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Background The p.Arg14del (c.40_42delAGA) phospholamban (PLN) pathogenic variant is a founder mutation that causes dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Carriers are at increased risk of malignant ventricular arrhythmias and heart failure, which has been ascribed to cardiac fibrosis. Importantly, cardiac fibrosis appears to be an early feature of the disease, occurring in many presymptomatic carriers before the onset of overt disease. As with most monogenic cardiomyopathies, no evidence-based treatment is available for presymptomatic carriers. Aims The PHOspholamban RElated CArdiomyopathy intervention STudy (iPHORECAST) is designed to demonstrate that pre-emptive treatment of presymptomatic PLN p.Arg14del carriers using eplerenone, a mineralocorticoid receptor antagonist with established antifibrotic effects, can reduce disease progression and postpone the onset of overt disease. Methods iPHORECAST has a multicentre, prospective, randomised, open-label, blinded endpoint (PROBE) design. Presymptomatic PLN p.Arg14del carriers are randomised to receive either 50 mg eplerenone once daily or no treatment. The primary endpoint of the study is a multiparametric assessment of disease progression including cardiac magnetic resonance parameters (left and right ventricular volumes, systolic function and fibrosis), electrocardiographic parameters (QRS voltage, ventricular ectopy), signs and/or symptoms related to DCM and ACM, and cardiovascular death. The follow-up duration is set at 3 years. Baseline results A total of 84 presymptomatic PLN p.Arg14del carriers (n = 42 per group) were included. By design, at baseline, all participants were in New York Heart Association (NHYA) class I and had a left ventricular ejection fraction > 45% and Conclusion iPHORECAST is a multicentre, prospective randomised controlled trial designed to address whether pre-emptive treatment of PLN p.Arg14del carriers with eplerenone can prevent or delay the onset of cardiomyopathy. iPHORECAST has been registered in the clinicaltrials.gov-register (number: NCT01857856).

Background/Introduction The founder mutation p.Arg14del in the gene encoding phospholamban (PLN) is a known cause of arrhythmogenic cardiomyopathy (ACM) with distinct clinical features, such as microvoltages on the ECG and right or left ventricular dysfunction or both. At present, risk stratification for ICD implantation in carriers of this mutation is based on left ventricular ejection fraction and previous ventricular arrhythmia's, which are not specific risk factors for the PLN p.Arg14del cardiomyopathy. Purpose Our goal is to develop a mutation specific prediction model for incident malignant ventricular arrhythmia to guide ICD implantation. Methods Data were collected from p.Arg14del carriers with no history of malignant VA at baseline, identified between September 2009 and June 2018 in three Dutch university hospitals. Genetic analysis of PLN was performed in a clinical setting in index patients with clinical signs of DCM/ACM, or in family members of p.Arg14del carriers. We collected clinical data from the first cardiac evaluation and follow-ups. Malignant VAs were defined as sustained VA, appropriate ICD intervention or (aborted) sudden cardiac death. A prediction model was developed using Cox Proportional Hazard regression. Candidate baseline predictors were pre specified based on literature and clinical expertise. Age, sex, proband status, sudden cardiac death (SCD) in 1st degree relative, repolarization abnormalities, microvoltages, premature ventricular complexes (PVC) burden on 24hrs Holter monitoring, LVEF and non-sustained ventricular arrhythmias (NSVT) were considered. The multivariable model was fitted using stepwise backward selection based on Akaike's Information Criterion. Results We included 440 p.Arg14del carriers with a mean age of 41±18 years and 41% males. During a median follow-up of 4.7 years (IQR 1.7–7.3), 44 incident malignant VA occurred, 20 sustained VAs and 24 appropriate ICD therapies. The multivariable HR's of selected predictors were: 2.2 for minor and 4.5 for major repolarization abnormalities vs no abnormalities (p value respectively 0.06 and 0.01), 2.2 for LVEF 500PVC/24hrs (p value 0.003). Carriers with and without events could be accurately distinguished with this model, with an optimism corrected C-statistic of 0.81. The model calibrated well, with agreement of observed and predicted 5 year malignant VA risk. Risk groups were split into quintiles of predicted risk, figure 1 shows the Kaplan Meier curve of incident malignant VA per risk group. The 5 year risk of incident malignant VA in the lowest 3 quintiles was 0%, 9% in the 4th quintile and 25.2% in the highest quintile. Conclusion We created a PLN p.Arg14del mutation specific prediction model to estimate risk of incident malignant VA. With this model a clear distinction between high risk and low risk patients can be made and can be used to guide ICD implantation for primary prevention.

Contains fulltext : 204157.pdf (Publisher’s version ) (Closed access) BACKGROUND: Neuromuscular disorders (NMDs) are clinically and genetically heterogeneous. Accurate molecular genetic diagnosis can improve clinical management, provides appropriate genetic counseling and testing of relatives, and allows potential therapeutic trials. OBJECTIVE: To establish the clinical utility of panel-based whole exome sequencing (WES) in NMDs in a population with children and adults with various neuromuscular symptoms. METHODS: Clinical exome sequencing, followed by diagnostic interpretation of variants in genes associated with NMDs, was performed in a cohort of 396 patients suspected of having a genetic cause with a variable age of onset, neuromuscular phenotype, and inheritance pattern. Many had previously undergone targeted gene testing without results. RESULTS: Disease-causing variants were identified in 75/396 patients (19%), with variants in the three COL6-genes (COL6A1, COL6A2 and COL6A3) as the most common cause of the identified muscle disorder, followed by variants in the RYR1 gene. Together, these four genes account for almost 25% of cases in whom a definite genetic cause was identified. Furthermore, likely pathogenic variants and/or variants of uncertain significance were identified in 95 of the patients (24%), in whom functional and/or segregation analysis should be used to confirm or reject the pathogenicity. In 18% of the cases with a disease-causing variant of which we received additional clinical information, we identified a genetic cause in genes of which the associated phenotypes did not match that of the patients. Hence, the advantage of panel-based WES is its unbiased approach. CONCLUSION: Whole exome sequencing, followed by filtering for NMD genes, offers an unbiased approach for the genetic diagnostics of NMD patients. This approach could be used as a first-tier test in neuromuscular disorders with a high suspicion of a genetic cause. With uncertain results, functional testing and segregation analysis are needed to complete the evidence.

Pathogenic mutations in the phospholamban (PLN) gene may give rise to inherited cardiomyopathies due to its role in calcium homeostasis. Several PLN mutations have been identified, with the R14del mutation being the most prevalent cardiomyopathy-related mutation in the Netherlands. It is present in patients diagnosed with arrhythmogenic cardiomyopathy as well as dilated cardiomyopathy. Awareness of the phenotype of this PLN mutation is of great importance, since many carriers remain to be identified. Patients with the R14del mutation are characterised by older age at onset, low-voltage electrocardiograms and ahigh frequency of ventricular arrhythmias. Additionally, these patients have apoor prognosis often with left ventricular dysfunction and early-onset heart failure. Therefore, when there is asuspicion of aPLN mutation, cardiac and genetic screening is strongly recommended.

Background Mutations in the myosin heavy chain 7 (MYH7) gene commonly cause cardiomyopathy but are less frequently associated with congenital heart defects. Methods In this study, we describe a mutation in the MYH7 gene, c. 5754C > G; p. (Asn1918Lys), present in 15 probands and 65 family members. Results Of the 80 carriers (age range 0–88 years), 46 (57.5%) had cardiomyopathy (mainly dilated cardiomyopathy (DCM)) and seven (8.8%) had a congenital heart defect. Childhood onset of cardiomyopathy was present in almost 10% of carriers. However, in only a slight majority (53.7%) was the left ventricular ejection fraction reduced and almost no arrhythmias or conduction disorders were noted. Moreover, only one carrier required heart transplantation and nine (11.3%) an implantable cardioverter defibrillator. In addition, the standardised mortality ratio for MYH7 carriers was not significantly increased. Whole exome sequencing in several cases with paediatric onset of DCM and one with isolated congenital heart defects did not reveal additional known disease-causing variants. Haplotype analysis suggests that the MYH7 variant is a founder mutation, and is therefore the first Dutch founder mutation identified in the MYH7 gene. The mutation appears to have originated in the western region of the province of South Holland between 500 and 900 years ago. Conclusion Clinically, the p. (Asn1918Lys) mutation is associated with congenital heart defects and/or cardiomyopathy at young age but with a relatively benign course. Electronic supplementary material The online version of this article (10.1007/s12471-017-1037-5) contains supplementary material, which is available to authorized users.

Background/purpose: Peripartum cardiomyopathy (PPCM) is a cause of pregnancyassociated heart failure. It typically develops during the last month of pregnancy and up to six months after delivery in women without known cardiovascular disease. Recently, we showed that some cases of PPCM are part of the spectrum of familial dilated cardiomyopathy (DCM), presenting in the peripartum period. Inherited DCM is genetically highly heterogeneous; more than 50 genes are known to be involved. We performed haplotype sharing analysis in combination with exome sequencing in a family with familial PPCM/DCM in order to identify the underlying genetic defect. Methods: Haplotype analysis (250K SNP genotypes) was used to identify shared haplotypes between affected individuals of a family with two individuals with PPCM and four individuals with DCM (index patient, two siblings, one niece, two nephews). In order to identify the causal gene, exome sequencing was performed using DNA of the index patient and one of her affected nephews. Results: Haplotype analysis revealed a shared haplotype of 71 cM on chromosome 15, containing approximately 600 genes. This region does not contain any knownDCMgene. Exome sequencing and subsequent data analysis revealed several potential pathogenic variants in this region that were shared by the index patient and her nephew. Carriership analysis in affected individuals, healthy relatives and controls is in progress. Conclusions: We identified potential pathogenic variants in a novel candidate region on chromosome 15 in a family with familial PPCM/DCM. Our results underscore the applicability of the combined approach of haplotype sharing analysis and exome sequencing for the identification of a genetic cause in familial cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited cardiac disease with reduced penetrance and a highly variable expression. Mutations in the gene encoding the plakophilin-2 gene (PKP2) are detected in about 50% of ARVC/D patients. The p.Arg79X mutation in PKP2 has been identified in Europe and North America and has been functionally characterised. We evaluated the prevalence of the p.Arg79X mutation in PKP2 in the Dutch population.METHODS: Twelve index patients and 41 family members were evaluated in three university hospitals in the Netherlands. The diagnosis of ARVC/D was established according to the recently revised Task Force Criteria. Segregation of the p.Arg79X mutation was studied and haplotypes were reconstructed to determine whether the p.Arg79X mutation was a recurrent or a founder mutation.RESULTS: The p.Arg79X mutation in PKP2 was identified in 12 index patients. Haplotype analysis revealed a shared haplotype among Dutch p.Arg79X mutation carriers, indicating a common founder. Six index patients (50%) had a first- or second-degree relative who had died of sudden cardiac death below 40 years of age. At age 60, only 60% of the mutation carriers had experienced any symptoms. There was no significant difference in symptom-free survival and event-free survival between men and women.CONCLUSION: We have identified the largest series of patients with the same desmosome gene mutation in ARVC/D reported to date. This p.Arg79X mutation in PKP2 is a founder mutation in the Dutch population. The phenotypes of PKP2 p.Arg79X mutation carriers illustrate the clinical variability and reduced penetrance often seen in ARVC/D. (Neth Heart J 2010;18:583-91.).

Purpose: To examine the gross and microscopic pathological features of complete heart specimens from patients carrying the phospholamban (PLN) p.Arg14del mutation and to test our hypothesis that this mutation leads to aggregation and autophagy of mutant PLN protein in cardiomyocytes. Methods: Sixteen hearts were studied, acquired from 8 male and 8 female patients with a median age of 51.5 years (range 29-74). Eleven were obtained from autopsies and five were explants. Six autopsy cases had died suddenly. The other 10 patients were clinically evaluated on the basis of widely accepted criteria for ARVC and DCM. Immunohistochemistry (IHC) for PLN was performed to visualize protein aggregation. IHC for sequestosome-1 (SQSTM-1/p62) was applied to visualize autophagy. Double IHC staining was performed to study co-localization of PLN and SQSTM-1/p62 in aggregates. Results: All hearts had an ARVC phenotype with gross or microscopic fibrofatty replacement of the RV wall. LV involvement was present in 15/16 hearts, characterized by interstitial fibrosis and myocyte hypertrophy. Microscopic LV fibrofatty replacement was seen in 8/16 hearts. Clinically, 8/10 patients were diagnosed with combined ARVC/DCM and 2/10 had ARVC. Large perinuclear PLN protein aggregates were found in cardiomyocytes in both ventricles in all examined hearts. The median number of PLN aggregates was 8 per 5mm2 (range 1-19) in RV myocardium and 12 per 5mm2 (range 2-39) in LV myocardium. Co-localization of SQSTM-1/p62 and PLN was observed in these aggregates. Conclusions: In this series of 16 hearts, it appeared that PLN p.Arg14del-mutation related cardiomyopathy is a biventricular arrhythmogenic cardiomyopathy and a protein-aggregate associated disease characterized by large perinuclear PLN aggregates that are degraded by autophagy. ![Figure][1] Typical example gross pathological feat. [1]: pending:yes

The development of creatine kinase of skeletal muscles of fore legs, hind legs, heart, and brain of the rat has been studied. The activity of this enzyme of skeletal muscles of the fore legs was found to increase with age in a biphasic manner: a 7-fold increase was observed in the period 5–10 days after birth followed by a 3-fold increase in the period 15–20 days after birth. The development of enzymes involved in creatine biosynthesis has also been studied. The activity of glycine transaminidase of kidney cortex was found to be low in fetal life and increased about 10 times in the 30-day period after birth. The activity of guanidinoacetate transmethylase was high in fetal rat liver and decreased around birth to the adult level. The results indicate that the fetal rat has the capacity for the biosynthesis of creatine.