Deciphering DSC2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ECG and tailored drug therapy.

BACKGROUND: Severe ventricular rhythm disturbances are the hallmark of arrhythmogenic cardiomyopathy (ACM), and are often explained by structural conduction abnormalities. However, comprehensive investigations of ACM cell electrical instability are lacking. This study aimed to elucidate early electrical myogenic signature of ACM. METHODS: We investigated a 41-year-old ACM patient with a missense mutation (c.394C>T) in the DSC2 gene, which encodes desmocollin 2. Pathogenicity of this variant was confirmed using a zebrafish DSC2 model system. Control and DSC2 patient-derived pluripotent stem cells were reprogrammed and differentiated into cardiomyocytes (hiPSC-CM) to examine the specific electromechanical phenotype and its modulation by antiarrhythmic drugs (AADs). Samples of the patient's heart and hiPSC-CM were examined to identify molecular and cellular alterations. RESULTS: A shortened action potential duration was associated with reduced Ca2+ current density and increased K+ current density. This finding led to the elucidation of previously unknown abnormal repolarization dynamics in ACM patients. Moreover, the Ca2+ mobilised during transients was decreased, and the Ca2+ sparks frequency was increased. AAD testing revealed the following: (1) flecainide normalised Ca2+ transients and significantly decreased Ca2+ spark occurrence and (2) sotalol significantly lengthened the action potential and normalised the cells' contractile properties. CONCLUSIONS: Thorough analysis of hiPSC-CM derived from the DSC2 patient revealed abnormal repolarization dynamics, prompting the discovery of a short QT interval in some ACM patients. Overall, these results confirm a myogenic origin of ACM electrical instability and provide a rationale for prescribing class 1 and 3 AADs in ACM patients with increased ventricular repolarization reserve.

Functional characterization of putative novel splicing mutations in the cardiomyopathy-causing genes.

Molecular diagnosis of cardiomyopathies remains difficult not only because of the large number of causative genes and the high rate of private mutations but also due to the large number of unclassified variants (UVs) found in patients' DNA. This study reports the functional splicing impact of nine novel genomic variations previously identified in unrelated patients with cardiomyopathies. To identify splice variants among these UVs, a combination of in silico and in vitro hybrid minigene tools was used as transcript is not available. Using this two-step approach, these UVs were reclassified as splicing mutations (MYBPC3-c.655-25A>G, MYBPC3-c.1790G>A (p.Arg597Gln), MYBPC3-c.2414-36G>T) or as mutations with a majority of abnormally spliced transcripts (MYBPC3-c.1182C>A, TNNT2-c.460G>A (p.Glu154Lys), and TNNT2-c.822-3C>A) or as variations with a weak splicing effect (TNNT2-c.1000-38C>A). For the two remaining variations in intron 11 of the TNNT2 gene in the vicinity of the acceptor splice site (c.571-7G>A, c.571-29G>A), a minigene assay was inconclusive as exon 12 is neither recognized as an exon by HeLa nor by H9c2 cells. Our study highlights the importance of the combined use of in silico and in vitro splicing assays to improve the prediction of the functional splicing impact of identified genetic variants if the RNA sample from the patient is not easily available.

Homozygous mutation in MERTK causes severe autosomal recessive retinitis pigmentosa.

PURPOSE: Gene identification in retinitis pigmentosa is a prerequisite to future therapies. Accordingly, autosomal recessive retinitis pigmentosa families were genotyped to search for causative mutations. METHODS: Members of a consanguineous Moroccan family had standard ophthalmologic examination, optical coherence tomography-3 scan, autofluorescence testing, and electroretinogram. Their DNA was genotyped with the 250K SNP microchip (Affymetrix) and homozygosity mapping was done. MERTK exons were polymerase chain reaction amplified and sequenced. RESULTS: Two sisters and one brother out of 6 siblings had rod cone dystrophy type of retinitis pigmentosa. Salient features were night blindness starting in early infancy, dot-like whitish deposits in fovea and macula with corresponding autofluorescent dots in youngest patients, decreased visual acuity, and cone responses higher than rod responses at electroretinogram. The patients were homozygous in regions from chromosomes 2 and 8, but only that of chromosome 2 was inherited from a common ancestor. Sequencing of the MERTK gene belonging to the chromosome 2 region showed that the 3 affected patients carried a novel homozygous mutation in exon 17, c.2323C>T, leading to p.Arg775X, while their unaffected brothers and sister, parents, and paternal grandfather were heterozygous. CONCLUSIONS: MERTK mutations lead to severe retinitis pigmentosa with discrete dot-like autofluorescent deposits at early stages, which are a hallmark of this MERTK-specific dystrophy.