Compound Heterozygous FKTN Variants in a Patient with Dilated Cardiomyopathy Led to an Aberrant α-Dystroglycan Pattern.

Fukutin encoded by FKTN is a ribitol 5-phosphate transferase involved in glycosylation of α-dystroglycan. It is known that mutations in FKTN affect the glycosylation of α-dystroglycan, leading to a dystroglycanopathy. Dystroglycanopathies are a group of syndromes with a broad clinical spectrum including dilated cardiomyopathy and muscular dystrophy. In this study, we reported the case of a patient with muscular dystrophy, early onset dilated cardiomyopathy, and elevated creatine kinase levels who was a carrier of the compound heterozygous variants p.Ser299Arg and p.Asn442Ser in FKTN. Our work showed that compound heterozygous mutations in FKTN lead to a loss of fully glycosylated α-dystroglycan and result in cardiomyopathy and end-stage heart failure at a young age.

The Combined Human Genotype of Truncating TTN and RBM20 Mutations Is Associated with Severe and Early Onset of Dilated Cardiomyopathy.

A major cause of heart failure is cardiomyopathies, with dilated cardiomyopathy (DCM) as the most common form. Over 40 genes are linked to DCM, among them TTN and RBM20. Next Generation Sequencing in clinical DCM cohorts revealed truncating variants in TTN (TTNtv), accounting for up to 25% of familial DCM cases. Mutations in the cardiac splicing factor RNA binding motif protein 20 (RBM20) are also known to be associated with severe cardiomyopathies. TTN is one of the major RBM20 splicing targets. Most of the pathogenic RBM20 mutations are localized in the highly conserved arginine serine rich domain (RS), leading to a cytoplasmic mislocalization of mutant RBM20. Here, we present a patient with an early onset DCM carrying a combination of (likely) pathogenic TTN and RBM20 mutations. We show that the splicing of RBM20 target genes is affected in the mutation carrier. Furthermore, we reveal RBM20 haploinsufficiency presumably caused by the frameshift mutation in RBM20.

Cardiomyopathy-associated mutations in the RS domain affect nuclear localization of RBM20.

Mutations in RBM20 encoding the RNA-binding motif protein 20 (RBM20) are associated with an early onset and clinically severe forms of cardiomyopathies. Transcriptome analyses revealed RBM20 as an important regulator of cardiac alternative splicing. RBM20 mutations are especially localized in exons 9 and 11 including the highly conserved arginine and serine-rich domain (RS domain). Here, we investigated in several cardiomyopathy patients, the previously described RBM20-mutation p.Pro638Leu localized within the RS domain. In addition, we identified in a patient the novel mutation p.Val914Ala localized in the (glutamate-rich) Glu-rich domain of RBM20 encoded by exon 11. Its impact on the disease was investigated with a novel TTN- and RYR2-splicing assay based on the patients' cardiac messenger RNA. Furthermore, we showed in cell culture and in human cardiac tissue that mutant RBM20-p.Pro638Leu is not localized in the nuclei but causes an abnormal cytoplasmic localization of the protein. In contrast the splicing deficient RBM20-p.Val914Ala has no influence on the intracellular localization. These results indicate that disease-associated variants in RBM20 lead to aberrant splicing through different pathomechanisms dependent on the localization of the mutation. This might have an impact on the future development of therapeutic strategies for the treatment of RBM20-induced cardiomyopathies.

High proportion of genetic cases in patients with advanced cardiomyopathy including a novel homozygous Plakophilin 2-gene mutation.

Cardiomyopathies might lead to end-stage heart disease with the requirement of drastic treatments like bridging up to transplant or heart transplantation. A not precisely known proportion of these diseases are genetically determined. We genotyped 43 index-patients (30 DCM, 10 ARVC, 3 RCM) with advanced or end stage cardiomyopathy using a gene panel which covered 46 known cardiomyopathy disease genes. Fifty-three variants with possible impact on disease in 33 patients were identified. Of these 27 (51%) were classified as likely pathogenic or pathogenic in the MYH7, MYL2, MYL3, NEXN, TNNC1, TNNI3, DES, LMNA, PKP2, PLN, RBM20, TTN, and CRYAB genes. Fifty-six percent (n = 24) of index-patients carried a likely pathogenic or pathogenic mutation. Of these 75% (n = 18) were familial and 25% (n = 6) sporadic cases. However, severe cardiomyopathy seemed to be not characterized by a specific mutation profile. Remarkably, we identified a novel homozygous PKP2-missense variant in a large consanguineous family with sudden death in early childhood and several members with heart transplantation in adolescent age.

In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations.

BACKGROUND: Although numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate. METHODOLOGY/PRINCIPAL FINDINGS: To test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation. CONCLUSIONS/SIGNIFICANCE: The ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study.

In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.

BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

De novo desmin-mutation N116S is associated with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease, frequently accompanied by sudden cardiac death and terminal heart failure. Genotyping of ARVC patients might be used for palliative treatment of the affected family. We genotyped a cohort of 22 ARVC patients referred to molecular genetic screening in our heart center for mutations in the desmosomal candidate genes JUP, DSG2, DSC2, DSP and PKP2 known to be associated with ARVC. In 43% of the cohort, we found disease-associated sequence variants. In addition, we screened for desmin mutations and found a novel desmin-mutation p.N116S in a patient with ARVC and terminal heart failure, which is located in segment 1A of the desmin rod domain. The mutation leads to the aggresome formation in cardiac and skeletal muscle without signs of an overt clinical myopathy. Cardiac aggresomes appear to be prominent, especially in the right ventricle of the heart. Viscosimetry and atomic force microscopy of the desmin wild-type and N116S mutant isolated from recombinant Escherichia coli revealed severe impairment of the filament formation, which was supported by transfections in SW13 cells. Thus, the gene coding for desmin appears to be a novel ARVC gene, which should be included in molecular genetic screening of ARVC patients.