Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems. The LMNA gene generates two major transcripts encoding the nuclear lamina major components
lamin A and
lamin C by alternative splicing. Both haploinsuffiency and dominant negative effects have been proposed as disease mechanism for
premature termination codon (PTC) mutations in LMNA. These mechanisms however are still not clearly established. In this study, we used a representative LMNA
nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative
reverse transcriptase PCR and sequencing of these PCR products indicated that transcripts from the mutant allele were degraded by the nonsense-mediated mRNA decay (NMD) mechanism. The fact that no truncated
mutant protein was detectable in western blot (WB) analysis strengthens the notion that the mutant transcript is efficiently degraded. Furthermore, WB analysis showed that the expression of
lamin C protein was reduced by the expected approximately 50%. Clearly decreased
lamin A and
lamin C levels were also observed by immunofluorescence microscopy analysis. However, results from both WB and nano-liquid chromatography/mass spectrometry demonstrated that the levels of
lamin A protein were more reduced suggesting an effect on expression of
lamin A from the wild type allele. PCR analysis of the ratio of
lamin A to
lamin C transcripts showed unchanged relative amounts of
lamin A transcript suggesting that the effect on the wild type allele was operative at the
protein level. Immunofluorescence microscopy analysis showed no abnormal nuclear morphology of patient fibroblast cells. Based on these data, we propose that heterozygosity for the
nonsense mutation causes NMD degradation of the mutant transcripts blocking expression of the truncated
mutant protein and an additional trans effect on
lamin A protein levels expressed from the wild type allele. We discuss the possibility that skewing of the
lamin A to
lamin C ratio may contribute to ensuing processes that destabilize cardiomyocytes and trigger
cardiomyopathy.