Objective: To investigate thiopurine S-methyltransferase (TPMT) activity and gene promoter polymorphism to probe its significance of individual chemotherapy in acute lymphoblastic leukemia (ALL) children.
Methods: HPLC method was carried out to determine TPMT activity (n=100), which activity at newly diagnosed. At the same time determination of TPMT activity in healthy children (n=180), these children come from the health care clinic. Using online primer3 software design primers, PCR products were purified. To sequence TPMT gene of the patients with clinical events(n=30). According to the method to analysis of correlation between TPMT activity and toxicity.
Results: The average TPMT activities were (31.72±10.31) nmol·g⁻¹Hb·h⁻¹ and (30.70±9.67) nmol·g⁻¹Hb·h⁻¹ in ALL and healthy groups respectively, without gender differences of TPMT activities (P=0.45) in both groups. The TPMT activity with clinical events in newly diagnosed ALL patients (n=30) was (24.07±11.43) nmol·g⁻¹Hb·h⁻¹. There are significant differences of TPMT activities between severe bone marrow suppression [(20.96±7.24) nmol·g⁻¹Hb·h⁻¹] and ALL patients with clinical events groups (P<0.05). The TPMT activity of (40.46±8.18) nmol·g⁻¹Hb·h⁻¹ in recurrence children was also significantly different (P<0.05). TPMT activity in severe liver toxicity group was not significantly different (P=0. 930). Of TPMT gene sequencing in ALL patients with clinical events, only 3 children were heterozygosity mutations of TPMT*3C, while others homozygous genotype. There were significant differences of TPMT activities between heterozygosity genotype [(11.99±1.32) nmol·g⁻¹Hb·h⁻¹] and homozygous genotype groups [(24.95±11.32) nmol·g⁻¹Hb·h⁻¹] (P<0.05). There were five kinds of variations at the vicinity of the promoter region of -100 of tandem repeats (VNTR) polymorphism（*V3/*V3、*V3/*V4、*V4/*V4、*V5/*V5、*V4/*V6）without significant differences of TPMT activities among five kinds (P=0.186).
Conclusion: TPMT activity was related to the gene polymorphism. TPMT activity determination had prognostic value and guided individualized treatment.
目的 探讨巯基嘌呤甲基转移酶（TPMT）活性和其遗传多态性在儿童急性淋巴细胞白血病（ALL）个体化治疗中的临床意义。方法 运用高效液相色谱法（HPLC）对100例初诊ALL患儿、180名健康儿童进行TPMT活性检测。采用在线Primer3 软件设计引物,对30例具有临床事件的患儿进行TPMT基因DNA测序。分析TPMT活性、遗传多态性与临床不良反应的相关性。TPMT活性以在单位时间(1 h)内单位质量的血红蛋白(Hb,g)催化生成的2-氨基-6-甲基巯基嘌呤（6-MTG）的量(nmol)表示。结果 100例初诊ALL患儿TPMT活性为（31.72±10.31）nmol·g⁻¹Hb·h⁻¹,180名健康儿童TPMT活性为（30.70±9.67）nmol·g⁻¹Hb·h⁻¹,两者TPMT活性差异无统计学意义（P=0.450）。19例发生严重骨髓抑制的患儿TPMT活性为（20.96±7.24）nmol·g⁻¹Hb·h⁻¹,6例ALL复发者初诊时TPMT活性为（40.46±8.18）nmol·g⁻¹Hb·h⁻¹,两者分别与30例具有临床事件的ALL患儿初诊时活性[（24.07±11.43）nmol·g⁻¹Hb·h⁻¹]相比差异具有统计学意义（P值均<0.05）。5例化疗后发生严重肝脏毒性者初诊时TPMT活性为（23.60±7.48）nmol·g⁻¹Hb·h⁻¹,与30例ALL患儿初诊时活性相比差异无统计学意义（P=0.930）。对30例具有临床事件的ALL患儿TPMT基因DNA测序, 3例发生TPMT*3C杂合突变,其活性为（11.99±1.32 ）nmol·g⁻¹Hb·h⁻¹,27例呈野生纯合型,其活性为（24.95±11.32）nmol·g⁻¹Hb·h⁻¹,杂合子突变患儿TPMT活性明显低于野生纯合子型患儿,差异有统计学意义（P<0.05）。启动子-100附近区域发现5种数目变异的串联重复（VNTR）基因型（*V3/*V3、*V3/*V4、*V4/*V4、*V5/*V5、*V4/*V6）,其平均活性分别为19.35、25.06、22.61、32.16、11.85 nmol·g⁻¹Hb·h⁻¹,各组间活性差异无统计学意义（P=0.186）。结论 TPMT基因多态性与其活性有一定的相关性,TPMT活性高低对于儿童ALL预后的判断和指导个体化用药有重要的临床意义。