A152 INVESTIGATING THE ROLE OF NOVEL NUDT15 VARIANT IN AZATHIOPRINE-RELATED MYELOTOXICITY

Abstract

Abstract Background Azathioprine (AZA), an immunosuppressant, has classically been used to treat patients with inflammatory bowel disease (IBD). AZA inhibits purine synthesis, and its metabolism occurs via a pathway involving thiopurine methyltransferase (TPMT). While standard TPMT genetic screening is conducted for IBD patients initiating AZA treatment to minimize adverse drug effects (ADE), a majority of patients experiencing ADE have wildtype TPMT. Another gene, NUDT15, has been found to be associated with AZA-related myelotoxicity Aims In this study we report two novel variants in NUDT15 and aim to evaluate the impact of NUDT15 variation on its gene expression. We hypothesize that the mutations found within novel NUDT15 variant are detrimental either to the gene’s expression levels or its translation process, resulting in a lower amount of NUDT15 product present and hence translating to AZA-related myelotoxicity observed clinically. Methods IBD patients experiencing AZA-related myelotoxicity were recruited for this study. Patients were then genotyped and the NUDT15 variants were replicated through site-directed mutagenesis. The NUDT15 variants were subsequently transformed into mammalian cell lines then E. coli cells. DNA products were isolated, and transcription levels were assessed through RT-PCR. Results Patient cohort consisted of 27 AZA-exposed IBD patients who developed myelotoxicity despite their TPMT wildtype genotype. Two novel NUDT15 variants were found. The mutation in one of the variants was placed in 3’ UTR, and hence further research was not pursued. Further analysis was conducted for the variant with mutation in coding region. RT-PCR was conducted to assess and compare gene transcription levels between wildtype and variant NUDT15. Wildtype NUDT15 had a relative gene expression level of 0.8x107, whereas variant NUDT15’s relative gene expression level was at 1.1x107. The two groups were not significantly different in terms of gene expression. Conclusions Contrary to our initial hypothesis, it appears that the mutation in the start codon for variant NUDT15 gene does not significantly impact its gene expression as compared to the wildtype gene. We are currently pursuing protein expression analysis studies to assess for translational deficits possibly present in the novel NUDT15 variant. Funding Agencies SRTP - Schulich School of Medicine