Population-Specific Distribution of TPMT Deficiency Variants and Ancestry Proportions in Ecuadorian Ethnic Groups: Towards Personalized Medicine.

Purpose: Thiopurine S-methyltransferase (TPMT) is an enzyme that metabolizes purine analogs, agents used in the treatment of acute lymphoblastic leukemia. Improper drug metabolism leads to toxicity in chemotherapy patients and reduces treatment effectiveness. TPMT variants associated with reduced enzymatic activity vary across populations. Therefore, studying these variants in heterogeneous populations, such as Ecuadorians, can help identify molecular causes of deficiency for this enzyme. Methods: We sequenced the entire TPMT coding region in 550 Ecuadorian individuals from Afro-Ecuadorian, Indigenous, Mestizo, and Montubio ethnicities. Moreover, we conducted an ancestry analysis using 46 informative ancestry markers. Results: We identified 8 single nucleotide variants in the coding region of TPMT. The most prevalent alleles were TPMT*3A, TPMT*3B, and TPMT*3C, with frequencies of 0.055, 0.012, and 0.015, respectively. Additionally, we found rare alleles TPMT*4 and TPMT*8 with frequencies of 0.005 and 0.003. Correlating the ancestry proportions with TPMT-deficient genotypes, we observed that the Native American ancestry proportion influenced the distribution of the TPMT*1/TPMT*3A genotype (OR = 5.977, p = 0.002), while the contribution of African ancestral populations was associated with the TPMT*1/TPMT*3C genotype (OR = 9.769, p = 0.003). The rates of TPMT-deficient genotypes observed in Mestizo (f = 0.121) and Indigenous (f = 0.273) groups provide evidence for the influence of Native American ancestry and the prevalence of the TPMT*3A allele. In contrast, although Afro-Ecuadorian groups demonstrate similar deficiency rates (f = 0.160), the genetic factors involved are associated with contributions from African ancestral populations, specifically the prevalent TPMT*3C allele. Conclusion: The distribution of TPMT-deficient variants offers valuable insights into the populations under study, underscoring the necessity for genetic screening strategies to prevent thiopurine toxicity events among Latin American minority groups.

Implications of Tioguanine Dosing in IBD Patients with a TPMT Deficiency.

Tioguanine is metabolised by fewer enzymatic steps compared to azathioprine and mercaptopurine, without generating 6-methylmercaptopurine ribonucleotides. However, thiopurine S-methyl transferase (TPMT) plays a role in early toxicity in all thiopurines. We aimed to describe the hazards and opportunities of tioguanine use in inflammatory bowel disease (IBD) patients with aberrant TPMT metabolism and propose preventative measures to safely prescribe tioguanine in these patients. In this retrospective cohort study, all determined TPMT genotypes (2016-2021) were evaluated for aberrant metabolism (i.e., intermediate and poor TPMT metabolisers). Subsequently, all IBD patients on tioguanine with aberrant TPMT genotypes were evaluated for tioguanine dosages, adverse drug events, lab abnormalities, treatment duration and effectiveness. TPMT genotypes were determined in 485 patients, of whom, 50 (10.3%) and 4 patients (0.8%) were intermediate and poor metabolisers, respectively. Of these patients, 12 intermediate and 4 poor TPMT metabolisers had been prescribed tioguanine in varying doses. In one poor TPMT metaboliser, tioguanine 10 mg/day induced delayed pancytopenia. In general, reduced tioguanine dosages of 5 mg/day for intermediate TPMT metabolisers, and 10 mg two-weekly for poor TPMT metabolisers, resulted in a safe, long-term treatment strategy. Diminished or absent TPMT enzyme activity was related with a pharmacokinetic shift of tioguanine metabolism which is associated with relatively late-occurring myelotoxicity in patients on standard tioguanine dose. However, in strongly reduced dose regimens with strict therapeutic drug and safety monitoring, tioguanine treatment remained a safe and effective option in IBD patients with dysfunctional TPMT.

Case Report: Multifocal EBV-Associated Diffuse Large B-Cell Lymphoma in a Patient With 6-MP Associated Lymphopenia With TPMT Deficiency.

Introduction: EBV associated lymphoproliferative disorders (EBV LPD) are a known complication following solid organ or hematopoietic stem cell transplantation. The disturbance of the immune system leads to a lack of control of latent EBV-infected B-cells, as control by T-cells is mandatory to prevent uninhibited cell proliferation. EBV LPD in other settings is less frequent and etiology and pathogenesis are not completely understood. Case Presentation: We present the case of an 18-year old adolescent suffering from lymphoblastic T-cell lymphoma who developed a life-threatening EBV associated B-cell lymphoma while he was under therapy with 6-MP (6- mercaptopurine). An underlying homozygous TPMT (thiopurine S-methyltransferase) deficiency with subsequent insufficient degradation of 6-MP was identified as contributory for the development of a distinct lymphopenia leading to EBV LPD. The patient was successfully treated by discontinuation of 6-MP and initiating rituximab monotherapy. Discussion: Rare cases of EBV LPD during therapy with 6-MP are reported in patients with leukemia, but no data about TPMT pharmacogenomics are available. In contrast the disease development in the presented case may be explained by the iatrogenic immunosuppression in the context of TPMT deficiency. While using 6-MP testing of genetic variations is not required for every protocol, although the use of thiopurines in patients with TPMT deficiency can cause severe immunosuppression. Our case suggests that insufficient degradation of 6-MP can have significant consequences despite dose reduction. Conclusion: When using thiopurines, TPMT genetics should be initiated and strict drug monitoring and dose adjusting must be performed by a specialized center.

[Genetic polymorphisms of thiopurine methyltransferase and incidence of adverse events in patients with medical indication of azathioprine]. / Variantes genéticas de la tiopurina metiltransferasa e incidencia de eventos adversos en pacientes con indicación de azatioprina.

Azathioprine is a thiopurine which has a narrow therapeutic index and marked hematological and hepatic toxicity. Thiopurine s-methyltransferase is an enzyme involved in the metabolism of thiopurines. Mutations in the gene that encodes the enzyme may augment the risk of adverse events. For that reason, pharmacogenetic determinations prior to the initiation of therapy can provide useful information for the future therapeutic strategy. Nevertheless, its utility in the local environment is not completely established. Forty-five subjects (13 men) who had been prescribed azathioprine were included. The presence of *2, *3A, *3B and *3C mutations were determined by PCR-RFLP, and the relationship between genotype and incidence of adverse events related to the drug was analyzed. Nine carried at least one non-functional allele, one of them with *3A/*3A genotype. Among the eighteen patients who initiated treatment with azathioprine, toxicity was detected in 3 cases: 2 mild events were observed in patients with normal genotype, and the only serious event (bone marrow suppression) occurred in the individual with homozygous mutant genotype. The only homozygous mutant patient developed the most severe of the registered events, in spite of being under treatment with low doses of azathioprine. This is the reason why enzymatic determination could be of utility, even though it does not replace clinical and biochemical follow-up in patients under thiopurine treatment.

Variantes genéticas de la tiopurina metiltransferasa e incidencia de eventos adversos en pacientes con indicación de azatioprina / Genetic polymorphisms of thiopurine methyltransferase and incidence of adverse events in patients with medical indication of azathioprine

La azatioprina es una tiopurina que presenta rango terapéutico estrecho y marcada toxicidad hematológica y hepática. La tiopurina S-metiltransferasa es una enzima que metaboliza ese grupo de drogas. Mutaciones en el gen que codifica dicha enzima aumentan el riesgo de presentar eventos adversos, por lo que su estudio farmacogenético permite contar con información para el diseño de la estrategia terapéutica. Sin embargo, su utilidad en el medio local no está completamente establecida. Fueron incluidos 45 sujetos (13 hombres) con indicación de azatioprina. Se determinó la presencia de las mutaciones *2, *3A, *3B y *3C de TMPT por PCR-RFLP y se analizó la relación entre el genotipo y la incidencia de eventos adversos relacionados al fármaco. Nueve portaban al menos un alelo no funcional, uno de ellos con genotipo *3A/*3A. Se detectó toxicidad en 3 de los 18 que iniciaron tratamiento con azatioprina: 2 pacientes con genotipo normal presentaron eventos adversos leves, y el único evento adverso de gravedad (aplasia medular) ocurrió en el sujeto con genotipo homocigota mutado. El único que presentó genotipo homocigota mutado desarrolló el más grave de los eventos adversos registrados, a pesar de estar en tratamiento con dosis bajas de azatioprina. Por este motivo, la determinación del genotipo de la tiopurina metiltransferasa pareciera ser de utilidad, pero no reemplaza la necesidad de seguimiento clínico y bioquímico en pacientes en tratamiento con tiopurinas.

Azathioprine is a thiopurine which has a narrow therapeutic index and marked hematological and hepatic toxicity. Thiopurine s-methyltransferase is an enzyme involved in the metabolism of thiopurines. Mutations in the gene that encodes the enzyme may augment the risk of adverse events. For that reason, pharmacogenetic determinations prior to the initiation of therapy can provide useful information for the future therapeutic strategy. Nevertheless, its utility in the local environment is not completely established. Forty-five subjects (13 men) who had been prescribed azathioprine were included. The presence of *2, *3A, *3B and *3C mutations were determined by PCR-RFLP, and the relationship between genotype and incidence of adverse events related to the drug was analyzed. Nine carried at least one non-functional allele, one of them with *3A/*3A genotype. Among the eighteen patients who initiated treatment with azathioprine, toxicity was detected in 3 cases: 2 mild events were observed in patients with normal genotype, and the only serious event (bone marrow suppression) occurred in the individual with homozygous mutant genotype. The only homozygous mutant patient developed the most severe of the registered events, in spite of being under treatment with low doses of azathioprine. This is the reason why enzymatic determination could be of utility, even though it does not replace clinical and biochemical follow-up in patients under thiopurine treatment.

Genotipo y fenotipo de la enzima tiopurina metiltransferasa en población chilena / Phenotype and genotype of thiopurine methyltransferase in Chilean individuals

Background: Thiopurines (azathioprine and 6-mercaptopurine) are highly effective medications but with potential adverse effects. Thiopurine methyltransferase (TMPT) is the key enzyme in their pharmacokinetics and is genetically regulated. A low activity of TPMT is associated with myelotoxicity. The genotype and enzyme activity can vary by ethnicity. Aim: To study the activity and genotype of TPMT in a group of Chilean subjects. Material and Methods: In 200 healthy adult blood donors, TPMT activity was determined by high performance liquid chromatography (HPLC). Deficient, low, normal or high levels were defined when enzymatic activity was < 5, 6-24,25-55 and > 56 nmol/grHb/h, respectively. Genotyping of TPMT (*1, *2, *3A, *3B, *3C) was performed by PCR. Results: Seventy seven women (38.5%) and 123 men (61.5%), with an average age of 34.9 years were studied. Eighteen subjects (9%) had a low enzymatic activity, 178 (89%) had normal activity, 4 (2%) had high activity and no genotype deficient subjects were identified. The wild type genotype (*1) was found in 184 (92%) individuals and 16 (8%) were heterozygous for the variants: *2 (n = 2), *3A (n = 13) and *3C (n = 1). No homozygous subjects for these variants were identified. Wild type genotype had an increased enzymatic activity (40.8 ± 7.2 nmol/gHb/h) compared to heterozygous group (21.2 ± 3 nmol/ gHb/h; p < 0.001). Conclusions: Less than 10% of a Chilean population sample has a low enzymatic activity or allelic variants in the TPMT gene, supporting the use of thiopurines according to international recommendations.