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 Wessels; Ronald H Lekanne Dit Deprez; Diane Fatkin; Luisa Mestroni; M Benjamin Shoemaker

doi:10.1016/j.hrthm.2023.05.006

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

Heart Rhythm. 2023 Aug;20(8):1158-1166. doi: 10.1016/j.hrthm.2023.05.006. Epub 2023 May 9.

Authors

Matthew J O'Neill¹, Suet Nee Chen², Lynne Rumping³, Renee Johnson⁴, Marjon van Slegtenhorst⁵, Andrew M Glazer⁶, Tao Yang⁶, Joseph F Solus⁶, Julie Laudeman⁶, Devyn W Mitchell⁶, Loren R Vanags⁶, Brett M Kroncke⁶, Katherine Anderson⁷, Shanshan Gao², Job A J Verdonschot⁸, Han Brunner⁸, Debby Hellebrekers⁸, Matthew R G Taylor³, Dan M Roden⁹, Marja W Wessels⁵, Ronald H Lekanne Dit Deprez³, Diane Fatkin¹⁰, Luisa Mestroni², M Benjamin Shoemaker¹¹

Affiliations

¹ Vanderbilt University School of Medicine, Medical Scientist Training Program, Vanderbilt University, Nashville, Tennessee.
² Cardiovascular Institute and Adult Medical Genetics Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
³ Department of Human Genetics, Amsterdam UMC, Amsterdam, The Netherlands.
⁴ Molecular Cardiology Division, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia; School of Clinical Medicine, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW, Australia.
⁵ Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands.
⁶ Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee.
⁷ Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.
⁸ Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands.
⁹ Departments of Medicine, Pharmacology, and Biomedical Informatics, Vanderbilt University Medical Center, Nashville, Tennessee.
¹⁰ Molecular Cardiology Division, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia; School of Clinical Medicine, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW, Australia; Cardiology Department, St. Vincent's Hospital, Sydney, NSW, Australia.
¹¹ Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee. Electronic address: moore.b.shoemaker@vumc.org.

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.

Keywords: Arrhythmia; Arrhythmogenic cardiomyopathy; Functional genetics; Nonsense-mediated decay; Splicing.

Publication types

Multicenter Study
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cardiomyopathies* / genetics
Codon, Nonsense
Filamins / genetics
Humans
Mutation
Myocytes, Cardiac
RNA / genetics

Substances

Codon, Nonsense
Filamins
RNA
FLNC protein, human

Abstract

Publication types

MeSH terms

Substances

Grants and funding