Diplotype analysis of NUDT15 using digital PCR in pediatric acute lymphoblastic leukemia

Diplotype analysis of NUDT15 using digital PCR in pediatric acute lymphoblastic leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Diplotype analysis of NUDT15 using digital PCR in pediatric acute lymphoblastic leukemia Yasunori Iida, Reiji Fukano, Takuya Ichimura, Sachiyo Kamimura, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8438183/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Patients with NUDT15 variants exhibit intolerance to 6-mercaptopurine (6-MP), which can cause severe myelosuppression and increase the risk of second malignancies, especially in those with bi-allelic variants. However, no reliable analytical method is available to determine the diplotype of NUDT15 . Therefore, we aimed to develop a practical diplotyping method using digital PCR to improve clinical outcomes and reduce complications. We analyzed NUDT15 exon 1 and 3 variants in 38 children with acute lymphoblastic leukemia (ALL) who received 6-MP during maintenance therapy between 2010 and 2021. Variants were genotyped by Sanger sequencing, and tolerated 6-MP doses were assessed according to genotype. For patients carrying multiple variants, germline RNA was extracted, synthesized into cDNA, and analyzed using four variant-specific dPCR probes. Nine patients carried single variants, and two carried multiple variants, and required markedly lower 6-MP doses. The dPCR successfully resolved the phase of the variants and identified a compound heterozygous diplotype in one patient, highlighting its ability to allow the assignment of variant combinations to specific alleles. Our findings demonstrate that dPCR is a practical tool for NUDT15 diplotyping and may facilitate optimized 6-MP therapy by reducing toxicity risk and improving treatment precision for pediatric ALL. Biological sciences/Cancer Health sciences/Diseases Biological sciences/Genetics Biological sciences/Molecular biology Health sciences/Molecular medicine Health sciences/Oncology NUDT15 variants diplotype analysis digital polymerase chain reaction variant-specific probe acute lymphoblastic leukemia 6-mercaptopurine sensitivity. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Acute lymphoblastic leukemia (ALL) is the most common form of leukemia in Japan. Despite a long-term survival rate of approximately 90% for pediatric patients with ALL [ 1 ], enhancing treatment outcomes and minimizing associated complications continue to pose significant challenges. The treatment of pediatric ALL typically involves a multifaceted approach incorporating multidrug chemotherapy regimens. A key drug in both consolidation and maintenance therapies is the thiopurine derivative 6-mercaptopurine (6-MP) [ 2 – 4 ]. However, the toxicity of 6-MP varies considerably among patients and often requires dose reductions or interruptions for those exhibiting heightened sensitivity [ 5 ]. Variability in 6-MP sensitivity is strongly correlated with specific variants of the thiopurine S-methyltransferase ( TPMT ) [ 6 ] and/or nucleoside diphosphate-linked moiety X-type motif 15 ( NUDT15 ) genes [ 7 , 8 ]. The influence of NUDT15 variants is especially pronounced in East Asian and Hispanic populations and significantly affects 6-MP sensitivity [ 9 ]. Genetic variants of NUDT15 can lead to severe side effects, such as bone marrow suppression [ 10 , 11 ]. Additionally, excessive dosing in patients with these variants increases the risk of developing secondary malignancies [ 12 ]. Currently, the Clinical Pharmacogenetics Implementation Consortium guidelines (Fig. 1 ) recognize nine alleles of NUDT15 variants, mainly located in exons 1 and 3 [ 5 ]. Patients harboring these variants can experience varying degrees of bone marrow suppression depending on the specific variant. Some variants cause a complete loss of function (*2, *3, *9), whereas others have uncertain functionality (*4–*8), leading to a complex and inconsistent impact on patient outcomes [ 5 ]. Sensitivity to 6-MP is determined by both the variant type and the number of alleles carrying the variant. Patients with biallelic variants require a significant reduction in 6-MP dosage owing to the increased risk of severe bone marrow suppression compared with those with monoallelic variants [ 13 ]. Thus, NUDT15 diplotyping is vital to prevent severe bone marrow suppression and reduce the risk of secondary cancers in patients with multiple heterozygous variants. Although several methods for NUDT15 diplotyping have been reported [ 14 – 16 ], a practical method for evaluating all variants remains to be established. Therefore, we aimed to develop a practical diplotyping analysis method using digital PCR for clinical applications based on previously reported approaches. Results Marked reduction in 6-MP dosage for patients with multiple variants A total of 38 patients were included in the study: 21 patients treated at Yamaguchi University Hospital, and 17 patients treated at cooperating institutions who required a dose reduction of 6-MP to < 50% of the standard dose. Among the 38 analyzed cases, 27 displayed wild-type alleles for both exons 1 and 3, whereas 11 harbored variants. The 11 variant-harboring cases are summarized in Table 1 . Cases 1 –9 were identified as single-variant cases, and cases 10 and 11 were classified as multiple-variant cases. The average 6-MP dosage was 34.2 mg/m 2 /day for patients with wild-type genotypes, 22.0 mg/m 2 /day for those with single variants, and 8.1 mg/m 2 /day for those with multiple variants. Patients with multiple variants required significantly lower 6-MP doses than those with wild-type (P = 0.0062) or single-variant genotypes (P = 0.0213) (Fig. 2 ). Table 1 Data of patients with variants among the ALL cohort Of the eleven cases with variants, nine had a single variant, and two had multiple variants. Abbreviations: M, Male; F, Female; BCP, B-cell precursor; 6-MP, 6-mercaptopurine No. variants age sex phenotype risk 6-MP dosage (mg/m 2 ) 1 c.415C > T (hetero) 1 male T-ALL SR 25.8 2 c.415C > T (hetero) 1 male BCP-ALL SR 34.1 3 c.415C > T (hetero) 1 female BCP-ALL SR 11.0 4 c.415C > T (hetero) 1 female BCP-ALL SR 27.0 5 c.415C > T (hetero) 2 male BCP-ALL SR 34.0 6 c.415C > T (hetero) 7 male BCP-ALL SR 7.2 7 c.415C > T (hetero) 7 female BCP-ALL SR 1.5 8 c.52G > A (hetero) 10 male BCP-ALL HR 45.1 9 c.415C > T (hetero) 12 male BCP-ALL IR 12.6 10 c.52G > A (hetero) c.415C > T (hetero) 2 female BCP-ALL SR 12.0 11 55_56insGAGTCG (hetero) c.415C > T (homo) 10 male T-ALL HR 4.2 Cases 1 9, each harboring a single heterozygous variant, were categorized as monoallelic variants. Case 11, which had a heterozygous variant in exon 1 and a homozygous variant in exon 3, was classified as a biallelic variant. Case 10 exhibited heterozygous variants in both exons 1 and 3, creating uncertainty about whether it represented a monoallelic or biallelic variant. Consequently, we performed diplotype analysis using digital PCR for Case 10. Digital PCR analysis reveals compound heterozygosity in NUDT15 Initially, we confirmed the specificity of the probes for their intended sequences using artificially synthesized plasmids (Fig. 3 ). One plasmid contained exon 1 with the c.52G > A variant and exon 3 as wild-type, whereas the other plasmid contained exon 1 as wild-type and exon 3 with the c.415C > T variant. The FAM probe, which is specific to the c.52G > A variant, yielded a positive signal only with the plasmid containing the c.52G > A variant in exon 1, while remaining negative for wild-type exon 1. Similarly, the VIC probe for wild-type exon 1, the JUN probe for wild-type exon 3, and the ABY probe for the c.415C > T variant in exon 3 all functioned as intended. Based on these results, cut-off values were established to distinguish between positive and negative signals for each fluorescent color. Digital PCR was subsequently performed using cDNA from Case 10, and results were presented in two-color analyses for each probe combination (Fig. 4 ). Signals for VIC and ABY were double positive, suggesting that wild-type exon 1 and the c.415C > T variant in exon 3 were present on the same allele (Fig. 4 a). Wild-type exons 1 and 3 appeared to reside on opposite alleles, as indicated by distinct fluorescence populations (Fig. 4 b). The c.52G > A variant in exon 1 and the c.415C > T variant in exon 3 were located on opposite alleles (Fig. 4 c), whereas the c.52G > A variant in exon 1 and wild-type exon 3 were found on the same allele (Fig. 4 d). These findings confirmed that the variants were located on separate alleles, indicating that Case 10 harbored a compound heterozygous variant. Based on the diplotyping analysis, of the 11 cases with variants, nine cases with single variants (Cases 1 –9) were categorized as the monoallelic variant group, whereas two cases with multiple variants—Case 10 with compound heterozygous variants and Case 11 with a heterozygous variant in exon 1 and a homozygous variant in exon 3—were categorized as the biallelic variant group. Regarding tolerable 6-MP dosages, patients with single variants were classified as monoallelic variant cases, whereas those with multiple variants were classified as biallelic variant cases. As reported previously [ 13 ], patients with biallelic variants tolerated significantly lower 6-MP doses than those with wild-type or monoallelic variants. Validation of compound heterozygosity through cloning analysis To further validate these findings, we performed a cloning experiment. Of the nine cloned colonies, five contained the c.52G > A variant in exon 1 and the wild-type exon 3, whereas four displayed wild-type exon 1 and the c.415C > T variant in exon 3 (Fig. 5 ). These results corroborated the diplotype determined through digital PCR analysis. Discussion NUDT15 diplotyping analysis has been performed using several methods, including paired-end sequencing using next-generation sequencing [ 14 ], allele-specific PCR [ 15 ], and digital PCR [ 16 ]. Cloning methods have been used in previous studies [ 9 ]. Yu et al. successfully performed diplotyping analysis of NUDT15 in 37 patients with ALL using next-generation sequencing paired-end sequencing [ 14 ]. Although next-generation sequencing delivers highly accurate results through direct sequence reading, its high cost and extensive time requirements limit its practicality for routine clinical application. Allele-specific PCR targets the 3ʹ base of a single nucleotide polymorphism allele in patient DNA using specific primers [ 15 ]. Under stringent conditions, only the complementary strand hybridizes with the target sequence, whereas the non-complementary strand is not amplified. The presence of a variant is confirmed when the complementary primer sequences are successfully amplified. In one report, allele-specific PCR was used to evaluate diplotypes in a case involving both c.55_56insGAGTCG and c.415C > T variants, although its utility for other variants was not discussed [ 15 ]. Tsujimoto et al. used digital PCR to analyze NUDT15 diplotypes in patients carrying c.55_56insGAGTCG and c.415C > T variants [ 16 ]. However, they could not distinguish between the compound heterozygous variants c.52G > A and c.415C > T, as in our case. Although the potential of digital PCR for diplotyping was demonstrated in this study, the method was ineffective for all variants. In our study, we successfully performed diplotype analysis using digital PCR for a previously undetectable variant. Our results showed that wild-type probes indicated the presence of wild-type sequences in exons 1 and 3 of opposite alleles. This outcome may be attributed to the adjustment of the probe and primer-binding sites. These findings suggest that the patient harbored compound heterozygous variants. To confirm the variant-specific binding of these probes, we used four types of probes in various combinations, consistently demonstrating the presence of compound heterozygous variants in the patient. Our findings highlight that digital PCR with variant-specific probes enables the evaluation of compound heterozygous variants of c.52G > A and c.415C > T, which were previously difficult to assess. The results of the present study are applicable across different races or environments in which digital PCR is available. Digital PCR offers several advantages, including ease of use, clinical feasibility, and cost-effectiveness [ 16 ]. However, further studies are required to determine whether this method can be applied to other variants. We aim to develop this approach to enable NUDT15 diplotype analysis for all patients with ALL at the time of diagnosis. We intend to use this strategy to tailor 6-MP treatment to minimize bone marrow suppression and reduce the risk of secondary malignancies. The potential carcinogenic effects of thiopurines have been demonstrated [ 17 ], and among Asians with NUDT15 variants, an increased incidence of secondary malignancies has been observed [ 12 ]. This suggests that 6-MP cytotoxicity may increase the risk of developing secondary malignancies [ 12 ]. To address these concerns, we plan to prospectively assess whether the use of this diplotyping strategy to guide treatment adjustments can help mitigate these adverse effects. Beyond its role in guiding adjustments to 6-MP dosing, this test may have broader applications for other diseases. Nudix hydrolase 15 reportedly affects the metabolism of medications such as azathioprine [ 7 ] and antiviral agents such as acyclovir and ganciclovir [ 18 ]. Thus, determining NUDT15 diplotypes could benefit patients who are being considered for these treatments. Nonetheless, this study has certain limitations. The small sample size and retrospective study design may have introduced bias. Moreover, even among patients with the NUDT15 wild-type or single-variant genotype, some tolerate substantially low 6-MP doses. This intolerance suggests that other factors may influence 6-MP sensitivity. By analyzing the characteristics of this group, we identified additional factors affecting 6-MP tolerance. Ultimately, a comprehensive analysis of all factors contributing to 6-MP sensitivity may enable personalized dosing, thereby improving treatment outcomes. In this study, we identified NUDT15 variants in pediatric patients with ALL using direct sequencing. For cases with multiple heterozygous variants, diplotype analysis was performed using digital PCR. This approach enabled accurate identification of variants such as c.52G > A and c.415C > T, which are often challenging to detect using conventional methods. Consistent with previous studies, we observed a significant reduction in the average dose of 6-MP in individuals with biallelic variants of NUDT15 compared with those with wild-type or monoallelic variants. Collectively, our findings suggest that, for cases with multiple heterozygous variants, diplotype analysis is crucial for preventing severe bone marrow suppression caused by 6-MP. Digital PCR is a practical method for NUDT15 diplotyping in clinical settings. Future studies are warranted to explore the applicability of this method to other variants. Methods Study design and patient enrollment In this retrospective observational study, we included 38 pediatric ALL cases diagnosed between January 2010 and December 2021. Among these, 21 patients received 6-MP during maintenance therapy at Yamaguchi University Hospital, Ube, Japan, whereas 17 patients were treated at Kagoshima University Hospital, University of Miyazaki Hospital, Kurume University Hospital, National Hospital Organization Kyushu Cancer Center, and the National Hospital Organization Kumamoto Medical Center. In maintenance therapy, the standard dose of 6-MP was 50 mg/m 2 . The 6-MP dose was adjusted to maintain the white blood cell count at 2000–3000/µL. For inclusion in this study, the 17 patients required to have a reduction in their 6-MP dosage to < 50%. We specifically selected patients from other hospitals who were administered a 6-MP dose of < 50% because we anticipated that many of them would have variants. The patients included in the study ranged in age from 1 year to 13 years at diagnosis; 22 males and 16 females were included in the study. We ensured that this study complied with the guidelines of the Declaration of Helsinki and Ethical Guidelines for Medical and Biological Research Involving Human Subjects and received approval from the Institutional Review Board of Yamaguchi University Hospital (approval no. H2022-060). Written informed consent was obtained from the parents of all participants. NUDT15 genotyping and dose assessment DNA was extracted from normal white blood cells in the peripheral blood using the QIAamp® DNA Blood Mini Kit (catalog number: 51104; QIAGEN, Hilden, Germany). NUDT15 variants on exons 1 and 3 were amplified using KAPA2G Robust HotStart® ReadyMix with dye (2×) (catalog number: KK5707, Roche, Basel, Switzerland) and AmpliTaq Gold Fast PCR Master Mix (catalog number: 4390939, Applied Biosystems, Waltham, MA, USA). These reagents were used according to the manufacturer’s protocols. PCR experiments were performed using a Takara PCR Thermal Cycler, and the amplified products were analyzed via Sanger sequencing. The primers used for amplification were as follows: exon 1 forward primer: 5ʹ-CAAAGCACAACTGTAAGCGACT-3ʹ; exon 1 reverse primer: 5ʹ-GAAAGACCCAGCTAGCAAAGAC-3ʹ; exon 3 forward primer: 5ʹ-AAGCAAATGCAAAGCATCAC-3ʹ; and exon 3 reverse primer: 5ʹ-GGCTGAAAGAGTGGGGGATA-3ʹ. Thermal cycling conditions included an initial denaturation step at 95°C for 3 min (1 cycle), followed by 35 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 5 s, with a final extension at 72°C for 3 min. The amplified PCR products were analyzed for the presence of specific NUDT15 variants using Sanger sequencing. The average 6-MP dosage was calculated for patients with wild-type, single-variant, and multiple-variant genotypes. The average 6-MP dose in each NUDT15 genotype was estimated using the Kruskal–Wallis test with JMP Pro 16 (SAS Institute Inc., Cary, NC, USA). Each box includes data between the 25th and 75th percentiles, with the horizontal line indicating the median. The mean doses for each group were compared. Diplotyping analysis using digital PCR Diplotyping analysis was performed on patients with multiple variants. RNA was extracted from peripheral blood, followed by complementary DNA (cDNA) synthesis using SuperScript IV® Reverse Transcriptase (catalog number: 18090050, Invitrogen, Waltham, MA, USA), RNase Out® Recombinant Ribonuclease Inhibitor (catalog number: 10777019, Invitrogen), Random Hexamers® (catalog number: N8080127, Invitrogen), and a 10- mM dNTP mix® (catalog number: 18427088, Invitrogen). Digital PCR analysis was conducted using synthesized cDNA and the QuantStudio Absolute Q Digital PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The primers used for digital PCR were: Common exon 1 forward primer: 5ʹ-GGCGGCCAGGAGTCG-3ʹ; Common exon 1 reverse primer: 5ʹ-CAACGCGGATGCTTGCA-3ʹ; Common exon 3 forward primer: 5ʹ-CCCCTGGACCAGCTTTTCTG-3ʹ; and Common exon 3 reverse primer: 5ʹ-CCACCAGATGGTTCAGATCTTCTTTAAA-3ʹ. Specific probes included: Wild-type exon 1 probe (VIC): 5ʹ-TCACCACGACTCCGAC-3ʹ; c.52G > A variant probe for exon 1 labeled with FAM: 5ʹ-CACCACGATTCCGAC-3ʹ; Wild-type probe for exon 3 labeled with JUN: 5ʹ-AAACAACGCAGTCCC-3ʹ; and c.415C > T variant probe for exon 3 labeled with ABY: 5ʹ-TTTAAACAACACAGTCCC-3ʹ. For all 9-µL PCR reaction mixtures, Absolute Q™ Universal DNA Digital PCR Master Mix (5×) (catalog number: A72710, Applied Biosystems) was used, with primers at a final concentration of 0.9 µM and probes at a final concentration of 0.2 µM. Synthesized cDNA was diluted 100,000-fold, and 0.25 µL of diluted cDNA was added to the reaction mixture. Thermal cycling conditions included an initial denaturation step at 96°C for 10 min (1 cycle), followed by 40 cycles at 96°C for 5 s (ramp rate undisclosed) and 64°C for 30 s (ramp rate undisclosed). We recorded the fluorescence intensity of each microarray after PCR amplification. PCR and fluorescence intensity measurements were performed using the PCR device described above. Cloning and Sanger sequencing for diplotype confirmation To confirm the diplotype, the synthesized cDNA was cloned using the Zero Blunt TOPO™ PCR Cloning Kit (catalog number: K280040; Invitrogen). The cDNA was cloned into a vector and transformed into TOP10 competent cells provided in the kit. Single colonies were cultured in Luria–Bertani medium containing kanamycin (50 µg/mL) at 37°C for 15 h, and plasmids were extracted using the QIAprep Spin Miniprep Kit (catalog number: 27104, Qiagen). Sanger sequencing was performed using the forward primer 5ʹ-GTGAGCGCGTCACTTCCTGC-3ʹ and reverse primer 5ʹ-ATCAAATCTTCTCGGCCACCTAGAG-3ʹ. Nine colonies were selected and sequenced to determine the NUDT15 diplotypes. Declarations Competing interests The authors have no conflicts of interest to disclose. This study was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS), Grant Number 22K15944 (I.T.). Funding This study was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japanese Society for the Promotion of Science (JSPS), Grant Number 22K15944 (I.T.). No other funding was received for this study. Author Contribution YI, RF, TI, and SH were responsible for designing the protocol, writing the protocol and report, conducting the search, screening potentially eligible studies, extracting and analyzing data, interpreting results, and updating the reference lists. SK, TN, SO, HM, YO, HN, and MM collected samples from patients who met the inclusion criteria of this study and submitted them for the research. YS and TY provided advice and support regarding the implementation of digital PCR. Acknowledgement We would like to express our gratitude to the technical support team at Thermo Fisher Scientific for their assistance with the digital PCR analysis. We thank Dr. Masanori Yoshida from St Jude Children’s Research Hospital, Dr. Shin-Ichi Tsujimoto from Yokohama City University, and Dr. Motohiro Kato from The University of Tokyo for their insightful advice on NUDT15 analysis. Data Availability The data were generated using the methods described in the Subjects and Methods section of this article. Data are not available to the public except as noted above but may be accessed upon reasonable request to the corresponding author. References Conter, V. et al. Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. Blood 115 , 3206–3214 (2010). Kato, M. & Manabe, A. Treatment and biology of pediatric acute lymphoblastic leukemia. Pediatr. Int. 60 , 4–12 (2018). Karran, P. & Attard, N. Thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer. Nat. Rev. Cancer . 8 , 24–36 (2008). Koren, G. et al. Systemic exposure to mercaptopurine as a prognostic factor in acute lymphocytic leukemia in children. N Engl. J. Med. 323 , 17–21 (1990). Relling, M. V. et al. Clinical Pharmacogenetics Implementation Consortium Guideline for thiopurine dosing based on TPMT and NUDT15 genotypes: 2018 Update. Clin. Pharmacol. Ther. 105 , 1095–1105 (2019). Schmiegelow, K. et al. Thiopurine methyltransferase activity is related to the risk of relapse of childhood acute lymphoblastic leukemia: results from the NOPHO ALL-92 study. Leukemia 23 , 557–564 (2009). Yang, S. K. et al. A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia. Nat. Genet. 46 , 1017–1020 (2014). Moriyama, T. et al. Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry. Blood 130 , 1209–1212 (2017). Yang, J. J. et al. Inherited NUDT15 variant is a genetic determinant of mercaptopurine intolerance in children with acute lymphoblastic leukemia. J. Clin. Oncol. 33 , 1235–1242 (2015). Moriyama, T. et al. NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity. Nat. Genet. 48 , 367–373 (2016). Yoshida, M. et al. Low NUDT15 expression levels due to biallelic NUDT15 variants and 6-mercaptopurine intolerance. Br. J. Haematol. 199 , 270–276 (2022). Yoshida, M. et al. NUDT15 variants confer high incidence of second malignancies in children with acute lymphoblastic leukemia. Blood Adv. 5 , 5420–5428 (2021). Tanaka, Y. et al. An international retrospective study for tolerability of 6-mercaptopurine on NUDT15 bi-allelic variants in children with acute lymphoblastic leukemia. Haematologica 106 , 2026–2029 (2021). Yu, C. H. et al. Determination of NUDT15 variants by targeted sequencing can identify compound heterozygosity in pediatric acute lymphoblastic leukemia patients. Sci. Rep. 10 , 14400 (2020). Yu, C. H. et al. Allele-specific polymerase chain reaction can determine the diplotype of NUDT15 variants in patients with childhood acute lymphoblastic leukemia. Sci. Rep. 13 , 490 (2023). Tsujimoto, S. et al. Diplotype analysis of NUDT15 variants and 6-mercaptopurine sensitivity in pediatric lymphoid neoplasms. Leukemia 32 , 2710–2714 (2018). Kandiel, A., Fraser, A. G., Korelitz, B. I., Brensinger, C. & Lewis, J. D. Increased risk of lymphoma among inflammatory bowel disease patients treated with azathioprine and 6-mercaptopurine. Gut 54 , 1121–1125 (2005). Nishii, R. et al. NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir. Nat. Commun. 12 , 4181 (2021). Additional Declarations No competing interests reported. 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Center","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Migita","suffix":""},{"id":569735514,"identity":"901ce0c2-e0ee-4c02-a19e-60160272dcf7","order_by":12,"name":"Shunji Hasegawa","email":"","orcid":"","institution":"Yamaguchi University","correspondingAuthor":false,"prefix":"","firstName":"Shunji","middleName":"","lastName":"Hasegawa","suffix":""}],"badges":[],"createdAt":"2025-12-24 02:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8438183/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8438183/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99813791,"identity":"80950223-963f-4099-8549-81ebe7ea9f30","added_by":"auto","created_at":"2026-01-08 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14:40:03","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49455,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/e5eb06a0b663aa9ac34caa5c.png"},{"id":99814250,"identity":"e120e9fb-b5b6-4e24-b065-6a911906ee45","added_by":"auto","created_at":"2026-01-08 14:40:42","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69203,"visible":true,"origin":"","legend":"","description":"","filename":"1530fe23260548f0be6269ae0ae0ca301structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/97ea3c7ee520ccc4ec9b4bd3.xml"},{"id":99814086,"identity":"26914ec8-e9ff-4d43-98f9-38c2e555e8bc","added_by":"auto","created_at":"2026-01-08 14:40:17","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79785,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/447ec075147d4e90ee60d8c3.html"},{"id":99814163,"identity":"d4f13b6f-ec61-47dd-9829-c26f3e0f130e","added_by":"auto","created_at":"2026-01-08 14:40:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14573,"visible":true,"origin":"","legend":"\u003cp\u003eCurrently reported \u003cem\u003eNUDT15\u003c/em\u003e haplotypes, created based on Reference 5.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/5bd6950c5bd688dd6f02fd00.png"},{"id":99813788,"identity":"c19cbf0b-9819-48f0-be15-1a2297f4691b","added_by":"auto","created_at":"2026-01-08 14:39:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15034,"visible":true,"origin":"","legend":"\u003cp\u003eAverage dose of 6-MP during maintenance therapy evaluated by the number of variants. Although no significant differences were observed, patients with a single variant had a lower average dose of 6-MP than that of patients with the wild-type variant. Patients with multiple variants had a significantly lower average dose than those with wild-type or single variants. Single-variant cases were classified as monoallelic variants, and multiple-variant cases were classified as biallelic variants based on diplotyping, aligning with previously reported findings.\u003c/p\u003e\n\u003cp\u003eAbbreviation: 6-MP, 6-mercaptopurine\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/121fce886901cdc43844d2b2.png"},{"id":99814001,"identity":"ecfd1e7f-b6ca-41c2-aa35-8ef8ddc0be5b","added_by":"auto","created_at":"2026-01-08 14:40:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51895,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of sequence-specific probes created for digital PCR, confirming exclusive binding to target sequences. The FAM probe for the exon 1 c.52G\u0026gt;A variant showed positive results only with the plasmid containing the sequence and negative results with the plasmid lacking the sequence. Similarly, other fluorescent probes demonstrated binding to their respective target sequences.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/0270769990eb25485ac29262.png"},{"id":99813915,"identity":"4bcb8c16-2cf4-4e0e-80d1-162973694c9d","added_by":"auto","created_at":"2026-01-08 14:40:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54165,"visible":true,"origin":"","legend":"\u003cp\u003eResults of digital PCR using the specific probe from Figure 3, indicating compound heterozygosity in this case.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/9ed579f8ad92da5a0b10f1b2.png"},{"id":99813651,"identity":"39fa2afa-5e90-4048-afc0-9e2a80606512","added_by":"auto","created_at":"2026-01-08 14:39:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38262,"visible":true,"origin":"","legend":"\u003cp\u003eResults of Sanger sequencing performed on nine \u003cem\u003eE. coli\u003c/em\u003e colonies into which \u003cem\u003eNUDT15\u003c/em\u003e was introduced via cloning. Among the colonies, five exhibited the c.52G\u0026gt;A variant only in exon 1, and the other four exhibited the c.415C\u0026gt;T variant only in exon 3. These findings indicate compound heterozygosity in this case.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/c3993724abfb6ccea4d80fa3.png"},{"id":99816381,"identity":"e1c15799-7e56-44a1-9227-51934fb77e2b","added_by":"auto","created_at":"2026-01-08 14:46:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":827880,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8438183/v1/4aa30f5b-0147-405b-b260-6728cbde642d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diplotype analysis of NUDT15 using digital PCR in pediatric acute lymphoblastic leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute lymphoblastic leukemia (ALL) is the most common form of leukemia in Japan. Despite a long-term survival rate of approximately 90% for pediatric patients with ALL [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], enhancing treatment outcomes and minimizing associated complications continue to pose significant challenges.\u003c/p\u003e \u003cp\u003eThe treatment of pediatric ALL typically involves a multifaceted approach incorporating multidrug chemotherapy regimens. A key drug in both consolidation and maintenance therapies is the thiopurine derivative 6-mercaptopurine (6-MP) [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the toxicity of 6-MP varies considerably among patients and often requires dose reductions or interruptions for those exhibiting heightened sensitivity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Variability in 6-MP sensitivity is strongly correlated with specific variants of the thiopurine S-methyltransferase (\u003cem\u003eTPMT\u003c/em\u003e) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and/or nucleoside diphosphate-linked moiety X-type motif 15 (\u003cem\u003eNUDT15\u003c/em\u003e) genes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The influence of \u003cem\u003eNUDT15\u003c/em\u003e variants is especially pronounced in East Asian and Hispanic populations and significantly affects 6-MP sensitivity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Genetic variants of \u003cem\u003eNUDT15\u003c/em\u003e can lead to severe side effects, such as bone marrow suppression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, excessive dosing in patients with these variants increases the risk of developing secondary malignancies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the Clinical Pharmacogenetics Implementation Consortium guidelines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) recognize nine alleles of \u003cem\u003eNUDT15\u003c/em\u003e variants, mainly located in exons 1 and 3 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Patients harboring these variants can experience varying degrees of bone marrow suppression depending on the specific variant. Some variants cause a complete loss of function (*2, *3, *9), whereas others have uncertain functionality (*4\u0026ndash;*8), leading to a complex and inconsistent impact on patient outcomes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSensitivity to 6-MP is determined by both the variant type and the number of alleles carrying the variant. Patients with biallelic variants require a significant reduction in 6-MP dosage owing to the increased risk of severe bone marrow suppression compared with those with monoallelic variants [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, \u003cem\u003eNUDT15\u003c/em\u003e diplotyping is vital to prevent severe bone marrow suppression and reduce the risk of secondary cancers in patients with multiple heterozygous variants.\u003c/p\u003e \u003cp\u003eAlthough several methods for \u003cem\u003eNUDT15\u003c/em\u003e diplotyping have been reported [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], a practical method for evaluating all variants remains to be established. Therefore, we aimed to develop a practical diplotyping analysis method using digital PCR for clinical applications based on previously reported approaches.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMarked reduction in 6-MP dosage for patients with multiple variants\u003c/h2\u003e \u003cp\u003eA total of 38 patients were included in the study: 21 patients treated at Yamaguchi University Hospital, and 17 patients treated at cooperating institutions who required a dose reduction of 6-MP to \u0026lt;\u0026thinsp;50% of the standard dose. Among the 38 analyzed cases, 27 displayed wild-type alleles for both exons 1 and 3, whereas 11 harbored variants. The 11 variant-harboring cases are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Cases \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;9 were identified as single-variant cases, and cases 10 and 11 were classified as multiple-variant cases. The average 6-MP dosage was 34.2 mg/m\u003csup\u003e2\u003c/sup\u003e/day for patients with wild-type genotypes, 22.0 mg/m\u003csup\u003e2\u003c/sup\u003e/day for those with single variants, and 8.1 mg/m\u003csup\u003e2\u003c/sup\u003e/day for those with multiple variants. Patients with multiple variants required significantly lower 6-MP doses than those with wild-type (P\u0026thinsp;=\u0026thinsp;0.0062) or single-variant genotypes (P\u0026thinsp;=\u0026thinsp;0.0213) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eData of patients with variants among the ALL cohort Of the eleven cases with variants, nine had a single variant, and two had multiple variants. Abbreviations: M, Male; F, Female; BCP, B-cell precursor; 6-MP, 6-mercaptopurine\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003evariants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003esex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ephenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003erisk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6-MP dosage\u003c/p\u003e \u003cp\u003e(mg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e25.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e27.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.52G\u0026thinsp;\u0026gt;\u0026thinsp;A (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e45.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.52G\u0026thinsp;\u0026gt;\u0026thinsp;A (hetero)\u003c/p\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (hetero)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBCP-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55_56insGAGTCG (hetero)\u003c/p\u003e \u003cp\u003ec.415C\u0026thinsp;\u0026gt;\u0026thinsp;T (homo)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT-ALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCases 1\u003c/strong\u003e \u003cp\u003e9, each harboring a single heterozygous variant, were categorized as monoallelic variants. Case 11, which had a heterozygous variant in exon 1 and a homozygous variant in exon 3, was classified as a biallelic variant. Case 10 exhibited heterozygous variants in both exons 1 and 3, creating uncertainty about whether it represented a monoallelic or biallelic variant. Consequently, we performed diplotype analysis using digital PCR for Case 10.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDigital PCR analysis reveals compound heterozygosity in\u003c/b\u003e \u003cb\u003eNUDT15\u003c/b\u003e\u003c/p\u003e \u003cp\u003eInitially, we confirmed the specificity of the probes for their intended sequences using artificially synthesized plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). One plasmid contained exon 1 with the c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A variant and exon 3 as wild-type, whereas the other plasmid contained exon 1 as wild-type and exon 3 with the c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variant. The FAM probe, which is specific to the c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A variant, yielded a positive signal only with the plasmid containing the c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A variant in exon 1, while remaining negative for wild-type exon 1. Similarly, the VIC probe for wild-type exon 1, the JUN probe for wild-type exon 3, and the ABY probe for the c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in exon 3 all functioned as intended. Based on these results, cut-off values were established to distinguish between positive and negative signals for each fluorescent color.\u003c/p\u003e \u003cp\u003eDigital PCR was subsequently performed using cDNA from Case 10, and results were presented in two-color analyses for each probe combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Signals for VIC and ABY were double positive, suggesting that wild-type exon 1 and the c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in exon 3 were present on the same allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Wild-type exons 1 and 3 appeared to reside on opposite alleles, as indicated by distinct fluorescence populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A variant in exon 1 and the c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in exon 3 were located on opposite alleles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), whereas the c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A variant in exon 1 and wild-type exon 3 were found on the same allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These findings confirmed that the variants were located on separate alleles, indicating that Case 10 harbored a compound heterozygous variant.\u003c/p\u003e \u003cp\u003eBased on the diplotyping analysis, of the 11 cases with variants, nine cases with single variants (Cases \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;9) were categorized as the monoallelic variant group, whereas two cases with multiple variants\u0026mdash;Case 10 with compound heterozygous variants and Case 11 with a heterozygous variant in exon 1 and a homozygous variant in exon 3\u0026mdash;were categorized as the biallelic variant group. Regarding tolerable 6-MP dosages, patients with single variants were classified as monoallelic variant cases, whereas those with multiple variants were classified as biallelic variant cases. As reported previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], patients with biallelic variants tolerated significantly lower 6-MP doses than those with wild-type or monoallelic variants.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eValidation of compound heterozygosity through cloning analysis\u003c/h3\u003e\n\u003cp\u003eTo further validate these findings, we performed a cloning experiment. Of the nine cloned colonies, five contained the c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A variant in exon 1 and the wild-type exon 3, whereas four displayed wild-type exon 1 and the c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in exon 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results corroborated the diplotype determined through digital PCR analysis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eNUDT15\u003c/em\u003e diplotyping analysis has been performed using several methods, including paired-end sequencing using next-generation sequencing [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], allele-specific PCR [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and digital PCR [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Cloning methods have been used in previous studies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Yu et al. successfully performed diplotyping analysis of \u003cem\u003eNUDT15\u003c/em\u003e in 37 patients with ALL using next-generation sequencing paired-end sequencing [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although next-generation sequencing delivers highly accurate results through direct sequence reading, its high cost and extensive time requirements limit its practicality for routine clinical application.\u003c/p\u003e \u003cp\u003eAllele-specific PCR targets the 3ʹ base of a single nucleotide polymorphism allele in patient DNA using specific primers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Under stringent conditions, only the complementary strand hybridizes with the target sequence, whereas the non-complementary strand is not amplified. The presence of a variant is confirmed when the complementary primer sequences are successfully amplified. In one report, allele-specific PCR was used to evaluate diplotypes in a case involving both c.55_56insGAGTCG and c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variants, although its utility for other variants was not discussed [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTsujimoto et al. used digital PCR to analyze \u003cem\u003eNUDT15\u003c/em\u003e diplotypes in patients carrying c.55_56insGAGTCG and c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variants [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, they could not distinguish between the compound heterozygous variants c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T, as in our case. Although the potential of digital PCR for diplotyping was demonstrated in this study, the method was ineffective for all variants.\u003c/p\u003e \u003cp\u003eIn our study, we successfully performed diplotype analysis using digital PCR for a previously undetectable variant. Our results showed that wild-type probes indicated the presence of wild-type sequences in exons 1 and 3 of opposite alleles. This outcome may be attributed to the adjustment of the probe and primer-binding sites. These findings suggest that the patient harbored compound heterozygous variants. To confirm the variant-specific binding of these probes, we used four types of probes in various combinations, consistently demonstrating the presence of compound heterozygous variants in the patient.\u003c/p\u003e \u003cp\u003eOur findings highlight that digital PCR with variant-specific probes enables the evaluation of compound heterozygous variants of c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T, which were previously difficult to assess. The results of the present study are applicable across different races or environments in which digital PCR is available. Digital PCR offers several advantages, including ease of use, clinical feasibility, and cost-effectiveness [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, further studies are required to determine whether this method can be applied to other variants.\u003c/p\u003e \u003cp\u003eWe aim to develop this approach to enable \u003cem\u003eNUDT15\u003c/em\u003e diplotype analysis for all patients with ALL at the time of diagnosis. We intend to use this strategy to tailor 6-MP treatment to minimize bone marrow suppression and reduce the risk of secondary malignancies.\u003c/p\u003e \u003cp\u003eThe potential carcinogenic effects of thiopurines have been demonstrated [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and among Asians with \u003cem\u003eNUDT15\u003c/em\u003e variants, an increased incidence of secondary malignancies has been observed [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This suggests that 6-MP cytotoxicity may increase the risk of developing secondary malignancies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To address these concerns, we plan to prospectively assess whether the use of this diplotyping strategy to guide treatment adjustments can help mitigate these adverse effects. Beyond its role in guiding adjustments to 6-MP dosing, this test may have broader applications for other diseases. Nudix hydrolase 15 reportedly affects the metabolism of medications such as azathioprine [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and antiviral agents such as acyclovir and ganciclovir [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, determining \u003cem\u003eNUDT15\u003c/em\u003e diplotypes could benefit patients who are being considered for these treatments.\u003c/p\u003e \u003cp\u003eNonetheless, this study has certain limitations. The small sample size and retrospective study design may have introduced bias. Moreover, even among patients with the \u003cem\u003eNUDT15\u003c/em\u003e wild-type or single-variant genotype, some tolerate substantially low 6-MP doses. This intolerance suggests that other factors may influence 6-MP sensitivity. By analyzing the characteristics of this group, we identified additional factors affecting 6-MP tolerance. Ultimately, a comprehensive analysis of all factors contributing to 6-MP sensitivity may enable personalized dosing, thereby improving treatment outcomes.\u003c/p\u003e \u003cp\u003eIn this study, we identified \u003cem\u003eNUDT15\u003c/em\u003e variants in pediatric patients with ALL using direct sequencing. For cases with multiple heterozygous variants, diplotype analysis was performed using digital PCR. This approach enabled accurate identification of variants such as c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T, which are often challenging to detect using conventional methods. Consistent with previous studies, we observed a significant reduction in the average dose of 6-MP in individuals with biallelic variants of \u003cem\u003eNUDT15\u003c/em\u003e compared with those with wild-type or monoallelic variants. Collectively, our findings suggest that, for cases with multiple heterozygous variants, diplotype analysis is crucial for preventing severe bone marrow suppression caused by 6-MP. Digital PCR is a practical method for \u003cem\u003eNUDT15\u003c/em\u003e diplotyping in clinical settings. Future studies are warranted to explore the applicability of this method to other variants.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003e\u003cstrong\u003eStudy design and patient enrollment\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eIn this retrospective observational study, we included 38 pediatric ALL cases diagnosed between January 2010 and December 2021. Among these, 21 patients received 6-MP during maintenance therapy at Yamaguchi University Hospital, Ube, Japan, whereas 17 patients were treated at Kagoshima University Hospital, University of Miyazaki Hospital, Kurume University Hospital, National Hospital Organization Kyushu Cancer Center, and the National Hospital Organization Kumamoto Medical Center. In maintenance therapy, the standard dose of 6-MP was 50 mg/m\u003csup\u003e2\u003c/sup\u003e. The 6-MP dose was adjusted to maintain the white blood cell count at 2000\u0026ndash;3000/\u0026micro;L. For inclusion in this study, the 17 patients required to have a reduction in their 6-MP dosage to \u0026lt;\u0026thinsp;50%. We specifically selected patients from other hospitals who were administered a 6-MP dose of \u0026lt;\u0026thinsp;50% because we anticipated that many of them would have variants. The patients included in the study ranged in age from 1 year to 13 years at diagnosis; 22 males and 16 females were included in the study. We ensured that this study complied with the guidelines of the Declaration of Helsinki and Ethical Guidelines for Medical and Biological Research Involving Human Subjects and received approval from the Institutional Review Board of Yamaguchi University Hospital (approval no. H2022-060). Written informed consent was obtained from the parents of all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNUDT15 genotyping and dose assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from normal white blood cells in the peripheral blood using the QIAamp\u0026reg; DNA Blood Mini Kit (catalog number: 51104; QIAGEN, Hilden, Germany). \u003cem\u003eNUDT15\u003c/em\u003e variants on exons 1 and 3 were amplified using KAPA2G Robust HotStart\u0026reg; ReadyMix with dye (2\u0026times;) (catalog number: KK5707, Roche, Basel, Switzerland) and AmpliTaq Gold Fast PCR Master Mix (catalog number: 4390939, Applied Biosystems, Waltham, MA, USA). These reagents were used according to the manufacturer\u0026rsquo;s protocols. PCR experiments were performed using a Takara PCR Thermal Cycler, and the amplified products were analyzed via Sanger sequencing. The primers used for amplification were as follows: exon 1 forward primer: 5ʹ-CAAAGCACAACTGTAAGCGACT-3ʹ; exon 1 reverse primer: 5ʹ-GAAAGACCCAGCTAGCAAAGAC-3ʹ; exon 3 forward primer: 5ʹ-AAGCAAATGCAAAGCATCAC-3ʹ; and exon 3 reverse primer: 5ʹ-GGCTGAAAGAGTGGGGGATA-3ʹ.\u003c/p\u003e\n\u003cp\u003eThermal cycling conditions included an initial denaturation step at 95\u0026deg;C for 3 min (1 cycle), followed by 35 cycles at 95\u0026deg;C for 10 s, 60\u0026deg;C for 15 s, and 72\u0026deg;C for 5 s, with a final extension at 72\u0026deg;C for 3 min. The amplified PCR products were analyzed for the presence of specific \u003cem\u003eNUDT15\u003c/em\u003e variants using Sanger sequencing. The average 6-MP dosage was calculated for patients with wild-type, single-variant, and multiple-variant genotypes. The average 6-MP dose in each \u003cem\u003eNUDT15\u003c/em\u003e genotype was estimated using the Kruskal\u0026ndash;Wallis test with JMP Pro 16 (SAS Institute Inc., Cary, NC, USA). Each box includes data between the 25th and 75th percentiles, with the horizontal line indicating the median. The mean doses for each group were compared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiplotyping analysis using digital PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiplotyping analysis was performed on patients with multiple variants. RNA was extracted from peripheral blood, followed by complementary DNA (cDNA) synthesis using SuperScript IV\u0026reg; Reverse Transcriptase (catalog number: 18090050, Invitrogen, Waltham, MA, USA), RNase Out\u0026reg; Recombinant Ribonuclease Inhibitor (catalog number: 10777019, Invitrogen), Random Hexamers\u0026reg; (catalog number: N8080127, Invitrogen), and a 10- mM dNTP mix\u0026reg; (catalog number: 18427088, Invitrogen). Digital PCR analysis was conducted using synthesized cDNA and the QuantStudio Absolute Q Digital PCR System (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003eThe primers used for digital PCR were: Common exon 1 forward primer: 5ʹ-GGCGGCCAGGAGTCG-3ʹ; Common exon 1 reverse primer: 5ʹ-CAACGCGGATGCTTGCA-3ʹ; Common exon 3 forward primer: 5ʹ-CCCCTGGACCAGCTTTTCTG-3ʹ; and Common exon 3 reverse primer: 5ʹ-CCACCAGATGGTTCAGATCTTCTTTAAA-3ʹ. Specific probes included: Wild-type exon 1 probe (VIC): 5ʹ-TCACCACGACTCCGAC-3ʹ; c.52G\u0026thinsp;\u0026gt;\u0026thinsp;A variant probe for exon 1 labeled with FAM: 5ʹ-CACCACGATTCCGAC-3ʹ; Wild-type probe for exon 3 labeled with JUN: 5ʹ-AAACAACGCAGTCCC-3ʹ; and c.415C\u0026thinsp;\u0026gt;\u0026thinsp;T variant probe for exon 3 labeled with ABY: 5ʹ-TTTAAACAACACAGTCCC-3ʹ.\u003c/p\u003e\n\u003cp\u003eFor all 9-\u0026micro;L PCR reaction mixtures, Absolute Q\u0026trade; Universal DNA Digital PCR Master Mix (5\u0026times;) (catalog number: A72710, Applied Biosystems) was used, with primers at a final concentration of 0.9 \u0026micro;M and probes at a final concentration of 0.2 \u0026micro;M. Synthesized cDNA was diluted 100,000-fold, and 0.25 \u0026micro;L of diluted cDNA was added to the reaction mixture. Thermal cycling conditions included an initial denaturation step at 96\u0026deg;C for 10 min (1 cycle), followed by 40 cycles at 96\u0026deg;C for 5 s (ramp rate undisclosed) and 64\u0026deg;C for 30 s (ramp rate undisclosed). We recorded the fluorescence intensity of each microarray after PCR amplification. PCR and fluorescence intensity measurements were performed using the PCR device described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning and Sanger sequencing for diplotype confirmation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the diplotype, the synthesized cDNA was cloned using the Zero Blunt TOPO\u0026trade; PCR Cloning Kit (catalog number: K280040; Invitrogen). The cDNA was cloned into a vector and transformed into TOP10 competent cells provided in the kit. Single colonies were cultured in Luria\u0026ndash;Bertani medium containing kanamycin (50 \u0026micro;g/mL) at 37\u0026deg;C for 15 h, and plasmids were extracted using the QIAprep Spin Miniprep Kit (catalog number: 27104, Qiagen). Sanger sequencing was performed using the forward primer 5ʹ-GTGAGCGCGTCACTTCCTGC-3ʹ and reverse primer 5ʹ-ATCAAATCTTCTCGGCCACCTAGAG-3ʹ. Nine colonies were selected and sequenced to determine the \u003cem\u003eNUDT15\u003c/em\u003e diplotypes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no conflicts of interest to disclose. This study was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS), Grant Number 22K15944 (I.T.).\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japanese Society for the Promotion of Science (JSPS), Grant Number 22K15944 (I.T.). No other funding was received for this study.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eYI, RF, TI, and SH were responsible for designing the protocol, writing the protocol and report, conducting the search, screening potentially eligible studies, extracting and analyzing data, interpreting results, and updating the reference lists. SK, TN, SO, HM, YO, HN, and MM collected samples from patients who met the inclusion criteria of this study and submitted them for the research. YS and TY provided advice and support regarding the implementation of digital PCR.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe would like to express our gratitude to the technical support team at Thermo Fisher Scientific for their assistance with the digital PCR analysis. We thank Dr. Masanori Yoshida from St Jude Children\u0026rsquo;s Research Hospital, Dr. Shin-Ichi Tsujimoto from Yokohama City University, and Dr. Motohiro Kato from The University of Tokyo for their insightful advice on NUDT15 analysis.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data were generated using the methods described in the Subjects and Methods section of this article. Data are not available to the public except as noted above but may be accessed upon reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eConter, V. et al. Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e115\u003c/b\u003e, 3206\u0026ndash;3214 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKato, M. \u0026amp; Manabe, A. Treatment and biology of pediatric acute lymphoblastic leukemia. \u003cem\u003ePediatr. Int.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e, 4\u0026ndash;12 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarran, P. \u0026amp; Attard, N. Thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer. \u003cem\u003eNat. Rev. Cancer\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 24\u0026ndash;36 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoren, G. et al. Systemic exposure to mercaptopurine as a prognostic factor in acute lymphocytic leukemia in children. \u003cem\u003eN Engl. J. Med.\u003c/em\u003e \u003cb\u003e323\u003c/b\u003e, 17\u0026ndash;21 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRelling, M. V. et al. Clinical Pharmacogenetics Implementation Consortium Guideline for thiopurine dosing based on TPMT and NUDT15 genotypes: 2018 Update. \u003cem\u003eClin. Pharmacol. Ther.\u003c/em\u003e \u003cb\u003e105\u003c/b\u003e, 1095\u0026ndash;1105 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmiegelow, K. et al. Thiopurine methyltransferase activity is related to the risk of relapse of childhood acute lymphoblastic leukemia: results from the NOPHO ALL-92 study. \u003cem\u003eLeukemia\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 557\u0026ndash;564 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, S. K. et al. A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia. \u003cem\u003eNat. Genet.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 1017\u0026ndash;1020 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoriyama, T. et al. Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry. \u003cem\u003eBlood\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e, 1209\u0026ndash;1212 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, J. J. et al. Inherited NUDT15 variant is a genetic determinant of mercaptopurine intolerance in children with acute lymphoblastic leukemia. \u003cem\u003eJ. Clin. Oncol.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 1235\u0026ndash;1242 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoriyama, T. et al. NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity. \u003cem\u003eNat. Genet.\u003c/em\u003e \u003cb\u003e48\u003c/b\u003e, 367\u0026ndash;373 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida, M. et al. Low NUDT15 expression levels due to biallelic NUDT15 variants and 6-mercaptopurine intolerance. \u003cem\u003eBr. J. Haematol.\u003c/em\u003e \u003cb\u003e199\u003c/b\u003e, 270\u0026ndash;276 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida, M. et al. NUDT15 variants confer high incidence of second malignancies in children with acute lymphoblastic leukemia. \u003cem\u003eBlood Adv.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 5420\u0026ndash;5428 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, Y. et al. An international retrospective study for tolerability of 6-mercaptopurine on NUDT15 bi-allelic variants in children with acute lymphoblastic leukemia. \u003cem\u003eHaematologica\u003c/em\u003e \u003cb\u003e106\u003c/b\u003e, 2026\u0026ndash;2029 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, C. H. et al. Determination of NUDT15 variants by targeted sequencing can identify compound heterozygosity in pediatric acute lymphoblastic leukemia patients. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 14400 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, C. H. et al. Allele-specific polymerase chain reaction can determine the diplotype of NUDT15 variants in patients with childhood acute lymphoblastic leukemia. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 490 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsujimoto, S. et al. Diplotype analysis of NUDT15 variants and 6-mercaptopurine sensitivity in pediatric lymphoid neoplasms. \u003cem\u003eLeukemia\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 2710\u0026ndash;2714 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKandiel, A., Fraser, A. G., Korelitz, B. I., Brensinger, C. \u0026amp; Lewis, J. D. Increased risk of lymphoma among inflammatory bowel disease patients treated with azathioprine and 6-mercaptopurine. \u003cem\u003eGut\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 1121\u0026ndash;1125 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishii, R. et al. NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 4181 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NUDT15 variants, diplotype analysis, digital polymerase chain reaction, variant-specific probe, acute lymphoblastic leukemia, 6-mercaptopurine sensitivity.","lastPublishedDoi":"10.21203/rs.3.rs-8438183/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8438183/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePatients with \u003cem\u003eNUDT15\u003c/em\u003e variants exhibit intolerance to 6-mercaptopurine (6-MP), which can cause severe myelosuppression and increase the risk of second malignancies, especially in those with bi-allelic variants. However, no reliable analytical method is available to determine the diplotype of \u003cem\u003eNUDT15\u003c/em\u003e. Therefore, we aimed to develop a practical diplotyping method using digital PCR to improve clinical outcomes and reduce complications. We analyzed \u003cem\u003eNUDT15\u003c/em\u003e exon 1 and 3 variants in 38 children with acute lymphoblastic leukemia (ALL) who received 6-MP during maintenance therapy between 2010 and 2021. Variants were genotyped by Sanger sequencing, and tolerated 6-MP doses were assessed according to genotype. For patients carrying multiple variants, germline RNA was extracted, synthesized into cDNA, and analyzed using four variant-specific dPCR probes. Nine patients carried single variants, and two carried multiple variants, and required markedly lower 6-MP doses. The dPCR successfully resolved the phase of the variants and identified a compound heterozygous diplotype in one patient, highlighting its ability to allow the assignment of variant combinations to specific alleles. Our findings demonstrate that dPCR is a practical tool for NUDT15 diplotyping and may facilitate optimized 6-MP therapy by reducing toxicity risk and improving treatment precision for pediatric ALL.\u003c/p\u003e","manuscriptTitle":"Diplotype analysis of NUDT15 using digital PCR in pediatric acute lymphoblastic leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 14:08:04","doi":"10.21203/rs.3.rs-8438183/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-03T16:51:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T09:17:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193381182126248607499614545361631665596","date":"2026-02-12T01:08:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66185414791916844682045756458409912015","date":"2026-02-11T13:51:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-23T15:18:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310416885134990574463180654350536033716","date":"2026-01-07T18:41:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-05T16:39:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-31T09:05:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-27T05:13:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-27T05:12:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-24T02:37:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cadef0a-f228-4a57-9f57-fb798905a2da","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60621850,"name":"Biological sciences/Cancer"},{"id":60621851,"name":"Health sciences/Diseases"},{"id":60621852,"name":"Biological sciences/Genetics"},{"id":60621853,"name":"Biological sciences/Molecular biology"},{"id":60621854,"name":"Health sciences/Molecular medicine"},{"id":60621855,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-07T13:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 14:08:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8438183","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8438183","identity":"rs-8438183","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}