NUDT15基因多态性与巯嘌呤类药物所致白细胞减少的研究进展

硫唑嘌呤（azathioprine,AZA）和6-巯基嘌呤（6-mercaptopurine,6-MP）被广泛用于炎症性肠病、急性淋巴细胞白血病的治疗,但以白细胞减少为主的不良反应一直是困扰临床安全用药的难题。研究发现个体间的基因差异与该不良反应密切相关,美国食品药品监督管理局早在2005年就推荐服药前进行巯嘌呤类药物代谢相关的巯基嘌呤甲基转移酶（TPMT）的基因型检测以评估白细胞减少不良反应的发生,但该位点不能很好地预防亚洲人群中该不良反应的频发。新近研究提示NUDT15基因位点与亚洲人群的白细胞减少更密切,本文将综述NUDT15位点与亚洲人群服用AZA/6-MP后出现的白细胞减少之间的相关性研究。     

反相高效液相色谱法测定红细胞中巯嘌呤甲基转移酶的活性

目的:研究中国健康人群和急性白血病患者红细胞内巯嘌呤甲基转移酶(TPMT)活性。方法:测定TPMT活性的基础是,应用非放射性S-腺苷蛋氨酸(SAM)作为甲基供体,6-MP经TPMT甲基化为6-MeMP。用反相高效液相色谱技术(RP-HPLC),样品经乙酸苯汞加合物(PMA)提取6-MeMP。色谱柱为Dopond C18(10 μm,4.6 mm×250 mm),流动相为甲醇-水(20:80),流速1.5 mL·min～1,检测波长303nm。结果:6-巯基嘌呤(6-MP)和甲基巯嘌呤(6-MeMP)的保留时间分别为3.9 min和5.2 min,日内和日间RSD均小于2％,重复性好。结论:本方法简便、快速、准确,适用于巯嘌呤类药物细胞内药理学研究。     

急性淋巴细胞白血病患儿的rs116855232基因多态性与巯嘌呤所致不良反应相关性

摘要：目的:了解NUDT15 rs116855232基因多态性与急性淋巴细胞白血病（ALL）患儿巯嘌呤化疗后不良反应的相关性。方法:94例ALL患儿采用荧光染色原位杂交测序法检测NUDT15 rs116855232基因型,比较维持治疗阶段不同基因型患儿巯嘌呤所致不良反应发生情况。结果:维持治疗阶段,大部分患儿出现骨髓抑制、肝脏毒性和口腔黏膜/胃肠道反应等不良反应。与NUDT15 rs116855232 CC基因型ALL患儿相比,CT、TT基因型患儿发生白细胞减少、血红蛋白下降和血小板减少的比例更高（P<0.05）。而NUDT15 rs116855232不同基因型患儿发生中性粒细胞减少、肝脏毒性和口腔黏膜/胃肠道反应差异均无统计学意义（P>0.05）。结论:ALL患儿NUDT15 rs116855232基因多态性可能与巯嘌呤所致白细胞减少等骨髓抑制有关。ALL患儿可根据NUDT15 rs116855232基因检测结果调整巯嘌呤剂量,以减少骨髓抑制等不良反应的发生。     

高效液相色谱法测定急性淋巴细胞白血病患者巯嘌呤甲基转移酶活性

目的:建立一种简易的非放射性方法检测成人急性淋巴细胞白血病患者血红细胞中巯嘌呤甲基转移酶(TPMT)活性。方法:以非放射性S-腺苷-L-甲硫氨酸(SAM)为甲基供体,完成6-巯基嘌呤(6-MP)向6-甲基巯基嘌呤(6-MeMP)的甲基化转变。孵育结束时,6-MeMP经醋酸苯汞混和物抽提。色谱柱为hypersil ODS C18(250mm×4.6mm,5μm),流动相为甲醇-水(加入三乙胺并以磷酸调至pH3.2)(70∶30,v/v),流速为1mL.min-1,检测波长为290nm。结果:线性范围为20~1 280μg.L-1(r=0.999 5,n=7),高、中、低3种浓度的平均回收率为92.3%。结论:该法简便、快速、准确,适用于TPMT药动学研究和临床监测要求。     

巯嘌呤甲基转移酶基因检测用于指导6-巯基嘌呤在中国儿童急性淋巴细胞白血病中个体化治疗的循证评价

目的　系统评价6-巯基嘌呤(6-mercaptopurine, 6-MP)治疗亚洲儿童急性淋巴细胞白血病(acute lymphoblastic leukemia, ALL)出现骨髓抑制与巯嘌呤甲基转移酶(thiopurine methyltransferase, TPMT)基因多态性的相关性,并分析中国ALL患儿TPMT基因检测的经济性。方法　采用循证医学方法搜集6-MP治疗亚洲儿童ALL相关骨髓抑制与TPMT基因多态性相关的随机对照试验或观察性研究进行Meta分析。借助决策树模型针对中国ALL患儿,对两种6-MP初始给药剂量方案,进行成本效果分析。结果　最终纳入6个观察性研究,共577例亚洲患儿。Meta分析结果显示：TPMT基因多态性与骨髓毒性［OR=5.61,95%CI（2.05,15.34）,P=0.0008］发生有关。在基础数据分析中,针对中国ALL患儿,以上两个方案以严重骨髓抑制发生率为效果指标,增量成本-效果比为10403.83,敏感性分析显示结果稳定。结论　亚洲ALL患儿的TPMT基因多态性与6-MP的骨髓毒性显著相关。决策树模型结果显示,在中国,ALL患儿通过TPMT基因检测调整6-MP初始剂量并不优于标准剂量给药。     

巯基嘌呤甲基转移酶在嘌呤类药物代谢中的作用及其多态性的研究进展

对巯基嘌呤甲基转移酶在巯嘌呤类药物体内代谢过程中的作用、其活性水平与药物活性物质在体内的浓度与药效、药物的毒副作用的关系、酶活性多态性种族差异及基因遗传多态性关系进行综述. 为指导临床合理用药,避免药物不良反应及个体化用药提供参考.     

药物代谢酶基因多态性对嘌呤类药物反应的影响

硫唑嘌呤(AZA)、6-巯基嘌呤(6-MP)和6-硫鸟嘌呤(6-TG)等嘌呤类药物在临床应用已50余年,至今仍然作为免疫抑制药用于器官移植、自身免疫性疾病和慢性炎症性肠病(IBD)的治疗,或作为抗肿瘤药用于急性淋巴细胞性白血病(ALL)的化疗。该类药物治疗窗窄,严重不良反应如骨髓抑制、肝脏损害和胰腺炎等发生率较高。约1/3患者因不良反应导致治疗无效,约1/5患者中断用药。因此,如何减少不良反应发生,成为该类药物临床应     

儿童急性淋巴细胞性白血病维持治疗期间巯嘌呤的减量及对远期预后的影响

目的:探讨儿童急性淋巴细胞白血病(ALL)维持治疗中,依据外周血白细胞、中性粒细胞计数调低巯嘌呤(6-MP)用量或停药对远期预后的影响.方法:选取2011年1月至2016年1月郑州儿童医院收治的120例ALL患儿为研究对象,依据6-MP的用量将患儿分为标准剂量治疗组、半剂量及以上治疗组、半剂量以下治疗组及停药组,比较各...     

急性淋巴细胞白血病患儿亚甲基四氢叶酸还原酶基因检测在临床治疗中的意义

目的:探讨亚甲基四氢叶酸还原酶(MTHFR)基因检测在急性淋巴细胞白血病患儿应用大剂量甲氨蝶呤治疗中的临床意义。方法:收集50例急性淋巴细胞白血病患儿在应用大剂量甲氨蝶呤前的外周血,进行MTHFR基因单核苷酸多态性位点基因型检测,同时观察大剂量甲氨蝶呤应用后的毒副反应,分析各基因型与甲氨蝶呤毒副反应之间的关系。结果:具有突变基因型的患儿,在大剂量甲氨蝶呤应用后毒副反应的风险大。结论:根据MTHFR基因的检测结果,可指导临床在急性淋巴细胞白血病患儿治疗中大剂量甲氨蝶呤的应用。     

硫嘌呤甲基转移酶遗传多态性检测在6-MP个体化治疗中的意义

目的：研究硫嘌呤甲基转移酶活性和基因型检测对6-巯基嘌呤（6-MP）的个体化治疗的意义。方法-94例口服6-MP维持治疗的白血病患者，19例口服6-Mp后出现血液系统危象的为观察组，同时服用6-MP没有出现血液系统损害的75患者为对照组，采用高效液相色谱法测定硫嘌呤甲基转移酶（TPMT）活性，等位基因特异性PCR和限制性片段长度多态性（RFLP）的方法检测TPMT＊2、TPMT＊3A、TPMT＊3B和TPMT＊3C的等位基因频率。通过临床白细胞记数和骨髓象检查及临床表现判定巯嘌呤的疗效和不良反应。结果：观察组TPMT活性是（6．1±2．1）U·mL^-1 pRBCs显著低于对照组（15．3±2．3）U·mL^-1 pRBCs，而观察组的基因突变率10．7％高于对照组1．2％。结论：TPMT活性低下和／或TPMT基因突变型个体，在服用标准剂量的巯嘌呤后，发生不良反应的危险较高，应及时发现此类患者并积极调整剂量实现安全有效的治疗。     

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

Purpose

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.

Methods

Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.

Results

Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (?9.2 %; p?=?0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).

Conclusions

Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.     

Pharmacogenetic determinants of mercaptopurine disposition in children with acute lymphoblastic leukemia

BACKGROUND: The backbone of drug therapy used in acute lymphoblastic leukemia (ALL) in children includes 6-mercaptopurine (6-MP). Intracellular metabolism of this prodrug is a key component of the therapeutic response. Many metabolizing enzymes are involved in 6-MP disposition and active 6-MP metabolites are represented by 6-thioguanine nucleotides (6-TGN) and methylated metabolites primarily methylated by the thiopurine S-methyltransferase enzyme (TPMT). The genetic polymorphism affecting TPMT activity displays an important inter-subject variability in metabolites pharmacokinetics and influences the balance between 6-MP efficacy and toxicity: patients with high 6-TGN levels are at risk of myelosuppression while patients with high levels of methylated derivates are at hepatotoxic risk. However, the genetic TPMT polymorphism does not explain all 6-MP adverse events and some severe toxicities leading to life-threatening conditions remain unexplained. Additional single nucleotide polymorphisms (SNPs) in genes encoding enzymes involved in 6-MP metabolism and 6-MP transporters may also be responsible for this inter-individual 6-MP response variability. AIM: This review presents the pharmacogenetic aspects of 6-MP metabolism in great detail. We have focused on published data on ALL treatment supporting the great potential of 6-MP pharmacogenetics to improve efficacy, tolerance, and event-free survival rates in children with ALL.     

Analysis of thiopurine S-methyltransferase polymorphism in the population of Serbia and Montenegro and mercaptopurine therapy tolerance in childhood acute lymphoblastic leukemia

Thiopurine S-methyltransferase (TPMT) is an enzyme that converts thiopurine drugs into inactive metabolites. It is now well established that interindividual variation in sensitivity to thiopurines can be the result of the presence of genetic polymorphisms in the TPMT gene. The aim of this study was to determine the frequency and type of TPMT polymorphisms in the population of Serbia and Montenegro and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Blood samples from 100 healthy adults and 100 children with ALL were analyzed for common mutations in the TPMT gene using polymerase chain reaction-based assays. The results revealed that allelic frequencies were 0.2% for TPMT*2, 3.2% for TPMT*3A, and 0.5% for TPMT*3B. A rare TPMT*3B allele was detected in 2 families. No TPMT*3C allele was found. The general pattern of TPMT-variant allele distribution as well as their frequencies in the population of Serbia and Montenegro, is similar to those determined for other Slavic and Mediterranean populations. The ability to tolerate 6-mercaptopurine (6-MP) -based maintenance therapy was used as a surrogate marker of hematologic toxicity. In the study of 50 patients with childhood ALL treated according to the BFM-like protocol, it was found that even TPMT-heterozygous patients are at greater risk of thiopurine drug-related leukopenia (mean duration of period when children missed therapy as a result of leukopenia for TPMT-heterozygous patients was 11.3 weeks vs 3.4 weeks for wild-type genotype patients, P < 0.01). In another group of 50 patients, the TPMT genotype was determined prospectively. The therapy protocol was modified considering their TPMT genotype. Administering reduced 6-MP dosages in the initial phase of maintenance allowed TPMT-heterozygous patients to later receive full protocol doses of both 6-MP and nonthiopurine therapy without omitting therapy resulting from myelotoxicity. These results justify performing TPMT genotyping before initiating thiopurine therapy in all children with ALL to minimize consequent toxicity.     

Thiopurine S-methyltransferase phenotype-genotype correlation in children with acute lymphoblastic leukemia

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly as a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. The aim of this study was to determine the TPMT genotype and phenotype (activity) and investigate the correlation between TPMT genotype and enzyme activity in 43 Polish children receiving 6-MP during maintenance therapy in course of acute lymphoblastic leukemia (ALL), in 16 children with ALL at diagnosis and 39 healthy controls. TPMT activity was measured in RBC by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant allelesusing PCR-RFLP and allele-specific PCR methods. In the group of children with ALL during maintenance therapy, median TPMT activity (29.3 nmol 6-mMP g(-1) Hb h(-1)) was significantly higher compared to the group of children with ALL at diagnosis (20.6 nmol 6-mMP g(-1) Hb h(-1), p = 0.0028), as well as to the control group (22.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.0002). Percentages of individuals heterozygous for TPMT variant allele in respective groups were: 9.3, 6.2 and 15.5% (p > 0.05). In all the study groups heterozygous patients manifested a significantly lower TPMT activity as compared to the wild type homozygotes (16.7 +/- 2.1 vs. 31.2 +/- 6.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.002, in children during maintenance therapy, 11.9 +/- 2.7 vs. 24.6 +/- 9.5, p = 0.0003, in the combined group of children with ALL at diagnosis and controls). The results present that commencement of the thiopurine therapy caused an increase in the TPMT activity in RBCs by approximately 20%. All patients heterozygous for the TPMT variant allele revealed decreased TPMT activity compared to TPMT wild-type patients. Since decreased TPMT activity is associated with higher risk for toxicity after receiving standard doses of thiopurine drugs, pretreatment determination of TPMT status, with phenotypic or genetic assay, should be performed routinely, also in Poland.     

Epistatic interactions between thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) variations determine 6-mercaptopurine toxicity in Indian children with acute lymphoblastic leukemia

Purpose  

To explore the role of genetic variants of thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) in 6-mercaptopurine (6-MP)-induced toxicity in Indian children with acute lymphoblastic leukemia (ALL).     

S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6-mercaptopurine cytotoxicity in MOLT lymphoblasts

Six-mercaptopurine (6-MP) is a pro-drug widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL). Side-effects of thiopurine therapy have been correlated with thiopurine methyltransferase (TPMT) activity.We propose a novel TPMT-mediated mechanism of S-adenosylmethionine (SAM)-specific effects on 6-mercaptopurine (6-MP) induced cytotoxicity in a model cell line for acute lymphoblastic leukemia (MOLT). Our results show that exogenous SAM (10-50 μM) rescues cells from the toxic effects of 6-MP (5 μM) by delaying the onset of apoptosis. We prove that the extent of methylthioinosine monophosphate (MeTIMP) induced inhibition of de novo purine synthesis (DNPS) determines the concentrations of intracellular ATP, and consequently SAM, which acts as a positive modulator of TPMT activity. This leads to a greater conversion of 6-MP to inactive 6-methylmercaptopurine, and thus lower availability of thioinosine monophosphate for the biotransformation to cytotoxic thioguanine nucleotides (TGNs) and MeTIMP. We further show that the addition of exogenous SAM to 6-MP treated cells maintains intracellular SAM levels, TPMT activity and protein levels, all of which are diminished in cells incubated with 6-MP. Since TPMT mRNA levels remained unaltered, the effect of SAM appears to be restricted to protein stabilisation rather than an increase of TPMT expression. We thus propose that SAM reverses the extent of 6-MP cytotoxicity, by acting as a TPMT-stabilizing factor.This study provides new insights into the pharmacogenetics of thiopurine drugs. Identification of SAM as critical modulator of TPMT activity and consequently thiopurine toxicity may set novel grounds for the rationalization of thiopurine therapy.     

Effect of age on 6-mercaptopurine metabolic profile during the maintenance phase in children with acute lymphoblastic leukaemia

INTRODUCTION: 6-Mercaptopurine (6-MP) is a thiopurine analogue administered for the treatment of acute lymphoblastic leukaemia (ALL). It is an inactive pro-drug that undergoes extensive metabolism resulting in the formation of active metabolites 6-thioguanine nucleotides (6-TGN) and inactive 6-mercaptopurine methylated metabolites (6-MMP) under the genetic control of the enzyme thiopurine methyltransferase (TPMT). 6-MP metabolic profile (6-MMP/6-TGN) was proposed as a tool to     

Genetic variants of thiopurine and folate metabolic pathways determine 6-MP-mediated hematological toxicity in childhood ALL

Aim: The rationale of this study was to explore the contribution of genetic variants of the folate pathway to toxicity of 6-mercaptopurine (6-MP)-mediated hematological toxicity in children with acute lymphoblastic leukemia (ALL) and to explore the interaction of these variants with TPMT and ITPA haplotypes using multifactor dimensionality reduction analysis. Materials & methods: Children with ALL (n = 96) were screened for GCPII C1561T, RFC1 G80A, cSHMT C1420T, TYMS 5′-UTR 2R3R, TYMS 3′-UTR ins6/del6, MTHFR C677T, MTR A2756G polymorphisms using PCR-RFLP and PCR-amplified fragment length polymorphism techniques. Results: GCPII C1561T showed independent association with toxicity. The following synergetic interactions appeared to increase the toxicity of 6-mercaptopurine: TPMT*12 × RFC1 G80A; TPMT CTTAT haplotype × RFC1 G80A; TPMT CTTAT haplotype × RFC1 G80A × TYMS 2R3R. The genetic variants of thiopurine and folate pathway cumulatively appeared to increase the predictability of toxicity (r(2) = 0.41) in a multiple linear regression model. For the observed toxicity grades of 1, 2, 3 and 4, the respective predicted toxicity grades were 1.65 ± 0.29, 1.68 ± 0.24, 2.56 ± 0.58 and 2.99 ± 1.03, p(trend) < 0.0001. Conclusion: Gene-gene interaction between thiopurine and folate pathways inflate the 6-MP-mediated toxicity in Indian children with ALL illustrating the importance of ethnicity in the toxicity of 6-MP. Original submitted 3 January 2012; Revision submitted 23 April 2012.