Thymidylate Synthase Enhancer Region Research Articles

Abstract 5805: Novel approach to thymidylate synthase genotype determination and utility as a biomarker of severe toxicity to 5-FU chemotherapy

Abstract The primary mechanism of action for the chemotherapeutic 5-fluorouracil (5-FU) is inhibition of thymidylate synthase (TS), which leads to nucleotide imbalance and subsequent cellular death. Expression of TS has been suggested to be determined by a variable number of tandem repeats located within the TS enhancer region (TSER). TSER genotype has been inconsistently linked to 5-FU-related adverse events, including severe grade ≥3 drug-related toxicity. Our recent studies demonstrated that the number of repeats within the TSER, as well as the presence or absence of a polymorphism within each repeat, was significantly associated with 5-FU toxicity in 629 patients. Our findings are consistent with a model in which the number of upstream stimulatory factor (USF1) transcription factor binding sites in the TSER, which is determined both by repeat status and genotype within each repeat, defines TYMS expression and contributions to 5-FU toxicity risk. We present an updated nomenclature that concisely and unambiguously identifies both the number of repeats and the number of USF1 binding sites. The difficulty in obtaining clear genotypes within this highly repetitive and variable region limits the ability to integrate this biomarker into predictive tests. We present methodologies for assigning TSER genotypes using conventional and high-throughput sequencing, including whole genome sequence data. Our results are expected to promote the improved study of TSER genotype in future clinical studies and the integration of TSER status into clinical predictive tests for severe 5-FU toxicity. Citation Format: Huixing Huang, Dominic Schaerer, Remington Schmidt, Ting Zhang, Tanja K Froehlich, Kelly Bouchonville, Robert B Diasio, Ursula Amstutz, Carlo Largiadèr, Steven M Offer. Novel approach to thymidylate synthase genotype determination and utility as a biomarker of severe toxicity to 5-FU chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5805.

Polymorphism in Thymidylate Synthase Gene Predicts Survival and Toxicity in North Indian Lung Cancer Patients Undergoing Platinum-Based Doublet Chemotherapy.

With an estimated 1.8 million deaths, lung cancer is one of the widely reported malignancies, with substantial morbidity and mortality rates. Thymidylate synthase (TS) is an important drug target for platinum-based doublet chemotherapy as it is the only de novo source of thymidylate production in the cell. TS polymorphisms in the 5'UTR of Thymidylate synthase enhancer region (TSER) 2R/3R and 3'- UTR 1494del6 are investigated in this study. A total of 700 lung cancer patients with platinum-based doublet chemotherapy were recruited in this study. TSER (2R/3R) and TS 1494del6 polymorphisms in North Indian lung cancer patients were examined, and statistical analysis was performed. According to our findings, patients with the wild genotype (2R/2R) for the TSER polymorphism had a longer median survival time as compared to patients harboring the mutant type genotype (3R/3R) [MST=9.77 vs. 7.57 months; p=0.04]. On the contrary, patients with the mutant 14946del6 polymorphism (-6/-6) had a longer survival time than patients with the wild-type genotype (+6/+6) [MST=7.23 vs. 9 months]. Further, our findings elucidated that the patients with heterozygous genotype (2R3R) for TSER polymorphism had a 2.30-fold increased risk of developing leukopenia (AOR=2.30, 95% CI=0.96-5.52; p=0.05). A substantial risk of 5.14-fold constipation was found in heterozygous genotype (2R3R) when intermediate grade 2 toxicity was compared with low toxicity (grade 1) (p=0.007).An increased risk of nausea/vomiting was observed in patients with mutant genotype (-6/-6bp) for 1494 ins/del6 polymorphism compared to patients with wild-type genotype (+6/+6bp) (AOR= 2.77; 95%CI=1.10-6.96, p=0.03). According to our findings, TSER and the 1494del6 polymorphism may operate as a prognostic marker in lung cancer patients receiving platinum-based chemotherapy. Furthermore, TS polymorphisms may influence the onset of platinum-related toxicity, such as hematological and gastrointestinal toxicity. These findings might facilitate therapeutic decisions for individualized therapy in lung cancer patients.

A Novel Nomenclature for Repeat Motifs in the Thymidylate Synthase Enhancer Region and Its Relevance for Pharmacogenetic Studies.

Inhibition of thymidylate synthase (TS) is the primary mode of action for 5-fluorouracil (5FU) chemotherapy. TS expression is modulated by a variable number of tandem repeats in the TS enhancer region (TSER) located upstream of the TS gene (TYMS). Variability in the TSER has been suggested to contribute to 5FU-induced adverse events. However, the precise genetic associations remain largely undefined due to high polymorphism and ambiguity in defining genotypes. To assess toxicity associations, we sequenced the TSER in 629 cancer patients treated with 5FU. Of the 13 alleles identified, few could be unambiguously named using current TSER-nomenclature. We devised a concise and unambiguous systematic naming approach for TSER-alleles that encompasses all known variants. After applying this comprehensive naming system to our data, we demonstrated that the number of upstream stimulatory factor (USF1-)binding sites in the TSER was significantly associated with gastrointestinal toxicity in 5FU treatment.

5-Fluorouracil degradation rate as a predictive biomarker of toxicity in breast cancer patients treated with capecitabine

Capecitabine is an oral prodrug of 5-fluorouracil with a relevant role in the treatment of breast cancer. Severe and unexpected toxicities related to capecitabine are not rare, and the identification of biomarkers is challenging. We evaluate the relationship between dihydropyrimidine dehydrogenase, thymidylate synthase enhancer region and methylenetetrahydrofolate reductase polymorphisms, 5-fluorouracil degradation rate and the onset of G3-4 toxicities in breast cancer patients. Genetic polymorphisms and the 5-fluorouracil degradation rate of breast cancer patients treated with capecitabine were retrospectively studied. Genetic markers and the 5-fluorouracil degradation rate were correlated with the reported toxicities. Thirty-seven patients with a median age of 58 years old treated with capecitabine for stages II-IV breast cancer were included in this study. Overall, 34 (91.9%) patients suffered from at least an episode of any grade toxicity while nine patients had G3-4 toxicity. Homozygous methylenetetrahydrofolate reductase 677TT was found to be significantly related to haematological toxicity (OR = 6.5 [95% IC 1.1-37.5], P = 0.04). Three patients had a degradation rate less than 0.86 ng/mL/106 cells/min and three patients greater than 2.1 ng/mL/106 cells/min. At a univariate logistic regression analysis, an altered value of 5-fluorouracil degradation rate (values < 0.86 or >2.10 ng/mL/106 cells/min) increased the risk of G3-4 adverse events (OR = 10.40 [95% IC: 1.48-7.99], P = 0.02). A multivariate logistic regression analysis, adjusted for age, comorbidity and CAPE-regimen, confirmed the role of 5-fluorouracil degradation rate as a predictor of G3-4 toxicity occurrence (OR = 10.9 [95% IC 1.2-96.2], P = 0.03). The pre-treatment evaluation of 5-fluorouracil degradation rate allows to identify breast cancer patients at high risk for severe 5-FU toxicity.

Associations of MTRR and TSER polymorphisms related to folate metabolism with susceptibility to metabolic syndrome.

Hyperhomocysteinemia is a potential risk factor for the development of metabolic syndrome (MetS). Among genes involved in homocysteine metabolism, polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene are known to be associated with MetS incidence. However, effects of polymorphisms of other folate metabolism-related genes on MetS susceptibility are not well known yet. This study was to determine whether methionine synthase (MTR) 2756A > G, methionine synthase reductase (MTRR) 66A > G, and thymidylate synthase enhancer region (TSER) 2R/3R polymorphisms might be associated with risks of MetS development in the Korean population. Genotype analysis of the three polymorphisms was performed for a total of 483 subjects including 236 MetS patients and 247 unrelated healthy controls using polymerase chain reaction-restriction fragment length polymorphism technique. The present study revealed that MTRR and TSER polymorphisms were associated with susceptibility to MetS. Several genotypes and allele combinations from the three polymorphisms were also related to the MetS prevalence. When polymorphism data were stratified according to the risk components of MetS, MTR polymorphism was significantly associated with an increased risk of MetS in subjects with systolic blood pressure < 132.7mmHg (AOR 1.842, 95% CI 1.039-3.266, P = 0.037) and fasting blood glucose level < 106.3mg/dL (AOR 1.772, 95% CI 1.069-2.937, P = 0.027). MTRR polymorphism was significantly associated with a decreased risk of MetS in subjects with triglyceride level < 216.3mg/dL (AOR 0.616, 95% CI 0.399-0.951, P = 0.029). To the best of our knowledge, this is the first to provide reliable evidence about the association of other folate metabolism-related gene polymorphisms besides MTHFR with MetS susceptibility and its risk factors. Results of this study suggest that MTRR and TSER polymorphisms might be potential genetic markers for the risk of MetS development in Korean population.

The thymidylate synthase enhancer region (TSER) polymorphism increases the risk of thymic lymphoid hyperplasia in patients with Myasthenia Gravis

BackgroundMyasthenia Gravis (MG) is caused, in approximately 80% of the patients, by autoantibodies against the nicotinic acetylcholine receptor (AChR). The disease is often associated with pathological changes of the thymus: thymic epithelial tumours are present in about 10–20% of the patients, while up to 80% of the patients with early disease onset have thymic hyperplasia. Folate metabolism is required for the production of DNA precursors and for proper DNA methylation reactions, and impaired folate metabolism has been often associated with cellular growth and cancer. MethodsWe investigated if major polymorphisms of folate-related genes, namely MTHFR c.677C>T, MTR c.2756A>G, MTRR c.66A>G and TYMS TSER (a 28-bp tandem repeat in the 5′ promoter enhancer region of TYMS) increase the risk of pathological changes of the thymus in AChR+ MG patients. A total of 526 AChR+ MG patients, including 132 patients with normal (involuted) thymus, 146 patients with thymic hyperplasia, and 248 patients with a thymoma were included in the study. Allele and genotype comparisons were performed among the three study groups, after correcting for multiple testing. ResultsThe frequency of the TYMS TSER 3R allele was significantly higher in MG patients with thymic hyperplasia (P=0.004), and the TYMS TSER 3R3R genotype was significantly associated with increased risk of thymic hyperplasia [OR 2.71 (95% CI: 1.34–5.47)]. ConclusionsThe 3R allele in the thymidylate synthase promoter enhancer region results in increased protein production, required for the synthesis of DNA precursors. The present study suggests that the TYMS TSER 3R allele increases the risk of thymic lymphoid hyperplasia in AChR+ MG patients.

TSER polymorphism is not associated with risk of pediatric acute lymphoblastic leukemia

Accumulating studies have explored the effect of thymidylate synthase enhancer region (TSER) variation on risk of pediatric acute lymphoblastic leukemia (ALL) with controversial results. Therefore, this quantitative meta-analysis was performed to assess synthetically the association of TSER variation with susceptibility to develop pediatric ALL. The PubMed, ScienceDirect, Google Scholar, Wanfang Database, and China National Knowledge Infrastructure were systematically retrieved to obtain the published case-control studies about the relationship between TSER variation and pediatric ALL risk. The quality assessment of the included studies was preformed and relevant information was collected. Odds ratios (ORs) and 95% confidence intervals (CIs) were applied to evaluate the strength of association. This meta-analysis finally included 2681 children with ALL and 3854 matched controls from 11 investigations. The quantitative synthesis results found no significant association between TSER variation and susceptibility to pediatric ALL in overall comparisons under 5 genetic models (2R/3R vs 3R/3R: OR = 0.95, 95% CI = 0.84-1.07, P = 0.41; 2R/2R vs 3R/3R: OR = 0.99, 95% CI = 0.84-1.16, P = 0.90; 2R2R vs 3R/3R+2R/3R: OR = 1.05, 95% CI = 0.92-1.21, P = 0.45; 2R/3R+2R/2R vs 3R/3R: OR = 0.97, 95% CI = 0.87-1.09, P = 0.63; 2R vs 3R: OR = 1.03, 95% CI = 0.92-1.15, P = 0.61). Similarly, there was no significant association existed in the stratification analyses according to ethnicity, control source, and quality score. This meta-analysis shows that TSER variation is not related to the development risk of pediatric ALL.

High frequency of the 3R/3R polymorphism in the thymidylate synthase enhancer region in Indonesian childhood acute lymphoblastic leukemia

Background Deoxyuridylate monophosphate (dTMP) is neces-sary for DNA synthesis and thymidylate synthase (TS) is an im-portant target of cancer chemotherapy. Ethnic variations of thepolymorphic tandem repeat sequence in the enhancer region ofthe TS promoter has previously been described to influence theoutcome of acute lymphoblastic leukemia (ALL). A triple repeat isassociated with a higher TS gene expression than a double re-peat, resulting in poorer outcome of ALL patients treated with anti-folate methotrexate (MTX).Objective In this study, we determined the incidences of TS andmethylenetetrahydrofolate reductase (MTHFR) polymorphism andethnic variations between Indonesian and Caucasian ALL cellsamples obtained at diagnosis. Furthermore, we determined theinvolvement of TS polymorphisms in MTX sensitivity using athymidilate synthase inhibition assay (TSIA).Methods ALL cell samples were obtained at diagnosis from 101Indonesian and 157 Caucasian children treated with MTX prospec-tively. Genotyping for TS and MTHFR was analyzed by Genescanand Lightcycler. TS polymorphism was determined by PCR assayand MTHFR polymorphism and was analyzed by melting curveanalyses on lightcycler.Results Homozygous TS triple repeats were more than twice ascommon in Indonesian samples (76.3%) than in Caucasian samples(33.1%). Heterozygotes of the MTHFR mutations were seen in 15%of the screened Indonesian samples.Conclusion There are significant ethnic variations in TS generegulatory elements of leukemic cells. A difference was found be-tween the MTX sensitivity and a double or triple repeat in the Cau-casian ALL group. The samples with a triple repeat show a shift intheir distribution towards hypersensitivity to MTX. Further investi-gation on Indonesian samples may give insight in the role of poly-morphisms in MTX sensitivity

Association between folate metabolism-related polymorphisms and colorectal cancer risk.

Folate has essential roles in DNA synthesis, repair and methylation. Folate metabolism-related gene variants may modulate the levels of this vitamin and affect the cancer risk. Thus, whether these polymorphisms play an important role in carcinogenesis, particularly colorectal cancer (CRC) development, has been a subject interest. The present study investigated the association between polymorphisms in the methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS) and the reduced folate carrier 1 (RFC1) genes and CRC risk. Polymorphisms in MTHFR (677C>T and 1298A>C), TS [1494del6 and the TS enhancer region (TSER)] and RFC1 (-43T>C, 80G>A and 696C>T) were characterized using polymerase chain reaction-restriction fragment length polymorphism in 477 CRC cases and 514 controls. Although no polymorphisms were significantly associated with the CRC risk in the overall sample, significant associations between folate metabolism-related polymorphisms and CRC risk were identified in the stratified analyses. The MTHFR 677CT/1298AC and MTHFR 1298AC+CC/TSER 2R3R genotypes in the presence of plasma folate levels ≤4.12 ng/ml were associated with significantly increased CRC risk. In addition, individuals with the MTHFR 677TT/TSER 3R3R or MTHFR 677/TSER 3R3R/TS 1494 0bp6bp+6bp6bp genotypes and diabetes mellitus (DM) were at an increased risk for CRC. Therefore, the data suggest that i) MTHFR polymorphisms combined with low plasma folate levels and ii) polymorphisms in folate metabolism-related genes combined with metabolic syndrome risk factors (hypertension and DM) increase the odds of developing CRC.

Thymidylate synthase and methylenetetrahy-drofolate reductase gene polymorphisms and gastric cancer susceptibility in a population of Northern Brazil.

The folate metabolic pathway, which is involved in DNA synthesis and methylation, is associated with individual susceptibility to several diseases, including gastric tumors. In this study, we investigated four polymorphisms [thymidylate synthase enhancer region, single nucleotide polymorphism thymidylate synthase 5' (TS5'), TS3' untranslated region, and methylenetetrahydrofolate reductase (MTHFR) 677C> T] in 2 genes related to the folate pathway, TS and MTHFR, and their possible association with the risk gastric cancer development in a population from Pará state, Brazil. For the TS enhancer region, TS3' untranslated region, and single nucleotide polymorphism TS5' polymorphisms, no significant results were obtained. For the MTHFR 677C>T polymorphism, TT genotype carriers had a higher risk of developing tumors in the antrum (P = 0.19 vs CC and P = 0.02 vs CT) and intestine (odds ratio = 4.18, 95% confidence interval = 0.66-26.41; P = 0.252 vs CC and odds ratio = 2.25, 95% confidence interval = 0.32-15.75; P = 0.725 vs CT). Those carrying at least 1 T allele had an increased risk of lymph node metastasis (odds ratio = 3.00, 95% confidence interval = 0.88-10.12; P = 0.133). Our results suggest that polymorphisms in MTHFR affect the susceptibility to gastric tumors in the Brazilian population and may be a factor causing poor prognosis in such patients.

Polymorphisms of folate metabolism-related genes and survival of patients with colorectal cancer in the Korean population

Background5-Fluorouracil (5-FU) is a cornerstone of chemotherapy for colorectal cancer (CRC), and the major targets of 5-FU are thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), and reduced folate carrier 1 (RFC1). We hypothesized that polymorphisms in the genes encoding these proteins would be associated with CRC patient survival. Patients and methodsWe genotyped the following polymorphisms in 372 CRC patients: TS enhancer region (TSER), TS 1494del6, MTHFR 677C>T and 1298A>C, and RFC1 −43T>C, 80G>A, and 696C>T. Using Kaplan–Meier curves, log-rank tests, and Cox proportional hazard models, we evaluated associations between these polymorphisms and overall survival (OS). ResultsThe combined TS 1494 0bp6bp+6bp6bp genotype was associated with reduced OS compared to the TS 1494 0bp0bp genotype. Among rectal cancer patients, the RFC1 −43CC and 80AA genotypes were associated with favorable OS. ConclusionsOur data suggest that TS and RFC1 polymorphisms are associated with CRC prognosis in Korean patients. Further studies are needed to verify these findings.

Thymidylate Synthase Gene Polymorphism and Survival of Colorectal Cancer Patients Receiving Adjuvant 5-Fluorouracil

Limited studies indicate a possible association of 5'-UTR thymidylate synthase enhancer region polymorphism and treatment outcome in patients medicated with 5-fluorouracil (5-FU). The study was designed to verify the relationship in patients with colorectal cancer (CRC), a Polish population that received 5-FU-based adjuvant chemotherapy. The study analyzed 145 Astler-Coller B2 and C CRC patients. Genotyping for a variable number of tandem repeats and G to C single-nucleotide polymorphism in the 5'-UTR of the thymidylate synthase (TS) gene was carried out. TS genotypes were classified into high expression (high TS) and low expression types (low TS). High TS was found in 22.8% of patients. The right-side tumors were more frequently associated with high TS than the left-side tumors (p=0.024). High TS was only found in 9.3% of rectal tumors, but in 29.7% of colon cancers (p=0.0042). Disease-free survival after 20 months (DFS 20) was longer in subjects with low TS than in high TS (p=0.043). Patients who underwent chemotherapy had longer DFS 20 in the low TS than in the high TS subgroup (p=0.051). The low TS was found to be an independent good prognostic factor for DFS 20 in the whole group as well as in the subgroup treated with chemotherapy (p=0.024 and p=0.034, respectively). Patients with low TS did not show any differences in DFS 20 whether they were treated with adjuvant chemotherapy or not. Proximal CRC tumors are characterized by higher TS expression genotypes than distal tumors, and are at significantly greater risk of early recurrence during the first 20 months after surgery.

Association of methylenetetrahydrofolate reductase (MTHFR 677C&gt;T) and thymidylate synthase (TSER and TS 1494del6) polymorphisms with premature ovarian failure in Korean women

The aim of our study was to investigate whether methylenetetrahydrofolate reductase (MTHFR) gene variant (MTHFR 677C>T) and thymidylate synthase (TS) gene variants (TS enhancer region [TSER] and TS 1494del6) confer a risk for premature ovarian failure (POF). We genotyped 136 POF patients and 236 controls among Korean women for the three single nucleotide polymorphism sites using polymerase chain reaction restriction fragment length polymorphism analysis. Differences in the MTHFR 677C>T, TSER, and TS 1494del6 genotype frequencies between POF patients and controls were compared, and odds ratios (ORs) and 95% CIs were determined as a measure of the strength of the association between genotypes and POF. The MTHFR 677CT and CT + TT variant genotypes were more frequent in POF patients than in controls (OR, 2.249; 95% CI, 1.317-3.843; and OR, 2.132; 95% CI, 1.268-3.585, respectively). The combined genotype frequencies of MTHFR 677CT + TT/TSER 3R3R and 677CT + TT/TS 1494del6 del6/del6 were higher in patients than in controls (OR, 2.300; 95% CI, 1.219-4.337; and OR, 3.314; 95% CI, 1.623-6.767, respectively). The T-3R-del6 and T-2R-del6 (MTHFR 677C>T/TSER/TS 1494del6) haplotypes were more frequent in patients (OR, 1.450; 95% CI, 1.050-2.002; and OR, 2.911; 95% CI, 1.191-7.117, respectively), whereas the C-2R-del6 haplotype was less frequent in patients (OR, 0.372; 95% CI, 0.152-0.912). The T-del6 (MTHFR 677/TS 1494del6) haplotype frequency was higher among patients (OR, 1.653; 95% CI, 1.206-2.266), whereas the C-del6 haplotype frequency was lower among patients (OR, 0.700; 95% CI, 0.516-0.950). We did not find an association between TSER or TS 1494del6 polymorphisms and POF. Our data suggest that the MTHFR 677T allele may increase the risk for POF, which could lead to the development of novel genetic markers for predicting the risk of POF in patients.

Erratum to: Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) polymorphisms with osteoporotic vertebral compression fracture (OVCF) in postmenopausal Korean women

Hyperhomocysteinemia is associated with the risk of skeletal health problems, such as osteoporosis, low body mineral density, and fracture. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) are involved in homocysteine metabolism. We hypothesized that certain genetic polymorphisms of MTHFR and TS that cause altered enzyme activity may lead to hyperhomocysteinemia and affect bone metabolism. Therefore, we determined whether MTHFR 677C>T, MTHFR 1298A>C, TS enhancer region (TSER), and TS 3′-UTR 6 bp insertion/deletion polymorphisms are associated with osteoporotic vertebral compression fracture (OVCF) in postmenopausal Korean women. A total of 308 postmenopausal Korean women were enrolled as study subjects. Among them, 84 were patients with OVCF and 224 were controls. The polymorphisms were analysed by PCR-RFLP methods. Single mutations of MTHFR or TS were not associated with the occurrence of OVCF. However, the combined genotypes 2R3R+2R2R/0bp6bp+6bp6bp (TSER/TS 3′-UTR) and AC+CC/0bp6bp+6bp6bp (MTHFR 1298A>C/TS 3′-UTR) were associated with decreased risk for OVCF. 2R-0bp and 2R-6bp haplotype frequencies of TS were significantly different between the cases and controls. In the present study, the combined genotype of TSER/TS 3′-UTR and MTHFR 1298A>C/TS 3′-UTR was associated with a decreased risk for OVCF in postmenopausal Korean women. However, due to the several limitations of our study including the moderately small sample size, our findings should be considered with caution and further research is needed to draw more definitive conclusions.

The association between two polymorphisms in the TS gene and risk of cancer: A systematic review and pooled analysis

Thymidylate synthase (TS) is an important enzyme involved in folate metabolism and catalyzes methylation of deoxyuridine monophosphate to deoxythymidine monophosphate, which is essential for DNA replication. Thymidylate synthase enhancer region (TSER) and TS1494del6, two functionally important and ethnically diverse polymorphisms mapping to its gene region, are the most extensively studied. Considering the potential influence of altering TS activity, it is plausible that TS polymorphisms might play a role in the development of cancer. Although the effects of TS polymorphisms on susceptibility to human cancer have been investigated in many studies, the results remain conflicting rather than conclusive. To resolve these conflicts, we performed a quantitative synthesis of the evidence on the association between these two polymorphisms and cancer risk, including 63 studies (19,707 cases and 27,398 controls) for TSER polymorphism and 39 studies (13,489 cases and 16,297 controls) for TS1494del6 polymorphism. Our meta-analysis suggested that these two polymorphisms are not associated with cancer risk when all studies were pooled together. In the stratified analyses, we found that individuals with 2R/2R genotype had a significantly higher cancer risks among Asians (2R/2R vs. 3R/3R: odds ratio [OR] = 1.24, 95% confidence interval (95% CI) = 1.05-1.45; recessive model: OR = 1.23, 95% CI = 1.05-1.44). Further analyses revealed that 2R/2R genotype was significantly associated with an increased risk of gastroesophageal cancer among Asians, whereas it might provide protecting effects against colorectal cancer risk in a dominant genetic model for Caucasians. Additionally, TS1494del6 polymorphism may contribute to genetic susceptibility of breast cancer among Asians.

Thymidylate synthase enhancer region polymorphism not related to susceptibility to acute lymphoblastic leukemia in the Kashmir population

Thymidylate synthase (TS) is a crucial enzyme in folate metabolism and plays a vital role in DNA synthesis and repair. The most common polymorphism in TS is a unique double (2R) or triple (3R) 28-bp tandem repeat sequence in the enhancer region of the TS gene (TSER). This genetic variation in TSER has been widely investigated and has been implicated as a risk factor for the development of various cancers, including acute lymphoblastic leukemia. It has also been found to influence sensitivity to anti-cancer drugs, such as methotrexate. We evaluated this polymorphism in acute lymphoblastic leukemia patients in the Kashmir population. In order to determine whether a double (2R2R) versus a triple (3R3R) 28-bp tandem repeat in the TSER modulates risk for acute lymphoblastic leukemia, 72 acute lymphoblastic leukemia cases and 144 age and gender matched, unrelated healthy individuals from the Kashmir region of India were evaluated for this polymorphism by PCR and direct sequencing. We found the frequency of the TS 2R allele to be 32.6 and 26.0%, in cases and controls, respectively. The TS 2R/2R genotype was found to be present in 15.27% of the cases and 9.72% of the controls, the 2R/3R variant in 34.72% of the cases and 32.63% of the controls, and the 3R/3R genotype in 50.0% of the cases and 57.63% of the controls. There was a significant association between the TS 2R/2R genotype and gender of acute lymphoblastic leukemia patients with males harboring the 2R/2R genotype exhibiting a higher risk of developing acute lymphoblastic leukemia than females (P = 0.009) We concluded that the TSER polymorphism appears not to be a risk factor for susceptibility to acute lymphoblastic leukemia in the Kashmir population.

Polymorphisms of Folate Pathway Enzymes (Methylenetetrahydrofolate Reductase and Thymidylate Synthase) and Their Relationship with Thymidylate Synthase Expression in Human Astrocytic Tumors

Two important polymorphisms of folate cycle enzymes, methylenetetrahydrofolate reductase (MTHFR) C677T and thymidylate synthase (TS) enhancer region (TSER) 28-bp tandem repeat, are related to risk of various types of cancer, including brain tumors, although there are few studies on this subject. A case-control study of these two polymorphisms in astrocytomas of different grades was carried out using polymerase chain reaction-restriction fragment length polymorphism, also determining the immunohistochemical expression of TS. The MTHFR 677 TT genotype was less associated with astrocytic tumors (odds ratio [OR]=0.00; p=0.0238), but the TSER polymorphism did not show any significant association. Combined genotype TT-double repeats/triple repeats (2R/3R) had a protective effect against astrocytomas (OR=0.00; p=0.0388). Expression of TS protein was observed in the majority of cases, with grade IV tumors being the exception. Moreover, the median H-score for the pilocytic astrocytomas was significantly higher when compared with that for diffuse tumors. There was an inverse correlation between the 2R/2R genotype and the highest TS-expressing tumors, and 3R/3R was relatively more frequent among the tumors grouped in the third and fourth quartiles. Our results provide support for the role of MTHFR and TS polymorphism in gliomagenesis, possibly because of the alteration of DNA methylation and repair status. Moreover, high levels of TS expression were detected in these tumors.

Thymidylate synthase gene polymorphisms and markers of DNA methylation capacity

Background DNA methylation plays a critical role in gene regulation and has been implicated in the etiology of chronic disease including atherosclerosis, neural degeneration and cancer. One-carbon metabolism serves two critically important functions: one concerning the production of purines and thymidine for DNA synthesis and the other related to the provision of methyl groups through the metabolism of methionine. Critical intermediates of methionine metabolism relevant to DNA methylation include S-adenosylmethionine (SAM), a universal methyl donor, and S-adenosylhomocysteine (SAH), a potent inhibitor of most methylation reactions. Thymidine synthesis, catalyzed by the crucial enzyme thymidylate synthase (TS), competes with methionine metabolism for a common substrate. Three functional polymorphisms in the TS gene have been identified including: (i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and (ii) the G to C single nucleotide polymorphism (G/C SNP) both of which occur in the 5′untranslated region (UTR) of the TS gene; and (iii) the 6-bp deletion at base pair 1494 (TS1494del6) located in the 3′UTR. Purpose The purpose of this research was to investigate the relationship between TS polymorphisms and concentrations of SAM and SAH, markers of DNA methylation capacity. Methods The study population consisted of 395 healthy male and female volunteers from Kingston, Ontario and Halifax, Nova Scotia, Canada between 2006 and 2008. The effect of each TS polymorphism on SAM and SAH concentrations was investigated, and further analyses were conducted on categorization of polymorphisms based on 5′ or 3′UTR. The combined effect of TS polymorphisms on SAM and SAH concentrations was also investigated, in addition to interactions between polymorphisms in TS and MTHFR 677C>T and interactions between TS polymorphisms and serum folate and vitamin B 12 status. Results No associations were observed between TS polymorphisms and concentrations of SAM and SAH. Analysis of interaction between TS and MTHFR polymorphisms on SAH levels revealed a significant interaction with TS 3′polymorphism and MTHFR C677T ( p = 0.03). As well, interactions between TS 3′polymorphism and serum folate ( p = 0.03) and the combined effect of TS polymorphisms and serum folate on SAH levels ( p = 0.04) were found. Conclusions The findings of this research provide evidence that SAH, a marker of methylation capacity, is influenced by genetic and environmental factors and their interactions.

Thymidylate Synthase Gene Polymorphism Affects the Response to Preoperative 5-Fluorouracil Chemoradiation Therapy in Patients With Rectal Cancer

This study aims to correlate thymidylate synthase (TS) gene polymorphisms with the tumor response to preoperative 5-fluorouracil (5-FU)-based chemoradiation therapy (CRT) in patients with rectal cancer. Forty-four patients with rectal cancer treated with 5-FU-based preoperative CRT were prospectively enrolled in this study. Thymidylate synthase expression and TS gene polymorphisms were evaluated in tumor obtained before preoperative CRT and were correlated with the pathologic response, as assessed by histopathologic staging (pTNM) and tumor regression grade. Patients exhibited 2R/3R and 3R/3R tandem repeat polymorphisms in the TS gene. With regard to TS expression in these genotypes, 2R/3RC and 3RC/3RC were defined as the low-expression group and 2R/3RG, 3RC/3RG, and 3RG/3RG as the high-expression group. There was no significant correlation between TS expression and tumor response. There was no significant difference in the tumor response between patients homozygous for 3R/3R and patients heterozygous for 2R/3R. However, 13 of 14 patients in the low-expression group with a G>C single-nucleotide polymorphism (SNP) (2R/3RC [n = 5] or 3RC/3RC [n = 9]) exhibited a significantly greater tumor downstaging rate, as compared with only 12 of 30 patients in the high-expression group without the SNP (2R/3RG [n = 10], 3RC/3RG [n = 9], or 3RG/3RG [n = 11]) (p = 0.001). The nodal downstaging rate was also significantly greater in this low-expression group, as compared with the high-expression group (12 of 14 vs. 14 of 30, p = 0.014). However, there was no significant difference in the tumor regression grade between these groups. This study suggests that SNPs within the TS enhancer region affect the tumor response to preoperative 5-FU-based CRT in rectal cancer.

Combination of polymorphisms between MTHFR and TS gene modulates survival after 5-fluorouracil-based therapy in colorectal cancer patients

The major targets of 5-fluorouracil (5-FU) are thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR). Therefore, we hypothesized that the variable number of tandem repeat (VNTR) of the thymidylate synthase enhancer region (TSER) together with methylenetetrahydrofolate reductase (MTHFR 677C>T) polymorphism could alter drug activity and predict drug toxicity or efficacy. This study was designed to investigate the influence of TSER and MTHFR polymorphisms on the clinical outcomes of patients with colorectal cancer receiving 5-FU-based chemotherapy. Genomic DNA was isolated from 103 samples of colorectal cancer patients. The genotypes of two common polymorphisms (TSER and MTHFR 677C>T) were determined by polymerase chain reaction-restriction fragment length polymorphism. Patient prognoses were compared with genotype groups and analyzed according to tumor location and gender. There were no differences in prognosis between genotypes or functional groups when the TSER and MTHFR groups were considered separately. However, analysis of combined genotypes of the TSER and the MTHFR 677C>T polymorphisms were associated with the survival rate of colorectal cancer patients who received 5-FU-based chemotherapy (P=0.028). Prognosis of colorectal cancer patients was significantly different between proximal colon and distal colon cancers (P=0.002). However, prognosis did not receive any effect of the combined genotype when analyzed according to tumor location, such as proximal and distal colon cancer. The male group also showed a significant difference between low and high risk types of TSER and MTHFR combined genotypes when stratified according to gender (P=0.019). In conclusion, the combined TSER and MTHFR 677C>T genotypes can be potential prognostic factors in colorectal cancer with 5-FU-based chemotherapy, especially in male gender in Koreans.