Background Thiopurines are widely used in different treatments. However, exposure to this drug can cause toxicity in the patient due to polymorphisms in the gene coding NUDT15, an enzyme involved in the metabolic activity of thiopurines. Carriers of defective alleles of NUDT15 accumulate large amounts of active metabolites which can cause serious damage to DNA.
Purpose To develop a methodology to identify mutations that abrogate or reduce the activity of NUDT15. In this way a more personalised therapy to the patient can be applied with associated low cost.
Material and methods Genetic variants of NUDT15 that cause a decrease or absence of total enzyme activity were identified: rs116855232 (called *6), rs147390019 (*7), rs186364861 (*8) and rs554405994 (*9). Determination of polymorphisms was performed by PCR and subsequent sequencing. For this purpose, pairs of primers that flank the region of interest were designed. Given the proximity of the polymorphisms on the chromosome, each primer pair amplified a region containing two of the four polymorphisms, so only two pairs of amplification primers were designed: F:GCA TCA CTA TGA GTT TAT TAG TAG C/R:CAC CAG ATG GTT CAG ATC TTC for *6 and *7 and F:ACG CAT TAC GCA CCG C/R:GCT CAC CCG AAC TCC AGA T for *8 and *9 polymorphisms. A primer was also designed within each amplified region for sequencing: CAC TAT GAG TTT ATT AGT AGC AAG (*6 and *7) and CGC TAT GAC GGC CAG (*8 and *9) .
Primer design was performed with the GeneRunner programme and with the online analysis tool Primer-Blast, to confirm the specificity of pairs of primers. Reading the chromatograms was carried out with the programme Mega. DNA extraction was performed from a drop of blood deposited on paper Whatman 9031.1
Results Regions encompassing the four polymorphisms were amplified using only two primer pairs. The yield of the reaction was optimum, allowing its subsequent sequencing. The direct costs associated with determination of four markers was €27 (€6.75 for each polymorphism).
Conclusion Genotyping NUDT15 allowed individualised treatment doses, as a carrier for any of these mutations. Future studies will determine the initial dose of the drug. We wished to show a simple and economical method, accessible to any laboratory with basic equipment in molecular biology, which allows detection of mutations in patients that adversely affect the metabolism of thiopurines.
References and/or acknowledgements 1. Ramos-Díaz R, et al. Cancer Chemother Pharmacol2015;75:1095–8.
No conflict of interest
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