严重不耐受巯嘌呤的急性淋巴细胞白血病患儿TPMT基因序列分析

Thiopurine S-Methyltransferase Gene Sequence Analysis of ALL Children Severely Intolerant to 6-Mercaptopurine

本研究分析严重不耐受巯嘌呤(6-MP)的急性淋巴细胞白血病(ALL)患儿巯嘌呤甲基转移酶(TPMT)基因序列,以探讨ALL儿童6-MP耐受性差异的原因,为更安全合理地应用6-MP提供依据。对2004-10-1-2007-9-30在我院规范应用BCH-2003-ALL化疗方案的ALL儿童,按NCI-CTC V2.0评价药物不良反应。分析处于6-MP维持化疗期,出现3-4度不良反应(即严重不耐受)ALL患儿的TPMT基因序列。为保证测序的准确性,将TPMT基因(NM_000367)编码区的738 bp片段分3段分别进行双向测序。结果表明:在调查的133例ALL患儿中,61例为严重不耐受6-MP,剔除其中2例无标本患儿,对59例进行了TPMT基因测序。其中单纯骨髓不良反应37例,肝脏和骨髓不良反应9例,单纯肝脏不良反应12例,皮肤不良反应1例。59例中男39例,女20例,中位年龄67个月(17-183个月)。在59例患儿中,57例发现C474T变异,变异率为96.6%,其中杂合突变21例,纯合突变36例。又对10例6-MP耐受性好的ALL患儿进行TPMT基因测序。结果显示,8例也出现C474T变异,均为纯合突变,变异率为80%,说明TPMT基因C474T变异与6-MP严重不耐受无关。结论:TPMT基因(NM_000367)编码区的738 bp片段中C474T变异率极高,但与6-MP耐受性无关,提示ALL患儿6-MP的严重不耐受可能与TPMT编码区基因的变异无关。

This study was aimed to analyze the thiopurine S-methyltransferase （TPMT） gene sequence in acute lyrnphoblastic leukemia （ALL） children severely intolerant to 6-mercaptopurine （6-MP） and to investigate the causes resulting in tolerance difference to 6-MP in ALL children so as to provide evidence for safe and rational use of 6-MP. The adverse reactions of drug was evaluated in ALL children treated with BCH-2003-ALL chemotherapeutic protocol during 2004-10-1 to 2007-9-30 according to NCI-CTC V2.0. The TPMT gene sequences of ALL children with 3 - 4 grade of severe toxicity during the maintenance therapy were analyzed by PCR and direct DNA sequencing. To assure the accuracy of sequencing, the 738 bp fragment of coding region in TPMT gene （ NM_000367 ） was divided into 3 subfragments and bidirectionaly sequenced. The results indicated that among 133 ALL children, 61 were severely intolerant to 6-MP. The direct DNA sequencing showed that among 59 patients （excluding 2 cases without RNA samples）, the simple myelotoxicity was found in 37 cases, hepato-myelotoxicity was observed in 9 cases, hepatotoxicity along appeared in 12 cases, 1 case showed skin rash. Out of 59 ALL children, the C474T mutation was found in 57 cases, with mutation rate 96.6%, including 21 cases with heterozygous mutation and 36 cases with homozygosis mutation. The TPMT gene sequencing of 10 cases tolerant to 6-MP indicated that C474T mutation was detected in 8 cases which was homozygous mutation. It is concluded that the C474T mutation in 738 bp fragment of coding region in TPMT gene is very frequant, but it is not related with tolerance to 6-MP, suggesting that severe intolerance to 6-MP in ALL children may be not related with the mutation of coding region in TPMT gene.