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Genetic characterization of biopsy-proven myocarditis: a pilot study Background Myocarditis is characterized by the presence of an inflammatory infiltrate in the myocardium with degenerative/necrotic changes of cardiomyocytes, not-related to ischemic damage. Its clinical presentation is extremely heterogenous. Endomyocardial Biopsy (EMB) is the diagnostic gold standard and provides etiopathogenetic diagnosis. Biopsy-proven myocarditis may be infectious, mainly viral, toxic or non-infectious immune-mediated/autoimmune. A complex interplay between genetic factors, environmental triggers (i.e. viral infection) and the immune response of the host is postulated at the basis of different disease evolution. Purpose To determine the prevalence of pathogenic/likely pathogenic (P/LP) variants in cardiomyopathy-related genes in a well-characterized cohort of biopsy-proven myocarditis. Methods Seventy-three biopsy-proven myocarditis-affected patients (mean age 51±9, 41 males) underwent screening of 200 genes related to inherited cardiomyopathies. Definite/moderate gene association with Dilated Cardiomyopathy (DCM) and Arrhythmogenic Cardiomyopathy was based on the ClinGen framework. Variant prioritization was carried out using American College of Medical Genetics and Genomics rules. Correlation with presence of virus on endomyocardial biopsy by polymerase chain reaction, family history and serum anti-heart autoantibodies (AHA) and/or aintiintercalated-disk autoantibodies (AIDA), among other clinical features, was appraised. Results Nineteen of the 73 biopsy-proven myocarditis patients (26%) carried a P/LP variant in cardiac-related genes. Titin (TTN) was the most overrepresented gene accounting for 11% of cases (8/73), followed by Myosin Binding Protein C3 (MYBPC3) and Desmoplakin (DSP), each accounting for 3% of cases (2/73), respectively. No differences were observed in the distribution of disease onset, heart failure classification (NYHA class), ejection fraction, presence of virus and presence of anti-heart autoantibodies between genotype-positive and genotype-negative cohorts. However, 12 of the 19 genotype-positive index cases (63%) referred family history for cardiomyopathy and/or sudden cardiac death; whereas only 4 of the 54 genotype-negative declared positive family history, demonstrating a statistically significant difference. On the other hand, 37% of genotype positive patients declared negative family history, suggesting a non-familial or sporadic form of myocarditis. Conclusion The prevalence of clinically actionable P/LP variants in cardiomyopathy-related genes is nearly one third of biopsy-proven viral or autoimmune myocarditis patients, most of them associated with DCM, but clinical parameters do not differ between genotype-positive and genotype-negative patients. On the other hand, positive family history in genotype-positive patients indicate that family history assessment is mandatory to identify the genetic substrate of inherited inflammatory cardiomyopathies.

Supplemental Digital Content is available in the text., Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease characterized by ventricular arrhythmias and progressive ventricular dysfunction. Genetic testing is recommended, and a pathogenic variant in an ARVC-associated gene is a major criterion for diagnosis according to the 2010 Task Force Criteria. As incorrect attribution of a gene to ARVC can contribute to misdiagnosis, we assembled an international multidisciplinary ARVC Clinical Genome Resource Gene Curation Expert Panel to reappraise all reported ARVC genes. Methods: Following a comprehensive literature search, six 2-member teams conducted blinded independent curation of reported ARVC genes using the semiquantitative Clinical Genome Resource framework. Results: Of 26 reported ARVC genes, only 6 (PKP2, DSP, DSG2, DSC2, JUP, and TMEM43) had strong evidence and were classified as definitive for ARVC causation. There was moderate evidence for 2 genes, DES and PLN. The remaining 18 genes had limited or no evidence. RYR2 was refuted as an ARVC gene since clinical data and model systems exhibited a catecholaminergic polymorphic ventricular tachycardia phenotype. In ClinVar, only 5 pathogenic/likely pathogenic variants (1.1%) in limited evidence genes had been reported in ARVC cases in contrast to 450 desmosome gene variants (97.4%). Conclusions: Using the Clinical Genome Resource approach to gene-disease curation, only 8 genes (PKP2, DSP, DSG2, DSC2, JUP, TMEM43, PLN, and DES) had definitive or moderate evidence for ARVC, and these genes accounted for nearly all pathogenic/likely pathogenic ARVC variants in ClinVar. Therefore, only pathogenic/likely pathogenic variants in these 8 genes should yield a major criterion for ARVC diagnosis. Pathogenic/likely pathogenic variants identified in other genes in a patient should prompt further phenotyping as variants in many of these genes are associated with other cardiovascular conditions.

Aims The aim of this study is to evaluate the clinical features of patients affected by arrhythmogenic cardiomyopathy (AC), presenting with chest pain and myocardial enzyme release in the setting of normal coronary arteries (‘hot phase’). Methods and results We collected detailed anamnestic, clinical, instrumental, genetic, and histopathological findings as well as follow-up data in a series of AC patients who experienced a hot phase. A total of 23 subjects (12 males, mean age at the first episode 27 ± 16 years) were identified among 560 AC probands and family members (5%). At first episode, 10 patients (43%) already fulfilled AC diagnostic criteria. Twelve-lead electrocardiogram recorded during symptoms showed ST-segment elevation in 11 patients (48%). Endomyocardial biopsy was performed in 11 patients, 8 of them during the acute phase showing histologic evidence of virus-negative myocarditis in 88%. Cardiac magnetic resonance was performed in 21 patients, 12 of them during the acute phase; oedema and/or hyperaemia were detected in 7 (58%) and late gadolinium enhancement in 11 (92%). At the end of follow-up (mean 17 years, range 1–32), 12 additional patients achieved an AC diagnosis. Genetic testing was positive in 77% of cases and pathogenic mutations in desmoplakin gene were the most frequent. No patient complained of sustained ventricular arrhythmias or died suddenly during the ‘hot phase’. Conclusion ‘Hot phase’ represents an uncommon clinical presentation of AC, which often occurs in paediatric patients and carriers of desmoplakin gene mutations. Tissue characterization, family history, and genetic test represent fundamental diagnostic tools for differential diagnosis.

Aims To study the impact of genotype on the performance of the 2019 risk model for arrhythmogenic right ventricular cardiomyopathy (ARVC). Methods and results The study cohort comprised 554 patients with a definite diagnosis of ARVC and no history of sustained ventricular arrhythmia (VA). During a median follow-up of 6.0 (3.1,12.5) years, 100 patients (18%) experienced the primary VA outcome (sustained ventricular tachycardia, appropriate implantable cardioverter defibrillator intervention, aborted sudden cardiac arrest, or sudden cardiac death) corresponding to an annual event rate of 2.6% [95% confidence interval (CI) 1.9–3.3]. Risk estimates for VA using the 2019 ARVC risk model showed reasonable discriminative ability but with overestimation of risk. The ARVC risk model was compared in four gene groups: PKP2 (n = 118, 21%); desmoplakin (DSP) (n = 79, 14%); other desmosomal (n = 59, 11%); and gene elusive (n = 160, 29%). Discrimination and calibration were highest for PKP2 and lowest for the gene-elusive group. Univariable analyses revealed the variable performance of individual clinical risk markers in the different gene groups, e.g. right ventricular dimensions and systolic function are significant risk markers in PKP2 but not in DSP patients and the opposite is true for left ventricular systolic function. Conclusion The 2019 ARVC risk model performs reasonably well in gene-positive ARVC (particularly for PKP2) but is more limited in gene-elusive patients. Genotype should be included in future risk models for ARVC.

Desmoplakin (DSP) genetic variants have been reported in arrhythmogenic cardiomyopathy with particular regard to predominant left ventricular (LV) involvement.The purpose of this study was to improve our understanding of clinical phenotype and outcome of DSP variant carriers.The clinical picture and outcome of 73 patients (36% probands) harboring a pathogenic/likely pathogenic DSP variant were evaluated.The phenotype during follow-up (mean 11 years; range 1-39 years) changed in 25 patients (35%), arrhythmogenic LV cardiomyopathy (ALVC) forms being the most frequent (n = 26 [36%]), followed by biventricular (BIV; n = 20 [27%]) and arrhythmogenic right ventricular cardiomyopathy (ARVC; n = 16 [22%]) forms. Major ventricular arrhythmias were detected in 21 patients (29%), and they were more common in ARVC (n = 6, 56%) and BIV forms (n = 8, 40%) than in ALVC forms (n = 4, 15%). In patients with ALVC, major ventricular arrhythmias occurred in the setting of a normal/mildly reduced systolic function. Heart failure (HF) occurred in 6 patients (8%); none affected with ALVC. Females showed more commonly LV involvement, while ARVC forms were more frequently detected in males (21 [61%] vs 15 [38%]; P = .147). Males showed a higher incidence of major ventricular arrhythmias (18 [52%] vs 9 [24%]; P = .036), HF (11 [31%] vs 1 [3%]; P = .004), and cardiac death (11 [31%] vs 0 [0%]; P.001).The clinical phenotype in pathogenic/likely pathogenic DSP variant carriers is wide. Although most patients show LV involvement, 16 (22%) has right ventricular abnormalities in keeping with a "classical" arrhythmogenic cardiomyopathy form. In ALVC, HF and major ventricular arrhythmias seem less common than in right ventricular and BIV variants. Females show more frequently LV involvement and a better outcome.

Background Mutations in filamin-C (FLNC) are involved in the pathogenesis of arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy (DCM), and have been associated with a left ventricular (LV) phenotype, characterized by nonischemic LV fibrosis, ventricular arrhythmias, and sudden cardiac death (SCD). Objective The purpose of this study was to investigate the prevalence of FLNC variants in a gene-negative ACM population and to evaluate the clinical phenotype and SCD risk factors in FLNC-associated cardiomyopathies. Methods ACM probands who tested negative for mutations in ACM-related genes underwent FLNC genetic screening. Clinical and genetic data were collected and pooled together with those of previously published FLNC-ACM and FLNC-DCM patients. Results In a cohort of 270 gene-elusive ACM probands, 12 (4.4%) had FLNC variants, and 13 additional family members carried the same mutation. Eighteen FLNC variant carriers (72%) had a diagnosis of ACM (72% male; mean age 45 years). On pooled analysis, 145 patients with FLNC-associated cardiomyopathies were included. Electrocardiographic (ECG) low QRS voltages were detected in 37%, and T-wave inversion (TWI) in inferolateral/lateral leads in 24%. Among 67 patients who had cardiac magnetic resonance (CMR), LV nonischemic late gadolinium enhancement (LGE) was found in 75%. SCD occurred in 28 patients (19%), 15 of whom showed LV nonischemic LGE/fibrosis. Compared with patients with no SCD, those who experienced SCD more frequently had inferolateral/lateral TWI (P = .013) and LV LGE/fibrosis (P = .033). Conclusion Clinical phenotype of FLNC cardiomyopathies is characterized by late-onset presentation and typical ECG and CMR features. SCD is associated with the presence of LV LGE/fibrosis but not with severe LV systolic dysfunction.

Background Arrhythmogenic Cardiomyopathy (AC) is a genetic cardiac disorder, mainly caused by mutations in genes encoding desmosomal proteins, and accounts for most stress-related arrhythmic sudden cardiac deaths (SCD) in the young and athletes. The AC myocardium is hallmarked by cardiomyocyte (CM) death and fibro-fatty replacement, which generate a pro-arrhythmogenic substrate. Several pathogenetic factors in AC remain obscure and better understanding of the disease mechanisms is required to develop novel efficacious therapies to prevent SCD, which are sorely missing. The lexical analogy between desmosomes and desmosomal proteins has originally biased AC research towards CMs, the paradigmatic desmosome-bearing cells in heart. However, the myocardium is composed by different cell types, many of which express desmosomal proteins, albeit in the absence of desmosomes, including CMs, sympathetic neurons, vascular cells and fibroblasts. Notably, AC mutations are transmitted at germline, and thus may manifest in all cell types expressing desmosomal proteins. This might explain why the majority of preclinical AC models, using CM specific over-expression or deletion of the disease-causing mutation, failed to fully recapitulate the human disease phenotype. Hypothesis On these bases, we aimed to generate a knock-in (KI) AC mouse model for comprehensively studying AC pathogenesis. Methods As Desmoplakin (DSP) mutations occur in a large part of the Italian AC population, we used CRISP/Cas9 to generate a KI mouse strain harboring the Serine-to-Alanine substitution of S311, the murine homolog of human S299 [Bauce et al, 2005]. We successfully obtained DSPS311A/WT KI founders, which were viable and fertile and after backcrossing for >10 generations, used to expand the new mouse strain. Mouse cardiac phenotype was characterized, at different stages (1,2,4,6,9 mo.) by functional (i.e. ECHO, telemetry-ECG, chronic exercise) and structural (i.e. EM, standard histology, confocal IF, TUNEL assay) analyses. Molecular/biochemical analyses probed the state of the main pathways involved in AC. Results Our analyses showed that, starting from 4 mo., DSP homozygous KI mice display contractile dysfunction, worsening during aging, and fibrotic myocardial remodelling with focal fatty lesions, accompanied by frequent arrhythmic beats, which become sustained ventricular arrhythmias upon Noradrenaline administration. Hearts showed desmosome alterations, particularly at advanced disease stages, and lateralization of cx43, which corresponded to the phenotype of human AC hearts. Heterozygous mice showed similar alterations, which only took longer to appear. Exercise accelerated disease progression and increased the incidence of SCD (DSPS311A: SCD=63%, n=11; ctrls: SCD=8%, n=12). Conclusion Our KI mice replicate the clinical and pathological phenotype of DSP-linked biventricular AC and are thus suited for the mechanistic study of the multicellular origin of the disease. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PRIN Miur 2015

Background: Each of the cardiomyopathies, classically categorized as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy, has a signature genetic theme. Hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning >10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. Methods: An international panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The panel used the Clinical Genome Resource semiquantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories on the basis of the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. Results: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from 8 gene ontologies were classified as having definitive ( BAG3 , DES , FLNC , LMNA , MYH7 , PLN , RBM20 , SCN5A , TNNC1 , TNNT2 , TTN ) or strong ( DSP ) evidence. Seven genes (14%; ACTC1 , ACTN2 , JPH2 , NEXN , TNNI3 , TPM1 , VCL ) including 2 additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, 6 were similarly classified for hypertrophic cardiomyopathy and 3 for arrhythmogenic right ventricular cardiomyopathy. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of the 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. Conclusions: In the curation of 51 genes, 19 had high evidence (12 definitive/strong, 7 moderate). It is notable that these 19 genes explain only a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high-evidence genes; however, genes lacking robust evidence are also commonly included. We recommend that high-evidence DCM genes be used for clinical practice and that caution be exercised in the interpretation of variants in variable-evidence DCM genes.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

Purpose The genetic architecture of Plakophilin 2 (PKP2) cardiomyopathy can inform our understanding of its variant pathogenicity and protein function. Methods We assess the gene-wide and regional association of truncating and missense variants in PKP2 with arrhythmogenic cardiomyopathy (ACM), and arrhythmogenic right ventricular cardiomyopathy (ARVC) specifically. A discovery data set compares genetic testing requisitions to gnomAD. Validation is performed in a rigorously phenotyped definite ARVC cohort and non-ACM individuals in the Geisinger MyCode cohort. Results The etiologic fraction (EF) of ACM-related diagnoses from truncating variants in PKP2 is significant (0.85 [0.80,0.88], p < 2 x 10(-16)), increases for ARVC specifically (EF = 0.96 [0.94,0.97], p < 2 x 10(-16)), and is highest in definite ARVC versus non-ACM individuals (EF = 1.00 [1.00,1.00], p < 2 x 10(-16)). Regions of missense variation enriched for ACM probands include known functional domains and the C-terminus, which was not previously known to contain a functional domain. No regional enrichment was identified for truncating variants. Conclusion This multicohort evaluation of the genetic architecture of PKP2 demonstrates the specificity of PKP2 truncating variants for ARVC within the ACM disease spectrum. We identify the PKP2 C-terminus as a potential functional domain and find that truncating variants likely cause disease irrespective of transcript position.

Background: The cardiomyopathies are classically categorized as hypertrophic (HCM), dilated (DCM), and arrhythmogenic right ventricular (ARVC), and each have a signature genetic theme. HCM and ARVC are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning more than 10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. Methods: An international Panel with clinical and scientific expertise in DCM genetics was assembled to evaluate evidence supporting monogenic relationships of genes with idiopathic DCM. The Panel utilized the ClinGen semi-quantitative gene-disease clinical validity classification framework. Results: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from eight gene ontologies were classified as having definitive (BAG3, DES, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNT2, TTN) or strong (DSP) evidence. Seven genes (14%) (ACTC1, ACTN2, JPH2, NEXN, TNNI3, TPM1, VCL) including two additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of the 19 genes classified as definitive, strong or moderate, six were similarly classified for HCM and three for ARVC. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of 16 commercially available genetic testing panels evaluated, most definitive genes were included, but panels also included numerous genes with minimal human evidence. Conclusions: In a systematic curation of published evidence for genes considered relevant for monogenic DCM, 12 were classified as definitive or strong and seven as moderate evidence spanning 10 gene ontologies. Notably, these 19 genes only explain a minority of DCM cases, leaving the remainder of DCM genetic architecture incompletely addressed. While clinical genetic testing panels include most high evidence genes, genes lacking robust evidence are also commonly included. Until the genetic architecture of DCM is more fully defined, care should be taken in the interpretation of variable evidence DCM genes in clinical practice.

Background Arrhythmogenic cardiomyopathy (AC) is a myocardial disease due to desmosomal mutations whose pathogenesis is incompletely understood. Objective The purpose of this study was to identify molecular pathways underlying early AC by gene expression profiling in both humans and animal models. Methods RNA sequencing for differentially expressed genes (DEGs) was performed on the myocardium of transgenic mice overexpressing the Desmoglein2-N271S mutation before phenotype onset. Zebrafish signaling reporters were used for in vivo validation. Whole exome sequencing was undertaken in 10 genotype-negative AC patients and subsequent direct sequencing in 140 AC index cases. Results Among 29 DEGs identified at early disease stages, Lgals3/GAL3 (lectin, galactoside-binding, soluble, 3) showed reduced cardiac expression in transgenic mice and in 3 AC patients who suffered sudden cardiac death without overt structural remodeling. Four rare missense variants of LGALS3 were identified in 5 human AC probands. Pharmacologic inhibition of Lgals3 in zebrafish reduced Wnt and transforming growth factor-β signaling, increased Hippo/YAP-TAZ signaling, and induced alterations in desmoplakin membrane localization, desmosome integrity and stability. Increased LGALS3 plasma expression in genotype-positive AC patients and CD98 activation supported the galectin-3 (GAL3) release by circulating macrophages pointing toward the stabilization of desmosomal assembly at the injured regions. Conclusion GAL3 plays a crucial role in early AC onset through regulation of Wnt/β-catenin signaling and intercellular adhesion.

Arrhythmogenic cardiomyopathy (AC) is an inherited cardiac disease characterized by a progressive fibro-fatty replacement of the working myocardium and by life-threatening arrhythmias and risk of sudden cardiac death. Pathogenic variants are identified in nearly 50% of affected patients mostly in genes encoding for desmosomal proteins. AC incomplete penetrance and phenotypic variability advocate that other factors than genetics may modulate the disease, such as microRNAs (miRNAs). MiRNAs are small noncoding RNAs with a primary role in gene expression regulation and network of cellular processes. The implication of miRNAs in AC pathogenesis and their role as biomarkers for early disease detection or differential diagnosis has been the objective of multiple studies employing diverse designs and methodologies to detect miRNAs and measure their expression levels. Here we summarize experiments, evidence, and flaws of the different studies and hitherto knowledge of the implication of miRNAs in AC pathogenesis and diagnosis.

Arrhythmogenic Cardiomyopathy (AC) is a clinically and genetically heterogeneous myocardial disease. Half of AC patients harbour private desmosomal gene variants. Although microRNAs (miRNAs) have emerged as key regulator molecules in cardiovascular diseases and their involvement, correlated to phenotypic variability or to non-invasive biomarkers, has been advanced also in AC, no data are available in larger disease cohorts. Here, we propose the largest AC cohort unbiased by technical and biological factors. MiRNA profiling on nine right ventricular tissue, nine blood samples of AC patients, and four controls highlighted 10 differentially expressed miRNAs in common. Six of these were validated in a 90-AC patient cohort independent from genetic status: miR-122-5p, miR-133a-3p, miR-133b, miR-142-3p, miR-182-5p, and miR-183-5p. This six-miRNA set showed high discriminatory diagnostic power in AC patients when compared to controls (AUC-0.995), non-affected family members of AC probands carrying a desmosomal pathogenic variant (AUC-0.825), and other cardiomyopathy groups (Hypertrophic Cardiomyopathy: AUC-0.804, Dilated Cardiomyopathy: AUC-0.917, Brugada Syndrome: AUC-0.981, myocarditis: AUC-0.978). AC-related signalling pathways were targeted by this set of miRNAs. A unique set of six-miRNAs was found both in heart-tissue and blood samples of AC probands, supporting its involvement in disease pathogenesis and its possible role as a non-invasive AC diagnostic biomarker.

PURPOSE: To develop and assess a novel custom next-generation sequencing (NGS) panel for male infertility genetic diagnosis. METHODS: A total of 241 subjects with diagnosis of idiopathic infertility ranging from azoospermia to normozoospermia were sequenced by a custom NGS panel including AR, FSHB, FSHR, KLHL10, NR5A1, NANOS1, SEPT12, SYCP3, TEX11 genes. Variants with minor allele frequency

The genetic architecture of Plakophilin 2 (PKP2) cardiomyopathy can inform our understanding of its variant pathogenicity and protein function.We assess the gene-wide and regional association of truncating and missense variants in PKP2 with arrhythmogenic cardiomyopathy (ACM), and arrhythmogenic right ventricular cardiomyopathy (ARVC) specifically. A discovery data set compares genetic testing requisitions to gnomAD. Validation is performed in a rigorously phenotyped definite ARVC cohort and non-ACM individuals in the Geisinger MyCode cohort.The etiologic fraction (EF) of ACM-related diagnoses from truncating variants in PKP2 is significant (0.85 [0.80,0.88], p 2 × 10This multicohort evaluation of the genetic architecture of PKP2 demonstrates the specificity of PKP2 truncating variants for ARVC within the ACM disease spectrum. We identify the PKP2 C-terminus as a potential functional domain and find that truncating variants likely cause disease irrespective of transcript position.

Cardiovascular diseases (CVDs) are a wide group of disorders affecting the heart and blood vessels, including coronary artery, valve, pericardial, conduction system, myocardial and vascular diseases, either congenital or acquired, which can be also heritable. The advent of next generation sequencing (NGS) was accompanied by quick advances in understanding the genetic basis of human diseases, prompting translation of genetics to the clinic. Precision medicine is based on these findings and on the role of genetic testing to improve the diagnosis, to identify individuals with previously unrecognized disease and family members at risk of future disease development which require longitudinal follow-up. However, the probabilistic nature of genetic testing and the subjectivity of genetic variants classification weighted on current evidence, making this powerful clinical tool difficult to be applied in precision diagnostics and therapeutics. Here, we reviewed systematically the genetic basis of CVDs with special emphasis on the current role of NGS in clinical diagnosis and risk assessment, underlying the need of multidisciplinary cardio-genetic referral centers.

Arrhythmogenic cardiomyopathy (AC) is characterized by progressive non-ischaemic cardiomyocyte death and fibro-fatty repair, triggering ventricular arrhythmias and sudden death. A familial background consistent with an autosomal dominant trait of inheritance is described in nearly half of AC patients, even if recessive variants have also been reported, either associated or not with palmoplantar keratosis and woolly hair. Advances in DNA sequencing have led to the identification of more than 350 pathogenic mutations in 15 disease-causing genes associated with AC. Nearly half of patients with AC harbour mutations in genes encoding desmosomal proteins (plakoglobin, JUP; desmoplakin, DSP; plakophillin-2, PKP2; desmoglein-2, DSG2; and desmocollin-2, DSC2) and less than 1% carry mutations in non-desmosomal genes (transforming growth factor beta-3, TGFB3; desmin, DES; alpha-T catenin, CTNNA3; phospholamban, PLN; lamin A/C, LMNA; titin, and TTN; ryanodine receptor-2, RYR2). The inheritance pattern of AC is more complex than previously appreciated, with frequent requirement of more than one mutation for fully penetrant disease. Compound/digenic heterozygosity was identified in up to 25% of AC-causing desmosomal gene mutation carriers, explaining in part the phenotypic variability. In the era of next-generation sequencing, parallel analysis of thousands of genes is feasible and many rare variants can be detected (nearly 1000 nucleotide variants of unknown significance have been linked to AC), so the challenge is to distinguish causative mutations from the genetic noise since genetic testing may be a precious tool in distinguishing AC patients at risk of disease onset and of sudden death.

Background: Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disease associated with point mutations in genes encoding for cardiac desmosome proteins. Conventional mutation screening is positive in ≈50% of probands. Copy number variations (CNVs) have recently been linked to AC pointing to the need to determine the prevalence of CNVs in desmosomal genes and to evaluate disease penetrance by cosegregation analysis in family members. Methods and Results: A total of 160 AC genotype-negative probands for 5 AC desmosomal genes by conventional mutation screening underwent multiplex ligation-dependent probe amplification. Nine heterozygous CNVs were identified in 11 (6.9%) of the 160 probands. Five carried a deletion of the entire plakophilin-2 ( PKP2 ) gene, 2 a deletion of only PKP2 exon 4, 1 a deletion of the PKP2 exons 6 to 11, 1 a PKP2 duplication of 5′ untranslated region till exon 1, 1 the desmocollin-2 ( DSC2 ) duplication of exons 7 to 9, and 1 a large deletion of chromosome 18 comprising both DSC2 and desmoglein-2 genes. All probands were affected by moderate-severe forms of the disease, whereas 10 (32%) of the 31 family members carrying one of these deletions fulfilled the diagnostic criteria. Conclusions: Genomic rearrangements were detected in ≈7% of AC probands negative for pathogenic point mutations in desmosomal genes, highlighting the potential of CNVs analysis to substantially increase the diagnostic yield of genetic testing. Genotype–phenotype correlation demonstrated the presence of the disease in about one third of family members carrying the CNV, underlying the role of other factors in the development and progression of the disease.

Dysregulation of the transforming growth factor beta (TGF-β) 1 pathway has been associated with either syndromic or isolated mitral valve (MV) prolapse due to myxoid degeneration (floppy MV). The activation of Smad receptor-mediated intracellular TGF-β pathway and its effect on adherens junction (AJ) molecular pattern of activated valvular interstitial cells (VICs) in MV prolapse are herein investigated.Floppy MV leaflets were obtained from 30 patients (24 males, mean age 55.5±12.7 years) who underwent surgical repair, and 10 age- and sex-matched Homograft Tissue Bank samples served as controls. MV leaflet cellular and extracellular matrix composition, including collagen I and III, was evaluated by histology and transmission electron microscopy. Smad2 active phosphorylated form (p-Smad2), α-smooth muscle actin (α-SMA), and junctional proteins (N-cadherin, cadherin-11, β-catenin, plakoglobin, plakophilin-2) in VICs were assessed by immunohistochemistry and immunofluorescence and confirmed by immunoblotting. Quantitative real-time polymerase chain reaction was carried out for components of TGF-β pathway cascade and filamin A (FLN-A).Floppy MV leaflets were thicker (P.001) and had higher α-SMA+ cell density (P=.002) and collagen III expression (P.001) than controls. Enhanced p-Smad2 nuclear immunoreactivity (P.001) and TGF-β1 gene (P=.045), TIMP1 (P=.020), and CTGF (P=.047) expression but no differences in FLN-A and total Smad2 gene expression levels were found between floppy MV and controls. Higher expression of cadherin-11, either exclusively or in colocalization with N-cadherin, and aberrant presence of plakophilin-2 at the AJ were found in floppy MV vs.TGF-β1 pathway activation in nonsyndromic MV prolapse induces VICs differentiation into contractile myofibroblasts and is associated with changes in the molecular pattern of the AJ, with increased cadherin-11 and aberrant plakophilin-2 expression. AJ reinforcement might promote latent TGF-β1 activation leading to extracellular matrix remodeling in floppy MV.

470 Extracardiac endothelium patterns embryonic coronary arterio-venous connections {#article-title-2} Recent reports suggest that mammalian embryonic coronary endothelium (CoE) develops from the sinus venosus and ventricular endocardium. Although several studies in non-mammalian vertebrates have proven that extracardiac endothelial cells also participate in CoE development, the contribution of such cells to mammalian CoE is regarded to be minor and non-significant. Using classic (Wt1Cre) and novel (G2-Gata4Cre) mouse transgenic models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardiun (ST/PE)-derived endothelial cells are required for proper coronary development. Our results indicate that, at least, 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE. Conditional deletion of the epicardial lineage gene Wt1 in the ST/PE (G2-Gata4Cre) or the endothelium (Tie2Cre) reveals a critical role for these ST/PE-derived endothelial cells in the establishment of transmural ventricular coronary arterio-venous connections. Taken together, our results confirm that embryonic CoE is a developmental mosaic forming from different endothelial sources, and that extracardiac cells are necessary for the completion of coronary morphogenesis. These data contribute to our understanding of some coronary congenital anomalies and suggest embryonic CoE heterogeneity as an ontogenetic factor for adult coronary disease. # 471 DCM-associated RBM20-mutations lead to aberrant splicing of titin and ryanodin receptor 2 in the human myocardium {#article-title-3} Background: Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy (DCM). RBM20 is predominantly expressed in the heart. Within the nucleus it partially colocalises with other splice factors and binds RNA. Deep sequencing of the cardiac transcriptome of a human RBM20 mutant and of a RBM20 deficient rat model revealed RBM20 dependent regulation of myocardial alternative splicing. We have identified the previously published RBM20 mutation, p.P638L, which is localised in the highly conserved RS-region, in a large German family. The mutation is highly penetrant leading to terminal heart failure and/or to sudden cardiac death. Additionally we have discovered the new RBM20 mutation p.V914A localised outside the RS-region. Purpose: Though it is investigated that RBM20 is involved in alternative splicing in the rat heart little is known on aberrant myocardial splicing in human RBM20-mutation carriers. Goal of this study was to gain insight into the mechanisms leading to cardiomyopathy by analysing explanted human ventricular myocardium of RBM20-mutation carriers. Material and Methods: Cardiac tissue from two RBM20-p.P638L- and one RBM20-p.V914A-DCM-patients and a patient with the RBM20-variant p.D888N was obtained during heart transplantation or the implantation of a ventricular assist device, respectively. Ventricular myocardium was used for RNA extraction, cDNA synthesis and splicing analysis of TTN, RYR2, LDB3, CAMK2D, NEXN and TRDN by Real-Time-PCR. The alternative splicing of Titin was additionally examined by Western Blot using cardiac tissue lysates. Results: We identified aberrant RYR2 splicing in the RBM20 mutant myocardium characterised by an increased inclusion of an additional 24bp-exon. Furthermore we found a decrease of the major cardiac titin splice variant N2B by real-time PCR and Western Blot. Titin Western Blots revealed an increase of the splice variant N2BA in the RBM20 mutants. Of note, an influence on splicing could be detected not only for the RBM20-p.P638L-mutant but also for the mutation p.PV914A localised outside of the conserved RS-domain. The variant p.D888N showed no influence on the splicing of the genes analysed so far. In contrast to the rat hearts, in the human myocardium of RBM20-mutation carriers we did not find splicing defects in TRDN and NEXN. Conclusion: We present here first data on a molecular defect of a novel RBM20-mutation localised outside the conserved RS-region. Furthermore we show the influence of mutant RBM20 on splicing of RYR2 and TTN in the human explanted myocardium of three mutation carriers. The identification of the molecular pathomechanisms of the RBM20-mutations in the affected patients allows a differentiated insight into the disease mechanisms and might offer potential targets for curative treatment in the future. Furthermore the analysis of the splicing pattern helps to distinguish pathogenic variants like RBM20-p.PV914A from rare polymorphisms. # 472 The impact of missense versus nonsense mutations in arrhythmogenic cardiomyopathy phenotype {#article-title-4} Background: Arrhythmogenic Cardiomyopathy (AC) is an inherited heart muscle disease associated with mutations in five genes mainly encoding desmosomal components. Missense mutations leading to a single nucleotide substitution have disputed pathogenicity in inherited cardiomyopathies whereas truncating and splice site mutations are considered disease-causing due to their impact on form and function of the resulting protein. Purpose: Herein we describe the clinical features of two probands and their family members in whom a missense and a nonsense mutation in two desmosome-encoding genes co-segregate. Methods: A Next Generation Sequencing-based strategy comprising 150 inherited cardiomyopathy related genes was applied on the two index female cases (63y, 70y). Libraries were prepared according to the manufacturer’s instructions and sequenced on a MiSeq platform (Illumina). The Illumina Variant Studio software was used to filter and prioritize variants on the basis of genotype quality, allele frequencies, predicted pathogenicity, literature information. Subsequent conventional sequencing was used for cascade mutation screening. HL-1 cells have been transfected to ascertain localization of the mutated proteins. Results: Two unrelated female probands (63 y-old, 70 y-old) carried a missense mutation in DSG2 (p.Asn266Ser). The 63 y-old proband underwent cardiac transplantation (family A). The 70 y-old proband carried an additional nonsense mutation in DSP (c.88G>A, p.Gln113X), as well as her 64 y-old sister. Eight of the fifteen relatives from both families carried the DSG2 mutation: seven of them exhibited typical clinical AC features, whereas two young family members were unaffected at the ages of 6 and 12, respectively. The two DSP mutation carriers of family B were unaffected at the age of 44 and 66. No sudden cardiac death events occurred in both families. Haplotype analysis indicated that all the DSG2 mutation carriers shared the same haplotype suggesting a common founder allele. In vitro experiments on the cardiomyocyte cell line HL-1 showed correct localization of the mutated DSG2, whereas the mutated DSP was absent at the membrane level. Conclusions: This study is calling into question whether nonsense mutations are of high relevance with respect to the missense ones in the pathogenesis of AC. Our results highlight the dominant negative effect of a known missense DSG2 mutation with a presumed common founder effect, whereas the pathogenic role of the DSP nonsense mutation needs further studies to elucidate its contribution to the disease phenotype.

Background: Arrhythmogenic Cardiomyopathy (ACM) is an inherited myocardial disease characterized by fibro-fatty replacement of myocardium. Patients are at risk of ventricular arrhythmias and sudden death. Screening of desmosomal genes currently results in a diagnostic yield of about 50%. Aim: The aim of this study is to investigate whether detection of large deletions/duplications of the plakophilin-2 gene (PKP2) increase the yield of genetic screening in ACM. Methods: 60 index cases with a clinical diagnosis of ACM consecutively enrolled at our referral center underwent bidirectional sequencing using ABI-PRISM 3730 (Life Technologies) for all desmosomal-encoding genes. Genotype-negative patients were additionally screened for copy number variations (CNVs) in PKP2 by Multiplex Ligation-dependant Probe Amplification (MLPA) using SALSA MLPA kit P168 ARVC-PKP2 (MRC-Holland) and by quantitative Real-Time PCR on a Roche Light Cycler 480 platform. Results: Conventional sequencing of the ACM patients identified disease-causing nucleotide variants in 36 of the 60 ACM-patients (60%); 8% (n=5) of the cohort presented mutation in Desmoglein-2 gene, 23% (n=14) in Desmoplakin, 12% (n=7) in PKP2, 3% (n=2) in Desmocollin-2, 3% in Plakoglobin (n=2) whereas 10% (n=6) were multiple mutations carriers. MLPA analysis successfully identified a heterozygous deletion of exon 4 in an otherwise genotype-negative case, which was further confirmed by qPCR analysis showing a reduction of PKP2 copy number when compared to control samples. Conclusions: This study highlights the potential of increasing the diagnostic genetic yield - in our population by nearly 2% for just one gene- by searching CNVs in ACM patients. This will be also achieved by extending the analysis to all ACM disease-causing genes as well as to genotype-positive patients.

Introduction Arrhythmogenic cardiomyopathy (AC) is characterized by myocyte necrosis and progressive fibro-fatty substitution. Recently, truncated mutations on Filamin C gene have been correlated with AC and a peculiar phenotype characterized by a prominent left ventricular fibrosis and high risk of sudden death. Purpose To evaluate clinical and instrumental features of subjects affected by AC in whom genetic study identified presence of truncating and missense mutations on FLNC gene. Materials and methods A population of 192 probands affected by AC according to 2010 Task Force Criteria or McKenna's proposed criteria for left dominant AC were evaluated for FLNC variants. In positive probands and families anamnestic and clinical data (ECG, echocardiographic and cardiac magnetic resonance (CMR), twenty-four-hours ECG monitoring) were evaluated. Results A total of 19 subjects (9 probands and 10 family members) were identified as carrier of nine different FLNC mutations (5 truncating and 4 missense). In 3 patients (23%) clinical onset was characterized by major arrhythmic episodes and in one (8%) by sudden death. In 6 (46%) ECG was unremarkable and the most common abnormalities were low QRS voltages in peripheral leads (85%), followed by T wave inversion in lateral (15%) and in inferior leads (16%). Twenty-four-hours ECG monitoring revealed a high arrhythmic burden (PVC >500/die) in 6 cases (46%). CMR was performed in all patients. Four of them (31%) showed a LV dilatation, while in 2 cases (15%) a RV dilatation was present. In 8 (61%) a fatty infiltration was detected mainly affecting the left ventricle (6 cases, 46%). Moreover, late enhancement was present in 8 cases (62%), with a LV distribution. Conclusions This is the first studied population in which both truncating and missense variants were evaluated as causative of AC, confirming that FLNC gene mutations are rare (4% of probands without known AC causative mutations) and the prevalent clinical expression is a left dominant phenotype with a high degree of electrical instability and recurrence of sudden familial cardiac death. Funding Acknowledgement Type of funding source: None