The authors have no conflict of interest.
A Common Methylenetetrahydrofolate Reductase (C677T) Polymorphism Is Associated With Low Bone Mineral Density and Increased Fracture Incidence After Menopause: Longitudinal Data From the Danish Osteoporosis Prevention Study†
Article first published online: 1 APR 2003
Copyright © 2003 ASBMR
Journal of Bone and Mineral Research
Volume 18, Issue 4, pages 723–729, April 2003
How to Cite
Abrahamsen, B., Madsen, J. S., Landbo Tofteng, C., Stilgren, L., Bladbjerg, E. M., Kristensen, S. R., Brixen, K. and Mosekilde, L. (2003), A Common Methylenetetrahydrofolate Reductase (C677T) Polymorphism Is Associated With Low Bone Mineral Density and Increased Fracture Incidence After Menopause: Longitudinal Data From the Danish Osteoporosis Prevention Study. J Bone Miner Res, 18: 723–729. doi: 10.1359/jbmr.2003.18.4.723
- Issue published online: 2 DEC 2009
- Article first published online: 1 APR 2003
- Manuscript Accepted: 15 OCT 2002
- Manuscript Revised: 23 AUG 2002
- Manuscript Received: 8 NOV 2001
- bone mineral density;
- methylenetetrahydrofolate reductase genotype;
- homocysteine metabolism
A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR) has recently been associated with bone mineral density (BMD) in postmenopausal Japanese women. It is not known whether this effect is also present in European populations and whether it is caused by lower peak bone mass or accelerated postmenopausal bone loss. MTHFR genotyping was done in 1748 healthy postmenopausal Danish women participating in a prospective study of risk factors for osteoporosis. At the time of enrollment, 3–24 months after last menstrual period, the less prevalent genotype (TT, 8.7% of the population) was associated with significantly lower BMD at the femoral neck (ANOVA, p < 0.05), total hip (p < 0.01), and spine (p < 0.05 adjusted for lifestyle covariates, p = 0.06 without adjustment). The mean difference was between 0.1 and 0.3 SD, depending on measurement site. MTHFR genotype added significantly to prediction of BMD by weight and age. Fracture incidence was increased more than 2-fold in subjects with the TT genotype (risk ratio [RR], 2.6; 95% CI 1.2–5.6). This remained significant when the Cox analysis was controlled for BMD (RR, 2.4; 95% CI 1.1–5.2). No differences in serum osteocalcin, bone-specific alkaline phosphatase, and 25-OH-vitamin D were found between genotypes. The response to hormone replacement therapy (HRT) did not differ, but the association of the TT genotype with reduced BMD was maintained at the total hip after 5 years of HRT. The MTHFR TT genotype is associated with low BMD and increased fracture incidence in early postmenopausal women.
FAMILY AND TWIN studies have revealed significant genetic influence on peak bone mass, the rate of bone loss, and the risk of developing osteoporosis.(1,2) Recently, an allelic polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR) was found to be associated with reduced bone mineral density (BMD) in postmenopausal women with the TT genotype.(3) This genotype is associated with slightly higher plasma homocysteine (HCy) levels, which could affect collagen maturation.(4) The present study was undertaken to test whether this MTHFR polymorphism influenced BMD and fracture risk in a European population, whether the effect was related to peak bone mass or postmenopausal bone loss rates, and whether hormone replacement therapy (HRT) modified the impact of MTHFR genotype. This possibility was raised after previous observations(5) in a subset of the participants indicated that HRT may reduce plasma HCy levels.
The Danish Osteoporosis Prevention Study (DOPS) is a nationwide 20-year multicenter study of risk factors for osteoporosis, comprised of 2016 healthy postmenopausal women. It is an open study with a randomized (HRT or no treatment) and a nonrandomized arm (HRT or not by personal choice) and a planned duration of 20 years.(6) In women with an intact uterus, first line hormone replacement consisted of cyclic Trisekvens (estradiol and norethisterone acetate; Novo Nordisk, Malmö, Sweden). Hysterectomized women received unopposed estradiol (estrofem, Novo Nordisk). A number of second line alternatives were used as previously described.(7) Women were eligible for inclusion, provided they were 45-58 years of age and either3-24 months past last menstrual bleeding or still menstruating but exhibiting perimenopausal symptoms, including menstrual irregularities with a serum follicle stimulating hormone (FSH) level more than 2 SD above the premenopausal mean. All participants gave informed consent before entry in the study, which was conducted in accordance with the Helsinki II declaration and approved by the local Ethics Committees. Exclusion criteria were (1) metabolic bone disease, including osteoporosis defined as nontraumatic vertebral fractures on X-ray; (2) current estrogen use or estrogen use within the past 3 months; (3) current or past treatment with glucocorticoids for >6 months; (4) current or past malignancy; (5) newly diagnosed or uncontrolled chronic disease; and (6) alcohol or drug addiction. Of the 2016 participants, 1795 gave permission for DNA to be stored and used for analysis of genes, which could be associated with BMD. DNA from 1748 participants was available for the current analysis (Table 1). Of these, 1119 entered the untreated arms of the study, while 629 entered the HRT arms by randomization or personal choice. Drop-out and noncompliance to treatment allocation was as follows. In the untreated group, 98 participants (8.8%) left the study within the first year. The remaining 1021 participants remained in the study and all attended the 1-, 2-, and 5-year visits. Among initially untreated women, 152 had since elected to begin HRT because of climacteric symptoms, leaving 869 for analysis. In the HRT group, 55 women (8.7%) left the study in the first year, and the remaining 574 participants attended the 1-, 2-, and 5-year visits, with 473 having taken HRT throughout. Thus, 869 MTHFR genotyped subjects in the control group and 473 subjects in the HRT group remained compliant to their initial allocation of HRT or no treatment and were available for 5-year analysis. The study population had a mean folate intake of 293 μg/day based on 7-day diet records. Plasma folate, determined in a subset of 209 patients, was 9.4 nM.(5)
Measurement of BMD
BMD of the spine, hip, forearm, and whole body was measured using cross-calibrated QDR-1000/W and QDR-2000 densitometers (Hologic Inc., Waltham, MA, USA), as previously described.(8) Whole-body scans were done at inclusion and after 1, 2, and 5 years. All other measurements were done at inclusion and after 1, 2, 3, and 5 years. T-scores were calculated using the NHANES-modified Hologic reference range. The in vivo precision errors for BMD in the participating clinics were 1.5% (spine and total hip), 2.1% (femoral neck), 1.9% (ultradistal radius), and 0.7% (whole body). Long-term stability of the equipment was assessed by daily scans of an anthropometric phantom at each center. Changes in BMD were below 0.2% per year. Intercenter calibration differences were investigated by scanning one common anthropometric phantom at study start and once per year. Scans were analyzed locally at the four centers, following detailed guidelines. Bone loss rates were calculated in grams per centimeter squared per year using linear regression on all available serial BMD measurements from each participant. Rates were not calculated for women who left the study before the 5-year visit.
Reports of incident fractures were systematically collected from the participants at each visit to the clinic and subsequently verified through hospital notes, emergency room files, X-ray reports, and other relevant sources. In addition, lateral X-rays of the spine covering T4-L5 were performed at inclusion and after 5 years. Fractures were reviewed by investigators who did not have access to genotype data. 156 verified incident fractures in 140 subjects were included in the subsequent analysis. Fifteen reported fractures could not be verified. There were 51 verified forearm fractures, 6 fractures of the proximal humerus, 4 pelvic fractures, 2 hip fractures, and 1 vertebral fracture. The remaining verified fractures included rib fractures, toe fractures, and finger fractures. Finger and toe fractures were not included in the present analysis. Fracture-free survival by genotype was compared using Cox analysis; participants leaving the study before the 5-year visit remained in the fracture analysis up to the time of withdrawal from the study. The effects of HRT on fracture incidence were reported in detail previously.(9)
Serum and urine biochemistry
Blood samples were collected after overnight fasting, and serum was stored at −80°C for later analysis. Serum levels of 25-hydroxyvitamin D (25-OHD) were measured by radioimmunoassay (RIA), preceded by specific extraction procedures with ether, preliminary chromatography, and a competitive binding assay. This method includes both vitamin D2 and D3 metabolites. The limit of detection was 5 ng/ml; intra- and interassay coefficients of variation (CV) were 8.3% and 10.2%, respectively. Serum osteocalcin was analyzed by RIA. Intra-assay CV was 5% and interassay CV was 10%. Bone-specific alkaline phosphatase (BAP) was lectin-precipitated and analyzed spectrophotometrically. Intra-assay CV was 8% and interassay CV was 25%. Fasting second void urine samples were analyzed immediately for hydroxyproline using spectrophotometry with p-dimethylaminobenzaldehyde substrate. Values were expressed as a ratio relative to creatinine excretion (OHP/Cr). Plasma homocysteine concentrations were available in 206 of the participants (105 from the control group and 101 from the HRT group) from a previously published substudy.(5)
Genomic DNA was extracted from leukocytes by ammonium acetate precipitation. After amplification by polymerase chain reaction, the MTHFR C677T polymorphism was analyzed as described by Morita et al.(10) The polymorphism is located to nucleotide 677 in the MTHFR gene and is caused by a single base change (C T), leading to an amino acid replacement of alanine with valine at position 222 of the MTHFR enzyme.
The study had a power of 90% for detecting a difference in BMD between genotypes CC and TT of at least 0.05 g/cm2, with a 5% risk of a type I error. The power calculation was done following a pilot study in 272 participants, which had revealed a mean difference of 0.34 SD between these two genotypes and predicted a required study size of 1500 persons after adjustment for the low prevalence (7% in the pilot sample) of the TT homozygote genotype. The primary statistical analysis was ANOVA. If this was significant, two post hoc tests were performed: (1) independent samples t-test between the groups within the ANOVA table and (2) a linear regression analysis, testing for MTHFR allele dosage effects on BMD. Cox analysis of survival was used in the comparison of fracture risk by genotype, with and without adjustment for baseline BMD. This counts only the first fracture in patients with multiple fractures; p values below 0.05 were considered significant.
The genotype distribution among the 1748 participants was as follows: CC, 48.5%; CT, 42.8%; and TT, 8.7%. This was compatible with Hardy-Weinberg equilibrium (p = 0.9) and a genotype frequency of 0.7 for the C allele and 0.3 for the T allele.
MTHFR genotype and BMD at menopause
The less common genotype TT was associated with significantly lower BMD at the femoral neck and total hip (ANOVA, p < 0.05 and p < 0.01), with post hoc t-tests showing significant differences between the TT genotype and the CC genotype and the combined CC/CT genotypes. Similarly, BMD adjusted for lifestyle factors and anthropometric covariates (see Table 2) was significantly reduced in the TT genotype (ANOVA, p < 0.05 at the lumbar spine and femoral neck). The magnitude of the difference amounted to a 0.3 SD lower T-score at the spine, 0.2 SD at the total hip and forearm, and 0.1 SD at the femoral neck. MTHFR genotype had no overall association with BMD at the ultradistal forearm or whole body (ANOVA, not significant). However, whole-body BMD was lower in the TT genotype than the CC genotype (p < 0.05), and ultradistal (UD) forearm BMD was borderline (p = 0.06). For whole-body BMD, the difference was equivalent to 0.2 SD. The absolute BMD values are given in Table 2. Although BMD values were very similar for the common genotypes CC and CT, an allele dosage effect on BMD could not be ruled out by linear regression. In this analysis, each T allele was found to be associated with a reduction of spine BMD by 0.1 SD (r = 0.05; β-coefficient, −0.05; SEE, 1.3; p = 0.04). The association between the TT genotype and reduced BMD remained significant when controlling for body weight and age (β-coefficient, −0.05; total r = 0.32; SEE, 1.20; p = 0.04). Smoking status did not modify the effect of MTHFR genotype on BMD.
MTHFR genotype and postmenopausal bone loss
No significant genotype effects on untreated bone loss rates after menopause were observed (Table 3). A trend toward lower absolute bone loss rates in the TT genotype was inconsistent across regions and did not reach statistical significance (CC vs. TT: spine, p = 0.28; whole body, p = 0.10). Controlling bone loss rates for the covariates shown in Table 1 did not affect the results. There was no significant effect of genotype on BMD at 5 years in the untreated group.
MTHFR genotype and response to HRT
For all genotypes, HRT was associated with more positive rates of change for BMD (p < 0.001) and higher BMD at 5 years (p < 0.01) compared with the untreated group. BMD changes at all sites during HRT were independent of MTHFR genotype (Table 3). The relationship between MTHFR genotype and BMD was maintained after 5 years of HRT. This was significant at the total hip (CC vs. TT; p < 0.05; mean difference, −0.3 SD) and whole body (−0.3 SD, p < 0.05), but not at the spine (−0.3 SD, p = 0.15). Significant genotype-by-HRT interaction in the prediction of BMD was not present (ΔF 0.93, p = 0.33, total population).
MTHFR genotype and biochemistry
MTHFR genotype was not associated with differences in serum 25-OH-vitamin D levels, or in the available bone formation- (osteocalcin and BAP) or resorption (urine hydroxyproline) markers. Plasma HCy levels were available(5) in 206 participants from the randomized arm at one center (Odense). Briefly, in untreated women, the TT genotype was associated with 53% higher (p = 0.07) median HCy levels than the CC and CT genotypes. In women receiving HRT, the corresponding difference was 47% (p < 0.05). HCy levels were not correlated with BMD or bone loss rates.
Forty-two women in the control group and 10 women receiving HRT sustained fractures (not including finger and toe fractures). Women with more than one fracture were only counted once. Nineteen percent of incident fractures in the control group occurred in women with the TT genotype. Thus, the observed rate of incident fracture cases in untreated participants was 1.0% per year in women with the CC genotype, 0.8% per year with the CT genotype, and 2.4% per year with the TT genotype (Table 4). The relative risk for fracture in women carrying the TT genotype was 2.6 (95% CI, 1.2-5.6; Fig. 1). When fracture-free survival was controlled for BMD in the Cox analysis, the TT genotype remained a significant indicator of increased fracture risk (RR, 2.4; 95% CI, 1.1-5.2). The MTHFR attributable risk at the individual level was 1.6% per year, and the attributable proportion at the population level was 11.5%, indicating that one in nine fractures could be explained by MTHFR genotype. Within the HRT group, there was no increased fracture risk in women with the TT genotype. Analysis of the impact of HCy levels on fracture risk was not feasible in this study because only two patients with fractures had HCy measurements.
This study showed that the association between MTHFR genotype and BMD, recently described in a cohort of postmenopausal Japanese women, is also present in postmenopausal European women. Fracture incidence was monitored in the first 5 years after the menopause, and a 2-fold higher fracture rate in women with the BMD-adverse genotype TT was observed. The impact of the BMD-adverse genotype seemed to consist of a lower menopausal BMD—which is likely to be an indicator of a lower peak bone mass—but not in accelerated early postmenopausal bone loss, as assessed by serial BMD measurements over 5 years after menopause. The effects of HRT on fracture incidence and BMD in the parent study have been reported previously.(9) The adverse genotype was less common in our population (8.7%) than in the Japanese cohort (18.6%).(3) An estimated 30% of the Danish population and 43% of the Japanese population carry this allelic polymorphism.
A link between homocysteine metabolism and BMD may seem puzzling at first. However, the relationship between collagen, BMD, and bone strength is well established, as is the relationship between HCy and collagen maturation. Indeed, defects in bone collagen synthesis underlie inherited diseases with strongly reduced BMD and bone strength such as osteogenesis imperfecta and Ehlers-Danloss syndrome. In the general population, a restriction fragment length polymorphism in the gene encoding the α-chain of type I collagen has been shown to influence BMD.(11–13) Collagen metabolism is affected by HCy, which interferes with cross-link formation in newly formed collagen.(4) Accordingly, patients with pathologically raised plasma HCy as part of homocystinuria exhibit skeletal abnormalities and low BMD.(14)
HCy can be metabolized through two pathways: re-methylation back to methionine and a transsulfuration of sulfur from HCy to serine, resulting in formation of cysteine. Conversion of HCy to cysteine only takes place in certain tissues and organs. It is a two-stage process that involves two vitamin B6-dependent reactions.(15) By contrast, most cells are able to re-methylate HCy to methionine through the action of vitamin B12-dependent methionine synthase. In this process, 5-methylene-tetrahydrofolate donates the methyl group, and the production of 5-methylenetetra-hydrofolate is catalyzed by MTHFR. Thus, severe deficiency in or absence of MTHFR leads to homocystinuria, whereas less pronounced enzyme defects are associated with mild-to-moderate hyperhomocysteinanemia. At least 18 rare mutations in MTHFR have been found in homocystinuria.(16,17) In addition, five common polymorphisms in MTHFR have been described.(18) Only one, the 677 C T (causing a substitution of valine for alanine) has been shown to affect Hcy levels. This genotype is associated with a thermolabile MTHFR variant, leading to moderately raised plasma total HCy levels when folate intake is suboptimal. This polymorphism is associated with low BMD in Japanese women,(3) and as demonstrated in the present study, in European women. Thus, a modest reduction in BMD of 0.2-0.3 SD was observed in subjects with the BMD-adverse genotype TT. The difference was statistically significant at the femoral neck, total hip, and—after adjusting for lifestyle covariates potentially exerting influence on BMD—the lumbar spine. Interestingly, this effect persisted after HRT, whereas it was attenuated in women who experienced estrogen unopposed postmenopausal bone loss. This was not because of any lack of response to HRT in the TT genotype; the response to HRT was independent of genotype. Instead, it seems that the MTHFR genotype exerts its influence chiefly on premenopausal bone mass and that the effect could be diminished once accelerated postmenopausal bone loss sets in.
The MTHFR genotype was not predictive of bone loss rates, but the study was not powered to rule out such an effect, as bone loss rates are subject to greater measurement error than BMD itself. While one may speculate that the TT genotype may not translate to increased risk of fracture late in life—a recent Danish case-control study indicates that it may even carry a reduced risk in the elderly(19)—fracture risk in the first years after menopause is substantially increased in women with the BMD-adverse genotype, and one of nine fractures in this age group can be attributed to this gene alone.
It should also be stressed that women with the TT genotype respond normally to HRT. No increased fracture risk with this genotype could be demonstrated in subjects in the HRT arm of the study, although the difference in BMD persisted. The ability of HRT to reduce the impact of the MTHFR genotype without abolishing the deficit in BMD suggests that the increased fracture risk may not be explained by BMD alone. In support of this, the impact of the MTHFR genotype on fracture risk in untreated women is only slightly reduced when the survival analysis is controlled for BMD. It is possible that HCy levels—which were not correlated with BMD but respond to HRT(5)—have direct influences on bone quality that are not fully mirrored by calcium content. Additional work is required to address the impact of the MTHFR genotype on BMD in men and on bone loss rates in older postmenopausal women, as well as the influence of folate levels and intake. With regard to the latter, our results point toward an effect of MTHFR levels on peak bone mass with early effects on fracture risk. Thus, assessing the relationship between genotype, folate intake, and BMD in childhood and young adulthood could provide additional valuable information. Studies with measurement of HCy levels on a larger scale and using more sensitive markers of bone resorption and formation than those available in the present study may be able to further illuminate the underlying mechanism. The availability of about 200 HCy measurements in the study population enabled us to confirm that the TT genotype was associated with higher HCy levels but not to assess relationships between HCy levels and fracture risk. In conclusion, women who are homozygous for the common allelic MTHFR (C677T) polymorphism (8.7% of the population) have lower BMD of the hip and spine at menopause and twice the risk of peripheral fractures in the first years after menopause.
The Danish Osteoporosis Prevention Study is financially supported by grants from the Karen Elise Jensen Foundation and from Novo Nordisk Farmaka (Lyngby, Denmark). The present investigation was supported by grants from the Danish Medical Research Council, the Institute of Clinical Research University of Southern Denmark, the Faculty of Health Sciences University of Southern Denmark, “danmark” Sundhedsfond, and Overlægerådets legat, Odense University Hospital. The authors are grateful to all technicians and secretarial staff who contributed to the study. Participating centers include the following: Aarhus University Hospital: Professor L. Mosekilde (center leader), Dr P. Charles. Hvidovre Hospital: Dr O.H. Sørensen (center leader). Hillerød Central Hospital: Dr S.Pors Nielsen (center leader). Odense University Hospital: Professor H. Beck-Nielsen (center leader).
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