Genes & Genomics (2012) 34: 257-263 DOI 10.1007/s13258-011-0202-2
RESEARCH ARTICLE
Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) polymorphisms with osteoporotic vertebral compression fracture (OVCF) in postmenopausal Korean women Young Sun Chung ․ Young Joo Jeon ․ Dong Eun Shin ․ Kyung Tae Min ․ Yu Shik Shin ․ Keun Soo Won ․ Young Cho Koh ․ Seung Ho Hong ․ Nam Keun Kim 1)
Received: 05 October 2011 / Accepted: 29 December 2011 / Published online: 31 May 2012 © The Genetics Society of Korea and Springer 2012
Abstract Hyperhomocysteinemia is associated with the risk of skeletal health problems, such as osteoporosis, low body mineral density, and fracture. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) are involved in homocysteine metabolism. We hypothesized that certain genetic polymorphisms of MTHFR and TS that cause altered enzyme activity may lead to hyperhomocysteinemia and affect bone metabolism. Therefore, we determined whether MTHFR 677C>T, MTHFR 1298A>C, TS enhancer region (TSER), and TS 3'-UTR 6 bp insertion/deletion polymorphisms are associated with osteoporotic vertebral compression fracture (OVCF) in postmenopausal Korean women. A total of 308 postmenopausal Korean women were enrolled as study subjects. Among them, 84 were patients with OVCF and 224 were controls. The polymorphisms were analysed by PCR-RFLP methods. Single mutations of MTHFR or TS were not associated with the occurrence of OVCF. However, the combined genotypes 2R3R+2R2R/0bp6bp+6bp6bp (TSER/TS 3'-UTR) and AC+CC/0bp6bp+6bp6bp (MTHFR 1298A>C/TS 3'-UTR) Y. S. Chung ․ Y. S. Shin ․ K. S. Won ․ Y. C. Koh Department of Neurosurgery, KonKuk University School of Medicine, Korea Y. J. Jeon ․ K. T. Min ․ N. K. Kim ( ) Institute for Clinical Research, CHA Bundang Medical Center, School of Medicine, CHA University, Seongnam, Korea e-mail:
[email protected] D. E. Shin Department of Orthopedic Surgery, CHA Bundang Medical Center, School of Medicine, CHA University, Seongnam, Korea S. H. Hong ( ) Department of Science Education, Teachers College, Jeju National University, Jeju, Korea e-mail:
[email protected]
were associated with decreased risk for OVCF. 2R-0bp and 2R-6bp haplotype frequencies of TS were significantly different between the cases and controls. In the present study, the combined genotype of TSER/TS 3'-UTR and MTHFR 1298A>C/TS 3'-UTR was associated with a decreased risk for OVCF in postmenopausal Korean women. However, due to the several limitations of our study including the moderately small sample size, our findings should be considered with caution and further research is needed to draw more definitive conclusions. Keywords Thymidylate synthase (TS); Osteoporotic vertebral compression fracture (OVCF); Methylenetetrahydrofolate reductase (MTHFR); Homocysteine (Hcy); Polymorphism
Introduction Hyperhomocysteinemia is an independent risk factor for osteoporotic fractures, silent brain infarction, recurrent pregnancy loss, and cancer (Nelen et al., 2000; Kim et al., 2003; van Meurs et al., 2004; Sato et al., 2005). Treatment with folate and vitamin B12, which lower homocysteine (Hcy) levels, markedly decreases the risk of hip fracture in stroke patients (Heijmans et al, 2003), suggesting that Hcy plays an important role in the development of osteoporotic fractures. Osteoporosis is a disease caused by several factors, including metabolic abnormality of Hcy/folate. Thymidylate synthase (TS) is one enzyme involved in Hcy/folate metabolism (Lieberman et al., 2001; Liu et al., 2002), as it is a critical enzyme in thymidylate and DNA synthesis. 5, 10-methylenetetrahydrofolate is required for this process (Mclean et al., 2004). TS polymorphisms influence the Hcy conversion to methionine because TS competes with 5, 10-methylenetetrahydrofolate reductase (MTHFR) for 5, 10-methylenetetrahydrofolate as a substrate (Trinh et al., 2002; Kim et al., 2006). However, osteoporosis is more strongly influenced by genetic factors than
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by environmental influences. Twin and pedigree studies have shown that genetic influences account for 50-80% of the interindividual variability of bone mineral density (BMD) in young adults (Dequecker et al., 1987; Pocock et al., 1987; Slemenda et al., 1991). The gene encoding human TS is located on chromosome 18p11.32 and consists of seven exons (Tischer et al., 1991). Three polymorphisms in the 3' and 5'-untranslated regions (UTRs) of the TS gene have been reported. These included a 28-bp variable number of tandem repeat (VNTR) in thymidylate synthase enhancer region (TSER) of the 5'-UTR (Marsh et al., 2001), and a 6-bp insertion(ins)/deletion(del) polymorphism in the 3'-UTR (Mandola et al., 2004). Polymorphism studies of osteoporosis have been performed in several populations with candidate genes, such as vitamin D receptor, estrogen receptor, calcitonin receptor, MTHFR, and peroxisome proliferators-activated receptor (Baldock et al., 2004; Ferrari et al., 2004). However, there are conflicting data for the risk of osteoporotic fracture among populations. No studies on the association of the TS polymorphisms and osteoporotic vertebral compression fracture (OVCF) in postmenopausal women have been reported. Therefore, we investigated the association of TS and MTHFR gene polymorphisms with OVCF patients in postmenopausal Korean women.
Materials and Methods Study subjects This was a case-control study. The case group consisted of 84 osteoporotic postmenopausal women (mean age ± SD, 69.74±9.69 years; age range, 46-89 years), defined by a t-score of -2.5 or lower and/or at least one non-traumatic fracture of the spine, recruited from March 2005 to December 2008 in the Neurosurgery and Orthopedic Surgery Departments at CHA Bundang Medical Center, CHA University of South Korea. The control group consisted of 224 postmenopausal women (mean age ± SD, 68.37±8.01 years; age range, 46-91 years) without disease or medications known to influence bone mass and bone turnover. BMD at the lumbar spine was measured using dual-energy X-ray (Hologic Discovery W, Waltham, MA, USA). Vertebral compression fracture (VCF) was defined as at least 15% or more reduction of the anterior, posterior, or central height of vertebra (Voormolen et al., 2006). All examinations were performed by a trained neurosurgeon and orthopedic surgeon, according to previously described methods (Villadsen et al., 2005). The study was approved by the Institutional Review Board (IRB) of CHA Bundang Medical Center, CHA University, and written informed consent was obtained from all study subjects. Measurements of Hcy levels Whole blood was collected into potassium EDTA tubes from
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individuals after an overnight fast, and the plasma was then separated by centrifugation. Plasma Hcy levels were determined as total Hcy by fluorescence polarization immunoassay (FPIA). Serum folate level was determined by competitive immunoassay (ACS:180; Bayer, Tarrytown, NY, USA). Genotyping of the MTHFR and TS polymorphisms Genomic DNA was extracted from anti-coagulated peripheral blood using the G-DEX blood extraction kit (Intron, Seongnam, South Korea). Polymorphism analysis was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods. The primers and PCR conditions for each polymorphism analysis have been previously described (Kim et al., 2006; Yim et al., 2005). For each of the MTHFR and TS polymorphisms, 20% of the PCR assays were randomly chosen for a second PCR assay followed by DNA sequencing to validate the RFLP findings. Sequencing was performed using an ABI3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). The concordance of the quality control samples was 100%. Statistical analysis Odd ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the relative risk associated with ischemic stroke and the TS genotypes. We used the Mann-Whitney test for continuous data (age and Hcy levels) between patients and controls. Logistic regression was performed using age. Statistical significance was accepted at the P < 0.05 level. Statistical analyses were performed with GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA, USA) and SNPAlyzeTM, version 5.10 (DYNACOM Co., Ltd., Yokohama, Japan). We used the false-positive report probability (FPRP) statistical tool to evaluate the significance of the MTHFR and TSER association using the method described by Wacholder et al. (2004).
Results Information on controls and patients is presented in Table 1. Hcy levels were not significantly different between controls and OVCF patients. Table 2 shows a comparison of genotype frequencies for the MTHFR and TS polymorphisms between the case and control groups. The overall genotype distributions in the case and control groups for each polymorphism were in Hardy-Weinberg equilibrium. There were no associations of genotype and allele frequencies between OVCF and control groups. The combined genotype frequencies of MTHFR and TS polymorphisms are presented in Table 3. Among the various combinations, 2R3R+2R2R/0bp6bp+6bp6bp of TSER/TS 3'-UTR (OR, 0.415; 95% CI, 0.188-0.914; P=0.029) and
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Table 1. Baseline characteristics of osteoporotic vertebral compression fracture (OVCF) patients and control subjects. Characteristics N Age (years) Homocysteine (μmol/L, n)
Controls 224 68.37±8.01 9.82±3.55 (224)
OVCF patients 84 69.74±9.69 10.08±3.77 (81)
P 0.120 0.602
Values are mean±S.D. P-values were calculated using the Mann-Whitney test for continuous data.
Table 2. Genotype frequencies of MTHFR 677C>T, 1298A>C, TSER, and TS 3'-UTR ins/del 6bp in OVCF patients and control subjects. Characteristics Controls (n=224) MTHFR 677C>T CC 76 (33.9) CT 108 (48.2) TT 40 (17.9) Dominant (CC vs. CT+TT) Recessive (CC+CT vs. TT) MTHFR 1298A>C AA 156 (69.6) AC 65 (29.0) CC 3 (1.3) Dominant (AA vs. AC+CC) Recessive (AA+AC vs. CC) TSER 3R3R 163 (72.8) 2R3R 58 (25.9) 2R2R 3 (1.4) Dominant (3R3R vs. 2R3R+2R2R) Recessive (3R3R+2R3R vs. 2R2R) TS 3'-UTR 0bp0bp 117 (52.2) 0bp6bp 89 (39.7) 6bp6bp 18 (8.0) Dominant (0bp0bp vs. 0bp6bp+6bp6bp) Recessive (0bp0bp+0bp6bp vs. 6bp6bp)
OVCF patients (n=84)
AOR (95% CI)
P*
25 (29.8) 44 (52.4) 15 (17.9)
1.000 1.196 1.137 1.175 1.012
(reference) (0.671 - 2.129) (0.539 - 2.398) (0.680 - 2.031) (0.525 - 1.953)
0.544 0.737 0.563 0.971
66 (78.6) 18 (21.4) 0 (0.0)
1.000 (reference) 0.651 (0.358 - 1.183) 0.622 (0.343 - 1.129) -
0.159 0.118 -
66 (78.6) 15 (17.9) 3 (3.6)
1.000 0.632 2.463 0.719 2.686
(reference) (0.334 - 1.196) (0.484 - 12.525) (0.394 - 1.311) (0.528 - 13.664)
0.158 0.277 0.282 0.234
54 (64.3) 28 (33.3) 2 (2.4)
1.000 0.672 0.238 0.606 0.301
(reference) (0.394 - 1.147) (0.053 - 1.076) (0.361 - 1.018) (0.068 - 1.338)
0.145 0.062 0.058 0.115
*Adjusted for age.
AC+CC/0bp6bp+6bp6bp of MTHFR 1298A>C/TS 3'-UTR (OR, 0.345; 95% CI, 0.133-0.892; P=0.028) were significantly decreased in OVCF patients. We constructed the haplotypes of the TS gene, listed in Table 4. 2R-0bp (OR, 2.806; 95% CI, 1.273-6.187; P=0.008) and 2R-6bp (OR, 0.389; 95% CI, 0.181-0.839; P=0.013) of the TS gene were significantly associated with OVCF risk. We evaluated the influence of TSER 2R3R+2R2R/TS 3'-UTR 0bp6bp+6bp6bp and MTHFR 1298AC+CC/TS 3'-UTR 0bp6bp+6bp6bp on plasma Hcy levels, because hyperhomocysteinemia is a risk factor of fracture (van Meurs et al., 2004). However, TSER 2R3R+2R2R/TS 3'-UTR 0bp6bp+6bp6bp and MTHFR 1298AC+CC/TS 3'-UTR 0bp6bp+6bp6bp were not associated with plasma Hcy levels (Table 5). We calculated false-positive probability values for the TSER 2R3R+2R2R/TS 3'-UTR 0bp6bp+6bp6bp and MTHFR 1298AC+CC/TS 3’-UTR 0bp6bp+6bp6bp combined genotypes
(Table 6). When the assumption of prior probability was 0.25 or 0.1, TSER 2R3R+2R2R/TS 3'-UTR 0bp6bp+6bp6bp had 85.2% and 65.7% true association, respectively. The false-positive probability (FPRP) approach advocated that these results were significant (FPRP < 0.4).
Discussion Osteoporosis is known to be influenced by environmental factors, such as life style or dietary habits. In spite of these facts, genetic factors may have a stronger influence on the occurrence of this disease. Polymorphisms of many genes, such as vitamin D receptor, estrogen receptor, vascular endothelial growth factor, and MTHFR are known to influence skeletal health (Baldock et al., 2004; Ferrari et al., 2004; Chung et al., 2010). TS is involved in Hcy/folate metabolism, and Hcy
260 Table 3. Combined genotype frequencies subjects. Characteristics MTHFR 677/1298 CC/AA CC/AC CC/CC CT/AA CT/AC TT/AA TSER/TS 3'-UTR 3R3R/0bp0bp 3R3R/0bp6bp+6bp6bp 2R3R+2R2R/0bp0bp 2R3R+2R2R/0bp6bp+6bp6bp MTHFR 677/TSER TT/3R3R TT/2R3R+2R2R CC+CT/3R3R CC+CT/2R3R+2R2R MTHFR 1298/TSER AA/3R3R AA/2R3R+2R2R AC+CC/3R3R AC+CC/2R3R+2R2R MTHFR 677/TS 3'-UTR TT/0bp0bp TT/0bp6bp+6bp6bp CC+CT/0bp0bp CC+CT/0bp6bp+6bp6bp MTHFR 1298/TS 3'-UTR AA/0bp0bp AA/0bp6bp+6bp6bp AC+CC/0bp0bp AC+CC/0bp6bp+6bp6bp
Genes & Genomics (2012) 34: 257-263 of MTHFR 677C>T, 1298A>C, TSER, and TS 3'-UTR ins/del 6bp in OVCF patients and control Controls (n=224)
OVCF patients (n=84)
AOR (95% CI)
P*
42 (18.8) 31 (13.8) 3 (1.3) 74 (33.0) 34 (15.2) 40 (17.9)
14 (16.7) 11 (13.1) 0 (0.0) 37 (44.0) 7 (8.3) 15 (17.9)
1.000 1.071 1.395 0.596 1.103
(reference) (0.428 - 2.681) (0.671 - 2.902) (0.213 - 1.667) (0.471 - 2.583)
0.884 0.373 0.324 0.822
108 (48.2) 55 (24.6) 9 (4.0) 52 (23.2)
45 (53.6) 21 (25.0) 9 (10.7) 9 (10.7)
1.000 0.918 2.507 0.415
(reference) (0.498 - 1.694) (0.918 - 6.841) (0.188 - 0.914)
0.785 0.073 0.029
27 (12.1) 13 (5.8) 136 (60.7) 48 (21.4)
11 (13.1) 4 (4.8) 55 (65.5) 14 (16.7)
1.000 0.658 0.999 0.645
(reference) (0.168 - 2.582) (0.461 - 2.163) (0.250 - 1.661)
0.549 0.997 0.363
112 (50.0) 44 (19.6) 51 (22.8) 17 (7.6)
53 (63.1) 13 (15.5) 13 (15.5) 5 (6.0)
1.000 0.595 0.539 0.624
(reference) (0.293 - 1.207) (0.270 - 1.077) (0.218 - 1.784)
0.150 0.080 0.379
21 19 96 88
(9.4) (8.5) (42.9) (39.3)
10 (11.9) 5 (6.0) 44 (52.4) 25 (29.8)
1.000 0.542 0.967 0.563
(reference) (0.156 - 1.885) (0.420 - 2.227) (0.230 - 1.379)
0.335 0.937 0.209
82 74 35 33
(36.6) (33.0) (15.6) (14.7)
42 (50.0) 24 (28.6) 12 (14.3) 6 (7.1)
1.000 0.641 0.666 0.345
(reference) (0.353 - 1.163) (0.313 - 1.417) (0.133 - 0.892)
0.144 0.292 0.028
*Adjusted for age.
Table 4. Haplotype frequencies of the TS gene in OVCF patients and control subjects. Haplotype Overall (n=308) Control (n=224) Case (n=84) OR (95% CI) 3R-0bp 0.7021 0.6909 0.7349 1.217 (0.819 - 1.808) 3R-6bp 0.1599 0.1662 0.1401 0.842 (0.511 - 1.387) 2R-6bp 0.0950 0.1128 0.0503 0.389 (0.181 - 0.839) 2R-0bp 0.0430 0.0301 0.0747 2.806 (1.273 - 6.187)
P* 0.331 0.500 0.013 0.008
P** 0.371 0.532 0.013 0.013
* Chi-square test. ** After 10,000-permutation test.
levels are associated with the risk of skeletal health problems, such as osteoporosis and fracture. However, no study of the association between TS gene polymorphisms and skeletal disorders has been reported. Thus, we investigated whether MTHFR and TS polymorphisms are associated with OVCF in postmenopausal Korean women. A number of studies have suggested an effect of the MTHFR 677C>T polymorphism in hyperhomocysteinemia. In our
study, the MTHFR 677C>T polymorphism was also associated with a change in plasma Hcy levels (data not shown). However, the occurrence of OVCF was not influenced by MTHFR 677TT, despite reports of an association with skeletal health. On the other hand, in combination with the TS 3’-UTR 6bp polymorphism, the frequency of MTHFR 1298A>C mutation was significantly different between controls and OVCF patients. Because the MTHFR 1298A>C mutation results in
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Table 5. Comparison of Hcy levels according to TSER/TS 3'-UTR or MTHFR 1298/TS 3'-UTR combined genotypes in the OVCF patients and control subjects. Characteristics Total (n=305) Controls (n=224) OVCF patients (n=81) P* TSER/TS 3'-UTR 3R3R/0bp0bp 10.02±3.72 10.03±3.88 10.00±3.34 0.800 3R3R/0bp6bp+6bp6bp 10.32±4.07 10.33±4.13 10.28±4.02 0.885 2R3R+2R2R/0bp0bp 9.65±4.43 8.69±2.01 10.62±5.97 0.546 2R3R+2R2R/0bp6bp+6bp6bp 9.08±2.01 9.03±1.91 9.41±2.70 0.803 P** 0.631 0.673 0.989 MTHFR 1298/TS 3'-UTR AA/0bp0bp 10.15±3.80 10.11±3.87 10.23±3.69 0.793 AA/0bp6bp+6bp6bp 10.02±3.58 9.87±3.60 10.52±3.55 0.433 AC+CC/0bp0bp 9.52±3.77 9.49±3.56 9.61±4.57 0.887 AC+CC/0bp6bp+6bp6bp 9.15±2.73 9.32±2.50 8.19±3.92 0.403 P** 0.283 0.565 0.429 * P-values between controls and OVCF patients were calculated using Mann-Whitney test. ** P-values among TSER/TS 3'-UTR or MTHFR 1298/TS 3'-UTR combined genotypes were calculated using Kruskal-Wallis test.
Table 6. False positive report probability (FPRP) values for associations between OVCF patients and control subjects. Prior probability* Characteristics AOR (95% CI) 0.25 0.1 0.01 0.001 0.0001 TSER/TS 3'-UTR 2R3R+2R2R/0bp6bp+6bp6bp 0.415 (0.188 - 0.914) 0.148 0.343 0.852 0.983 0.998 MTHFR 1298/TS 3'-UTR AC+CC/0bp6bp+6bp6bp 0.345 (0.133 - 0.892) 0.144 0.336 0.848 0.983 0.998
0.00001 1.000 1.000
* The most likely range of prior probabilities is presented in bold type for each polymorphism.
reduced MTHFR activity, this suggests a possible association for Hcy/folate metabolism with OVCF occurrence. The UTR of a gene is often important for the regulation of gene transcription or translation (Maeng et al., 1998; Goto et al., 2001; Mill et al., 2002; Moor et al., 2005). Regulatory roles of the polymorphisms in gene UTRs have been elucidated in several previous studies (Maeng et al., 1998; Moor et al., 2005). In this study, two polymorphisms of the TS gene are located in an UTR. The TSER polymorphism exists in the TS enhancer region of the 5'-UTR. It is classified into genotypes, such as double (2R) or triple (3R) according to the number of 28-bp repeats. The 3R-bearing genotype has a more active transcriptional or translational efficiency than the 2R (Horie et al., 1995; Trinh et al., 2002). Our data show that the 3R allele frequency is higher than that of the 2R allele. The 3R allele frequency was higher in the case group than in controls, although the difference was not significant. Thus, the 2R allele may be protective for OVCF in Koreans. The 6-bp ins/del polymorphism is located in the 3'-UTR, and the 6-bp allele was shown to result in longer mRNA stability (Kawakami et al., 1999; Marsh et al., 2001). Polymorphisms in the UTRs can contribute to differences in susceptibility to, and severity of a disease between individuals. The effect may be seen with polymorphism alone or in combination with other polymorphisms. Haplotype or combined genotype analyses, which are currently the focus of intense genetic research ef-
forts, will make risk estimates more specific than single locus analyses. In the present study, although the single mutations of TSER and TS 3'-UTR were not associated with the risk of OVCF, the combined TSER 2R3R+2R2R/TS 3'-UTR 0bp6bp+6bp6bp genotype and the 2R-6bp haplotype were associated with decreased risk for OVCF. The combined genotypes or haplotypes of TSER and 3'-UTR polymorphisms can influence the level of TS gene expression, suggesting that these polymorphic effects can be applied to susceptibility and diagnosis of OVCF. The high transcription levels of 3R and 6-bp alleles enhance TS enzyme activity, and the raised activity can increase Hcy levels due to the shortage of 5, 10-methlylenetetrahydrofolate. For example, the increased TS activity raised the levels of blood Hcy or the Hcy/folate ratio in healthy Chinese from Singapore (Ulrich et al., 2000; Saw et al., 2001). Kealey et al (2005) also reported that the 3'-UTR ins/del polymorphism of the TS gene is a determinant of red blood cell folate and Hcy levels in young subjects from Northern Ireland, while TSER polymorphism is not associated with Hcy levels in the same population (Brown et al., 2004). In the present study, the Hcy levels were not significantly different between the case and control groups. Similarly, when Hcy levels were divided by the lower 20th percentile or the upper 20th percentile, the frequencies of the MTHFR 1298A>C/TS 3'-UTR and TSER/TS 3'-UTR combined genotypes were not significantly
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different between the lower and upper 20th percentile groups (Supplemental Tables 1 and 2). The reason for this may be high levels of Hcy due to hormonal change in both controls and cases of postmenopausal women. Yim et al. (2005) reported, from a comparison of the TS polymorphism data in healthy populations obtained from the literature, that the Asian population shows a higher frequency of the 3R3R genotype than the 2R2R or 2R3R genotype in the TSER polymorphism, and of 0bp0bp rather than 6bp6bp in the ins/del polymorphism. This may explain ethnic variations in the effect of the TS polymorphisms among the various populations studied. On the other hand, the 2R2R, 2R3R, and 6bp6bp genotypes have relatively high frequencies in American Caucasian, Hispanic, and European populations. A possible interaction between the 5' and 3'-UTR polymorphisms is suggested by the 3R3R/6bp6bp frequency of the TS gene, according to ethnic groups, and 3R3R/6bp6bp may have an essential influence on elevated Hcy levels. Further study is required to investigate the effect of 3R3R/6bp6bp on Hcy metabolism in other ethnic populations, as well as in the Asian population. In conclusion, the combination of MTHFR 1298A>C/TS 3'-UTR and TSER/TS 3'-UTR was associated with a decreased risk for OVCF in postmenopausal Korean women. To our knowledge, this is the first report on the association between TS polymorphisms and OVCF in postmenopausal women. However, there are limitations of the present study. First, the study could not be performed with sufficient sample sizes due to the IRB approval. Second, to confirm our association data, molecular biological and biochemical studies, such as transcription and enzyme activity measurement should be performed on the TS variants. Furthermore, these data should be confirmed in other ethnic or racial populations with sufficient samples sizes. Acknowledgements This work was supported by Konkuk University in 2011.
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