Multicenter clinical and functional evidence reclassifies a recurrent noncanonical filamin C splice-altering variant

BACKGROUND Truncating variants in filamin C (FLNC) can cause ar-rhythmogenic cardiomyopathy (ACM) through haploinsufficiency. Non-canonical splice-altering variants may contribute to this phenotype. OBJECTIVE The purpose of this study was to investigate the clinical and functional consequences of a recurrent FLNC intronic variant of uncertain significance (VUS), c.970-4A>G.METHODS Clinical data in 9 variant heterozygotes from 4 kindreds were obtained from 5 tertiary health care centers. We used in silico pre-dictors and functional studies with peripheral blood and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Isolated RNA was studied by reverse transcription polymerase chain reaction. iPSC-CMs were further characterized at baseline and af-ter nonsense-mediated decay (NMD) inhibition, using quantitative po-lymerase chain reaction (qPCR), RNA-sequencing, and cellular electrophysiology. American College of Medical Genetics and Genomics (ACMG) criteria were used to adjudicate variant pathogenicity.RESULTS Variant heterozygotes displayed a spectrum of disease phenotypes, spanning from mild ventricular dysfunction with palpi- tations to severe ventricular arrhythmias requiring device shocks or progressive cardiomyopathy requiring heart transplantation. Consistent with in silico predictors, the c.970-4A>G FLNC variant activated a cryptic splice acceptor site, introducing a 3-bp insertion containing a premature termination codon. NMD inhibition upregu-lated aberrantly spliced transcripts by qPCR and RNA-sequencing. Patch clamp studies revealed irregular spontaneous action poten-tials, increased action potential duration, and increased sodium late current in proband-derived iPSC-CMs. These findings fulfilled multiple ACMG criteria for pathogenicity.CONCLUSION Clinical, in silico, and functional evidence support the prediction that the intronic c.970-4A>G VUS disrupts splicing and drives ACM, enabling reclassification from VUS to pathogenic.

Automated summary

This study investigates a recurrent FLNC intronic variant of uncertain significance and its clinical and functional consequences in arrhythmogenic cardiomyopathy. Through clinical data, in silico prediction, and functional studies, it is found that the variant disrupts splicing and drives the disease. These findings suggest reclassification of the variant from uncertain significance to pathogenic.