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Ethnic Variation in the Thymidylate Synthase Enhancer RegionPolymorphism among Caucasian and Asian Populations

Sharon Marsh,* Elaina S. R. Collie-Duguid,* Tao Li,†,‡ Xiehe Liu,‡ and Howard L. McLeod*,1

*Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD,United Kingdom; †Section of Molecular Genetics, The Institute of Psychiatry, De Crespigny Park, Denmark Hill, London, SE5 8AF,

United Kingdom; and ‡West China University of Medical Sciences, Chengdu, Sichuan 610041, People’s Republic of China

Received February 18, 1999

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Thymidylate synthase (TS) regulates the productionf DNA synthesis precursors and is an important tar-et of cancer chemotherapy. A tandem repeat se-uence in a TS promoter enhancer region (TSER) wasecently identified. Polymorphic variation affected initro expression levels of the gene. We evaluated thenfluence of ethnicity on TSER genotype. Allele fre-uency was similar in Caucasian and Southwest Asianubjects. However, homozygous triple repeat subjectsere twice as common in Chinese subjects (67%) than

n Caucasian subjects (38%). This demonstrates signif-cant ethnic variation in a TS gene regulatory elementhich may have significant impact on pyrimidine ho-eostasis and drug therapy. © 1999 Academic Press

Key Words: thymidylate synthase; polymorphism;thnic groups; Asian; pharmacogenetics.

Thymidylate synthase (TS) catalyzes the intracellu-ar conversion of dUMP to dTMP (1). This is the only deovo source of thymidylate, an essential precursor ofNA biosynthesis. TS is also an important target for

ancer chemotherapy drugs such as 5-fluorouracil andaltitrexed (1, 2). Therefore, the in vivo regulation ofS is important both in normal tissue biology and inancer therapeutics. The human TS promoter has re-ently been characterized, identifying several impor-ant mechanisms for gene regulation (3). Tandem re-eat sequences were identified near the initiation startite (4). This region contains no putative transcriptionactor binding sites, but acts as a cis-acting enhancerlement in the TS gene (5). This TS enhancer region

Sequence data from this article have been deposited with theMBL/GenBank Data Libraries under Accession Nos. AF127519 andF127520.

1 To whom correspondence should be addressed. Fax: 44 122473066. E-mail: h.l.mcleod@abdn.ac.uk.

enomics 58, 310–312 (1999)rticle ID geno.1999.5833

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888-7543/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

her two or three 28-bp tandem repeats (4). In vitroxpression studies suggest that the presence of a tripleepeat results in 2.6-fold greater TS expression than aouble repeat (4). As we have identified ethnic differ-nces in a diverse range of genetic polymorphisms (6,), we evaluated the presence and frequency of TSERlleles in Caucasian, Southwest Asian, and Chineseubjects.Polymerase chain reaction (PCR) analysis was per-

ormed in genomic DNA from 96 Caucasian (33 female,3 male), 95 Southwest Asian (49 female, 46 male), and6 Chinese (28 female, 68 male) healthy unrelatedolunteers (6–8) using primer 1 (sense), 59-GTGGCTC-TGCGTTTCCCCC-39, and primer 2 (antisense), 59-CTCCGAGCCGGCCACAGGCATGGCGCGG-39, asreviously described (4). The Caucasian subjects werelood donors from Aberdeen and Glasgow, Unitedingdom (7). The Southwest Asian subjects were indi-iduals of Indian, Pakistani, Sri Lankan, and Nepaliirth working in Aberdeen (6). The Chinese populationas Han Chinese from Chengdu, Sichuan Province,hina (8). DNA samples from two unrelated multigen-ration families were obtained from Mr. A. Cumming,niversity of Aberdeen. This study was approved by

he local ethical committee and written informed con-ent was obtained from all subjects.Two products of approximately 220 and 250 bp were

learly distinguished on a 4% agarose gel after PCRmplification. Sequencing studies confirmed the pres-nce of either two repeats (TSER*2; Genbank Acces-ion No. AF127519) or three repeats (TSER*3; Gen-ank Accession No. AF127520) of a 28-bp sequence.nalysis in two unrelated multigeneration familiesrovided the first evidence that the repeats are inher-ted in an autosomal recessive manner and are not aesult of random recombination or PCR artifact (Fig. 1).

Both TSER genotype and allele frequency wereearly identical in Caucasian and Southwest Asian

subjects (Table 1). In contrast, the TSER genotype wassjrTtC0Cdsqp

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ignificantly different in Chinese and Caucasian sub-ects: 67% of Chinese were homozygous for the tripleepeat compared with 38% of Caucasians (P , 0.001;able 1). The allele frequency was also different in thewo populations: the double repeat constituted 40% ofaucasian alleles, but only 18% of Chinese alleles (P ,.001; Table 1). The allele frequency observed in the 96hinese subjects is nearly identical to that previouslyescribed in 21 Japanese subjects (Table 1) (4). Thisuggests that there is a relatively stable allele fre-uency for the TSER polymorphism among East Asianopulations.There are a number of possible explanations for eth-

ic differences in the TSER. Recent cladistic analysisuggests multidirectional population movements be-ween Africa and East Asia prior to population expan-ion into Southwest Asia and Europe (9). The TSERolymorphism may be a by-product of migration ratherhan true allele evolution. However, the allele frequen-ies for the TSER were not significantly different be-ween Chinese and Japanese subjects, ethnic groupshat usually display significant genetic differences (10,1). Environmental and/or other epigenetic factorsay have an important role in influencing this poly-orphism. It is possible that regional dietary varia-

ions could have provided the selective pressure toause a shift in allele frequencies for different popula-ions. If thymidine intake was low among East Asianopulations, subjects with higher thymidylate syn-hase, due to a triple repeat in their TSER, wouldaximize their conversion of dUMP to dTMP, provid-

ng this important precursor for DNA synthesis andiving these individuals a selective advantage.These data also provide an important starting point

or pharmacogenomic analysis in cancer chemother-py. Prospective validation of the clinical impact of the

SER polymorphism will bring us closer to providingseful molecular tools for optimizing drug therapy in

ndividual patients.

ACKNOWLEDGMENTS

The efforts of Debbie Ismail and Julieann Sludden were greatlyppreciated. Many thanks to Dr. David A. Collier for his encourage-ent in our population analysis. This work was supported in part by

he Medical Research Council, a grant from the Wellcome Trust (No.46607), Aberdeen Royal Hospitals NHS Trust Endowments, and aniversity of Aberdeen equipment grant.

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TS Enhancer Region Genotype and Allele Frequencyin Caucasian, Southwest Asian, and Chinese Subjectsa

Ethnic group n

GenotypeAllele

frequency

2/2 2/3 3/3 2 3

aucasian 96 19% 43% 38% 0.40 0.60outhwest Asian 95 16% 44% 40% 0.38 0.62hinese 96 2% 31% 67% 0.18 0.82apanese (4) 21 n/a n/a n/a 0.19 0.81

a 2/2 5 homozygous for the double repeat, 2/3 5 heterozygous forhe double and triple repeat, 3/3 5 homozygous for the triple repeat,/a 5 results not available, n 5 number of subjects analyzed.

FIG. 1. Inheritance of the double and triple repeat alleles in theS enhancer region in two multigeneration families. 2/2 5 homozy-ous for the double repeat, 2/3 5 heterozygous, 3/3 5 homozygous forhe triple repeat, n/a 5 DNA not available.

Frequency of the codon 108/158 low activity allele is lower in and Zegura, S. L. (1998). Out of Africa and back again: Nested

1

1

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9. Hammer, M. F., Karafet, T., Rasanayagam, A., Wood, E. T.,Altheide, T. K., Jenkins, T., Griffiths, R. C., Templeton, A. R.,

cladistic analysis of human Y chromosome variation. Mol. Biol.Evol. 15, 427–441.

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