Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies. Cardiovasc Res 2004 Oct 01;64(1):40-51
Date
09/15/2004Pubmed ID
15364612DOI
10.1016/j.cardiores.2004.06.004Scopus ID
2-s2.0-4444223413 (requires institutional sign-in at Scopus site) 94 CitationsAbstract
OBJECTIVE: There has been considerable interest in understanding determinants of the diverse cardiac phenotypes associated with heterozygous NKX2.5 mutations. We hypothesized that analysis of functional properties of NKX2.5 mutant proteins would result in the ability to classify mutations according to function in a scheme that would help to clarify genotype-phenotype correlations. We analyzed missense mutations in the conserved homeodomain.
METHODS: We studied in vitro biochemical characteristics, including nuclear localization, DNA binding, transcriptional activation and protein-protein interaction with transcriptional partners (GATA4, TBX5 and NKX2.5 itself), of eight homeodomain missense mutations. Associated phenotypes include atrioventricular (AV) block (98% penetrance), atrial septal defect (83% penetrance), and additional varied heart malformations.
RESULTS: Mutations were present at varied homeodomain locations in the putative nuclear localizing signal (1), helix 2 (1), a turn between helix 2 and 3 (1) and helix 3 (5); a spectrum of biochemical phenotypes was observed. All mutants localized to the nuclei but some exhibited anomalous nuclear distribution. While all mutants exhibited markedly decreased DNA binding and reduced transcriptional activation, interaction with transcriptional partners was varied.
CONCLUSION: Each mutant protein had a unique spectrum of observed properties, but our data show that while dominant negative properties could be demonstrated in vitro, the best correlation with clinical phenotypes resulted from the markedly reduced DNA binding shared by all eight homeodomain mutations. This suggests that the principle determinant of the two most common phenotypes associated with homeodomain missense mutations is the total dose of NKX2.5 capable of binding to DNA.
Author List
Kasahara H, Benson DWMESH terms used to index this publication - Major topics in bold
Cell NucleusDNA
Electrophoretic Mobility Shift Assay
Female
Genes, Homeobox
Genotype
Heart Block
Heart Defects, Congenital
Homeobox Protein Nkx-2.5
Homeodomain Proteins
Humans
Male
Mutation, Missense
Myocardium
Pedigree
Phenotype
Protein Binding
Transcription Factors
Transcriptional Activation
