Primary cardiomyopathies (CMPs) are monogenic but multi-allelic disorders with dozens of genes involved in pathogenesis. The implementation of next-generation sequencing (NGS) approaches has resulted in more time- and cost-efficient DNA diagnostics of cardiomyopathies. However, the diagnostic yield of genetic testing for each subtype of CMP fails to exceed 60%. The aim of this study was to demonstrate that allelic dropout (ADO) is a common phenomenon that reduces the diagnostic yield in primary cardiomyopathy genetic testing based on targeted gene panels assayed on the Ion Torrent platform. We performed mutational screening with three custom targeted gene panels based on sets of oligoprimers designed automatically using AmpliSeq Designer® containing 1049 primer pairs for 37 genes with a total length of 153 kb. DNA samples from 232 patients were screened with at least one of these targeted gene panels. We detected six ADO events in both IonTorrent PGM (three cases) and capillary Sanger sequencing (three cases) data, identifying ADO-causing variants in all cases. All ADO events occurred due to common or rare single nucleotide variants (SNVs) in the oligoprimer binding sites and were detected because of the presence of “marker” SNVs in the target DNA fragment. We ultimately identified that PCR-based NGS involves a risk of ADO that necessitates the use of Sanger sequencing to validate NGS results. We assume that oligoprimer design without ADO data affects the amplification efficiency of up to 0.77% of amplicons.

The cardiac voltage-gated sodium channel Na_v1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Na_v1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Na_v1.5 NTD: Y87C–here newly identified–, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Na_v1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Na_v1.5 at the cell surface and in whole-cell lysates. Localization of Na_v1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Na_v1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Na_v1.5 construct missing residues 80–105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Na_v1.5 variant Y87C and present first evidence that calmodulin binds to the Na_v1.5 NTD, which seems to be a determinant for the DNE.

We present 38-years male patient. He has suffered from muscle weakness since 5 years. Arrhythmias appeared at the age of 32. In 37 years he had sick sinus syndrome, transient AV block II degree, paroxysmal atrial fibrillation, atrial flutter, ventricular arrhythmias. At this time, dilated cardiomyopathy was also detected. The evaluation revealed knees and elbows contractures, increased level of creatine kinase. The genetic testing revealed a frame shift deletion c.del619C in the emerin (EMD) gene and c.IVS4-13T> A in the lamin (LMNA) gene, and c.del619C deletion in the heterozygous state in a patient`s mother. Radiofrequency ablation of cavotricuspid isthmus, implantable cardioverter-defibrillator (ICD) implantation were performed. Heart transplantation was performed nine months later, due to severe heart failure and electrical storm. A morphological evaluation revealed sclerosis, atrophy and hypertrophy of cardiomyocytes. He underwent an induction therapy with (basiliximab) methylprednisolone, tacrolimus, mycophenolate after heart transplantation. During 40 months after transplantation, patient`s condition is satisfactory. heart failure in Emery-Dreifuss muscular dystrophy can progress quickly unless the previously stable condition. The use of correct regimens of immunosuppression therapy provides good long-term results of the heart transplantation.

We characterized a novel genetic variant c.292G > A (p.E98K) in the TPM1 gene encoding cardiac tropomyosin 1.1 isoform (Tpm1.1), found in a proband with a phenotype of complex cardiomyopathy with conduction dysfunction and slow progressive neuromuscular involvement. To understand the molecular mechanism by which this mutation impairs cardiac function, we produced recombinant Tpm1.1 carrying an E98K substitution and studied how this substitution affects the structure of the Tpm1.1 molecule and its functional properties. The results showed that the E98K substitution in the N-terminal part of the Tpm molecule significantly destabilizes the C-terminal part of Tpm, thus indicating a long-distance destabilizing effect of the substitution on the Tpm coiled-coil structure. The E98K substitution did not noticeably affect Tpm’s affinity for F-actin but significantly impaired Tpm’s regulatory properties. It increased the Ca²⁺ sensitivity of the sliding velocity of regulated thin filaments over cardiac myosin in an in vitro motility assay and caused an incomplete block of the thin filament sliding at low Ca²⁺ concentrations. The incomplete motility block in the absence of Ca²⁺ can be explained by the loosening of the Tpm interaction with troponin I (TnI), thus increasing Tpm mobility on the surface of an actin filament that partially unlocks the myosin binding sites. This hypothesis is supported by the molecular dynamics (MD) simulation that showed that the E98 Tpm residue is involved in hydrogen bonding with the C-terminal part of TnI. Thus, the results allowed us to explain the mechanism by which the E98K Tpm mutation impairs sarcomeric function and myocardial relaxation.

Background. A few cases of combination of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) with left ventricular noncompaction (LVNC) have been described. Aims to study the genetics, diagnostical features and clinical course of the combination of ARVC with LVNC. Methods. 58 patients with ARVC diagnosis (26 men; mean age 39.1 14.2 years; mean follow-up period 21.5 [6; 60] months) and 125 patients with LVNC (74 men; mean age 46.4 15.1 years; mean follow-up period 14 [3; 40] months). All patients underwent electrocardiogram (ECG), echocardiography, 24-h ECG monitoring. Heart MRI was performed in 53 (91.4%) patients with ARVC and 60 (48%) with LVNC, heart CT in 18 (31%) patients with ARVC and 89 (71.2%) with LVNC. For all patients with combination of ARVC and LVNC DNA-diagnostic was performed using both ARVC (PKP2, DSG2, DSP, DSC2, JUP, TMEM43, TGFB3, PLN, LMNA, DES, CTTNA3, EMD, SCN5A, LDB3, CRYAB, FLNC) and LVNC (MYH7, MYBPC3, TAZ, TPM1, LDB3, MYL2, MYL3, ACTC1, TNNT2, TNI3) gene panels. Results. Combination of ARVC and LVNC was found in 9 patients (15.5% of patients form ARVC cohort and 7.2% from LVNC cohort). These patients were distinguished from patients with isolated ARVC or LVNC by aggressive ventricular arrhythmias (frequent premature ventricular beats, sustained ventricular tachycardia, significantly worse antiarrhythmic therapy effect, appropriate shocks of implanted cardioverter-defibrillators (ICD) in all patients with ICD). Patients with combination of ARVC + LVNC were also distinguished from patients with isolated LVNC by the dilatation of RV, low QRS voltage on ECG, presence of AV block, absence of signs of LV hypertrophy on ECG. LV dilatation with reduction of its ejection fraction distinguished patients with mixed cardiomyopathy from patients with isolated ARVC. Potentially pathogenic variants (IVV classes of pathogenicity) and variants of unclear clinical significance (III class of pathogenicity) were found in both desmosomal and non-desmosomal genes in 78% of patients, including 3 (33%) in DSP gene. Conclusions. The combination of ARVC and LVNC can be caused by mutations in both desmosomal and non-desmosomal genes and has typical features: aggressive, resistant ventricular rhythm abnormalities leading to appropriate ICD shocks and a high risk of sudden cardiac death.

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST-segment elevation in the right precordial leads on electrocardiograms (ECG), and high risk of life-threatening ventricular arrhythmia and sudden cardiac death (SCD). Mutations in the responsible genes have not been fully characterized in the BrS patients, except for the SCN5A gene. We identified a new genetic variant, c.1189C>T (p.R397C), in the KCNH2 gene in the asymptomatic male proband diagnosed with BrS and mild QTc shortening. We hypothesize that this variant could alter I_Kr-current and may be causative for the rare non-SCN5A-related form of BrS. To assess its pathogenicity, we performed patch-clamp analysis on I_Kr reconstituted with this KCNH2 mutation in the Chinese hamster ovary cells and compared the phenotype with the wild type. It appeared that the R397C mutation does not affect the I_Kr density, but facilitates activation, hampers inactivation of the hERG channels, and increases magnitude of the window current suggesting that the p.R397C is a gain-of-function mutation. In silico modeling demonstrated that this missense mutation potentially leads to the shortening of action potential in the heart.