Nucleoside Diphosphate-linked Moiety X-type Motif Research Articles

A NUDT15 Genetic Polymorphism Study in Adult Acute Leukemia Patients Integrating Pharmacogenomics and Clinical Practice: A Single Centre Experience from South India

Background: Severe myelosuppression secondary to thiopurines such as 6-Mercaptopurine (6-MP) in patients with acute leukemia undergoing maintenance therapy is frequently attributed to inherited genetic polymorphisms of Thiopurine Methyltransferase (TPMT); however, this is rare in the Asian population. Nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) is also responsible for encoding critical enzymes involved in 6-MP metabolism. We aimed to determine the frequency of genetic polymorphisms of NUDT15 in the South Indian population and the impact of the omission of 6-MP from the treatment protocol of these patients. Methods: We retrospectively reviewed 17 patients diagnosed with acute leukemia between May 2018 and May 2020 and analyzed the prevalence of NUDT15 genetic polymorphisms in these patients. The NUDT15 mutation study (NUDT15 415C>T) used allele-specific Real-Time PCR in peripheral blood. 6-Mercaptopurine (6-MP) was excluded from the treatment regimen of patients with NUDT15 genetic polymorphisms and the impact of its exclusion was also studied. Results: Among the 17 patients for whom we cared, 10 were diagnosed with B-cell Acute Lymphoblastic Leukemia (B-ALL), three with T-cell Acute Lymphoblastic Leukemia (T-ALL), one with Mixed Phenotype Acute Leukemia (MPAL), and one with Acute Promyelocytic Leukemia (APML). Treatment records could not be retrieved for 2 patients. NUDT15 mutations were present in six patients (36%) (see fig1), of whom two had homozygous (33%) polymorphisms and four had heterozygous (67%) polymorphisms. In the heterozygous group, 2 were Ph. (Philadelphia chromosome) negative B-ALL; one was treated with BFM and the other with hyper-CVAD, one Ph. positive B-ALL was treated with BFM + Ponatinib, and one High-risk APML was treated with the APML 4 protocol. In the homozygous group, treatment details could not be retrieved for one case, and the other patient had T-ALL, which was treated with hyper-CVAD. 6-MP was omitted from the treatment protocol for all NUDT15 mutation-positive patients, both homozygous and heterozygous. Of these patients, two developed grade 1 myelosuppression, one developed grade 2 myelosuppression, one developed dyselectrolytemia, and one patient did not develop any toxicity. The incidence of severe or grade 3 and 4 myelosuppression in NUDT15 polymorphisms receiving 6MP can be as high as 50% (Khaeso K et.al Front Pharmacol. 2021). In comparison, none of our patients developed grade 3 or 4 myelosuppression. Overall, treatment-related adverse events included grade 1-2 myelosuppression (n=6), grade 1-2 hepatotoxicity (n=1), deep vein thrombosis (n=2), and sepsis (n=2). Five of the 6 patients with NUDT15 mutations achieved complete remission (CR). Conclusion: The prevalence of NUDT15 polymorphisms was high in our study population. Based on a meta-analysis, the prevalence of NUDT15 in the South Indian population was 16.5%, and in patients with adverse effects, the pooled prevalence of NUDT15 polymorphisms was 49.51% (Anuraag Jena et.al. Expert Review of Clinical Pharmacology 2021). Based on our results, although limited by the small number of cases, we suggest that pre-emptive testing to detect these polymorphisms should be undertaken in all adult acute leukemia patients of South Indian origin, given their high prevalence in this population. The omission of 6-MP from treatment protocols can help prevent severe myelosuppression and treatment interruption or discontinuation, which in turn can improve treatment outcomes.

Loss of Nudt15 thiopurine detoxification increases direct DNA damage in hematopoietic stem cells

Thiopurines, such as 6-mercaptopurine (6-MP), are widely used as cytotoxic agents and immunosuppressants for leukemia and autoimmune or inflammatory diseases. A nonsynonymous single nucleotide polymorphism (p.Arg139Cys; R139C) of the nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene causes the loss of thiopurine detoxification, inducing myelosuppression. To understand such hematotoxicity, we investigate the effects of NUDT15 R139C on hematopoietic stem cells (HSCs) upon thiopurine administration. Using previously established Nudt15R138C knock-in mice, which mimic myelosuppression in NUDT15R139C homozygous or heterozygous patients following thiopurine administration, we investigated the numerical changes of HSCs and hematopoietic progenitor cells following 6-MP administration using in vivo flowcytometry and ex vivo HSC expansion. Genes differentially expressed between Nudt15+/+ HSCs and Nudt15R138C/R138C HSCs were identified using RNA-sequencing before the emergence of 6-MP-induced HSC-damage. Gene Ontology (GO) and Transcriptional Regulatory Relationships Unraveled by Sentence-based Text Mining (TRRUST) analyses were performed to elucidate the molecular effects of 6-MP on HSCs. In Nudt15R138C/R138C mice, 6-MP induced exhaustion of HSCs faster than that of multipotent progenitors and as fast as that of myeloid-committed progenitors. Ex vivo-expanded Nudt15R138C/R138C HSCs were dose- and time-dependently damaged by 6-MP. GO analysis identified the DNA damage response and cell cycle process as the most strongly influenced processes in Nudt15R138C/R138C HSCs. TRRUST analysis revealed that the Trp53-regulated transcriptional regulatory network is influenced prior to HSC exhaustion in Nudt15R138C/R138C HSCs. The loss of NUDT15 thiopurine detoxification enhances thiopurine-mediated DNA damage via the Trp53 networks in HSCs. Therefore, caution is required in long-term thiopurine use in patients with NUDT15 R139C in view of its adverse effects on HSCs in the form of DNA damage.

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.

Single-Nucleotide Polymorphisms, c.415C > T (Arg139Cys) and c.416G > A (Arg139His), in the NUDT15 Gene Are Associated with Thiopurine-Induced Leukopenia.

While nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene polymorphism Arg139Cys (rs116855232) is known to be a risk factor for thiopurine-induced severe leukopenia, association with the NUDT15 gene polymorphism Arg139His (rs147390019) has not yet been clarified. In addition, the accuracy of TaqMan PCR to assess these two polymorphisms has not been investigated. In this study, we evaluated TaqMan PCR for detection of the NUDT15 single-nucleotide polymorphisms (SNPs) and examined the clinical impact of Arg139His on thiopurine-induced leukopenia. First, we demonstrated that a TaqMan PCR assay successfully detected the Arg139His polymorphism of NUDT15 in clinical samples. Next, the NUDT15 gene polymorphisms (Arg139Cys and Arg139His) were separately analyzed by TaqMan Real-Time PCR in 189 patients from August 2018 to July 2019. The incidences of leukopenia within 2 years were 16.2, 57.9, and 100% for arginine (Arg)/Arg, Arg/cysteine (Cys), and Arg/histidine (His), respectively. The leukopenia was significantly increased in Arg/Cys and Arg/His compared with Arg/Arg. This retrospective clinical study indicated that, in addition to Arg139Cys, Arg139His may be clinically associated with a high risk of leukopenia. Pharmacogenomics will help in selecting drugs and determining the individualized dosage of thiopurine drugs.

The role of therapeutic drug monitoring in the management of inflammatory bowel disease

Therapeutic drug monitoring (TDM) is performed when a drug has a narrow therapeutic range (e.g., thiopurines) or there is no linear drug response relationship (biologic agents). TDM is the measurement of drug and/or antibody concentration and individualizing the dose to maintain the drug concentration in the target range. Maintaining serum drug concentration in a therapeutic range is associated with a higher rate of favorable outcomes. Reactive drug monitoring is performed when the disease is active, and proactive drug monitoring is performed when the disease is in remission. Thiopurines still form an important therapeutic armamentarium in the treatment of inflammatory bowel disease (IBD) but have a narrow therapeutic range. Preemptive thiopurine methyl transferase and nucleoside diphosphate-linked moiety X-type motif (NUDT) genotype measurement can reduce the toxicity with appropriate dose initiation and thiopurine metabolite measurement can optimize the dose and reduce the toxicity. Antitumor necrosis factor agents brought about a paradigm shift in the management of moderate-to-severe IBD at the turn of the past century. Despite their efficacy, up to half the patients discontinue the biologic agents at the end of 1 year due to primary nonresponse or secondary loss of response. TDM can optimize the drug level and improve clinical response, reduce antibody formation, reduce cost, and can improve corticosteroid- free clinical remission. Despite postulated benefits and guidelines, the evidence for objective benefits from meta-analysis is mixed. This article reviews the current concepts regarding TDM.

NUDT15 is a key genetic factor for prediction of hematotoxicity in pediatric patients who received a standard low dosage regimen of 6-mercaptopurine

6-Mercaptopurine (6-MP) is commonly used for treatment of acute lymphoblastic leukemia (ALL). The incidence of hematotoxicity caused by this drug is quite high in Asians even using a standard low dosage regimen. The present study was aimed to elucidate the impact of thiopurine S-methyltransferase (TPMT), a nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15), inosine triphosphatase (ITPA) and ATP Binding Cassette Subfamily C Member 4 (ABCC4) polymorphisms on hematotoxicity in pediatric patients who received a standard low starting dose of 6-MP. One hundred and sixty-nine pediatric patients were enrolled and their genotypes were determined. Patients who carried NUDT15∗3 and NUDT15∗2 genotypes were at a 10–15 fold higher risk of severe neutropenia than those of the wild-type during the early months of the maintenance phase. Risk of neutropenia was not significantly increased in patients with other NUDT15 variants as well as in patients with TPMT, ITPA or ABCC4 variants. These results suggest that NUDT15 polymorphisms particularly, NUDT15∗3 and NUDT15∗2, play major roles in 6-MP-induced severe hematotoxicity even when using a standard low dosage of 6-MP and genotyping of these variants is necessary in order to obtain precise tolerance doses and avoid severe hematotoxicity in pediatric patients.

Heterozygous NUDT15 Gene Polymorphism Would Not Associate with the Severity of 6-Mercaptopurine Side Effects in Early Intensification Therapy for Childhood Acute Lymphoblastic Leukemia

BackgroundIn the treatment of childhood acute lymphoblastic leukemia (ALL), 6-mercaptopurine (6-MP) is essential for early intensification and maintenance therapy. Recently, it has been reported that a gene polymorphism of nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) rs116855232 is associated with the 6-MP induced severe myelotoxicity. Since the NUDT15 rs116855232 polymorphism is relatively common in East Asian and Hispanic populations, it is important to evaluate the association between the polymorphism and 6-MP for determining the dose of effective therapy and avoiding the side effect in Japan. However, there are few reports on the association between NUDT15 polymorphisms and the therapeutic efficacy or side effects of 6-MP in the early intensification; most previous reports focused on the maintenance therapy so far. The purpose of this study is to clarify the association between NUDT15 polymorphism and the actual treatment status with 6-MP or its side effects, therefore to contribute to the effective and safe treatment of childhood ALL in Japan.MethodsTwenty-four patients with ALL who received early intensification therapy according to the JPLSG ALL-B12 or JPLSG ALL-T11 protocol at the Department of Pediatrics, Hokkaido University Hospital, between April 2013 and May 2021 were included in the study. We retrospectively collected the clinical and laboratory data from the clinical records. And we also performed the sequence analysis of the exon 1 and 3 in NUDT15 geneResultsGenetic analysis of NUDT15 showed no nucleotide changes other than rs116855232. Twenty patients were CC (wild-type), 4 patients were CT (heterozygous-variant), and 0 patients were TT (homozygous-variant). Of the 4 patients with heterozygous-variant, one patient received a reduced dose of 6-MP because of early onset myelotoxicity during early intensification therapy. In this patient, the total dose of 6-MP was 70% of the standard dose. All the 24 patients showed myelotoxicity and hepatotoxicity during early intensification therapy. Correlations between NUDT15 haplotype and side effects as myelotoxicity and hepatotoxicity were not significant.DiscussionIn the heterozygous-variant of NUDT15 rs116855232, the incidence and degree of myelotoxicity and hepatotoxicity during early intensification therapy did not differ from the wild-type. The international consortium (Clinical Pharmacogenetics Implementation Consortium Guideline, 2018) recommends that the dose of 6-MP should be reduced to 10mg/m2/day in patients with homozygous variant of NUDT15, however, the reduction of the starting dose of 6-MP in those with heterozygous variant is not recommended. Of note, these recommendations were made based on the data in the maintenance therapy. Our study suggests that the reduction of the starting dose may not be necessary in children with heterozygous variant. The NUDT15 rs116855232 polymorphism is common in Japan, with 10% of heterozygotes and 1.1% of homozygotes. In future study, a larger study on the association between NUDT15 polymorphism and treatment outcome including side effects in the early intensification is needed in East Asia to confirm the results of our study. DisclosuresNo relevant conflicts of interest to declare.

HIF2α promotes tumour growth in clear cell renal cell carcinoma by increasing the expression of NUDT1 to reduce oxidative stress.

BackgroundThe key role of hypoxia‐inducible factor 2alpha (HIF2α) in the process of renal cancer has been confirmed. In the field of tumour research, oxidative stress is also considered to be an important influencing factor. However, the relationship and biological benefits of oxidative stress and HIF2α in ccRCC remain unclear. This research attempts to explore the effect of oxidative stress on the cancer‐promoting effect of HIF2α in ccRCC and reveal its mechanism of action.MethodsThe bioinformatics analysis for ccRCC is based on whole transcriptome sequencing and TCGA database. The detection of the expression level of related molecules is realised by western blot and PCR. The expression of Nucleoside diphosphate‐linked moiety X‐type motif 1 (NUDT1) was knocked down by lentiviral infection technology. The functional role of NUDT1 were further investigated by CCK8 assays, transwell assays and cell oxidative stress indicator detection. The exploration of related molecular mechanisms is realised by Luciferase assays and Chromatin immunoprecipitation (ChIP) assays.ResultsMolecular screening based on knockdown HIF2α sequencing data and oxidative stress related data sets showed that NUDT1 is considered to be an important molecule for the interaction of HIF2α with oxidative stress. Subsequent experimental results showed that NUDT1 can cooperate with HIF2α to promote the progression of ccRCC. And this biological effect was found to be caused by the oxidative stress regulated by NUDT1. Mechanistically, HIF2α transcription activates the expression of NUDT1, thereby inhibiting oxidative stress and promoting the progression of ccRCC.ConclusionsThis research clarified a novel mechanism by which HIF2α stabilises sirtuin 3 (SIRT3) through direct transcriptional activation of NUDT1, thereby inhibiting oxidative stress to promote the development of ccRCC. It provided the possibility for the selection of new therapeutic targets for ccRCC and the study of combination medication regimens.

Thiopurine pharmacogenomics and pregnancy in inflammatory bowel disease

The thiopurine drugs azathioprine and 6-mercaptopurine are widely used for the maintenance of clinical remission in steroid-dependent inflammatory bowel disease (IBD). Thiopurines are recommended to be continued throughout pregnancy in IBD patients, but conclusive safety data in pregnant patients remain still insufficient. On the other hand, a strong association between a genetic variant of nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15 p.Arg139Cys) and thiopurine-induced myelotoxicity has been identified. Pharmacokinetic studies have revealed that thiopurine metabolism is altered in pregnant IBD patients and suggested that the fetus may be exposed to the active-thiopurine metabolite, 6-thioguaninetriphosphate, in the uterus. A recent study using knock-in mice harboring the p.Arg138Cys mutation which corresponds to human p.Arg139Cys showed that oral administration of 6-MP at clinical dose induces a severe toxic effect on the fetus harboring the homozygous or heterozygous risk allele. This suggests that NUDT15 genotyping may be required in both women with IBD who are planning pregnancy (or pregnant) and their partner to avoid adverse outcomes for their infant. The risk to the fetus due to maternal thiopurine use is minimal but there are some concerns that are yet to be clarified. In particular, a pharmacogenomic approach to the fetus is considered necessary.

Genetic Polymorphisms of Drug-Metabolizing Enzymes Involved in 6-Mercaptopurine-Induced Myelosuppression in Thai Pediatric Acute Lymphoblastic Leukemia Patients.

Genetic polymorphisms of thiopurine S-methyltransferase (TPMT) and nucleoside diphosphate-linked moiety X-type motif 15 ( NUDT15 ) genes have been proposed as key determinants of 6-mercaptopurine (6-MP)-induced myelosuppression in pediatric acute lymphoblastic leukemia (ALL). In the present study, genotypes of TPMT and NUDT15 were investigated in 178 Thai pediatric patients with ALL by the TaqMan SNP genotyping assay and DNA sequencing. The frequency of TPMT*3C was 0.034. Among NUDT15 variants, NUDT15*3 is the most common variant with the allele frequency of 0.073, whereas those of NUDT15*2 , NUDT15*5 , and NUDT15*6 variants were 0.022, 0.011, and 0.039. These data suggest that a high proportion of Thai pediatric ALL patients may be at risk of thiopurine-induced myelosuppression.

NUDT15 Variants Confer High Incidence of Secondary Malignancies of ALL in Children

Background: Secondary or subsequent malignant neoplasms (SMNs) are one of the most serious late complications in children and adolescents after treatment for acute lymphoblastic leukemia (ALL). Several studies have reported that excess dosing of 6-mercaptopurine (6-MP) therapy and polymorphisms of 6-MP metabolizing enzyme coding gene might influence the risk of SMNs. Recently, nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) variants have been identified as a cause of high sensitivity to 6-MP, which reflects a high sensitivity to 6MP in East Asians. Therefore, we investigated an association between NUDT15 genotype and incidence of SMNs in pediatric ALL. Methods: Germline samples including bone marrow samples, peripheral blood samples and buccal swab samples during remission were obtained from 13 cases who suffered from SMN after childhood ALL. The prevalence of major NUDT15 non-functional alleles (*2 and *3), which is categorized intermediate or poor metabolizer of 6-MP in Clinical Pharmacogenetics Implementation Consortium Guideline (Relling M et al. Clin Pharmacol Ther 2019), was evaluated by using Sanger sequencing and/or whole exome sequencing. The NUDT15 variants prevalence in SMN patients with primary ALL was compared to those without SMN, who were registered in the TCCSG L04-16 study, which was conducted by the Tokyo Children's Cancer Study Group (TCCSG). Results: Patients' profiles and NUDT15 genotypes were summarized in table 1. Median ages at diagnoses of ALL and SMNs were 4 (1-15) and 13 (5-44) years, respectively. Median duration between ALL and SMNs was 8 (1-30) years. SMNs included acute myeloblastic leukemia (AML) / myelodysplastic syndrome (MDS) (n = 8), brain tumor (n = 1), basal cell carcinoma (n = 1), bladder cancer (n = 1), Ewing sarcoma (n = 1) and osteosarcoma (n = 1). Among the 13 SMN cases, six patients (46.2%) possessed NUDT15 non-functional alleles. The SMNs of these cases included AML / MDS (n = 4), bladder cancer (n = 1) and osteosarcoma (n = 1). Among them, 3 patients, whose SMNs were AML, bladder cancer and osteosarcoma, were treated with radiation therapy against primary ALL. All NUDT15 genotypes were heterozygous variants (3 were *1/*2 and 3 were *1/*3) and seemed to be intermediate metabolizers. In patients without known SMNs in TCCSG ALL L04-16 cohort study, 73 of 437 (16.7%) possessed NUDT15 non-functional alleles. Three of them had bi-allelic variants (2 cases were *3/*3, 1 case was *2/*3), and the others were all heterozygous. The prevalence of NUDT15 non-functional alleles was significantly higher in SMNs compared to that of non-SMN cases (P = 0.012). Conclusion: Our study indicated the NUDT15 hypomorphic variant alleles could be a risk of SMNs after ALL treatment with 6-MP. NUDT15 heterozygotic variants carriers are generally tolerable of 6-MP, and are usually treated with normal or slightly low-dose of 6-MP. However, our results suggested that standard adjustment of 6-MP in maintenance therapy might cause potential overdose of 6-MP for cases with NUDT15 variants, leading to increased risk of SMNs. To certify it, a higher number of study samples and somatic samples analysis of 6-MP induced SMNs are required. To obtain the best long-term event-free survival probability, it might be necessary to reduce the risk of SMNs development without compromising therapeutic effect by further optimal adjustment of 6-MP dose (or alternative agents). Disclosures No relevant conflicts of interest to declare.

Nucleoside diphosphate-linked moiety X-type motif 15 R139C genotypes impact 6-thioguanine nucleotide cut-off levels to predict thiopurine-induced leukopenia in Crohn's disease patients.

BACKGROUNDThiopurine-induced leukopenia (TIL) is a life-threatening toxicity and occurs with a high frequency in the Asian population. Although nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) variants significantly improve the predictive sensitivity of TIL, more than 50% of cases of this toxicity cannot be predicted by this mutation. The potential use of the 6-thioguanine nucleotide (6TGN) level to predict TIL has been explored, but no decisive conclusion has been reached. Can we increase the predictive sensitivity based on 6TGN by subgrouping patients according to their NUDT15 R139C genotypes?AIMTo determine the 6TGN cut-off levels after dividing patients into subgroups according to their NUDT15 R139C genotypes.METHODSPatients’ clinical and epidemiological characteristics were collected from medical records from July 2014 to February 2017. NUDT15 R139C, thiopurine S-methyltransferase, and 6TGN concentrations were measured.RESULTSA total of 411 Crohn’s disease patients were included. TIL was observed in 72 individuals with a median 6TGN level of 323.4 pmol/8 × 108 red blood cells (RBC), which was not different from that of patients without TIL (P = 0.071). Then, we compared the 6TGN levels based on NUDT15 R139C. For CC (n = 342) and CT (n = 65) genotypes, the median 6TGN level in patients with TIL was significantly higher than that in patients without (474.8 vs 306.0 pmol/8 × 108 RBC, P = 9.4 × 10-5; 291.7 vs 217.6 pmol/8 × 108 RBC, P = 0.039, respectively). The four TT carriers developed TIL, with a median 6TGN concentration of 135.8 pmol/8 × 108 RBC. The 6TGN cut-off levels were 411.5 and 319.2 pmol/8 × 108 RBC for the CC and CT groups, respectively.CONCLUSIONThe predictive sensitivity of TIL based on 6TGN is dramatically increased after subgrouping according to NUDT15 R139C genotypes. Applying 6TGN cut-off levels to adjust thiopurine therapies based on NUDT15 is strongly recommended.

Long-term effect of NUDT15 R139C on hematologic indices in inflammatory bowel disease patients treated with thiopurine.

A missense variant of the nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene (R139C) predisposes Asian patients with inflammatory bowel disease (IBD) to thiopurine-induced leukopenia. This study evaluates the long-term effect of NUDT15 R139C heterozygosity on hematological parameters during thiopurine administration. We enrolled 83 Japanese IBD patients who were on anti-tumor necrosis factor-α agents and had used thiopurine. NUDT15 R139C was genotyped by polymerase chain reaction. We retrospectively reviewed patient clinical charts to collect data on white blood cell (WBC) count, mean corpuscular volume (MCV), hemoglobin, and platelet count during the 24months following thiopurine initiation. The included patients had either Crohn's disease (54; 65.1%) or ulcerative colitis (29; 34.9%). Genotyping of NUDT15 R139C identified 62 patients (74.7%) of genotype C/C and 21 (25.3%) of genotype C/T. The median dose of thiopurine was lower in the C/T group than in the C/C group after starting thiopurine. At 6months, the mean WBC count of the C/T group became significantly lower than that of the C/C group (P = 0.008) and remained lower through the 24months. The C/T group developed grade 2-4 leukopenia by 6months, which persisted through 12-24months. The mean MCV in the C/T group became higher than that of the C/C group after 3months. NUDT15 R139C heterozygosity affected the WBC count and MCV for 24months after thiopurine administration. Our results indicate that careful monitoring of leukopenia and dose adjustment are necessary throughout treatment in IBD patients heterozygous for the NUDT15 R139C.

Alopecia as the first manifestation of azathioprine myelosuppression in a genetically predisposed patient

Azathioprine, an analog of mercaptopurine, is used for various inflammatory/autoimmune diseases in diverse fields such as rheumatology, ophthalmology, gastroenterology, and neurology. One of the dreaded side effects of this drug is myelosuppression, which can be either dose related or “idiosyncratic.” The latter group often manifests with sudden cytopenias and is often secondary to a genetic predisposition. Mutations in the genes thiopurine S-methyltransferase (TPMT) and nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) have been identified to predispose to severe myelosuppression with azathioprine. An important clinical clue that can precede the onset of myelosuppression is alopecia. Here, we describe the case of a young lady treated with azathioprine, who developed alopecia (progressing to alopecia totalis), which was followed by severe cytopenia – both of which recovered on stopping the drug. On genetic analysis, she was found to be homozygous for mutation in NUDT15 (C415T) but was negative for mutation in TPMT. We review various other cases reporting alopecia as a marker of azathioprine-induced myelosuppression and the genetic mutations described.

Association of NUDT15 c.415C>T allele and thiopurine-induced leukocytopenia in Asians: a systematic review and meta-analysis.

Thiopurines, commonly used to treat autoimmune conditions and cancer, can be limited by life-threatening leucopenia. However, whether NUDT15 (nucleoside diphosphate-linked moiety X-type motif 15) is associated with thiopurine-induced leucopenia in Asians is controversial. Relevant studies in English that were published until July 10, 2016 were identified through PubMed, EMbase, and other web knowledge databases. Study quality was assessed according to the Newcastle-Ottawa Scale (NOS) criteria. Summary risk ratio (RR) and 95% confidence intervals (CI) were estimated based on a fixed-effects model or a random-effects model, depending on the absence or presence of significant heterogeneity. Seven studies of 1138 patients met our inclusion criteria. Random-effects model meta-analysis provided evidence that T carriers of NUDT15 c.415C>T were significantly correlated with high incidences of thiopurine-induced leukocytopenia [CT + TT vs. CC: RR=3.79, 95%CI (2.64~5.44), P<0.00001]. This correlation was especially strong in TT patients, where it was found to be significantly increased by 6.54-fold compared with CC patients [TT vs. CC: RR=6.54, 95%CI (3.34~12.82), P<0.00001]. We also found that the NUDT15 c.415C>T variant was common in Asians and Hispanics, but rare in Europeans and Africans; the frequency of the NUDT15 c.415C>T distribution varied substantially by race/ethnicity. The results of this meta-analysis confirm that NUDT15 c.415C>T may be an important predictor of thiopurine-induced leukocytopenia in Asians. Genotype targeting of NUDT15 c.415C>T before initiating thiopurine treatment may be useful to limit leukocytopenia.

Nucleoside diphosphate-linked moiety X-type motif 15 C415T variant as a predictor for thiopurine-induced toxicity in Indian patients.

Interindividual variation seen in the thiopurine metabolism is attributed to the genetic variant in thiopurine methyltransferase (TPMT) gene leading to myelosuppression. In Asians, the thiopurine-induced toxicity is not completely explained by TPMT variants. Literature indicates that a newer genetic variant in nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene is associated with thiopurine intolerance. We aimed to determine the risk allele frequency of NUDT15 genetic variant and its association with thiopurine-induced toxicity in Indian patients. In this pilot study, 69 patients on thiopurine therapy were analyzed. The frequencies of thiopurine-induced leukopenia were recorded. NUDT15 (C415T) and TPMT (*2, *3A, *3B, and *3C) genotyping was performed using amplification refractory mutation system-polymerase chain reaction and restriction fragment length polymorphism technique. Results were validated by DNA sequencing. The NUDT15 CC, CT, and TT genotypes were found to be 86.9%, 11.5%, and 1.5%, respectively, whereas TPMT genetic variants were absent. Of 60 patients without NUDT15 variant, none developed leukopenia, whereas of nine patients with NUDT15 variant, six developed leukopenia (P-value < 0.0001). The mean thiopurine dose of 1.01 and 0.73 mg/kg/day for patients with wild and mutant NUDT15 alleles, respectively, was statistically significant (P < 0.01). The sensitivity and specificity for NUDT15 variant were 100% and 95.2%, respectively. The NUDT15 risk allele frequency was 7.2%. There are 6/69 (8.7%) patients who developed leukopenia and harbored NUDT15 variant, thus showing a strong association for thiopurine-induced toxicity. Hence, NUDT15 genotyping may be considered before thiopurine therapy in Indian patients.

Review article: recent advances in pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy ininflammatory bowel disease.

Azathioprine and mercaptopurine have a pivotal role in the treatment of inflammatory bowel disease (IBD). However, because of their complex metabolism and potential toxicities, optimal use of biomarkers to predict adverse effects and therapeutic response is paramount. To provide a comprehensive review focused on pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy in IBD. A literature search up to July 2015 was performed in PubMed using a combination of relevant MeSH terms. Pre-treatment thiopurine S-methyltransferase typing plus measurement of 6-tioguanine nucleotides and 6-methylmercaptopurine ribonucleotides levels during treatment have emerged with key roles in facilitating safe and effective thiopurine therapy. Optimal use of these tools has been shown to reduce the risk of adverse effects by 3-7%, and to improve efficacy by 15-30%. For the introduction of aldehyde oxidase (AOX) into clinical practice, the association between AOX activity and AZA dose requirements should be positively confirmed. Inosine triphosphatase assessment associated with adverse effects also shows promise. Nucleoside diphosphate-linked moiety X-type motif 15 variants have been shown to predict myelotoxicity on thiopurines in East Asian patients. However, the impact of assessments of xanthine oxidase, glutathione S-transferase, hypoxanthine guanine phosphoribosyltransferase and inosine monophosphate dehydrogenase appears too low to favour incorporation into clinical practice. Measurement of thiopurine-related enzymes and metabolites reduces the risk of adverse effects and improves efficacy, and should be considered part of standard management. However, this approach will not predict or avoid all adverse effects, and careful clinical and laboratory monitoring of patients receiving thiopurines remains essential.

Three-dimensional Reconstruction Using Transmission Electron Microscopy Reveals a Swollen, Bell-shaped Structure of Transient Receptor Potential Melastatin Type 2 Cation Channel

Transient receptor potential melastatin type 2 (TRPM2) is a redox-sensitive, calcium-permeable cation channel activated by various signals, such as adenosine diphosphate ribose (ADPR) acting on the ADPR pyrophosphatase (ADPRase) domain, and cyclic ADPR. Here, we purified the FLAG-tagged tetrameric TRPM2 channel, analyzed it using negatively stained electron microscopy, and reconstructed the three-dimensional structure at 2.8-nm resolution. This multimodal sensor molecule has a bell-like shape of 18 nm in width and 25 nm in height. The overall structure is similar to another multimodal sensor channel, TRP canonical type 3 (TRPC3). In both structures, the small extracellular domain is a dense half-dome, whereas the large cytoplasmic domain has a sparse, double-layered structure with multiple internal cavities. However, a unique square prism protuberance was observed under the cytoplasmic domain of TRPM2. The FLAG epitope, fused at the C terminus of the ADPRase domain, was assigned by the antibody to a position close to the protuberance. This indicates that the agonist-binding ADPRase domain and the ion gate in the transmembrane region are separately located in the molecule.