广东连南汉族和瑶族儿童硫嘌呤甲基转移酶研究

目的本文对硫嘌呤甲基转移酶(Thiopurine S-methyltransferase,TPMT)在广东连南地区健康汉族儿童(n=87)和瑶族儿童(n=1 26)中的活性分布和4种常见TPMT基因突变的等位基因频率进行研究.方法采用高效液相色谱法测定红细胞的TPMT活性;采用等位基因特异性的PCR(allelespecific PCR,ASPCR)方法和限制性片断长度多态性(PCR-restrictionfragmentlength polymorphism,RFLP)的方法检测TPMT*2(G238C)、TPMT*3A(A719G/G460A)、TPMT*3B(G460A)和TPMT*3C(A719G)的等位基因频率.结果健康汉族儿童和瑶族儿童的TPMT活性都呈正态分布,活性的平均值分别为13.01±2.80U.ml-1pRBC和13.54±3.89U.ml-1 pRBC,两者的差异无显著性.在健康汉族儿童中只找到1例TPMT*3C杂合子(该汉族儿童的TPMT活性为12.36U.ml-1 pRBC),没有找到TPMT*2、TPMT*3A和TPMT*3B,汉族儿童总的TPMT基因突变频率是0.6%.在健康瑶族儿童中没有找到这几种突变.结论广东连南地区汉族和瑶族儿童TPMT的活性分布和总的TPMT基因突变频率没有显著性差异.     

中国新疆维吾尔族硫嘌呤甲基转移酶基因突变研究

目的 研究硫嘌呤甲基转移酶(thiopurine S-methyltransferase，TPMT)在新疆维吾尔族中的基因突变频率。方法 用等位基因特异性的PCR方法和限制性片断长度多态性的方法检测4种常见的导致酶活性降低的突变类型：TPMT*2、TPMT*3A、TPMT*3B和TPMT*3C。结果 在160名维吾尔族中发现了1例TPMT*3A(A719G／G460A)杂合子、5例TPMT*3C(A719G)杂合子，TPMT*3A和TPMT*3C的等位基因频率分别是0．3％和1．6％。结论 维吾尔族总的TPMT突变等位基因频率(1．9％)同中国其他民族相近；TPMT*3C是维吾尔族最主要的突变类型。     

中国维吾尔族和哈萨克族人硫嘌呤甲基转移酶活性的分布

目的研究硫嘌呤甲基转移酶(TPMT)在中国新疆维吾尔族和哈萨克族人中的分布。方法运用改良的RP-HPLC法,测定红细胞中TPMT活性。结果160名维吾尔族成年人TPMT活性呈正态分布,活性均值为(11．57±3．52)U·mL-1 RBC (3．35-29．07 U·mL-1RBC)。327名哈萨克族成年人的TPMT活性呈正态分布,TPMT活性均值(12．27±3．42)U·mL-1 RBC (3．29-24．67 U·mL-1RBC)。在中国新疆维吾尔族和哈萨克族中没有发现酶缺乏者,TPMT活性不存在性别差异。维吾尔族、哈萨克族成年人与汉族TPMT活性相比较,两者差异无统计学意义。结论中国新疆维吾尔族和哈萨克族人TPMT活性呈正态分布。     

河南地区汉族人群药物代谢酶CYP2C19，NAT2和TPMT基因多态性分析(英文)

目的：了解细胞色素P450(cytochromes P450,CYP)2C19,N-乙酰基转移酶2(arylamine N- acetyltransferase 2,NAT2)和硫嘌呤甲基转移酶(thiopurine S-methyltransferase,TPMT)基因常见的遗传多态性在河南地区汉族人群中的分布及其频率。方法：应用聚合酶链反应-限制性片段长度多态性分析(PCR-RFLP)对210名河南地区汉族人群的CYP2C19突变基因(*2和*3)、NAT2突变基因(*6和*7)和TPMT突变基因(*3A,*3B和*3C)进行检测。用聚合酶链反应-等位基因特异性扩增(PCR-ASA)对NAT2突变基因(*5)和TPMT突变基因(*2)进行检测。结果：CYP2C19*2和*3等位基因分布频率分别为34．76%和6．4%,同时携带2个等位突变基因的慢基因型频率占14．8%。NAT2*4(wt),*5(341C),*6(590A)和*7(857A)等位基因分布频率分别为59．1%,4．1%,26．4%和9．5%,慢基因型分布频率占19．5%。TPMT*3C等位基因分布频率为1．2%,未发现TPMT*2,TPMT*3A或TPMT*3B。结论：CYP2C19,NAT2和TPMT基因常见的遗传多态性在汉族人群中的分布及其频率与白人存在明显差异,这将有助于我国汉族人群临床药动学研究和给药剂量的确定。     

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。     

肾移植受者硫嘌呤甲基转移酶基因多态性与硫唑嘌呤不良反应关系的研究

目的:探讨硫嘌呤甲基转移酶(thiopurine S-methyltransferase,TPMT)表型和基因多态性与硫唑嘌呤(AZA)所致不良反应的关系。方法:应用高效液相色谱法(HPLC)测定150例肾移植患者红细胞TPMT活性,采用等位基因特异性的PCR和限制性片断长度多态性的方法检测TPMT*2、*3A、*3B和*3C四种基因型,分析TPMT活性和基因多态性与AZA所致不良反应的关系。结果:30例(20%)患者由于发生了不良反应而停用AZA或减少了AZA的用量,其中12例患者发生了血液毒性,另外18例发生了肝脏毒性。将未发生不良反应的患者作为对照组,其红细胞TPMT活性范围为16.63~68.25 U,平均为(38.43±11.59)U。发生了血液毒性的患者红细胞TPMT活性平均为(24.16±9.84)U,明显低于未发生不良反应的患者(P=0.0003)。另外18例发生了肝脏毒性的患者TPMT活性离散度较大,与对照组比较差异无统计学意义(P=0.145)。本研究未发现TPMT活性缺乏者。共发现7例(4.7%)TPMT*3C杂合子患者,这7例患者均为TPMT中等活性13.04~19.21 U,平均为(...     

新疆维吾尔族和哈萨克族健康人群CYP2C9和VKORC1基因多态性研究

目的：了解细胞色素P450（CYP）2C9、维生素K环氧化还原酶复合体1（VKORC1）基因单核苷酸多态性在新疆维吾尔族和哈萨克族健康人群中的频率分布及与其他不同民族之间的差异,为新疆维吾尔族和哈萨克族人群实施华法林个体化用药剂量提供理论依据。方法：采用BaiO华法林敏感性基因检测试剂盒的芯片检测技术对506位新疆维吾尔族和哈萨克族健康者的CYP2C9＊2和CYP2C9＊3位点以及VKORCI（-1639A/G和1173T/C）位点基因多态性进行检测,统计其等位基因和基因型频率,并与国外多个民族的这两个基因多态性分布进行比较。结果：506份样本中共检测到CYP2C9的3种等位基因CYP2C9＊1、CYP2C9＊2和CYP2C9＊3,等位基因频率分别为89.6%、2.8%和7.6%;5种等位基因型分别为CYP2C9＊1/＊1,CYP2C9＊1/＊2,CYP2C9＊1/＊3,CYP2C9＊2/＊3和CYP2C9＊3/＊3。VKORC1-1639A/G检测出两种等位基因A和G,其频率分别为63.8%和36.2%;三种等位基因型AA（41.7%）、AG（44.3%）和GG（14%）的频率分别与1173T/C的TT、TC和CC型完全相同。结论：新疆维吾尔族和哈萨克族人群CYP2C9和VKORC1存在明显的基因多态性,其分布与中国汉族等亚洲人群以及欧美人群均存在较大差异。     

贵州地区急性淋巴细胞白血病儿童TPMT、NUDT15基因多态性与6-MP耐受性分析

目的 观察贵州地区ALL儿童TPMT、NUDT15基因多态性,探讨其与6-MP耐受性的关系。方法 收集贵阳市儿童医院血液科住院的贵州地区ALL儿童。Sanger法检测患者NUDT15c.415C>T和TPMT*2、TPMT*3A、TPMT*3B、TPMT*3C基因型。所有ALL儿童均按CCLG-2008方案化疗,每周1次监测血常规及肝肾功能。根据2016新编WHO化疗药物毒性反应分度标准,出现Ⅲ～Ⅳ度与6-MP相关的毒性反应,称为6-MP不耐受(除外感染、其他药物影响)。分析TPMT、NUDT15基因多态性与6-MP不耐受的相关性。结果 共纳入患者60例,检测到TPMT突变(中间代谢型)3例(5%),正常代谢型57例(95%)。NUDT15基因CT型12例(20%),TT型1例(1.7%),CC型(野生型) 47例(78.3%)。NUDT15基因突变率21.7%(13/60)显著高于TPMT基因突变率5%(3/60),差异有统计学意义(P=0.007)。TPMT突变型3例均发生6-MP不耐受(100%),NUDT15突变型13例中11例发生6-MP不耐受(84.6%)。结论 TP...     

中国汉族和维吾尔族药物代谢酶CYP3A4、CYP2C9、CYP2C19、CYP2D6基因多态性分析

目的对中国汉族、维吾尔族健康人群CYP3A4、CYP2C9、CYP2C19、CYP2D6进行基因多态性分析,并对汉族和维吾尔族人群等位基因频率和基因型频率进行比较。方法聚合酶链反应一限制性片段长度多态性(PCR-RFLP)法对CYP3A4、CYP2C9、CYP2C19、CYP2D6进行分型。结果汉族、维吾尔族健康人群CYP3A4*5等位基因频率为0,CYP3A4*18等位基因频率分别为0.183 8、0.140 2;CYP2C9*2等位基因频率分别为0.011 0、0.095 8,CYP2C9*13等位基因频率分别为0、0.002 3;CYP2C19*2等位基因频率分别为0.386 0、0.324 8,CYP2C19*3等位基因频率分别为0.051 5、0.021 0;CYP2D6*10等位基因频率分别为0.573 5、0.224 3。结论本研究在汉族、维吾尔族健康人群中未发现CYP3A4*5等位基因。汉族、维吾尔族健康人群CYP3A4*18、CYP2C9*13、CYP2C19*2、CYP2C19*3等位基因频率差异均无统计学意义。维吾尔族健康人群CYP2C9*2等位基因频率远高于汉族(P<0.01);CYP2D6*10等位基因频率远低于汉族(P<0.01),存在民族差异。     

中国汉族和回族药物代谢酶细胞色素P450(CYP)3A4、CYP2C9、CYP2C19及CYP2D6基因多态性分析

目的对中国汉族、回族健康人群细胞色素P450(CYP)3A4、CYP2C9、CYP2C19及CYP2D6进行基因多态性分析,比较汉族和回族健康人群基因表型和基因频率分布。方法多聚酶链反应-限制性片段长度多态性(PCR-RFLP)法,对300名志愿者的几种基因进行分型。结果汉族、回族CYP3A4*5等位基因频率均为0,CYP3A4*18等位基因频率分别为0.18,0.19;汉族、回族CYP2C9*2等位基因频率分别为0.01,0.05;CYP2C9*13等位基因频率均为0;汉族、回族CYP2C19*2等位基因频率分别为0.39,0.50;CYP2C19*3等位基因频率分别为0.05,0.05;汉族、回族CYP2D6*10等位基因频率分别为0.57,0.39。结论汉族、回族健康人群的CYP3A4*18、CYP2C9*2、CYP2C19*2、CYP2C19*3均没有显著性差异;在汉族、回族健康人群中未发现CYP3A4*5和CYP2C9*13突变;汉族、回族CYP2D6*10等位基因频率有显著性差异(P<0.01);回族人群CYP2D6中速代谢型(*10/*10)频率为13.4%,明显低于汉族的33.1%(P<0.01)。     

The frequency and distribution of thiopurine methyltransferase alleles in Caucasian and Asian populations

Thiopurine methyltransferase metabolizes 6-mercaptopurine, thioguanine and azathioprine, thereby regulating cytotoxicity and clinical response to these thiopurine drugs. In healthy Caucasian populations, 89-94% of individuals have high thiopurine methyltransferase activity, 6-11% intermediate and 0.3% low, resulting from genetic polymorphism. Four variant thiopurine methyltransferase alleles were detected in over 80% of individuals with low or intermediate thiopurine methyltransferase activity. The wild-type allele is defined as TPMT*1 and the mutant alleles are TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3B (A719G). The frequency of these alleles in different ethnic groups is not well defined. In this study, DNA from 199 British Caucasian, 99 British South West Asian and 192 Chinese individuals was analysed for the presence of these variant alleles using polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction based assays. The frequency of individuals with a variant thiopurine methyltransferase genotype was: Caucasians 10.1% (20/199), South West Asians 2.0% (2/99) and Chinese 4.7% (9/192). Two TPMT*2 heterozygotes were identified in the Caucasian population, but this allele was not found in the two Asian populations. TPMT*3A was the only mutant allele found in the South West Asians (two heterozygotes). This was also the most common mutant allele in the Caucasians (16 heterozygotes and one homozygote) but was not found in the Chinese. All mutant alleles identified in the Chinese population were TPMT*3C (nine heterozygotes). This allele was found at a low frequency in the Caucasians (one heterozygote). This suggests that A719G is the oldest mutation, with G460A being acquired later to form the TPMT*3A allele in the Caucasian and South West Asian populations. TPMT*2 appears to be a more recent allele, which has only been detected in Caucasians to date. These ethnic differences may be important in the clinical use of thiopurine drugs.     

Allelic variants of the thiopurine methyltransferase (TPMT) gene in the Colombian population

Thiopurine methyltransferase (TPMT) catalyzes the inactivation of thiopurine drugs (mercaptopurine, thioguanine and azathioprine) used to treat acute lymphoblastic leukemia, autoimmune diseases and recipients of transplanted organs. No endogenous substrates for this enzyme are known. The TPMT polymorphism is a major determinant of individual differences in the toxicity or therapeutic efficacy of these drugs. The molecular basis of this polymorphism has been established in Caucasians, Africans, African-Americans and Asians, but not yet in the heterogeneous Latin American groups, including the Colombian population. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G), were determined in 140 Colombian volunteers of Mestizo origin, using allele-specific PCR and PCR-RFLP assays. The *3A allele was found in 10 samples and the *2 allele in one, all heterozygotes; neither homozygous mutant genotypes nor the *3B and *3C alleles were detected. In agreement with these results, 92.1% and 7.9% of the Colombian population correspond to the phenotypes high and intermediate methylators, respectively. These results show that the frequency of mutations and the allelic distribution of the TPMT gene in the Colombian population are similar to the genetic profile found among US and European Caucasian populations, where the *3A allele is prevalent and the *2 allele is currently present.     

Phenotyping and genotyping study of thiopurine S-methyltransferase in healthy Chinese children: a comparison of Han and Yao ethnic groups

AIMS: Ethnicity is an important variable influencing drug response. Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs. Previous population studies have identified ethnic variations in both phenotype and genotype of TPMT, but limited information is available within Chinese population that comprises at least 56 ethnic groups. The current study was conducted to compare both phenotype and genotype of TPMT in healthy Han and Yao Chinese children. METHODS: TPMT activity was measured in healthy Chinese children by a HPLC assay (n = 213, 87 Han Chinese and 126 Yao Chinese). Allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) were used to determine the frequency of TPMT mutant alleles (TPMT*2, TPMT*3 A, TPMT*3B and TPMT*3C) in these children. RESULTS: There was no significant difference in the mean TPMT activity between Han and Yao Chinese children. A unimodal distribution of TPMT activity in Chinese children was found and the mean TPMT activity was 13.32 +/- 3.49 U ml(-1) RBC. TPMT activity was not found to differ with gender, but tended to increase with age in Yao Chinese children. TPMT*2, TPMT*3B and TPMT*3A were not detected, and only one TPMT*3C heterozygote (Han child) was identified in 213 Chinese children. Erythrocyte TPMT activity of this TPMT*3C heterozygote was 12.36 U ml(-1) RBC. The frequency of the known mutant TPMT alleles was 0.2%[1/426] in Chinese children. CONCLUSION: The frequency distribution of RBC TPMT activity was unimodal. The frequency of the known mutant TPMT alleles in Chinese Children is low and TPMT*3C appears to be the most prevalent among the tested mutant TPMT alleles in this population.     

Thiopurine methyltransferase genotype distribution in patients with Crohn's disease

BACKGROUND: Inter-individual response to azathioprine is partly due to inter-individual variation in the thiopurine methyltransferase (TPMT) activity. The TPMT genotype, which reflects the TPMT activity, has previously been studied in healthy Caucasians, with the most common variant allele being TPMT*3A. TPMT genotyping in adult patients with Crohn's disease has never been performed systematically. AIM: To determine the TPMT genotype distribution in adult patients with Crohn's disease. METHODS: One hundred and twenty randomly selected Danish patients (64 females and 56 males) with azathioprine-dependent Crohn's disease were included, and a polymerase chain reaction assay was used for TPMT genotyping. The patients were genotyped for the low-level genotype G460-->A and A719-->G transitions. RESULTS: One hundred and nine patients (90.3%; 95% confidence interval, 84.1-95.3) had a wild-type/ wild-type genotype, whereas 10 patients (8.3%; 95% confidence interval, 4.1-14.8) had one non-functional mutant allele and one patient (0.8%; 95% confidence interval, 0.02-4.6) had two non-functional mutant alleles. Only the TPMT*3A variant allele was found. CONCLUSIONS: The study showed a TPMT genotype distribution amongst adult Danish patients with Crohn's disease which was similar to the distribution of TPMT variant alleles normally found in healthy Caucasians.     

Explaining TPMT genotype/phenotype discrepancy by haplotyping of TPMT*3A and identification of a novel sequence variant, TPMT*23

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2-*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patient's relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.     

Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity

The polymorphic enzyme thiopurine methyltransferase (TPMT) is involved in the methylation of thiopurines. On comparing the phenotype with the genotype in Swedish patients with inflammatory bowel disease and healthy individuals, we found two discordant cases with low TPMT enzyme activity (0.3 and 0.4 U/ml packed red blood cells (pRBC). Genotyping by pyrosequencing revealed that they carried the nucleotide substitutions 460G>A and 719A>G, giving two possible genotypes (TPMT*1/*3A or TPMT*3B/*3C). DNA sequencing of exon III to X was performed in the patients and their parents. We identified an A>G transition in the start codon (exon III, 1A>G, Met>Val, TPMT*14) in one of the patients and her father (6.3 U/ml pRBC). The mother in this family carried the 460G>A and 719A>G nucleotide substitutions (TPMT*1/*3A; 5.0 U/ml pRBC). In the second family, sequencing revealed a G>A transition in the acceptor splice site in intron VII/exon VIII (IVS7 -1G>A, TPMT*15) in the patient and his mother (6.9 U/ml pRBC). His father was genotyped as TPMT*1/*3A (6.0 U/ml pRBC). Hence, we report the identification of two novel sequence variants, present in highly conserved nucleotide positions of the human TPMT gene, resulting in a loss of enzyme activity.     

Gene mutation of thiopurine<Emphasis Type="Italic"> S</Emphasis>-methyltransferase in Uygur Chinese

Objective This study was to investigate the gene mutation of thiopurine S-methyltransferase (TPMT) in Uygur Chinese.Methods Polymerase chain reaction-based methods were used to analyze three commonly reported inactivating mutations—G238C, G460A and A719G.Results One TPMT*3A heterozygote and five TPMT*3C heterozygotes were found in 160 Uygur Chinese subjects, and allele frequencies of TPMT*3A and TPMT*3C were 0.3% and 1.6%, respectively.Conclusion TPMT*3C is a common mutant allele in Uygur Chinese, while TPMT*3A is a rare mutant allele in Uygur Chinese.     

Phenotyping and Genotyping Studies of Thiopurine <Emphasis Type="BoldItalic">S</Emphasis>-Methyltransferase in Kazaks

Objective This study was conducted to investigate the thiopurine S-methyltransferase (TPMT) activity distribution and gene mutations in Kazaks, and compared the results with those of other ethnic groups. Methods Erythrocyte TPMT activity was measured in Kazaks (n = 327) via a validated high-performance liquid chromatography assay. Polymerase chain reaction-based methods were used to analyze three commonly reporter-inactivating mutations: G238C, G460A, and A719G. Results Unimodal distribution of TPMT activity was found in Kazaks. Six TPMT*3C heterozygotes and two TPMT*3A heterozygotes were found in 327 Kazaks, with allele frequencies of 0.9 and 0.3%, respectively. The subjects with TPMT*3A and TPMT*3C heterogygotes had substantial TPMT activity over the range of 6.40–11.75 U/ml RBC. Conclusion Unlike in most Caucasians, TPMT*3C is a common mutant allele in Kazaks, whereas TPMT*3A is a rare mutant allele. Further studies are needed to explore the clinical impact of these TPMT mutants to thiopurine therapy in Kazak patients.     

Thiopurine methyltransferase phenotypes and genotypes in Brazilians

The polymorphism of thiopurine methyltransferase (TPMT) was studied in 306 healthy Brazilians who were classed, on the basis of self-declared colour and ancestry, as Euro-derived (n = 81), Afro-derived (n = 18) or having interethnic admixture (n = 204). TPMT activity (range 0.17-25.93 U) displayed a trimodal distribution of high (> 11.3 U; 9% of individuals), intermediate (5-11.3 U; 9.8%) and low (0.17 U; 0.3%) phenotypes. The occurrence of the TPMT mutations 238G>C, 460G>A and 719A>G was investigated in all individuals with low or intermediate phenotype, and in 43 with high-activity phenotype. None and two mutant alleles were associated with high- or low-activity phenotypes, respectively, whereas one mutant allele was detected in 26 of the 30 intermediate phenotype individuals. The allele frequencies of TPMT*2, TPMT*3A and TPMT*3C did not differ between individuals classed as Euro-derived (0.76%, 2.03% and 2.54%, respectively) or having interethnic admixture (0.60%, 1.81% and 1.81%, respectively). Furthermore, within each of these groups, the frequencies of TPMT*3A and TPMT*3C were not significantly different.     

Thiopurine methyltransferase pharmacogenetics: alternative molecular diagnosis and preliminary data from Northern Portugal.

The thiopurine methyltransferase (TPMT) genetic polymorphism has been shown to have a highly significant clinical impact, namely in the therapeutic efficiency of thiopurine drugs used in the treatment of a wide range of diseases. Available diagnostic methods, although reproducible and sensitive, are relatively laborious. Thus population studies are still very scarce. In this work we describe a new polymerase chain reaction-single strand confirmational analysis based protocol for TPMT specific detection which introduces a substantial technical simplification avoiding the use of restriction enzyme treatment after polymerase chain reaction amplification. Additionally, the use of this protocol allows the simultaneous detection of a T474 to C substitution, a frequent silent mutation in the North Portuguese population (TPMT*1S = 0.215). In a sample of 310 unrelated Northern Portuguese individuals, 15 were found to be heterozygous for the TPMT*3A allele (defined by the presence of two transitions, G460 to A and A719 to G) which is associated with TPMT enzymatic deficiency; the corresponding gene frequency estimate was 0.024. We also attempted to evaluate the relationship between the molecular TPMT genotype and the reaction to treatments involving thiopurine drugs by analysing a sample of 24 children submitted to curative therapy of acute lymphoblastic leukaemia. Four of them were shown to be heterozygous for the TPMT*3A allele. An examination of their clinical histories showed that all four patients exhibited signs of severe hepatic toxicity during treatment.