Development of a New Molecular Approach for Detecting Genetic Variants Predisposing to Thiopurine Toxicity in Pediatric ALL

Introduction: Thiopurine-associated toxicity in pediatric acute lymphoblastic leukemia (ALL) is related to polymorphisms in the TPMT and NUDT15 genes.Conventional screening techniques, such as allele-specific PCR, detect only recurrent variants, excluding rare ones of clinical relevance.Thus, this study proposes an alternative methodology based on Sanger sequencing of complementary DNA (cDNA), aiming to expand the detection of functional variants.Methodology: This was a retrospective and prospective longitudinal cohort study, in which samples from all patients diagnosed with B-ALL and treated at the Hospital da Criança de Brasília between 2024 and 2025 were included.RNA was extracted and converted into cDNA, followed by PCR with specific primers and sequencing of the TPMT and NUDT15 genes.The data were analyzed using bioinformatics software and classified according to the CPIC guidelines.The project was approved by the Research Ethics Committee (CAAE: 44796221.9.0000.0144).Results: Primers covering all exons of the TPMT and NUDT15 genes were synthesized and validated by PCR and 1.5% agarose gel electrophoresis.Ten samples have already been sequenced for the TPMT gene: two were classified as intermediate metabolizers, three as wild-type, and five carried the TPMT:c.474C>Tvariant, considered to have no impact on 6-mercaptopurine metabolism.Conclusions: Primer validation and preliminary sequencing results demonstrate the feasibility of the strategy.Sanger sequencing, widely used for diagnosing recurrent alterations in ALL, is also applicable for investigating variants associated with thiopurine toxicity.This study shows high potential but requires a larger cohort to corroborate the findings.