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Background- Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited disease with incomplete penetrance and variable expression. Causative mutations in genes encoding 5 desmosomal proteins are found in ~=50% of ARVD/C index patients. Previous genotype-phenotype relation studies involved mainly overt ARVD/C index patients, so follow-up data on relatives are scarce. Methods and Results- One hundred forty-nine ARVD/C index patients (111 male patients; age, 49±13 years) according to 2010 Task Force criteria and 302 relatives from 93 families (282 asymptomatic; 135 male patients; age, 44±13 years) were clinically and genetically characterized. DNA analysis comprised sequencing of plakophilin-2 (PKP2), desmocollin-2, desmoglein-2, desmoplakin, and plakoglobin and multiplex ligation-dependent probe amplification to identify large deletions in PKP2. Pathogenic mutations were found in 87 index patients (58%), mainly truncating PKP2 mutations, including 3 cases with multiple mutations. Multiplex ligation-dependent probe amplification revealed 3 PKP2 exon deletions. ARVD/C was diagnosed in 31% of initially asymptomatic mutation-carrying relatives and 5% of initially asymptomatic relatives of index patients without mutation. Prolonged terminal activation duration was observed more than negative T waves in V(1) to V(3), especially in mutation-carrying relatives <20 years of age. In 45% of screened families, >=1 affected relatives were identified (90% with mutations). Conclusions- Pathogenic desmosomal gene mutations, mainly truncating PKP2 mutations, underlie ARVD/C in the majority (58%) of Dutch index patients and even 90% of familial cases. Additional multiplex ligation-dependent probe amplification analysis contributed to discovering pathogenic mutations underlying ARVD/C. Discovering pathogenic mutations in index patients enables those relatives who have a 6-fold increased risk of ARVD/C diagnosis to be identified. Prolonged terminal activation duration seems to be a first sign of ARVD/C in young asymptomatic relatives. [ABSTRACT FROM AUTHOR]

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.Typical example gross pathological feat. [ABSTRACT FROM PUBLISHER]

Purpose: Bone scintigraphy is key to non-invasively diagnosing wild-type transthyretin (ATTRwt) amyloidosis, and is mainly used to assess cardiac radiotracer uptake. However, extracardiac radiotracer uptake is also observed. We investigated whether intensity of soft tissue radiotracer uptake is associated with amyloid load in subcutaneous abdominal fat tissue and with mortality., Methods: This prospective cohort study included 94 ATTRwt amyloidosis patients and 26 amyloid-negative heart failure controls who underwent whole-body [ 99m Tc]Tc-hydroxydiphosphonate scintigraphy. Site-to-background ratios were calculated for heart, elbows, subcutaneous tissue, shoulders and wrists on anterior planar bone scintigraphy images using rib and whole-body radiotracer uptake as background. Fat tissue aspirates were stained with Congo red to grade amyloid load. Site-to-rib ratios were compared between ATTRwt amyloidosis patients and controls, and associations of site-to-background ratio with Congo red score and all-cause mortality were studied., Results: ATTRwt amyloidosis patients had higher soft tissue-to-rib, heart-to-rib and heart-to-whole body ratios compared with controls. The intensity of soft tissue uptake was positively associated with amyloid load in fat tissue in ATTRwt amyloidosis patients. Estimated glomerular filtration rate, N-terminal brain natriuretic propeptide, high-sensitivity cardiac troponin T (hs-cTnT), and the prognostic Mayo and NAC staging system were associated with all-cause mortality in univariable models. Soft tissue/rib ratio, hs-cTnT and the prognostic staging systems were the only two variables that were independently associated withall-cause mortality., Conclusion: Soft tissue radiotracer uptake on bone scintigraphy in ATTRwt amyloidosis patients is positively associated with amyloid load in abdominal fat tissue and is independently associated with mortality., (© 2024. The Author(s).)

POPDC2 encodes for the Popeye domain-containing protein 2 which has an important role in cardiac pacemaking and conduction, due in part to its cAMP-dependent binding and regulation of TREK-1 potassium channels. Loss of Popdc2 in mice results in sinus pauses and bradycardia and morpholino knockdown of popdc2 in zebrafish results in atrioventricular (AV) block. We identified bi-allelic variants in POPDC2 in 4 families that presented with a phenotypic spectrum consisting of sinus node dysfunction, AV conduction defects and hypertrophic cardiomyopathy. Using homology modelling we show that the identified POPDC2 variants are predicted to diminish the ability of POPDC2 to bind cAMP. In in vitro electrophysiological studies we demonstrated that, while co-expression of wild-type POPDC2 with TREK-1 increased TREK-1 current density, POPDC2 variants found in the patients failed to increase TREK-1 current density. While patient muscle biopsy did not show clear myopathic disease, it showed significant reduction of the expression of both POPDC1 and POPDC2, suggesting that stability and/or membrane trafficking of the POPDC1-POPDC2 complex is impaired by pathogenic variants in any of the two proteins. Single-cell RNA sequencing from human hearts demonstrated that co-expression of POPDC1 and 2 was most prevalent in AV node, AV node pacemaker and AV bundle cells. Sinoatrial node cells expressed POPDC2 abundantly, but expression of POPDC1 was sparse. Together, these results concur with predisposition to AV node disease in humans with loss-of-function variants in POPDC1 and POPDC2 and presence of sinus node disease in POPDC2, but not in POPDC1 related disease in human. Using population-level genetic data of more than 1 million individuals we showed that none of the familial variants were associated with clinical outcomes in heterozygous state, suggesting that heterozygous family members are unlikely to develop clinical manifestations and therefore might not necessitate clinical follow-up. Our findings provide evidence for POPDC2 as the cause of a novel Mendelian autosomal recessive cardiac syndrome, consistent with previous work showing that mice and zebrafish deficient in functional POPDC2 display sinus and AV node dysfunction.

Background: Phospholamban (PLN) p.(Arg14del) variant carriers are at risk for development of malignant ventricular arrhythmia (MVA). Accurate risk stratification allows timely implantation of intracardiac defibrillators and is currently performed with a multimodality prediction model., Objective: This study aimed to investigate whether an explainable deep learning-based approach allows risk prediction with only electrocardiogram (ECG) data., Methods: A total of 679 PLN p.(Arg14del) carriers without MVA at baseline were identified. A deep learning-based variational auto-encoder, trained on 1.1 million ECGs, was used to convert the 12-lead baseline ECG into its FactorECG, a compressed version of the ECG that summarizes it into 32 explainable factors. Prediction models were developed by Cox regression., Results: The deep learning-based ECG-only approach was able to predict MVA with a C statistic of 0.79 (95% CI, 0.76-0.83), comparable to the current prediction model (C statistic, 0.83 [95% CI, 0.79-0.88]; P = .054) and outperforming a model based on conventional ECG parameters (low-voltage ECG and negative T waves; C statistic, 0.65 [95% CI, 0.58-0.73]; P < .001). Clinical simulations showed that a 2-step approach, with ECG-only screening followed by a full workup, resulted in 60% less additional diagnostics while outperforming the multimodal prediction model in all patients. A visualization tool was created to provide interactive visualizations (https://pln.ecgx.ai)., Conclusion: Our deep learning-based algorithm based on ECG data only accurately predicts the occurrence of MVA in PLN p.(Arg14del) carriers, enabling more efficient stratification of patients who need additional diagnostic testing and follow-up., Competing Interests: Disclosures The UMC Groningen, which employs several of the authors, received research grants and/or fees from AstraZeneca, Abbott, Boehringer Ingelheim, Cardior Pharmaceuticals GmbH, Ionis Pharmaceuticals, Inc, Novo Nordisk, and Roche (outside the submitted work). Rudolf A. de Boer has had speaker engagements with Abbott, AstraZeneca, Bayer, Bristol Myers Squibb, Novartis, and Roche (outside the submitted work). Rutger R. van de Leur and René van Es are cofounders, shareholders, and board members of Cordys Analytics B.V., a spin-off of the UMC Utrecht that has licensed AI-ECG algorithms, not including the algorithm studied in the current manuscript. The UMC Utrecht receives royalties from Cordys Analytics for potential future revenues. Pieter A. Doevendans is founder and shareholder of HeartEye B.V., an ECG-device company. The other authors declare that there is no conflict of interest., (Copyright © 2024 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate serum neurofilament light chain (sNfL) as biomarker of disease onset, progression and treatment effect in hereditary transthyretin (ATTRv) amyloidosis patients and TTR variant ( TTR v) carriers., Methods: sNfL levels were assessed longitudinally in persistently asymptomatic TTR v carriers ( N  = 12), persistently asymptomatic ATTRv amyloidosis patients (defined as asymptomatic patients but with amyloid detectable in subcutaneous abdominal fat tissue) ( N  = 8), in TTR v carriers who developed polyneuropathy ( N  = 7) and in ATTRv amyloidosis patients with polyneuropathy on treatment (TTR-stabiliser ( N  = 20) or TTR-silencer ( N  = 18)). Polyneuropathy was confirmed by nerve conduction studies or quantitative sensory testing. sNfL was analysed using a single-molecule array assay., Results: sNfL increased over 2 years in persistently asymptomatic ATTRv amyloidosis patients, but did not change in persistently asymptomatic TTR v carriers. In all TTR v carriers who developed polyneuropathy, sNfL increased from 8.4 to 49.8 pg/mL before the onset of symptoms and before polyneuropathy could be confirmed neurophysiologically. In symptomatic ATTRv amyloidosis patients on a TTR-stabiliser, sNfL remained stable over 2 years. In patients on a TTR-silencer, sNfL decreased after 1 year of treatment., Conclusion: sNfL is a biomarker of early neuronal damage in ATTRv amyloidosis already before the onset of polyneuropathy. Current data support the use of sNfL in screening asymptomatic TTR v carriers and in monitoring of disease progression and treatment effect.

Aims: Recently, a genetic variant-specific prediction model for phospholamban (PLN) p.(Arg14del)-positive individuals was developed to predict individual major ventricular arrhythmia (VA) risk to support decision-making for primary prevention implantable cardioverter defibrillator (ICD) implantation. This model predicts major VA risk from baseline data, but iterative evaluation of major VA risk may be warranted considering that the risk factors for major VA are progressive. Our aim is to evaluate the diagnostic performance of the PLN p.(Arg14del) risk model at 3-year follow-up., Methods and Results: We performed a landmark analysis 3 years after presentation and selected only patients with no prior major VA. Data were collected of 268 PLN p.(Arg14del)-positive subjects, aged 43.5 ± 16.3 years, 38.9% male. After the 3 years landmark, subjects had a mean follow-up of 4.0 years (± 3.5 years) and 28 (10%) subjects experienced major VA with an annual event rate of 2.6% [95% confidence interval (CI) 1.6-3.6], defined as sustained VA, appropriate ICD intervention, or (aborted) sudden cardiac death. The PLN p.(Arg14del) risk score yielded good discrimination in the 3 years landmark cohort with a C-statistic of 0.83 (95% CI 0.79-0.87) and calibration slope of 0.97., Conclusion: The PLN p.(Arg14del) risk model has sustained good model performance up to 3 years follow-up in PLN p.(Arg14del)-positive subjects with no history of major VA. It may therefore be used to support decision-making for primary prevention ICD implantation not merely at presentation but also up to at least 3 years of follow-up., Competing Interests: Conflict of interest: The UMCG, which employs several of the authors, has received research grants and/or fees from AstraZeneca, Abbott, Boehringer Ingelheim, Cardior Pharmaceuticals GmbH, Ionis Pharmaceuticals, Inc., Novo Nordisk, and Roche. R.A.deB. has had speaker engagements with Abbott, AstraZeneca, Bayer, Bristol Myers Squibb, Novartis, and Roche. T.G. has had speaker engagements with Bristol Myers Squibb. M.Y.C.vd.H. and A.A.M.W. report grants from PLN foundation, grants from CVON Netherlands Heart Foundation (PREDICT2) and ZonMw (PSIDER-Heart) during this study., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Purpose: There is a need for early quantitative markers of potential treatment response in patients with hereditary transthyretin (ATTRv) amyloidosis to guide therapy. This study aims to evaluate changes in cardiac tracer uptake on bone scintigraphy in ATTRv amyloidosis patients on different treatments., Methods: In this retrospective cohort study, outcomes of 20 patients treated with the transthyretin (TTR) gene silencer patisiran were compared to 12 patients treated with a TTR-stabilizer. Changes in NYHA class, cardiac biomarkers in serum, wall thickness, and diastolic parameters on echocardiography and NYHA class during treatment were evaluated., Results: Median heart/whole-body (H/WB) ratio on bone scintigraphy decreased from 4.84 [4.00 to 5.31] to 4.16 [3.66 to 4.81] (p < .001) in patients treated with patisiran for 29 [15-34] months. No changes in the other follow-up parameters were observed. In patients treated with a TTR-stabilizer for 24 [20 to 30] months, H/WB ratio increased from 4.46 [3.24 to 5.13] to 4.96 [ 3.39 to 5.80] (p = .010), and troponin T increased from 19.5 [9.3 to 34.0] ng/L to 20.0 [11.8 to 47.8] ng/L (p = .025). All other parameters did not change during treatment with a TTR-stabilizer., Conclusion: A change in cardiac tracer uptake on bone scintigraphy may be an early marker of treatment-specific response or disease progression in ATTRv amyloidosis patients., (© 2023. The Author(s).)

(1) Background: Individuals carrying a pathogenic transthyretin gene variant ( TTRv ) are at high risk for developing hereditary transthyretin (ATTRv) amyloidosis and are routinely screened for the development of cardiomyopathy (ATTRv-CM). This study aims to evaluate whether the cardiac biomarkers N-terminal pro B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT) can be used to rule out ATTRv-CM. (2) Methods: In this retrospective case-control study, data from 46 ATTRv-CM patients and 101 TTRv carriers and ATTRv amyloidosis patients without cardiomyopathy were included. Binary logistic regression models were used to assess the ability of NT-proBNP and hs-cTnT to predict the diagnosis of ATTRv-CM. An optimal cutoff for the relevant biomarker(s) was determined based on a sensitivity of ≥99% and the highest possible percentage of additional tests avoided (%ATA) in the index dataset. (3) Results: Hs-cTnT demonstrated the highest predictive capabilities for ATTRv-CM. The addition of NT-proBNP did not improve the predictive model. A hs-cTnT cutoff of <6 ng/L resulted in a 97% sensitivity and a negative predictive value of 95% with a %ATA of 30% in the validation dataset. (4) Conclusion: In conclusion, hs-cTnT is a useful biomarker for excluding cardiac involvement in TTRv carriers and ATTRv amyloidosis patients and it has the potential to prevent unnecessary diagnostic procedures.

The c.40&#95;42delAGA variant in the phospholamban gene (PLN) has been associated with dilated and arrhythmogenic cardiomyopathy, with up to 70% of carriers experiencing a major cardiac event by age 70. However, there are carriers who remain asymptomatic at older ages. To understand the mechanisms behind this incomplete penetrance, we evaluated potential phenotypic and genetic modifiers in 74 PLN:c.40&#95;42delAGA carriers identified in 36,339 participants of the Lifelines population cohort. Asymptomatic carriers (N = 48) showed shorter QRS duration (- 5.73 ms, q value = 0.001) compared to asymptomatic non-carriers, an effect we could replicate in two different independent cohorts. Furthermore, symptomatic carriers showed a higher correlation (r Pearson  = 0.17) between polygenic predisposition to higher QRS (PGS QRS ) and QRS (p value = 1.98 × 10 -8 ), suggesting that the effect of the genetic variation on cardiac rhythm might be increased in symptomatic carriers. Our results allow for improved clinical interpretation for asymptomatic carriers, while our approach could guide future studies on genetic diseases with incomplete penetrance., (© 2023. The Author(s).)

Background: Dilated cardiomyopathy (DCM) can be caused by truncating variants in the filamin C gene (FLNC). A new pathogenic FLNC variant, c.6864&#95;6867dup, p.(Val2290Argfs∗23), was recently identified in Dutch patients with DCM., Objectives: The report aimed to evaluate the phenotype of FLNC variant carriers and to determine whether this variant is a founder variant., Methods: Clinical and genetic data were retrospectively collected from variant carriers. Cardiovascular magnetic resonance studies were reassessed. Haplotypes were reconstructed to determine a founder effect. The geographical distribution and age of the variant were determined., Results: Thirty-three individuals (of whom 23 [70%] were female) from 9 families were identified. Sudden cardiac death was the first presentation in a carrier at the age of 28 years. The median age at diagnosis was 41 years (range 19-67 years). The phenotype was heterogeneous. DCM with left ventricular dilation and reduced ejection fraction (<45%) was present in 11 (33%) individuals, 3 (9%) of whom underwent heart transplantation. Cardiovascular magnetic resonance showed late gadolinium enhancement in 13 (65%) of the assessed individuals, primarily in a ringlike distribution. Nonsustained ventricular arrhythmias were detected in 6 (18%), and 5 (15%) individuals received an implantable cardioverter-defibrillator. A shared haplotype spanning 2.1 Mb was found in all haplotyped individuals. The variant originated between 275 and 650 years ago., Conclusion: The pathogenic FLNC variant c.6864&#95;6867dup, p.(Val2290Argfs∗23) is a founder variant originating from the south of the Netherlands. Carriers are susceptible to developing heart failure and ventricular arrhythmias. The cardiac phenotype is characterized by ringlike late gadolinium enhancement, even in individuals without significantly reduced left ventricular function., (Copyright © 2023. Published by Elsevier Inc.)

Competing Interests: Conflict of interest: R.A.d.B. is president-elect of the Dutch Cardiac Society (NVVC), member of the Executive Committee of the DELIVER-trial and has received grants from AstraZeneca, Abbott, Boehringer Ingelheim, Cardior Pharmaceuticals GmbH, Ionis Pharmaceuticals, Inc., Novo Nordisk, and Roche in the last 36 months. R.A.d.B. has received payment or honoraria from Abbott, AstraZeneca, Bayer, Bristol Myers Squibb, Novartis, and Roche in the last 36 months. J.P.v.T. has received consulting fees from StrideBio in the last 36 months. A.A.M.W. has received grants from the Dutch Heart Foundation and consulting fees from LQT Therapeutics and ARMGO in the last 36 months. A.A.M.W. additionally is a member of the advisory board of the Leap Trial.

Background: Endurance and frequent exercise are associated with earlier onset of arrhythmogenic right ventricular cardiomyopathy (ARVC) and ventricular arrhythmias (VA) in desmosomal gene variant carriers. Individuals with the pathogenic c.40&#95;42del; p.(Arg14del) variant in the PLN gene are frequently diagnosed with ARVC or dilated cardiomyopathy (DCM). The aim of this study was to evaluate the effect of exercise in PLN p.(Arg14del) carriers., Methods: In total, 207 adult PLN p.(Arg14del) carriers (39.1% male; mean age 53 ± 15 years) were interviewed on their regular physical activity since the age of 10 years. The association of exercise with diagnosis of ARVC, DCM, sustained VA and hospitalisation for heart failure (HF) was studied., Results: Individuals participated in regular physical activities with a median of 1661 metabolic equivalent of task (MET) hours per year (31.9 MET-hours per week) until clinical presentation. The 50% most and least active individuals had a similar frequency of sustained VA (18.3% vs 18.4%; p = 0.974) and hospitalisation for HF (9.6% vs 8.7%; p = 0.827). There was no relationship between exercise and survival free from (incident) sustained VA (p = 0.65), hospitalisation for HF (p = 0.81), diagnosis of ARVC (p = 0.67) or DCM (p = 0.39) during follow-up. In multivariate analyses, exercise was not associated with sustained VA or HF hospitalisation during follow-up in this relatively not-active cohort., Conclusion: There was no association between the amount of exercise and the susceptibility to develop ARVC, DCM, VA or HF in PLN p.(Arg14del) carriers. This suggested unaffected PLN p.(Arg14del) carriers can safely perform mild-moderate exercise, in contrast to desmosomal variant carriers and ARVC patients., (© 2023. The Author(s).)

Background: Splice prediction algorithms currently used in routine DNA diagnostics have limited sensitivity and specificity, therefore many potential splice variants are classified as variants of uncertain significance (VUSs). However, functional assessment of VUSs to test splicing is labour-intensive and time-consuming. We developed a decision tree to prioritise potential splice variants for functional studies and functionally verified the outcome of the decision tree., Materials and Methods: We built the decision tree, SEPT-GD, by setting thresholds for the splice prediction programs implemented in Alamut. A set of 343 variants with known effects on splicing was used as control for sensitivity and specificity. We tested SEPT-GD using variants from a Dutch cardiomyopathy cohort of 2002 patients that were previously classified as VUS and predicted to have a splice effect according to diagnostic rules. We then selected 12 VUSs ranked by SEPT-GD to functionally verify the predicted effect on splicing using a minigene assay: 10 variants predicted to have a strong effect and 2 with a weak effect. RT-PCR was performed for nine variants. Variant classification was re-evaluated based on the functional test outcome., Results: Compared to similar individually tested algorithms, SEPT-GD shows higher sensitivity (91 %) and comparable specificity (88 %) for both consensus (dinucleotides at the start and end of the intron, GT at the 5' end and AG at the 3' end) and non-consensus splice-site variants (excluding middle of exon variants). Using clinical diagnostic criteria, 1295 unique variants in our cardiomyopathy cohort had originally been classified as VUSs, with 57 predicted by Alamut to have an effect on splicing. Using SEPT-GD, we prioritised 31 variants in 40 patients. In the minigene assay, all 12 variants showed results concordant with SEPT-GD predictions. RT-PCR confirmed the minigene results for two variants, TMEM43 c.1000 + 5G > T and TTN c.25922-6 T > G. Based on all outcomes, the SGCD c.4-1G > A and CSRP3 c.282-5&#95;285del variants were reclassified as likely pathogenic., Conclusion: SEPT-GD outperforms the tools commonly used for RNA splicing prediction and improves prioritisation of variants in cardiomyopathy genes for functional splicing analysis in a diagnostic setting., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: In the molecular genetic diagnostics of Mendelian disorders, solutions are needed for the major challenge of dealing with the large number of variants of uncertain significance (VUSs) identified using next-generation sequencing (NGS). Recently, promising approaches using constraint metrics to calculate case excess scores (CE), etiological fractions (EF), and gnomAD-derived constraint scores have been reported that estimate the likelihood of rare variants in specific genes or regions that are pathogenic. Our objective is to study the usability of these constraint data into variant interpretation in a diagnostic setting, using our cardiomyopathy cohort. Methods and Results: Patients (N = 2002) referred for clinical genetic diagnostics underwent NGS testing of 55-61 genes associated with cardiomyopathies. Previously classified likely pathogenic (LP) and pathogenic (P) variants were used to validate the use of data from CE, EF, and gnomAD constraint analyses for (re)classification of associated variant types in specific cardiomyopathy subtype-related genes. The classifications corroborated in 94% (354/378) of cases. Next, we reclassified 23 unique VUSs to LP, increasing the diagnostic yield by 1.2%. In addition, 106 unique VUSs (5.3% of patients) were prioritized for co-segregation or functional analyses. Conclusions: Our analysis confirms that the use of constraint metrics data can improve variant interpretation, and we, therefore, recommend using constraint scores on other cohorts and disorders and its inclusion in variant interpretation protocols., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alimohamed, Westers, Vos, Van der Velde, Sijmons, Van der Zwaag, Sikkema-Raddatz and Jongbloed.)

Background: A pathogenic variant in the gene encoding phospholamban (PLN), a protein that regulates calcium homeostasis of cardiomyocytes, causes PLN cardiomyopathy. It is characterized by a high arrhythmic burden and can progress to severe cardiomyopathy. Risk assessment guides implantable cardioverter-defibrillator therapy and benefits from personalization. Whether sex-specific differences in PLN cardiomyopathy exist is unknown., Objective: The purpose of this study was to improve the accuracy of PLN cardiomyopathy diagnosis and risk assessment by investigating sex-specific aspects., Methods: We analyzed a multicenter cohort of 933 patients (412 male, 521 female) with the PLN p.(Arg14del) pathogenic variant following up on a recently developed PLN risk model. Sex-specific differences in the incidence of risk model components were investigated: low-voltage electrocardiogram (ECG), premature ventricular contractions, negative T waves, and left ventricular ejection fraction., Results: Sustained ventricular arrhythmias (VAs) occurred in 77 males (18.7%) and 61 females (11.7%) (P = .004). Of the 933 cohort members, 287 (31%) had ≥1 low-voltage ECG during follow-up (180 females [63%], 107 males [37%]; P = .006). Female sex, age, age at clinical presentation, and proband status predicted low-voltage ECG during follow-up (area under the curve: 0.78). Sustained VA-free survival was lowest in males with low-voltage ECG (P <.001)., Conclusion: Low-voltage ECGs predict sustained VA and are a component of the PLN risk model. Low-voltage ECGs are more common in females, yet prognostic value is greater in males. Future studies should determine the impact of this difference on the risk prediction of PLN cardiomyopathy and possibly other cardiomyopathies., (Copyright © 2021 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: The p.Arg14del (c.40&#95;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 < 2500 ventricular premature contractions during 24-hour Holter monitoring. There were no statistically significant differences between the two groups in any of the baseline characteristics. The study is currently well underway, with the last participants expected to finish in 2021., 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)., (© 2021. The Author(s).)

Aims: This study aims to improve risk stratification for primary prevention implantable cardioverter defibrillator (ICD) implantation by developing a new mutation-specific prediction model for malignant ventricular arrhythmia (VA) in phospholamban (PLN) p.Arg14del mutation carriers. The proposed model is compared to an existing PLN risk model., Methods and Results: Data were collected from PLN p.Arg14del mutation carriers with no history of malignant VA at baseline, identified between 2009 and 2020. Malignant VA was defined as sustained VA, appropriate ICD intervention, or (aborted) sudden cardiac death. A prediction model was developed using Cox regression. The study cohort consisted of 679 PLN p.Arg14del mutation carriers, with a minority of index patients (17%) and male sex (43%), and a median age of 42 years [interquartile range (IQR) 27-55]. During a median follow-up of 4.3 years (IQR 1.7-7.4), 72 (10.6%) carriers experienced malignant VA. Significant predictors were left ventricular ejection fraction, premature ventricular contraction count/24 h, amount of negative T waves, and presence of low-voltage electrocardiogram. The multivariable model had an excellent discriminative ability {C-statistic 0.83 [95% confidence interval (CI) 0.78-0.88]}. Applying the existing PLN risk model to the complete cohort yielded a C-statistic of 0.68 (95% CI 0.61-0.75)., Conclusion: This new mutation-specific prediction model for individual VA risk in PLN p.Arg14del mutation carriers is superior to the existing PLN risk model, suggesting that risk prediction using mutation-specific phenotypic features can improve accuracy compared to a more generic approach., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background - Variants within the alpha-tropomyosin gene (TPM1) cause dominantly inherited cardiomyopathies, including dilated (DCM), hypertrophic (HCM) and restrictive (RCM) cardiomyopathy. Here we investigated whether TPM1 variants observed in DCM and HCM patients affect cardiomyocyte physiology differently. Methods - We identified a large family with DCM carrying a recently identified TPM1 gene variant (T201M) and a child with RCM with compound heterozygote TPM1 variants (E62Q and M281T) whose family members carrying single variants show diastolic dysfunction and HCM. The effects of TPM1 variants (T201M, E62Q or M281T) and of a plasmid containing both the E62Q and M281T variants on single-cell Ca 2+ transients (CaT) in HL-1 cardiomyocytes were studied. To define toxic threshold levels, we performed dose-dependent transfection of TPM1 variants. In addition, cardiomyocyte structure was studied in human cardiac biopsies with TPM1 variants. Results - Overexpression of TPM1 variants led to time-dependent progressive deterioration of CaT, with the smallest effect seen for E62Q and larger and similar effects seen for the T201M and M281T variants. Overexpression of E62Q/M281T did not exacerbate the effects seen with overexpression of a single TPM1 variant. T201M (DCM) replaced endogenous tropomyosin dose-dependently, while M281T (HCM) did not. Human cardiac biopsies with TPM1 variants revealed loss of sarcomeric structures. Conclusion - All TPM1 variants result in reduced cardiomyocyte CaT amplitudes and loss of sarcomeric structures. These effects may underlie pathophysiology of different cardiomyopathy phenotypes., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Phospholamban (PLN) plays a role in cardiomyocyte calcium handling as primary inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). The p.(Arg14del) pathogenic variant in the PLN gene results in a high risk of developing dilated or arrhythmogenic cardiomyopathy with heart failure. There is no established treatment other than standard heart failure therapy or heart transplantation. In this study, we generated a novel mouse model with the PLN-R14del pathogenic variant, performed detailed phenotyping, and tested the efficacy of established heart failure therapies eplerenone or metoprolol. Heterozygous PLN-R14del mice demonstrated increased susceptibility to ex vivo induced arrhythmias, and cardiomyopathy at 18 months of age, which was not accelerated by isoproterenol infusion. Homozygous PLN-R14del mice exhibited an accelerated phenotype including cardiac dilatation, contractile dysfunction, decreased ECG potentials, high susceptibility to ex vivo induced arrhythmias, myocardial fibrosis, PLN protein aggregation, and early mortality. Neither eplerenone nor metoprolol administration improved cardiac function or survival. In conclusion, our novel PLN-R14del mouse model exhibits most features of human disease. Administration of standard heart failure therapy did not rescue the phenotype, underscoring the need for better understanding of the pathophysiology of PLN-R14del-associated cardiomyopathy. This model provides a great opportunity to study the pathophysiology, and to screen for potential therapeutic treatments.

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 (O 2 -· ) into H 2 O 2 . Measurement of hydroethidine oxidation showed a significant increase in O 2 -· 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., Competing Interests: Competing interests: PHGMW is scientific advisor of Khondrion, Nijmegen, the Netherlands. This SME had no involvement in the data collection, analysis and interpretation, writing of the manuscript and in the decision to submit the manuscript for publication., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Clinical research on arrhythmogenic cardiomyopathy (ACM) is typically limited by small patient numbers, retrospective study designs, and inconsistent definitions., Aim: To create a large national ACM patient cohort with a vast amount of uniformly collected high-quality data that is readily available for future research., Methods: This is a multicentre, longitudinal, observational cohort study that includes (1) patients with a definite ACM diagnosis, (2) at-risk relatives of ACM patients, and (3) ACM-associated mutation carriers. At baseline and every follow-up visit, a medical history as well information regarding (non-)invasive tests is collected (e. g. electrocardiograms, Holter recordings, imaging and electrophysiological studies, pathology reports, etc.). Outcome data include (non-)sustained ventricular and atrial arrhythmias, heart failure, and (cardiac) death. Data are collected on a research electronic data capture (REDCap) platform in which every participating centre has its own restricted data access group, thus empowering local studies while facilitating data sharing., Discussion: The Netherlands ACM Registry is a national observational cohort study of ACM patients and relatives. Prospective and retrospective data are obtained at multiple time points, enabling both cross-sectional and longitudinal research in a hypothesis-generating approach that extends beyond one specific research question. In so doing, this registry aims to (1) increase the scientific knowledge base on disease mechanisms, genetics, and novel diagnostic and treatment strategies of ACM; and (2) provide education for physicians and patients concerning ACM, e. g. through our website ( www.acmregistry.nl ) and patient conferences.

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.

The diagnostic yield of exome and genome sequencing remains low (8-70%), due to incomplete knowledge on the genes that cause disease. To improve this, we use RNA-seq data from 31,499 samples to predict which genes cause specific disease phenotypes, and develop GeneNetwork Assisted Diagnostic Optimization (GADO). We show that this unbiased method, which does not rely upon specific knowledge on individual genes, is effective in both identifying previously unknown disease gene associations, and flagging genes that have previously been incorrectly implicated in disease. GADO can be run on www.genenetwork.nl by supplying HPO-terms and a list of genes that contain candidate variants. Finally, applying GADO to a cohort of 61 patients for whom exome-sequencing analysis had not resulted in a genetic diagnosis, yields likely causative genes for ten cases.

Phospholamban (PLN) p.Arg14del cardiomyopathy is characterized by a distinct arrhythmogenic biventricular phenotype that can be predominantly left ventricular, right ventricular, or both. Our aim was to further elucidate distinct features of this cardiomyopathy with respect to the distribution of desmosomal proteins observed by immunofluorescence (IF) in comparison to desmosomal arrhythmogenic cardiomyopathy and co-existent genetic variants. We studied eight explanted heart specimens from PLN p.Arg14del mutation carriers. Macro- and microscopic examination revealed biventricular presence of fibrofatty replacement and interstitial fibrosis. Five out of 8 (63%) patients met consensus criteria for both arrhythmogenic right ventricular cardiomyopathy (ARVC) and dilated cardiomyopathy (DCM). In four cases, targeted next-generation sequencing revealed one additional pathogenic variant and six variants of unknown significance. IF showed diminished junction plakoglobin signal intensity at the intercalated disks in 4 (67%) out of 6 cases fulfilling ARVC criteria but normal intensity in both cases fulfilling only DCM criteria. Notably, the four cases with diminished junction plakoglobin were also those where an additional gene variant was detected. IF for two proteins recently investigated in desmosomal arrhythmogenic cardiomyopathy (ACM), synapse-associated protein 97 and glycogen synthase kinase-3 beta, showed a distinct distributional pattern in comparison to desmosomal ACM. In 7 (88%) out of 8 cases we observed both a strong synapse-associated protein 97 signal at the sarcomeres and no glycogen synthase kinase-3 beta translocation to the intercalated discs. Phospholamban p.Arg14del cardiomyopathy is characterized by a distinct molecular signature compared to desmosomal ACM, specifically a different desmosomal protein distribution. This study substantiates the idea that additional genetic variants play a role in the phenotypical heterogeneity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent advancements in next generation sequencing (NGS) technology have led to the identification of the giant sarcomere gene, titin (TTN), as a major human disease gene. Truncating variants of TTN (TTNtv) especially in the A-band region account for 20% of dilated cardiomyopathy (DCM) cases. Much attention has been focused on assessment and interpretation of TTNtv in human disease; however, missense and non-frameshifting insertions/deletions (NFS-INDELs) are difficult to assess and interpret in clinical diagnostic workflow. Targeted sequencing covering all exons of TTN was performed on a cohort of 530 primary DCM patients from three cardiogenetic centres across Europe. Using stringent bioinformatic filtering, twenty-nine and two rare TTN missense and NFS-INDELs variants predicted deleterious were identified in 6.98% and 0.38% of DCM patients, respectively. However, when compared with those identified in the largest available reference population database, no significant enrichment of such variants was identified in DCM patients. Moreover, DCM patients and reference individuals had comparable frequencies of splice-region missense variants with predicted splicing alteration. DCM patients and reference populations had comparable frequencies of rare predicted deleterious TTN missense variants including splice-region missense variants suggesting that these variants are not independently causative for DCM. Hence, these variants should be classified as likely benign in the clinical diagnostic workflow, although a modifier effect cannot be excluded at this stage.

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 a high frequency of ventricular arrhythmias. Additionally, these patients have a poor prognosis often with left ventricular dysfunction and early-onset heart failure. Therefore, when there is a suspicion of a PLN mutation, cardiac and genetic screening is strongly recommended.

Aims: The p.Arg14del founder mutation in the gene encoding phospholamban (PLN) is associated with an increased risk of malignant ventricular arrhythmia (VA) and heart failure. It has been shown to lead to calcium overload, cardiomyocyte damage, and eventually to myocardial fibrosis. This study sought to investigate ventricular function, the extent and localization of myocardial fibrosis and the associations with ECG features and VA in PLN p.Arg14del mutation carriers., Methods and Results: Cardiovascular magnetic resonance (CMR) data of 150 mutation carriers were analysed retrospectively. Left ventricular (LV) and right ventricular (RV) volumes, mass, and ejection fraction were measured. The extent of late gadolinium enhancement (LGE) was expressed as a percentage of myocardial mass. All standard ECG parameters were measured. Occurrence of VA was analysed on ambulatory 24-h and/or exercise electrocardiography, if available. Mean age was 40 ± 15 years, 42% males, and 7% were index patients while 93% were pre-symptomatic carriers identified after family cascade screening. Mean LV ejection fraction (LVEF) and RV ejection fraction were 58 ± 9% and 55 ± 9%, respectively. LV-LGE was present in 91% of mutation carriers with reduced LVEF (<45%) and in 30% of carriers with preserved LVEF. In carriers with positive LV-LGE, its median extent was 5.9% (interquartile range 3.2-12.7). LGE was mainly observed in the inferolateral wall. Carriers with inverted T-waves in the lateral ECG leads more often had LV-LGE (P < 0.01) than carriers without. Finally, the presence of LV-LGE, but not attenuated R-waves and inverted lateral T-waves, was independently associated with VA., Conclusion: LV myocardial fibrosis is present in many PLN p.Arg14del mutation carriers, and who still have a preserved LVEF. It is seen predominantly in the LV inferolateral wall and corresponds with electrocardiographic repolarization abnormalities. Although preliminary, myocardial fibrosis was found to be independently associated with VA. Our findings support the use of CMR with LGE early in the diagnostic work-up.

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.

Purpose: We evaluated the diagnostic yield in pediatric dilated cardiomyopathy (DCM) of combining exome sequencing (ES)-based targeted analysis and genome-wide copy-number variation (CNV) analysis. Based on our findings, we retrospectively designed an effective approach for genetic testing in pediatric DCM., Methods: We identified 95 patients (in 85 families) with pediatric onset of DCM. We initially excluded 13 of these families because they already had a genetic diagnosis, leaving a total of 31 probands for single-nucleotide polymorphism (SNP) array and trio-ES. We used Human Phenotype Ontology (HPO)-based filtering for our data analysis., Results: We reached a genetic diagnosis in 15/31 (48.4%) families. ES yielded a diagnosis in 13 probands (13/15; 86.7%), with most variants being found in genes encoding structural cardiomyocyte components. Two large deletions were identified using SNP array. If we had included the 13 excluded families, our estimated yield would have been 54%., Conclusion: We propose a standardized, stepwise analysis of (i) well-known cardiomyopathy genes, (ii) CNVs, (iii) all genes assigned to HPO cardiomyopathy, and (iv) if appropriate, genes assigned to other HPO terms. This diagnostic approach yields the highest increase at each subsequent step and reduces analytic effort, cost, the number of variants of unknown clinical significance, and the chance of incidental findings.

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.

Background: Accurate estimates of survival are indispensable for cardiologists, clinical geneticists, and genetic counselors dealing with families with an inherited cardiac disease. However, a bias towards a more severe disease with a worse outcome in the first publications may not accurately represent the actual survival forecast. We, therefore, evaluated the effect of ascertainment bias on survival in 3 different inherited cardiac diseases (idiopathic ventricular fibrillation, SCN5A overlap syndrome, and arrhythmogenic cardiomyopathy) caused by a founder mutation., Methods: We collected mortality data from mutation-positive subjects with either DPP6-associated idiopathic ventricular fibrillation, SCN5A overlap syndrome, and PLN-R14del-mediated arrhythmogenic cardiomyopathy >2 to 10 years of ongoing clinical/cascade genetic screening., Results: The median age of survival in DPP6 mutation-positive subjects increased from 44.6 years in the original cohort from 2008 (n=60; 95% CI, 36.8-52.4 years) to 68.2 years in the extended cohort from 2012 (n=235; 95% CI, 64.6-71.7 years; P<0.001). In the SCN5A overlap syndrome, survival increased from 56.1 years in 1999 (n=86; 95% CI, 48.0-64.2 years) to 69.7 years in 2009 (n=197; 95% CI, 61.3-78.2 years; P=0.049). In PLN-R14del positive patients, the median age of survival increased from 63.5 years in 2010 (n=89; 95% CI, 59.1-68.0 years) to 65.2 years in 2012 (n=370; 95% CI, 62.0-68.3 years; P=0.046)., Conclusions: The median age of survival in 3 different inherited cardiac diseases with an established pathogenic substrate significantly increased once genetic testing and cascade screening extended, after the first publication that elucidated the discovery of the disease-susceptibility gene/mutation. This underscores the direct and negative influence of ascertainment bias on survival forecasts and the importance of ongoing clinical/genetic follow-up to establish the most accurate disease prognosis for genetically mediated heart diseases.

Rationale: Orthostatic hypotension is a common clinical problem, but the underlying mechanisms have not been fully delineated., Objective: We describe 2 families, with 4 patients in total, experiencing severe life-threatening orthostatic hypotension because of a novel cause., Methods and Results: As in dopamine β-hydroxylase deficiency, concentrations of norepinephrine and epinephrine in the patients were low. Plasma dopamine β-hydroxylase activity, however, was normal, and the DBH gene had no mutations. Molecular genetic analysis was performed to determine the underlying genetic cause. Homozygosity mapping and exome and Sanger sequencing revealed pathogenic homozygous mutations in the gene encoding cytochrome b561 ( CYB561 ); a missense variant c.262G>A, p.Gly88Arg in exon 3 in the Dutch family and a nonsense mutation (c.131G>A, p.Trp44*) in exon 2 in the American family. Expression of CYB561 was investigated using RNA from different human adult and fetal tissues, transcription of RNA into cDNA, and real-time quantitative polymerase chain reaction. The CYB561 gene was found to be expressed in many human tissues, in particular the brain. The CYB561 protein defect leads to a shortage of ascorbate inside the catecholamine secretory vesicles leading to a functional dopamine β-hydroxylase deficiency. The concentration of the catecholamines and downstream metabolites was measured in brain and adrenal tissue of 6 CYB561 knockout mice (reporter-tagged deletion allele [post-Cre], genetic background C57BL/6NTac). The concentration of norepinephrine and normetanephrine was decreased in whole-brain homogenates of the CYB561 (- /- ) mice compared with wild-type mice ( P <0.01), and the concentration of normetanephrine and metanephrine was decreased in adrenal glands ( P <0.01), recapitulating the clinical phenotype. The patients responded favorably to treatment with l-dihydroxyphenylserine, which can be converted directly to norepinephrine., Conclusions: This study is the first to implicate cytochrome b561 in disease by showing that pathogenic mutations in CYB561 cause an as yet unknown disease in neurotransmitter metabolism causing orthostatic hypotension., (© 2018 American Heart Association, Inc.)

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.