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

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

Haplotype structure of the <i>TYMS</i> genetic region.

<p><b>A.</b> Linear map of the 80 kb <i>TYMS</i> genetic region covering the <i>TYMS</i> gene (coordinates 657,604–673,499), upstream region (600,000–657,603) and downstream region (673,500–680,000). All coordinate positions are according to UCSC genomic build GRCh37/hg19. SNPs along this region were selected from the HapMap database (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034426#s4" target="_blank">Materials and Methods</a>) and from our sequence analysis. <b>B.</b> Nine haplotype blocks (in triangular shape), numbered 1 to 9, were obtained by haplotype analysis using Haploview (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034426#s4" target="_blank">Materials and Methods</a>). The reference SNP numbers (rs) are indicated on top. The linkage disequilibrium (D′) is indicated in the small boxes colored red or blue (a color legend is provided). Some newly discovered SNPs that were not in the public database at the time of analysis were named as TYMS_SG 1, 2, 3, 16,19, 22, and 24. At the time of submission of the new SNPs, we noticed they were deposited by others and had assigned SNP numbers of rs12964837, rs11872762, rs11877806, rs36124867, rs75363899, rs2853533, and rs72634355 respectively. <b>C.</b> The largest haplotype block spanning the <i>TYMS</i> gene and some parts in the 5′ UTR, including the VNTR and the mononucleotide repeats, and the 3′UTR, is expanded. Blocks 1 and 2 in this figure corresponds to blocks 8 and 9 respectively, of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034426#pone-0034426-g001" target="_blank">Figure 1B</a>. The unmatched marker 87 corresponding to SNP number rs3826626 (in panel B) was removed in this figure. The locations of the VNTR, MR (mononucleotide repeats), and the 6-bp deletion/insertion polymorphism are shown. The <i>TYMS</i> translational start codon is 13 bp downstream of the VNTR. Enlarged versions of figures B and C are provided in supporting information as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034426#pone.0034426.s002" target="_blank">figure S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034426#pone.0034426.s003" target="_blank">figure S3</a>, respectively.</p

VNTR genotypes among 40 genomes.

<p>VNTR genotypes among 40 genomes.</p

Length variability (polymorphism) within the MR2 repeat.

1<p>Machine-generated measurement of DNA length at the top of the observed peak, in nucleotide scale.</p>2<p>Peak width at half-maximal heights of both peak slopes, in nucleotide scale.</p>3<p>The peak width was between 4 and 5 nucleotides (half-maximal peak height for this sample was more than 4 nucleotides but less than 5). Analysis of the graphs from mixtures made of differing ratios produced a migration of the peak as expected, confirming a variation in the polymorphic forms of samples PN9 and PN104.</p

The nine most common SNP haplotypes.

<p>Common haplotypes and estimated haplotype frequencies as determined using Haploview across the region under survey. Numbers on top of the figure indicate the ‘SNP number’ from the 80 kb analyzed region (refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034426#pone-0034426-g001" target="_blank">Figure 1A</a>) as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034426#pone.0034426.s005" target="_blank">Table S2</a>. Numbers in the middle reflect frequencies of the individual haplotype. These frequencies sum up to the numbers at the bottom because they reflect only fairly common haplotypes (i.e., the number at the end ‘0.94’, explains frequencies of 94% of individuals, the rest of the individuals have rare haplotypes).</p