Structural, biophysical and cellular assessment of filamin C M82K: A test case for VUS interpretation in cardiomyopathy.

Filamin C (FLNC) is a key Z-disc protein that anchors actin filaments to membrane-bound complexes and is essential for sarcomeric stability and mechanotransduction. Genetic variants in FLNC are implicated in cardiomyopathy, a hereditary disease affecting heart muscle function. However, the structural and functional consequences of FLNC missense variants remain poorly defined, particularly those classified as variants of uncertain significance (VUS). Here, we used an integrated approach to investigate the FLNC missense variant M82K, identified in two unrelated patients presenting with dilated cardiomyopathy and associated clinical symptoms. Using structural modelling of the FLNC actin-binding domain (ABD), we predicted that the M82K variant may destabilise the ABD. Biophysical analyses of the E. coli-expressed FLNC-ABD-M82K variant displayed a ~ 10 °C decrease in melting temperature, accelerated thermolysin-mediated proteolysis, decreased solubility, and reduced actin-binding affinity. Further analysis using size exclusion chromatography coupled with and without small-angle X-ray scattering (SEC and SEC-SAXS), native PAGE, and mass photometry, all demonstrated a pronounced shift of mutant protein toward high-molecular-weight aggregates, with thermal SAXS confirming aggregation across elevated temperatures. Cellular analysis of GFP-tagged full-length FLNC-M82K showed reduced protein stability using cycloheximide. In neonatal rat cardiomyocytes there was no clear evidence of aggregation relative to wild-type in short-term transient transfections. Together, these findings support a pathogenic classification of FLNC-M82K and provide mechanistic insight into how FLNC destabilisation could lead to cardiomyopathy. More broadly, this multi-pronged strategy provides a framework for interpreting VUSs in cardiomyopathy-associated genes.