摘要目的 识别先天性室间隔缺损(VSD)患者新的分子遗传缺陷.方法 收集2006年3月至2008年6月在上海儿童医学中心、上海同济医院和上海市儿童医院160例先天性VSD患者的临床资料和血标本,以200名健康者为对照.应用聚合酶链反应扩增NKX2-5基因的全部外显子,采用双脱氧核苷链末端合成终止法对全部扩增片段进行测序.借助BLAST程序将所测序列与GenBank中的已知序列进行比对以识别基因突变,并用Clustal W软件分析突变氨基酸的保守性.结果 在3例VSD患者的NKX2-5基因识别出2个新的杂合错义突变,即2例VSD患者的第179位密码子TCC变为TTC,导致第179位的丝氨酸变为苯丙氨酸,1例VSD患者的第36位密码子CGC变为AGC,导致第36位的精氨酸变为丝氨酸.200名健康对照者均无此突变,多物种NKX2-5序列比对显示突变氨基酸在进化上均高度保守.此外,还发现了2个不改变氨基酸的单核甘酸多态,即常见的c.63A>G多态和少见的c.606G>C多态,但这些多态在VSD患者和健康对照者间的分布差异均无统计学意义(c.63A>G多态:X2=3.403、P=0.0651,c.606G>C多态:X2=3.278、P=0.0702).结论 在先天性VSD患者识别出新的NKX2-5基因突变,揭示了VSD新的分子病因.

abstractsObjective 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: X2=3.403, P=0.0651; c. 606G>C: X2=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.