title: Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32610, USA. hkasahar@phys.med.ufl.edu
authors.forename: Hideko
authors.initials: H
authors.lastname: Kasahara
authors.forename: D Woodrow
authors.initials: DW
authors.lastname: Benson
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D005801:Genes, Homeobox; D005838:Genotype; D006327:Heart Block / Q000235:genetics* / Q000378:metabolism; D006330:Heart Defects, Congenital / Q000235:genetics* / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D009206:Myocardium / Q000378:metabolism; D010375:Pedigree; D010641:Phenotype; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D014157:Transcription Factors; D004247:DNA