Shikshya Shrestha

Quantitative Contribution of rs75017182 to Dihydropyrimidine Dehydrogenase mRNA Splicing and Enzyme Activity

Dihydropyrimidine dehydrogenase (DPD; DPYD gene) variants have emerged as reliable predictors of adverse toxicity to the chemotherapy agent 5‐fluorouracil (5‐FU). The intronic DPYD variant rs75017182 has been recently suggested to promote alternative splicing of DPYD. However, both the extent of alternative splicing and the true contribution of rs75017182 to DPD function remain unclear. In the present study we quantified alternative splicing and DPD enzyme activity in rs75017182 carriers utilizing healthy volunteer specimens from the Mayo Clinic Biobank. Although the alternatively spliced transcript was uniquely detected in rs75017182 carriers, canonically spliced DPYD levels were only reduced by 30% (P = 2.8 × 10‐6) relative to controls. Similarly, DPD enzyme function was reduced by 35% (P = 0.025). Carriers of the well‐studied toxicity‐associated variant rs67376798 displayed similar reductions in DPD activity (31% reduction). The modest effects on splicing and function suggest that rs75017182 may have clinical utility as a predictor of 5‐FU toxicity similar to rs67376798.

Novel Deleterious Dihydropyrimidine Dehydrogenase Variants May Contribute to 5‐Fluorouracil Sensitivity in an East African Population

Clinical studies have identified specific genetic variants in dihydropyrimidine dehydrogenase (DPD; DPYD gene) as predictors of severe adverse toxicity to the commonly used chemotherapeutic 5‐fluorouracil (5‐FU); however, these studies have focused on European and European‐American populations. Our laboratory recently demonstrated that additional variants in non‐European haplotypes are predictive of 5‐FU toxicity. The objective of this study was to identify potential risk variants in an understudied East African population relevant to our institutions catchment area. The DPYD protein‐coding region was sequenced in 588 individuals of Somali or Kenyan ancestry living in central/southeast Minnesota. Twelve novel nonsynonymous variants were identified, seven of which significantly decreased DPD activity in vitro. The commonly reported toxicity‐associated variants, *2A, D949V, and I560S, were not detected in any individuals. Overall, this study demonstrates a critical limitation in our knowledge of pharmacogenetic predictors of 5‐FU toxicity, which has been based on clinical studies conducted in populations of limited diversity.

Histone H3K27 trimethylation modulates 5-fluorouracil resistance by inhibiting PU.1 binding to the DPYD promoter

The antimetabolite 5-fluorouracil (5-FU) is one of the most widely used chemotherapy drugs. Dihydropyrimidine dehydrogenase (DPD) is a major determinant of 5-FU response and toxicity. Although DPYD variants may affect 5-FU metabolism, they do not completely explain the reported variability in DPD function or the resultant differences in treatment response. Here, we report that H3K27 trimethylation (H3K27me3) at the DPYD promoter regulated by Ezh2 and UTX suppresses DPYD expression by inhibiting transcription factor PU.1 binding, leading to increased resistance to 5-FU. Enrichment of H3K27me3 at the DPYD promoter was negatively correlated with both DPYD expression and DPD enzyme activity in peripheral blood specimens from healthy volunteers. Lastly, tumor expression data suggest that DPYD repression by Ezh2 predicts poor survival in 5-FU-treated cancers. Collectively, the findings of the present article suggest that a previously uncharacterized mechanism regulates DPD expression and may contribute to tumor resistance to 5-FU. Cancer Res; 76(21); 6362-73. ©2016 AACR.

Gene‐Specific Variant Classifier (DPYD‐Varifier) to Identify Deleterious Alleles of Dihydropyrimidine Dehydrogenase

Deleterious variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) can be highly predictive of clinical toxicity to the widely prescribed chemotherapeutic 5‐fluorouracil (5‐FU). However, there are very limited data pertaining to the functional consequences of the >450 reported no‐synonymous DPYD variants. We developed a DPYD‐specific variant classifier (DPYD‐Varifier) using machine learning and in vitro functional data for 156 missense DPYD variants. The developed model showed 85% accuracy and outperformed other in silico prediction tools. An examination of feature importance within the model provided additional insight into functional aspects of the DPD protein relevant to 5‐FU toxicity. In the absence of clinical data for unstudied variants, prediction tools like DPYD‐Varifier have great potential to individualize medicine and improve the clinical decision‐making process.

Epigenetic Regulations of GABAergic Neurotransmission: Relevance for Neurological Disorders and Epigenetic Therapy

The GABAergic neurotransmission is a highly conserved system that has been attributed to various regulatory events. There has been a notable number of studies on the importance of GABAergic neurotransmission, both excitatory and inhibitory, in neurogenesis and central nervous system development including its control of neuronal cell proliferation and migration, synaptogenesis, dendrite formation and branching, and new neuronal cell integration in the adult brain. There has been remarkable progress in understanding the epigenetic regulations of GABAergic genes and their aberrant expressions in various neurological disorders such as autism spectrum disorder, Retts syndrome, schizophrenia and PWS. The roles of histone modifications, chromatin looping and gene methylation have been implicated in altered regulations of key genes in the GABAergic pathway. Taken together, they affect the functioning of GABAergic neurotransmission and disrupt various events in brain development. Here, we focus on the role of GABAergic neurotransmission in brain development and on how various genetic and epigenetic events regulate the GABAergic genes in pre- and postnatal brain. We also discuss how these regulatory mechanisms contribute to the pathogenesis of neurological disorders and, therefore, can be used in the development of potential epigenetic therapy for these diseases.

Abstract 3893: Quantitative contribution of rs75017182 to dihydropyrimidine dehydrogenase mRNA splicing and enzyme activity

5-Fluorouracil (5-FU) has remained a widely prescribed anti-cancer drug for decades. However, 5-FU–based treatments show inter-individual variability, with up to 34% of 5-FU–treated patients developing severe (grade 3+) adverse toxicity. Dihydropyrimidine dehydrogenase (DPD; DPYD gene) is the rate-limiting enzyme of 5-FU catabolism and converts 80–85% of 5-FU into its inactive metabolites. Three deleterious genetic variations in DPYD are well-established predictors of severe 5-FU toxicity (i.e., *2A, p. I560S, and p. D949V); however, these variants only explain 30–35% of adverse cases. Recently, an intronic variant (rs75017182) has been suggested to contribute to 5-FU–related toxicity by promoting the alternative splicing of DPYD. However, clinical studies are conflicting as to the degree to which rs75017182 affects DPD enzyme function, and the true contribution of the rs75017182 variant towards DPD function and 5-FU toxicity remains unclear and inconclusive. In the present study, we directly examined the effect of the intronic SNP rs75017182 on DPYD mRNA splicing, DPYD expression, and DPD enzyme function. We genotyped 3950 healthy Caucasian volunteers from the Mayo Clinic Biobank to identify the rs75017182, *2A, p.I560S, and p.D949V variants. From the initial cohort, 204 volunteers who were rs75017182 heterozygous carriers were recruited as our study population. Alternative splicing and DPD activity in rs75017182 carriers were measured. The findings were confirmed using a novel mini-gene reporter system in vitro. A moderate, but significant, reduction (30%) was detected in canonically spliced DPYD expression in rs75017182 carriers compared to non-carriers. A correlative reduction (35%) in DPD enzyme activity was observed in those carriers, which was similar to that of D949V carriers (31%). The results demonstrated an association of this deep intronic variant with decreased DPD activity and suggested that rs75017182 may be a predictor of 5-FU toxicity similar to D949V. Citation Format: Qian Nie, Shikshya Shrestha, Erin E. Tapper, Colbren S. Trogstad-Isaacson, Kelly J. Bouchonville, Steven M. Offer, Robert B. Diasio. Quantitative contribution of rs75017182 to dihydropyrimidine dehydrogenase mRNA splicing and enzyme activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3893.

Abstract LB-195: The intronic DPYD variant rs75017182 correlates with a modest reduction in DPD enzyme activity

Nearly one-third of cancer patients that receive the commonly prescribed chemotherapy drug 5-fluorouracil (5-FU) experience severe (grade 3+) adverse toxicity related to treatment. Approximately 1,300 deaths are directly attributed to 5-FU toxicity annually in the U.S. While three deleterious genetic variations in the dihydropyrimidine dehydrogenase (DPD) gene (DPYD) are well-established predictors of severe toxicity (i.e., *2A, p.I560S, p.D949V), they only explain 30-35% of adverse cases of 5-FU toxicity. Recently, an intronic variant (rs75017182) has been suggested to contribute to 5-FU toxicity risk by promoting the alternative splicing of DPYD. However, recent clinical studies are conflicting as to the degree to which rs75017182 affects DPD enzyme function, and it remains unclear as to the extent to which DPYD splicing is affected in carriers. The primary aim of this study was to directly determine correlations between rs75017182, DPD enzyme function, and DPYD splicing. Genotyping for rs75017182, *2A, p.I560S, and p.D949V was performed on DNA obtained from 3950 healthy participants in the Mayo Clinic Biobank. Individuals carrying one or more of the genotyped variants, as well as matched non-carriers, were asked to provide an additional blood sample that was used to measure DPYD expression, DPYD splicing relevant to rs75017182, and DPD enzyme activity (n = 204). A moderate, but significant, reduction was noted in the expression of canonically spliced DPYD in rs75017182 carriers compared to non-carriers. DPD enzyme activity in peripheral blood mononuclear cells was similarly reduced in rs75017182 carriers. The degree to which DPD activity was reduced in rs75017182 carriers was not as severe as was noted for carriers of the toxicity-associated variants *2A, p.I560S, or p.D949V. Collectively, our data support a model in which rs75017182 leads to alternative splicing of a fraction of primary transcripts that harbor the variant, which in turn causes a modest reduction in the levels of functional DPD enzyme. The modest phenotypes noted may partially explain some of the discrepant results noted between various clinical studies that have attempted to evaluate the variant. Citation Format: Shikshya Shrestha, Steven M. Offer, Robert B. Diasio. The intronic DPYD variant rs75017182 correlates with a modest reduction in DPD enzyme activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-195.