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

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.