Analysis of Polymorphisms and Haplotype Structure of the Human Thymidylate Synthase Genetic Region: A Tool for Pharmacogenetic Studies

Soma Ghosh,Roxann G Ingersoll,Scott E Kern,Michael Borges,Michael G Goggins,Alison P Klein,M Zulfiquer Hossain,James R Eshleman,Anthony W I Lo

doi:10.1371/journal.pone.0034426

Abstract

5-fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5′-untranslated region (5′-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.

Highlights

We examined an 80 kb stretch from the TYMS genetic region (Figure 1A) to infer the haplotype structure from the single nucleotide polymorphisms (SNPs) in the region and to determine whether the SNPs were in linkage disequilibrium (D9, a numerical representation of correlation with other forms of polymorphism [25])
Several clinical trials were analyzed on the basis of the concept that genotyping the TYMS variable number of tandem repeats (VNTR) alone would give an estimate of thymidylate synthase (Tyms) protein levels in normal tissues and tumors and help in dosing patients with 5FU
Minisatellites, or VNTRs, can arise readily due to mispairing of repeats in an array [32], by slipped-strand mispairing during DNA replication, by unequal sister chromatin exchange (USCE) during mitosis or meiosis, or by unequal crossover between homologous chromosomes during meiosis

Summary

Introduction

5-Fluorouracil (5FU) was developed by Heidelberger and colleagues [1] as the first generation of chemotherapeutic agent active for gastric cancer. A 6-bp insertion/deletion polymorphism in the 39-UTR of the gene is described [12] Prior studies supported these 59UTR repeats as important determinants of Tyms expression in tumors and normal tissues and proposed that the TYMS VNTR genotypes could be used to help decide fluoropyrimidine dosage in patients [13,14,15]. We report three types of polymorphisms in this region: the known VNTRs, mononucleotide repeats of the promoter region, and the SNPs of the wider genomic context These polymorphisms when taken together, but not in isolation, define the genotype of an individual, and knowledge of linkage disequilibrium among them might provide clues to understand variation in 5FU-related toxicity in patients

Results

Discussion

Materials and Methods