Novel Mutation in FLNC (Filamin C) Causes Familial Restrictive Cardiomyopathy

Abstract

Background: Restrictive cardiomyopathy (RCM) is a rare cardiomyopathy characterized by impaired diastolic ventricular function resulting in a poor clinical prognosis. Rarely, heritable forms of RCM have been reported, and mutations underlying RCM have been identified in genes that govern the contractile function of the cardiomyocytes.

Methods and results: We evaluated 8 family members across 4 generations by history, physical examination, electrocardiography, and echocardiography. Affected individuals presented with a pleitropic syndrome of progressive RCM, atrioventricular septal defects, and a high prevalence of atrial fibrillation. Exome sequencing of 5 affected members identified a single novel missense variant in a highly conserved residue of FLNC (filamin C; p.V2297M). FLNC encodes filamin C-a protein that acts as both a scaffold for the assembly and organization of the central contractile unit of striated muscle and also as a mechanosensitive signaling molecule during cell migration and shear stress. Immunohistochemical analysis of FLNC localization in cardiac tissue from an affected family member revealed a diminished localization at the z disk, whereas traditional localization at the intercalated disk was preserved. Stem cell-derived cardiomyocytes mutated to carry the effect allele had diminished contractile activity when compared with controls.

Conclusion: We have identified a novel variant in FLNC as pathogenic variant for familial RCM-a finding that further expands on the genetic basis of this rare and morbid cardiomyopathy.

Keywords: atrial fibrillation; cardiomyopathy, restrictive; mutation; stem cells; ventricular function.

Figure 1. Transmission of the FLNC p.V2297M variant with cardiac phenotypes

A: Pedigree detailing the spectrum of myocardial phenotypes observed in the family. +/− denotes carrier versus wildtype genotype at amino acid 2297. B: Chromatogram obtained from Sanger sequencing reaction of amplicon obtained from the genomic DNA of II-5. C: Amino acid sequence alignment of FLNC in vertebrate species. Red denotes the p.V2297M variant observed. D: Depiction of FLNC functional domains overlain with known variants and associated phenotypes.

Figure 2. Clinical and pathological findings for patient II-5

A. Electrocardiogram is notable for coarse atrial fibrillation, ventricular, hypertrophy and incomplete right bundle branch block pattern. B. Echocardiogram notable for massively enlarged atria with left atrial dimensions of 46×51×82mm. Histologically the right atrial myocardium demonstrated myocyte hypertrophy with enlarged nuclei on hematoxylin and eosin stain (C) and extensive fibrosis (blue) on trichrome stain (D). On Congo red stains, there was no amyloid in the left ventricle (E) but focal amyloid in the right atrium (F) indicating isolated atrial amyloid. By transmission electron microscopy of the left ventricle myocardium (G and enlargements in panels a, and b) there was no evidence of a storage disorder or appreciable alteration in sarcomeric architecture.

Figure 3. Cellular consequences of FLNC p.V2297M variant

A: Localization of FLNC (green) and α-MHC (red) in ventricular free wall tissue obtained from a control sample and in the failing heart of II-5. Images were obtained from independent tissue sections B: Quantification of fluorescence intensity for intercalated disk resident FLNC, sarcomeric FLNC, and sarcomeric α-actin. Images from all samples were obtained with equivalent acquisition conditions. N represents number of independent intercalated disks or myofibrils examined. C: Left panel: Comparison of fractional shortening measured from singularized stem cell derived cardiomyocytes. N represents the combined number of cells analyzed in three independent experiments. Error bars represent the SEM. Right panel: Representative images of isolated cardiomyocytes. Red line represents the major axis for fractional shortening measurement. Scale bar represents 20μm. Lower images represent line scans of the major axes for wild-type and mutant cardiomyocytes displayed over time.