RBM20 Mutations Research Articles

Generation of an RBM20-mutation-associated left-ventricular non-compaction cardiomyopathy iPSC line (UMGi255-A) into a DCM genetic background to investigate monogenetic cardiomyopathies

RBM20 mutations account for 3 % of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient.

Malignant arrhythmias and advanced heart failure in patients with RBM20 cardiomyopathy: a single center cohort study and review of the literature

Abstract Background Sudden cardiac death (SCD), and death rate attributable to heart failure (HF) are common causes of death in the general population. Both are seen in cardiomyopathy patients. RBM20 mutations associated with a cardiomyopathy phenotype and a high risk of SCD and HF. Purpose The aim of this study is to investigate the genotype-phenotype correlations, and clinical outcomes of the RBM20 mutations. Methods Peripheral blood from patients was drawn as a routine diagnostic procedure and genomic DNA extracted according to standard protocols. Patients were tested for at least 45 cardiomyopathy-related genes included in the Dutch core panel. 23 patients with a likely pathogenic or pathogenic RBM20 mutation and a cardiomyopathy phenotype were retrospectively enrolled in a single-center cohort. Patient data on clinical characteristics, imaging and pathology were extracted retrospectively. Furthermore, a literature search was conducted for a literature review on RBM20 mutations in cardiomyopathies. Peer reviewed studies with patients aged 18 or older, a RBM20 mutation, and a cardiomyopathy were included. Results Whole genome sequencing found 5 different RBM20 protein altering mutations. The c.1900C>T mutation was the most frequently seen in 18 patients. Four RBM20 mutations were novel mutations; c.1912, c.2919, c.414 and c.1118_1119 (Table1). The 23 patients had a mean age of 52 ± 15 years, and 13 (59%) were male. The phenotype was predominantly dilated cardiomyopathy (DCM). This was seen in 18 (82%)patients compared to 2 (12%) noncompaction cardiomyopathy (NCCM) patients. Nine (41%) patients had experienced malignant ventricular arrhythmia (VA). The mean age of first VA was 49 ± 10 years. An implantable cardioverter-defibrillator (ICD) was implanted in 13 (57%) patients, and gave appropriate therapy in 5 (23%) patients compared to 1 (9%) patient receiving inappropriate therapy. Ten (46%) patients were diagnosed with heart failure (HF) at a mean age of 50 ± 11 years. One patient died of heart failure, while 5 (25%) patients underwent heart transplantation (HTx). Delayed enhancement (DE) was shown on MRI in 5 (50%) patients, of which 3 had DE located in the mid-myocardial and inferolateral segments. They all had c.1900C>T, but did not differ in clinical outcomes compared to patients without DE. The literature review included 32 studies with 394 total patients. Five studies reported on c.1900C>T, of which 4 case reports/series and 1 cohort study. The mean age at diagnosis was lower in 4 studies and varied between 26 and 52 years compared to 51 ± 15 years in our c.1900C>T group. The prevalence of VA was 66%-100% in the case reports/series, and 28% in the cohort study. In our cohort had 5 (42%) patients a VA. Conclusion This study found 4 novel RBM20 mutations. Moreover, the RBM20 mutation phenotype was predominantly DCM combined with a high prevalence of malignant VA and/or HF, which both had an peak age of early onset between 45-54 years.

Deep phenotyping of two preclinical mouse models and a cohort of RBM20 mutation carriers reveals no sex-dependent disease severity in RBM20 cardiomyopathy.

RBM20 cardiomyopathy is an arrhythmogenic form of dilated cardiomyopathy caused by mutations in the splicing factor RBM20. A recent study found a more severe phenotype in male patients with RBM20 cardiomyopathy patients than in female patients. Here, we aim to determine sex differences in an animal model of RBM20 cardiomyopathy and investigate potential underlying mechanisms. In addition, we aim to determine sex and gender differences in clinical parameters in a novel RBM20 cardiomyopathy patient cohort. We characterized an Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, cardiac function, and arrhythmia susceptibility do not differ between sexes. Next, we performed deep phenotyping of these mice, and show that male and female Rbm20-KO mice possess transcriptomic and phosphoproteomic differences. Hypothesizing that these differences may influence the heart's ability to compensate for stress, we exposed Rbm20-KO mice to acute catecholaminergic stimulation and again found no functional differences. We also replicate the lack of functional differences in a mouse model with the Rbm20-R636Q mutation. Lastly, we present a patient cohort of 33 RBM20 cardiomyopathy patients and show that these patients do not possess sex and gender differences in disease severity. Current mouse models of RBM20 cardiomyopathy show more pronounced changes in gene expression and phosphorylation of cardiac proteins in male mice, but no sex differences in cardiac morphology and function. Moreover, other than reported before, male RBM20 cardiomyopathy patients do not present with worse cardiac function in a patient cohort from Germany and the Netherlands.NEW & NOTEWORTHY Optimal management of the cardiac disease is increasingly personalized, partly because of differences in outcomes between sexes. RBM20 cardiomyopathy has been described to be more severe in male patients, and this carries the risk that male patients are more scrutinized in the clinic than female patients. Our findings do not support this observation and suggest that treatment should not differ between male and female RBM20 cardiomyopathy patients, but instead should focus on the underlying disease mechanism.

Abstract 14462: RBM20 Regulation In Healthy And Diseased Cardiomyocytes: A New Opportunity For Targeted Therapeutics

Introduction: Mutations in the splicing factor RBM20 cause a severe form of dilated cardiomyopathy (DCM) characterized by early onset, fast disease progression, and high risk of sudden death. We have previously shown that modulation of RBM20 expression is a promising strategy for patient with mutations in RBM20. However, our understanding of its endogenous regulation is limited. Hypothesis: DCM-causing mutations in RBM20 interfere with phosphorylation of RBM20 RS domain and with localization of the protein. We hypothesize that modulation of RBM20 phosphorylation could be used to normalize function in the presence of mutations that impair it. We sought to uncover the pathways responsible for such regulation and test their therapeutic potential. Methods: We developed a fluorescent in vitro reporter system that allows for the quantification of RBM20 expression and localization in human cardiomyocytes. We generated an induced Pluripotent Stem cells (iPSC) line in which the endogenous RBM20 is tagged at the C-term with an in-frame mCherry (RBM20-mCh). The tag leaves the regulatory regions at the 5’ end of the gene intact. RBM20-mCh hiPSC were differentiated into cardiomyocytes (iPSC-CMs) and matured for 4 weeks. We screened 2-month-old matured iPSC-CM on Matrigel-coated 384-well plates against a library of siRNA targeting all human kinases and phosphatases. We tested the hits in a contractility assay Results: We identified 35 kinases and 16 phosphatases that alter RBM20 expression and localization. Functional studies showed that the kinases that upregulated RBM20 expression increased cardiomyocyte contractility while downregulating kinases reduced contractility. Conclusions: In conclusion, understanding the kinase regulation of RBM20 provides insight into control of mRNA splicing of proteins such as Titin and may point to new targeted therapeutic strategies for RBM20 mutant DCM.

Abstract 13431: Gain-of-Function Mutations in RNA Binding Motif 20 Result in Cardiac Remodelling and Pathogenesis in Mice

Gain-of-function mutations of a gene result in new molecular function. Genetic studies show that mutations in a gene named as RNA binding motif 20 (RBM20) are associated with dilated cardiomyopathy (DCM). To test whether Rbm20 mutations are responsible for cardiac remodelling and potential underlying mechanisms, we selected two mutations (S637A and S639G) located in arginine/serine-rich domain of RBM20 and generated two knock-in mouse models ( Rbm20 S637A and Rbm20 S639G ) respectively. Both mouse lines were subjected to histological and anatomical analysis as well as cardiac function assessment with echocardiography. The results revealed that heterozygous (HT) and homozygous (HM) male and female mice from both lines displayed DCM phenotype with enlarged left ventricular (LV) chamber, thinner LV walls and reduced ejection fraction. Interestingly, we observed that Rbm20 S639G mice manifested more severe phenotype than that in Rbm20 S637A mice. Rbm20 S639G HM mice exhibited higher mortality rate (~50%) than that (~34%) in Rbm20 S637A HM mice at an early age (<100 days). Differentially expressed and spliced genes from RNA-seq data are involved in arrhythmia, cardiomyopathy, and sudden death based on Human Phenotype Ontology enrichment analysis (FDR≤0.05) in both lines. Immunofluorescent analysis in tissues and single cardiomyocytes respectively showed both mutations promote RBM20 nucleocytoplasmic trafficking and protein condensates, suggesting they are gain-of-function mutations, leading to high mortality rate at younger age. Both mutations also interrupted the alternative splicing of TTN and calcium handling genes. Force measurements and Ca 2+ transient evaluation of intact myocytes showed that the reduced cellular diastolic stiffness and impaired contractility in HT and HM mice. Studies suggested that phosphorylation of RS domain is critical for protein trafficking. We then performed mass spectrometry on the in vitro expressed RBM20 and identified phosphorylation on S637 and S639. Phosphomimetic assay showed that phosphorylation was not responsible for RBM20 trafficking and condensates. Together, we reported a new mechanism by which cardiac genetic mutations causes protein trafficking and aggregation in cardiac remodelling.

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

RBM20-mutations induce disturbed splicing of calcium relevant genes and guides clinically therapy in different cardiomyopathies

Abstract Background Mutations in the splice factor RBM20 account for ∼3% of genetic cardiomyopathies. Mutations at position R634 in the hotspot RS-domain were found to cause dilative cardiomyopathy (DCM) (R634W) or left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remain unknown. Purpose We aimed here to identify the molecular events caused by the distinct RBM20 mutations from DCM and LVNC patients using a patient-specific induced stem cell model (iPSC) and test if the currently clinically used β-blockers (Metroprolol) are suitable for different RBM20-dependent cardiomyopathies. Methods We generated iPSC-cardiomyocytes of 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC). We investigated alternative splicing, sarcomeric regularity, cAMP-level, kinase-specific phosphorylation of Ca2+ players and Ca2+ handling. To investigate the impact of the genetic background, isogenic rescue lines were generated by CRISPR/Cas9. Different clinical drugs as Metoprolol and Verapamil were used to analyze the pharmacological improvement in vitro. Results We investigated the splicing pattern of the 2 RBM20 mutations in DCM and LVNC iPSC-CMs and observed common isoform changes in titin and a 24bp-insertion in the gene RYR2. The Ca2+ handling gene triadin is misspliced in LVNC-CMs, whereas the structural gene LDB3 is misspliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes, both DCM and LVNC-CMs exhibited an irregular sarcomeric structure. The Ca2+ handling gene CAMK2δ was predominantly misspliced in LVNC-CMs leading to CAMK2δ-dependent hyperphosphorylation of its target PLN-Thr17 and subsequently to shortened Ca2+ elimination time and weakened response to β-adrenergic stimulation. By contrast, DCM-CMs exhibited increased Ca2+ sparks and decreased systolic and diastolic Ca2+ levels. RBM20 expression itself was decreased in LVNC-CMs, but not in DCM-CMs. This highlights that 2 distinct RBM20 mutations can lead to different pathological Ca2+ phenotypes. Isogenic CRISPR/Cas9 repair of both RBM20 mutations in LVNC and DCM demonstrated a rescue in gene missplicing, sarcomeric regularity and the Ca2+ handling aberrations and underscored the causative nature of the 2 mutations and their diverging effects. Ca2+ channel blockage with Verapamil showed a significant improvement of some of the LVNC disease characteristics compared to commonly clinically used β-blocker Metoprolol and underpins the potential clinical use of this drug in patients with LVNC. Conclusion We show the first iPSC-model of splice-defect associated RBM20-dependent LVNC and DCM. In summary, our results suggest that the molecular aberrations in alternative splicing differ depending on the distinct mutation in RBM20 and lead to shared and differential pathologies. Verapamil could be a good candidate in the treatment of RBM20-dependent LVNC. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Bunderministerium für Bildung und Forschung BMBFGerman Center for Cardiovascular Research DZHK

Phenotype and progression among patients with dilated cardiomyopathy and RBM20 mutations

BackgroundOver 70 genes that encode different cell components have been involved in the aetiology of dilated cardiomyopathy. Genotype-phenotype interactions are an unsolved problem, and to a large extent the effects of mutations in the expression mechanisms involved in the disease remain unknown, although associations are increasingly being established which have clinical and prognostic implications. Methods and resultsThe objective of our work was to describe our population that has cardiomyopathy associated with mutations in the gene RBM20, and study the genotype-phenotype relationship. We studied 8 cases undergoing follow-up at our Unit, and collected data for demographic, clinical and diagnostic testing variables. The mean age on diagnosis was 55 years [52–59], with a median follow-up of 31.5 months [26.0–67.3]. It is worth noting that 62.5% of the patients in our group had a history of cardiomyopathy in first degree relatives, and 37.5% of them had a family history of sudden death. One of the genetic variations of the sample was shared by three subjects who had no apparent family relationship with each other, and this variation had not been described in controls. It is also interesting that arrhythmic events were found in 37.5% of the sample, and 50% of patients had an indication for implantable cardiac defibrillator. ConclusionThis is the first analysis of patients with RBM20 mutations conducted in our country, and it indicates a profile with prominent arrhythmogenesis, a high penetrance of familial cardiomyopathy, and sudden death.

Familial dilated cardiomyopathy with RBM20 mutation in an Indian patient: a case report

BackgroundDilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by ventricular dilation and a left ventricular ejection fraction of less than 40%. Unlike hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC), DCM-causing mutations are present in a large number of genes. In the present study, we report a case of the early age of onset of DCM associated with a pathogenic variant in the RBM20 gene in a patient from India.Case presentationA 19-year-old Indian male diagnosed with DCM was suggested for heart transplantation. His ECG showed LBBB and echocardiography showed an ejection fraction of 14%. He had a sudden cardiac death. A detailed family history revealed it to be a case of familial DCM. Genetic screening identified the c.1900C>T variant in the RBM20 gene which led to a missense variant of amino acid 634 (p.Arg634Trp).ConclusionTo the best of our knowledge, the variant p.Arg634Trp has been earlier reported in the Western population, but this is the first case of p.Arg634Trp in an Indian patient. The variant has been reported to be pathogenic at an early age of onset; therefore, close clinical follow-up should be done for the family members caring for the variant.

Genetics of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), which include genetic and nongenetic forms, is the most common form of cardiomyopathy. DCM is characterized by left ventricular or biventricular dilation with impaired contraction. In the United States, DCM is a burden to healthcare that accounts for approximately 10,000 deaths and 46,000 hospitalizations annually. In this review, we will focus on the genetic forms of DCM and on recent advances in the understanding of cytoskeletal, sarcomeric, desmosomal, nuclear membrane, and RNA binding genes that contribute to the complexity and genetic heterogeneity of DCM. Although mutations in TTN remain the most common identifiable cause of genetic DCM, there is a growing appreciation for arrhythmogenic-prone DCM due to mutations in LMNA, desmosomal genes, and the recently described FLNC gene encoding the structural filamin C protein. Mutations in RBM20 highlight the relevance of RNA splicing regulation in the pathogenesis of DCM. Although expanded genetic testing has improved access to genetic diagnostic studies for many patients, the molecular mechanisms in the pathogenesis of the disease remained largely unknown. : The identification of the molecular causes and subsequent insight into the molecular mechanisms of DCM is expanding our understanding of DCM pathogenesis and highlights the complexity of DCM and the need to develop multifaceted strategies to treat the various causes of DCM.

From the Literature

From the Literature

RBM20-mutations induce disturbed splicing of calcium relevant genes in patient-specific stem cell models of cardiomyopathies

Abstract Background and aim Mutations in the splice factor RBM20 have been identified to account for ∼3% of cardiomyopathies. In particular, the highly conserved RS-domain is a hotspot for disease-associated mutations. Distinct mutations at position 634 in the RS-domain were already described to be associated to dilative cardiomyopathy (DCM) (R634W) or to left ventricular non-compaction cardiomyopathy (LVNC) (R634L), but the molecular mechanisms that govern the heterogenic entity of DCM and LVNC remain largely unknown. We aimed to analyze the molecular driver behind the RBM20 mutation-based DCM and LVNC in a patient-specific stem cell model. Methods Human somatic cells from 2 DCM- and 2 LVNC-patients harboring the RBM20-mutations R634W (DCM) or R634L (LVNC) were reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into functional cardiomyocytes (CM). Gene expression, alternative splicing activity, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of important Ca2+ players, and physiological cardiac functions as Ca2+ homeostasis were analyzed (Fluo3 and Fura4). Isogenic rescue lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations to the cardiac phenotype. Results We investigated the role of RBM20 mutations in DCM and LVNC-iPSC-CMs RBM20-splicing and observed common splice defects in titin-isoform-switch or a 24bp insertion in the gene ryanodine receptor 2 (RYR2).. In contrast, the calcium-handling gene Camk2δ was predominantly mis-spliced in LVNC-CMs, whereas the structural gene LDB3 was mis-spliced in DCM-CMs. As a possible consequence of splice defects in sarcomeric genes both DCM and LVNC-CMs exhibited an irregular sarcomeric structure at the Z-disk and M-line. Interestingly, the LVNC-CMs showed faster Ca2+ transient decay time and weakened response to β-adrenergic stimulation. In contrast, the DCM-CMs did exhibit increased Ca2+-sparks and decreased systolic and diastolic Ca2+ highlighting that two distinct missense mutations can lead to different pathological Ca2+ phenotypes. Ca2+ kinetic defects in LVNC-iPSC-CMs were independent of cAMP, but in line with Camk2δ-dependent hyperphosphorylation of the specific target PLN. Isogenic WT-iPSC lines were generated using CRISPR/Cas9 technology and underscored the role of RBM20-mutations in cardiomyopathies as the sarcomeric defects, Ca2+ cycling and leakage were rescued for both LVNC-CMs and DCM-CMs. Conclusion We show the first iPSC-model of splice-defect-associated RBM20-dependent LVNC and DCM. Our data demonstrate that RBM20-R634L induce mis-splicing of Camk2δ leading to hyperphosphorylation of PLN-Thr17 along with increased Ca2+ kinetics in LVNC, whereas RBM20-R634W induced RYR2-dependent Ca2+ leak with disturbed systolic and diastolic Ca2+in DCM. Taken together these results suggest that the molecular aberrations in alternative splicing differ depending on the distinct missense mutation in RBM20. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): BMBF, DZHK German Center for Cardiovascular research

A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice

Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20S637A protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes.

Upregulating RBM20 as a therapy for DCM.

A study in an in vitro model of RBM20-deficient dilated cardiomyopathy suggests that pharmacological upregulation of RBM20 with all-trans retinoic acid is a potential therapeutic strategy in patients with a heterozygous RBM20 mutation.

IPSC Modeling of RBM20-Deficient DCM Identifies Upregulation of RBM20 as a Therapeutic Strategy

SUMMARYRecent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20.

The Emerging Role of the RBM20 and PTBP1 Ribonucleoproteins in Heart Development and Cardiovascular Diseases.

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies.

Can Genetic Profile of Patients Undergoing Heart Transplantation Alter Clinical Decisions?

Although a significant proportion of cardiomyopathies are determined by genetic factors, the genetic testing of these patients has not been introduced routinely into clinical practice. The aim of this study is the genotyping of patients with advanced cardiomyopathy who have been bridged for heart transplantation or have been already transplanted, in order to evaluate the usefulness of genetic testing in the early diagnosis of the disease, and in the clinical management of these patients and their relatives. Fifteen heart transplanted patients and 5 patients bridged for heart transplantation due to familial dilated, hypertrophic or restrictive cardiomyopathy were genotyped with next generation sequencing, using Illumina's Trusight Cardio sequencing panel, covering 174 clinically relevant genes to cardiac diseases. Detected variants were characterized according to the recommendations of the American College of Medical Genetics and Genomics. Fifteen patients (75%) were found positive for rare single mutations after genotyping. Nine of these mutations (45% of patients) in PLN, LAMP2, RBM20, TNNI3, LMNA and TTN genes were characterized as pathogenic or likely pathogenic and 7 of them were previously described in the literature. A new likely pathogenic mutation in LMNA gene (p.Arg50Cys) and a new frameshift mutation in TTN gene characterized as pathogenic (p.Ile29015Thr*15), were identified in two families with DCM. The PLN gene mutation (p.Leu39*) was detected in 3 of the 20 patients (15%) indicating that it could be a founder mutation in the Greek population. Six patients (30%) carried variants of unknown significance (vus), while 5 patients (25%) didn't harbor any mutation. One of the vus in the TPM1 gene is of great interest since it was detected in three heart transplanted members of a DCM family. A significant proportion of rare variants in genes associated with cardiomyopathies has been identified in a cohort of patients undergoing heart transplantation. Detection of these variants may be critical in early diagnosis and management of the patient and his family and might prevent sudden cardiac death or alter heart transplantation or VAD implantation clinical decisions.

Whole exome sequencing in a large pedigree with DCM identifies a novel mutation in RBM20

Background: Familial dilated cardiomyopathy (DCM) is genetically heterogeneous and is associated with mutations in at least 40 different genes. Apart from TTN encoding the giant protein Titin, none of these genes have an expected diagnostic yield of more than 5% complicating genetic diagnosis. Whole exome sequencing (WES) is a powerful alternative for the identification of the causal gene, however variant interpretation remains challenging. We report on WES in a large family with autosomal dominant DCM complicated by end stage heart failure and non-sustained ventricular arrhythmias in whom no causative mutation was identified using a targeted gene panel including 28 genes.Methods and results: WES was applied on 2 affected cousins. Stringent filtering of the identified genetic variants was performed including population variant frequencies, in silico analysis, orthologous and paralogous conservation. Subsequently Sanger sequencing was performed for 10 potential disease causing variants in order to confirm the presence of the variant and to evaluate co-segregation. Only one variant in exon 9 of the RBM20 gene (c.2714T > A, p.Met950Lys, NM_001334363) showed full co-segregation in the 7 affected family members resulting in a maximum 2-point LOD score of 2.1 and suggesting this as the pathogenic mutation responsible for the phenotype. Recently mutations in RBM20 have been linked to arrhythmogenic dilated cardiomyopathy caused by defective splicing of the giant sarcomere protein titin and abnormal calcium handling.Conclusions: We report the identification of a novel mutation in RBM20 by WES in a large pedigree with DCM.

Megaesophagus Is a Major Pathological Condition in Rats With a Large Deletion in the Rbm20 Gene.

A spontaneously arising, loss-of-function mutation in the RNA binding motif protein 20 (Rbm20) gene, which encodes a nuclear splicing protein, was previously identified as the underlying reason for expression of an abnormally large TITIN (TTN) protein in a rat model of cardiomyopathy. An outbreak of Pseudomonas aeruginosa led to submission of rats with dyspnea, sneezing, lethargy, nasal discharge, and/or unexpected death for diagnostic evaluation. Necropsy revealed underlying megaesophagus in Rbm20–/– rats. Further phenotyping of this rat strain and determination of the size of esophageal TTN was undertaken. The Rbm20-defective rats developed megaesophagus at an early age (26 weeks) with high frequency (13/32, 41%). They also often exhibited secondary rhinitis (9/32, 28%), aspiration pneumonia (8/32, 25%), and otitis media/interna (6/32, 19%). In addition, these rats had a high prevalence of hydronephrosis (13/32, 41%). RBM20 is involved in splicing multiple RNA transcripts, one of which is the muscle-specific protein TTN. Rbm20 mutations are a significant cause of dilated cardiomyopathy in humans. In Rbm20-defective rats, TTN size was significantly increased in the skeletal muscle of the esophagus. Megaesophagus in this rat strain (maintained on a mixed genetic background) is hypothesized to result from altered TTN stretch signaling in esophageal skeletal muscle. This study describes a novel mechanism for the development of megaesophagus, which may be useful for understanding the pathogenesis of megaesophagus in humans and offers insights into potential myogenic causes of this condition. This is the first report of megaesophagus and other noncardiac pathogenic changes associated with mutation of Rbm20 in any species.

Clinical and genetic insights into non-compaction: a meta-analysis and systematic review on 7598 individuals.

Left ventricular non-compaction has been increasingly diagnosed in recent years. However, it is still debated whether non-compaction is a pathological condition or a physiological trait. In this meta-analysis and systematic review, we compare studies, which investigated these two different perspectives. Furthermore, we provide a comprehensive overview on the clinical outcome as well as genetic background of left ventricular non-compaction cardiomyopathy in adult patients. We retrieved PubMed/Medline literatures in English language from 2000 to 19/09/2018 on clinical outcome and genotype of patients with non-compaction. We summarized and extensively reviewed all studies that passed selection criteria and performed a meta-analysis on key phenotypic parameters. Altogether, 35 studies with 2271 non-compaction patients were included in our meta-analysis. The mean age at diagnosis was the mid of their fifth decade. Two-thirds of patients were male. Congenital heart diseases including atrial or ventricular septum defect or Ebstein anomaly were reported in 7% of patients. Twenty-four percent presented with family history of cardiomyopathy. The mean frequency of neuromuscular diseases was 5%. Heart rhythm abnormalities were reported frequently: conduction disease in 26%, supraventricular tachycardia in 17%, and sustained or non-sustained ventricular tachycardia in 18% of patients. Three important outcome measures were reported including systemic thromboembolic events with a mean frequency of 9%, heart transplantation with 4%, and adequate ICD therapy with 15%. Nine studies investigated the genetics of non-compaction cardiomyopathy. The most frequently mutated gene was TTN with a pooled frequency of 11%. The average frequency of MYH7 mutations was 9%, for MYBPC3 mutations 5%, and for CASQ2 and LDB3 3% each. TPM1, MIB1, ACTC1, and LMNA mutations had an average frequency of 2% each. Mutations in PLN, HCN4, TAZ, DTNA, TNNT2, and RBM20 were reported with a frequency of 1% each. We also summarized the results of eight studies investigating the non-compaction in altogether 5327 athletes, pregnant women, patients with sickle cell disease, as well as individuals from population-based cohorts, in which the presence of left ventricular hypertrabeculation ranged from 1.3 to 37%. The summarized data indicate that non-compaction may lead to unfavorable outcome in different cardiomyopathy entities. The presence of key features in a multimodal diagnostic approach could distinguish between benign morphological trait and manifest cardiomyopathy.