摘要目的 本研究旨在识别先天性房间隔缺损(atrial septal defect,ASD)患者的分子遗传缺陷,为其早期防治奠定基础.方法 收集180例特发性ASD患者(其中12例有阳性ASD家族史)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基因的全部外显子和外显子两侧的部分内含子,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ClustalW软件分析突变氨基酸的保守性.结果 在1例家族史阳性的ASD患者的NKX2-5基因识别出一个新的杂合突变,即编码核苷酸序列第536位的胞嘧啶变为胸腺嘧啶,导致第179位的丝氨酸变为苯丙氨酸.该突变也存在于这个家系中的另外3位患病成员,但不存在于这一家系中的健康成员和200名正常对照者,多物种NKX2-5基因编码氨基酸序列比对显示突变氨基酸在进化上高度保守.此外,还发现了一个常见的单核苷酸多态,即编码核苷酸序列第63位的腺嘌呤变为鸟嘌呤,但这种多态在ASD患者和健康对照者间的频率分布差异无统计学意义(χ~2=2.8641,P=0.0906).结论 NKX2-5基因突变能够导致家族性ASD.

abstractsObjective The purpose of this investigation was to identify the novel genetic mutations in patients with a congenital atrial septal defect (ASD). Methods Clinical data and blood specimens from a total of 12 unrelated ASD pedigrees and a cohort of 168 unrelated subjects with sporadic ASD were collected and evaluated in contrast to 200 healthy individuals. The whole exons and partial 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 programme BLAST to identify the sequence variations. The software ClustalW was applied for analysis of the conservative of the altered amino acids. Results A novel heterozygous mutation of NKX2-5 gene, i.e., a substitution of thymine for cytosine at nucleotide 536, predicting the conversion of serine into phenylalanine at amino acid residue 179, was identified initially in a proband. The same missense mutation was thereafter detected in other 3 affected members of the identical family but neither in the healthy members of the kindred nor in the 200 normal controls. A cross-species alignment of the protein sequences encoded by NKX2-5 gene displayed that the mutated amino acid was highly conserved evolutionarily. No mutation of NKX2-5 gene was observed in the other 11 ASD pedigrees or in 168 patients with sporadic ASD. Additionally, a common synonymous single nucleotide polymorphism, a transition of adenine (A) into guanine (G) at nucleotide 63, was found in NKX2-5 gene. However, there were no significant differences in the prevalence of alleles A and G between ASD patients and healthy controls (χ~2=2.8641, P=0.0906). Conclusion A novel mutation of NKX2-5 gent identified in an ASD family suggests that mutated NKX2-5 gene is responsible for familial ASD.