RT-PCR-DGGE检测急性白血病儿童胸苷酸合成酶基因多态性研究

Polymorphisms of thymidylate synthase gene detected by RT-PCR-denaturing gradient gel electrophoresis in children with acute leukemia

目的胸苷酸合成酶(TS)是DNA合成的关键酶,在DNA合成与修复中起重要作用,是叶酸代谢循环中起中心作用的酶类之一,也是以5-氟尿嘧啶(5-FU)为基础化疗的靶酶,TS的遗传多态性在抗白血病等肿瘤疾病5-FU类药物的体内代谢中起着重要作用。该文研究胸苷酸合成酶基因编码区单核苷酸多态性(cSNP)及基因突变在急性白血病(AL)患儿和正常儿童中的分布情况,以探讨TS基因变异与AL患儿5-FU类药物化疗效应之间可能的相关性。方法应用RT-PCR-变性梯度凝胶电泳(RT-PCR-DGGE)结合DNA直接测序技术,对53例AL患儿和115例正常儿童的cDNAs进行了TS基因cSNP及基因突变的筛查与鉴定,分析各基因型在两组之间的分布差异。结果首次鉴定了儿童TS 381 A>G(E127E)cSNP位点,其在AL患儿及正常儿童中的等位基因频率分别为12.3%,13.5%,与国际SNP文库中12.3%基本一致。该cSNP在两组人群的等位基因频率差异无显著性意义,与白血病的易感性无相关性。研究未发现T349C,G470T及C500T变异。结论采用RT-PCR-DGGE结合DNA测序法首次确定了儿童TS基因存在381 A>G cSNP位点,其等位基因频率为13.1%,基因型分布频率在AL患儿及正常儿童两组人群中差异无显著性。

Objective Thymidylate synthase （TS） catalyses the conversion of deoxy-uridylate to deoxy-thymidylate and is a key enzyme for DNA synthesis. TS is the target enzyme of 5-fluorouracil （5-FU） and involved in folate metabolism. TS gene polymorphisms play an important role in the efficiency of fluorouracil activity in vivo. This study investigated the allelic frequencies and distribution characters of single-nueleotide polymorphisms within the coding region （cSNPs） of TS gene in Chinese children with acute leukemia （AL） and normal control children in order to explore the possible relationship between the cSNP in human TS gene and chemotherapeutic effects of 5-fluorouracils. Methods Bone marrow samples from 53 children with AL and peripheral blood samples from 115 normal children were obtained to prepare complementary DNAs （cDNAs）. The cDNAs were analyzed for the polymorphisms in TS gene by reverse transcriptase （RT）-polymerase chain reaction （PCR）-denaturing gradient gel electrophoresis （DGGE） and direct sequencing. The distributive difference of each genotype between AL children and control children was evaluated. Results A polymorphism 381 A 〉 G （E127E） in the coding region of TS gene was firstly identified in the Chinese population. The 381 A 〉 G allelic frequency in AL children and control children was 12.3% and 13.5% respectively （P 〉0.05） ,which were similar to that in the International SNP Bank （ 12.3% ）. The allelic frequency of cSNPs was not associated with the susceptibility to AL. Conclusions A polymorphism 381 A 〉 G （E127E） in TS gene was successfully identified in children using RT-PCR- DGGE combined with DNA sequencing. There was no significant difference in the allelic frequency of cSNPs in AL children and normal children.