Preliminary functional characterization of the LMNA c.929 A > G (p.Gln310Arg) variant identified in a patient with dilated cardiomyopathy

Variants in LMNA are established causes of inherited dilated cardiomyopathy (DCM); however, the clinical significance of many rare missense variants remains uncertain. We identified the rare LMNA c.929 A > G (p.Gln310Arg) variant in a patient with progressive DCM and performed preliminary variant-specific functional characterization in an AC16 transient overexpression model.

Duo whole-exome sequencing and copy-number variation analysis were performed in the proband and his son. Wild-type and p.Gln310Arg lamin A expression constructs were transiently transfected into AC16 cells. Flag-tagged protein expression, Flag-based transfection efficiency, and lamin A/C distribution were assessed by western blotting and immunofluorescence staining. Mitochondrial membrane potential and intracellular ROS-associated fluorescence were assessed using JC-1 and DCFH-DA staining, respectively. ERK1/2, JNK, and p38 MAPK phosphorylation was evaluated by western blotting. Hypertrophy-associated NPPA and NPPB expression was measured by RT-qPCR, and cell area was quantified after phalloidin staining.

The LMNA c.929 A > G variant was classified as a variant of uncertain significance. Wild-type and p.Gln310Arg lamin A constructs showed comparable Flag-tagged protein expression and Flag-based transfection efficiency. Expression of p.Gln310Arg lamin A was associated with an increased proportion of cells displaying ring-like lamin A/C staining patterns. Variant-expressing cells showed increased intracellular DCF fluorescence, reduced JC-1 red-to-green fluorescence ratio, and selectively increased ERK1/2 phosphorylation, whereas no significant changes were detected in JNK or p38 MAPK phosphorylation. The same cells also showed a hypertrophy-like phenotype characterized by increased NPPA and NPPB expression and enlarged cell area.

In an AC16 transient overexpression model, expression of p.Gln310Arg lamin A was associated with altered lamin A/C distribution, increased intracellular ROS-associated fluorescence, reduced mitochondrial membrane potential, selectively increased ERK1/2 phosphorylation, and a hypertrophy-like cellular phenotype. These findings provide preliminary variant-specific functional evidence but do not establish the pathogenicity of the variant or the causal relationships among the observed cellular abnormalities. Further validation in endogenous and physiologically relevant cardac models is required.