MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #376 (2000) Online

© 2000 WILEY-LISS, INC.

Received 29 May 2000; Revised manuscript accepted 14 September 2000.

Novel Thymidylate Synthase Enhancer Region Allelesin African PopulationsSharon Marsh1, Margaret M. Ameyaw1,2, Jessie Githang’a3, Anne Indalo3, David Ofori-Adjei2,and Howard L. McLeod1

1 Department of Medicine & Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill,Aberdeen AB25 2ZD, United Kingdom

2 Centre for Tropical Clinical Pharmacology, University of Ghana Medical School, Accra, Ghana

3 Kenyatta National Hospital, University of Nairobi, Kenya

Correspondence to: Dr. Howard L. McLeod, Department of Medicine, Washington University School of Medicine,660 S. Euclid Ave., Campus Box 8069, St Louis, MO 63110-1093. Fax: 1-314-747-2797. E-mail:hmcleod@imgate.wustl.edu

Contract grant sponsor: Medical Research Council Research Studentship, Wellcome Trust project grant (046607),Commonwealth Scholarship Commission award, a grant from the British Council-Kenya.

Communicated by Riccardo Fodde

ABSTRACT

Thymidylate synthase (TS) regulates the production of DNA synthesis precursors and is animportant target of cancer chemotherapy. A polymorphic tandem repeat sequence in theenhancer region of the TS promoter was previously described, where the triple repeat giveshigher in vitro gene expression than a double repeat. We recently identified ethnicdifferences in allele frequencies between Caucasian and Asian populations. We now describeassessment of genotype and allele frequencies of the TS polymorphism in 640 African(African American, Ghanaian and Kenyan) and Caucasian (UK, USA) subjects. The doubleand triple repeat were the predominant alleles in all populations studied. The frequency ofthe triple repeat allele was similar between Kenyan (49%), Ghanaian (56%), AfricanAmerican (52%), American Caucasian (54%) and British Caucasian (54%) subjects.However, two novel alleles contained 4 and 9 copies of the tandem repeat. These novel alleleswere found at a higher allele frequency in African populations (Kenyan 7%, Ghanaian 3%,African American 2%) than Caucasians (UK 1%, USA 0%). The novel alleles identified inthis study decrease in frequency with Western migration, while the common alleles arerelatively stable. This is a unique example suggesting the influence of multiple selectionpressures within individual populations. ©2000 Wiley-Liss, Inc.

KEY WORDS: thymidylate synthase; TS, TYMS; ethnic; PCR; tandem repeat;.African, cancer

Genbank accession numbers: TSER*2: AF127519; TSER*3: AF127520; TSER*4: AF134214; TSER*9: AF134215.

2 Marsh et al.

INTRODUCTION

Thymidylate synthase (TS; TYMS; MIM# 188350) catalyses the intracellular conversion of dUMP to dTMP(Rustum et al., 1997). This is the only de novo source of thymidylate, an essential precursor of DNA biosynthesis.TS is also an important target for cancer chemotherapy drugs, such as 5-fluorouracil and raltitrexed (Rustum et al.,1997; Leichman et al., 1997). Therefore, the in vivo regulation of TS is important both in normal tissue biology andin cancer therapeutics. Analysis of the 5’-untranslated region of the TS gene identified a tandem repeat sequencethat is a cis-acting enhancer element (Kaneda et al., 1987). The TS promoter enhancer region (TSER) was shownto contain either two or three 28bp tandem repeats (Horie et al., 1995). In vitro expression studies found that thepresence of a triple repeat (TSER*3) results in 2.6 fold greater mRNA expression than a double repeat (TSER*2)(Horie et al., 1995) and in vivo studies in human gastrointestinal tumour have shown a significant increase in TSprotein levels and functional activity in patients with TSER*3 compared to individuals with TSER*2 (Kawakami etal., 1999a).

We have demonstrated ethnic variation in TSER genotype, with Caucasians (TSER*3/TSER*3 = 28%) andSouthwest Asians (TSER*3/TSER*3 = 40%) significantly different (p<0.001) from Chinese subjects(TSER*3/TSER*3 = 67%) (Marsh et al., 1999). From a population genetics and allelic evolution standpoint, themigration from Africa often sheds light on the mechanisms by which allele preference is maintained (Cavalli-Sforza, 1998). Therefore, we assessed TSER in 640 Kenyan, Ghanaian, African American, American Caucasianand British Caucasian subjects.

MATERIALS AND METHODS

Subjects Genomic DNA was extracted from whole blood of 98 Kenyans, 249 Ghanaians, 92 African Americans,104 American Caucasians and 97 British Caucasians, as previously described (McLeod et al., 1998, Ameyaw etal., 1999). The Kenyan subjects were blood donors from Kenyatta National Hospital, University of Nairobi,Kenya. Ghanaian subjects were blood donors and volunteers from Accra, Ghana. African American and AmericanCaucasian subjects were blood donors from Memphis, TN, USA. British Caucasian subjects were blood donorsfrom Aberdeen and Glasgow, UK. Ethical approval for this study was obtained each clinical center. Writteninformed consent was obtained from all subjects.

PCR Genomic DNA was resuspended in 10mM Tris/1mM EDTA pH 8 and used directly for PCR analysis.Assessment of the TSER, using primers specific for the 5-untranslated region of the TS gene, was carried out aspreviously described (Marsh et al., 1999).

DNA sequencing PCR products corresponding to the 4 alleles were dissected out of the agarose gel and purifiedusing the Qiagen gel extraction kit (Qiagen, West Sussex, UK). 4µl purified DNA was mixed with 2.5µl big dyemix (Applied Biosystems, California, USA), 1.6 pmole of either the forward or reverse primer and made up to10µl with sterile H2O. Samples were overlayed with mineral oil and amplified for 25 cycles of 10 seconds at 95°C,5 seconds at 50°C and 4 minutes at 60°C. Products were sequenced and analyzed using an ABI377 automatedsequencer and SeqEd software.

Statistical analysis CLUMP software (Sham and Curtis, 1995) was used to determine that the TSER genotypefrequencies for each population were in Hardy-Weinberg equilibrium. 95% Confidence Intervals (95%CI) werecalculated for all observed allele frequencies and chi-squared analysis was carried out to determine any differencesin allele frequencies between the populations in this study.

RESULTS

PCR amplification produced two bands corresponding to TSER*2 and TSER*3 in the majority of individuals(Figure 1). In addition, two novel alleles were also identified, with a size of approximately 280bp and 430bp(Figure 1). Sequencing data showed the novel PCR bands to correspond to 4 (TSER*4) and 9 (TSER*9) identicalcopies of the tandem repeat. The frequency of TSER*4 was higher in Kenyans and Ghanaians (0.07, 0.03; Table 1;Figure 2) than African Americans and British Caucasians (0.02 and 0.01; Table 1; Figure 2). TSER*4 was notidentified among 208 American Caucasian alleles and TSER*9 was only found in the Ghanaian population (Table

TS Polymorphism in Africans and Caucasians 3

1a). TSER*4 was found heterozygous with either TSER*2 or TSER*3 at an overall frequency of 13% in Kenyans,7% in Ghanaians, 3% in African Americans and 1% in British Caucasians (Table 1a). TSER*9 was likewise foundas heterozygous with either TSER*2 or TSER*3 at an overall frequency of 2% in Ghanaians (Table 1a). TSER*9was distributed among different Ghanaian tribes (data not shown). No significant difference was found betweenexpected and observed TSER genotype values in any of the populations studied with (p>0.05), indicating that thealleles are in Hardy-Weinberg equilibrium. No significant difference in allele frequencies were observed betweenthe populations in this study (p>0.05 in all cases).

Figure 1: Analysis of the TSER polymorphism by PCR. Examples are shown fromgenomic DNA containing all the genotypes found. Lane 1 = 100bp marker, 2/2=homozygous TSER*2, 3/3 = homozygous TSER*3, 2/3 = heterozygous for TSER*2and TSER*3, 2/4 = heterozygous for TSER*2 and TSER*4, 3/4 = heterozygous forTSER*3 and TSER*4, 3/9 = heterozygous for TSER*3 and TSER*9, 2/9 =heterozygous for TSER*2 and TSER*9.

Figure 2: Distinct differences in the rare TSER alleles, TSER*4 and TSER*9 (- - -) but notTSER*3 ( ) are observed for the African and Caucasian populations, whereas TSER*3 ismuch higher in Chinese subjects (Marsh et al., 1999)

2/2 3/3 2/3 2/4 3/4 3/9 2/9

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4 Marsh et al.

Hardy-Weinberg analysis predicts an allele frequency of 0.0001 for homozygous TSER*4 genotype in BritishCaucasians, 0.0004 in African Americans, 0.005 in Kenyans and 0.0009 in Ghanaians. In the Ghanaian population,homozygous TSER*9 is predicted to occur at a frequency of 0.0001 and heterozygous TSER*4/TSER*9 at afrequency of 0.0006. Although TSER*4 was not found in American Caucasian, Southwest Asian and Chinesepopulations (Table 1b), and TSER*9 was not found in any population other than the Ghanaians (Table 1b), it ispossible that these alleles do exist in these populations at a frequency too low to be observed in a study of thisscale.

Table 1a: Observed TS enhancer region genotype in Kenyan, Ghanaian,African American, American Caucasian and British Caucasian populations

Population Sample TSER genotypeEthnic Group n 2/2 2/3 3/3 2/4 3/4 2/9 3/9Kenyan 98 17% 44% 26% 10% 3% ND NDGhanaian 249 14% 48% 29% 3% 4% 0.4% 1.6%African American 92 20% 53% 24% ND 3% ND NDAmerican Caucasian 104 19% 54% 27% ND ND ND NDBritish Caucasian 97 18% 53% 28% 1% ND ND ND2/2 = homozygous for TSER*2, 2/3 = heterozygous for TSER*2 and TSER*3, 3/3 =homozygous for TSER*3, 2/4 = heterozygous for TSER*2 and TSER*4, 3/4 =heterozygous for TSER*3 and TSER*4, 2/9 = heterozygous for TSER*2 and TSER*9,3/9 = heterozygous for TSER*3 and TSER*9, n = number of subjects analyzed, ND =not detected.

Table 1b: Observed TSER allele frequency in Kenyan, Ghanaian, African American, American Caucasian,British Caucasian, Southwest Asian (Marsh et al., 1999) and Chinese populations (Marsh et al., 1999)

Population Sample Allele FrequencyEthnic Group n TSER*2 TSER*3 TSER*4 TSER*9

Kenyan 196 0.44 (0.34-0.54) 0.49 (0.39-0.59) 0.07 (0.02-0.12) NDGhanaian 496 0.40 (0.34-0.46) 0.56 (0.50-0.62) 0.03 (0.01-0.05) 0.01 (0*-0.02)African American 184 0.46 (0.36-0.56) 0.52 (0.42-0.62) 0.02 (0*-0.05) NDAmerican Caucasian 208 0.46 (0.36-0.56) 0.54 (0.44-0.64) ND NDBritish Caucasian 194 0.45 (0.35-0.55) 0.54 (0.44-0.64) 0.01 (0*-0.03) NDSouthwest Asian 190 0.38 (0.28-0.48) 0.62 (0.52-0.72) ND NDChinese 192 0.18 (0.10-0.26) 0.82 (0.74-0.90) ND ND

n = number of alleles, numbers in brackets = 95% Confidence Intervals. (* Lower 95% CI values truncated at 0)

DISCUSSION

In this study, 640 Kenyan, Ghanaian, African American, American Caucasian and British Caucasian individualswere assessed for the TSER polymorphism. No significant difference was observed in the TSER*2 and TSER*3allele frequencies between Ghanaian (TSER*2 = 0.40), Kenyan (TSER*2 = 0.44), African American (TSER*2 =0.46), British Caucasian (TSER*2= 0.45) and American Caucasian (TSER*2= 0.46) subjects (Table 1). This is incontrast to previous population studies where a shift in the predominant allele is usually observed during Westernmigration. For example, a polymorphism in base -308 of the TNF-α gene promoter shows significant variationbetween African (Gambian TNF2 = 0.16) and Caucasian (USA TNF2 = 0.31) populations (McGuire et al., 1994;Wingerchuk et al., 1997). Also, frequency of the high activity g1947G allele for catechol-O-methyltransferasevaried from 0.68 in Kenyans to 0.45 in Caucasians (McLeod et al., 1998). In addition, most models of populationgenetics define clear differences between African and Caucasian subjects (Cavalli-Sforza, 1996). This suggeststhat a similar degree of selective pressure for TSER has been maintained in these diverse populations.

TS Polymorphism in Africans and Caucasians 5

In the Chinese population, TSER*3 is 4.6 fold more common than TSER*2 (Marsh et al., 1999), implying aselective pressure has maintained the high levels of TSER*3 in this population. However, the same pressure is notevident in Caucasian and African populations in this study, as TSER*2 and TSER*3 are found in roughly equalproportions. This pattern is also found in thiopurine methyltransferase, where 9.4% of African American (Hon etal., 1999), 10.1% of Caucasian (McLeod et al., 1999), 10.9% Kenyans (McLeod et al., 1999) and 14.8% ofGhanaian subjects (Ameyaw et al., 1999) showed variant alleles compared to 4.7% of Chinese subjects (Collie-Duguid et al., 1999). This supports the hypothesis that multiple selective pressures will have distinct influenceswithin populations (Cavalli-Sforza, 1996).

Two novel TSER alleles were identified in this study. These alleles contained 4 and 9 copies of the TSER repeatand were primarily confined to African populations. TSER*4 was found as 2-7% of African alleles (Table 1b;Figure 2). However, TSER*4 was also found in a British Caucasian subject but not among the American Caucasianpopulation studied. This suggests that TSER*4 occurs at low frequency in Caucasian populations. AlthoughTSER*9 appears to be unique to the Ghanaian population, the sample size evaluated in this study cannot dismissthe presence of this allele in other African populations. The presence of TSER*9 in different Ghanaian tribessuggests this allele has been lost during migration rather than a recent recombination event.

An increase in TSER up to 5 tandem repeats is associated with increased TS expression in vitro (Horie et al.,1995; Kawakami et al., 1999b) and TS protein levels in vivo (Kawakami et al., 1999a). The functional significanceof TSER*9 is under evaluation. Over-expression of TS is associated with poor prognosis (Lenz et al., 1995;Johnston et al., 1994) and resistance to TS targeted chemotherapy agents such as 5FU and Tomudex (Lenz et al.,1995; Leichman et al., 1997). However, there is no data in the literature that evaluates the role of ethnicity inresponse to TS inhibitor chemotherapy. Increased TS expression from the TSER polymorphism may reduceefficiency of TS inhibitors. Direct analysis is now needed to assess the impact of these ethnic variations on the useof TS targeted chemotherapy agents.

ACKNOWLEDGEMENTS

The efforts of Julieann Sludden, Dr. Phil Carter, Kenny Reay, Jerome Breen, Caleb Buernortey, Mary Kimani,Mr. Ngugi and Dr. Jill Mollison were greatly appreciated. Many thanks to Professor William Evans for access tothe American DNA samples.

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