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Jean Mayer-U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA;
* Department of Internal Medicine, University of Messina, Messina, Italy;
Department of Clinical and Experimental Medicine, Policlinico G.B. Rossi, University of Verona, Verona, Italy;
** Boston University School of Medicine and The Framingham Heart Study, National Heart, Lung, and Blood Institute, Framingham, MA
3To whom correspondence should be addressed. E-mail: paul.jacques{at}tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • homocysteine • methylenetetrahydrofolate reductase • age • sex • folate • genetics
As circulating total homocysteine (tHcy)3 concentrations increase so does the risk of occlusive vascular disease (1,2). A recent meta-analysis demonstrated that a 3 µmol/L or 25% lower tHcy concentration was associated with an 11% lower risk of ischemic heart disease and a 19% lower risk of stroke (3). Many factors are related to circulating tHcy concentrations (4). Fasting plasma tHcy concentration is consistently higher in men than in women, and increases with age (5–10). The male-female difference has been attributed mainly to sex differences in muscle mass (6,11,12) and circulating sex hormones (10,11,13). tHcy concentrations are strongly dependent on renal function (6,14–17); thus, impaired renal function associated with aging may account in part for the increased plasma tHcy concentrations in older subjects. The nutritional status of vitamin B-12, B-6 and folate is a major determinant of tHcy concentrations (18), and other recently identified nutritional and lifestyle factors may also influence circulating tHcy levels (19–21).
Genetic background also affects tHcy concentrations. A common gene variant of methylenetetrahydrofolate reductase (MTHFR, EC 1.7.99.5), which synthesizes N5-methyltetrahydrofolate, the methyl donor for methylation of homocysteine to methionine, is the most frequent genetic cause of mild hyperhomocysteinemia. The molecular defect responsible for this MTHFR genetic variant, first described as "thermolabile" MTHFR by Kang et al. (22) because of its decreased stability under specific heat conditions, resides in a cytosine to thymine transition at nucleotide 677 (C677T) in the MTHFR gene locus (23,24). This, in turn, determines an alanine to valine substitution in the folate-binding site of the enzyme. Those bearing two copies of the mutant T allele have circulating tHcy concentrations that are on average between 1.5 and 2.6 µmol/L (
15–25%) higher than subjects carrying the C allele (25,26), but the association of the thermolabile variant with mild hyperhomocysteinemia appears to be contingent on folate availability (25–28). When folate status is low, homozygotes for this mutation have tHcy concentrations that range from 2.3 to 4.3 µmol/L (25–50%) higher than concentrations in those without the T allele (25). If folate concentrations are adequate, however, MTHFR genotype does not affect tHcy levels. A recent study from our group (29) suggested that riboflavin, a cofactor for MTHFR, also interacts with the MTHFR genotype in determining plasma tHcy concentrations, but only in subjects with lower folate status.
Because tHcy concentrations are strongly modulated by gender and age, interactions among these factors, folate status and the C677T mutation in MTHFR gene locus may explain the inconsistent association of this polymorphism with the risk of occlusive vascular disease (25,26). Moreover, age and sex interactions with polymorphic mutations were reported recently for several gene loci associated with cardiovascular disease, such as apolipoprotein (apo) E, apoA1, lipoprotein lipase and apoC3 (30–33).
The aim of the present study was to determine whether the association of the common MTHFR C677T mutation with plasma tHcy concentrations varies by age and gender in a large cohort of free-living U.S. Caucasian subjects, well characterized for folate status and other major determinants of plasma tHcy concentrations.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The details of the design and methods of the Framingham Offspring Study were presented elsewhere (34). Briefly, the Framingham Heart Study, an epidemiologic study of heart disease, was established in Framingham, Massachusetts between 1948 and 1950 with a cohort of 5209 men and women, aged 30 to 59 y (35). By 1971, the original cohort included 1644 husband-wife pairs and 378 individuals, who had developed cardiovascular disease. The offspring of these subjects and the offsprings’ spouses were invited to participate in the Framingham Offspring Study, and 5135 of the 6838 eligible individuals participated in the first study examination (34). The offspring cohort has undergone repeat examinations at 3- to 4-y cycles. Nearly all subjects were Caucasians (34,36). The fifth examination of the offspring cohort began in January 1991 and was completed in December 1994. This study was approved by the Human Investigations Review Committee at New England Medical Center and by the Institutional Review Board for Human Research at Boston University Medical Center.
Biochemical analyses.
Twelve-hour fasting venous blood samples were collected in tubes containing 0.1% EDTA from subjects who attended the 5th examination visit of the Framingham Offspring Study. Plasma was separated from blood cells by centrifugation at 1850 x g for 30 min at 4°C and immediately used for the measurement of lipids. Plasma total cholesterol, HDL cholesterol and triglyceride concentrations were measured as previously described (37). HDL cholesterol was measured after precipitation of apo B-containing lipoproteins with heparin-manganese chloride (38). LDL cholesterol concentrations were estimated with the equation of Friedewald et al. (39). The CV for total cholesterol, HDL cholesterol, and triglyceride measurements were each <5%. Serum creatinine levels were measured by the Jaffe method (40), adapted for autoanalyzers (Roche Diagnostics, Indianapolis, IN). The CV was 2.7% for creatinine.
tHcy, folate, vitamin B-12 and pyridoxal-5'-phosphate (PLP, the active circulating form of vitamin B-6) were determined on plasma samples from the 5th examination cycle. tHcy in plasma was determined by HPLC with fluorescence detection (41), plasma folate by a 96-well plate microbial (Lactobacillus casei) assay (42,43), plasma PL-5'-phosphate by the tyrosine decarboxylase apoenzyme method (44) and plasma vitamin B-12 by a radioassay (BioRad Quantaphase II, Hercules, CA). The CV for these assays were 8% for homocysteine, 13% for folate, 16% for PLP and 7% for vitamin B-12.
MTHFR genotyping.
Blood samples for DNA analysis were collected between 1987 and 1991. DNA was isolated from peripheral blood leukocytes by standard methods (45). MTHFR C677T genotyping was performed using the Perkin-Elmer/Applied Biosystems 7700 Sequence Detection Systems and TaqMan reagents (Applied Biosystems, Foster City, CA). The technique involves the use of two probes labeled at the 5' end with different reporter fluorescent dyes, one complementary to the wild-type DNA strand and the other complementary to the strand with the C to T mutation, and at the 3' end with a fluorescent quencher. The sample was genotyped as CC if there was fluorescence only from the wild-type reporter, TT if there was fluorescence only from the reporter for the mutant allele, and CT if there was an intermediate fluorescence from both reporters (46,47).
Statistical analysis.
Because plasma tHcy concentration was positively skewed, analyses were done using natural logarithm transformations. Inverse transformations were performed to provide geometric mean tHcy concentrations and their 95% CI. Other subject characteristics with positively skewed distributions were also transformed using either the natural logarithm (plasma folate, plasma PLP, serum creatinine, plasma HDL cholesterol and plasma triglycerides) or a square-root transformation (plasma vitamin B-12).
Differences in tHcy concentrations and various participant characteristics between men and women were determined using t tests. We performed an analysis of covariance (ANCOVA) to determine the mean values for tHcy concentrations and other participant characteristics in each MTHFR C677T genotype and compared the means in each genotype category to the others using the Tukey procedure for multiple contrasts. The means were adjusted for age, sex and serum creatinine concentrations. The P-values presented in the tables represent the overall significance of the test for differences across MTHFR genotype (based on the F-statistic).
We also used ANCOVA to test for the established interaction between genotype and folate status (as the logarithm of plasma folate) adjusting for age, sex and serum creatinine concentrations. As a consequence of a significant interaction between genotype and folate status, we stratified subsequent analyses relating genotype to tHcy concentrations by folate status dividing plasma folate concentration at its median value of 12.5 nmol/L. Analyses relating genotype to tHcy concentrations within folate strata were adjusted for age, sex, serum creatinine concentrations and plasma folate concentrations (as a continuous variable within folate strata). Within the low folate stratum, we also tested for age and sex interactions. As a consequence of a significant interaction, we stratified the analysis of the relation between genotype and tHcy concentration by both sex and age, dividing age at its median value of 55 y. These analyses were adjusted for age (as a continuous variable within the age strata), serum creatinine and plasma folate (as a continuous variable with the low folate stratum).
All analyses were performed with SAS statistical software (48). Unless specified, differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the study sample as a whole, plasma tHcy and folate concentrations were significantly related to MTHFR genotype (Table 2). tHcy concentrations in those with the CC genotype (9.4 µmol/L) were lower than concentrations in those with the CT (9.8 µmol/L, P = 0.02) and TT (10.2 µmol/L, P < 0.001) genotypes, after adjusting for sex, age and serum creatinine. Geometric mean plasma folate concentrations were higher in the CC (14.3 nmol/L) genotype category than in the CT (12.6 nmol/L, P < 0.001) and the TT (12.3 nmol/L, P = 0.007) genotype categories. Plasma PLP, vitamin B-12, creatinine and lipid concentrations were not associated with MTHFR genotype.
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the sample median, but for subjects with plasma folate <12.5 nmol/L, mean tHcy was higher in those with the TT genotype (P = 0.001) than in those with the CC or CT genotypes.
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55 y old), the relationship between MTHFR genotype and plasma tHcy concentration was not significant in the low folate status group. However, in younger men (<55 y old) with low folate concentrations, those who were homozygous for the T allele had significantly higher tHcy concentrations than those carrying the C allele (P < 0.001). The prevalence of tHcy concentrations >13 µmol/L among the younger men with low folate concentrations was <20% for those with at least one C allele, whereas 45% of the homozygotes for the T allele had tHcy concentrations >13 µmol/L. The effect of genotype on the tHcy distribution in these younger men with low folate concentrations was not confined to the upper tail. For example, the 25th percentile value for tHcy in the homozygotes for the T allele, 10.5 µmol/L, was equal to the median tHcy value for those carrying the C allele.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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25% (3.6 µmol/L) lower than those of subjects with the TT genotype. Meta-analyses from the Homocysteine Studies Collaboration (3) suggest that this difference in tHcy concentrations corresponds to an 10–20% lower risk of ischemic heart disease and stroke, respectively.
Our study is the first to examine the simultaneous influence of age and sex on the relation between tHcy and MTHFR C677T genotype. Only a few of the many earlier studies that evaluated the effect of MTHFR genotypes on plasma tHcy concentrations conducted gender (49–54) or age (55–57) comparisons, and the results were mixed. Two of these studies reported associations in young and middle-aged adult men but not in women of comparable ages (49,50). Three studies found no association in men or women aged 70 y (51), 
65 y (52) or 19–90 y (53). One study of subjects aged 40–59 y demonstrated an association in both sexes (54). None of these studies took into account the effect of folate status on the relation between tHcy and genotype. Three studies examined the association between tHcy and genotype at different ages in mixed samples of men and women (55,56) and separate samples of men (57) and women (55). The association generally diminished with age (55,57) but it persisted in older coronary artery disease patients (56). Two of these studies examined the influence of folate status (56,57), demonstrating that the association was present only in those with low folate status.
Information on the association between tHcy and MTHFR C677T genotype might also be gleaned from studies limited to a single sex or restricted age range. For example, Passaro et al. (58) reported that the association was present in a group of women aged 55–80 y, but these women were highly selected to exclude anyone with known cardiovascular risk factors. Brown and et al. (59) observed the association to be present in postmenopausal women receiving HRT, but not in similarly aged women who were not receiving therapy. In a study of women aged 18–44 y, the association was present in those with low folate status (<8.4 nmol/L), but not among women with adequate folate (15.6 nmol/L) (60). Gudnason et al. (61) provided evidence of a strong association in a cohort of young men aged 22–25 y, with genotype accounting for 12% of variance in tHcy concentrations.
The findings of Kauwell et al. (62) may help to explain some of the inconsistencies regarding the influence of age and sex on the relationship between MTHFR C677T genotype and tHcy concentrations. Consistent with our findings, Kauwell et al. observed no cross-sectional relationship between MTHFR genotype and tHcy concentrations in a sample of women aged 60–85 y. However, after 7 wk of consuming a low folate diet, the relation between genotype and tHcy concentrations was apparent. This observation suggests that the relationship between genotype and tHcy is present in women and persists into older ages. Cross-sectional studies such as ours may fail to find true relationships between genotype and tHcy concentrations in the elderly and women because of the increased influence in those population subgroups of age- and gender-related risk factors for hyperhomocysteinemia. There is an abundant literature on the relation between impaired renal function and elevated tHcy concentrations (14), and there is growing evidence that plasma tHcy concentration is influenced by estrogen status in women (8,10,13,63–65). The analytical consequence of additional determinants such as these might be a diminished amount of variance in tHcy concentrations attributable to genotype, which could affect the ability of cross-sectional studies to detect genotype-tHcy associations. Because the other tHcy determinants are essentially held constant in short-term intervention studies such as that of Kauwell et al. (62), the true genotype-tHcy associations may be more easily demonstrated.
Our understanding of the relationships between genetic and environmental determinants of homocysteine concentrations remains incomplete. Because fairly modest elevations in tHcy concentrations are associated with a higher risk of vascular disease, it is crucial to better understand the interplay among factors that affect tHcy levels. Whether the observed effect of age and sex on the relation between MTHFR and tHcy concentrations is real or a consequence of our inability to detect the association against a background of other tHcy determinants, this finding could be of particular relevance in the light of the renewed debate on the role of the MTHFR mutation as a candidate risk factor for coronary heart disease (26). In conclusion, our data suggest that the influence of folate status, age and sex on this relationship between the MTHFR C677T mutation and tHcy requires further examination, especially in elderly cohorts, to critically assess the effect of this mutation on the risk of vascular disease.
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FOOTNOTES |
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2 Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
4 Abbreviations used: ANCOVA, analysis of covariance; apo, apolipoprotein; C, cytosine; C677T, cytosine to thymine transition at base pair 677 of the methylenetetrahydrofolate reductase gene; HRT, hormonal replacement therapy; MTHFR, methylenetetrahydrofolate reductase; PLP, pyridoxal-5'-phosphate; T, thymine; tHcy, total homocysteine.
Manuscript received 30 May 2003. Initial review completed 24 June 2003. Revision accepted 1 September 2003.
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