摘要目的 识别先天性心脏病(CHD)患者新的分子病因.方法 收集120例特发性CHD患者的临床资料和血标本,以100名健康者为对照.应用聚合酶链反应扩增GATA4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 3例CHD患者的GATA4基因各识别出1个新的杂合错义突变,即第90、95和329位的密码子分别由AGC、GAC和AAG变为AGA、GAG和AAT,导致第90、95和329位的氨基酸分别由丝氨酸、天冬氨酸和赖氨酸变为精氨酸、谷氨酸和天冬酰胺,即S90R、D95E和K329N突变.这些突变在正常对照者中均不存在,多物种GATA4序列比对显示第329位的赖氨酸在进化上高度保守.此外,还发现了1个不改变氨基酸的单核甘酸多态,即c.99 G>T多态,但这些多态在CHD患者和健康对照者间的频率分布差异无统计学意义(P>0.05).结论 特发性CHD具有显著的遗传异质性,GATA4基因突变可能是部分特发性CHD患者的分子病因.

abstractsObjective To identify the novel genetic determinants in patients with congenital heart disease(CHD).Methods The clinical data and peripheral venous blood samples from 120 unrelated individuals with idiopathic CHD were collected and evaluated compared to 100 unrelated healthy controls.The complete coding exons and the partial flanking introns of GATA4 gene were amplified by polymerase chain reaction and sequenced by di-deoxynucleotide chain termination.The generated sequences were aligned with those retrieyed from GenBank with the aid of programme BLAST to identify the sequence variations.The software Clustal W was utilized to analyze the conservation of altered amino acids.Results Three novel heterozygous missense CATA4 mutations were identified in 3 of 120 CHD cases. Namely,the triplet substitutions of AGA for AGC at codon 90,GAG for GAC at codon 95,and AAT for AAG at codon 329,predicting the conversions of serine into arginine at amino acid residue 90(S90R),aspartic acid into glutamic acid at amino acid residue 95(D95E)and lysine into agparagine at amino acid residue 329 (K329N), were detected.None of these three mutations were probed in 100 controls.A cross-species alignment of GATA4 encoded protein sequences displayed that the lysine at amino acid residue 329 was completely conserved evolutionarily.Additionally.a single nucleotide polymorphism c.99G>T was observed.However.the polymorphic frequency distribution in CHD patients was not statistically different from that in controls (for genotypes,χ~2=0.2640,P=0.6074;for alleles,χ~2=0.2514,P=0.6161). Conclusion The idiopathic CHD has a marked heterogeneity and the mutated GATA4.A4 gene may be responsible for CHD in a subset of patients.