Delimiting Allelic Imbalance of TYMS by Allele-Specific Analysis

Abstract

Allelic imbalance of thymidylate synthase (TYMS) is attributed to polymorphisms in the 5′- and 3′-untranslated region (UTR). These polymorphisms have been related to the risk of suffering different cancers, for example leukemia, breast or gastric cancer, and response to different drugs, among which are methotrexate glutamates, stavudine, and specifically 5-fluorouracil (5-FU), as TYMS is its direct target. A vast literature has been published in relation to 5-FU, even suggesting the sole use of these polymorphisms to effectively manage 5-FU dosage. Estimates of the extent to which these polymorphisms influence in TYMS expression have in the past been based on functional analysis by luciferase assays and quantification of TYMS mRNA, but both these studies, as the association studies with cancer risk or with toxicity or response to 5-FU, are very contradictory. Regarding functional assays, the artificial genetic environment created in luciferase assay and the problems derived from quantitative polymerase chain reactions (qPCRs), for example the use of a reference gene, may have distorted the results. To avoid these sources of interference, we have analyzed the allelic imbalance of TYMS by allelic-specific analysis in peripheral blood mononuclear cells (PBMCs) from patients.Allelic imbalance in PBMCs, taken from 40 patients with suspected myeloproliferative haematological diseases, was determined by fluorescent fragment analysis (for the 3′-UTR polymorphism), Sanger sequencing and allelic-specific qPCR in multiplex (for the 5′-UTR polymorphisms).For neither the 3′- nor the 5′-UTR polymorphisms did the observed allelic imbalance exceed 1.5 fold. None of the TYMS polymorphisms is statistically associated with allelic imbalance.The results acquired allow us to deny the previously established assertion of an influence of 2 to 4 fold of the rs45445694 and rs2853542 polymorphisms in the expression of TYMS and narrow its allelic imbalance to 1.5 fold, in our population. These data circumscribe the influence of these polymorphisms in the clinical outcome of 5-FU and question their use for establishing 5-FU dosage, above all when additional genetic factors are not considered.

TYMS 2R3R polymorphism and DPYD [IVS]14+1G>A mutation genes in Mexican colorectal cancer patients

“…Nevertheless, TYMS 2R/2R genotype was assessed to be a risk factor for hematological toxicity, and the heterozygous 2R3R TYMS genotype was found in CRC patients with stage III-IV tumors. Previous studies have observed that chemotherapy influences drug response, toxicities and clinical outcomes in patients (Wang et al, 2012;Rosmarin et al, 2015;Zhao et al, 2015;Balboa et al, 2015;Wang et al, 2016). Capecitabine (Xeloda, Roche, oral drug) and 5-FU (pro-drug) are chemotherapeutic drugs commonly given to patients with colorectal cancer (CRC).…”

“…In addition to these mechanisms, several factors may influence the development of toxicities or the clinical outcome of CRC, including other gene polymorphisms and alternative cytotoxic paths that are capable of increasing activity and inducing changes in cell physiology to result in neoplastic progression (Gutiérrez et al, 2016). Different studies have observed an association of TYMS 2R/3R with different clinical outcome manifestations (Wang et al, 2012;Rosmarin et al, 2015;Zhao et al, 2015;Balboa et al, 2015;Wang et al, 2016). In this study, we also observed an association of gastric and hematological toxicities in the non-chemotherapy responses of patients carrying 2R/2R-2R/3R genotypes.…”

“…Chemotherapy resistance has been associated with factors that can influence the therapeutic potential of a given drug (Tecza et al, 2016). The thymidylate synthase enzyme (TS) has been reported to participate in the synthesis of pyrimidines, which is essential for DNA synthesis (Balboa et al, 2015). Mechanisms of DNA hypomethylation and hypermetilation are important for the colon carcinogenesis process.…”

“…The TYMS 2R/3R and DPYD [IVS]14+1G>A polymorphisms have displayed a significant association between the response to and/or toxicity of capecitabine and 5-fluorouracil (5-FU) in some studies (Balboa et al, 2015;Wang et al, 2016;Romiti et al, 2016). However, in the Mexican population, these associations remain unknown.…”

SNPs in predicting clinical efficacy and toxicity of chemotherapy: walking through the quicksand

“…Although knockdown or amplification experiments succeeded in demonstrating the in vitro importance of TYMS in 5-FU resistance and toxicity, clinical studies on the predictive role of these polymorphisms have been controversial so far [ 68 ]. For example, no significant association between TYMS genotype and response rate or overall survival (OS) has been observed in patients with gastric cancer treated with platinum/5-FU combinations.…”