ORIGINAL ARTICLE   

Minerva Biotecnologica 2020 September;32(3):95-100

DOI: 10.23736/S1120-4826.20.02620-8

Copyright © 2020 EDIZIONI MINERVA MEDICA

language: English

Evaluation of the polymorphism NUDT15c.415C>T real-time PCR on the CFX96 real-time PCR system and 7500 real-time PCR system

Valentina DAPRÀ ^{1, 2}, Ilaria GALLIANO ¹, Carla ALLIAUDI ¹, Elena ZANIOL ¹, Elisa GRAZIANO ¹, Cristina CALVI ¹, Paola MONTANARI ¹, Massimiliano BERGALLO ^{1, 2} ✉

¹ Department of Public Health and Pediatrics, School of Medicine, University of Turin, Turin, Italy; ² BioMole srl, Turin, Italy

BACKGROUND: Thiopurine-related hematoxicity is clinically relevant and therapy-limiting in treatment of patients with acute lymphoblastic leukemia (ALL) or inflammatory bowel disease (IBD). For clinical implementation, the Clinical Pharmacogenetics Implementation Consortium (CPIC ) published peer-reviewed and evidence-based gene/drug clinical practice guidelines indicating preemptive TPMT genotype-guided thiopurine-dose individualization to mitigate drug toxicity. Recently loss-of-function germline variants in NUDT15 have been identified as additional genetic determinants of thiopurine intolerance.
METHODS: We used a TaqMAMA genotyping PCR assay NUDT15-MAMAPCR real time cod. BioMole 020 for detection of c.415 C-to-T transition (rs116855232) variant of the nucleoside diphosphate-linked moiety X motif 15 (NUDT15) gene on 7500 Real-Time PCR System and CFX96 Real-Time PCR System. Subsequently we used this assay in subject with thiopurine treatment.
RESULTS: Homozygotes and heterozygotes condition were tested. We reported the ΔCt obtained from mimic homozygotes C/C (CtsC 22.31, CtaT 28.90 with ΔCt 6.59) and T/T (CtsT 22.75, CtaC 31.01 with ΔCt 8.26) and heterozygotes C/T conditions (CtsC 23.33, CtsT 23.72 with ΔCt 0.38). For the clinical validation of the techniques we analyzed a population of 31 thiopurine treated pediatric patients blood samples (13 male, 18 female). All 31 samples were tested with 31/31 (100%) homozygotes (31 CC ).
CONCLUSIONS: The CFX96 Real-Time PCR System is a suitable instrument to use in conjunction with the NUDT15-MAMAPCR real time to generate predictive information about genotyping assay of rs116855232, C415T polymorphism in children with thiopurine treatment.

KEY WORDS: Real-time polymerase chain reaction; Toxicity tests; Polymorphism, genetic