosis in people at risk and directing clinical management. Next-generation sequencing (NGS) provides unprecedented opportunities to maximize mutation yields and improve clinical management, genetic counseling and monitoring of patients. AREAS COVERED IN THIS REVIEW: Recent NGS applications are reviewed, focusing on methods relevant for molecular diagnostics in cardiogenetics. Requirements for reliable implementation in clinical practice and challenges that clinicians, bioinfomaticians and molecular diagnosticians must deal with in analyzing resulting data are discussed. WHAT THE READER WILL GAIN: Readers will be introduced to recent developments, techniques and applications in NGS. They will learn about possibilities of using it in clinical diagnostics. They will become acquainted with difficulties and challenges in interpreting the data and considerations around communicating these issues to patients and the community. TAKE HOME MESSAGE: Although several obstacles are still to be overcome and there is much still to learn, NGS will revolutionize clinical molecular diagnostics of inherited and congenital cardiac diseases, maximizing mutation yields and leading to optimized diagnostic and clinical care.