Penetrance and Prognosis of MYH7 Variant-Associated Cardiomyopathies: Results From a Dutch Multicenter Cohort Study.

BACKGROUND: MYH7 variants cause hypertrophic cardiomyopathy (HCM), noncompaction cardiomyopathy (NCCM), and dilated cardiomyopathy (DCM). Screening of relatives of patients with genetic cardiomyopathy is recommended from 10 to 12 years of age onward, irrespective of the affected gene. OBJECTIVES: This study sought to study the penetrance and prognosis of MYH7 variant-associated cardiomyopathies. METHODS: In this multicenter cohort study, penetrance and major cardiomyopathy-related events (MCEs) were assessed in carriers of (likely) pathogenic MYH7 variants by using Kaplan-Meier curves and log-rank tests. Prognostic factors were evaluated using Cox regression with time-dependent coefficients. RESULTS: In total, 581 subjects (30.1% index patients, 48.4% male, median age 37.0 years [IQR: 19.5-50.2 years]) were included. HCM was diagnosed in 226 subjects, NCCM in 70, and DCM in 55. Early penetrance and MCEs (age <12 years) were common among NCCM-associated variant carriers (21.2% and 12.0%, respectively) and DCM-associated variant carriers (15.3% and 10.0%, respectively), compared with HCM-associated variant carriers (2.9% and 2.1%, respectively). Penetrance was significantly increased in carriers of converter region variants (adjusted HR: 1.87; 95% CI: 1.15-3.04; P = 0.012) and at age ≤1 year in NCCM-associated or DCM-associated variant carriers (adjusted HR: 21.17; 95% CI: 4.81-93.20; P < 0.001) and subjects with a family history of early MCEs (adjusted HR: 2.45; 95% CI: 1.09-5.50; P = 0.030). The risk of MCE was increased in subjects with a family history of early MCEs (adjusted HR: 1.82; 95% CI: 1.15-2.87; P = 0.010) and at age ≤5 years in NCCM-associated or DCM-associated variant carriers (adjusted HR: 38.82; 95% CI: 5.16-291.88; P < 0.001). CONCLUSIONS: MYH7 variants can cause cardiomyopathies and MCEs at a young age. Screening at younger ages may be warranted, particularly in carriers of NCCM- or DCM-associated variants and/or with a family history of MCEs at <12 years.

The genetic basis of apparently idiopathic ventricular fibrillation: a retrospective overview.

AIMS: During the diagnostic work-up of patients with idiopathic ventricular fibrillation (VF), next-generation sequencing panels can be considered to identify genotypes associated with arrhythmias. However, consensus for gene panel testing is still lacking, and variants of uncertain significance (VUS) are often identified. The aim of this study was to evaluate genetic testing and its results in idiopathic VF patients. METHODS AND RESULTS: We investigated 419 patients with available medical records from the Dutch Idiopathic VF Registry. Genetic testing was performed in 379 (91%) patients [median age at event 39 years (27-51), 60% male]. Single-gene testing was performed in 87 patients (23%) and was initiated more often in patients with idiopathic VF before 2010. Panel testing was performed in 292 patients (77%). The majority of causal (likely) pathogenic variants (LP/P, n = 56, 15%) entailed the DPP6 risk haplotype (n = 39, 70%). Moreover, 10 LP/P variants were found in cardiomyopathy genes (FLNC, MYL2, MYH7, PLN (two), TTN (four), RBM20), and 7 LP/P variants were identified in genes associated with cardiac arrhythmias (KCNQ1, SCN5A (2), RYR2 (four)). For eight patients (2%), identification of an LP/P variant resulted in a change of diagnosis. In 113 patients (30%), a VUS was identified. Broad panel testing resulted in a higher incidence of VUS in comparison to single-gene testing (38% vs. 3%, P < 0.001). CONCLUSION: Almost all patients from the registry underwent, albeit not broad, genetic testing. The genetic yield of causal LP/P variants in idiopathic VF patients is 5%, increasing to 15% when including DPP6. In specific cases, the LP/P variant is the underlying diagnosis. A gene panel specifically for idiopathic VF patients is proposed.

Identification of novel genetic risk factors of dilated cardiomyopathy: from canine to human.

BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening heart disease and a common cause of heart failure due to systolic dysfunction and subsequent left or biventricular dilatation. A significant number of cases have a genetic etiology; however, as a complex disease, the exact genetic risk factors are largely unknown, and many patients remain without a molecular diagnosis. METHODS: We performed GWAS followed by whole-genome, transcriptome, and immunohistochemical analyses in a spontaneously occurring canine model of DCM. Canine gene discovery was followed up in three human DCM cohorts. RESULTS: Our results revealed two independent additive loci associated with the typical DCM phenotype comprising left ventricular systolic dysfunction and dilatation. We highlight two novel candidate genes, RNF207 and PRKAA2, known for their involvement in cardiac action potentials, energy homeostasis, and morphology. We further illustrate the distinct genetic etiologies underlying the typical DCM phenotype and ventricular premature contractions. Finally, we followed up on the canine discoveries in human DCM patients and discovered candidate variants in our two novel genes. CONCLUSIONS: Collectively, our study yields insight into the molecular pathophysiology of DCM and provides a large animal model for preclinical studies.

A Systematic Analysis of the Clinical Outcome Associated with Multiple Reclassified Desmosomal Gene Variants in Arrhythmogenic Right Ventricular Cardiomyopathy Patients.

The presence of multiple pathogenic variants in desmosomal genes (DSC2, DSG2, DSP, JUP, and PKP2) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to a severe phenotype. However, the pathogenicity of variants is reclassified frequently, which may result in a changed clinical risk prediction. Here, we present the collection, reclassification, and clinical outcome correlation for the largest series of ARVC patients carrying multiple desmosomal pathogenic variants to date (n = 331). After reclassification, only 29% of patients remained carriers of two (likely) pathogenic variants. They reached the composite endpoint (ventricular arrhythmias, heart failure, and death) significantly earlier than patients with one or no remaining reclassified variant (hazard ratios of 1.9 and 1.8, respectively). Periodic reclassification of variants contributes to more accurate risk stratification and subsequent clinical management strategy. Graphical Abstract.

The arrhythmogenic cardiomyopathy phenotype associated with PKP2 c.1211dup variant.

BACKGROUND: The arrhythmogenic cardiomyopathy (ACM) phenotype, with life-threatening ventricular arrhythmias and heart failure, varies according to genetic aetiology. We aimed to characterise the phenotype associated with the variant c.1211dup (p.Val406Serfs*4) in the plakophilin­2 gene (PKP2) and compare it with previously reported Dutch PKP2 founder variants. METHODS: Clinical data were collected retrospectively from medical records of 106 PKP2 c.1211dup heterozygous carriers. Using data from the Netherlands ACM Registry, c.1211dup was compared with 3 other truncating PKP2 variants (c.235C > T (p.Arg79*), c.397C > T (p.Gln133*) and c.2489+1G > A (p.?)). RESULTS: Of the 106 carriers, 47 (44%) were diagnosed with ACM, at a mean age of 41 years. By the end of follow-up, 29 (27%) had experienced sustained ventricular arrhythmias and 12 (11%) had developed heart failure, with male carriers showing significantly higher risks than females on these endpoints (p < 0.05). Based on available cardiac magnetic resonance imaging and echocardiographic data, 46% of the carriers showed either right ventricular dilatation and/or dysfunction, whereas a substantial minority (37%) had some form of left ventricular involvement. Both geographical distribution of carriers and haplotype analysis suggested PKP2 c.1211dup to be a founder variant originating from the South-Western coast of the Netherlands. Finally, a Cox proportional hazards model suggested significant differences in ventricular arrhythmia-free survival between 4 PKP2 founder variants, including c.1211dup. CONCLUSIONS: The PKP2 c.1211dup variant is a Dutch founder variant associated with a typical right-dominant ACM phenotype, but also left ventricular involvement, and a possibly more severe phenotype than other Dutch PKP2 founder variants.

MYH7 p.(Arg1712Gln) is pathogenic founder variant causing hypertrophic cardiomyopathy with overall relatively delayed onset.

INTRODUCTION: The MYH7 c.5135G > A p.(Arg1712Gln) variant has been identified in several patients worldwide and is classified as pathogenic in the ClinVar database. We aimed to delineate its associated phenotype and evaluate a potential founder effect. METHODS: We retrospectively collected clinical and genetic data of 22 probands and 74 family members from an international cohort. RESULTS: In total, 53 individuals carried the MYH7 p.(Arg1712Gln) variant, of whom 38 (72%) were diagnosed with hypertrophic cardiomyopathy (HCM). Mean age at HCM diagnosis was 48.8 years (standard deviation: 18.1; range: 8-74). The clinical presentation ranged from asymptomatic HCM to arrhythmias (atrial fibrillation and malignant ventricular arrhythmias). Aborted sudden cardiac death (SCD) leading to the diagnosis of HCM occurred in one proband at the age of 68 years, and a family history of SCD was reported by 39% (5/13) probands. Neither heart failure deaths nor heart transplants were reported. Women had a generally later-onset disease, with 14% of female carriers diagnosed with HCM at age 50 years compared with 54% of male carriers. In both sexes, the disease was fully penetrant by age 75 years. Haplotypes were reconstructed for 35 patients and showed a founder effect in a subset of patients. CONCLUSION: MYH7 p.(Arg1712Gln) is a pathogenic founder variant with a consistent HCM phenotype that may present with delayed penetrance. This suggested that clinical follow-up should be pursued after the seventh decade in healthy carriers and that longer intervals between screening may be justified in healthy women < 30 years.

Defining the clinical validity of genes reported to cause pulmonary arterial hypertension.

PURPOSE: Pulmonary arterial hypertension (PAH) is a rare, progressive vasculopathy with significant cardiopulmonary morbidity and mortality. Genetic testing is currently recommended for adults diagnosed with heritable, idiopathic, anorexigen-, hereditary hemorrhagic telangiectasia-, and congenital heart disease-associated PAH, PAH with overt features of venous/capillary involvement, and all children diagnosed with PAH. Variants in at least 27 genes have putative evidence for PAH causality. Rigorous assessment of the evidence is needed to inform genetic testing. METHODS: An international panel of experts in PAH applied a semi-quantitative scoring system developed by the NIH Clinical Genome Resource to classify the relative strength of evidence supporting PAH gene-disease relationships based on genetic and experimental evidence. RESULTS: Twelve genes (BMPR2, ACVRL1, ATP13A3, CAV1, EIF2AK4, ENG, GDF2, KCNK3, KDR, SMAD9, SOX17, and TBX4) were classified as having definitive evidence and 3 genes (ABCC8, GGCX, and TET2) with moderate evidence. Six genes (AQP1, BMP10, FBLN2, KLF2, KLK1, and PDGFD) were classified as having limited evidence for causal effects of variants. TOPBP1 was classified as having no known PAH relationship. Five genes (BMPR1A, BMPR1B, NOTCH3, SMAD1, and SMAD4) were disputed because of a paucity of genetic evidence over time. CONCLUSION: We recommend that genetic testing includes all genes with definitive evidence and that caution be taken in the interpretation of variants identified in genes with moderate or limited evidence. Genes with no known evidence for PAH or disputed genes should not be included in genetic testing.

Red flags and adjusted suspicion index for distinguishing hereditary transthyretin amyloid polyneuropathy from idiopathic axonal polyneuropathy.

BACKGROUND: Early diagnosis of hereditary ATTR polyneuropathy (ATTRv-PN) is important since treatment options have become available, which are most effective early in the disease course. ATTRv-PN is likely underdiagnosed as patients might be misdiagnosed with idiopathic polyneuropathy. It is uncertain if it is useful to test for TTR gene mutations in patients with a typical presentation for chronic idiopathic axonal polyneuropathy (CIAP) and which are the distinguishing clinical features. METHODS: We carried out a retrospective cohort study to assess the yield of TTR gene sequencing in patients with polyneuropathy and assessed if the identified patients with ATTRv-PN had a clinical presentation typical of CIAP. Additionally, we assessed which clinical features, including previously defined red flag symptoms, can differentiate between patients with CIAP and ATTRv-PN and assessed the performance of the TTR suspicion index. RESULTS: Out of 338 patients with polyneuropathy, 10 patients had a pathogenic TTR gene mutation (all p.Val50Met) and none had a clinical presentation typical of CIAP. Patients with ATTRv-PN more often had bilateral CTS, motor involvement of arms, cardiac involvement, family history suggestive of hATTRv, and autonomic symptoms than patients with CIAP. All patients with ATTRv-PN as well as 70% of patients with CIAP fulfilled the suspicion index. CONCLUSION: Routine TTR gene sequencing in patients with a typical presentation for CIAP is not useful. However, red flag symptoms can differentiate patients with ATTRv-PN from patients with CIAP. We propose an adjusted version of the TTR suspicion index to increase diagnostic yield.

Untargeted Metabolomics Identifies Potential Hypertrophic Cardiomyopathy Biomarkers in Carriers of MYBPC3 Founder Variants.

Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disease, commonly caused by pathogenic MYBPC3 variants, and a significant cause of sudden cardiac death. Severity is highly variable, with incomplete penetrance among genotype-positive family members. Previous studies demonstrated metabolic changes in HCM. We aimed to identify metabolite profiles associated with disease severity in carriers of MYBPC3 founder variants using direct-infusion high-resolution mass spectrometry in plasma of 30 carriers with a severe phenotype (maximum wall thickness ≥20 mm, septal reduction therapy, congestive heart failure, left ventricular ejection fraction <50%, or malignant ventricular arrhythmia) and 30 age- and sex-matched carriers with no or a mild phenotype. Of the top 25 mass spectrometry peaks selected by sparse partial least squares discriminant analysis, XGBoost gradient boosted trees, and Lasso logistic regression (42 total), 36 associated with severe HCM at a p < 0.05, 20 at p < 0.01, and 3 at p < 0.001. These peaks could be clustered to several metabolic pathways, including acylcarnitine, histidine, lysine, purine and steroid hormone metabolism, and proteolysis. In conclusion, this exploratory case-control study identified metabolites associated with severe phenotypes in MYBPC3 founder variant carriers. Future studies should assess whether these biomarkers contribute to HCM pathogenesis and evaluate their contribution to risk stratification.

Whole genome sequencing analysis reveals post-zygotic mutation variability in monozygotic twins discordant for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a heterogeneous, fatal neurodegenerative disease, characterized by motor neuron loss and in 50% of cases also by cognitive and/or behavioral changes. Mendelian forms of ALS comprise approximately 10-15% of cases. The majority is however considered sporadic, but also with a high contribution of genetic risk factors. To explore the contribution of somatic mutations and/or epigenetic changes to disease risk, we performed whole genome sequencing and methylation analyses using samples from multiple tissues on a cohort of 26 monozygotic twins discordant for ALS, followed by in-depth validation and replication experiments. The results of these analyses implicate several mechanisms in ALS pathophysiology, which include a role for de novo mutations, defects in DNA damage repair and accelerated aging.

Prevalence and Disease Expression of Pathogenic and Likely Pathogenic Variants Associated With Inherited Cardiomyopathies in the General Population.

BACKGROUND: Pathogenic and likely pathogenic variants associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), dilated cardiomyopathy (DCM), and hypertrophic cardiomyopathy (HCM) are recommended to be reported as secondary findings in genome sequencing studies. This provides opportunities for early diagnosis, but also fuels uncertainty in variant carriers (G+), since disease penetrance is incomplete. We assessed the prevalence and disease expression of G+ in the general population. METHODS: We identified pathogenic and likely pathogenic variants associated with ARVC, DCM and/or HCM in 200 643 UK Biobank individuals, who underwent whole exome sequencing. We calculated the prevalence of G+ and analyzed the frequency of cardiomyopathy/heart failure diagnosis. In undiagnosed individuals, we analyzed early signs of disease expression using available electrocardiography and cardiac magnetic resonance imaging data. RESULTS: We found a prevalence of 1:578, 1:251, and 1:149 for pathogenic and likely pathogenic variants associated with ARVC, DCM and HCM respectively. Compared with controls, cardiovascular mortality was higher in DCM G+ (odds ratio 1.67 [95% CI 1.04; 2.59], P=0.030), but similar in ARVC and HCM G+ (P≥0.100). Cardiomyopathy or heart failure diagnosis were more frequent in DCM G+ (odds ratio 3.66 [95% CI 2.24; 5.81], P=4.9×10-7) and HCM G+ (odds ratio 3.03 [95% CI 1.98; 4.56], P=5.8×10-7), but comparable in ARVC G+ (P=0.172). In contrast, ARVC G+ had more ventricular arrhythmias (P=3.3×10-4). In undiagnosed individuals, left ventricular ejection fraction was reduced in DCM G+ (P=0.009). CONCLUSIONS: In the general population, pathogenic and likely pathogenic variants associated with ARVC, DCM, or HCM are not uncommon. Although G+ have increased mortality and morbidity, disease penetrance in these carriers from the general population remains low (1.2-3.1%). Follow-up decisions in case of incidental findings should not be based solely on a variant, but on multiple factors, including family history and disease expression.

Clinical Characteristics and Follow-Up of Pediatric-Onset Arrhythmogenic Right Ventricular Cardiomyopathy.

OBJECTIVES: The goal of this study was to describe characteristics, cascade screening results, and predictors of adverse outcome in pediatric-onset arrhythmogenic right ventricular cardiomyopathy (ARVC). BACKGROUND: Although ARVC is increasingly recognized in children, pediatric ARVC cohorts remain underrepresented in the literature. METHODS: This study included 12 probands with pediatric-onset ARVC (aged <18 years at diagnosis) and 68 pediatric relatives (aged <18 years at first evaluation) referred for cascade screening. ARVC diagnosis was based on 2010 Task Force Criteria. Clinical presentation, diagnostic testing, and outcomes (sustained ventricular tachycardia [VT]; heart failure) were ascertained. Predictors of adverse outcome were determined by using univariable logistic regression. RESULTS: Pediatric-onset ARVC was diagnosed in 12 probands and 12 (18%) relatives at a median age of 16.6 years (interquartile range: 13.8-17.4 years), whereas 12 (18%) relatives reached ARVC diagnosis as adults (median age, 22.0 years; interquartile range: 20.0-26.7 years). Sudden cardiac death/arrest was the first disease manifestation in 3 (25%) probands and 3 (4%) relatives. In patients without ARVC diagnosis at presentation (n = 61), electrocardiogram and Holter monitoring abnormalities occurred before development of imaging Task Force Criteria (7.3 ± 5.0 years vs 8.4 ± 5.0 years). Clinical course was characterized by sustained VT (91%) and heart failure (36%) in probands, which were rare in relatives (2% and 0%, respectively). Male sex (P < 0.01), T-wave inversion V1-V3 (P < 0.01), premature ventricular complexes/runs (P ≤ 0.01), and decrease in biventricular ejection fraction (P ≤ 0.01) were associated with VT occurrence. CONCLUSIONS: Pediatric ARVC carries high arrhythmic risk, especially in probands. Disease progression is particularly observed on electrocardiogram or Holter monitoring. Arrhythmic events are associated with male sex, T-wave inversions, premature ventricular complexes/runs, and reduced biventricular ejection fraction.

P62-positive aggregates are homogenously distributed in the myocardium and associated with the type of mutation in genetic cardiomyopathy.

Genetic cardiomyopathy is caused by mutations in various genes. The accumulation of potentially proteotoxic mutant protein aggregates due to insufficient autophagy is a possible mechanism of disease development. The objective of this study was to investigate the distribution in the myocardium of such aggregates in relation to specific pathogenic genetic mutations in cardiomyopathy hearts. Hearts from 32 genetic cardiomyopathy patients, 4 non-genetic cardiomyopathy patients and 5 controls were studied. Microscopic slices from an entire midventricular heart slice were stained for p62 (sequestosome-1, marker for aggregated proteins destined for autophagy). The percentage of cardiomyocytes with p62 accumulation was higher in cardiomyopathy hearts (median 3.3%) than in healthy controls (0.3%; P < .0001). p62 accumulation was highest in the desmin (15.6%) and phospholamban (7.2%) groups. P62 accumulation was homogeneously distributed in the myocardium. Fibrosis was not associated with p62 accumulation in subgroup analysis of phospholamban hearts. In conclusion, accumulation of p62-positive protein aggregates is homogeneously distributed in the myocardium independently of fibrosis distribution and associated with desmin and phospholamban cardiomyopathy. Proteotoxic protein accumulation is a diffuse process in the myocardium while a more localized second hit, such as local strain during exercise, might determine whether this leads to regional myocyte decay.

A hereditary spastic paraplegia predominant phenotype caused by variants in the NEFL gene.

INTRODUCTION: This study reports a large series of patients with a clinical picture dominated by spastic paraplegia in whom variants in the NEFL gene, a known cause for Charcot-Marie-Tooth disease, were identified. METHODS: Index patients referred for a suspicion of hereditary spastic paraplegia (HSP) were clinically assessed and genetic analysis by next-generation sequencing was undertaken. Additional family members were clinically examined and subjected to targeted testing. RESULTS: We identified two different heterozygous dominant variants in the NEFL gene in 25 patients from 14 families. Most of them (21/25) had a clinical diagnosis of HSP, often with a concomitant clinical diagnosis of polyneuropathy (16/21). Two patients were identified with a polyneuropathy with a pyramidal reflex pattern, but without spasticity. Two patients had isolated polyneuropathy. Out of the 21 patients with a diagnosis of HSP, two had co-occurring cerebellar signs. The c.262A > C p.(Thr88Pro) variant was detected in 13 families. Genealogical analysis showed shared ancestors or a similar geographical origin in 12, suggesting a founder effect. The other variant, c.296A > C p.(Asp99Ala), was found in only one family, in which limited segregation analysis could be performed. DISCUSSION: Variants in the NEFL gene can cause HSP, with or without co-existing polyneuropathy, and should be included in diagnostic testing strategies for HSP patients.

A mutation update for the FLNC gene in myopathies and cardiomyopathies.

Filamin C (FLNC) variants are associated with cardiac and muscular phenotypes. Originally, FLNC variants were described in myofibrillar myopathy (MFM) patients. Later, high-throughput screening in cardiomyopathy cohorts determined a prominent role for FLNC in isolated hypertrophic and dilated cardiomyopathies (HCM and DCM). FLNC variants are now among the more prevalent causes of genetic DCM. FLNC-associated DCM is associated with a malignant clinical course and a high risk of sudden cardiac death. The clinical spectrum of FLNC suggests different pathomechanisms related to variant types and their location in the gene. The appropriate functioning of FLNC is crucial for structural integrity and cell signaling of the sarcomere. The secondary protein structure of FLNC is critical to ensure this function. Truncating variants with subsequent haploinsufficiency are associated with DCM and cardiac arrhythmias. Interference with the dimerization and folding of the protein leads to aggregate formation detrimental for muscle function, as found in HCM and MFM. Variants associated with HCM are predominantly missense variants, which cluster in the ROD2 domain. This domain is important for binding to the sarcomere and to ensure appropriate cell signaling. We here review FLNC genotype-phenotype correlations based on available evidence.

Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmosomal Variants Are Rarely De Novo.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with pathogenic/likely pathogenic (P/LP) variants in genes encoding the cardiac desmosomal proteins. Origin of these variants, including de novo mutation rate and extent of founder versus recurrent variants has implications for variant adjudication and clinical care, yet this has never been systematically investigated. METHODS: We identified arrhythmogenic right ventricular cardiomyopathy probands who met 2010 Task Force Criteria and had undergone genotyping that included sequencing of the desmosomal genes (PKP2, DSP, DSG2, DSC2, and JUP) from 3 arrhythmogenic right ventricular cardiomyopathy registries in America and Europe. We classified the desmosomal variants, defined the contribution of unique versus nonunique (ie, not family-specific) P/LP variants, and identified the frequency and characteristics of de novo variants. Next, we haplotyped nonunique variants to determine how often they likely represent a single mutation event in a common ancestor (implied by shared haplotypes) versus multiple mutation events at the same genetic location. RESULTS: Of 501 arrhythmogenic right ventricular cardiomyopathy probands, 322 (64.3%) carried 327 desmosomal P/LP variants. Most variants (n=247, 75.6%, in 245 patients) were identified in more than one proband and, therefore, considered nonunique. For 212/327 variants (64.8%) genetic cascade screening was performed extensively enough to identify the parental origin of the P/LP variant. Only 3 variants were de novo, 2 of which were whole gene deletions. For 24 nonunique P/LP PKP2 variants, haplotyping was conducted in 183 available families. For all 24 variants, multiple seemingly unrelated families sharing identical haplotypes were identified, suggesting that these variants originate from common founders. CONCLUSIONS: Most desmosomal P/LP variants are inherited, nonunique, and originate from ancient founders. Two of 3 de novo variants were large deletions. These observations inform genetic testing, cascade screening, and variant adjudication.

Mortality Risk Associated With Truncating Founder Mutations in Titin.

Background Truncating titin variants (TTNtv) are the most prevalent genetic cause of dilated cardiomyopathy, found in ≤25% of familial cases. Moreover, TTNtv associated with dilated cardiomyopathy are estimated to be present in 0.5% of the general population. The prognosis of asymptomatic carriers of TTNtv is poorly understood because TTNtv are associated with a highly variable phenotype. We aim to assess the natural history and clinical relevance of TTNtv by analyzing standardized mortality ratios (SMR) in multigenerational pedigrees and in close relatives of present-day patients. Methods Haplotype and genealogical analyses were performed on 3 recurrent TTNtv. Subsequently, the family tree mortality ratio method was used to compare all-cause mortality of subjects at an a priori 50% risk of carrying TTNtv to the general Dutch population. SMRs were stratified for sex, age, and calendar period. Subgroups were compared with Poisson regression. Similarly, SMRs were calculated in parents of 128 present-day dilated cardiomyopathy probands with TTNtv using the reverse parent-offspring method. Results The TTNtv were established as founder mutations and traced to 18th century ancestors. In 20 522 person-years, overall mortality was not significantly increased (SMR, 1.06; 95% CI, 0.95-1.18; P=0.162). However, mortality was significantly increased in subjects living after 1965 (SMR, 1.27; 95% CI, 1.04-1.53; P=0.009) and aged ≥60 years (SMR, 1.17; 95% CI, 1.01-1.35; P=0.02). The reverse parent-offspring analysis showed overall excess mortality (SMR, 1.26; 95% CI, 1.07-1.48; P=0.003), driven by subjects aged ≥60 years. Conclusions The natural history of the analyzed TTNtv shows a relatively mild disease course with significant excess mortality in elderly patients. With increasing life expectancy, TTNtv-associated morbidity and mortality will likely become more prevalent.

Glycogen Storage Disease Type IV: A Rare Cause for Neuromuscular Disorders or Often Missed?

Advancements in genetic testing now allow early identification of previously unresolved neuromuscular phenotypes. To illustrate this, we here present diagnoses of glycogen storage disease IV (GSD IV) in two patients with hypotonia and delayed development of gross motor skills. Patient 1 was diagnosed with congenital myopathy based on a muscle biopsy at the age of 6 years. The genetic cause of his disorder (two compound heterozygous missense mutations in GBE1 (c.[760A>G] p.[Thr254Ala] and c.[1063C>T] p.[Arg355Cys])), however, was only identified at the age of 17, after panel sequencing of 314 genes associated with neuromuscular disorders. Thanks to the availability of next-generation sequencing, patient 2 was diagnosed before the age of 2 with two compound heterozygous mutations in GBE1 (c.[691+2T>C] (splice donor variant) and the same c.[760A>G] p.[Thr254Ala] mutation as patient 1). GSD IV is an autosomal recessive metabolic disorder with a broad and expanding clinical spectrum, which hampers targeted diagnostics. The current cases illustrate the value of novel genetic testing for rare genetic disorders with neuromuscular phenotypes, especially in case of clinical heterogeneity. We argue that genetic testing by gene panels or whole exome sequencing should be considered early in the diagnostic procedure of unresolved neuromuscular disorders.

The homozygous K280N troponin T mutation alters cross-bridge kinetics and energetics in human HCM.

Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (k ACT) and the rate constant of isometric relaxation (slow k REL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces k ACT, slow k REL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases k ACT, slow k REL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.

Lack of evidence for a causal role of CALR3 in monogenic cardiomyopathy.

The pathogenicity of previously published disease-associated genes and variants is sometimes questionable. Large-scale, population-based sequencing studies have uncovered numerous false assignments of pathogenicity. Misinterpretation of sequence variants may have serious implications for the patients and families involved, as genetic test results are increasingly being used in medical decision making. In this study, we assessed the role of the calreticulin-3 gene (CALR3) in cardiomyopathy. CALR3 has been included in several cardiomyopathy gene panels worldwide. Its inclusion is based on a single publication describing two missense variants in patients with hypertrophic cardiomyopathy. In our national cardiomyopathy cohort (n = 6154), we identified 17 unique, rare heterozygous CALR3 variants in 48 probands. Overall, our patient cohort contained a significantly higher number of rare CALR3 variants compared to the ExAC population (p = 0.0036). However, after removing a potential Dutch founder variant, no statistically significant difference was found (p = 0.89). In nine probands, the CALR3 variant was accompanied by a disease-causing variant in another, well-known cardiomyopathy gene. In three families, the CALR3 variant did not segregate with the disease. Furthermore, we could not demonstrate calreticulin-3 protein expression in myocardial tissues at various ages. On the basis of these findings, it seems highly questionable that variants in CALR3 are a monogenic cause of cardiomyopathy.