Thiopurine S-methyltransferase Variants Research Articles

Pharmacogenomic Assessment of Genes Implicated in Thiopurine Metabolism and Toxicity in a UK Cohort of Pediatric Patients With Inflammatory Bowel Disease.

Thiopurine drugs are effective treatment options in inflammatory bowel disease and other conditions but discontinued in some patients due to toxicity. We investigated thiopurine-induced toxicity in a pediatric inflammatory bowel disease cohort by utilizing exome sequencing data across a panel of 46 genes, including TPMT and NUDT15. The cohort included 487 patients with a median age of 13.1 years. Of the 396 patients exposed to thiopurines, myelosuppression was observed in 11%, gastroenterological intolerance in 11%, hepatotoxicity in 4.5%, pancreatitis in 1.8%, and "other" adverse effects in 2.8%. TPMT (thiopurine S-methyltransferase) enzyme activity was normal in 87.4%, intermediate 12.3%, and deficient in 0.2%; 26% of patients with intermediate activity developed toxicity to thiopurines. Routinely genotyped TPMT alleles associated with defective enzyme activity were identified in 28 (7%) patients: TPMT*3A in 4.5%, *3B in 1%, and *3C in 1.5%. Of these, only 6 (21%) patients developed toxic responses. Three rare TPMT alleles (*3D, *39, and *40) not assessed on routine genotyping were identified in 3 patients, who all developed toxic responses. The missense variant p.R139C (NUDT15*3 allele) was identified in 4 patients (azathioprine 1.6mg/kg/d), but only 1 developed toxicity. One patient with an in-frame deletion variant p.G13del in NUDT15 developed myelosuppression at low doses. Per-gene deleteriousness score GenePy identified a significant association for toxicity in the AOX1 and DHFR genes. A significant association for toxicity was observed in the AOX1 and DHFR genes in individuals negative for the TPMT and NUDT15 variants. Patients harboring the NUDT15*3 allele, which is associated with myelosuppression, did not show an increased risk of toxicity.

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.

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

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

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.

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.

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.

Population-scale predictions of DPD and TPMT phenotypes using a quantitative pharmacogene-specific ensemble classifier

BackgroundInter-individual differences in dihydropyrimidine dehydrogenase (DPYD encoding DPD) and thiopurine S-methyltransferase (TPMT) activity are important predictors for fluoropyrimidine and thiopurine toxicity. While several variants in these genes are known to decrease enzyme activities, many additional genetic variations with unclear functional consequences have been identified, complicating informed clinical decision-making in the respective carriers.MethodsWe used a novel pharmacogenetically trained ensemble classifier to analyse DPYD and TPMT genetic variability based on sequencing data from 138,842 individuals across eight populations.ResultsThe algorithm accurately predicted in vivo consequences of DPYD and TPMT variants (accuracy 91.4% compared to 95.3% in vitro). Further analysis showed high genetic complexity of DPD deficiency, advocating for sequencing-based DPYD profiling, whereas genotyping of four variants in TPMT was sufficient to explain >95% of phenotypic TPMT variability. Lastly, we provided population-scale profiles of ethnogeographic variability in DPD and TPMT phenotypes, and revealed striking interethnic differences in frequency and genetic constitution of DPD and TPMT deficiency.ConclusionThese results provide the most comprehensive data set of DPYD and TPMT variability published to date with important implications for population-adjusted genetic profiling strategies of fluoropyrimidine and thiopurine risk factors and precision public health.

Dosage of 6-Mercaptopurine in Relation to Genetic TPMT and ITPA Variants: Toward Individualized Pediatric Acute Lymphoblastic Leukemia Maintenance Treatment.

6-mercaptopurine (6-MP) is the mainstay in pediatric acute lymphoblastic leukemia (ALL) maintenance treatment. Variants in genes coding for thiopurine S-methyl transferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) are known to influence 6-MP metabolism. We determined TPMT and ITPA genotype and enzyme activity and the mean 6-MP doses during maintenance treatment in 40 children treated for ALL according to the Dutch Childhood Oncology Group (DCOG)-ALL11 protocol in the Radboudumc Amalia Children's Hospital, Nijmegen, The Netherlands. Patients with genetic variants in TPMT (N=3) had significantly lower TPMT enzyme activity (mean 0.46 vs. 0.72 µmol/mmol hemoglobin/h, P=0.005). Although the difference was not statistically significant, they were treated with lower mean 6-MP doses (28.1 mg/m [SD 25.5 mg/m] vs. 41.3 mg/m [SD 17.2 mg/m], P=0.375). In patients with genetic ITPA variants (N=21), ITPA enzyme activity was significantly lowered (mean 3.67 vs. 6.84 mmol/mmol hemoglobin/h, P<0.0005). The mean 6-MP doses did not differ between patients with and without variants in ITPA (40.0 mg/m [SD 20.3 mg/m] vs. 40.6 mg/m [SD 14.9 mg/m], P=0.663). The TPMT genotype, but not the ITPA genotype, should be considered as part of standard evaluation before starting ALL maintenance treatment.

An intronic FTO variant rs16952570 confers protection against thiopurine-induced myelotoxicities in multiethnic Asian IBD patients

Thiopurines are used in the treatment of inflammatory bowel disease (IBD) but remain clinically challenging to manage due to wide interpatient variability in clinical outcomes and adverse events. Apart from genetic variants in thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15) genes, polymorphisms in FTO alpha-ketoglutarate dependent dioxygenase (FTO) were found predictive of thiopurine-induced leukopenia, albeit with conflicting results. To clarify the role of FTO variants in a multiethnic Asian IBD cohort, we recruited 149 patients on thiopurine-based therapy and genotyped two FTO variants p.Ala134Thr (rs79206939) and rs16952570 T > C using Sanger sequencing. FTO p.Ala134Thr (rs79206939) was non-polymorphic and absent whereas intronic rs16952570 T > C was equally prevalent in Chinese (22%) and Indians (18%) and higher in Malays (28%). Higher nadir white blood cell (WBC) and absolute neutrophil count (ANC) levels were observed in patients harboring FTO rs16952570 CC genotypes compared with TT carriers at 4, 8, and 12 weeks after start of thiopurine therapy (P < 0.05). A similar trend was observed in patients carrying the previously well-characterized NUDT15 rs116855232 wild-type CC genotypes. Further in silico analysis suggests that FTO variants linked to rs16952570, particularly rs74018601, may play a regulatory role in altering the FTO expression. The findings from this study indicate a novel protective association with the FTO variant rs16952570 CC genotype and hematological parameters.

Impact of NUDT15 genetics on severe thiopurine-related hematotoxicity in patients with European ancestry

PurposeSevere hematotoxicity in patients with thiopurine therapy has been associated with genetic polymorphisms in the thiopurine S-methyltransferase (TPMT). While TPMT genetic testing is clinically implemented for dose individualization, alterations in the nudix hydrolase 15 (NUDT15) emerged as independent determinant of thiopurine-related hematotoxicity. Because data for European patients are limited, we investigated the relevance of NUDT15 in Europeans. MethodsAdditionally to TPMT phenotyping/genotyping, we performed in-depth Sanger sequencing analyses of NUDT15 coding region in 107 European patients who developed severe thiopurine-related hematotoxicity as extreme phenotype. Moreover, genotyping for NUDT15 variants in 689 acute lymphoblastic leukemia (ALL) patients was performed. ResultsAs expected TPMT was the main cause of severe hematotoxicity in 31% of patients, who were either TPMT deficient (10%) or heterozygous carriers of TPMT variants (21%). By comparison, NUDT15 genetic polymorphism was identified in 14 (13%) patients including one novel variant (p.Met1Ile). Six percent of patients with severe toxicity carried variants in both TPMT and NUDT15. Among patients who developed toxicity within 3 months of treatment, 13% were found to be carriers of NUDT15 variants. ConclusionTaken together, NUDT15 and TPMT genetics explain ~50% of severe thiopurine-related hematotoxicity, providing a compelling rationale for additional preemptive testing of NUDT15 genetics not only in Asians, but also in Europeans.

Retrospective Data Analysis of the Influence of Age and Sex on TPMT Activity and Its Phenotype-Genotype Correlation.

Therapeutic efficacy and toxicity of thiopurine drugs (used as anticancer and immunosuppressant agents) are affected by thiopurine S-methyltransferase (TPMT) enzyme activity. TPMT genotype and/or phenotype is used to predict the risk for adverse effects before drug administration. Inosine triphosphate pyrophosphatase (ITPA) is another enzyme involved in thiopurine metabolism. In this study, we aimed to evaluate (a) frequency of various TPMT phenotypes and genotypes, (b) correlations between them, (c) influence of age and sex on TPMT activity, and (d) distribution of ITPA variants among various TPMT subgroups. TPMT enzyme activity was determined by LC-MS/MS. TPMT (*2,*3A-C) and ITPA (rs1127354, rs7270101) genotypes were determined using a customized TaqMan® OpenArray®. TPMT enzyme activity varied largely (6.3-90 U/mL). The frequency of low, intermediate, normal, and high activity was 0.5% (n = 230), 13.1% (n = 5998), 86.1% (n = 39448), and 0.28% (n = 126), respectively. No significant difference in TPMT activity in relation to age and sex was found. Genotype analysis revealed the frequency of variant TPMT alleles was 6.73% (*3A, n = 344), 0.05% (*3B, n = 2), 2.22% (*3C, n = 95), and 0.42% (*2, n = 19). Analysis of paired phenotype and genotype showed that TPMT activity in samples with variant allele(s) was significantly lower than those without variant alleles. Lastly, an equal distribution of ITPA variants was found among normal and abnormal TPMT activity. This retrospective data analysis demonstrated a clustering of variant TPMT genotypes with phenotypes, no significant influence of age and sex on TPMT activity, and an equal distribution of ITPA variants among various TPMT subgroups.

Elevation in Cell Cycle and Protein Metabolism Gene Transcription in Inactive Colonic Tissue From Icelandic Patients With Ulcerative Colitis.

BackgroundA combination of genetic and environmental factors is thought to be involved in the pathogenesis of ulcerative colitis (UC). In Iceland, the incidence of UC is one of the highest in the world. The aim of this study was to characterize patients with UC and identify potential germline mutations and pathways that could be associated with UC in this population.MethodsExome sequencing and genome-wide microarray analysis on macroscopically noninflamed colonic mucosa from patients and controls were performed. Exome sequence data were examined for very rare or novel mutations that were over-represented in the UC cohort. Combined matching of variant analysis and downstream influence on transcriptomic expression in the rectum were analyzed.ResultsOne thousand eight hundred thirty-eight genes were differentially expressed in rectal tissue from UC patients and identified an upregulation in genes associated with cell cycle control and protein processing in the endoplasmic reticulum (ER). Two missense mutations in thiopurine S-methyltransferase (TPMT) with a minor allele frequency of 0.22 in the UC patients compared with a reported 0.062 in the Icelandic population were identified. A predicted damaging mutation in the gene SLC26A3 is potentially associated with increased expression of DUOX2 and DUOXA2 in rectal tissue.ConclusionsColonic mucosa of UC patients demonstrates evidence of an elevation in genes involving cell proliferation and processing of proteins within the ER. Exome sequencing identified a possible increased prevalence of 2 damaging TPMT variants within the UC population, suggesting screening the UC population before initiation of thiopurine analogue therapy to avoid toxicity associated with these mutations.

P087 Allele frequency of Thiopurine S-methyltransferase (TPMT) in Hong Kong population

Aim Azathioprine, 6-Mercaptopurine and 6-Thioguanine are commonly used in the management of acute leukemia, autoimmune diseases and as the immunosuppressants in organ transplantation. These drugs belong to the class of thiopurine which can inhibit DNA synthesis and cell proliferation of fast growing lymphocytes by suppressing de novo purine synthesis. Thioguanine nucleotides (TGNs) are the active metabolites in which accumulation of high level cytotoxic TGNs can lead to myelosuppression. It has been demonstrated that Thiopurine S-methyltransferase (TPMT) is the key enzyme responsible for catalytic conversion of TGNs to non-cytotoxic methyl-thioguanine. The TPMT gene is polymorphic and TPMT protein variants with reduced enzyme activity are associated with inefficient removal of TGNs. In this study, we aim to investigate the allele frequency of the most common TPMT variants (TPMT*2 and *3C) in Hong Kong population. Methods Genomic DNA from whole blood was extracted. The presence of TPMT*2 and *3C mutant alleles were determined using Amplification-Refractory Mutation System (ARMS) PCR method. Beta-2-microglobulin was used as the internal control. All mutant alleles were confirmed by Sanger’s sequencing. Hong Kong healthy individuals (n = 300) were included in this study. Rare TPMT alleles were not determined. Results TPMT*1/3C genotype was detected in 9 out of 300 healthy subjects while TPMT*2 allele was absent. The allele frequency of TPMT*3C in Hong Kong population is 1.5%. Conclusions This study provides the first analysis of the allele frequency of TPMT variants in Hong Kong population and our finding is in accordance with the reported frequency range in Chinese population. TPMT*3C is the most common TPMT variant in Hong Kong subjects. The establishment of TPMT genotyping assay for patient requires thiopurine therapy will provide additional information to guide the reduction of drug dose to minimize the risk of myelosuppression.

472 - NUDT15 Variants Contribute to Thiopurine-Induced Myelosuppression in European Populations

Introduction Thiopurines are commonly used in the maintenance treatment of inflammatory bowel disease (IBD) but this is limited by myelosuppression in 7% of patients.1 Thiopurine S-methyltransferase (TPMT) variants only explain 20% of thiopurine-induced myelosuppression (TIM) in Europeans suggesting the presence of other genetic determinants.2 We aimed to identify the clinical features of TIM and to identify novel variants associated with TIM through a genome wide association study and whole exome sequencing. Method We recruited 491 IBD patients with TIM from 82 hospitals. Inclusion criteria included drug exposure in the 7 days prior to TIM, white blood cell count ≤2.5 ×109/L, or neutrophil count ≤1.0 ×109/L and that TIM necessitated dose reduction/drug withdrawal. After expert adjudication, 329 cases assigned a high likelihood of causality were genotyped and exome sequenced with 635 thiopurine-tolerant IBD controls for use in the final analyses. Results We first confirmed an association of TIM with TPMT in an initial GWAS. We then, using exome sequencing, discovered a novel 6 bp in-frame deletion (rs746071566; AGGAGTC/A,p.Gly17_Val18del) at position 48611918 of chromosome 13 in exon 1 of NUDT15 (5.8% cases, 0.2% controls, OR=38.2,p=1.33×10-8). This deletion was replicated in an independent cohort (3/75 [4%] cases vs. 2/785 [0.3%] thiopurine-tolerant IBD controls; OR=16.2, p=0.006). All NUDT15 coding variants were confirmed with complete concordance by Sanger sequencing. Multivariable logistic regression demonstrated that adjusted dose (OR 2.2,p=9.4×10-11), NUDT15 genotype (OR 21.7,p=2.0×10-8) and TPMT genotype (OR 2.2,p=2.6×10-4 for MUT/WT and OR 51.2,p=1.8×10-4 for MUT/MUT) were independently associated with TIM. Conclusion These are the largest ever clinical and genetic analyses of thiopurine-induced myelosuppression. We identified NUDT15 coding variants, including a novel 6 bp deletion; carriage of any coding variant confers a 22-fold increase in the odds of TIM, independent of TMPT genotype and thiopurine dose. Although NUDT15 variants are less common than TPMT variants, their effect size for heterozygotes is greater. The number of patients needed to genotype to prevent TIM due to NUDT15 coding variant heterozygosity is 100. The estimated absolute risk of TIM in NUDT15 heterozygotes is 59%. A future clinical decision tool incorporating NUDT15 and TPMT genotypes will offer personalised thiopurine therapy and prevent the considerable morbidity and costs associated with severe bone marrow toxicity. Reference . Gisbert JP & Gomollon F.doi10.1111/j.1572-0241.2008.01848.x (2008) . Yang SK et al. doi10.1038/ng.3060 (2014) Disclosure of Interest None Declared

Association Between Thiopurine S-Methyltransferase (TPMT) Genetic Variants and Infection in Pediatric Heart Transplant Recipients Treated With Azathioprine: A Multi-Institutional Analysis.

Bone marrow suppression is a common adverse effect of the immunosuppressive drug azathioprine. Polymorphisms in the gene encoding thiopurine S-methyltransferase (TPMT) can alter the metabolism of azathioprine, resulting in marrow toxicity and life-threatening infection. In a multicenter cohort of pediatric heart transplant (HT) recipients, we determined the frequency of TPMT genetic variation and assessed whether azathioprine-treated recipients with TPMT variants were at increased risk of infection. We genotyped TPMT in 264 pediatric HT recipients for the presence of the TPMT*2, TPMT*3A, and TPMT*3C variant alleles. Data on infection episodes and azathioprine use were collected as part of each patient's participation in the Pediatric Heart Transplant Study. We performed unadjusted Kaplan-Meier analyses comparing infection outcomes between groups. TPMT variants were identified in 26 pediatric HT recipients (10%): *3A (n = 17), *3C (n = 8), and *2 (n = 1). Among those with a variant allele, *3C was most prevalent in black patients (4 of 5) and *3A most prevalent among white and Hispanic patients (16 of 20). Among 175 recipients (66%) who received azathioprine as part of the initial immunosuppressive regimen, we found no difference in the number of infections at 1 year after HT (0.7 ± 1.3; range, 0-6 versus 0.5 ± 0.9; range, 0-3; p = 0.60) or in freedom from infection and bacterial infection between non-variant and variant carriers. There was 1 infection-related death in each group. In this multicenter cohort of pediatric HT recipients, the prevalence of TPMT variants was similar across racial/ethnic groups to what has been previously reported in non-pediatric HT populations. We found no association between variant alleles and infection in the first year after HT. Because clinically detected cytopenia could have prompted dose adjustment or cessation, we recommend future studies assess the relationship of genotype to leukopenia/neutropenia in the pediatric transplantation population.

Prevalence of thiopurine S-methyltransferase gene polymorphisms in patients with inflammatory bowel disease from the island of Crete, Greece.

There is evidence that genotyping for the thiopurine S-methyltransferase (TPMT) gene variants is useful for the prediction of response to thiopurine analogs (azathioprine and 6-mercaptopurine) in patients with inflammatory bowel disease (IBD). The aim of the present study was to determine the prevalence of TPMT gene polymorphisms in a genetic homogenous population of IBD patients in Crete and to correlate the results with adverse reactions to thiopurine drugs. Genotyping for the most common TPMT variants TPMT*2, TPMT*3A, TPMT3*C, and TPMT*3B was performed using the PCR-restriction fragment length polymorphism method in 223 consecutive IBD patients and 119 age-matched and sex-matched healthy controls. The hospital medical records were reviewed for thiopurine use in these patients and related adverse events. The prevalence of TPMT variants TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3C was 1.8, 2.7, 1.3, and 1.8%, respectively. The G238C mutation was detected in four (1.8%) out of 223 patients, three (1.3%) patients were carriers of the G460A mutation, four (1.8%) of the A719G mutation, and six (2.7%) of both G460A and A719G mutations. In healthy controls, only one (0.8%) carried both the G460A and the A719G mutation, whereas TPMT*2, TPMT*3C, and TPMT*3B were not detected. None of the genotypes was homozygous. A statistically significant correlation between the presence of the G460A mutation and the development of leucopenia after the administration of thiopurines was observed (P=0.048). This study showed a lower frequency of total TPMT variants and a higher frequency of TPMT*3B in Cretan IBD patients compared with other Caucasian populations. The presence of the G460A mutation is associated with the development of leukopenia.

In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT*6 and TPMT*8.

Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

Enrichment of Genetic Variants in the Glucocorticoid Receptor Signalling Pathway in Autoimmune Hepatitis with Failure of Standard Treatment.

The standard treatment for autoimmune hepatitis (AIH) is predniso(lo)ne for remission induction, tapered and followed by azathioprine, which effectively controls the disease in the majority of patients. However, some patients prove to be unresponsive or non-tolerant and require alternative immunosuppressive regimens for disease control. We aimed to investigate whether these AIH patients who experience failure of standard treatment have a genomic basis for their problem in the form of pharmacogenetic variants. Fifty-six consecutive patients with AIH [41 female and 15 male; median age 42 years (12-76)] were retrospectively stratified according to being responders to standard therapy (n = 33) or patients with failure of standard therapy (n = 23). Their blood DNA was exome-captured and sequenced. Genomic variants were filtered and compared between the groups using Ingenuity Variant Analysis (3.1.20150407). The exome sequencing and data processing revealed glucocorticoid receptor signalling to be the most prominently affected pathway among the patients with failure of standard therapy (highly significant). Standard treatment failure was not associated with thiopurine S-methyltransferase variants or the HLA-DRB1*03 genotype. In conclusion, enrichment of variants related to glucocorticoid receptor signalling was a particular genomic trait of the AIH patients in whom standard treatment failed and alternative immunosuppression was required. If confirmed, a future application of this finding may be to identify prospective cases of failure of standard treatment already at diagnosis.

P154 Complete sequence-based NUDT15 and TPMT variants for predicting thiopurine-induced leukopenia in patients with Crohn's disease

Background: Thiopurine-induced leukopenia is a life-threatening complication in Asians. Other than previously known thiopurine S-methyltransferase (TPMT) mutation, the NUDT15 genetic variant was recently identified to have a strong association with thiopurine-induced leukopenia. We investigated the association of NUDT15, TPMT, metabolites of thiopurines on leukopenia in patients with Crohn's disease (CD). Methods: We investigated the data of 168 adult patients with CD undergoing thiopurine treatment. Clinical evaluation and laboratory examinations were performed every 1–3 months. We carried out a complete sequencing analysis for evaluating NUDT15 and TPMT variants and measured thiopurine metabolites levels. Results: Of 168 patients, 34 patients (20.2%) had NUDT15 variant alleles. NUDT15 heterozygous variants were found in 28 patients (16.7%) and NUDT15 homozygous variants were found in 6 patients (3.6%). Among the 168 patients, 35 (20.8%) patients experienced leukopenia during first year of thiopurine treatment. NUDT15 variant types were strongly associated with developing leukopenia [10.9% wild type as reference; 48.5% heterozygous variant genotype; 100% homozygous variant genotype, odds ratio (OR) 3.44 (95% CI, 1.21–9.78)]. Table 1. The risk of leukopenia according to NUDT15 and TPMT genotype However, TPMT genotype was not associated with developing leukopenia (OR 0.58, 95% CI 0.07–4.96). Patients with NUDT15 homozygous variant genotype developed severe early leukopenia with average 88.2% (range, 84–94%) reduction from baseline WBC count at 4 weeks. Figure 1. WBC count as a percentage of baseline at 4 weeks after thiopurines treatment. In subgroup of NUDT15 variant type, there was no significant difference of 6-TGN levels between patients with and without leukopenia [298.5 (205–407) vs. 280.8 (174–327) pmol/8×108 RBC, p=0.344]. Table 2. Thiopurine metabolites levels and thiopurine-induced leukopenia in NUDT15 variant genotype subgroup Conclusions: This is the first complete sequence-based analysis for NUDT15 variants in Asian populations. Our findings suggest that NUDT15 genotype should be detected rather than TPMT before initiating thiopurines. Thiopurines treatment should not be recommended to patients with NUDT15 homozygous variant genotype due to severe early leukopenia.

P407. Clinical manifestations of thiopurine S-methyltransferase variants in Korean paediatric patients with inflammatory bowel disease

P407. Clinical manifestations of thiopurine S-methyltransferase variants in Korean paediatric patients with inflammatory bowel disease