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目的 了解先天性巨结肠与巨结肠同源病RET基因突变及其突变的差异。方法 30例散发性先天性巨结肠 ,14例巨结肠同源病 ,10例正常对照。取外周血 ,提取DNA ,聚合酶链反应(PCR)扩增RET基因第 13外显子 ;单链构象多态 (SSCP)分析外显子突变 ,并通过测序明确突变的位点和类型。结果 30例散发性先天性巨结肠RET基因 13外显子检测 5例存在基因突变 ,共发现三种突变类型 :有 3例在碱基 18888位点 ,胸苷酸残基T被鸟苷酸残基G置换 ,导致亮氨酸的静默突变 ;有 1例在碱基 18919位点 ,腺苷酸残基A被鸟苷酸残基G置换 ,导致赖氨酸突变为谷氨酸 ,为错义突变 ;有 1例在碱基 18974位点 ,插入一个鸟苷酸残基G ,导致框架移位突变。RET基因 13外显子突变率为 16 .7% (5 /30 )。巨结肠同源病和正常对照组未见RET基因 13外显子突变。结论 先天性巨结肠与RET基因突变有关 ,而巨结肠同源病未发现RET基因突变 ,提示这两种疾病具有分子遗传学差异。
Objective To understand the mutations of RET gene and its mutations in homology of Hirschsprung’s disease and megacolon. Methods Thirty cases of sporadic Hirschsprung’s disease, 14 cases of Hirschsprung’s disease, and 10 cases of normal control. The exon 13 of RET gene was amplified by polymerase chain reaction (PCR) and peripheral blood was extracted. The single nucleotide conformation polymorphism (SSCP) was used to analyze exon mutations. The site and type of mutations were identified by sequencing. Results There were five mutations in exon 13 of RET gene in sporadic Hirschsprung ’s disease. There were three types of mutation in the RET gene exon 13: 3 in 18888 bases, Base G substitution, resulting in a silent mutation of leucine; 1 case at base 18919, adenylate residue A was replaced by guanylic acid residue G, resulting in the mutation of lysine to glutamic acid, missense 1 case was inserted at base 18974, a guanylic acid residue G was inserted, leading to frame shift mutation. The exon mutation rate of RET gene was 16.7% (5/30). There was no mutation of exon 13 of RET gene in megakaryocyte homologous disease group and normal control group. Conclusions Hirschsprung ’s disease is related to the mutation of RET gene, whereas RET gene mutation is not found in Homologus homomorphosis, suggesting that these two diseases have molecular genetic differences.