Whole Sequencing of Most Prevalent Dilated Cardiomyopathy-Causing Genes as a Molecular Strategy to Improve Molecular Diagnosis Efficiency?

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and one of the most common causes of heart failure. TTN-truncating variants represent the most common cause of DCM. Similarly, among other prevalent DCM-causing genes, truncating variants were also frequently detected in BAG3, DSP, FLNC, and LMNA. For these four genes, the current study aims to determine the prevalence of deep intronic pathogenic variants that could lead to splice defects. A next-generation sequencing (NGS) workflow based on whole gene sequencing of BAG3, DSP, FLNC, and LMNA of a cohort of 95 DCM patients, for whom no putatively causative point mutations were identified after NGS of a panel of 48 cardiomyopathy-causing genes, was thus performed. Our approach did not lead us to reconsider the molecular diagnosis of any patient of the cohort. This study suggests that deep splice mutations do not account for a significant proportion of DCM cases. In contrast with MYBPC3 in hypertrophic cardiomyopathy cases, NGS of BAG3, DSP, FLNC, and LMNA whole intronic sequences would not significantly improve the efficiency of molecular diagnosis of DCM probands.

Whole MYBPC3 NGS sequencing as a molecular strategy to improve the efficiency of molecular diagnosis of patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiomyopathy, historically believed to affect 1 of 500 people. MYBPC3 pathogenic variations are the most frequent cause of familial HCM and more than 90% of them introduce a premature termination codon. The current study aims to determine the prevalence of deep intronic MYBPC3 pathogenic variations that could lead to splice mutations. To improve molecular diagnosis, a next-generation sequencing (NGS) workflow based on whole MYBPC3 sequencing of a cohort of 93 HCM patients, for whom no putatively causative point mutations were identified after NGS sequencing of a panel of 48 cardiomyopathy-causing genes, was performed. Our approach led us to reconsider the molecular diagnosis of six patients of the cohort (6.5%). These HCM probands were carriers of either a new large MYBPC3 rearrangement or splice intronic variations (five cases). Four pathogenic intronic variations, including three novel ones, were detected. Among them, the prevalence of one of them (NM_000256.3:c.1927+ 600 C>T) was estimated at about 0.35% by the screening of 1,040 unrelated HCM individuals. This study suggests that deep MYBPC3 splice mutations account for a significant proportion of HCM cases (6.5% of this cohort). Consequently, NGS sequencing of MYBPC3 intronic sequences have to be performed systematically.

TRPM4 mutations to cause autosomal recessive and not autosomal dominant Brugada type 1 syndrome.

Cardiac channelopathies, mainly Long QT and Brugada syndromes, are genetic disorders for which genotype/phenotypes relationships remains to be improved. To provide new insights into the Brugada syndrome pathophysiology, a mutational study was performed on a 64-year-old man presented with isolated exertional dyspnea (NYHA class: II-III), hypertension, chronic kidney disease, coronary disease, an electrocardiogram suggesting a Brugada type 1-like pattern with ST-segment elevation in leads V1-V2. Molecular diagnosis study was performed using molecular strategy based on the sequencing of a panel of 19 Brugada-associated genes. The proband was carrier of 2 TRPM4 null alleles [IVS9+1G > A and p. Trp525X] resulting in the absence of functional hTRPM4 proteins. Due to this unexpected genotype, meta-analysis of previously reported TRPM4 variations associated with cardiac pathologies was performed using ACMG guidelines. All were detected in a heterozygous status. This additional meta-analysis indicated that most of them could not be considered definitely as pathogen. In conclusion, our study reports, for the first time, identification of compound heterozygous TRPM4 null mutations in a proband with, at an arrhythmogenic level, only a Brugada type 1-like electrocardiogram. By combining the genotype/phenotype relationship of this case and analysis of previously reported TRPM4 variations, we suggest that loss-of-function TRPM4 variations, in a heterozygous status, could not be considered as pathogenic or likely pathogenic mutations in cardiac channelopathies such as Long QT syndrome or Brugada syndrome.

A fast and cost-effective molecular diagnostic tool for genetic diseases involved in sudden cardiac death.

BACKGROUND: Cardiomyopathies and arrhythmia syndromes are common genetic cardiac diseases that account for a significant number of sudden cardiac death (SCD) cases. METHODS: NGS workflow based on a panel of 95 genes was developed on Illumina NextSeq500™ sequencer for sequencing prevalent SCD-causing genes. A cohort of 90 patients (56 genotype-positive, 27 genotype-negative and 7 new cases) was screened to evaluate this strategy in terms of sensitivity, specificity, practicability and cost. In silico analysis were performed using a pipeline based on NextGENe® software and a personalized Sophia Genetics pipeline. RESULTS: Using our panel custom, 100% of targeted sequences were efficiently covered and all previously identified genetic variants were readily detected. Applied to 27 genotype-negative patients, this molecular strategy allowed the identification of pathogenic or likely pathogenic variants into 12 cases. It confirmed the involvement of HCN4 mutations in the combined bradycardia­myocardial non-compaction phenotype, and also suggested, for the first time, the involvement of PKP2, usually associated with arrhythmogenic right ventricular dysplasia, in ventricular non-compaction. CONCLUSION: This NGS approach is a fast, cheap, sensitive and high-throughput mutation detection method that is ready to be deployed in clinical laboratories and would provide new insights on physiopathology of SCD, more particularly of cardiomyopathies and arrhythmia syndromes.

Evaluation of a new NGS method based on a custom AmpliSeq library and Ion Torrent PGM sequencing for the fast detection of genetic variations in cardiomyopathies.

BACKGROUND: Hypertrophic and dilated cardiomyopathies are common genetic cardiac diseases. Due to large cohorts to investigate, large number of causative genes and high rate of private mutations, mutational screening must be performed using an extremely sensitive and specific detection method. METHODS: NGS workflow based on a custom AmpliSeq panel was designed for sequencing most prevalent cardiomyopathy-causing genes on the Ion PGM™ Sequencer. A cohort of 75 previously studied patients was screened to evaluate this strategy in terms of sensibility, specificity, practicability and cost. In silico analysis was performed using the NextGENe® software. RESULTS: Our AmpliSeq custom panel allowed us to efficiently explore 96% of targeted sequences. Using adjusted alignment settings, all genetic variants (57 substitutions, 34 indels) present in covered regions and previously detected by HRM/sequencing were readily identified except a 73-bp MYBPC3 deletion (analytical sensitivity: 98.9%). Uncovered targeted regions were further analysed by a HRM/sequencing strategy. Complete molecular investigation was performed faster and cheaper than with previously used mutation detection methods. CONCLUSION: Finally, these results suggested that our new NGS approach based on Ampliseq libraries and Ion PGM sequencing is a highly efficient, fast and cheap high-throughput mutation detection method that is ready to be deployed in clinical laboratories.

Evaluation of a new high-throughput next-generation sequencing method based on a custom AmpliSeq™ library and ion torrent PGM™ sequencing for the rapid detection of genetic variations in long QT syndrome.

BACKGROUND AND OBJECTIVE: Inherited long QT syndrome (LQTS) is a cardiac channelopathy associated with a high risk of sudden death. The prevalence has been estimated at close to 1:2,000. Due to large cohorts to investigate and high rate of private mutations, mutational screening must be performed using an extremely sensitive and specific detection method. Mutational screening is crucial as this may have implications for therapy and management of LQTS patients. METHODS: Next-generation sequencing (NGS) workflow based on a custom AmpliSeq™ panel was designed for sequencing the five most prevalent cardiomyopathy-causing genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2) on Ion PGM™ Sequencer. A cohort of 30 previously studied patients was screened to evaluate this strategy in terms of sensitivity, specificity, practicability, and cost. In silico analysis was performed using NextGENe(®) software. RESULTS: Our AmpliSeq™ custom panel allowed us to explore 86 % of targeted sequences efficiently. Using adjusted alignment settings, all genetic variants (40 substitutions, 17 indels) present in covered regions and previously detected by high-resolution melt (HRM)/sequencing were readily identified. Uncovered targeted regions, which were mainly located in KCNH2, were further analyzed by HRM/sequencing strategy. Complete molecular investigation was performed faster and cheaper than with previously used mutation detection methods. CONCLUSION: Finally, these results suggested that our new NGS approach based on AmpliSeq™ libraries and Ion PGM™ sequencing is a highly efficient, fast, and cheap high-throughput mutation detection method that is ready to be deployed in clinical laboratories. This method will allow fast identification of LQTS mutations that will have further implications for therapeutics.

Next-generation sequencing (NGS) as a fast molecular diagnosis tool for left ventricular noncompaction in an infant with compound mutations in the MYBPC3 gene.

Left ventricular noncompaction (LVNC) is a clinically heterogeneous disorder characterized by a trabecular meshwork and deep intertrabecular myocardial recesses that communicate with the left ventricular cavity. LVNC is classified as a rare genetic cardiomyopathy. Molecular diagnosis is a challenge for the medical community as the condition shares morphologic features of hypertrophic and dilated cardiomyopathies. Several genetic causes of LVNC have been reported, with variable modes of inheritance, including autosomal dominant and X-linked inheritance, but relatively few responsible genes have been identified. In this report, we describe a case of a severe form of LVNC leading to death at 6 months of life. NGS sequencing using a custom design for hypertrophic cardiomyopathy panel allowed us to identify compound heterozygosity in the MYBPC3 gene (p.Lys505del, p.Pro955fs) in 3 days, confirming NGS sequencing as a fast molecular diagnosis tool. Other studies have reported neonatal presentation of cardiomyopathies associated with compound heterozygous or homozygous MYBPC3 mutations. In this family and in families in which parental truncating MYBPC3 mutations are identified, preimplantation or prenatal genetic screening should be considered as these genotypes leads to neonatal mortality and morbidity.

Development of a high resolution melting method for the detection of genetic variations in Long QT Syndrome.

BACKGROUND: Inherited Long QT Syndrome (LQTS) is a cardiac channelopathy associated with a high risk of sudden death. The prevalence has been estimated at close to 1:2000. Due to large cohorts to investigate, the size of the 3 prevalent mutated genes, and the presence of a large spectrum of private mutations, mutational screening requires an extremely sensitive and specific scanning method. METHODS: Efficiency of high resolution melting (HRM) analysis was evaluated for the most prevalent LQTS-causing genes (KCNQ1, KCNH2) using control DNAs and DNAs carrying previously identified gene variants. A cohort of 34 patients with a suspicion of LQTS was further blindly screened. To evaluate HRM sensitivity, this cohort was also screened using an optimized DHPLC strategy. RESULTS: HRM analysis was successfully optimized for KCNQ1 but optimisation of KCNH2 was more laborious as only 3 KCNH2 exons could be finally optimized. Remaining KCNH2 exons were analysed by direct sequencing. This molecular approach, which combined HRM and direct sequencing, was applied on the cohort of 34 cases and 9 putative mutations were identified. Using this approach, molecular investigation was completed faster and cheaper than using DHPLC strategy. CONCLUSIONS: This HRM/sequencing procedure represents an inexpensive, highly sensitive and high-throughput method to allow identification of mutations in the coding sequences of prevalent LQTS genes.

Development of a high resolution melting method for the detection of genetic variations in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease affecting 1 in 500 people. Due to large cohorts to investigate, the number of disease-causing genes, the size of the 2 prevalent mutated genes, and the presence of a large spectrum of private mutations, mutational screening must be performed using an extremely sensitive and specific scanning method. METHODS: High Resolution Melting (HRM) analysis was developed for prevalent HCM-causing genes (MYBPC3, MYH7, TNNT2, and TNNI3) using control DNAs and DNAs carrying previously identified gene variants. A cohort of 34 HCM patients was further blindly screened. To evaluate HRM sensitivity, this cohort was also screened using an optimized DHPLC methodology. RESULTS: All gene variants detected by DHPLC were also readily identified as abnormal by HRM analysis. Mutational screening of a cohort of 34 HCM cases led to identification of 19 mutated alleles. Complete molecular investigation was completed two times faster and cheaper than using DHPLC strategy. CONCLUSIONS: HRM analysis represents an inexpensive, highly sensitive and high-throughput method to allow identification of mutations in the coding sequences of prevalent HCM genes. Identification of more HCM mutations will provide new insights into genotype/phenotype relationships and will allow a better knowledge of the HCM physiopathology.

Validation of high-resolution DNA melting analysis for mutation scanning of the LMNA gene.

OBJECTIVES: LMNA mutations lead to a wide spectrum of disorders now called laminopathies. Due to large cohorts to investigate, mutational screening must be performed using an extremely sensitive and specific scanning method. DESIGN AND METHODS: High Resolution Melting (HRM) analysis was developed for LMNA mutation detection. A cohort of 64 patients with dilated cardiomyopathy was prospectively screened using both HRM and DHPLC methodologies. RESULTS: All gene variants detected by DHPLC or by direct sequencing were also readily identified as abnormal by HRM analysis. Mutations were identified in 7 patients (approximately 11%). Complete molecular LMNA investigation was completed two times faster and cheaper than using DHPLC strategy. CONCLUSIONS: HRM analysis represents an inexpensive, highly sensitive and high-throughput method to identify LMNA genetic variants. The discovery of novel LMNA mutations will provide new insights into the pathophysiology of dilated cardiomyopathy and in all other laminopathies.

Rapid, sensitive and inexpensive detection of SCN5A genetic variations by high resolution melting analysis.

OBJECTIVES: SCN5A mutations lead to a wide spectrum of cardiovascular disorders. Due to large cohorts to investigate and the large gene size, mutational screening must be performed using an extremely sensitive and specific scanning method. DESIGN AND METHODS: High Resolution Melting (HRM) analysis was developed for SCN5A mutation detection using control DNAs and DNAs carrying previously identified gene variants. A cohort of 40 patients was further screened. To evaluate HRM sensitivity, this cohort was also screened using an optimized DHPLC methodology. RESULTS: All gene variants detected by DHPLC were also readily identified as abnormal by HRM analysis. Mutations were identified for 5 patients. Complete molecular SCN5A investigation was completed two times faster and cheaper than using DHPLC strategy. CONCLUSIONS: HRM analysis represents an inexpensive, highly sensitive and high-throughput method to allow identification of SCN5A gene variants. Identification of more SCN5A mutations could provide new insights into the pathophysiology of SCN5A-linked diseases syndromes.