Risks of Ventricular Arrhythmia and Heart Failure in Carriers of RBM20 Variants

BACKGROUND: Variants in RBM20 are reported in 2% to 6% of familial cases of dilated cardiomyopathy and may be associated with fatal ventricular arrhythmia and rapid heart failure progression. We sought to determine the risk of adverse events in RBM20 variant carriers and the impact of sex on outcomes. METHODS: Consecutive probands and relatives carrying RBM20 variants were retrospectively recruited from 12 cardiomyopathy units. The primary end point was a composite of malignant ventricular arrhythmia (MVA) and end-stage heart failure (ESHF). MVA and ESHF end points were also analyzed separately and men and women compared. Left ventricular ejection fraction (LVEF) contemporary to MVA was examined. RBM20 variant carriers with left ventricular systolic dysfunction (RBM20LVSD) were compared with variant-elusive patients with idiopathic left ventricular systolic dysfunction. RESULTS: Longitudinal follow-up data were available for 143 RBM20 variant carriers (71 men; median age, 35.5 years); 7 of 143 had an MVA event at baseline. Thirty of 136 without baseline MVA (22.0%) reached the primary end point, and 16 of 136 (11.8%) had new MVA with no significant difference between men and women (log-rank P=0.07 and P=0.98, respectively). Twenty of 143 (14.0%) developed ESHF (17 men and 3 women; log-rank P<0.001). Four of 10 variant carriers with available LVEF contemporary to MVA had an LVEF >35%. At 5 years, 15 of 67 (22.4%) RBM20LVSD versus 7 of 197 (3.6%) patients with idiopathic left ventricular systolic dysfunction had reached the primary end point (log-rank P<0.001). RBM20 variant carriage conferred a 6.0-fold increase in risk of the primary end point. CONCLUSIONS: RBM20 variants are associated with a high risk of MVA and ESHF compared with idiopathic left ventricular systolic dysfunction. The risk of MVA in male and female RBM20 variant carriers is similar, but male sex is strongly associated with ESHF.

was retrospectively selected from the University College London cardiomyopathy database (iLVSD cohort) for comparison with RBM20 variant-carriers with LVSD (RBM20LVSD cohort).

Genetic Testing and Variant Classification
For the RBM20 cohort, genetic testing in probands was undertaken using predominantly nextgeneration targeted panels at participating centers or accredited diagnostic laboratories.In 2 families (3 RBM20 variant-carriers), genetic testing was conducted with gene panels in which FLNC was not included.In remaining families, genetic testing included all genes in which variants are associated with strong or definitive evidence of association with a DCM phenotype. 4Variants were classified using American College of Medical Genetics (ACMG) criteria (Supplemental Table II). 18Where additional evidence of pathogenicity (e.g.segregation data) was available from the contributing center, variants were reclassified accordingly.Only LP/P RBM20 variants were included in the analysis.RBM20 variantcarriers with additional LP/P variants in other genes associated with cardiomyopathy were excluded.Sanger sequencing was used for cascade screening of relatives.
For the iLVSD cohort, next-generation targeted sequencing using panels with a minimum of 81 genes (Supplemental Table IX) was undertaken at an accredited diagnostic laboratory.
These panels included all genes in which variants are associated with strong or definitive evidence of association with a DCM phenotype. 4

Data Collection and Study Variables
Study data were collected independently by each center and managed using REDCap (Research Electronic Data Capture) tools hosted at University College London. 17Standard data collection procedures, in accordance with general data protection regulation, were followed.Baseline demographics, comorbidities, symptoms, 12-lead electrocardiogram (ECG), transthoracic echocardiogram (TTE), ambulatory Holter ECG recordings and cardiac magnetic resonance (CMR) scan data were collected from clinical records.The baseline phenotypic data comprised the primary dataset used for most analyses.

Study Endpoints
Follow-up time was calculated from date of first evaluation to date of most recent evaluation, heart transplantation or death from any cause.The primary endpoint was a composite of malignant ventricular arrhythmia (MVA) (defined as SCD, aborted SCD, appropriate implantable cardiac defibrillator (ICD) therapy or sustained ventricular tachycardia (VT) or fibrillation (VF)) and end-stage heart failure (ESHF) (left ventricular assist device implantation (LVAD), heart transplantation or heart failure-related death). 19MVA and ESHF endpoints were also analysed separately as secondary endpoints.Patients were censored at the time of their first endpoint event during follow-up or at their last evaluation.New onset phenotype development in variant-carriers with normal baseline evaluation was defined as an LVEF < 50% on echocardiogram or CMR scan or occurrence of MVA.

Statistical Analysis
All data were anonymised and statistical analyses performed using SPSS (version 27.0) 20 and the Python programming language (Version 3.8, Python Software Foundation). 21Continuous variables were tested for normality of distribution by visual inspection of histograms and statistical normality tests (Shapiro-Wilk).Normally distributed variables are expressed as mean ± SD and non-normally distributed variables as median [25 th , 75 th percentiles].
Categorical variables are reported as counts and percentages, as appropriate.
The TableOne library was used for the construction of summary statistics tables and for statistical comparisons. 22The Seaborn and Matplotlib libraries were used for data visualization. 23The zEpid library was used to calculate incidence rates. 24e Lifelines library was used for all time-to-event analyses. 25Kaplan-Meier plots were used to display the cumulative probability of the occurrence of endpoints, stratified by sex, within the RBM20 cohort and to compare endpoints between the iLVSD and RBM20LVSD cohorts.
The log-rank test was used to compare survival.The impact of competing risk from heart transplantation on the incidence of MVA was assessed using Aalen-Johansen and Fine-Gray analyses. 26,27P-values < 0.05 were considered significant.A cumulative probability plot was used to examine age at disease penetrance.
Univariable Cox regression was used to assess the association of baseline variables with endpoints after observation that the proportional hazards assumption was supported by the data.Where variables had less than 10% of data missing, a stochastic imputation method was used to allow inclusion in multivariable models.The Sklearn library (impute.IterativeImputer) was used to perform a total of 10 imputation rounds before returning the imputations computed during the final round. 28A round was a single imputation of each feature with missing values.Forward stepwise regression using Cox proportional hazards was used to identify multivariable models associated with adverse events during follow-up and calculate hazard ratios (HR) and 95% confidence intervals (CIs).Indicator variables were created for the three most prevalent RBM20 variants (p.Arg636Ser, p.Arg636His and p.Arg634Gln) and included in the forward stepwise regression model.The robust sandwich estimator was used to obtain standard errors to deal with correlations within families.
Propensity scores for RBM20 variant carriage were estimated using plausible modifiers of phenotype (sex, age at presentation, hypertension and diabetes).These scores were included in a multivariable Cox model in order to adjust for differences in baseline characteristics between the RBM20LVSD and iLVSD groups, with bootstrapping estimation (with 1000 replications) of standard errors to allow for uncertainty in the estimated propensity scores.

Table I :
Enrolment details of the 32 probands within the RBM20 cohort.
Supplemental TableII: RBM20 variants within the study cohort and their ACMG classification with supporting criteria.Variant nomenclature according to Ensembl transcript ID no: ENST00000369519.*PP1_moderate called for three variants based on available segregation data showing 5 or more informative meioses.Abbreviations: ACMG, American College of Medical Genetics and Genomics; LP, likely pathogenic; MAF, minor allele frequency; P, pathogenic; PM, moderate evidence for pathogenicity; PP, supporting evidence for pathogenicity; PS, dysfunction; MVA, malignant ventricular arrhythmia; NYHA, New York Heart Association; RV, right ventricle; VE, ventricular ectopics.dysfunction; MVA, malignant ventricular arrhythmia; NYHA, New York Heart Association; RV, right ventricle; VE, ventricular ectopics.

Table V :
RBM20 variant carriers with LVEF available contemporary to MVA event.Table showing the details of ten RBM20 variant carriers with LVEF available within 6 months of an MVA event.Four patients had a contemporary LVEF > 35%.LVEF, left ventricular ejection fraction; MVA, malignant ventricular arrhythmia; ICD, implantable cardiac defibrillator; VT, ventricular tachycardia.