Thiopurine Methyltransferase Polymorphisms Research Articles

Association of TPMT and NUDT15 gene polymorphisms with azathioprine-induced leukopenia: A case–control study in Eastern India

Abstract: BACKGROUND: Azathioprine (AZA) is a widely used immunosuppressant drug. Leukopenia is a serious adverse effect of the drug which often necessitates dose reduction or drug withdrawal. Predictors of leukopenia include genetic and nongenetic factors. Genetic polymorphism of AZA-metabolizing enzyme, thiopurine S-methyltransferase (TPMT) is well established. There is inconclusive evidence about the role of Nudix hydrolase (NUDT15) gene polymorphism. This case–control study assessed the association of genetic polymorphisms of NUDT15 and TPMT with leukopenia induced by AZA. MATERIALS AND METHODS: Cases were patients on AZA who developed leukopenia (white blood cell count <4000/μl) within 1 year of treatment initiation that necessitated dose reduction or drug withdrawal. Age and gender-matched patients without leukopenia within 1 year of treatment with AZA served as controls. TPMT (3 loci: c238G to C, c460G to A, c719A to G) and NUDT15 (c 415C to T, rs116855232) genotyping were done using TPMT strip assay and polymerase chain reaction–restriction fragment length polymorphism, respectively. Genotype frequencies were noted, and the odds ratio was calculated to determine the association between genotypes and leukopenia. RESULTS: Twenty-nine subjects (15 cases and 14 controls) were enrolled. Statistically significant differences were not observed in the TPMT genotype (*1/*1 and *1/*3C) (P = 0.23) between cases and controls. NUDT15 genotypes (*1/*1 and *1/*3) (P = 0.65) also showed no statistically significant difference between cases and controls. CONCLUSION: The above genotypes do not appear to be associated with AZA-induced leukopenia in an eastern Indian population.

The c.415C>T polymorphism in NUDT15 is more frequent than the polymorphisms in TPMT in Chilean patients who use thiopurine drugs.

Azathioprine (AZA) and 6-mercaptopurine (6-MP) are drugs widely used in the treatment of autoimmune diseases. Among the enzymes involved in the metabolism of AZA and 6-MP are thiopurine methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15). The existence of single nucleotide polymorphisms in the genes that code for these enzymes could decreased enzymatic activity AND lead to severe myelosuppression. The most relevant polymorphism is NUDT15*3 (rs116855232), where the replacement of cytosine for thymine at position 415, which in turn leads to a loss of enzymatic activity. In a previous study, it was identified that together the polymorphisms in the TPMT gene reach an allelic frequency of 3.81%. There is no information regarding the rs116855232 polymorphism in the NUDT15 gene, so this corresponds to the objective of this report. Blood samples from Chilean adult patients with indications for the use of AZA or 6-MP for different pathologies and who had undergone a TPMT gene polymorphism study were retrospectively analyzed. A total of 253 blood samples were analyzed. Of the 253 patients, 47 presented the c.415C>T polymorphism in the NUDT15 gene, 3 being homozygous and 44 heterozygous. Four of the heterozygous patients for NUDT15 also had the *3A variant in the TPMT gene, also heterozygous. The allelic frequency of the minor T allele found (9.88%) was very similar to that found in patients of Asian origin, and much higher than that reported for the European Caucasian or Latin American population.

Genetic Polymorphism of Thiopurine S-Methyltransferase in Children with Acute Lymphoblastic Leukemia

Background: 6-Mercaptopurine (6-MP), a widely used anti-metabolite for the maintenance phase of childhood acute lymphoblastic leukemia (ALL), has been observed to cause myelotoxicity due to genetic polymorphism of thiopurine methyl transferase (TPMT), one of the drug-metabolizing enzymes. This study aimed to determine the frequency of thiopurine S-methyl transferase (TPMT) polymorphic variants in a group of Pakistani children with acute lymphoblastic leukemia (ALL) using 6-mercaptopurine (6-MP). Methods: Diagnosed cases (n=100) of ALL, either Pre-B or T Cell, between the ages of 2 to 18 years were randomly selected from OPD of Children Cancer Hospital and National Institute of Child Health (NICH), Karachi. The subjects were under BFM (Berlin Frankfurt Munster) protocol. Expired and relapsed patients (2/100) were excluded. Blood samples were drawn and DNA was extracted from serum to determine the genotype of TPMT (*2, *3a, *3B and *3C) using allele-specific PCR (AS-PCR) and RFLP. PCR assays were done to detect the G238C transversion in TPMT*2 and the G460A and A719G transition in TMPT*3 Alleles. Results: Polymorphism in TMPT was found 100% (98/98) in ALL patients belonging to a heterozygous group. Out of them, 74.5% of ALL patients showed myelotoxicity. Furthermore, *1/*3B was the most prevalent variant TPMT allele observed with the highest frequency reported at 97%. The variant allele *1/*3A was found in only 3 patients, whereas, TMPT*2 and *3C Alleles were not found. Conclusion: The majority of our patients displayed a distinct TPMT genetic polymorphism TPMT*1/*3B which is comparatively rare in other parts of the world. Keywords: 6-Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Genetic Polymorphism; Alleles; Pakistan.

Association of genetic polymorphism of NUDT15, TPMT and ITPA gene in the toxicity and efficacy of azathioprine-based regimen in Egyptian inflammatory bowel disease patients

BackgroundInflammatory Bowel disease (IBD) is a chronic progressive condition that prompts generous physical and mental morbidity. Choosing the best kind of management and medication dosage prevents new episodes of high disease activity during therapy because of adverse drug reactions (ADRs). This can lead to cessation or inefficacy of the treatment, or complete non-responsiveness to specific medications. Pharmacogenetics (PGx) is a well-established aspect in IBD. One of the exemplary instances of PGx is thiopurines, which are frequently utilized as IBD therapy. This study aimed to evaluate specific gene polymorphism involved in the toxicity and efficacy of Azathioprine (AZA) use in the management in Egyptian patients and to find the correlation between the polymorphism of Nudix Hydrolase15 (NUDT15) gene (rs116855232), The Thiopurine methyltransferase (TPMT) gene (rs1800460) and Inosine Triphosphatase (ITPA) gene (rs1127354) which are involved in the metabolism of the medications utilized in IBD management.MethodsThis prospective study was performed in 150 patients with IBD. All patients were treated with 2 mg/kg per day AZA (Imuran, GlaxoSmithKline®) for at least 3 months at therapeutic doses to induce remission. Subsequent treatment of AZA. The minimum follow-up period for those who did not experience ADR was one year. Among the studied patients, one hundred twenty-nine patients were treated with combination regimen of steroids (oral prednisone 1 mg/kg/day).Also, treatment failure was considered among the patients who could not tolerate AZA side effects, or there was no improvement after dose modification.ResultsThe most identifiable adverse effect among the studied population was anemia followed by leukopenia and myelosuppression. SNPs genotype TPMT (rs1800460) and ITPA gene (rs1127354) were significantly related to adverse effects among IBD patients receiving Azathioprine treatment. There was a lack of any variants in the NUDT15 genotype among the Egyptian population.ConclusionFurther research is required in to clarify the relationship between NUDT15 PGx and AZA-ADRs. The effect of NUDT15 PGx on toxicity and ADRs as yet necessitates to be elucidated. Studies with a larger sample size and involving different ethnicities are also necessary.

ITPA polymorphisms do not predict additional risk beyond TPMT and NUDT15 for thiopurine-induced cytopenia in inflammatory bowel disease

Introduction and aimThiopurine-related leukopenia is associated with polymorphisms in the thiopurine methyltransferase (TPMT) and nucleoside diphosphate-linked moiety X type motif 15 (NUDT15) genes. However, those polymorphisms explain only a fraction of thiopurine-related leukopenia. Our aim was to study the role of an inosine triphosphate pyrophosphatase (ITPA) polymorphism in patients with inflammatory bowel disease (IBD) and thiopurine-related leukopenia that was unexplained by the TPMT and NUDT15 polymorphisms. Material and methodsWe enrolled consecutive IBD patients on thiopurines (azathioprine or 6-mercaptopurine) from January 2019–March 2020, at a tertiary care center in North India. The presence of the ITPA (C.94C > A) polymorphism was evaluated in all patients, along with its association with thiopurine-related leukopenia. ResultsOf the 33 patients (from a total of 119 patients) that developed leukopenia, 8 had the TPMT (n = 1) or NUDT15 (n = 7) polymorphism. Of the remaining 111 patients, their mean age was 36.36 ± 13.54 years and 57 (51.3%) were males. Twenty-five (21.01%) had unexplained leukopenia. The ITPA polymorphism was detected in 4 (16%) patients in the unexplained leukopenia group and 24 (27.9%) patients in the non-leukopenia group (p = 0.228). The odds ratio for predicting leukopenia with the ITPA polymorphism was 0.4921 (95% CI 0.1520–1.5830, p = 0.234). ConclusionThe ITPA (C.94C > A) polymorphism was frequently detected in the study population but was not predictive for leukopenia in patients with IBD on thiopurine therapy.

A bioinformatics approach to the identification of novel deleterious mutations of human TPMT through validated screening and molecular dynamics

Polymorphisms of Thiopurine S-methyltransferase (TPMT) are known to be associated with leukemia, inflammatory bowel diseases, and more. The objective of the present study was to identify novel deleterious missense SNPs of TPMT through a comprehensive in silico protocol. The initial SNP screening protocol used to identify deleterious SNPs from the pool of all TPMT SNPs in the dbSNP database yielded an accuracy of 83.33% in identifying extremely dangerous variants. Five novel deleterious missense SNPs (W33G, W78R, V89E, W150G, and L182P) of TPMT were identified through the aforementioned screening protocol. These 5 SNPs were then subjected to conservation analysis, interaction analysis, oncogenic and phenotypic analysis, structural analysis, PTM analysis, and molecular dynamics simulations (MDS) analysis to further assess and analyze their deleterious nature. Oncogenic analysis revealed that all five SNPs are oncogenic. MDS analysis revealed that all SNPs are deleterious due to the alterations they cause in the binding energy of the wild-type protein. Plasticity-induced instability caused by most of the mutations as indicated by the MDS results has been hypothesized to be the reason for this alteration. While in vivo or in vitro protocols are more conclusive, they are often more challenging and expensive. Hence, future research endeavors targeted at TPMT polymorphisms and/or their consequences in relevant disease progressions or treatments, through in vitro or in vivo means can give a higher priority to these SNPs rather than considering the massive pool of all SNPs of TPMT.

Thiopurine S-Methyltransferase Polymorphisms Predict Hepatotoxicity in Azathioprine-Treated Patients with Autoimmune Diseases.

Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in Azathioprine (AZA) metabolization. Although studies have discussed the association between the TPMT polymorphisms and myelosuppression, the data about the relationship between TPMT genotypes and hepatoxicity in Asian patients remain limited. This study investigated the correlation between TPMT polymorphisms and AZA-related hepatotoxicity. This study enrolled the patients who had prior exposure to AZA from the Taichung Veterans General Hospital (TCVGH)-Taiwan Precision Medicine Initiative (TPMI) cohort. Genetic variants were determined using a single nucleotide polymorphism (SNP) array. Participants were accordingly categorized into normal metabolizer (NM) and non-normal metabolizer (non-NM) groups. From the TCVGH-TPMI cohort, we included 50 TPMT non-NM patients, including 1 poor metabolizer (PM), 49 intermediate metabolizers (IMs), and 1000 NM patients. The non-NM genotype was associated with hepatotoxicity compared with the NM genotype (hazard ratio (HR): 3.85, 95% confidence interval (CI): 1.83–8.10). In the non-NM group, the 3-year cumulative incidence of hepatotoxicity was higher than that in the NM group at 8.5% in the first year and 18.6% in the second and third years (p < 0.001). A TPMT non-NM genotype was associated with the occurrence of hepatotoxicity following AZA therapy. Preemptive testing helps individualize AZA therapy by minimizing the risk of hepatotoxicity.

Los polimorfismos de ITPA no predicen un riesgo adicional más allá de TPMT y NUDT15 para citopenia inducida por tiopurina en la enfermedad inflamatoria intestinal

Introduction and aimThiopurine-related leukopenia is associated with polymorphisms in the thiopurine methyltransferase (TPMT) and nucleoside diphosphate-linked moiety X type motif 15 (NUDT15) genes. However, those polymorphisms explain only a fraction of thiopurine-related leukopenia. Our aim was to study the role of an inosine triphosphate pyrophosphatase (ITPA) polymorphism in patients with inflammatory bowel disease (IBD) and thiopurine-related leukopenia that was unexplained by the TPMT and NUDT15 polymorphisms. Material and methodsWe enrolled consecutive IBD patients on thiopurines (azathioprine or 6-mercaptopurine) from January 2019-March 2020, at a tertiary care center in North India. The presence of the ITPA (C.94C>A) polymorphism was evaluated in all patients, along with its association with thiopurine-related leukopenia. ResultsOf the 33 patients (from a total of 119 patients) that developed leukopenia, 8 had the TPMT (n=1) or NUDT15 (n=7) polymorphism. Of the remaining 111 patients, their mean age was 36.36±13.54 years and 57 (51.3%) were males. Twenty-five (21.01%) had unexplained leukopenia. The ITPA polymorphism was detected in 4 (16%) patients in the unexplained leukopenia group and 24 (27.9%) patients in the non-leukopenia group (P=.228). The odds ratio for predicting leukopenia with the ITPA polymorphism was 0.4921 (95% CI: 0.1520-1.5830; P=.234). ConclusionThe ITPA (C.94C>A) polymorphism was frequently detected in the study population but was not predictive for leukopenia in patients with IBD on thiopurine therapy.

Passive smoking and thiopurine methyl transferase genotyping in 6-mercaptopurine-induced myelosuppression in Egyptian children with acute lymphoblastic leukemia

6-mercaptopurine (6-MP) is a crucial component drug used to treat childhood acute lymphoblastic leukemia (ALL) in the maintenance phase. Thiopurine S-methyltransferase (TPMT) is an enzyme that metabolizes thiopurine drugs like 6-MP. In patients taking thiopurine medications, lack of TPMT activity is linked to an increased risk of myelosuppression. Patients with TPMT polymorphisms are more likely to develop mercaptopurine-induced myelosuppression. The goal of this study was to see how passive smoking and TPMT genetic variations affected 6-MP-induced myelosuppression in Egyptian ALL children. This study included 100 children: 50 with acute lymphoblastic leukemia in the maintenance phase of treatment who had a history of passive smoking and 50 age-matched healthy controls. 6-MP was given to patients in the maintenance phase, and myelosuppression was monitored at the end of this phase by comparing WBC counts to those obtained at the start of treatment. After six months of 6-MP administration, there was no significant association between cotinine levels and WBC count (as evidence of myelosuppression), although there was a significant correlation between TPMT genotypes and WBC count, with 75% of hetero genotype patients suffering from severe declines in WBC count and 91% of wild genotype people undergoing mild to moderate decreases in WBC count.

Genome‐Wide Association Study for the Genetic Determinants of Thiopurine S‐Methyltransferase Protein Expression in the Liver

HypothesisSinge nucleotide polymorphisms will be associated with differential expression of Thiopurine S‐Methyltransferase (TPMT) in the liver.MethodsNormal human liver samples (n=288) were acquired from University of Minnesota Liver Tissue Cell Distribution System, the Cooperative Human Tissue Network, and XenoTech LCC (Lenexa, KS, USA). Absolute quantification of TPMT in liver microsomes was calculated using a DIA total protein approach (DIA‐TPA). Human liver samples were genotyped using the Illumina Multi‐Ethnic Global Assay (Illumina, Miami, USA).ResultsAfter accounting for population stratification, genome‐wide association study (GWAS) analysis was conducted on 243 human liver samples. There were 30 genetic variants, all located on chromosome 6, that passed our genome‐wide threshold (P &lt; 5.0 x 10‐8) after Bonferroni correction. Further GWAS analysis conditioning on rs1142345 (TPMT*3C/TPMT*3A), the strongest signal within the dataset and a known nonfunctional TPMT allele, showed no other independent signals. The presumed homozygous wild‐type TPMT subjects had a 1.97‐fold higher expression of TPMT than the TPMT*3A heterozygotes (0.1056 pmol/mg ± 0.0289 pmol/mg vs. 0.0536 pmol/mg ± 0.0143 pmol/mg, p‐value: 8.4e‐14).When comparing TPMT expression by ancestry, European ancestry donors (n= 214) had significantly higher expression than African ancestry donors (n= 34) when removing TPMT*3A carriers (0.1085 pmol/mg ± 0.0260 pmol/mg vs. 0.0860 pmol/mg ± 0.0416 pmol/mg, p‐value: 0.004.ConclusionFor the first time, we quantified absolute TPMT protein expression in the liver using a liquid chromatography tandem mass spectrometry based DIA‐TPA proteomics approach and conducted a GWAS to identify genetic variants affecting hepatic TPMT protein expression. The only independent genetic signal from the GWAS was from the TPMT*3A haplotype, which is comprised of two nonsynonymous SNPs rs1800460 and rs1142345. Previous in vitrostudies investigating the effect of common polymorphisms on TPMT expression in COS‐1 cells demonstrated that TPMT*3A is almost a complete loss of detectable TPMT due to its rapid degradation rate.1 This is in line with our expression data, where heterozygous carriers of TPMT*3A had about half the amount of TPMT as wild‐type subjects.We also discovered that donors with African ancestry had significantly less TPMT expression than donors with European ancestry after removing samples that had known TPMT polymorphisms (TPMT*3A/*3B/*3C). A previous study reported patients of Afro‐Caribbean ancestry had significantly lower TPMT activity than those of European or South Asian descent.2 Future studies need to be conducted to determine if the difference in TPMT expression by ancestry is due to genetic polymorphisms or other factors.

Understanding thiopurine methyltransferase polymorphisms for the targeted treatment of hematologic malignancies

ABSTRACT Introduction Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines (mercaptopurine (MP) and tioguanine (TG)), chemotherapeutic agents used in the treatment of acute lymphoblastic leukemia (ALL). Polymorphisms in TPMT gene encode diminished activity enzyme, enhancing accumulation of active metabolites, and partially explaining the inter-individual differences in patients’ clinical response. Areas covered This review gives an overview on TPMT gene and function, and discusses the pharmacogenomic implications of TPMT variants in the prevention of severe thiopurine-induced hematological toxicities and the less known implication on TG-induced sinusoidal obstruction syndrome. Additional genetic and non-genetic factors impairing TPMT activity are considered. Literature search was done in PubMed for English articles published since1990, and on PharmGKB. Expert opinion To titrate thiopurines safely and effectively, achieve the right degree of lymphotoxic effect and avoid excessive myelosuppression, the optimal management will combine a preemptive TPMT genotyping to establish a safe initial dose with a close phenotypic monitoring of TPMT activity and/or of active metabolites during long-term treatment. Compared to current ALL protocols, replacement of TG by MP during reinduction phase in TPMT heterozygotes and novel individualized TG regimens in maintenance for TPMT wild-type subjects could be investigated to improve outcomes while avoiding risk of severe hepatotoxicity.

TPMT and NUDT15 polymorphisms in thiopurine induced leucopenia in inflammatory bowel disease: a prospective study from India

BackgroundPolymorphisms in thiopurine methyltransferase (TPMT) and Nudix hydrolase-15 (NUDT15) have been implicated as the predominant cause of thiopurine induced leukopenia in the Western countries and East Asia respectively. Exact role of these polymorphisms in South Asian population with inflammatory bowel disease (IBD) is uncertain.MethodsWe included consecutive patients with IBD who were initiated on thiopurines at a center in North India. The dosage of thiopurines was titrated using regular monitoring of hemogram and liver function tests. Three TPMT polymorphisms (c.238 G > C, c.460 G > A, and c.719A > G) and one NUDT15 polymorphism (c.415 C > T) were assessed. Comparison regarding incidence of leukopenia and maximum tolerated thiopurine dosage was performed between those with wild polymorphism and those with TPMT and NUDT15 polymorphisms, respectively.ResultsOf the 119 patients (61 males, mean age 36.8 ± 13.5 years), 105 (88.2%) had ulcerative colitis and 14 (11.8%) had Crohn’s disease. Leukopenia was noted in 33 (27.7%), gastrointestinal intolerance in 5 (4.2%) and pancreatitis in 2 (1.6%). TPMT polymorphisms were detected amongst five patients of whom 1 developed leukopenia. NUDT15 polymorphism was noted in 13 patients of whom 7 had leukopenia. The odds of developing leukopenia in TPMT polymorphism were non-significant (0.77, 95% CI:0.0822 to 7.2134, P = 0.819) but were significantly higher in those with NUDT15 polymorphism (3.5933, 1.1041 to 11.6951, P value: = 0.0336).ConclusionNUDT15 polymorphism was more frequent than TPMT polymorphisms and was associated with thiopurine induced leukopenia. However, the tested polymorphisms account for only 24.2% of the risk of thiopurine induced leukopenia.

NUDT15 polymorphism in healthy children with Bai nationality in Yunnan of China

Thiopurine methyltransferase (TPMT) polymorphism is one of the causes of the toxicity of thiopurines, but this is rarely seen in Asian populations. Rather, the nucleoside diphosphate-linked X-component motif 15 (NUDT15) gene is frequently linked to mercaptopurine (MP) intolerance and myelotoxicity in children with acute lymphoblastic leukemia (ALL) in East Asians; however, little is known about the NUDT15 polymorphism in healthy children, especially in ethnic minorities in China. A total of 162 cases of healthy children with Bai nationality were enrolled for NUDT15 genotyping. Three coding variants were identified in the NUDT15 gene including rs186364861, rs746071566 and rs116855232. Notably, the rs746071566 and rs116855232 in NUDT15 showed much higher frequencies in healthy children with Bai nationality compared with healthy East Asian populations, suggesting a concentrated distribution of these variants in the Bai ethnic group. This finding reveals the genetic polymorphism of NUDT15 in children with Chinese Bai nationality, providing a biological genetic background for the individualized therapy of thiopurines for children with Bai nationality in China.

TPMT polymorphisms and minimal residual disease after 6-mercaptopurine post-remission consolidation therapy of childhood acute lymphoblastic leukaemia

Bone marrow minimal residual disease (MRD) is the strongest predictor of relapse in children with acute lymphoblastic leukemia (ALL). 6-mercaptopurine (6MP) in ALL therapy has wide inter-individual variation in disposition and is strongly influenced by polymorphisms in the thiopurine methyltransferase (TPMT) gene. In 952 patients treated according to the NOPHO ALL2008 protocol, we explored the association between thiopurine disposition, TPMT genotypes and MRD levels after consolidation therapy with 6MP, high-dose methotrexate (HD-MTX), asparaginase, and vincristine. The levels of the cytotoxic DNA-incorporated thioguanine were significantly higher on day 70-79 in G460A/A719G TPMT heterozygous (TPMT HZ) compared to TPMT wild type (TPMT WT) patients (mean: 230.7 vs. 149.7 fmol/µg DNA, p = 0.002). In contrast, TPMT genotype did not associate with the end of consolidation MRD levels irrespective of randomization of the patients to fixed dose (25 mg/m2/day) or 6MP escalation (up to 50 or 75 mg/m2/day) during consolidation therapy.

Influence of TPMT polymorphisms on azathioprine-induced myelosuppression in Bangladeshi patients with systemic lupus erythematosus

Thiopurine methyltransferase (TPMT) enzymes play a crucial role in azathioprine metabolism. Mutations in the enzyme initiate generation of excess thioguanine, which causes suppression of various cell lineages. The objectives of this study were to investigate the prevalence of TPMT mutation in Bangladeshi patients with systemic lupus erythematosus (SLE) requiring azathioprine therapy and its relation to the development of myelosuppression. 250 patients with SLE were enrolled, then monitored for myelosuppression adverse events for 4 months. TPMT*3C (rs1142345), TPMT*3B (rs1800460), and TPMT*2 (rs1800462) polymorphisms were analyzed using polymerase chain reaction–restriction fragment length polymorphism. The risk of myelosuppression (i.e., leukopenia, thrombocytopenia, and anemia) was estimated as the odds ratio (OR) with 95% confidence intervals (CIs) and p values. Of the 250 patients, 17 (6.8%) were heterozygous for the TPMT*3 mutation and 233 (93.2%) were homozygous for the wild type; no patients carried a homozygous mutation. Grade III/IV leukopenia, thrombocytopenia, and anemia occurred in 12.0%, 12.0%, and 27.9% of wild-type TPMT patients respectively; corresponding rates in heterozygous TPMT*3C patients were 70.6%, 64.7%, and 5.9%. Patients with Grade III/IV azathioprine-induced leukopenia were significantly more likely to have the heterozygous TPMT*3C genotype than the wild-type variant (OR 17.6; 95% CI 5.8–53.6; p < 0.0001), with comparable results for Grade III/IV azathioprine-induced thrombocytopenia (OR 13.4; 95% CI 4.6–39.2; p < 0.0001). Patients with SLE carrying the TPMT*3C heterozygous genotype are at risk of azathioprine-induced myelosuppression.

Thiopurine Methyltransferase Genetic Polymorphisms and Activity and Metabolic Products of Azathioprine in Patients with Inflammatory Bowel Disease.

Thiopurine S-methyltransferase (TPMT) is a cytoplasmic enzyme that catalyzes thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. There is a correlation between thiopurine drug metabolism, response, and toxicity and genetic polymorphism of TPMT. The aim of this study is to assess TPMT genetic polymorphisms activity and metabolic products of AZA in patients with IBD. Blood samples were obtained from 50 IBD unrelated patients from a private laboratory. We used polymerase chain reaction-restriction length polymorphism (PCR-RFLP) and allele-specific PCRbased assays to determine the TPMT gene for the different variants. A high-performance liquid chromatography system (HPLC) was carried out to determine the whole blood 6-TGN concentration. Determination of serum TMPT activity was done by ELISA kit. In IBD patients, 46/50 (92%) subjects were homozygous for the wild-type allele (TPMT*1/*1). Mutant TPMT*1/*2 and TPMT*1/*3C alleles were found in 4/46 (8%) and 3/47 (6%) of IBD patients, respectively. TPMT*1/*3B variant was not detected in any of the IBD patients. TPMT enzyme activity was higher in wild-type than that mutant variants TPMT*1/*2 and TPMT*1/*3C, suggesting that there are statistically significant differences between 6-TG levels and polymorphisms of TMPT enzyme. 6-TG levels significantly increased in IBD patients mutant variants TPMT*1/*2 and TPMT*1/*3C. Our results showed that TPMT polymorphisms are associated with 6-TGN levels in patients using AZA. This study suggests that AZA dosage may be determined according to the high or low prevalence of a TPMT genotype. Moreover, the results present the determination of metabolite for assessing possible safe effective dosage of the drug.

Frequency of serious adverse events of thiopurine treatment in normal thiopurine S-methyltransferase genotype children with inflammatory bowel disease

Genetic polymorphism of thiopurine S-methyltransferase, the key enzyme in metabolism of thiopurines (azathioprine and 6-mercaptopurine) used in the treatment of inflammatory bowel disease, results in different enzyme activities. Decreased enzyme activity causes myelosuppression whereas abnormally high activity results in hepatotoxicity at standard thiopurine doses. Four allele variants (TMPT*2, TMPT*3A, TPMT*3B and TPMT*3C) account for decreased activity in more than 95% of cases. To evaluate the frequency of severe side effects, such as myelosuppression and hepatotoxicity, at standard or decreased azathioprine doses in children with inflammatory bowel disease who do not exhibit any of the four most common variant alleles. Retrospective analysis of children with inflammatory bowel disease treated with azathioprine at a single tertiary referral center. 51 patients were identified (44: Crohn's disease, 7: ulcerative colitis; male ratio: 28/51; mean age at diagnosis: 12.4 years). Two patients discontinued azathioprine arbitrarily whereas in one patient it was stopped due to serious pancreatitis and in another one because of severe flu-like symptoms. None of the remaining 47 patients exhibited hepatotoxicity suggesting abnormally high thiopurine S-methyltransferase activity. Four patients (8.5%) had profound myelosuppression on less than 1 mg/kg/day azathioprine requiring discontinuation of the drug, and all of them showed complete bone marrow recovery subsequently. No myelosuppression occurred in the remaining 43 patients on 2.17 ± 0.31 mg/kg/day (mean ± SD) azathioprine treatment. Regular blood tests are necessary on thiopurine therapy despite normal thiopurine S-methyltransferase genotype because of the risk of myelosuppression. The four most common variant alleles were identified in routine genotyping only, therefore most likely rare variant allele(s) or polymorphism of other enzymes involved in thiopurine metabolism account for the aforementioned four cases with profound bone marrow suppression. Thiopurine metabolite monitoring is the key for individualized treatment when optimal dosing can be achieved with the least side effects. Orv Hetil. 2019; 160(5): 179-185.

Thiopurine S-Methyltransferase Polymorphism in Iraqi Paediatric Patients with Acute Lymphoblastic Leukaemia

Thiopurine S-Methyltransferase Polymorphism in Iraqi Paediatric Patients with Acute Lymphoblastic Leukaemia

Detection of Thiopurine S-Methyltransferase (TPMT) Polymorphisms TPMT*3A, TPMT*3B and TPMT*3C in Children with Acute Lymphoblastic Leukemia

Background: Thiopurines are essential medications in Acute Lymphoblastic Leukemia (ALL) treatment protocols as anti-cancer agents since long time; however, their use might result in unexpected toxicities in ALL children due to the low thiopurine S-methyltransferase (TPMT) activity, a major enzyme involved in 6- mercaptopurine metabolism, which strongly correlates to the genetic polymorphism of the TPMT gene in those patients.&#x0D; Objective: To identify the most common TPMT polymorphisms in children with ALL and its frequencies.&#x0D; Methods: A cross sectional study enrolling eighty-one ALL children receiving mercaptopurine drug during their maintenance course of treatment according to UKALL – 2011 protocol, were enrolled in this study. After DNA extraction from whole blood TPMT genetic polymorphisms were detected by allele-specific multiplex-PCR analysis.&#x0D; Results: A total of 51 children with allele frequencies of (62.96%) were homozygous for the wild-type allele TPMT*1, 30 children with allelic frequency of (37.03%) were heterozygous for one of the two mutant alleles (TPMT*3A or TPMT*3C) with allele frequencies of 29.62% and 7.4% respectively, while no result was found homozygous for two mutant alleles or TPMT*3B allele.&#x0D; Conclusions: This is the first study in Iraq to identify the genetic polymorphism of TPMT in a group of ALL children being treated for ALL. The study revealed the presence of TPMT*3A and TPMT*3C genetic polymorphisms among the study sample, no TPMT*3B was identified in the study sample.

Influence of thiopurine S‐methyltransferase polymorphisms in mercaptopurine pharmacokinetics in healthy volunteers

Mercaptopurine is a drug commonly used in the treatment of different types of cancer, especially acute lymphoblastic leukaemia, and autoimmune diseases such as ulcerative colitis or Crohn's disease and in patients receiving organ transplants. It is metabolized by three cytosolic enzymes. One of them, thiopurine S-methyltransferase (TPMT), is responsible for catalysing the methylation reaction of mercaptopurine to 6-methylmercaptopurine, thus inactivating the drug. Individuals with TPMT loss-of-function alleles (*2, *3A, *3B or *3C) can be extremely sensitive to the effect of mercaptopurine, since it can be accumulated, therefore producing haematological toxicity. The objective of this study was to evaluate the role of TPMT polymorphisms on the pharmacokinetics of mercaptopurine. For that purpose, we used collected pharmacokinetic data from 48 healthy volunteers (all males) who received a single oral dose of mercaptopurine 50 mg in two bioequivalence studies. The volunteers were subsequently genotyped for TPMT *2, *3A, *3B and *3C alleles by real-time PCR. There were four carriers (8.3%) of TPMT*2 and TPMT*3A alleles. Mercaptopurine elimination was affected by TPMT loss-of-function polymorphisms, since heterozygous subjects show 18% higher half-life compared to wild-type individuals. This fact is consistent with the expected since the presence of loss-of-function alleles decreases TPMT enzymatic activity and, thus, affects mercaptopurine elimination. Moreover, mercaptopurine pharmacokinetic parameters were different among races, since Latins showed higher plasma concentrations and lower clearance compared to Caucasians. This fact might be due to a different distribution of polymorphisms in genes, other than TPMT, that also influence the pharmacokinetics of mercaptopurine.