Functional characterization of a novel mutation in NKX2-5 associated with congenital heart disease and adult-onset cardiomyopathy.

BACKGROUND The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown. METHODS AND RESULTS Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein. CONCLUSIONS Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity. (NHMRC grants 573732,546133 and 573705, National Heart Foundation, St Vincent’s Clinic Foundation).