Functional Validation of a Pathogenic Missense Variant in Human Filamin C Cardiomyopathy through Disruption of a Zebrafish Homologue Recapitulates Cardiac Disease

Purpose We recently performed whole-genome sequencing (WGS) of an East Asian proband and family members with diverse cardiac diseases, including arrhythmias, cardiomyopathies, premature heart failure requiring transplant, and identified a pathogenic missense variant (p.G2151S) in exon 39 of the filamin C gene, FLNC. In this study, we aim to demonstrate recapitulation of the pathogenic effects of the variant using a zebrafish (Danio rerio) model. Methods The proband, 3 other affected and 3 unaffected family members of a four-generation nonconsanguineous Hong Kong Chinese pedigree of 25 individuals were sequenced by WGS with 30X coverage. Single nucleotide variants and insertion-deletions that met our inclusion criteria were shortlisted, and bioinformatic analysis and variant prioritization were performed using Kaviar, gnomAD and dbNSFP databases to identify damaging variants in known cardiac-specific genes. The study was approved by the university/hospital's institutional review board. Sequence alignment of the pathogenic gene region with the zebrafish homologue was performed using the GENESTREAM network server, Institut de Genetique Humaine, Montpellier, France. Knockdown of the region was carried out using microinjection of morpholino oligonucleotides (MO), or control oligomers. Embryonic lethality, survival after birth, animal and cardiac morphology, heart rate, and histopathology were analyzed. Results Using a zebrafish model, we performed sequence alignment and identified the corresponding point mutation in flnca and flncb. Knockdown of the homologous sequences using antisense morpholino oligonucleotides resulted in stunted growth and development of the animal, cardiomegaly, pericardial edema, dose-dependent increase in embryonic lethality (survival rates of 95.8%, 46.6% and 9.7% at day 3 post-fertilization for control oligomer (n=24), 0.1 ng MO (n=88) and 0.5 ng MO (n=154), respectively), and reduced heart rate (134, 108 and 103 beats per minute, respectively; P Conclusion Damaging effects of the missense variant in human filamin C cardiomyopathy have been confirmed in the homologous region in this zebrafish model. The variable phenotypic manifestations within this East Asian family remain to be elucidated.