Effects of genetic and clinical factors on thiopurine drugs pharmacokinetics in Tunisian patients.

Aim: Thiopurine drugs are used in the treatment of various diseases including inflammatory bowel disease. Thiopurine-S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) are the crucial enzymes involved in thiopurines metabolism. The present study aims to investigate in Tunisian patients, the influence of genetic and nongenetic factors on thiopurine drugs pharmacokinetics.Experimental approach: We have included patients having received thiopurine drugs and have undergone 6-thioguanine nucleotides (6-TGN) concentration monitoring. The identification of TPMT and ITPA polymorphisms was performed using the polymerase chain reaction-restriction fragment length polymorphism method. The impact of both genetic and nongenetic factors on the variability of the 6-TGN C/D ratio was analyzed through a stepwise multiple regression model.Key results: One hundred and twenty-three patients were included in the study. For TPMT, the most frequent variant allele was TPMT*3B (3.3%). For ITPA, the predominant polymorphism was the c.IVS2 + 21A> C (7%). We have demonstrated that only gender, the TPMT*3A and TPMT*3C alleles are significantly involved on the variability of thiopurines pharmacokinetics.Conclusion: Our study is the first to evaluate, in African patients, the impact of both genetic and nongenetic factors on the thiopurine drugs pharmacokinetics. Considering the narrow therapeutic range of these drugs, TPMT genotyping combined with 6-TGN blood concentration monitoring may enhance their efficacy and safety.

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Personalization of thiopurine therapy: Current recommendations and future perspectives.

Despite great therapeutic advances in the field of biologics, small synthetic molecules such as thiopurines, including azathioprine, mercaptopurine, and thioguanine, remain an important therapeutic pillar in the treatment of inflammatory bowel disease, other autoimmune disorders, and cancer. This review presents the latest guidelines for thiopurine administration, highlighting the importance of individualized therapy guided by pharmacogenomics. It emphasizes dose adjustment based on nudix hydrolase 15 (NUDT15) and thiopurine S-methyltransferase (TPMT) genotype, along side thiopurine S-methyltransferase activity and thiopurine metabolic profile. In addition, the article takes a critical look at emerging research in the field of thiopurine pharmaco genomics featuring novel genetic markers and technological developments in genetic testing. Finally, the potential of integrated approaches that combine genetic, meta bolic, and clinical factors to further individualize thiopurine therapy is highlighted.

Influence of TPMT and NUDT15 Genetic Polymorphisms on Mercaptopurine Pharmacokinetics in Healthy Volunteers.

Aims: This study aimed to investigate the impact of genetic polymorphisms of thiopurine methyltransferase (TPMT) and NUDT15 on pharmacokinetics profile of mercaptopurine in healthy adults in China. Methods: Blood samples were obtained from 45 healthy adult volunteers who were administered azathioprine. Genomic DNA was extracted and sequenced for TPMT and NUDT15. The plasma concentrations of 6-mercaptopurine (6-MP) were determined by ultra-performance liquid chromatography-tandem mass spectrometry. Finally, pharmacokinetic parameters were calculated based on the time-concentration curve. Results: Among the 45 healthy adult volunteers enrolled in the study, two TPMT allelic variants and three NUDT15 allelic variants were detected. In total, six genotypes were identified, including TPMT*1/*1&NUDT15*1/*1, TPMT*1/*1&NUDT15*1/*2, TPMT*1/*1&NUDT15*1/*9, TPMT*1/*1&NUDT15*2/*5, TPMT*1/*6&NUDT15*1/*2, and TPMT*1/*3&NUDT15*1/*2. The results indicated that Area Under Curve (AUC) of 6-MP in volunteers with TPMT*1/*3&NUDT15*1/*2 and TPMT*1/*6&NUDT15*1/*2 were 1.57-1.62-fold higher than in individuals carrying the wild type (TPMT*1/*1&NUDT15*1/*1). Compared with wild type, the half-life (T1/2) of TPMT*1/*6&NUDT15*1/*2 was extended by 1.98 times, whereas T1/2 of TPMT*1/*3&NUDT15*1/*2 decreased by 67%. The maximum concentration (Cmax) of TPMT*1/*3&NUDT15*1/*2 increased significantly by 2.15-fold, whereas the corresponding clearance (CL/F) decreased significantly by 58.75%. Conclusion: The findings of this study corroborate the notion that various genotypes of TPMT and NUDT15 can impact the pharmacokinetics of mercaptopurine, potentially offering foundational insights for personalized mercaptopurine therapy.

Predicting Adverse Events to Thiopurines in IBD: Are We a Step Closer?

Thiopurines remain an important option in the treatment of IBD. However, the unpredictable and sometimes serious side effects and intolerance remain a major challenge. Pretreatment of extended genetic panel analysis, identification of novel variants, and monitoring of intermediate metabolites will help improve the overall outcome and reduce the toxicity.

Landscape of TPMT and NUDT15 Pharmacogenetic Variation in a Cohort of Canadian Pediatric Inflammatory Bowel Disease Patients.

BACKGROUND: Patients with inflammatory bowel disease (IBD) exhibit considerable interindividual variability in medication response, highlighting the need for precision medicine approaches to optimize and tailor treatment. Pharmacogenetics (PGx) offers the ability to individualize dosing by examining genetic factors underlying the metabolism of medications such as thiopurines. Pharmacogenetic testing can identify individuals who may be at risk for thiopurine dose-dependent adverse reactions including myelosuppression. We aimed to evaluate PGx variation in genes supported by clinical guidelines that inform dosing of thiopurines and characterize differences in the distribution of actionable PGx variation among diverse ancestral groups. METHODS: Pharmacogenetic variation in TPMT and NUDT15 was captured by genome-wide genotyping of 1083 pediatric IBD patients from a diverse Canadian cohort. Genetic ancestry was inferred using principal component analysis. The proportion of PGx variation and associated metabolizer status phenotypes was compared across 5 genetic ancestral groups within the cohort (Admixed American, African, East Asian, European, and South Asian) and to prior global estimates from corresponding populations. RESULTS: Collectively, 11% of the cohort was categorized as intermediate or poor metabolizers of thiopurines, which would warrant a significant dose reduction or selection of alternate therapy. Clinically actionable variation in TPMT was more prevalent in participants of European and Admixed American/Latino ancestry (8.7% and 7.5%, respectively), whereas variation in NUDT15 was more prevalent in participants of East Asian and Admixed American/Latino ancestry (16% and 15% respectively). CONCLUSIONS: These findings demonstrate the considerable interpopulation variability in PGx variation underlying thiopurine metabolism, which should be factored into testing diverse patient populations.

In a large, pediatric inflammatory bowel disease cohort comprised of 5 genetic ancestry groups, we evaluated the distribution of loss-of-function pharmacogenetic variants in TPMT and NUDT15 and predicted phenotypes (impact on thiopurine metabolism).

Assessing the Occurrence and Influence of Cancer Chemotherapy-Related Pharmacogenetic Alleles in the Chilean Population.

BACKGROUND: Pharmacogenomic knowledge as a biomarker for cancer care has transformed clinical practice, however, as current guidelines are primarily derived from Eurocentric populations, this limits their application in Latin America, particularly among Hispanic or Latino groups. Despite advancements, systemic chemotherapy still poses challenges in drug toxicity and suboptimal response. This study explores pharmacogenetic markers related to anticancer drugs in a Chilean cohort, filling a gap in Latin American research. Notably, the influence of native South American Mapuche-Huilliche ancestry. METHODS: To explore pharmacogenetic markers related to anticancer drugs, we utilized an ethnically Admixed Chilean genome-wide association studies (GWAS) dataset of 1095 unrelated individuals. Pharmacogenomic markers were selected from PharmGKB, totaling 36 level 1 and 2 evidence single nucleotide polymorphisms (SNPs) and 571 level 3 SNPs. Comparative analyses involved assessing SNP frequencies across diverse populations from the 1000 Genomes Project. Haplotypes were estimated, and linkage disequilibrium was examined. Ancestry-based association analyses explored relationships between SNPs and Mapuche-Huilliche and European ancestries. Chi-square distribution with p ≤ 0.05 and Bonferroni's multiple adjustment tests determined statistical differences between allele frequencies. RESULTS: Our study reveals significant disparities in SNP frequency within the Chilean population. Notably, dihydropyrimidine dehydrogenase (DPYD) variants (rs75017182 and rs67376798), linked to an increased risk of severe fluoropyrimidine toxicity, exhibit an exceptionally low frequency (minor allele frequency (MAF) < 0.005). Nudix hydrolase 15 (NUDT15) rs116855232, associated with hematological mercaptopurine toxicity, is relatively common (MAF = 0.062), and is further linked to Mapuche-Huilliche ancestry. Thiopurine methyltransferase enzyme (TPMT), implicated in severe toxicity to mercaptopurines, SNPs rs1142345 and rs1800460 of TMPT gene demonstrate higher MAFs in Admixed Americans and the Chilean population (MAF range 0.031-0.057). Finally, the variant in the UDP-glucuronosyltransferase 1 gene (UGT1A1) rs4148323, correlated with irinotecan neutropenia, exhibits the highest MAF in East Asian (MAF = 0.136) and Chilean (MAF = 0.025) populations, distinguishing them from other investigated populations. CONCLUSIONS: This study provides the first comprehensive pharmacogenetic characterization of cancer therapy-related SNPs and highlights significant disparities in SNP frequencies within the Chilean population. Our findings underscore the necessity for inclusive research and personalized therapeutic strategies to ensure the equitable and effective application of precision medicine across diverse global communities.

NUDT15 and TPMT polymorphisms in three distinct native populations of the Brazilian Amazon.

This is the first report of the distribution of TPMT and NUDT15 single nucleotide polymorphisms and metabolic phenotypes associated with cytotoxicity of thiopurine drugs, in indigenous groups of Brazilian Amazon: Munduruku, Paiter-Suruí and Yanomami. The minor allele frequency (MAF) of NUDT15 rs116855232 did not differ significantly across the groups; TPMT rs1800462 was absent, while rs1800460 and rs1142345 were in strong linkage disequilibrium, and 10- and 30-fold more common in Paiter-Suruí. Indeed, the MAFs in Paiter-Surui (0.193 and 0.188) are the largest report globally. The distribution of combined NUDT15/TPMT metabolic phenotypes differed significantly (p < 0.0001) and largely (Cramér´s V = 0.37) across cohorts. This has important pharmacogenetic implications: the Clinical Pharmacogenetics Implementation Consortium recommendations to reduce or consider reduction of thiopurine dose applies to 4.4% Yanomami, 5.6% Munduruku, versus 41% Paiter-Suruí. The proportion of Paiter-Suruí at risk of thiopurine intolerance is 3- to 4-fold higher than any other population worldwide.

SERS spectroscopy as a tool for the study of thiopurine drug pharmacokinetics in a model of human B leukemia cells.

Thiopurine drugs are immunomodulatory antimetabolites relevant for pediatric patients characterized by dose-dependent adverse effects such as myelosuppression and hepatotoxicity, often related to inter-individual differences, involving the activity of important enzymes at the basis of their biotransformation, such as thiopurine S-methyltransferase (TPMT). Surface Enhanced Raman Scattering (SERS) spectroscopy is emerging as a bioanalytical tool and represents a valid alternative in terms of affordable costs, shorter analysis time and easier sample preparation in comparison to the most employed methods for pharmacokinetic analysis of drugs. The aim of this study is to investigate mercaptopurine and thioguanine pharmacokinetics by SERS in cell lysates of a B-lymphoblastoid cell line (NALM-6), that did (TPMT*1) or did not (MOCK) overexpress the wild-type form of TPMT as an in vitro cellular lymphocyte model to discriminate between cells with different levels of TPMT activity on the base of the amount of thioguanosine nucleotides (TGN) metabolites formed. SERS analysis of the cell lysates was carried out using SERS substrates constituted by Ag nanoparticles deposited on paper and parallel samples were used for quantification of thiopurine nucleotides with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A direct SERS detection method has been set up that could be a tool to study thiopurine drug pharmacokinetics in in vitro cellular models to qualitatively discriminate between cells that do and do not overexpress the TPMT enzyme, as an alternative to other more laborious techniques. Results underlined decreased levels of TGN and increased levels of methylated metabolites when TPMT was overexpressed, both after mercaptopurine and thioguanine treatments. A strong positive correlation (Spearman's rank correlation coefficient rho = 0.96) exists between absolute quantification of TGMP (pmol/1 x 106 cells), obtained by LC-MS/MS, and SERS signal (intensity of TGN at 915 cm-1). In future studies, we aim to apply this method to investigate TPMT activity in pediatric patients' leukocytes.

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.

Azathioprine dose tailoring based on pharmacogenetic information: Insights of clinical implementation.

Azathioprine is commonly used as an immunosuppressive antimetabolite in the treatment of acute lymphoblastic leukemia, autoimmune disorders (such as Crohn's disease and rheumatoid arthritis), and in patients receiving organ transplants. Thiopurine-S-methyltransferase (TPMT) is a cytoplasmic trans-methylase catalyzing the S-methylation of thiopurines. The active metabolites obtained from thiopurines are hydrolyzed into inactive forms by the Nudix hydrolase 15 (NUDT15). The TPMT*2 (defined by rs1800462), *3A (defined by rs1800460 and rs1142345), *3B (defined by rs1800460), *3C (defined by rs1142345), *6 (defined by rs75543815), and NUDT15 rs116855232 genetic variant have been associated, with the highest level of evidence, with the response to azathioprine, and, the approved drug label for azathioprine and main pharmacogenetic dosing guidelines recommend starting with reduced initial doses in TPMT intermediate metabolizer (IM) patients and considering an alternative treatment in TPMT poor metabolizer (PM) patients. This study aims to assess the clinical impact of azathioprine dose tailoring based on TPMT genotyping studying the azathioprine toxicity and efficacy, treatment starts, and dose adjustments during follow-up, comparing TPMT IM/PM and normal metabolizer (NM) patients. It also studied the association of NUDT15 rs116855232 with response to azathioprine in patients receiving a tailored treatment based on TPMT and characterized the TMPT and NUDT15 studied variants in our population. Results show that azathioprine dose reduction in TPMT IM patients (TPMT*1/*2, *1/*3A, or *1/*3C genotypes) is related to lower toxicity events compared to TPMT NM (TPMT *1/*1 genotype), and lower azathioprine dose adjustments during follow-up without showing differences in the efficacy. The results support the hypothesis of existing other genetic variants affecting azathioprine toxicity.

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.

Preemptive TPMT Genotyping and Adherence to Genotype-Based Therapeutic Recommendations Reduces the Healthcare Cost in Patients Receiving Azathioprine or 6-Mercaptopurine for Autoimmune Diseases.

A cost analysis of thiopurine treatment was carried out in 257 patients, with 153 preemptively genotyped for TPMT and 104 retrospectively genotyped in a Spanish setting. The healthcare cost was significantly higher in patients retrospectively genotyped compared to those who were preemptively genotyped (p < 0.001). TPMT intermediate metabolizers (IMs) (n = 23) showed a 3.3-fold higher healthcare cost when compared to normal metabolizers (NMs) (p < 0.001). The healthcare cost in patients with a TPMT IM phenotype whose physician adhered to the genotype-informed recommendation was similar than the cost in TPMT NMs and was significantly lower than IMs whose physician did not adhere to the therapeutic recommendation (3.8-fold, p = 0.016). Myelotoxicity occurrence was significantly lower in patients preemptively vs. retrospectively genotyped (2.0% and 21.2%, respectively, p < 0.001). Patients who developed myelotoxicity showed a significantly higher healthcare cost than those who did not (4.10-fold, p < 0.001). Overall, 87% of patients whose dose was not adjusted despite being TPMT IMs suffered myelotoxicity, while only one of the eight patients (13%) whose dose was adjusted suffered myelotoxicity (p < 0.001). In conclusion, TPMT preemptive genotyping and physician adherence to genotype-informed therapeutic recommendations prevents myelotoxicity and significantly reduces the healthcare cost, and it is therefore essential for the sustainability of the Spanish healthcare system.

Genome-Wide Association Study for the Genetic Determinants of Thiopurine Methyltransferase Protein Expression in Human Livers and Racial Differences.

INTRODUCTION: Polymorphisms in the Thiopurine S-Methyltransferase (TPMT) gene are associated with decreased TPMT activity, but little is known about their impact on TPMT protein expression in the liver. This project is to conduct a genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) associated with altered TPMT protein expression in human livers and to determine if demographics affect hepatic TPMT protein expression. METHODS: Human liver samples (n = 287) were genotyped using a whole genome genotyping panel and quantified for TPMT protein expression using a Data-Independent Acquisition proteomics approach. RESULTS AND DISCUSSION: Thirty-one SNPs were found to be associated with differential expression of TPMT protein in the human livers. Subsequent analysis, conditioning on rs1142345, a SNP associated with the TPMT*3A and TPMT*3C alleles, showed no additional independent signals. Mean TPMT expression is significantly higher in wildtype donors compared to those carrying the known TPMT alleles, including TPMT*3A, TPMT*3C, and TPMT*24 (0.107 ± 0.028 vs. 0.052 ± 0.014 pmol/mg total protein, P = 2.2 × 10-16). After removing samples carrying the known TPMT variants, European ancestry donors exhibited significantly higher expression than African ancestry donors (0.109 ± 0.026 vs. 0.090 ± 0.041 pmol/mg total protein, P = 0.020). CONCLUSION: The GWAS identified 31 SNPs associated with TPMT protein expression in human livers. Hepatic TPMT protein expression was significantly lower in subjects carrying the TPMT*3A, TPMT*3C, and TPMT*24 alleles compared to non-carriers. European ancestry was associated with significantly higher hepatic TPMT protein expression than African ancestry, independent of known TPMT variants.

Pharmacogenetics of 6-mercaptopurine in a black Zimbabwean cohort treated for acute lymphoblastic leukaemia.

Background: 6-mercaptopurine usage is associated with myelotoxicity and increased risk in patients carrying metabolism-related genetic variations. This study aimed to determine the frequency of candidate gene polymorphisms and their association with 6-mercaptopurine intolerance. Methods: A total of 41 patients on acute lymphoblastic leukaemia treatment were genotyped for TPMT and NUDT15 (rs116855232) alleles, and their association with dose intensity was analyzed. Results: The defective TPMT*3C allele frequency was 9.8%. The median maintenance dose intensity for TPMT*1/*3C participants was considerably lower (47%) when compared with the TPMT*1/*1 wild-type (77%), although not statistically significant. Conclusion: This is the first pharmacogenetics study carried out in a black Zimbabwean leukemia patient cohort. The high defective TPMT*3C (9.8%) allele frequency points to the potential utility of pharmacogenetics testing for safe usage of 6-mercaptopurine in this population.

Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children in Zimbabwe and 6-mercaptopurine is frequently used as part of its treatment. However, 6-mercaptopurine is associated with side-effects such as severe neutropenia (a condition where you have a low number of white blood cells called neutrophils in your blood), with increased risk observed in patients carrying variants in genes involved in the metabolism of 6-mercaptopurine. Therefore, this study aimed to determine the frequency of polymorphisms in specific genes as well as their association with drug intolerance. A total of 41 patients on ALL treatment were studied. Review of treatment records was done to determine the cumulative 6-mercaptopurine dose and calculate dose intensity. Genotyping (to determine the versions of a gene a patient carries) for TPMT and NUDT15 (rs116855232) was performed and results correlated with drug dose intensity. The most frequent genotype was TPMT*1/*1, occurring in 80% of the participants. The remaining 20% were carriers with two different copies of TPMT (*1/*3C). The defective TPMT*3C variation occurred at 9.8% and none had TPMT*2, *3A, *3B or NUDT15 rs116855232 variants. Comparison analysis with dose intensity was done for 23 participants (56%) who had maintenance records available. The median dose intensity of 47% for TPMT*1/*3C participants was considerably low when compared to that of a normal TPMT*1/*1 patient, which was 77%. However, no statistically significant difference was observed between TPMT genotype and dose intensity. This is the first study in a group of leukemic Zimbabweans to investigate the frequency of TPMT and NUDT15 variants. With a high variation frequency of 9.8% for the defective TPMT*3C, pharmacogenetics testing for TPMT before treatment with 6-MP is recommended in the Zimbabwean population.

TPMT gene polymorphisms (c.238G>C, c.460G>A and c.719A>G) in a healthy Venezuelan population.

Background: The presence of polymorphisms in the TPMT gene is associated with adverse effects in patients treated with standard doses of thiopurine drugs. Scientific evidence recognizes significant ethnic differences in their frequencies and how their early identification can prevent clinical complications. Methods: 150 healthy residents of Aragua, Venezuela were enrolled. The SNPs c.460G>A and c.719A>G were detected by PCR-restriction fragment length polymorphism assay and c.238G>C by allele-specific PCR. Results: All genotype polymorphisms were heterozygous. TPMT*1/*3A, TPMT*1/*3C and TPMT*1/*2 genotypes were found in 4.0, 2.0 and 0.7%, respectively. Conclusion: 6.7% of individuals have an intermediate TPMT activity. These findings support the importance of prior genotyping of TPMT in Venezuelan patients who require thiopurine drug therapy.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AIMS: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation. RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. CONCLUSION: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

TPMT and NUDT15 testing for thiopurine therapy: A major tertiary hospital experience and lessons learned.

Variants in thiopurine methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15) are associated with an accumulation of cytotoxic metabolites leading to increased risk of drug-related toxicity with standard doses of thiopurine drugs. We established TPMT and NUDT15 genetic testing for clinical use and evaluated the utilization, service outcomes and potential value of multi-gene PGx testing for 210 patients that underwent pharmacogenetics (PGx) testing for thiopurine therapy with the aim to optimize service delivery for future prescribing. The test was most commonly ordered for Gastroenterology (40.0%) and Neurology (31.4%), with an average turnaround time of 2 days. Following testing, 24.3% patients were identified as intermediate or poor metabolizers, resulting in 51 recommendations for a drug or dose change in thiopurine therapy, which were implemented in 28 (54.9%) patients. In the remaining patients, 14 were not adjusted and 9 had no data available. Focusing on drug gene interactions available for testing in our laboratory, multi-gene PGx results would present opportunities for treatment optimization for at least 33.8% of these patients who were on 2 or more concurrent medications with actionable PGx guidance. However, the use of PGx panel testing in clinical practice will require the development of guidelines and education as revealed by a survey with the test providers. The evaluation demonstrated successful implementation of single gene PGx testing and this experience guides the transition to a pre-emptive multi-gene testing approach that provides the opportunity to improve clinical care.

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.

Side effects of steroid-sparing agents in patients with bullous pemphigoid and pemphigus: A systematic review.

Background: Systemic glucocorticoids are first-line treatment options for autoimmune blistering diseases; however, their long-term use is associated with significant toxicities. Objective: To evaluate the side effects of steroid-sparing agents and compare them with those of steroids. Methods: We searched Cochrane Reviews, Embase, MEDLINE, and Scopus between October 1978 and May 2020 using the keywords "bullous pemphigoid," "pemphigus," "autoimmune blistering diseases," and "side effects." A total of 31 randomized controlled trials and retrospective case series were critically appraised. Results: This review includes a total of 1685 patients with autoimmune blistering diseases, of whom 781 had bullous pemphigoid and 904 had either pemphigus vulgaris or pemphigus foliaceous. Limitations: A major limitation is that because adjuvants are generally used in combination with steroids, only 12 of the studies reviewed included a "steroid-only" arm to allow for a direct comparison of side effects. Additionally, there is inadequate literature and lack of standardized grade reporting of specific side effects of each steroid-sparing agent. Conclusion: In the future, researchers should consider implementing the Common Terminology Criteria for Adverse Events, version 5.0, for reporting of all side effects to allow for consistency and standardization. It would be useful to have an index similar to the Glucocorticoid Toxicity Index to quantify these side effects.