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

Matthew J. O'Neill, Suet Nee Chen, Lynne Rumping, Renee Johnson, Marjon van Slegtenhorst, Andrew M. Glazer, Tao Yang, Joseph F. Solus, Julie Laudeman, Devyn W. Mitchell, Loren R. Vanags, Brett M. Kroncke, Katherine Anderson, Shanshan Gao, Job A.J. Verdonschot, Han Brunner, Debby Hellebrekers, Matthew R.G. Taylor, Dan M. Roden, Marja W. WesselsRonald H. Lekanne Dit Deprez, Diane Fatkin, Luisa Mestroni, M. Benjamin Shoemaker*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)

Abstract

Background: Truncating variants in filamin C (FLNC) can cause arrhythmogenic cardiomyopathy (ACM) through haploinsufficiency. Noncanonical 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 predictors 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 after nonsense-mediated decay (NMD) inhibition, using quantitative polymerase 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 palpitations 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 upregulated aberrantly spliced transcripts by qPCR and RNA-sequencing. Patch clamp studies revealed irregular spontaneous action potentials, 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.

Original languageEnglish
Pages (from-to)1158-1166
Number of pages9
JournalHeart Rhythm
Volume20
Issue number8
Early online date9 May 2023
DOIs
Publication statusPublished - Aug 2023

Bibliographical note

Funding Information:
This research was funded by American Heart Association (AHA) 907581 to Matthew J. O’Neill; NIH 1F30HL163923-01 to Matthew J. O’Neill; R01HL149826 to Dr Roden; NSW Health and Australian Genomics to Dr Fatkin; R01HL147064 to Drs Taylor and Mestroni; Boettcher Investigator Webb-Waring Biomedical Research Award to Dr Chen; T32 Fellowship to Dr Gao; AHA 20SCG35540034 to Dr Shoemaker; and R01HL155197 to Dr Shoemaker. Flow Cytometry experiments were performed in the VMC Flow Cytometry Shared Resource, which is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404).

Funding Information:
Dr Shoemaker has received sponsored research funding from Roche Pharmaceuticals to Vanderbilt University Medical Center . All other author have no conflicts of interest to disclose.

Publisher Copyright:
© 2023

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