Influence of thymidylate synthase gene polymorphisms on total plasma homocysteine concentrations

Molecular Genetics and Metabolism 101 (2010) 18–24

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Influence of thymidylate synthase gene polymorphisms on total plasma homocysteine concentrations Vikki Ho a,*, Thomas E. Massey b, Will D. King a a b

Department of Community Health and Epidemiology, Queen’s University, Kingston, Ontario, Canada K7L3N6 Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada K7L3N6

a r t i c l e

i n f o

Article history: Received 12 April 2010 Received in revised form 20 May 2010 Accepted 20 May 2010 Available online 9 June 2010 Keywords: Thymidylate synthase Homocysteine Molecular epidemiology Gene–diet interaction Gene–gene interaction Genetic epidemiology

a b s t r a c t Background: Thymidylate synthase (TS) is a key enzyme that regulates the production of nucleotide synthesis by catalyzing the conversion of deoxyuridylate to thymidylate. Three functional polymorphisms in the TS gene have been identified including: (i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and (ii) the G to C single nucleotide polymorphism (G/C SNP) both of which occur in the 50 untranslated region (UTR) of the TS gene; and (iii) the 6 base pair deletion at base pair 1494 (TS1494del6) located in the 30 UTR. Purpose: The purpose of this research was to investigate the relationship between TS polymorphisms and total plasma homocysteine (tHcy) levels. Methods: The study population consisted of 396 healthy male and female volunteers from Kingston, Ontario and Halifax, Nova Scotia, Canada between 2006 and 2008. The effect of each TS polymorphism on tHcy concentrations was investigated and further analyses were conducted on categorization of polymorphisms based on 50 or 30 UTR. The combined effect of TS polymorphisms on tHcy concentration was also investigated, in addition to interactions between polymorphisms in TS and MTHFR 677C>T and interactions between TS polymorphisms and serum folate and vitamin B12 status. Results: An association between TS 50 polymorphisms and tHcy concentration was observed (p = 0.05). The combined effect of the TS polymorphisms was also found to be associated with tHcy concentration (p = 0.05). Additionally, an antagonistic interaction was observed between TS 50 polymorphism and MTHFR 677C>T on tHcy concentrations (p = 0.04). Conclusions: The findings of this research provide evidence of an association between TS polymorphisms and tHcy concentrations. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Homocysteine is a sulfur-containing amino acid produced in the metabolism of methionine [1]. In the methionine cycle, homocysteine is involved as an essential intermediate in the transfer of a

Abbreviations: tHcy, total plasma homocysteine; 5-methyl-THF, 5-methyltetrahydrofolate; MS, methionine synthase; SAM, S-adenosylmethionine; SAH, Sadenosylhomocysteine; TS, thymidylate synthase; UTR, untranslated region; TSER, thymidylate synthase enhancer region; G/C SNP, G to C single nucleotide polymorphism; bp, base pair; 3RG, three repeat with a guanine residue; +6, 6 base pair insertion; 2R, double repeat; 3R, triple repeat; 3RC, three repeat with a cytosine residue; 6, 6 base pair deletion; 5,10-methylene-THF, 5,10-methylenetetrahydrofolate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; DHFR, dihydrofolate reductase; MTHFR, methylenetetrahydrofolate reductase; PCR–RFLP, polymerase chain reaction–restriction fragment length polymorphism; 95% CI, 95% confidence interval. * Corresponding author. Fax: +1 613 533 6686. E-mail addresses: [email protected] (V. Ho), [email protected] (T.E. Massey), [email protected] (W.D. King). 1096-7192/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.05.010

methyl group to power methylation reactions in the body [2]. The intracellular concentration of homocysteine is strictly regulated and excess intracellular free homocysteine readily crosses the cell membrane and enters the plasma [3]. The term total plasma homocysteine (tHcy) concentration refers to all forms of homocysteine including both protein-bound and non-protein-bound homocysteine, homocysteine (the disulfide of homocysteine) and mixed disulfides of homocysteine found in plasma [4]. The occurrence of elevated tHcy levels is an indication that the methionine cycle has been compromised [5]. The suspected consequences of elevated tHcy include folate deficiency, production of reactive oxygen species and altered methylation capacity [5]. Evidence from experimental studies supports a biologically plausible association between high homocysteine levels and cellular damage, as well as impaired normal cellular and physiologic functions [6]. Elevated tHcy concentrations have been reported for several serious diseases; most notably associations with cardiovascular disease, pregnancy complications and cognitive impairment have been observed [6,7].

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Homocysteine exists at the critical junction between the methionine cycle and biosynthesis of the amino acids cysteine and taurine [5]. The methionine cycle closely associates with folate metabolism; thus, beyond genetic and metabolic control, disturbances in folate intake and metabolism can interfere with homeostasis of the methionine cycle [8]. Homocysteine metabolism occurs via two pathways: remethylation and transsulfuration [9] (Fig. 1). In remethylation, 5-methyltetrahydrofolate (5-methylTHF), a metabolite of folate metabolism, donates a methyl group to homocysteine to form methionine. This reaction occurs in all tissues, is catalyzed by methionine synthase (MS) and is vitamin B12 dependent [9]. A proportion of methionine produced from homocysteine is activated by adenosine triphosphate (ATP) to produce S-adenosylmethionine (SAM), a universal methyl donor. After SAM donates a methyl group, S-adenosylhomocysteine (SAH) is formed as a byproduct and is subsequently hydrolyzed to regenerate homocysteine, initiating a new cycle of one-carbon group transfer. Alternatively in the transsulfuration pathway, excess homocysteine is catabolized to form the amino acids cysteine and taurine in a series of irreversible reactions that are vitamin B6 dependent. Thymidylate synthase (TS; EC 2.1.1.45) is a crucial enzyme involved in folate metabolism and the production of thymidine, a nucleotide necessary for DNA synthesis and repair [11]. Three functional polymorphisms in the untranslated regions (UTR) of TS have been shown to alter TS enzymatic activity and mRNA stability. Two of these occur in the 50 UTR including: (1) a series of two (2R) or three (3R) 28 base pair (bp) tandem repeats found in the TS enhancer region (TSER); and (2) a G to C single nucleotide polymorphism (G/C SNP) occurring within the tandem repeats. A 6base pair (bp) deletion (TS1494del6) polymorphism in the 30 UTR has been reported to occur in linkage disequilibrium with the 50 polymorphisms. The three repeat with a guanine residue (3RG) and the 6-bp insertion (+6) confer higher transcriptional efficiency and greater mRNA stability in vitro than the double repeat or the three repeat with a cytosine residue (2R or 3RC) and the 6-bp

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deletion (6) [12,13]. Thymidine synthesis competes with the methionine cycle for the common substrate 5,10-methylenetetrahydrofolate (5,10-methylene-THF), a precursor of 5-methyl-THF. The reaction converts deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) and is catalyzed by TS. During the conversion of dUMP to dTMP, dihydrofolate is produced; dihydrofolate is then reduced by dihydrofolate reductase (DHFR) to tetrahydrofolate which can rejoin the pool of active folate co-factors. Consequently, it has been postulated that increasing TS activity (conferred by polymorphisms) may elevate homocysteine levels by diverting the supply of substrate towards thymidine synthesis and away from the remethylation of homocysteine to methionine, leading to homocysteine accumulation in the plasma [14,15]. An established determinant of homocysteine concentration is the activity of methylenetetrahydrofolate reductase (MTHFR), an enzyme that plays a central role in the provision of methyl groups for the methionine cycle and thymidine synthesis. MTHFR catalyzes the conversion of 5,10-methylene-THF to 5-methyl-THF. Thus, both the methionine cycle and thymidine synthesis are affected by the common polymorphism in the MTHFR gene (MTHFR 677C>T). Specifically, those homozygous for MTHFR TT express decreased enzymatic activity by 20–30% and thus, have been shown to associate with elevated homocysteine concentrations [14]. It is hypothesized that the influence of TS polymorphisms on homocysteine concentration is modified by MTHFR activity. Conflicting evidence on the effects of TS polymorphisms on tHcy levels has been reported. There are several gaps in knowledge that may have contributed to the inconsistent results. First, independent and combined effects of the three TS variants have not been considered within a single sample with respect to associations with homocysteine levels. The three TS polymorphisms occur in linkage disequilibrium, such that high activity conferred by polymorphisms found in the 50 UTR are associated with low activity conferred by TS1494del6 occurring in the 30 UTR. Second, the consequences of carrying combinations of polymorphisms (TS and

Fig. 1. Methionine–homocysteine cycle coupled with folate metabolism and thymidine production [10]. (Abbreviations: dUMP, deoxyuridine monophosphate; TS, thymidylate synthase; DHFR, dihydrofolate reductase; dTMP, deoxythymidine monophosphate; MTHFR, methylenetetrahydrofolate reductase; MS, methionine synthase; ATP, adenosine triphosphate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine).

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V. Ho et al. / Molecular Genetics and Metabolism 101 (2010) 18–24

MTHFR) on homocysteine concentration also require further investigation. Finally, the effect of co-factors in homocysteine remethylation such as vitamin B12 status on the TS-homocysteine relationship have been absent in the epidemiologic literature. Thus, we sought to examine the impact of TS polymorphisms on tHcy concentration in a healthy Canadian population between the ages of 20 and 50 years. Interactions between TS and relevant co-factors in homocysteine remethylation (serum folate and vitamin B12) and TS and MTHFR on tHcy concentrations were explored. Materials and methods Study population This cross-sectional study included 396 healthy male and female volunteers between the ages of 20 and 50 years from Kingston, Ontario and Halifax, Nova Scotia, Canada. Subjects with health conditions that may be related to homocysteine concentrations (e.g. history of angina or other vascular disease, cancer or diabetes), who were pregnant, or who had given birth in the previous year, were excluded from the study sample. Subjects provided a blood sample for genomic and biochemical analysis and completed a questionnaire. Ethics approval was granted by the Queen’s University Health Sciences Research Ethics Board and all subjects provided written informed consent. Biochemical analyses Fasting blood samples were collected for the determination of biochemical parameters and for DNA isolation. Concentrations of tHcy were quantified by the Abbott AxSYM immunoassay based on fluorescence polarization immunoassay technology (Abbott AxSYM, Abbott Laboratories, Abbott Park, Illinois, USA). Determination of serum folate and vitamin B12 status were assessed using a two-step competitive immuno-enzymatic assay with chemiluminescence detection (Beckman Coulter Inc., Fullerton, California, USA). All biochemical analyses were conducted by the Department of Pathology and Laboratory Medicine of the Ottawa General Hospital. Genotype determinations Genomic DNA was isolated from peripheral blood (200 lL) of the subjects using the Qiagen Blood Mini Kit. TSER, G/C SNP and TS1494del6 genotyping was accomplished by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses as described by Mandola et al. [12], Tan et al. [16] and Zhang et al. [17]. The MTHFR 677C>T genotyping method by PCR–RFLP has been reported previously [18]. To ensure reliability of all assays, genotyping was repeated for 10% of the samples and two samples of each genotype were randomly selected and subjected to direct DNA sequencing to verify genotype; concordance was found to be 100%. Statistical analysis The effect of each TS polymorphism on tHcy concentrations was assessed as a co-dominant model. Analyses of the effects of the 50 polymorphisms (TSER polymorphism and G/C SNP) and 30 polymorphism (TS1494del6) were further categorized according to TS functional activity. 50 Polymorphisms were categorized as ‘‘50 High” if genotype contained a functional 3R allele with a G residue (3RG/ 3RG, 3RG/3RC or 2R/3RG) versus ‘‘50 Low” if genotype contained a 2R allele or a 3R allele with a C residue (2R/2R, 2R/3RC or 3RC/ 3RC) [19]. 30 Polymorphism was categorized as ‘‘30 High” if genotype

was homozygous insertion (+6/+6) versus ‘‘30 Low” if genotype was heterozygous insertion/deletion (+6/6) or homozygous deletion (6/6) [20]. The combined effects of the three polymorphisms on tHcy concentrations were analyzed by grouping 50 and 30 genotypes according to functional status, yielding four functional TS categories (50 High & 30 High, 50 High & 30 Low, 50 Low & 30 High and 50 Low & 30 Low) [19]. MTHFR 677C>T genotypes were dichotomized to represent MTHFR activity, where those with MTHFR TT genotype were considered ‘‘Low MTHFR Expression” versus those with MTHFR CC or CT genotypes were considered ‘‘High MTHFR Expression” [14]. Serum folate status was dichotomized to ‘‘Low” if concentrations were less or equal to the 25th percentile and ‘‘Normal” if greater than the 25th percentile. Serum vitamin B12 status was dichotomized based on a clinical cut point of vitamin B12 deficiency in that those below 148 pmol/L were considered ‘‘Low” and those above were considered ‘‘Normal” [21]. Multiple linear regression was used to estimate adjusted means and 95% confidence intervals (95% CI) of tHcy concentrations within each stratum of TS genotype while controlling for strong predictors of homocysteine. Two-sided p-values based on F-tests are presented for each analysis of TS genotypes on levels of tHcy. Covariates included in each model were sex, age, study center and serum folate. Interactions between TS and MTHFR, TS and serum folate and TS and vitamin B12 levels on tHcy were explored by inclusion of product terms into the regression model. Mean tHcy concentration in this study population was determined to be 8.59 lmol/L (±1.96). With a sample size of 396 subjects and genotype frequencies of 30%, this study had 80% power to detect a difference in means of 0.62 lmol/L, with smaller detectable differences for genotype frequencies greater than 30%.

Results The total sample of this study included 396 participants. Analysis of the independent effects of TS polymorphisms on tHcy concentrations included 395 individuals since one individual with a variant genotype was omitted due to the uncertainty of the impact of this variant genotype (i.e. 3R/5R) on TS activity. Data for five individuals were missing for MTHFR polymorphism and analyses including this factor were based on 391 subjects. The frequency distributions and percentages for select characteristics and TS and MTHFR polymorphisms of the study population are presented in Table 1. Subject recruitment was designed to result in approximately balanced distribution by gender, age (in 10-year intervals) and study center. The final sample included equal representation by age and study center and a higher proportion of females (57%). The study population was primarily Caucasian (89%). The distributions of TS polymorphisms found in this study correspond well with those reported in the literature for Caucasian subjects [13,15,17,22–27]. The 50 and 30 polymorphisms occurred in linkage disequilibrium; where 50 High was more likely to associate with 30 Low (v2 p-value < 0.01). The genotype distributions of all polymorphisms were in agreement with Hardy–Weinberg equilibrium (p > 0.05). The distribution of tHcy concentrations was approximately normal with a range from 3.91 lmol/L to 17.53 lmol/L and a mean and standard deviation of 8.59 lmol/L and 1.96, respectively. The effects of each TS polymorphism and their combined effect on tHcy concentration are reported in Table 2. Individuals with 50 High genotypes had a higher mean tHcy levels than did subjects with 50 Low genotypes (p = 0.05). No association was observed between the independent effect of TS1494del6 polymorphism and tHcy concentration. Consideration of the combined effects of TS

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V. Ho et al. / Molecular Genetics and Metabolism 101 (2010) 18–24 Table 1 Distribution of select characteristics and TS and MTHFR polymorphisms in the study population.

Select characteristics

Sex Folate

a

Vitamin B12b Genotypes

TSER

TSER and G/C SNP

TS1494del6

50 Polymorphismsc 30 Polymorphismd Combined effects model

MTHFR 677C>Te

Categories

N

Percentage (%)

Female Male Normal (>22.36 nmol/L) Low (622.36 nmol/L) Normal (P148 pmol/L) Low (<148 pmol/L)

224 172 297 99 319 77

56.6 43.4 75.0 25.0 80.6 19.4

3R/3R 2R/3R 2R/2R Other 3RG/3RG 3RG/3RC 2R/3RG 3RC/3RC 2R/3RC 2R/2R Other +6/+6 +6/6 6/6 50 High 50 Low 30 High 30 Low 50 High & 30 High 50 High & 30 Low 50 Low & 30 High 50 Low & 30 Low CC CT TT

129 188 78 1 23 63 89 44 99 77 1 164 177 54 175 220 164 231 29 146 135 85 160 173 58

32.6 47.5 19.7 0.3 5.8 16.0 22.5 11.1 25.0 19.4 0.3 41.5 44.8 13.7 44.3 55.7 41.5 58.5 7.3 37.0 34.2 21.5 40.5 43.8 14.7

a

Dichotomized based on the 25th percentile. Dichotomized based on clinical cut point of vitamin B12 deficiency. c 50 Polymorphisms categorizes TSER and G/C SNP genotypes into 50 High (3RG/3RG, 3RG/3RC, 2R/3RG) and 50 Low (2R/2R, 2R/3RC, 3RC/3RC). d 30 Polymorphism categorizes TS1494del6 genotypes into 30 High (+6/+6) and 30 Low (+6/6, 6/6). e Missing MTHFR genotype data on five individuals. b

Table 2 Mean plasma homocysteine concentration expressed in lmol/L associated with TS and MTHFR polymorphisms, serum folate and vitamin B12 in the study population.

a

Polymorphism

TS genotype

Mean tHcy concentrationa (95% CI)

TSER and G/C SNP

3RG/3RG 3RG/3RC 2R/3RG 3RC/3RC 2R/3RC 2R/2R

8.4 9.0 8.7 8.4 8.5 8.4

9.2) 9.4) 9.1) 8.9) 8.9) 8.8)

0.22

50 Polymorphismsb

50 High 50 Low

8.8 (8.5, 9.1) 8.4 (8.2, 8.7)

0.05

30 Polymorphismc

30 High 30 Low

8.6 (8.3, 8.8) 8.6 (8.4, 8.8)

0.75

Combined effects

50 High & 30 High 50 High & 30 Low 50 Low & 30 High 50 Low & 30 Low

8.5 8.9 8.6 8.2

9.1) 9.1) 8.9) 8.6)

0.05

MTHFR

CC/CT TT

8.5 (8.3, 8.6) 9.4 (9.0, 9.9)

<0.01

Folate

Normal folate Low folate

8.3 (8.1, 8.5) 9.6 (9.3, 10.0)

<0.01

Vitamin B12

Normal B12 Low B12

8.4 (8.2, 8.6) 9.5 (9.1, 9.9)

<0.01

(7.7, (8.6, (8.4, (7.9, (8.2, (8.0,

(7.9, (8.6, (8.3, (7.8,

p-value

Adjusted for sex, age, study center and serum folate. 50 Polymorphisms categorizes TSER and G/C SNP genotypes into 50 High (3RG/ 3RG, 3RG/3RC, 2R/3RG) and 50 Low (2R/2R, 2R/3RC, 3RC/3RC). c 30 Polymorphism categorizes TS1494del6 genotypes into 30 High (+6/+6) and 30 Low (+6/6, 6/6). b

polymorphisms was suggestive of an interaction between the 50 and 30 TS polymorphisms (p-value interaction = 0.06). Combined effect analysis also revealed associations between TS genotypes and tHcy levels (p = 0.05). Mean tHcy levels were elevated among MTHFR TT individuals and those with low serum folate and vitamin B12 concentrations (p < 0.01). Since TS competes with MTHFR for the common substrate 5,10methylene-THF, we examined interactions between MTHFR 677C>T and TS polymorphisms on tHcy concentration (Table 3). Interaction was observed between TS 50 polymorphisms and MTHFR 677C>T (p-value interaction = 0.04). Specifically, the pattern among those with high MTHFR expression genotype was similar to that of the overall pattern for the independent effect of each TS polymorphism on homocysteine. Among those with low MTHFR expression genotype, the 50 High TS expression genotype had a lower mean tHcy concentration compared to 50 Low TS expression genotype. A suggestive interaction between the combined effects of TS genotypes and MTHFR polymorphism was observed (p-value interaction = 0.09). Finally, interactions between TS polymorphisms and serum folate and vitamin B12 were explored (Tables 4 and 5). Results were suggestive of an interaction between TS 50 polymorphisms and folate, where 50 High associated with a greater elevation of tHcy concentration among those with low folate levels compared to those with normal folate concentrations. No evidence of interactions with folate was observed for 30 polymorphism and the combined effects of 30 and 50 polymorphisms. Interaction between TS polymorphisms and vitamin B12 was explored, but no interaction was observed.

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V. Ho et al. / Molecular Genetics and Metabolism 101 (2010) 18–24

Table 3 Gene-gene interaction between TS and MTHFR polymorphisms on tHcy concentrations expressed in lmol/L. TS polymorphism

Genotype

Low expression MTHFR (TT) N

a b c

High expression MTHFR (CC or CT) a

Mean tHcy (95% CI)

Pinteraction a

N

Mean tHcy (95% CI)

50 Polymorphismsb

50 High 50 Low

33 25

9.2 (8.6, 9.7) 9.8 (9.2, 10.5)

141 192

8.7 (8.4, 9.0) 8.3 (8.0, 8.5)

0.04

30 Polymorphismc

30 High 30 Low

20 38

9.9 (9.2, 10.7) 9.2 (8.7, 9.7)

142 191

8.4 (8.1, 8.7) 8.5 (8.3, 8.8)

0.10

Combined effects

50 High & 30 High 50 High & 30 Low 50 Low & 30 High 50 Low & 30 Low

4 29 16 9

25 116 117 75

8.5 8.8 8.4 8.1

0.09

8.6 9.2 10.2 9.1

(7.0, (8.6, (9.4, (8.0,

10.3) 9.8) 11.1) 10.2)

(7.8, (8.4, (8.1, (7.7,

9.2) 9.1) 8.7) 8.5)

Adjusted for sex, age, study center and serum folate. 50 Polymorphisms categorizes TSER and G/C SNP genotypes into 50 High (3RG/3RG, 3RG/3RC, 2R/3RG) and 50 Low (2R/2R, 2R/3RC, 3RC/3RC). 30 Polymorphism categorizes TS1494del6 genotypes into 30 High (+6/+6) and 30 Low (+6/6, 6/6).

Table 4 Gene-diet interaction between TS polymorphisms and serum folate on tHcy concentrations expressed in lmol/L. TS polymorphism

a b c

Genotype

Low folate

Normal folate

Pinteraction

N

Mean tHcy (95% CI)a

N

Mean tHcy (95% CI)a

50 Polymorphismsb

50 High 50 Low

46 53

10.1 (9.6, 10.6) 9.2 (8.7, 9.7)

129 167

8.4 (8.1, 8.7) 8.2 (7.9, 8.5)

0.07

30 Polymorphismc

30 High 30 Low

43 56

9.4 (8.9, 10.0) 9.7 (9.3, 10.2)

121 175

8.3 (8.0, 8.6) 8.3 (8.0, 8.5)

0.41

Combined effects

50 High & 30 High 50 High & 30 Low 50 Low & 30 High 50 Low & 30 Low

10 36 33 20

19 110 102 65

8.1 8.4 8.3 8.0

0.26

9.7 10.2 9.4 8.9

(8.6, (9.6, (8.8, (8.1,

10.8) 10.8) 10.0) 9.7)

(7.3, (8.1, (8.0, (7.6,

8.8) 8.7) 8.7) 8.4)

Adjusted for sex, age and study center. 50 Polymorphisms categorizes TSER and G/C SNP genotypes into 50 High (3RG/3RG, 3RG/3RC, 2R/3RG) and 50 Low (2R/2R, 2R/3RC, 3RC/3RC). 30 Polymorphism categorizes TS1494del6 genotypes into 30 High (+6/+6) and 30 Low (+6/6, 6/6).

Table 5 Gene-diet interaction between TS polymorphisms and serum vitamin B12 on tHcy concentrations expressed in lmol/L. TS polymorphism

a b c

Genotype

Low vitamin B12

Normal vitamin B12

Pinteraction

N

Mean tHcy (95% CI)a

N

Mean tHcy (95% CI)a

50 Polymorphismsb

50 High 50 Low

40 37

9.6 (9.1, 10.2) 9.2 (8.6, 9.7)

135 183

8.6 (8.3, 8.9) 8.3 (8.0, 8.5)

0.73

30 Polymorphismca

30 High 30 Low

33 44

9.3 (8.8, 9.9) 9.5 (9.0, 10.0)

131 187

8.4 (8.1, 8.7) 8.4 (8.2, 8.7)

0.82

Combined effects

50 High & 30 High 50 High & 30 Low 50 Low & 30 High 50 Low & 30 Low

8 32 25 12

9.2 9.6 9.2 9.2

21 114 110 73

8.1 8.7 8.5 8.1

0.60

(8.6, (9.0, (8.5, (8.2,

11.0) 10.0) 9.8) 10.2)

(7.3, (8.3, (8.1, (7.7,

8.8) 9.0) 8.8) 8.4)

Adjusted for sex, age, study center and serum folate. 50 Polymorphisms categorizes TSER and G/C SNP genotypes into 50 High (3RG/3RG, 3RG/3RC, 2R/3RG) and 50 Low (2R/2R, 2R/3RC, 3RC/3RC). 30 Polymorphism categorizes TS1494del6 genotypes into 30 High (+6/+6) and 30 Low (+6/6, 6/6).

Discussion Overall, experimental and epidemiologic studies have provided evidence that elevated tHcy levels are associated with an increased risk of disease such as cardiovascular disease, pregnancy complications and cognitive impairment. In this study, we investigated the relationship between tHcy concentration and four common variants relevant to the regulation of tHcy concentration, including the TSER tandem repeat polymorphism, the TS G/C SNP, TS1494del6 polymorphism and MTHFR 677C>T polymorphism. An association was observed between 50 polymorphisms that confer higher TS activity (50 High) and elevated mean tHcy concentration. In addition, analysis of the combined effect of TS polymorphisms on tHcy levels revealed an association, whereby TS

genotypes that confer higher TS activity appear to associate with a higher mean tHcy concentrations. Finally, analysis of interaction between TS 50 polymorphisms and MTHFR revealed a significant antagonistic interaction. The consideration of the three functional polymorphisms as genetic determinants of tHcy concentration is novel. Of the studies that have examined the relationship between TS polymorphisms and homocysteine, none have considered all three functional polymorphisms within a single study sample and inconsistencies between studies were observed. In the studies that investigated the association between TSER and tHcy concentrations, some reported tHcy levels increased with the 3R/3R genotype [14,28] while others reported null findings [15,22,25] and one study found that tHcy decreased with the 3R/3R genotype [29]. To the best of our

V. Ho et al. / Molecular Genetics and Metabolism 101 (2010) 18–24

knowledge, no study has investigated the effect of TS G/C SNP on tHcy levels. Finally, among studies that investigated the effects of TS1494del6 on tHcy concentrations, null findings [22,25] and an association between 30 High (+6/+6) and higher tHcy levels were reported [27]. The inconsistent results observed in previous studies may be due in part to differences in ethnicity of the populations being studied. Specifically, important lifestyle, environmental or additional genetic factors that may interact with TS polymorphisms differ in their distributions between ethnic groups. Further, the lack of consideration of all functional polymorphisms of TS within the different study populations may have also contributed to the inconsistencies. In our study, the TSER polymorphism was not found to singularly associate with tHcy levels; however, consideration of the TSER polymorphism and the G/C SNP (50 polymorphisms) together revealed an association with tHcy concentrations. Thus, the results seen in our study provide some insight into the cause of the inconsistencies seen in the published literature. In our study population, having more tandem repeats with a guanine residue in the TS 50 UTR was associated with higher tHcy concentration. This is consistent with our hypothesis that polymorphisms that confer increased TS activity will associate with elevated tHcy concentrations. Also, the 50 and 30 polymorphisms were found to occur in linkage disequilibrium which is consistent with the published literature [19,30]. This is the first study to report a significant antagonistic interaction between TS 50 polymorphisms and MTHFR 677C>T on tHcy concentration since no previous study has considered the contribution of the G/C SNP within their study population. Specifically, among high expression MTHFR genotypes (CC and CT), the genotype that confers higher TS activity was associated with a higher mean tHcy level. In contrast, among individuals with low expression MTHFR genotype (TT), the relationship between TS 50 polymorphisms and homocysteine level was reversed such that the genotype that confers an increase in TS activity associated with a lower mean tHcy concentration. This finding was unexpected, as we anticipated that those with 50 polymorphisms conferring high TS activity and MTHFR TT genotype would have the highest tHcy levels. The direction of the observed interaction between TS and MTHFR polymorphisms in this study cannot be readily explained from the existing literature, but a combination of factors may be responsible. The existence of feedback mechanisms maintaining homeostasis between thymidine production, folate metabolism and the methionine cycle may have contributed to the unexpected finding [31]. Another possible contributing factor includes confounding by other genetic variants in enzymes which play a role in these complex pathways. In any case, replication of this finding is warranted to ensure that this is not a spurious result. Interactions between TS polymorphisms and serum folate and vitamin B12 on homocysteine concentrations were explored; however no interactions were observed (p-values >0.05). Results were suggestive of a synergistic interaction between TS 50 polymorphisms and serum folate, where among those with low folate levels, 50 High TS expression genotypes increased homocysteine concentration (relative to 50 Low TS expression genotype) to a greater extent than among those with normal folate levels. This result is consistent with our hypothesis since we conjectured that when folate stores are abundant it is possible that the impact of TS activity is limited given that there is enough folate to supply both thymidine production and homocysteine remethylation. However, in times of depletion, the activity of TS may play a greater role in funneling folate away from the remethylation of homocysteine and towards thymidine production causing homocysteine to accumulate. A priori, serum folate and vitamin B12 levels were postulated to modify genetic susceptibility con-

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ferred by TS polymorphisms to elevate homocysteine concentrations. However, it is recognized that due to the cross-sectional nature of this study, the intra-individual variability of serum folate and vitamin B12 concentration may challenge the validity of a single measurement in representing an individual’s average concentration and hence, limit our ability to study these interactions. Conclusion This study found that TS 50 polymorphisms are singularly associated with tHcy concentrations and interact with MTHFR 677C>T to influence tHcy levels. Results are suggestive that 50 polymorphisms that alter the transcriptional activity of the TS enzyme have a greater impact than the 30 polymorphism which affects the stability of mRNA on altering tHcy concentrations [12,13]. Consideration of the combined effects of the three functional polymorphisms in the TS gene on tHcy concentrations is novel. This study may have been underpowered to examine the interaction between combined effects of TS polymorphisms and MTHFR 677C>T on tHcy concentrations; thus future studies may need a larger sample size to investigate this interaction. In addition, consideration of other genetic factors relevant to the methionine cycle may further clarify the TS-homocysteine association. Finally, future studies should re-consider interactions between TS polymorphisms and folate and vitamin B12 by considering both dietary intake and multiple biological measures to assess the potential value of dietary interventions to lower homocysteine concentrations. In this research, we demonstrated that tHcy levels are influenced by genetic and environmental factors and their interactions. The identification of additional genetic modifiers of tHcy may help to clarify the role of homocysteine in the development of several disorders such as cardiovascular disease, pregnancy complications and cognitive impairment. Conflict of interest No potential conflicts of interest to disclose. Acknowledgments This work was supported by the Canadian Institutes of Health Research (CIHR) and student support was provided by the Queen’s University Terry Fox Foundation Training Program in Transdisciplinary Cancer Research in partnership with CIHR. References [1] A.L. Miller, The methionine–homocysteine cycle and its effects on cognitive diseases, Altern. Med. Rev. 8 (2003) 7–19. [2] J.B. Mason, Biomarkers of nutrient exposure and status in one-carbon (methyl) metabolism, J. Nutr. 133 (2003) 941S–947S. [3] S.J. James, S. Melnyk, M. Pogribna, I.P. Pogribny, M.A. Caudill, Elevation in Sadenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology, J. Nutr. 132 (2002) 2361S– 2366S. [4] B.A. de, W.M. Verschuren, D. Kromhout, L.A. Kluijtmans, H.J. Blom, Homocysteine determinants and the evidence to what extent homocysteine determines the risk of coronary heart disease, Pharmacol. Rev. 54 (2002) 599– 618. [5] M. Medina, J.L. Urdiales, M.I. Mores-Sanchez, Roles of homocysteine in cell metabolism: old and new functions, Eur. J. Biochem. 268 (2001) 3871–3882. [6] H. Refsum, E. Nurk, A.D. Smith, P.M. Ueland, C.G. Gjesdal, I. Bjelland, A. Tverdal, G.S. Tell, O. Nygard, S.E. Vollset, The Hordaland homocysteine study: a community-based study of homocysteine, its determinants, and associations with disease, J. Nutr. 136 (2006) 1731S–1740S. [7] H.K. Kuo, F.A. Sorond, J.H. Chen, A. Hashmi, W.P. Milberg, L.A. Lipsitz, The role of homocysteine in multisystem age-related problems: a systematic review, J. Gerontol. A Biol. Sci. Med. Sci. 60 (2005) 1190–1201. [8] L.B. Rasmussen, L. Ovesen, I. Bulow, N. Knudsen, P. Laurberg, H. Perrild, Folate intake, lifestyle factors, and homocysteine concentrations in younger and older women, Am. J. Clin. Nutr. 72 (2000) 1156–1163.

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