Objective: To identify the novel genetic defects in patients with congenital ventricular septal defect (VSD).
Methods: The clinical data and blood samples from 160 unrelated subjects with congenital VSD were collected and evaluated in comparison with 200 healthy individuals. The coding exons and flanking introns of NKX2-5 gene were amplified by polymerase chain reaction and sequenced using the di-deoxynucleotide chain termination approach. The acquired sequences were aligned with those publicized in GenBank by the aid of program BLAST to identify the sequence variations. The software Clustal W was applied to analyzing the conservation of altered amino acids.
Results: Two novel heterozygous missense NKX2-5 mutations were identified in 3 of 160 VSD patients. The same triplet substitution of TTC for TCC at codon 179, predicting the conversion of serine into phenylalanine at amino acid residue 179 (Ser179Phe), was detected in two cases. Another transition of CGC into AGC at codon 36, leading to the change of arginine into serine at amino acid residue 36 (Arg36Ser), was detected in a third case. These two mutations were not observed in 200 healthy controls. A cross-species alignment of NKX2-5 encoded protein sequences displayed that the mutated amino acids were highly conserved evolutionarily. Additionally, two single nucleotide polymorphisms including a common c. 63A > G and a rare c. 606G > C were observed. However, the polymorphic frequency distributions in VSD cases were not statistically different from those in healthy controls (c. 63A > G: chi(2) = 3.403, P = 0.0651; c. 606G > C: chi(2) = 3.278, P = 0.0702).
Conclusion: Novel NKX2-5 mutations are identified in patients with ASD. They may provide new insight into the molecular etiology responsible for VSD.