Thiopurine Drugs in the Treatment of Ulcerative Colitis: Identification of a Novel Deleterious Mutation in TPMT

Chronic inflammatory bowel disease (IBD) includes Crohn’s disease and ulcerative colitis. Both are characterized by inflammation of part of the digestive tract lining. Azathioprine (AZA) is a well-known immunosuppressant that has been known for many years for its ability to provide long-term disease remission in IBDs, but has important side effects, most of which are related to a single nucleotide polymorphism in the gene for thiopurine methyltransferase (TPMT), which ensures the degradation and efficacy of AZA. Since a direct correlation between TPMT gene polymorphisms and the haematological toxicity of the AZA treatment has been widely demonstrated, TPMT genotyping has been made necessary prior to any introduction of AZA. The monitoring of thiopurine metabolites presents one of the factors that limit wide adaptation of these thiopurines in clinical practice. Thus, identifying patients with asymmetric metabolism could help clinicians provide an ideal treatment recommendation to improve response and reduce adverse effects. Here, we review the role of AZA in the treatment of IBD and discuss the usefulness of TPMT genotyping to guide clinical decision-making. In addition, we report the identification of a new molecular alteration, never described, TPMT mutation affecting the TPMT activity and responsible for deleterious side effects in a clinical case of a 20-year-old woman patient.

Association between the TPMT*3C (rs1142345) Polymorphism and the Risk of Death in the Treatment of Acute Lymphoblastic Leukemia in Children from the Brazilian Amazon Region

Acute lymphoblastic leukemia (ALL) is the leading cause of death from pediatric cancer worldwide. However, marked ethnic disparities are found in the treatment of childhood ALL with less effective results and higher mortality rates being obtained in populations with a high level of Native American ancestry. Genetic variations of the patient can affect resistance to ALL chemotherapy and potentially play an important role in this disparity. In the present study, we investigated the association of 16 genetic polymorphisms with the cell and metabolic pathways of the chemotherapeutic agents used in the treatment of ALL with the risk of death in treating childhood ALL in patients with a high contribution of Amerindian ancestry, coming from the Brazilian Amazon. The study included 121 patients with B-cell ALL treated with the BFM-2002 protocol. We are the first to identify the association between the TPMT gene rs1142345 polymorphism and the high risk of death in treating childhood ALL. Patients with the CC genotype had an approximately 25.5 times higher risk of dying during treatment of the disease than patients with other genotypes (p = 0.019). These results may help elucidate how the patient’s genetic characteristics contribute to the mortality disparity in populations with a high contribution of Native American ancestry. The rs1142345 variant of the TPMT gene could be used as a potential marker to early stratify patients at high risk of death in treating childhood ALL in the investigated population.

Thiopurine S-methyltransferase genetic polymorphisms in adult patients with inflammatory bowel diseases in the Latvian population

Background: Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurines, and, for patients with inflammatory bowel disease (IBD), it is useful to perform TPMT genotyping prior to azathioprine (AZA) treatment. In this study, we determined TPMT gene polymorphisms in a cohort of IBD patients in Latvia. Methods: DNA samples were obtained from 244 IBD patients, and qPCR was performed for detection of rs1800462, rs1800460, and rs1142345 single-nucleotide polymorphisms (SNPs). Three common, non-functional TPMT alleles ( TPMT*2, *3B, and *3C) were identified (women, 51%; men, 49%). TPMT*2, *3A, *3B, and *3C allelic variants detected using qPCR were consistent with restriction fragment length polymorphism (RFLP) data. Results: Among patients, 78% had ulcerative colitis and 22% had Crohn’s disease, with 93.9% of the former carrying a wild-type homozygous TPMT*1/*1 genotype and 6.1% carrying heterozygous genotypes. The most frequent polymorphisms were TPMT*1/*3A (5.3%: two variants: TPMT*3B and TPMT*3C), TPMT*1/*3C (0.4%), and TPMT*1/*2 (0.4%). None of the patients carried a TPMT*3B polymorphism and no patients were homozygous for any mutation. Conclusion: This is the first study to identify TPMT gene polymorphisms in adult IBD patients in Latvia. The results indicate that the frequency of common TPMT alleles is similar to that of other European populations.

Correlation between Thiopurine S-Methyltransferase Genotype and Adverse Events in Inflammatory Bowel Disease Patients

Background and Objectives: In patients with inflammatory bowel diseases (IBD), the use of azathioprine results in adverse events at a rate of 5% to 20%. The aim of the study was to assess a possible correlation between genetic variability of the enzyme thiopurine S-methyltransferase (TPMT) and the development of toxicity to azathioprine. Materials and Methods: A retrospective, single center, blind, case-control study was conducted on 200 IBD patients, of whom 60 cases suspended azathioprine due to toxicity (leukopenia, pancreatitis, hepatitis, and nausea or vomiting), and 140 controls continued treatment with the drug without adverse events. Results: In the entire cohort, only 8 cases of heterozygous mutations of TPMT were observed, corresponding to 4% mutated haplotype rate, much lower than that reported in literature (close to 10%). No homozygous mutation was found. Regarding the TPMT allelic variants, we did not find any statistically significant difference between patients who tolerated azathioprine and those who suffered from adverse events. (OR = 0.77, 95% CI = 0.08–7.72; p = 0.82). Conclusions: According to our study, in IBD patients, the search for TPMT gene mutations before starting treatment with azathioprine is not helpful in predicting the occurrence of adverse events. Importantly, patients with allelic variants should not be denied the therapeutic option of azathioprine, as they may tolerate this drug.

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

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Analysis of polymorphisms in the TPMT gene in children with acute leukemia in the Krasnoyarsk Territory

Introduction.Leukemia accounts for 40 % of all malignant neoplasms under the age of 15 years in children. Determination of the genetic portrait of patients with acute lymphoblastic leukemia (ALL) helps to identify polymorphisms in the genes responsible for the metabolism of drugs included in standard treatment protocols.Materials and methods.51 children with confirmed diagnosis of acute lymphoid leukemia (ALL) were included in the analysis of the frequency of polymorphism of TPMT gene. The detection of polymorphisms TPMT*2, TPMT*3A and TPMT*3 was performed using a set of reagents “AmpliSens® Pyroskrin” & “FARMA-screen-2b”.Results.Polymorphisms in the TPMT gene of 51 patients were found in 6 (11.8 %) children. Of these, 4 patients have variant alleles TPMT*3A and TPMT*3C and 2 patients have only TPMT*3C. Of the 6 patients with TPMT polymorphism, two have a translocation t(12;21).