Thymidylate Synthase

Introduction

Background

Thymidylate synthase (TS) is a crucial enzyme in human biology, playing an indispensable role in the biosynthesis of deoxyribonucleic acid (DNA). This enzyme is fundamental for all dividing cells, making it particularly vital in biological processes that demand rapid cell growth, proliferation, and repair. The gene responsible for encoding this enzyme is known as TYMS.

Biological Basis

The primary biological function of thymidylate synthase is to catalyze a specific biochemical reaction: the reductive methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP). dTMP is subsequently converted into deoxythymidine triphosphate (dTTP), which is one of the four essential deoxynucleoside triphosphates required for the synthesis of new DNA strands. This particular step is a rate-limiting and critical reaction within the de novo pyrimidine synthesis pathway, and it relies on tetrahydrofolate as a necessary methyl donor. By efficiently producing dTMP, thymidylate synthase ensures a continuous and sufficient supply of thymidine, which is vital for accurate DNA replication and effective DNA repair mechanisms.

Clinical Relevance

Given its essential role in DNA synthesis, thymidylate synthasehas long been a significant and strategic target in the development of cancer chemotherapy agents. Many widely used anticancer drugs, such as 5-fluorouracil (5-FU) and its various derivatives, exert their therapeutic effects by directly inhibitingthymidylate synthase. This inhibition effectively blocks DNA replication in rapidly dividing cancer cells, ultimately leading to their programmed cell death. Genetic variations within theTYMS gene, particularly those found in its promoter region or coding sequences, can significantly influence the enzyme’s expression levels and overall activity. These genetic differences can impact an individual’s response to thymidylate synthase-targeting chemotherapy, potentially leading to variations in drug efficacy, the development of drug resistance, or the severity of associated side effects. Furthermore, dysregulated thymidylate synthase activity or expression has been linked to the initiation and progression of various types of cancers.

Social Importance

The comprehensive understanding of thymidylate synthase and the implications of its genetic variations carry substantial social importance, particularly within the evolving field of personalized medicine, especially in oncology. By identifying specific genetic markers related to the TYMSgene, healthcare professionals can potentially customize chemotherapy regimens for individual cancer patients. This personalized approach aims to optimize treatment outcomes, enhance therapeutic efficacy, and minimize adverse drug reactions, thereby improving the overall quality of life for patients undergoing arduous cancer treatments. Ongoing research intothymidylate synthasecontinues to yield valuable insights into mechanisms of drug resistance and paves the way for the development of novel and more targeted therapeutic strategies for cancer.

Limitations

Methodological and Statistical Constraints

Research into _thymidylate synthase_ (TS) often faces limitations stemming from study design and statistical power. Many studies, particularly early investigations or those focusing on rare variants, may suffer from insufficient sample sizes, which can inflate reported effect sizes and lead to findings that are difficult to replicate in larger, independent cohorts. The reliance on convenience samples or specific patient populations can also introduce cohort bias, potentially limiting the generalizability of observed associations between _thymidylate synthase_ genetic variations and clinical outcomes to broader populations. Furthermore, the absence of widespread replication studies for many reported associations leaves gaps in validating the robustness and true significance of findings, making it challenging to establish definitive links.

Population Diversity and Phenotypic Complexity

A significant limitation in understanding _thymidylate synthase_ function and its genetic variants pertains to ancestry and generalizability. Many studies are predominantly conducted in populations of European descent, which can lead to biased findings that do not accurately reflect the genetic landscape or clinical implications in more diverse ancestral groups. This lack of diversity can hinder the discovery of ancestry-specific variants or gene-environment interactions crucial for personalized medicine. Additionally, the definition and measurement of phenotypes related to _thymidylate synthase_ activity or drug response can be highly variable across studies, introducing inconsistencies that complicate meta-analyses and cross-study comparisons, thereby obscuring a comprehensive understanding of its biological roles.

Environmental Factors and Unaccounted Variability

The intricate interplay between genetic predispositions and environmental factors presents a substantial challenge in fully elucidating the role of _thymidylate synthase_. Environmental confounders, such as dietary folate intake, exposure to certain drugs, or lifestyle choices, can significantly modulate_thymidylate synthase_ activity and its impact on health, yet these are often not comprehensively captured or controlled for in research designs. The concept of “missing heritability” also applies, suggesting that a substantial portion of the variability in _thymidylate synthase_-related traits remains unexplained by currently identified genetic variants, pointing to complex gene-gene or gene-environment interactions that are yet to be fully characterized. Addressing these remaining knowledge gaps requires sophisticated study designs capable of integrating diverse data types, from genomic to exposomic, to unravel the multifaceted influences on _thymidylate synthase_ function.

Variants

The CFH (Complement Factor H) gene plays a critical role in regulating the complement system, a vital part of the innate immune response responsible for identifying and clearing pathogens and damaged cells. CFH acts as a soluble factor that prevents the complement system from attacking healthy host cells, thereby maintaining immune homeostasis and preventing excessive inflammation and tissue damage. ^[1] Variants within the CFH gene, such as rs488380 , can influence the efficiency of this regulatory function, potentially leading to dysregulation of the complement pathway. This particular variant is a common single nucleotide polymorphism that may alter the structure or expression of theCFH protein, thereby impacting its ability to control complement activation. ^[2]

Dysfunction in CFH regulation, often influenced by genetic variants like rs488380 , is associated with various inflammatory and autoimmune conditions. When CFH activity is compromised, the complement system can become overactive, leading to chronic inflammation and damage to host tissues. ^[3] This sustained inflammatory state can contribute to the progression of several diseases by creating a microenvironment conducive to cellular stress and altered metabolic pathways. The specific allele carried for rs488380 can influence an individual’s susceptibility to such inflammatory conditions, reflecting its impact on the delicate balance of immune regulation. ^[2]

The implications of CFH variants, including rs488380 , extend to processes involving thymidylate synthase (TYMS), an enzyme crucial for DNA synthesis and repair. TYMSis essential for providing deoxythymidine monophosphate (dTMP), a precursor for DNA replication and repair, and its activity is particularly high in rapidly dividing cells, such as those in inflammatory responses or cancer.^[4] Chronic inflammation, influenced by CFH dysfunction, can induce cellular stress and increase the demand for DNA repair, thereby indirectly affecting TYMS expression or activity. Consequently, variants like rs488380 in CFH may influence the cellular environment in ways that impact TYMS-dependent pathways, potentially altering cell proliferation, DNA integrity, and even the efficacy of therapeutic agents that target TYMS, such as certain chemotherapies. ^[3]

Key Variants

RS ID	Gene	Related Traits
rs488380	CFH	age-related macular degeneration nuclear receptor subfamily 1 group d member 1 measurement protein measurement mannan-binding lectin serine protease 1 amount thymidylate synthase measurement

Biological Background

Thymidylate Synthase: A Critical Node in DNA Replication

The enzyme thymidylate synthase (TYMS) plays an indispensable role in the de novo synthesis pathway of deoxythymidine monophosphate (dTMP), a crucial precursor for DNA replication and repair. Specifically, TYMS catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to dTMP, utilizing N5,N10-methylenetetrahydrofolate (MTHF) as a one-carbon donor. This reaction is the sole intracellular source of dTMP, highlighting TYMS’s bottleneck position in providing the building blocks necessary for cell division. ^[5] Without sufficient dTMP, cells cannot synthesize DNA, leading to a halt in proliferation and ultimately cell death. Consequently, the activity of TYMS is tightly regulated to match the cell’s demand for DNA synthesis, particularly during the S-phase of the cell cycle.

This metabolic process is intricately linked to folate metabolism, as MTHF is a derivative of tetrahydrofolate (THF), a crucial cofactor derived from dietary folate. After donating its methyl group, MTHF is oxidized to dihydrofolate (DHF), which must be reduced back to THF by dihydrofolate reductase (DHFR) to sustain the cycle. The interconnectedness of TYMS with folate metabolism means that disruptions in folate availability or the activity of related enzymes like DHFR can significantly impact DNA synthesis and cellular function. ^[6] This makes TYMSa central component of cellular metabolism, influencing both nucleotide synthesis and one-carbon metabolism.

Genetic Regulation and Expression of TYMS

The gene encoding thymidylate synthase,TYMS, is located on chromosome 18q11.2 and its expression is meticulously controlled at multiple levels to meet cellular demands. The TYMS gene contains specific regulatory elements in its promoter region that influence its transcription, often responding to signals related to cell proliferation and stress. Genetic variations, such as polymorphisms in the TYMS gene’s promoter region or 3’ untranslated region (3’UTR), can affect its mRNA stability and translation efficiency, leading to altered enzyme levels. ^[7] For instance, a common polymorphism involving a variable number tandem repeat (VNTR) in the 5’-untranslated region (5’-UTR) of TYMS has been shown to influence transcriptional activity, with higher repeat numbers generally correlating with increased gene expression.

Beyond transcriptional control, TYMS expression is also subject to post-transcriptional and translational regulation, including microRNA-mediated silencing and autoregulation by the TYMS protein itself. The TYMSmRNA contains unique structural features that allow the protein to bind to its own mRNA, thereby inhibiting its translation when dTMP levels are high. This feedback loop ensures that the enzyme’s production is finely tuned, preventing excessive dTMP synthesis while ensuring sufficient supply for DNA replication. Epigenetic modifications, such as DNA methylation in theTYMS promoter, can also impact gene expression, potentially silencing the gene and affecting cellular responses to various stimuli. ^[8]

TYMSin Pathophysiology and Cancer

Given its essential role in DNA synthesis, TYMSis a major target in the pathophysiology of diseases characterized by rapid cell proliferation, most notably cancer. Aberrant regulation or overexpression ofTYMS is frequently observed in various cancers, contributing to uncontrolled cell growth and providing a mechanism for tumor cells to evade therapeutic agents. High TYMSactivity ensures a constant supply of dTMP, allowing cancer cells to replicate their DNA efficiently and proliferate aggressively.^[9] This makes TYMS a key determinant of tumor growth and progression.

The critical function of TYMS has made it a prime target for chemotherapeutic drugs, particularly fluoropyrimidines like 5-fluorouracil (5-FU). These drugs act as suicide inhibitors, forming a covalent complex with TYMSand its folate cofactor, irreversibly blocking the conversion of dUMP to dTMP. This inhibition leads to a “thymineless death” as cells cannot synthesize new DNA, thereby halting cell division and inducing apoptosis in rapidly dividing cancer cells. However, genetic variations inTYMS or its regulatory pathways can influence patient response to these therapies, with some individuals exhibiting resistance due to increased TYMS expression or altered drug metabolism. ^[10]

Systemic Impact and Therapeutic Resistance

The widespread importance of TYMSin DNA synthesis means its activity has systemic consequences, particularly in tissues with high cellular turnover, such as the bone marrow, gastrointestinal tract, and hair follicles. Inhibition ofTYMSby chemotherapy, while effective against cancer, often results in significant side effects in these rapidly dividing healthy tissues, leading to myelosuppression, mucositis, and hair loss. Understanding the tissue-specific regulation and activity ofTYMS is crucial for developing more targeted therapies that minimize systemic toxicity. ^[11]

Furthermore, the development of therapeutic resistance to TYMS-targeting drugs is a major clinical challenge. Cancer cells can adapt by increasingTYMS gene amplification or expression, or by developing mutations that reduce drug binding affinity. Alternative salvage pathways for dTMP synthesis or altered folate metabolism can also contribute to resistance. Research continues to explore novel strategies to overcome TYMS-mediated resistance, including combination therapies and agents that target related metabolic pathways, aiming to improve treatment outcomes and reduce adverse effects across various cancers. ^[12]

Pathways and Mechanisms

Thymidylate Synthesis and Deoxynucleotide Metabolism

Thymidylate synthase (TS), encoded by the TYMS gene, is a pivotal enzyme in the metabolic pathway responsible for the de novo synthesis of deoxythymidine monophosphate (dTMP), a crucial precursor for DNA replication and repair. This enzyme catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to dTMP, utilizing N5,N10-methylenetetrahydrofolate (CH2-THF) as a one-carbon donor and a reductant. ^[13] This reaction is unique as CH2-THF is oxidized to dihydrofolate (DHF), making TS the sole enzyme that regenerates DHF, thereby linking dTMP synthesis directly to the folate metabolic pathway and influencing the cellular pool of various one-carbon units. ^[13] The tightly regulated flux through this pathway ensures an adequate supply of dTMP for DNA synthesis while preventing excessive accumulation, which could be mutagenic.

The activity of TSis intricately connected to other metabolic pathways, including purine and pyrimidine biosynthesis, amino acid metabolism (particularly serine and glycine), and the folate cycle. The supply of dUMP, a substrate forTS, is derived from the phosphorylation of deoxyuridine or the deamination of deoxycytidine, while the availability of CH2-THF depends on the intracellular folate status and the activity of enzymes like serine hydroxymethyltransferase (SHMT). ^[13] These metabolic interdependencies create a complex network where alterations in one pathway can significantly impact TS activity and, consequently, DNA synthesis. For example, disruptions in folate metabolism, whether due to nutritional deficiencies or genetic variations affecting folate-metabolizing enzymes, can directly impair TS function and lead to imbalances in deoxynucleotide pools. ^[14]

Transcriptional and Post-Translational Regulation of TS

The expression and activity of TS are subject to precise regulatory mechanisms, including gene regulation and post-translational modifications, which ensure its levels are appropriate for the cell’s proliferative state. Transcription of the TYMS gene is tightly controlled by several transcription factors, often responding to intracellular signaling cascades that signal the need for DNA synthesis, such as those activated during cell cycle progression. ^[15] For instance, the E2F family of transcription factors plays a significant role in upregulating TYMS expression as cells transition from G1 to S phase, preparing for DNA replication. ^[16] Furthermore, feedback loops exist where imbalances in deoxynucleotide pools can influence TYMS transcription, providing a homeostatic mechanism to adjust enzyme levels.

Beyond transcriptional control, TS activity is also modulated by various post-translational modifications and allosteric regulation. Phosphorylation, acetylation, and ubiquitination can alter the enzyme’s stability, catalytic efficiency, or interaction with other proteins. ^[17] For example, certain kinases activated during DNA damage response pathways can phosphorylate TS, potentially affecting its catalytic rate or its degradation. Allosteric control also plays a crucial role; dTMP itself, the product of the TS reaction, can act as a feedback inhibitor, binding to a regulatory site on the enzyme and reducing its activity when dTMP levels are high. ^[17] This multi-layered regulatory approach ensures that TS activity is finely tuned to the cell’s demands for DNA precursors, preventing both scarcity and overabundance.

Interconnectedness within Cellular Growth and Repair Pathways

The function of TS is not isolated but is deeply integrated into broader systems-level networks governing cell growth, proliferation, and DNA repair. As a key enzyme for DNA synthesis, TS activity is intimately linked to the cell cycle machinery and is a critical determinant of a cell’s ability to divide. Signaling pathways that control cell cycle progression, such as those involving cyclins and cyclin-dependent kinases, ultimately influence the need for and the production of dTMP via TS regulation. ^[18] This pathway crosstalk ensures that the resources for DNA synthesis are only mobilized when the cell is committed to division.

Moreover, TS plays a role in maintaining genomic integrity. Adequate dTMP levels are essential for accurate DNA replication, and imbalances can lead to misincorporation of uracil into DNA, which triggers DNA repair pathways. ^[19] The cellular response to DNA damage also impacts TS activity; for instance, activation of checkpoint pathways can transiently reduce TS expression or activity to halt DNA synthesis and allow time for repair. This hierarchical regulation ensures that DNA synthesis, repair, and cell cycle progression are coordinated, preventing the propagation of damaged DNA and maintaining cellular health. ^[19] The emergent properties of this integrated network include robust control over cell division and genomic stability.

Dysregulation of TSin Disease and Therapeutic Implications

Dysregulation of TSactivity is a significant mechanism underlying various disease states, particularly cancer, making it a critical therapeutic target. In many cancers,TS is overexpressed, leading to an increased capacity for DNA synthesis that supports uncontrolled cell proliferation and tumor growth. ^[20] This overexpression can be a compensatory mechanism in response to oncogenic signaling or can result from genetic alterations in the TYMS gene or its regulatory elements. The elevated TSlevels contribute to resistance against certain chemotherapeutic agents, as the enzyme efficiently replenishes dTMP, counteracting drugs that aim to deplete this nucleotide.

The central role of TS in DNA synthesis has made it a prime target for anticancer chemotherapy, notably with fluoropyrimidines like 5-fluorouracil (5-FU). These drugs act as suicide inhibitors, forming a covalent ternary complex with TS and its cofactor, N5,N10-methylenetetrahydrofolate, thereby irreversibly inhibiting the enzyme and depleting dTMP pools. ^[20] Understanding the mechanisms of TS dysregulation, including gene amplification, altered mRNA translation, or variations in its enzymatic activity due to polymorphisms like rs34743033 (a 28-bp tandem repeat in the TYMS promoter), is crucial for predicting therapeutic response and developing personalized treatment strategies. ^[21] Research continues to explore novel TS inhibitors and combination therapies to overcome resistance and improve patient outcomes.

Pharmacogenetics

TYMS Gene Polymorphisms and Fluoropyrimidine Pharmacodynamics

Variants within the TYMSgene, which encodes the thymidylate synthase enzyme, significantly influence the pharmacodynamic response to fluoropyrimidine chemotherapy drugs such as 5-fluorouracil (5-FU) and capecitabine. These drugs exert their anticancer effects by inhibitingTYMS, thereby disrupting DNA synthesis and repair. Key pharmacogenetic markers include a variable number tandem repeat (VNTR) in the 5’-untranslated region (5’-UTR) of the TYMSpromoter, commonly denoted as 2R/3R, and a single nucleotide polymorphism (SNP)rs34743033 (G>C) in the 3’-untranslated region (3’-UTR). ^[22] The 3R allele of the VNTR, associated with increased TYMS mRNA and protein expression, can lead to reduced drug efficacy and an increased risk of severe adverse reactions due to the need for higher drug concentrations to inhibit the enzyme. ^[23] Conversely, genotypes associated with lower TYMS expression may predict better therapeutic response but also potentially heightened toxicity if not carefully managed.

The functional impact of these TYMSvariants on enzyme expression directly modulates the sensitivity of cancer cells to fluoropyrimidine-based chemotherapy. Patients carrying genotypes that result in higherTYMS activity, such as the 3R/3R genotype, often exhibit resistance to standard 5-FU doses, necessitating alternative treatment strategies or dose adjustments. ^[24] This altered drug target availability means that the drug’s ability to bind and inhibit TYMS is compromised, leading to insufficient antitumour activity. Understanding these pharmacodynamic effects is crucial for predicting clinical outcomes, including tumor response rates and the likelihood of developing severe gastrointestinal toxicity or myelosuppression, which are common dose-limiting side effects of fluoropyrimidines. ^[25]

Interplay with Folate Metabolism and Methotrexate Response

TYMSplays a pivotal role in the folate metabolic pathway, making its genetic variants relevant to the pharmacodynamics of antifolate drugs like methotrexate. Methotrexate inhibits dihydrofolate reductase (DHFR), an enzyme upstream of TYMS in the folate synthesis pathway, thereby indirectly affecting the availability of the TYMS substrate, 5,10-methylenetetrahydrofolate. Polymorphisms in TYMS, particularly the 28-bp VNTR, can alter cellular folate pools and TYMS activity, influencing how cells respond to the broader disruption of folate metabolism caused by methotrexate. ^[26] This interplay highlights how TYMS variants contribute to the overall signaling pathway effects that determine therapeutic response to antifolates.

Specific TYMS genotypes, sometimes in conjunction with variants in other folate pathway genes such as MTHFR(methylenetetrahydrofolate reductase), have been investigated for their ability to predict methotrexate efficacy and toxicity in various conditions, including acute lymphoblastic leukemia and autoimmune diseases.^[27] For instance, certain TYMS genotypes may lead to differential accumulation of methotrexate polyglutamates or alter the balance of folate cofactors, impacting both the drug’s antineoplastic effects and the risk of adverse events like hepatotoxicity or mucositis. This complex interaction underscores that TYMS pharmacogenetics extends beyond its direct inhibitors to drugs that modulate related metabolic pathways, influencing therapeutic outcomes through intricate pharmacodynamic mechanisms. ^[28]

Clinical Application and Personalized Dosing Strategies

The pharmacogenetic insights into TYMS variants offer a foundation for implementing personalized prescribing strategies, particularly for patients undergoing fluoropyrimidine-based chemotherapy. Genotyping for TYMS polymorphisms, such as the 5’-UTR VNTR and 3’-UTR SNP, can help clinicians identify individuals at higher risk of treatment failure or severe adverse drug reactions. ^[29] For patients with genotypes associated with increased TYMS expression and thus potential resistance, dose escalation of fluoropyrimidines or selection of alternative agents might be considered to optimize therapeutic response. Conversely, those with genotypes suggesting lower TYMS activity could benefit from cautious dosing to mitigate toxicity while maintaining efficacy.

While TYMS genotyping is not yet universally incorporated into routine clinical guidelines for all indications, its potential for guiding drug selection and dose adjustments is increasingly recognized. Integrating TYMSpharmacogenetics into treatment algorithms aims to move towards more precise and effective cancer therapy, minimizing patient suffering from avoidable side effects and maximizing the chances of successful treatment.^[30] Ongoing research and clinical trials continue to refine the evidence base for TYMS testing, paving the way for its broader adoption in personalized medicine to improve patient outcomes and enhance the safety profile of critical anticancer agents. ^[31]

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