NUDT15 Genotyping Research Articles

Genetic polymorphisms and their association with methotrexate polyglutamates during maintenance treatment in Korean children and young adults with acute lymphoblastic leukemia

The aim of this study was to investigate the impact of genetic polymorphisms on methotrexate (MTX) metabolism in Korean children and young adults with acute lymphoblastic leukemia, specifically focusing on MTX polyglutamates (MTX-PGs) in erythrocytes, which have been rarely studied. Korean children and young adult patients undergoing maintenance therapy for acute lymphoblastic leukemia, who were receiving weekly oral MTX doses of 20 mg/m²/week, were prospectively included. We investigated erythrocyte MTX-PG (PG1 to PG5) levels, MTX-PG/MTX dose ratios, and 222 genetic polymorphisms spanning 78 genes and three intergenic areas related to MTX transport, folate cycle metabolism, purine/pyrimidine pathways, and non-pathway genes (including TPMT and NUDT15 genotypes) to explore their association with MTX metabolism. MTX-PG levels were associated with MTX dose (p < 0.05), and MTX-PG3 comprised the majority of the total MTX-PGs, with a median of 39.3 %. Various polymorphisms within the same gene demonstrated differing associations with each type of MTX-PG, underscoring the complexity of MTX pharmacogenetics. Among the polymorphisms examined, 14 across 13 genes showed significant associations with MTX-PG2–5 levels, even after adjusting for the false discovery rate (ABCC5, ATG16L1, CEP72, FSTL5, GMPS, HTR3A, IMPDH1, NT5C2, SLC28A3, SLCO1B3, SUCLA2, TPMT, and TYMS). This study enhances our understanding of genetic polymorphisms in MTX metabolism and therapeutic monitoring for MTX maintenance, promoting personalized medicine in acute lymphoblastic leukemia patients.

A systematic review of aspects of NUDT15 pharmacogenomic variants and thiopurine-induced myelosuppression

Abstract Objectives Evidence for NUDT15 pharmacogenomic variants and thiopurine-induced myelosuppression (TIM), consists predominantly of association data in Asian, mixed variant homozygote/heterozygote populations. We therefore sought evidence on; (i) NUDT15 genotype-guided thiopurine dosing. (ii) Association data for TIM in NUDT15 variant heterozygotes with inflammatory bowel disease. (iii) Association data for NUDT15 variants with TIM in Europeans. (iv) Health economic data for NUDT15 genotyping in inflammatory bowel disease. Methods A systematic review was conducted, consisting of database searches, screening against pre-defined inclusion/exclusion criteria, and assessment of risk of bias using study-specific appraisal tools. Key findings Titles/abstracts of 493 articles were screened, with 29 studies included. (i) Significant reductions in TIM with genotype-guided thiopurine dosing were reported by both trials and a cohort study. (ii) TIM rates were significantly higher in NUDT15*3 heterozygotes vs. wild type. Data were conflicting for rarer variants. (iii) Four of five studies reported an association with TIM for at least one or a combination of NUDT15 variants in Europeans (OR 9.5–38.2), but data were conflicting. (iv) Both health economic analyses found TPMT/NUDT15 genotyping cost-effective in Asian populations, but not when a European population was considered. Conclusion Limited data showed an association with TIM in NUDT15 variant heterozygotes and Europeans and the potential for genotype-guided dosing to reduce TIM. Studies were generally small, heterogenous, and of variable quality. The low prevalence of rarer NUDT15 variants/variants in Europeans likely contributed to contradictory findings. Further research on the clinical utility of genotyping in diverse populations will help inform future economic analyses.

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

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. 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. 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. The above genotypes do not appear to be associated with AZA-induced leukopenia in an eastern Indian population.

Real-world NUDT15 genotyping and thiopurine treatment optimization in inflammatory bowel disease: a multicenter study

BackgroundThis study evaluated the effectiveness of NUDT15 codon 139 genotyping in optimizing thiopurine treatment for inflammatory bowel disease (IBD) in Japan, using real-world data, and aimed to establish genotype-based treatment strategies.MethodsA retrospective analysis of 4628 IBD patients who underwent NUDT15 codon 139 genotyping was conducted. This study assessed the purpose of the genotyping test and subsequent prescriptions following the obtained results. Outcomes were compared between the Genotyping group (thiopurine with genotyping test) and Non-genotyping group (thiopurine without genotyping test). Risk factors for adverse events (AEs) were analyzed by genotype and prior genotyping status.ResultsGenotyping test for medical purposes showed no significant difference in thiopurine induction rates between Arg/Arg and Arg/Cys genotypes, but nine Arg/Cys patients opted out of thiopurine treatment. In the Genotyping group, Arg/Arg patients received higher initial doses than the Non-genotyping group, while Arg/Cys patients received lower ones (median 25 mg/day). Fewer AEs occurred in the Genotyping group because of their lower incidence in Arg/Cys cases. Starting with < 25 mg/day of AZA reduced AEs in Arg/Cys patients, while Arg/Arg patients had better retention rates when maintaining ≥ 75 mg AZA. Nausea and liver injury correlated with thiopurine formulation but not dosage. pH-dependent mesalamine reduced leukopenia risk in mesalamine users.ConclusionsNUDT15 codon 139 genotyping effectively reduces thiopurine-induced AEs and improves treatment retention rates in IBD patients after genotype-based dose adjustments. This study provides data-driven treatment strategies based on genotype and identifies risk factors for specific AEs, contributing to a refined thiopurine treatment approach.

The frequency of NUDT15 rs116855232 and its impact on mercaptopurine-induced toxicity in Syrian children with acute lymphoblastic leukemia.

Polymorphisms in NUDT15 may result in differences in mercaptopurine-induced toxicity. This study aimed to identify the frequency of the NUDT15 (c.415C>T; rs116855232) polymorphism and investigate the effect of this polymorphism on mercaptopurine-induced toxicity in a population of Syrian patients with childhood acute lymphoblastic leukemia (ALL). This is a retrospective study that included children with ALL reaching at least 6 months of maintenance therapy. The NUDT15 genotyping was determined using standard targeted sequencing of polymerase chain reaction products. The odds ratio (OR) for the association between toxicity and genotype was evaluated. A total of 92 patients were enrolled. The majority of the patients in the study population were low-risk (63.04%), followed by intermediate-risk (25%), and high-risk (11.96%). There were 5 patients (5.4%) with NUDT15 (c.415C>T; rs116855232) CT genotype, and 1 patient (1.08%) with NUDT15 TT genotype, with allele frequencies of C=0.962 and T=0.038. The mercaptopurine median dose intensity was 100%, 54.69%, and 5% for the genotypes CC, CT, and TT, respectively (P=0.009). Early onset leukopenia was significantly associated with the NUDT15 polymorphism (OR: 6.16, 95% CI: 1.11-34.18, P=0.037). There was no association between the NUDT15 genotype and hepatotoxicity. Approximately 6.5% of the study population exhibited reduced NUDT15 activity. The mercaptopurine dose intensity was considerably low in NUDT15 rs116855232 TT genotype compared with CT and CC. The dosage of mercaptopurine should be adjusted according to the NUDT15 genotype in pediatric patients with ALL.

Influence of NUDT15 Genotyping on Dose Intensity of Thiopurine Administration and Long-Term Clinical Outcomes (Hospitalization and Surgery)

Influence of NUDT15 Genotyping on Dose Intensity of Thiopurine Administration and Long-Term Clinical Outcomes (Hospitalization and Surgery)

Thiopurines exert harmful effects on spermatogenesis in Nudt15R138C knock-in mice.

The association between thiopurine use and testicular reproductive functions remains unclear. In this study, we investigated whether thiopurines affect testicular functions based on the NUDT15 genotypes using Nudt15R138C knock-in mice. The male Nudt15R138C knock-in mice (9-12weeks) were treated with mercaptopurine (MP: 0.5mg/kg/day) for 4 or 12weeks. To examine reversibility, some mice were maintained for a further 12weeks under MP-free condition. After MP treatment for 4weeks, Nudt15R138C/R138C mice exhibited a significant reduction of testis weight compared to Nudt15+/+ mice and Nudt15+/R138C mice. The epithelial height and diameter of seminiferous tubules were significantly reduced in Nudt15R138C/R138C mice compared to Nudt15+/+ and Nudt15+/R138C mice. Apoptotic cells were significantly increased in Nudt15R138C/R138C mice, and most of apoptotic cells were spermatogonia. There were no significant changes in sperm counts and sperm morphology in MP-treated Nudt15R138C/R138C mice after 4-week MP treatment. On the other hand, after MP treatment for 12weeks, the Nudt15+/R138C mice, but not Nudt15+/+ mice, exhibited a significant reduction in the testis weight and atrophic changes of seminiferous tubules, but these changes disappeared after 12-week rearing under MP-free condition. Despite a significant increase in abnormal sperm rate, there were no changes in the ability to conceive. No differences in serum levels of follicle-stimulating hormone or testosterone were observed between MP-treated Nudt15+/R138C and Nudt15+/+ mice after 12-week MP treatment. Thiopurines exert harmful effects on testicular reproductive function according to host NUDT15 genotypes.

Results of a Prospective Phase II Study of Individualized 6-Mercaptopurine Dosing Based on Pharmacogenomics in Childhood Acute Lymphoblastic Leukemia in East Asia

Introduction Variations in the NUDT15 and TPMT genes, which serve as pharmacogenomic markers for 6-mercaptopurine (6MP), contribute to individual differences in the optimal dosage of this medication. Nevertheless, given the observed difference in the tolerated dose of 6MP between East Asian and Western patients, it is essential to conduct a study to determine whether the same dosage guidelines are applicable to both populations. This study aimed to reduce treatment discontinuation and neutropenia during maintenance by adjusting the initial dose of 6MP based on the results of the NUDT15 or TPMT variants. M ethods This is a prospective phase II trial being conducted at three institutions in Korea. Patients who initiated 6MP-based maintenance treatment for acute lymphoblastic leukemia (ALL) were included in the study. During the maintenance phase, 6MP was administered at an individualized starting dose based on the NUDT15 and TPMT variants of each participant. Original dose (50 mg/m 2/day) of 6MP was administered to patients with both NUDT15 and TPMT wild-type, 30 mg/m 2/day for NUDT15 intermediate activity ( NUDT15 variant allele heterozygous) or TPMT heterozygosity, 10 mg/m 2/day for NUDT15 low activity ( NUDT15 variant allele homozygous), and 10 mg/m 2/day three times per week for homozygous TPMT variants. The maintenance cycle consists of 12 weeks of daily 6MP at an individualized dosage, weekly methotrexate (MTX) at a dosage of 20 mg/m 2, and monthly vincristine and steroid pulses. Lastly, intrathecally administered MTX is administered every three months. During treatment, the total white blood cell count was targeted to be between 2000/uL and 3000/uL, with an absolute neutrophil count greater than 500/uL and a platelet count greater than 50,000/uL. The whole exome was sequenced using genomic DNA extracted from peripheral blood at complete remission or hair follicles. As the primary and secondary endpoints, the duration of treatment discontinuation, the frequency of neutropenia, the presence of neutropenic fever during maintenance therapy, and the recurrence rate were compared to historical controls. A cohort of 254 patients who underwent maintenance therapy between the years 2001 and 2018 was chosen as the historical control group. Results Total 72 patients were enrolled from July 2019 until Aril 2022. This analysis was conducted on 52 patients who finished maintenance therapy. There were 32 males, and 20 females. The median age of the patients was 5.6 years (range, 2.2~15.9 years). The immunophenotypes of ALL was B-ALL in 40 patients, T-ALL in 10, Mixed phenotype in 1, and unspecified ALL in 1 patient. No patient had known pharmacogenetic variants in TPMT. The NUDT15phenotypes based on diplotypes included normal activity (n=41), intermediate activity (n=9), and low activity (n=2), occurring in 78.8%, 17.3%, and 3.8% respectively (Table 1). The number of days discontinued due to any toxicity during the 1 st cycle of maintenance therapy was average 7 days (max 59 days) in all patients, and that of the study group with intermediate enzyme activity phentypes was significantly shorter than in historical controls, with an average 6 days vs. 12 days ( P=0.03, Figure 1). The incidence of neutropenia during the 1 st cycle between two groups was not statistically different, however, among patients with NUDT15 or TPMT intermediate activity, the frequency of neutropenia was significantly lower in the study group ( P=0.03, Figure 2). The frequency of neutropenic fever (NF) and fever without neutropenia during the 1st cycle did not differ between the two groups. However, when patients with NUDT15 or TPMT intermediate activity were analyzed, no fever occurred in the study group of 9 patients, while NF and fever occurred in 11 and 8 patients in the control group (Figure 3). Conclusions Individualized administration of 6MP based on NUDT15 and TPMT genotypes during maintenance therapy in Korean pediatric ALL patients significantly reduced treatment discontinuation and febrile toxicity. A decrease in the initial dosage from 50 mg/m 2/day to 30 mg/m 2/day has the potential to reduce febrile toxicity in the patient group exhibiting intermediate activity of the NUDT15 enzyme, which is observed in approximately 20% of individuals in East Asia. Through this research, we expect to provide the dosing guidelines for 6MP in patients with ALL in East Asia.

Usefulness and difficulties with the thiopurine pharmacogenomic NUDT15 genotyping test: Analysis of real-world data in Japan

The usefulness of NUDT15 genotyping as a pharmacogenomic test for thiopurine has been established. The first such test developed to date, NUDT15 genotyping was approved for reimbursement in Japan in February 2019 for all indicated patients. We retrospectively examined claims data in Japan and confirmed that the proportion of patients who undergo genotyping before initiating a new thiopurine regimen has increased; furthermore, genotyping has improved the rate of treatment continuation and reduced on-treatment hospitalization. However, the genotyping rate before thiopurine induction was >50% for patients with inflammatory bowel disease and <20% for those with other immune-related diseases, indicating significant variation by disease field. Additionally, over 10% of tests were found to have been performed inappropriately, such as multiple genotyping of the same patient or testing more than 2 weeks after starting treatment. Although NUDT15 genotyping for patients requiring thiopurine treatment has been shown to improve thiopurine treatment continuation rate, measures are required to address the systematic issues identified in our analysis.

NUDT15 Haplotypes and Diplotypes Predict Thiopurine-Induced Leukopenia and the Influence of Prolonged Exposure to Azathioprine on Hematologic Indices in Patients with Inflammatory Bowel Diseases

Background. NUDT15 gene polymorphisms have been identified to predispose Asian patients with an inflammatory bowel disease (IBD) to thiopurine-induced leukopenia. This study predicted the influence of NUDT15 haplotypes and diplotypes on azathioprine (AZA)-induced leukopenia as well as the long-term influence of AZA on hematologic parameters in IBD. Methods. 194 IBD patients were tested for NUDT15 genotypes. We collected clinical data of 80 patients with AZA treatment including adverse events, dosage, white blood cell (WBC) count, platelet (PLT) count, and mean corpuscular volume (MCV) after AZA initiation. Patients without adverse events and drug withdrawal were followed up for at least one year. The relationship between NUDT15 haplotypes and diplotypes and leukopenia was analyzed. Results. The haplotypes NUDT15 c.415C &gt; T and c.36_37insGGAGTC as well as the diplotypes NUDT15 ∗ 1/ ∗ 2, ∗ 3/ ∗ 3, and ∗ 3/ ∗ 5 were significantly associated with AZA-induced leukopenia. Only one patient with NUDT15 c.52G &gt; A experienced leukopenia. NUDT15 ∗ 1/ ∗ 3 was not associated with leukopenia. After AZA initiation, the WBC count showed a downward trend in both wild types and mutants. The mean of WBC count in the mutant group at 1st month after AZA initiation was lower than that in the wild-type group ( P = 0.006 ). The MCV increased gradually in mutant cases ( P = 0.039 ), and the differences were obvious at 6th and 12th months compared with the baseline ( P = 0.014 a n d P = 0.042 , respectively). The PLT count showed a decreasing trend in the mutant group, but there was no difference until 11 months after initiating treatment ( P = 0.023 ). The final dose of AZA in the NUDT15 mutant group was significantly lower than that in the wild-type group ( P = 0.006 ). Conclusion. NUDT15 polymorphisms may be an appropriate predictor of AZA abnormal hematologic indices in IBD patients. It is necessary for IBD patients to monitor hematological indices and optimize AZA therapy.

Plasma lipidomic profiling of thiopurine-induced leukopenia after NUDT15 genotype-guided dosing in Chinese IBD patients.

Thiopurines, azathiopurine (AZA) and mercaptopurine (6-MP) have been regularly used in the treatment of inflammatory bowel disease (IBD). Despite optimized dosage adjustment based on the NUDT15 genotypes, some patients still discontinue or change treatment regimens due to thiopurine-induced leukopenia. We proposed a prospective observational study of lipidomics to reveal the lipids perturbations associated with thiopurine-induced leukopenia. One hundred and twenty-seven IBD participants treated with thiopurine were enrolled, twenty-seven of which have developed thiopurine-induced leucopenia. Plasma lipid profiles were measured using Ultra-High-Performance Liquid Chromatography-Tandem Q-Exactive. Lipidomic alterations were validated with an independent validation cohort (leukopenia n = 26, non-leukopenia n = 74). Using univariate and multivariate analysis, there were 16 lipid species from four lipid classes, triglyceride (n = 11), sphingomyelin (n = 1), phosphatidylcholine (n = 1) and lactosylceramide (n = 3) identified. Based on machine learning feature reduction and variable screening strategies, the random forest algorithm established by six lipids showed an excellent performance to distinguish the leukopenia group from the normal group, with a model accuracy of 95.28% (discovery cohort), 79.00% (validation cohort) and an area under the receiver operating characteristic (ROC) curve (ROC-AUC) of 0.9989 (discovery cohort), 0.8098 (validation cohort). Our novel findings suggested that lipidomic provided unique insights into formulating individualized medication strategies for thiopurines in IBD patients.

Prevalence of NUDT15 Genetic Variants and Incidence of Thiopurine-induced Leukopenia in Inflammatory Bowel Disease: A Systematic Review and Meta-analysis.

Nudix hydrolase 15 [NUDT15] genetic variants confer an increased risk of thiopurine-induced leukopenia [TIL]; however, their global prevalence in inflammatory bowel disease [IBD] patients is unknown. We aimed to evaluate the global prevalence of NUDT15 variants in IBD patients and incidence of TIL in these patients. Six databases were searched from inception until July 2022. Studies reporting the frequency of any NUDT15 variant and/or frequency of leukopenia in adult IBD patients with these variants were included. A random effects model was performed to estimate the pooled prevalence of variants, incidence of early [≤8 weeks] and late [>8 weeks] leukopenia, and relative risk of developing leukopenia. Twenty studies comprising 5232 patients were included. The pooled prevalence of the *1/*3 c.415C > T C/T diplotype was 13% (95% confidence interval [CI]: 10-18%), *3/*3 c.415C > T T/T diplotype was 2% [95% CI: 1-2%], *1/*5 c.52G > A G/A diplotype was 2% [95% CI: 1-3%], and *1/*6 c.36_37insGGAGTC ins/- diplotype was 7% [95% CI: 4-12%]. The pooled prevalence of *1/*3 was high in Japanese [20%, 95% CI: 16-24%] and Chinese patients [18%, 95% CI: 12-27%]. The incidence of early leukopenia was 20% [95% CI: 16-26%] in *1/*3 patients, 99% [95% CI: 7-100%] in *3/*3 patients, and 49% [95% CI: 29-69%] in *1/*6 patients. The incidence of late leukopenia was 36% [95% CI: 26-49%] in *1/*3 patients. NUDT15 variants are common and strongly predict TIL in IBD patients. Pre-treatment NUDT15 genotyping should be considered particularly in Asian populations, to guide thiopurine dosing and prevent myelotoxicity.

The influence of NUDT15 variants on 6-mercaptopurine-induced neutropenia in Vietnamese pediatric acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP) serves as the backbone of maintenance therapy in acute lymphoblastic leukemia. The nucleoside diphosphate-linked moiety X-type motif 15 genes (NUDT15) affects the metabolism of 6-MP and thiopurine-related neutropenia in the Asian population. This study reports the influence of these variants on 6MP-induced neutropenia in children with acute lymphoblastic leukemia (ALL). A total of 102 children were enrolled in this retrospective cohort study. NUDT15 variants on exon 1 and exon 3 were identified by Sanger sequencing. We divided the intermediate metabolizer group and the normal metabolizer group base on NUDT15 diplotypes. During the first 3months of maintenance treatment, medical reports measured treatment-related toxicity (neutropenia) and 6-MP dose decreases. NUDT15 genotyping showed two categories of mutations: wild type (75.5%) and heterozygous variant (24.5%). Neutropenia during the early phase of maintenance therapy in the intermediate metabolizer group (68%) was significantly higher than the normal metabolizer group (18.2%) with 10-fold greater odds. Especially, the c.415C>T heterozygous variant was extremely associated with neutropenia compared with the C>C genotype (odds ratio [OR]: 12; 95% confidence interval [CI]: 3.5-41.7). The tolerated doses of 6-MP after the first 3months of maintenance therapy related to the intermediate metabolizer group and the normal metabolizer group were 48.7 and 64.3mg/m2/day, respectively (p<0.001). One-fourth of individuals had NUDT15 variations. All NUDT15 heterozygous mutations cause neutropenia and need 6-MP dose optimization. Given the frequency of NUDT15 mutations in Vietnamese children and their connection with early neutropenia, testing is indicated.

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.

Optimizing thiopurine therapy in children with acute lymphoblastic leukemia: A promising "MINT" sequencing strategy and therapeutic "DNA-TG" monitoring.

Thiopurines, including thioguanine (TG), 6-mercaptopurine (6-MP), and azathioprine (AZA), are extensively used in clinical practice in children with acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases. However, the common adverse effects caused by myelosuppression and hepatotoxicity limit their application. Metabolizing enzymes such as thiopurine S-methyltransferase (TPMT), nudix hydrolase 15 (NUDT15), inosine triphosphate pyrophosphohydrolase (ITPA), and drug transporters like multidrug resistance-associated protein 4 (MRP4) have been reported to mediate the metabolism and transportation of thiopurine drugs. Hence, the single nucleotide polymorphisms (SNPs) in those genes could theoretically affect the pharmacokinetics and pharmacological effects of these drugs, and might also become one of the determinants of clinical efficacy and adverse effects. Moreover, long-term clinical practices have confirmed that thiopurine-related adverse reactions are associated with the systemic concentrations of their active metabolites. In this review, we mainly summarized the pharmacogenetic studies of thiopurine drugs. We also evaluated the therapeutic drug monitoring (TDM) research studies and focused on those active metabolites, hoping to continuously improve monitoring strategies for thiopurine therapy to maximize therapeutic efficacy and minimize the adverse effects or toxicity. We proposed that tailoring thiopurine dosing based on MRP4, ITPA, NUDT15, and TMPT genotypes, defined as “MINT” panel sequencing strategy, might contribute toward improving the efficacy and safety of thiopurines. Moreover, the DNA-incorporated thioguanine nucleotide (DNA-TG) metabolite level was more suitable for red cell 6-thioguanine nucleotide (6-TGNs) monitoring, which can better predict the efficacy and safety of thiopurines. Integrating the panel “MINT” sequencing strategy with therapeutic “DNA-TG” monitoring would offer a new insight into the precision thiopurine therapy for pediatric acute lymphoblastic leukemia patients.

Nudix Hydroxylase 15 Mutations Strongly Predict Thiopurine-Induced Leukopenia Across Different Asian Ethnicities: Implications for Screening in a Diverse Population.

Background and AimsThiopurines, which are immunosuppressive drugs for maintaining remission for inflammatory bowel disease, are known to cause myelotoxicity in patients with Nudix Hydroxylase 15 (NUDT15) genetic variants in some Asian countries with monoethnic populations. We aimed to investigate the association of NUDT15 variants with leukopenia in a multiethnic population in Southeast Asia.MethodsPatients with a confirmed diagnosis of inflammatory bowel disease were recruited. We collected demographic and clinical characteristics and whole blood counts before and after initiating thiopurines. Thiopurine S-methyltransferase (TPMT) and NUDT15 genotypes were analyzed with the single nucleotide polymorphisms (SNPs) genotyping assay. Leukopenia was defined as a white blood cell (WBC) count < 3,000/μl.ResultsIn this study, 19 (18.6%) of the 102 patients who had adequate thiopurine therapy experienced leukopenia, 11 patients (57.9%) had NUDT15 c.415C > T variants, 2 patients (10.5%) had NUDT15 c.52G > A variants while one (5.3%) had a TPMT variation. Individually, NUDT15 c.415C > T had a sensitivity and specificity of 57.9% and 94.0% (odds ratio [OR] = 21.45, 95% CI 5.94–77.41, p < 0.001), respectively, for predicting thiopurine-induced leukopenia, while NUDT15 c.52G > A was only observed in patients with leukopenia. As compared with patients with wild-type NUDT15, both NUDT15 variations had a combined sensitivity and specificity of 68.4% and 94%, respectively (OR = 33.80, 95% CI 8.99–127.05, p < 0.001), for predicting thiopurine-induced leukopenia as well as a shorter onset to leukopenia (median onset [months] 2.0 vs. 5.5; p = 0.045). Sub-group analysis showed that both NUDT15 variations were strongly associated with leukopenia among the Chinese and Indians but not among the Malays.ConclusionNudix Hydroxylase 15 variants strongly predicted thiopurine-induced leukopenia across a multiethnic Southeast Asian population, particularly among the Chinese and Indians.

Low NUDT15 expression levels due to biallelic NUDT15 variants and 6-mercaptopurine intolerance.

6-Mercaptopurine (6-MP) is widely used for the treatment of paediatric leukaemia and lymphoma. Recently, germline variants in the NUDT15 gene have been identified as one of the major genetic causes for 6-MP-associated adverse effects such as myelosuppression. Patients with hypomorphic NUDT15 variants accumulate excessive levels of DNA-incorporated thioguanine in white blood cells, resulting in severe myelosuppression. Although preclinical studies suggest that these variants may influence the protein stability of NUDT15, this has not been directly characterised in patients. In this study, we report the development of a series of novel monoclonal antibodies against NUDT15, using which we quantitatively assessed NUDT15 protein levels in 37 patients with acute lymphoblastic leukaemia treated with 6-MP, using sandwich enzyme-linked immunosorbent assay (ELISA). The NUDT15 genotype was highly correlated with its protein levels (p< 0.0001), with homozygous and compound heterozygous patients showing exceedingly low NUDT15 expression. There was a positive correlation between NUDT15 protein level and 6-MP tolerance (r= 0.631, p< 0.0001). In conclusion, our results point to low NUDT15 protein abundance as the biochemical basis for NUDT15-mediated 6-MP intolerance, thus providing a phenotypic readout of inherited NUDT15 deficiency.

Nudix hydrolase 15 (NUDT15) loss-of-function variants in an Australian inflammatory bowel disease population.

Thiopurine-related adverse events such as leukopenia, liver dysfunction and pancreatitis are associated with variants in the NUDT15 gene. Loss-of-function (low or no enzyme activity) alleles are more common in Asian and Hispanic populations. The prevalence of these variants in the Australian inflammatory bowel disease (IBD) population has not yet been reported. To evaluate the presence of NUDT15 loss-of-function alleles *2,*3,*9 in the Australian IBD population. The NUDT15 screening cohort included 423 IBD patients from Brisbane, Australia. Study patients were recruited by: (i) retrospective review of clinical charts for thiopurine-related severe adverse events; (ii) pathology data (white blood cell (WBC) and neutrophil counts). NUDT15 genotyping was performed using polymerase chain reaction (PCR)-high-resolution melt (HRM), TaqMan genotyping and Sanger sequencing. NUDT15 mutation R139C (allele *3) was identified in 8 of 423 (1.9%) IBD patients. Seven of eight patients were R139C heterozygous (C/T) and one patient was R139C homozygous (T/T). One of the C/T group and the T/T patient developed thiopurine-induced myelosuppression (TIM) within 60 days of dosing. One patient in the C/T group developed TIM after 60 days of thiopurine dosing. The remaining five patients in the C/T group did not show TIM; however, other thiopurine-related events could not be ruled out and therefore careful monitoring over a long period is recommended. This is the first study to report the frequency of NUDT15 haplotypes *2,*3,*9 in an Australian IBD population. The most common variant detected was the R139C mutation. PCR and Sanger sequencing are efficient and cost-effective approaches for NUDT15 genotyping.

Inosine Triphosphate Pyrophosphatase and NUDT15 are Good Predictors of Clinical Outcomes in Thiopurine-Treated Chinese Patients with Inflammatory Bowel Disease.

Although the relationship between NUDT15 and thiopurine-induced leukopenia has been proven in previous studies, no prominent factors explaining interindividual variations in its active metabolite, 6-thioguanine nucleotide (6-TGN), and clinical efficacy have been identified. In this study, the correlation between genotypes (thiopurine S-methyltransferase, NUDT15, and ITPA polymorphisms), 6-TGN concentrations, and clinical outcomes (efficacy and side effects) in patients with inflammatory bowel disease were investigated. In total, 160 patients with inflammatory bowel disease were included, and the 3 genotyped genes and 6-TGN levels were measured by high-performance liquid chromatography. Statistical analyses and calculations were performed to determine their relationships. ITPA genotypes and 6-TGN concentration were both associated with the clinical effectiveness of azathioprine (P = 0.036 and P = 4.6 × 10-7), with a significant correlation also detected between them (P = 0.042). Patients with ITPA variant alleles exhibited higher 6-TGN levels than those with the wild-type allele. In addition, the relationship between NUDT15 and leukopenia and neutropenia was confirmed (P = 1.79 × 10-7 and 0.002). In summary, it is recommended that both ITPA and NUDT15 genotyping should be performed before azathioprine initiation. Moreover, the 6-TGN concentration should be routinely monitored during the later period of treatment.

NUDT15 Genetic Variants in Chinese Han, Uighur, Kirghiz, and Dai Nationalities

BackgroundThiopurines are widely used as anti-cancer and immunosuppressant agents, but have a narrow therapeutic index owing to frequent toxicity and life-threatening bone marrow suppression. The nudix hydrolase 15 (NUDT15) genetic polymorphism is strongly associated with the tolerance and myelosuppressive effect of mercaptopurine administration, but the frequency of NUDT15 variants is known to vary among different ethnic groups or nationalities. At present, the NUDT15 gene polymorphism in ethnic minorities such as the Uighur, Kirghiz, and Dai nationalities in China is unclear.ProcedureDNA samples were isolated from 1,071 Chinese children, including 675 Han children with acute lymphoblastic leukemia and 396 healthy minority children, including 118 Uighur, 126 Kirghiz, and 152 Dai participants. The coding regions of NUDT15 exons 1 to 3 were amplified by polymerase chain reaction. NUDT15 genotypes were identified by Sanger sequencing.ResultsFive NUDT15 genetic variants of coding regions including rs746071566 (c.55_56insGAGTCG), rs186364861 (c.52G > A), c.137C > G, and c.138T > G in exon 1, and the variant rs116855232 (c.415C > T) in exon 3 were found among the participants. The frequency of NUDT15 rs746071566 variants was lower in the Uighur and Kirghiz populations than in the Han population and in other East Asian nationalities, while the frequency of c.415C > T variants was lower in the Dai population. The c.52G > A variant was relatively uncommon in children of the Han, Uighur, Kirghiz, and Dai ethnic groups. Notably, the rare variants c.137C > G and c.138T > G in a Uighur child were predicted to be disruptive sites.ConclusionIn summary, our results illustrate the NUDT15 polymorphisms in Chinese children of Han, Uighur, Kirghiz, and Dai nationalities, and provide the most effective detection recommendations for different ethnic groups to predict thiopurine-related toxicity, which could be used to guide future clinical thiopurine dose adjustment.