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目的研究巯嘌呤甲基转移酶(TPMT)基因型与酶活性的关系,为根据不同基因型、酶活性对巯嘌呤类药物的反应而改进治疗方案提供依据。方法应用以PCR为基础的限制性内切酶消化、变性高效液相色谱分析和SNaPshot定点的序列分析等方法,并结合DNA直接测序,对250例健康献血员、100例脐血标本和280例急性白血病患者,检测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。应用高效液相色谱分析技术,测定3组人群的红细胞内TPMT活性。结果本研究组人群TPMT基因外显子区3个位点的多态性频率均低,为3．5%。变异的位点为第10外显子A719G,且均为杂合变异。红细胞内TPMT活性波动范围为6～32 U,TPMT＞12 U者占95．1%(599例),6～12 U者占4．9%(31例)；未发现有TPMT活性缺乏者。健康献血员、脐血标本和急性白血病患者的TPMT杂合变异者,红细胞内平均TPMT活性分别为9．1 U、9．3 U和9．07 U,均分别低于其同组TPMT野生型者的17．6 U、17．67 U和18．6 U(P＜0．01)。在16例健康献血员和脐血标本中,在15例急性白血病患者的红细胞内TPMT低活性者中,分别有4例和6例未发现所检测的TPMT外显子3个SNP位点的变化,提示红细胞内TPMT活性还受其他因素的影响。结论TPMT基因多态性影响酶的活性,即TPMT杂合变异者,其TPMT活性降低,从而影响巯嘌呤类药物的治疗效应。根据不同TPMT基因型、酶活性对巯嘌呤类药物的反应制定治疗方案,可进一步提高治疗的有效性和安全性。
Objective To study the relationship between the enzyme activity and the genotype of thiopurine methyltransferase (TPMT), and to provide a basis for improving the treatment regimen according to the different genotypes and enzymatic activities of mercaptopurine. Methods PCR-based restriction endonuclease digestion, denaturing high performance liquid chromatography and SNaPshot site-directed sequencing and other methods, combined with DNA direct sequencing, 250 healthy blood donors, 100 cases of umbilical cord blood samples and 280 cases In patients with acute leukemia, three polymorphic sites of exon 5 G238C, exon 7 G460A and exon 10 A719G of TPMT gene were detected. TPMT activity in erythrocytes of three groups was determined by HPLC. Results The frequency of polymorphism in the three exons of TPMT gene in this study group was 3.5%. The mutation site was exon 10 A719G, and all were heterozygous variation. The TPMT activity in erythrocytes ranged from 6 to 32 U, 95.1% (599) with TPMT> 12 U, and 4.9% (6 cases) with 6 to 12 U; no lack of TPMT activity was found. The average TPMT activity in erythrocytes from healthy donors, umbilical cord blood samples and patients with acute leukemia was 9.1 U, 9.3 U and 9.07 U, respectively, which were lower than those of TPMT wild type 17.6 U, 17.67 U and 18.6 U (P <0.01). Of the 16 healthy blood donors and umbilical cord blood samples, among the 15 patients with acute leukemia who had low activity of TPMT in erythrocytes, there were 4 cases and 6 cases, respectively, in which no change of 3 SNPs was detected in the exons of TPMT detected , Suggesting that TPMT activity in erythrocytes is also affected by other factors. Conclusion TPMT gene polymorphism affects enzyme activity, that is, TPMT heterozygous mutation, its TPMT activity decreased, thus affecting the therapeutic effect of mercaptopurine drugs. According to different TPMT genotypes, enzyme activity of mercaptopurides in response to the development of treatment options, can further improve the effectiveness and safety of treatment.