Recent reports have implicated mutations in the transcription factor NKX2.5 as a cause of tetralogy of Fallot (TOF). To estimate the frequency of NKX2.5 mutations in TOF patients and to further investigate the genotype-phenotype correlation of NKX2.5 mutations, we genotyped 114 TOF patients. Patients were recruited prospectively (November 1992 through June 1999) and tested for a 22q11 deletion; those with 22q11 deletion or recognized chromosomal alteration were excluded from the present study. Patients were screened for NKX2.5 alterations by conformation-sensitive gel electrophoresis and sequencing of fragments with aberrant mobility. Four heterozygous mutations were identified in 6 unrelated patients with cases of TOF, including 3 with pulmonary atresia and 5 with right aortic arch; none had ECG evidence of PR interval prolongation. Three of 4 mutations (Glu21Gln, Arg216Cys, and Ala219Val) altered highly conserved amino acids, of which 2 mapped in the conserved NK2 domain. The fourth mutation (Arg25Cys) was identified in 3 unrelated probands in the present study and has been previously reported. No homeodomain mutations were identified. NKX2.5 mutations are the first gene defects identified in nonsyndromic TOF patients. NKX2.5 mutation is present in >/=4% of TOF patients. Mutations identified in the present study mapped outside of the homeodomain, were not associated with atrioventricular conduction disturbances, and were not fully penetrant, in contrast to mutations previously reported that impair homeodomain function.
