变性高效液相色谱分析巯嘌呤甲基转移酶基因多态性位点

Analysis of Thiopurine Methyltransferase Gene Polymorphism by Denaturing HPLC

目的 应用变性高效液相色谱技术（DHPLC）分析巯嘌呤甲基转移酶（TPMT）基因多态性位点.方法 用以PCR为基础的DHPLC,检测健康成人和脐血以及急性白血病病人,TPMT基因第5外显子G238C、第7外显子G460A和第10外显子A719G的3个多态性位点.结果 健康成人、脐血和急性白血病病人全部DNA样本,TPMT基因外显子区的3个位点的多态性频率为3.49%,远低于白种人和非洲人.变异的位点仅为第10外显子A719G,且均为杂合变异.TPMT第5外显子630例标本的色谱峰均为单峰.第7外显子有316例为单峰,214（34%）为杂合峰.将单峰标本进行1：1混合后重新进样也有杂合峰出现.经DNA测序分析,证实此区无序列变异;杂合峰标本在54和60℃为单峰,55～59℃均为杂合峰.而单峰标本峰形未随温度发生变化.说明DHPLC的检测误差并不是柱箱温度不合适引起的.第10外显子有608例为单峰,杂合峰为22例,其中10/22例标本进行DNA直接测序,证实为杂合变异.结论 DHPLC技术能快速筛选出TPMT第5和第10外显子区的多态性位点,但不能检测出第7外显子的多态性位点.因此,DHPLC不能作为单一方法进行TPMT基因多态性位点的检测.

OBJECTIVE To develop a denaturing high perfomance liquid chromatographic（DHPLC） assay for high throughput genotyping of thiopurine methyltransferase（ TPMT）. METHODS The TPMT genotype was determined in an unrelated population of Chinese healthy blood donors, cords blood and patients with acute leukemia. The TPMT genotyping assay was based on polymrase chin reaction（ PCR）, DHPLC and direct DNA sequencing. The genotype pulymorphism focused on the TPMT exon5 G238C, TPMT exon7 Gd60A and TPMT exon10 A719G. RESULTS The frequency of TPMT gene pulymorphism in the Han nationality of Chinese population was low. The frequency of the polymorphism was 3.49%. The varied alleles were TPMT exon10 A719G. All of them were heterozygote. According to the results of DHPLC, the screening of SNPs in TPMT exon-5 and-10 were the same as direct DNA sequencing. The variation of the heterozygotes in exon-7 was high, which was different from the results of direct DNA sequencing. After changing the Tm of DNA step by step, all the samples showed single peak when the temperature was 54 and 60℃. But this result was unbelievable because No heterozygote was found in exon 7 in positive control. CONCLUSION It was necessary to test the sensitivity and accuracy of DHPLC, though DHPLC is used as an effective method of SNPs screening. DHPLC ＇alone can not meet the need of clinics and research. The compensation of each other is very important.