Thiopurine S-methyltransferase Gene Research Articles

Novel variant in Nudix hydrolase 15 gene influences 6-mercaptopurine toxicity in childhood acute lymphoblastic leukemia patients.

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. 6-Mercaptopurine (6-MP) is a key component of ALL treatment. Its use, however, is also associated with adverse drug reactions, particularly myelosuppression. Thiopurine S-methyltransferase (TPMT) and, more recently, Nudix hydrolase 15 (NUDT15) deficiency, due to no-function variants in their respective genes, are well known for their role in the development of this toxicity. Two novel genetic variants, rs12199316 in TPMT and rs73189762 in the NUDT15 gene, were recently identified by targeted sequencing. The latter is particularly interesting because of its potential association with 6-MP intolerance. Here, we assessed the relationship of this variant with the risk of myelosuppression and 6-MP dose intensity in 275 patients treated with Dana Farber Cancer Institute ALL protocols at the Sainte Justine University Health Center. Carriers of the NUDT15 rs73189762 variant allele were at a higher risk of myelosuppression, as shown by absolute phagocyte count reduction during consolidation II and maintenance phases of therapy. Reduction in 6-MP dose intensity was observed in patients with both rs73189762 and known no-function variants in the NUDT15 and TPMT genes. This finding supports the initial observation and suggests that 6-MP dose reduction might be beneficial for individuals with this genotype combination.

P798 Study of the concordance between the enzymatic activity of Thiopurine S-methyltransferase and polymorphisms in the TPMT gene in patients with Inflammatory Bowel Disease

Abstract Background Thiopurine therapy is associated with an increased risk of serious adverse events in patients with Inflammatory Bowel Disease (IBD). Thiopurine S-methyltransferase (TPMT) is the enzyme responsible for metabolism, and its determination helps identify patients at risk of toxicity. This study aims to explore the level of correspondence between the TPMT genotype and enzymatic activity (phenotype) with the objective of preventing adverse events prior to the commencement of thiopurine therapy. Methods Consecutive patients with IBD, Crohn’s Disease (CD) or Ulcerative Colitis (UC), were included. Genotype was conducted for the most prevalent TPMT variants (TPMT*2, TPMT*3B, TPMT*3C), TPMT enzymatic activity assessment, and quantification of 6-thioguanine nucleotide (6-TGN) levels. Adverse effects were documented, along with the administration of biologics, mesalazine, ACE inhibitors or allopurinol. The primary objective was to evaluate the concordance between TPMT genotype and phenotype. Secondary objectives included determining the prevalence of genotype and phenotype within our patient cohort, 6-TGN serum levels, the prevalence of adverse events and identifying potential pharmacological inducers of the phenotype. Results A total of 218 patients were included (131 with CD and 81 with UC). Thirteen patients did not initiate thiopurines, and 34.63% (71/205) had discontinued treatment. The most observed adverse events were myelotoxicity (15.12%), gastrointestinal toxicity (12.20%), and hepatic toxicity (5.37%). TPMT activity exhibited a trimodal distribution, with 87.61% (191/218) having normal activity, 11.47% having intermediate activity, and 1% having low activity. Genotypically, 90.83% (198/218) had a wild-type genotype, 8.72% (18/218) had a heterozygous genotype, and 0.5% (1/218) had a homozygous genotype. The concordance index between phenotype and genotype was calculated at 0.57. Significantly, myelotoxicity was associated with the presence of genotypic variants (p=0.004). 6-TGN levels did not correlate with TPMT activity and concurrent medications did not affect TPMT activity . Conclusion The proactive assessment of TPMT (phenotype or genotype) before initiating thiopurine therapy effectively mitigates the occurrence of myelotoxicity in IBD patients. Importantly, the level of concordance between TPMT activity and genotype is moderate, with genotyping serving as the more reliable approach for preselecting patients susceptible to myelotoxic reactions.

Determination of Thiopurine S-Methyltransferase (TPMT) Activity in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine medications that are used in the treatment of multiple malignant and nonmalignant immunologic conditions. Polymorphisms in the TPMT gene associated with low enzyme activity can produce pronounced pharmacologic effects during therapy. The determination of TPMT erythrocyte activity is a valuable adjunct test to genotyping for the assignment of TPMT phenotype, especially in the presence of indeterminate genotypes. We present a method for the determination TPMT activity in human erythrocytes whereby we utilize LC-MS/MS to measure the amount of 6-methylmercaptopurine formed in red blood cell (RBC) lysates using 6-mercaptopurine as substrate and S-adenosylmethionine (SAM) as methyl donor. This method has been successfully implemented and proven to be reliable for use in human specimens.

Pharmacogenetic Effect of Thiopurine Methyl Transferase (TPMT) Gene Expression and Serum TNF on the Imuran Response in Ulcerative Colitis (UC) Iraqi Patients.

Ulcerative colitis (UC), a chronic inflammatory bowel disease (IBD), exerts its impact on both rectal and colonic mucosa, with a growing incidence. This study aims to explore the pharmacogenetic influence of thiopurine methyl transferase (TPMT) gene expression and serum tumor necrosis factor (TNF) levels on the response to Imuran in Iraqi patients with UC. Seventy individuals with chronic UC and 30 healthy controls were enrolled in this investigation. RNA extraction using the triazole method and enzyme-linked immunosorbent assay (ELISA) for TNF measurement were employed. Patients, aged 15-50 years, underwent Imuran treatment. Diverse responses to Imuran were observed among patients, with TPMT gene expression levels below 1 in 35 patients leading to side effects, while the remaining 35 patients exhibited positive responses with TPMT gene expression exceeding 1. Patients with varying degrees of severe, moderate, and mild UC associated with TNF showed a significant correlation with Imuran non-response. A distinct correlation was identified between TPMT gene expression and Imuran therapy outcomes in UC patients. Further investigation is warranted to elucidate the underlying mechanism, positioning the TPMT gene as a potential therapeutic target for mitigating the impact of UC.

B-225 Assessment of the Genotype Frequency of Thiopurine Methyltransferase (TPMT) Deficiency in a Large Cohort of Patients With Immune Mediated Inflammatory Disease and Cancer

Abstract Background Thiopurine methyltransferase (TPMT) alleles (*1/*2/*3A/*3B/*3C) are routinely genotyped to determine the metabolizer status of patients that plan to undergo thiopurine therapies (6-Mercaptopurine and Azathioprine). Screening of these alleles allows for personalized patient dosing and reduces the risk of severe or even fatal myelosuppression. The objective of this study was to determine the genotype frequency of TPMT deficiency in a large clinical cohort of patients with immune mediated inflammatory diseases and cancer. Methods All genotyping was performed in a reference clinical laboratory. Patient specimens were tested using genomic DNA isolated from whole blood. Clinical patient diagnoses were physician provided (international classification of disease, ICD-10). The genomic DNA was tested for the presence of three single nucleotide polymorphisms (SNPs) in the TPMT gene coding region and known to associate with reduced TPMT activity (rs1800462-Exon 5, rs1800460-Exon 7 and rs1142345-Exon 10). These polymorphisms determine the presence of *2/*3A/*3B/*3C alleles. Testing occurred from 10/26/2000 to 9/9/2022. Three different testing technologies were used during the 22 years of testing (PCR-RFLP from 2000 to 2003, Taqman™ chemistries from 2004 to 2015, and Luminex™ xMAP™ bead-based since 2016). Results A total of 219 129 patients were tested (mean age: 38 yrs, 56% female). As presented in the Table the genotype frequency for the wild type *1/*1 was 90.8% (95%CI: 90.7–90.9%), it was 9.0% (95%CI: 8.8–9.1%) for the heterozygous and 0.22% (95%CI: 0.020–0.024%) for homozygous variant, with similar frequency across disease states. The TPMT *1/*3B genotype frequency was very rare (<0.1%, n = 5) and the *3B/*3C homozygous variant (which cannot be distinguished from the *1/*3A) was calculated to have a frequency of 1:7 706 230 (95%CI: 1:7 043 228–1:8 369 232) and lower than initial estimates. Conclusion We estimated the frequency of TPMT genotypes in the largest cohort reported to date. The occurrence of the *3B/*3C genotype may be rarer than initially estimated.

The c.415C>T polymorphism in NUDT15 is more frequent than the polymorphisms in TPMT in Chilean patients who use thiopurine drugs.

Azathioprine (AZA) and 6-mercaptopurine (6-MP) are drugs widely used in the treatment of autoimmune diseases. Among the enzymes involved in the metabolism of AZA and 6-MP are thiopurine methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15). The existence of single nucleotide polymorphisms in the genes that code for these enzymes could decreased enzymatic activity AND lead to severe myelosuppression. The most relevant polymorphism is NUDT15*3 (rs116855232), where the replacement of cytosine for thymine at position 415, which in turn leads to a loss of enzymatic activity. In a previous study, it was identified that together the polymorphisms in the TPMT gene reach an allelic frequency of 3.81%. There is no information regarding the rs116855232 polymorphism in the NUDT15 gene, so this corresponds to the objective of this report. Blood samples from Chilean adult patients with indications for the use of AZA or 6-MP for different pathologies and who had undergone a TPMT gene polymorphism study were retrospectively analyzed. A total of 253 blood samples were analyzed. Of the 253 patients, 47 presented the c.415C>T polymorphism in the NUDT15 gene, 3 being homozygous and 44 heterozygous. Four of the heterozygous patients for NUDT15 also had the *3A variant in the TPMT gene, also heterozygous. The allelic frequency of the minor T allele found (9.88%) was very similar to that found in patients of Asian origin, and much higher than that reported for the European Caucasian or Latin American population.

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

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. 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. 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 ± 0.014pmol/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 ± 0.041pmol/mg total protein, P = 0.020). 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.

Impact of the homozygous mutation in Nudix hydrolase 15 on myelosuppression with 6-mercaptopurine in a European girl with acute lymphoblastic leukemia: A case report

A 6-year-old girl diagnosed with intermediate-risk acute lymphoblastic leukemia (ALL) presented with severe myelotoxicity and multiple infections during phase IB induction treatment with 6-mercaptopurine (6-MP). In the subsequent treatment phases, which included 6-MP, the patient continued to show bone marrow aplasia and neutropenia, necessitating numerous dose adjustments and interruptions. The recommended dose was eventually reduced to 5 %. A pharmacogenetic analysis, conducted in induction phase IB, detected three single-nucleotide polymorphisms (SNPs) of the thiopurine S-methyltransferase (TPMT) gene, and the phenotype of a normal metabolizer was observed. As a result of a second pharmacogenetic analysis, pathological polymorphisms were revealed in Nudix hydrolase 15 (NUDT15), which may explain the patient’s myelotoxicity. Hence, a pharmacogenetic analysis performed in advance would have been able to prevent her from suffering severe toxicity and/or treatment failure.

Genetic variants of genes involved in thiopurine metabolism pathway are associated with 6-mercaptopurine toxicity in pediatric acute lymphoblastic leukemia patients from Ethiopia.

Introduction: Genetic variation in the thiopurine S-methyltransferase (TPMT) gene by and large predicts variability in 6-mercaptopurine (6-MP) related toxicities. However, some individuals without genetic variants in TPMT still develop toxicity that necessitates 6-MP dose reduction or interruption. Genetic variants of other genes in the thiopurine pathway have been linked to 6-MP related toxicities previously. Objective: The aim of this study was to evaluate the effect of genetic variants in ITPA, TPMT, NUDT15, XDH, and ABCB1 on 6-MP related toxicities in patients with acute lymphoblastic leukemia (ALL) from Ethiopia. Methods: Genotyping of ITPA, and XDH was performed using KASP genotyping assay, while that of TPMT, NUDT15, and ABCB1 with TaqMan® SNP genotyping assays. Clinical profile of the patients was collected for the first 6 months of the maintenance phase treatment. The primary outcome was the incidence of grade 4 neutropenia. Bivariable followed by multivariable cox regression analysis was performed to identify genetic variants associated with the development of grade 4 neutropenia within the first 6 months of maintenance treatment. Results: In this study, genetic variants in XDH and ITPA were associated with 6-MP related grade 4 neutropenia and neutropenic fever, respectively. Multivariable analysis revealed that patients who are homozygous (CC) for XDH rs2281547 were 2.956 times (AHR 2.956, 95% CI = 1.494-5.849, p = 0.002) more likely to develop grade 4 neutropenia than those with the TT genotype. Conclusion: In conclusion, in this cohort, XDH rs2281547 was identified as a genetic risk factor for grade 4 hematologic toxicities in ALL patients treated with 6-MP. Genetic polymorphisms in enzymes other than TPMT involved in the 6-mercaptopurine pathway should be considered during its use to avoid hematological toxicity.

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.

Mercaptopurine induced myelosuppression in a child with a NUDT15 rs116855232 homozygous variant.

Mercaptopurine (6-MP) is the backbone of the consolidation and maintenance therapy for paediatric acute lymphoblastic leukaemia (ALL). Nevertheless, it can cause critical myelosuppression. Predicting adverse reactions to 6-MP often involves the investigation of pharmacogenetic variants; in particular thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15). Lately, NUDT15 variants have been shown to play a significant pharmacogenetic role in predicting 6-MP intolerance in children of Asian descent. We present a six-year-old male child of Indian origin with persistent cytopenia after treatment. This prompted targeted sequencing of the genes TPMT and NUD15. The results revealed two copies of the variant of NUD15 rs116855232, that is, NUDT15*2 genotype. Since the NUDT15*2 allele classified the patient as a poor metabolizer, he was restarted on a low dose of 6-MP, which he tolerated. Individuals with the NUDT15*2allele (*2/*2 genotype) are poor metabolizers of thiopurines which results in an adverse reaction to 6-MP. About 3.5% of Indians show variations in the TPMT gene as compared to 19.4% variations observed in NUDT15, which makes the latter a more reliable disease marker.

Genome‐Wide Association Study for the Genetic Determinants of Thiopurine S‐Methyltransferase Protein Expression in the Liver

HypothesisSinge nucleotide polymorphisms will be associated with differential expression of Thiopurine S‐Methyltransferase (TPMT) in the liver.MethodsNormal human liver samples (n=288) were acquired from University of Minnesota Liver Tissue Cell Distribution System, the Cooperative Human Tissue Network, and XenoTech LCC (Lenexa, KS, USA). Absolute quantification of TPMT in liver microsomes was calculated using a DIA total protein approach (DIA‐TPA). Human liver samples were genotyped using the Illumina Multi‐Ethnic Global Assay (Illumina, Miami, USA).ResultsAfter accounting for population stratification, genome‐wide association study (GWAS) analysis was conducted on 243 human liver samples. There were 30 genetic variants, all located on chromosome 6, that passed our genome‐wide threshold (P < 5.0 x 10‐8) after Bonferroni correction. Further GWAS analysis conditioning on rs1142345 (TPMT*3C/TPMT*3A), the strongest signal within the dataset and a known nonfunctional TPMT allele, showed no other independent signals. The presumed homozygous wild‐type TPMT subjects had a 1.97‐fold higher expression of TPMT than the TPMT*3A heterozygotes (0.1056 pmol/mg ± 0.0289 pmol/mg vs. 0.0536 pmol/mg ± 0.0143 pmol/mg, p‐value: 8.4e‐14).When comparing TPMT expression by ancestry, European ancestry donors (n= 214) had significantly higher expression than African ancestry donors (n= 34) when removing TPMT*3A carriers (0.1085 pmol/mg ± 0.0260 pmol/mg vs. 0.0860 pmol/mg ± 0.0416 pmol/mg, p‐value: 0.004.ConclusionFor the first time, we quantified absolute TPMT protein expression in the liver using a liquid chromatography tandem mass spectrometry based DIA‐TPA proteomics approach and conducted a GWAS to identify genetic variants affecting hepatic TPMT protein expression. The only independent genetic signal from the GWAS was from the TPMT*3A haplotype, which is comprised of two nonsynonymous SNPs rs1800460 and rs1142345. Previous in vitrostudies investigating the effect of common polymorphisms on TPMT expression in COS‐1 cells demonstrated that TPMT*3A is almost a complete loss of detectable TPMT due to its rapid degradation rate.1 This is in line with our expression data, where heterozygous carriers of TPMT*3A had about half the amount of TPMT as wild‐type subjects.We also discovered that donors with African ancestry had significantly less TPMT expression than donors with European ancestry after removing samples that had known TPMT polymorphisms (TPMT*3A/*3B/*3C). A previous study reported patients of Afro‐Caribbean ancestry had significantly lower TPMT activity than those of European or South Asian descent.2 Future studies need to be conducted to determine if the difference in TPMT expression by ancestry is due to genetic polymorphisms or other factors.

Understanding thiopurine methyltransferase polymorphisms for the targeted treatment of hematologic malignancies

ABSTRACT Introduction Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines (mercaptopurine (MP) and tioguanine (TG)), chemotherapeutic agents used in the treatment of acute lymphoblastic leukemia (ALL). Polymorphisms in TPMT gene encode diminished activity enzyme, enhancing accumulation of active metabolites, and partially explaining the inter-individual differences in patients’ clinical response. Areas covered This review gives an overview on TPMT gene and function, and discusses the pharmacogenomic implications of TPMT variants in the prevention of severe thiopurine-induced hematological toxicities and the less known implication on TG-induced sinusoidal obstruction syndrome. Additional genetic and non-genetic factors impairing TPMT activity are considered. Literature search was done in PubMed for English articles published since1990, and on PharmGKB. Expert opinion To titrate thiopurines safely and effectively, achieve the right degree of lymphotoxic effect and avoid excessive myelosuppression, the optimal management will combine a preemptive TPMT genotyping to establish a safe initial dose with a close phenotypic monitoring of TPMT activity and/or of active metabolites during long-term treatment. Compared to current ALL protocols, replacement of TG by MP during reinduction phase in TPMT heterozygotes and novel individualized TG regimens in maintenance for TPMT wild-type subjects could be investigated to improve outcomes while avoiding risk of severe hepatotoxicity.

TPMT polymorphisms and minimal residual disease after 6-mercaptopurine post-remission consolidation therapy of childhood acute lymphoblastic leukaemia

Bone marrow minimal residual disease (MRD) is the strongest predictor of relapse in children with acute lymphoblastic leukemia (ALL). 6-mercaptopurine (6MP) in ALL therapy has wide inter-individual variation in disposition and is strongly influenced by polymorphisms in the thiopurine methyltransferase (TPMT) gene. In 952 patients treated according to the NOPHO ALL2008 protocol, we explored the association between thiopurine disposition, TPMT genotypes and MRD levels after consolidation therapy with 6MP, high-dose methotrexate (HD-MTX), asparaginase, and vincristine. The levels of the cytotoxic DNA-incorporated thioguanine were significantly higher on day 70-79 in G460A/A719G TPMT heterozygous (TPMT HZ) compared to TPMT wild type (TPMT WT) patients (mean: 230.7 vs. 149.7 fmol/µg DNA, p = 0.002). In contrast, TPMT genotype did not associate with the end of consolidation MRD levels irrespective of randomization of the patients to fixed dose (25 mg/m2/day) or 6MP escalation (up to 50 or 75 mg/m2/day) during consolidation therapy.

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

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

S3509 Aplastic Anemia Following Autoimmune Hepatitis Treated With Azathioprine

INTRODUCTION: Aplastic anemia is a serious and potentially fatal disease where bone marrow suppression leads to pancytopenia. Azathioprine, an immunosuppressive drug used to treat various autoimmune disorders, can cause aplastic anemia. Here we present a case of aplastic anemia following azathioprine treatment of autoimmune hepatitis. CASE DESCRIPTION/METHODS: A 24 Y.O. Male presented with no significant past medical history presented with elevated liver enzymes and bilirubin. Ultrasound and CT confirmed mild hepatomegaly and diagnosis of acute hepatitis was given. A liver biopsy was performed and was noted for necrotic tissue. Acute hepatitis resolved spontaneously but relapsed 6 months later. Upon relapse, Anti Smooth Muscle Antibodies were found suggesting autoimmune hepatitis. The patient was treated with prednisone and 4 weeks later, azathioprine was started. 2 months after beginning azathioprine, labs revealed low hematocrit and platelet count, so the dose was lowered. By 4 months labs revealed pancytopenia and azathioprine treatment was stopped. The patient had a bone marrow biopsy done and was diagnosed with aplastic anemia. The patient began treatment to receive a bone marrow transplant but died of a systemic fungal infection. DISCUSSION: It is well documented that aplastic anemia, as a side effect of azathioprine treatment, is highly correlated to polymorphisms in the Thiopurine methyltransferase (TPMT) gene which occurs in 0.3%–0.5% of the population. Current treatment guidelines call for consideration of screening TPMT levels but considering the potential severity of the side effect and ease of testing, screening TPMT levels should be more common practice. However, it is also noted that the patient had low hematocrit levels starting about 1 month before the second episode of hepatitis. This suggests that the azathioprine may not have been the causative factor leading to pancytopenia, and it may have been a progression of Hepatitis Associated Aplastic Anemia (HAAA). HAAA is characterized by auto-immune hepatitis followed by aplastic anemia 2–3 months later. Testing for TPMT levels before starting azathioprine treatment for autoimmune hepatitis can help identify patients at risk for developing aplastic anemia as well as help rule out azathioprine as the causative agent for the aplastic anemia.

ALL-051: The Influence of Thiopurine Methyltransferase Gene Polymorphism on Egyptian Children with Acute Lymphoblastic Leukaemia

Context Gene polymorphisms involved in thiopurine methyltransferase (TPMT) transport, metabolism, or targeting are responsible for the variable drug response and regulates thiopurine therapeutic efficacy and toxicity. Objective To determine the frequency of TPMT gene polymorphism in Egyptian children with acute lymphoblastic leukaemia (ALL) and its influence on chemotherapy response and toxicity. This may allow identify patients who are at high risk for developing toxicity based on each patient's genetic constitution to individualized drug treatment. Design Paediatric patients with ALL, who were treated with BFM 90 or CCG 1991 standard risk protocol, and who experienced myelosuppression toxicity and required interruption and/or modification of thiopurine chemotherapy were recruited over a one-year period. Seventy healthy age- and sex-matched children served as controls. They subjected to clinical assessment, haematological panel investigations, and TPMT gene polymorphism for G238C, G460A, and A719G alleles assessment using PCR followed by RFLP analysis. Results Sixty-four ALL patients out of 150 who were regularly attending pediatric Hematology/Oncology clinic were recruited. The male: female ratio was 40 (62.5%): 24 (37.5%) and their mean age was 6.16 ± 3.16 years. Their diagnosis was T lineage (27%) and pre-B phenotype (73%). Thirty-two patients were on maintenance and another thirty-two were finished with their chemotherapy. Among the 64 patients and 70 control (288 examined allies), neither the studied leukemic patients nor the controls had the mutant TPMT variant alleles in either homozygous or heterozygous form. Myelosuppression toxicity in form of different degrees of neutropenia was detected in all patients, necessitating 6-MP dose modification for most of patients, either once (53.1%), twice (15.6%), or ≥three times (25.1%) during their maintenance course. Patients required stopping 6-MP for less than a week (62.5%), up to 2 weeks (28.1%), or >2 weeks (6.3%). Patients also developed infection that mostly (71%) necessitated hospitalization. Infections and febrile neutropenia were common causes of 6-MP dose modification and interruption. Conclusions The absence of any allelic variation responsible for the low TPMT activity in the studied leukemic patients, together with the fact that patients experienced signs of myelosuppression toxicity during the course of chemotherapy; would suggest the existence of additional factors in the development of 6-MP cytotoxicity.

Scorpion primer PCR analysis for genotyping of allele variants of thiopurine s‑methyltransferase*3.

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines. Mutations in the TPMT gene can affect drug activity, which may have adverse effects in humans. Thus, genotyping can help elucidate genetic determinants of drug response to thiopurines and optimize the selection of drug therapies for individual patients, effectively avoiding palindromia during maintenance treatment caused by insufficient dosing and the serious side effects caused by excessive doses. The current available detection methods used for TPMT*3B and TPMT*3C are complex, costly and time-consuming. Therefore, innovative detection methods for TPMT genotyping are urgently required. The aim of the present study was to establish and optimize a simple, specific and timesaving TPMT genotyping method. Using the principles of Web-based Allele-Specific PCR and competitive real-time fluorescent allele-specific PCR (CRAS-PCR), two pairs of Scorpion primers were designed for the detection of TPMT*3B and *3C, respectively, and a mutation in TPMT*3A was inferred based on data from TPMT*3B and *3C. In total, 226 samples from volunteers living in Chongqing were used for CRAS-PCR to detect TPMT*3 mutations. Results showed that nine (3.98%) were mutant (MT) heterozygotes and none were MT homozygotes for TPMT*3C, and no TPMT*3A and TPMT*3B mutations were found. Three TPMT*3C MT heterozygotes were randomly selected for DNA sequencing, and CRAS-PCR results were consistent with the sequencing results. In conclusion, in order to improve simplicity, specificity and efficiency, the present study established and optimized CRAS-PCR assays for commonly found mutant alleles of TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G).

Predictive Value of NUDT15 Variants on Neutropenia Among Han Chinese Patients with Dermatologic Diseases: A Single-Center Observational Study.

IntroductionAzathioprine is a synthetic purine analogue derived from 6-mercaptopurine which acts by disrupting nucleic acid synthesis and interfering with T cell activation. It is effective in dermatology diseases related to the immune system. However, its side effects, including severe neutropenia, kept patients from using it. Mutations in thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase (ITPA) genes account for the major genetic polymorphism markers for azathioprine adverse risk factors in Caucasians, but not in Asians. The predictive value of the nucleoside diphosphate-linked moiety X motif 15 gene (NUDT15) has been studied in various diseases among different populations. The aim of our study was to determine the contribution of NUDT15 mutations in azathioprine-induced neutropenia in Han Chinese patients with dermatologic diseases.MethodsThe study enrolled all consecutive patients, older than 13 years old, with dermatological diseases currently treated orally with azathioprine in our clinic. Samples were also collected from patients with documented leukopenia in our prior study that examined the association between TPMT, ITPA, and neutropenia after informed reconsent. Complete blood count, differential count, and hepatic and renal function were checked regularly. The DNA samples for NUDT15 genotype were obtained from the patients.ResultsIn total, we enrolled 56 patients (39 male, 17 female). The NUDT15 genotypes are mostly C/C (N = 36, 64.29%). Heterozygous variant (C/T) accounts for 30.36% (N = 17) and homozygous variant (T/T) accounts for 5.36% (N = 3). Among these patients, 15 patients (26.79%) developed neutropenia, including all three patients carry homozygous variant (T/T). The age-, sex-, and dose-adjusted risk of heterozygous variant compared to wild type is 9.383 (95% CI 1.32–66.96).ConclusionsPretreatment screening of NUDT15 might reduce the chance of azathioprine-induced neutropenia in Han Chinese patients with dermatologic diseases.

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

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