白血病患儿和健康儿童TPMT基因多态性检测分析

Studies on the mutation and polymorphism of the TPMT gene in Chinese children with acute leukemia

目的研究硫嘌呤甲基转移酶（thiopurine S-methyltransferase，TPMT）基因编码区突变及多态性在急性白血病（acute leukemia，AL）患儿和健康儿童中的分布情况。方法应用逆转录聚合酶链变性梯度凝胶电泳结合DNA直接测序技术，对53例AL患儿和115名健康儿童的cDNAs进行了TPMT突变及多态性的筛查与鉴定，分析各基因型在两组之间的分布差异。结果在健康儿童组TPM丁中发现2个国内外未见报道的基因突变位点：210C〉T（C70C）和622T〉C（F208L）杂合子各1例，提交至GenBankdbsNP，分别获NCBI—SS序列号：107796292、107795933。鉴定了2种已知的编码区单核苷酸多态性（single—nucleotide polymorphisms within the coding region，cSNPs），474T〉C（11581）即TPMT＊1S和719A〉G（T240C）即TPMT＊3C在中国AL患儿及健康儿童中的等位基因频率，分别为14．2％，2．83％与17．0％，3．04％，等位基因总频率分别为16．2％，2．99％。两种cSNPs在两组人群的等位基因频率差异无统计学意义。结论发现了两个新的基因突变位点，提供了为预测不同个体间对硫嘌呤类药物潜在敏感性差异的遗传学标志。确定了两种已知cSNPs在中国AL患儿及健康儿童中的基因型分布和等位基因频率，发现它们与白血病的易感性无相关性。

Objective To investigate the allelic frequencies and distribution of single-nucleotide polymorphisms within the coding region （cSNPs） of thiopurine S-methyltransferase gene （TPMT） in Chinese children with acute leukemia （AL） and healthy controls, in order to provide genetic references for individual chemotherapy for AL patients by studying the relationship between the cSNP in human TPMT and chemotherapeutic effect of thiopurine drugs. Methods The bone marrow samples from 53 children with AL and peripheral blood samples from 115 healthy children were obtained to prepare complementary DNAs （cDNAs）. The cDNAs were analyzed for the polymorphisms in the TPMT gene by reverse transcriptase- polymerase chain reaction （RT PCR）-denaturing gradient gel eleetrophoresis （DGGE） and DNA sequencing. The distribution of each genotype was evaluated. Results Two novel heterozygote mutations, 210C〉T （C70C,silent） and 622T〉C （F208L）, were identified in the coding region of the TPMT in a single sample, respectively. The mother of the child with mutation 622T〉C was confirmed as the same genotype by DGGE and sequencing （NCBI_ss accession numbers 107796292 and 107795933）. Two known polymorphisms, 474T〉C （silent） and 719A〉G （T240C）, were identified. The allelic frequencies were 14.2%, 2. 83% and 17. 0%, 3.04% in the AL children and control children respectively, with the total allelic frequencies of 16.2% （first reported in the Chinese Han population） and 2. 99% respectively. No association with susceptibility to disease was observed. Conclusion Two novel mutations and two known polymorphisms were identified in Chinese children by RT-PCR-DGGE combined with DNA sequencing, which provides the first step to identify genetic markers for predicting variability in response to and toxicity of thiopurine drugs.