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Association between osteoporosis and polymorphisms of the bone Gla protein, estrogen receptor 1, collagen 1-A1 and calcitonin receptor genes in Turkish postmenopausal women
Gene 515 (2013) 167–172
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Association between osteoporosis and polymorphisms of the bone Gla protein, estrogen receptor 1, collagen 1-A1 and calcitonin receptor genes in Turkish postmenopausal women Sengul Tural a,⁎, Nurten Kara a, Gamze Alayli b, Leman Tomak c a b c
Ondokuz Mayis University, Faculty of Medicine, Dept. of Medical Biology and Genetics, Samsun, Turkey Ondokuz Mayis University, Faculty of Medicine, Dept. of Physical Medicine and Rehabilitation, Samsun, Turkey Ondokuz Mayis University, Faculty of Medicine, Dept. of Biostatistics, Samsun, Turkey
a r t i c l e
i n f o
Article history: Accepted 10 October 2012 Available online 5 November 2012 Keywords: BGLAP ER1 COL1A1 CALCR Polymorphism Osteoporosis
a b s t r a c t In this study, we have investigated the association between osteoporosis and osteocalcin (BGLAP) −298 C>T, estrogen receptor 1 (ER1) 397 T>C, collagen type1 alpha 1 (Col1A1) 2046 G>T and calcitonin receptor (CALCR) 1340 T>C polymorphisms. Genomic DNA was obtained from 266 persons (158 osteoporotic and 108 healthy controls). Genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method and analyzed by PCR-RFLP. As a result, there was no statistically significant difference in the genotype and allele frequencies of patients and controls for BGLAP −298 C>T, Col1A1 2046 G>T, ER1 397 T>C and CALCR 1340 T>C polymorphisms. However, ER1 CC genotype compared with TT+ TC genotypes was found to increase the two fold the risk of osteoporosis [p=0.039, OR=2.156, 95% CI (1.083–4.293)] and CALCR CC genotype compared with TT+TC genotypes was found to have protective effect against osteoporosis [p=0.045, OR=0.471, 95% CI (0.237–0.9372)]. In the combined genotype analysis, ER1/CALCR TCCC combined genotype was estimated to have protective effect against osteoporosis [p=0.0125, OR=0.323, 95% CI (0.1383–0.755)] whereas BGLAP/Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were estimated as risk factors for osteoporosis in Turkish population (p=0.027, p =0.009 respectively). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis (OMIM166710) is a common skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue with increased susceptibility to fracture (Ralston and Uitterlinden, 2010). According to the International Osteoporosis Foundation (IOF), 30–50 % of women and 15–30 % of men will be afflicted in the course of their lives. It affects one in three postmenopausal women and the majority of the elderly. The incidence of osteoporotic fractures increases with age and it is higher in whites than blacks (Choi et al., 2012; Gennari et al., 2005; Lee et al., 2010; Ralston, 2010; Ralston and Uitterlinden, 2010).
Abbreviations: BGLAP, osteocalcin bone Gla protein; ER1, estrogen receptor 1; Col1A1, collagen type1 alpha 1; CALCR: CTR, calcitonin receptor; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; IOF, Osteoporosis Foundation; DEXA, dual energy X-ray absorptiometry; SNP, single nucleotide polymorphism; BMI, body mass index; WHO, World Health Organization; SD, standard deviation; Cis, confidence intervals; OP, osteoporosis. ⁎ Corresponding author at: Ondokuz Mayis University, Faculty of Medicine, Dept. of Medical Biology and Genetics, Turkey. Tel.: +90 5334925282/0362 3121919 3245. E-mail addresses: [email protected] (S. Tural), [email protected] (N. Kara), [email protected] (G. Alayli), [email protected] (L. Tomak). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.10.041
The major determinant of bone strength and osteoporotic fracture risk is bone mineral density (BMD), as assessed by dual photon absorptiometry or dual energy X-ray absorptiometry (DEXA). Osteoporosis is a multifactorial disorder with a strong genetic component. Twin and family studies suggest that about 50–85% of the variance in BMD is genetically determined (Ralston, 2010). There are several genes that play a role in the genetic determination of osteoporosis. In this regard, a large number of polymorphisms in multiple candidate genes have been investigated (Ralston, 2010). Osteocalcin (BGLAP) (also known as bone Gla protein, BGP) is one of the major noncollagenous proteins of bone (Chen et al., 2001). Noncollagenous proteins together with collagen, contribute to structural and mechanical properties of bone (Sroga and Vashishth, 2012). BGLAP also plays a role in bone resorption and remodeling (Wu et al., 2003). BGLAP gene −298 C>T polymorphic promoter region is important for the controlling expression of the BGLAP gene (containing osteocalcin box) (Wu et al., 2003). Various promoter elements lying less than a kilobase 5′ to the transcription initiation site contribute to basal expression and osteoblast specificity (Ivaska and Kaisa, 2005). −298 C>T polymorphism in the promoter region has been associated with osteoporosis in postmenopausal women (Chen et al., 2001). The other important and widely studied candidate gene for osteoporosis is estrogen receptor 1 (ER1). ER1 gene encodes ligand-activated
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transcription factor estrogen receptor alpha which belongs to nuclear receptor superfamily. Estrogen deficiency plays a major role in the pathogenesis of postmenopausal osteoporosis (Ivaska and Kaisa, 2005; Jeedigunta et al., 2010). The skeletal effects of estrogens are mediated by its binding to specific estrogen receptors. While many polymorphisms have been described in ESR1, the most widely studied are T397C and C351G polymorphisms located in the first intron. It is noted that the T>C transition associated with loss of the PvuII site results in a potential binding site for myb transcription factors. Thus, in some settings, the presence of the T allele might amplify ESR1 transcription (Gennari et al., 2005). Another important candidate gene for predisposition to osteoporosis is the collagen type1 alpha 1 (COL1A1) gene, which encodes the 1(I) protein chain of type I collagen. It makes up 90% of the organic matrix that has a role in bone mineralization and gives flexibility to the bone (Erdogan et al., 2010). It is a heterotrimer protein consisting of two α1 chains and one α2 chain of type 1 collagen. The Sp1 transcription factor binding site is located in the first intron of the COL1A1 gene, an important region for the regulation of collagen transcription (Hubacek et al., 2006). This is a single nucleotide polymorphism affecting the recognition site of the transcription factor Sp1. Presence of the T allele leads to abnormal production of the α-1collagen chain in comparison to the α-2 collagen chain, which has an adverse effect on bone composition and mechanical strength (Hubacek et al., 2006). The 1340 T>C polymorphism among human calcitonin receptor (CALCR) (also named CTR) gene polymorphism has generated interest precisely because of this single nucleotide polymorphism (SNP) in the coding region. It has been suggested that this locus modulates the susceptibility of postmenopausal women to osteoporotic phenotypes (Lee et al., 2010). CALCR is a member of the transmembrane receptor family and a point mutation polymorphism (1340 T>C) (codon 447) has been identified in the 3-region of the calcitonin receptor gene which included a Pro → Leu shift in the third intracellular domain of the protein (Masi and Brandi, 2007). This change may play a role in G-protein coupling and signal transduction (Wolfe et al., 2003). The absence of the proline residue could alter the secondary structure of the calcitonin receptor (Wolfe et al., 2003). The aim of this study is to investigate if there is an association between BGLAP − 298 C>T, ESR1 397 T>C, Col1A1 2046 G>T and CALCR 1340 T>C gene polymorphisms and osteoporosis risk in a Turkish population. 2. Materials and methods 2.1. Subjects 266 postmenopausal women with a mean age of 61.60 ±8.51 years were included in the study. Among them, a total of 158 had osteoporosis (T score b −2.5, mean age 63.65± 8.62 years) and 108 had normal BMD (T score>−1, mean age 58.81± 7.97 years). Subjects with a history of bone disease, metabolic or endocrine disorders such as hyperthyroidism and hyperparathyroidism, diabetes mellitus, liver disease,
renal disease, and medications known to affect bone metabolism (e.g., corticosteroids, anticonvulsants, and heparin sodium) were excluded. None of the women had a history of taking medicines for the treatment of osteoporosis, such as active vitamin D3, bisphosphonates, SERM, or calcium. All subjects were from the Black Sea Coastal region from Turkey. The subjects were interviewed using a standard questionnaire including demographics, body mass index (BMI), years since menopause, menarche age, menopause age and history. All the patients gave informed consent and the study was approved by the ethical committee of Ondokuz Mayıs University. 2.2. Bone mineral density measurements Dual-energy X-ray absorptiometry (Norland EXCELL, USA) was used to assess BMD. Left hip and posterior-anterior lumbar spine (L2–L3–L4) scans were performed with the patient lying supine on the imaging table using the protocols recommended by the manufacturer. Osteoporosis was defined according to the conventional World Health Organization (WHO) definition. 2.3. DNA extraction and determination of BGLAP, ER1, Col1A1 and CALCR genotypes Peripheral venous blood samples were obtained from all subjects, and genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method (Miller et al., 1988). Genotyping of BGLAP, ER1, Col1A1 and CALCR polymorphisms were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. PCR amplification was performed in thermal cycler (Techne Gradient, Cambridge, UK) and digestion of the PCR products was carried out with restriction enzymes. The primer sequences, restriction enzymes, and fragment lengths are given in Table 1. Osteocalcin and Col1A1 genes polymorphism identification were conducted by a slight modification based on the method of Chen et al. (2001) and Todhunter et al. (2005) respectively. ER1 and CALCR polymorphisms were determined by a slight modification of the polymerase chain reaction (PCR) described by Bandres et al. (2005). Amplification of the 253 bp fragment encompassing the BGLAP − 298 C>T polymorphic site was performed in 25 μl, 1 × PCR buffer containing 10 pmol of each primer (GmbH Biotech, Deutschland), 2 mM MgCl2, 200 μM of each dNTP (MBI, Fermentas, Lithuania), 200 ng DNA, and 1.5 U Taq polymerase (Promega, Madison, WI, USA). After initial denaturation at 94 °C for 8 min, amplification was performed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 60 s. Final extension was allowed to proceed at 72 °C for 10 min (Chen et al., 2001). Amplification of the 1361 bp fragment encompassing the ER1 397 T>C polymorphic site was performed in 25 μl, 1 × PCR buffer containing 20 pmol of each primer, 2 mM MgCl2, 200 μM of each dNTP, 200 ng DNA, and 1.5 U Taq polymerase (Promega). Following
Table 1 Single nucleotide polymorphisms (SNPs) investigated in this study and PCR primers for the genotyping of BGLAP, ER1, COL1A1 and CALCR. Gene and polymorphism
Region
BGLAP (−298C>T) ER1 (+397 T>C) Col1A1 (+2046 G>T) CALCR (+1377 T>C)
Promoter
rs1800247
Intron 1
rs2234693
Intron 1
rs1800012
Exon 13
rs1801197
a
F: forward primer, R: reverse primer.
rs number
Primer pairs
(F)5′CCGCAGCTCCCAACCACAATAAGCT-3′a (R)5′CAATAGGGCGAGGAGT-3′ (F)5′CTGCCACCCTATCTGTATCTTT-3 (R)5-ACCCTGGCGTCGATTATCTGA-3′ (F)5′CTGGACTATTTGCGGACTTTTTGG-3′ (R)5′GTCCAGCCCTCATCCTGGCC-3′ (F)5′AGGTCCAAACCACCGTGAAG-3′ (R)5′GCAGTGGGAGACTCCATTCC-3′
Method
Hind III based PCR-RFLP Pvu II based PCR-RFLP MscI/Bal I based PCR-RFLP Alu I based PCR-RFLP
Genotype Wild Ref
Heterozygote
Variant
CC
CT
TT
[5]
TT
TC
CC
[16]
GG
GT
TT
[15]
TT
TC
CC
[16]
S. Tural et al. / Gene 515 (2013) 167–172
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Fig. 1. Determination of BGLAP, ESR1, COL1A1 and CALCR gene polymorphisms by PCR-RFLP. (a) BGLAP genotypes (b) ER1 genotypes (c) COL1A1 genotypes (d) CALCR genotypes (M; puC19 DNA/MspI (HpaII). Marker for (a), (c) and (d), ΦX174 DNA/BsuRI (HaeIII) marker for (b).
initial denaturation at 94 °C for 10 min, amplification was performed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 64 °C for 60 s, and extension at 72 °C for 1.5 min. The reaction was terminated by final extension at 72 °C for 10 min (Bandres et al., 2005). Amplification of the 255 bp fragment encompassing the Col1A1 2046 G>T polymorphic site was performed in 25 μl, 1 × PCR buffer containing 20 pmol of each primer, 2 mM MgCl2, 200 μM of each dNTP, 200 ng DNA, and 1.25 U Taq polymerase (Promega) (Bandres et al., 2005). Following initial denaturation at 94 °C for 5 min, amplification was performed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 64 °C for 60 s, and extension at 72 °C for 1 min. The reaction was terminated by final extension at 72 °C for 7 min. Amplification of the 443 bp fragment encompassing the CALCR 2046 1340 T>C polymorphic site was performed in 25 μl, 1 × PCR buffer containing 20 pmol of each primer, 2 mM MgCl2, 200 μM of each dNTP, 200 ng DNA, and 1.25 U Taq polymerase (Promega) (Dean and Sullivan, 2010). Following initial denaturation at 94 °C for 10 min, amplification was performed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 45 s. The reaction was terminated by final extension at 72 °C for 7 min. The BGLAP, ER1 Col1A1 and CALCR gene PCR products were digested with HindIII, PvuII, and MscI/BalI restriction enzymes respectively at 37 °C for 16 h. PCR reaction products were separated on a 2–3% agarose gel and RFLP digested products were separated on 3% nu-micropore agarose gel stained with ethidium bromide (Fig. 1 ). In order to validate the accuracy and reproducibility of this method, each PCR reaction included internal controls for each
genotype. Second PCR was performed to confirm samples which results are not clear. Also, to confirm the accuracy of the genotyping, repeated analysis was performed on randomly selected samples. No discrepancies were found. 2.4. Statistical analysis Analysis of the data was performed using the computer software SPSS 13.0 (SPSS, Chicago, IL, USA) and OpenEpi Info software package program (Dean and Sullivan, 2010). Continuous data were given as mean ± SD (standard deviation) and median (min-max), categorical data were given as frequency (percent). The frequencies of the alleles and genotypes in patients and controls were compared with X 2 analysis. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated. The comparisons of two groups were performed by t test and Mann–Whitney test. P value smaller than 0.05 (two-tailed) was regarded as statistically significant. Power analysis was made by using Minitab 15.0 package program. 3. Results Clinical and laboratory findings of the patients and controls were given in Table 2. In power analysis, the power of ER1/CALCR CCTT genotype comparison between patients and controls was 82% in 95% confidence interval. The results of the genotype and allele frequencies of BGLAP − 298 C>T, ER1 397 T>C, Col1A1 2046 G>T and CALCR 1340 T>C for osteoporosis patients and control groups are presented in Table 3. The genotype frequencies of the BGLAP, Col1A1 and CALCR
Table 2 Clinical and laboratory findings. Characteristics
Patients (n = 158) (mean ± SD)
Median (min–max)
Controls (n = 108)
Median (min–max)
P value
BMI (kg/m2) Age at menopause (years) Years since menopause (years) Age at menarche (years) Number of birth (n) Serum calcium (mg/dl) Serum phosphorus (mg/dl) Serum PTH (pg/ml) Serum ALP (U/l)
28.04 ± 3.837 47.28 ± 5.527 15.37 ± 7.640 13.64 ± 1.529 3.74 ± 2.087 9.54 ± 0.550 3.99 ± 2.67 79.07 ± 53.91 189.02 ± 61.91
28.00 47.50 14.0 13.0 3.0 9.5 3.73 76.0 188.35
29.48 ± 4.622 46.76 ± 4.7243 9.67 ± 7.460 13.59 ± 1.309 2.63 ± 1.624 9.72 ± 0.4992 3.90 ± 0.69835 74.42 ± 28.583 188.43 ± 46.146
29 (22–39) 47 (33–59) 7 (1–26) 13 (11–17) 3 (0–6) 9.8 (8.5–11) 4 (2.07–5.51) 73.2 (31–178) 187.5 (97–313)
0.002 0.011 0.0002 0.856 0.003 0.022 0.047 0.560 0.991
The results that are statistically significant are typed in bold.
(19–39) (34–60) (2–35) (9–17) (0–10) (8.01–11.6) (2.2–26.20) (11.90–446) (2.34–410)
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Table 3 Comparison of genotype and allele frequencies of BGLAP, ER1, Col1A1 and CALCR gene polymorphisms between osteoporotic patients and controls. SNP
Genotype/allele
Patients (n = 158) (%)
Controls (n = 108) (%)
χ2
P value
BGLAP −298 (C>T) rs1800247
CC CT TT CC + CT:TT CC:TC + TT C T TT TC CC TT + TC:CC TT:TC + CC T C GG GT TT GG + GT:TT GG:GT + TT G T TT TC CC TT:TC + CC TT + TC:CC T C
87 (55.1) 51 (32.3) 20 (12.7) 138:20 87:71 225 (71.2) 91 (28.8) 30 (19) 92 (58.2) 36 (22.8) 122:36 30:128 152 (48.1) 164 (51.8) 71 (44.9) 61 (38.6) 26 (16.5) 132:26 71:87 203 (64.2) 113 (35.8) 53 (33.5) 88 (55.7) 17 (10.8) 53:105 141:17 194 (61.4) 122 (38.6)
51 (47.2) 45 (41.7) 12 (11.1) 96:12 51:57 147 (75) 69 (25) 24 (22.3) 71 (65.7) 13 (12) 95:13 24:84 119 (55) 97 (45) 48 (44.4) 49 (45.4) 11 (10.2) 97:11 48 :60 145 (67.1) 71 (32.9) 31 (28.7) 55 (50.9) 22 (20.4) 31:77 86:22 117 (54.2) 99 (45.8)
2.455
0.293
0.036 1.580 0.6042
0.850 0.209 0.437
4.944
0.084
4.242 0.239 2.59
0.039a 0.625 0.113
2.526
0.283
1.615 0.006 0.473
0.204 0.936 0.491
4.789
0.091
0.695 3.999 2.750
0.404 0.045 0.097
ER1 +397 (T>C) rs2234693
Col1A1 +2046 (G>T) rs1800012
CALCR +1340 (T>C) rs1801197
OR (95% CI)
1.159 (0.541–2.483) 0.730 (0.447–1.193) 0.8616 (0.5918–1.254)
2.156 (1.083–4.293) 1.219 (0.666–2.228) 0.755 (0.534–1.07)
1.737 (0.818–3.685) 0.980 (0.599–1.604) 1.137 (0.789–1.638)
1.254 (0.736–2.134) 0.471 (0.237–0.9372) 1.346 (0.948–1.910)
The results that are statistically significant are typed in bold. a Fisher exact test.
polymorphisms in the control group were in Hardy–Weinberg equilibrium. Only ER1 gene polymorphism was not in Hardy–Weinberg equilibrium. Our small sample size might be the reason of disequilibrium. Combined genotype analysis results were presented in Table 4. ER1 CC genotype compared with TT +TC genotypes was found to increase two fold the risk of osteoporosis [p=0.039, OR =2.156, 95% CI (1.083–4.293)]. CALCR CC genotype compared with TT +TC genotypes was found to have protective effect against osteoporosis [p =0,045, OR=0.471, 95% CI (0.237–0.9372)]. There was no statistically significant difference in the genotype and allele frequencies of patients and controls
for BGLAP −298 C>T (p=0.293 and p= 0.437, respectively) and Col1A1 2046 G>T polymorphisms (p= 0.283, p =0.491, respectively). In the combined genotype analysis, ER1/CALCR TCCC combined genotype was found to have protective effect against osteoporosis [p = 0.0125, OR = 0.323, 95% CI (0.1383–0.755)]. However, BGLAP/ Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were risk factors for osteoporosis (p = 0.027 and p = 0.009). ER1/CALCR TCCC combined genotype was estimated to have protective effect against osteoporosis (p = 0.0125). However, BGLAP/Col1A1 CCTT, ER1/CALCR TTTT combined genotypes were risk factors for osteoporosis (p =
Table 4 Comparison of combined genotypes of BGLAP, ER1, Col1A1 and CALCR gene polymorphisms between osteoporotic patients and controls. Genes
Combined genotypes
Patients (%)
Controls (%)
χ2
P value
OR (95% CI)
BGLAP /COL1A1 rs1800247/rs1800012
CCGG CCGT CCTT CTGG CTGT CTTT TTGG TTGT TTTT CCTT CCTC CCTT TCTT TCTC TCCC TTTT TTTC TTCC
40 (25.3) 33 (20.9) 14 (8.9) 22 (13.9) 22 (13.9) 7 (4.4) 9 (5.7) 6 (3.8) 5 (3.2) 19 (12) 12 (7.6) 5 (3.2) 26 (16.5) 57 (36.1) 9 (5.7) 8 (5.1) 19 (12) 3 (1.8) 158
27 (25.0) 22 (20.4) 2 (1.9) 15 (13.9) 24 (22.0) 6 (5.6) 6 (5.6) 3 (2.8) 3 (2.8) 3 (2.8) 8 (7.4) 2 (1.9) 21 (19.4) 33 (30.6) 17 (15.7) 7 (6.4) 14 (13)) 3 (2.8) 108
0.003 0.010
0.9350 0.9188 0.027a 0.8632 0.1110 0.8979 0.8245 0.4664a 0.8804b 0.009a 0.8573 0.0810 0.6427 0.3501 0.0125 0.8245 0.9365 0.9693
1.017 (0.578–1.788) 1.032 (0.5634–1.911)
BGLAP /CALCR rs2234693/rs1801197
Total The results that are statistically significant are typed in bold. a Fisher exact test. b Mid-P exact test.
0.029 2.536 0.016 0.049
0.0320 0.2152 0.8733 6.2440 0.0491 0.0014
1.003 0.566 0.788 1.027
(0.494–2.074) (2.988–1.073) (0.2486–2.566) (0.3504–3.187)
1.027 (0.405–2.604) 0.816 (0.4322–1.541) 1.283 (0.7606–2.163) 0.323 (0.1383–0.755) 0.769 (0.2706–2.189) 0.917 (0.4387–1.920)
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0.0139 and p = 0.009). There were no statistically significant differences in terms of BMI, years since menopause, tobacco smoking, menarche age or menopause age between the genotypes for each RFLP (data not shown). Additionally effect of combinations of three and four SNPs were analyzed and an association was not found (data not shown). 4. Discussion The results of the present study showed that polymorphic genotypes of BGLAP, ER1, Col1A1 and CALCR genes are not associated with osteoporosis in single form but associated in combined forms. In the present study, there was no statistically significant difference in the genotype and allele frequencies of patients and controls for BGLAP − 298 C>T, Col1A1 2046 G>T, ER1 397 T>C and CALCR 1340 T>C polymorphisms. However, ER1 CC genotype compared with TT + TC genotypes was found to increase two fold the risk of osteoporosis and CALCR CC genotype compared with TT + TC genotypes was found to have protective effect against osteoporosis. Osteocalcin is well known as a marker for differentiated mature osteoblasts and a determinant of the bone calcification process (Chen et al., 2001). BGLAP gene − 298C>T polymorphism in the promoter region has been found to be associated with osteoporosis in postmenopausal women (Dohi et al., 1998). In the present study, when we compared the genotype distribution and allele frequencies of patients and controls, we did not find a statistically significant difference, however in the combined genotype analysis, BGLAP/Col1A1 CCTT combined genotype was a risk factor for osteoporosis. The first research of BGLAP gene − 298C>T polymorphism and osteoporosis performed by Dohi et al. (1998) reported that C allele was associated with lower BMD in Japanese postmenopausal women. The following study in Taiwanese postmenopausal women supported these results (Chen et al., 2001). Another study was carried out in the Japanese women and significant association between lumbar spine, hip BMD and the BGLAP polymorphic genotypes was found only in postmenopausal women but not in pre-menopausal women (Yamada et al., 2002). In a recent study, McGuigan et al. (2010) (Yamada et al., 2002) did not find an association with 298C>T polymorphism and BMD in a large Swedish postmenopausal women population, but they observed a significant association with BMI. However a number of studies failed to detect a significant association (Jiang et al., 2007; Mo et al., 2004; Zhao et al., 2005). As seen in our combined analysis result, CC genotype not alone but together with COL1A1 TT genotype was a risk factor for osteoporosis. Our findings suggest that when certain genotypes of some genes act together, they influence each other and affect the person's osteoporosis risk. Estrogen functions as a potent regulator of peak bone mass and bone remodeling (Ivaska and Kaisa, 2005). ER1 gene 397 T>C (also named Pvu II polymorphism) is also associated with preeclampsia, endometriosis uterine fibroids and tardive dyskinesia in schizophrenia besides osteoporosis (Lai et al., 2002). The present study revealed that ER1 CC genotype was found to increase the risk of osteoporosis two fold when compared with TT + TC genotypes (p= 0.039). In the combined genotype analysis, ER1/CALCR TCCC combined genotype was estimated to have protective effects against osteoporosis and ER1/CALCR CCTT combined genotype was a risk factor for osteoporosis. As seen in our combined analysis result, ER1 CC genotype not alone but together with CALCR TT genotype was a risk factor for osteoporosis. Kobayashi et al. (1996) first demonstrated the association between 397 T>C polymorphism and BMD in Japanese women. A study conducted in Indian women indicated that average lumbar vertebra BMD value of TT genotypes was found to be higher than those of the CC genotype (Mitra et al., 2006). These findings are in agreement with our results. Our results are also in agreement with the findings of Erdogan et al.'s (2010) study which was carried out in 126 postmenopausal Turkish women and which revealed that average lumbar vertebra BMD value of women
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with TT genotype was significantly higher than that with CC genotype. Similarly, a recent study performed in a south Indian population from Andhra Pradesh declared that ER1 gene C allele was significantly higher in pre- and postmenopausal osteoporotics when compared to controls (Jeedigunta et al., 2010). A meta-analysis pooled the association between the ER1 polymorphisms and BMD in sixteen eligible studies involving 4,297 Chinese women and the data suggested that TT homozygotes had lower BMD than TC/CC genotype (Ralston, 2010). However, another meta-analysis (Ioannidis et al., 2002) including eight European centers did not find a significant association between ESR1 gene polymorphisms and BMD. Also there are several studies which revealed no association between 397 T>C polymorphism and osteoporosis (Bandres et al., 2005; Mitra et al., 2006). Col1A1 gene 2046 G>T (also named Sp1 polymorphism) has been associated with low BMD and an increased risk of osteoporotic fracture in several studies (Bandres et al., 2005; Hubacek et al., 2006). Col1A1 gene 2046 G>T polymorphism is also associated with osteosclerosis, Chron's disease and myocardial infarction (Speer et al., 2006). In our study, we have demonstrated that there were not statistically significant differences between the allele frequencies and genotype frequencies for Col1A1 gene 2046 G>T polymorphism. However BGLAP/Col1A1 CCTT combined genotype was a risk factor for osteoporosis. Grant et al. (1996) have first documented that unfavorable GT and TT genotypes are associated with low BMD and prevail in an osteoporotic fracture group compared to normal controls. In several studies, an association between osteoporosis and Col1A1 gene 2046 G>T polymorphism was not found (Long et al., 2004; Vidal et al., 2007). Our study was also similar to Hubacek et al.'s (2006) study in which there was no significant difference in terms of 2046 G>T polymorphism genotype frequency among women with normal, osteopenic, and osteoporotic bone mass in Caucasian women. Recently, a meta-analysis of 26 studies confirmed the association of the T allele of the 2046 G>T polymorphism with a modest reduction in BMD and a significant risk of osteoporotic fracture (Mann et al., 2001). COLIA1 TT genotype was associated with increased hip fracture risk in Caucasian women, and this association was independent of BMD and age (Nguyen et al., 2005). Bandres et al. (2005) described that the TT genotype was found to be overrepresented in Spanish women who had a personal history of fractures. Bustamante et al. (2007) reported that COL1A1-1997 G>T and 2046 G>T polymorphisms are associated with increased BMD for lumbar spine. Our results are in agreement with these results by our combined analysis. In contrast, a study carried out in post-menopausal Caucasian women from Canary Island showed that all patients with osteoporotic fractures carried the GG allele more frequently than TT homozygotic women (Navarro et al., 2007). Our results are also in agreement with Erdogan et al. (2010) who carried out a study in 126 postmenopausal Turkish women and revealed that there was no association between 2046 G>T polymorphism and osteoporosis. Ramirez reported that the frequency of T allele was significantly higher in Mexican women with OP than in women without OP (Drews et al., 2005). In a recent meta-analysis, for the 2046 G>T polymorphism, BMD values in TT homozygotes were lower at the spine and lower at the hip than GG homozygotes (Navarro et al., 2007). In the present study, BGLAP/Col1A1 CCTT combined genotype was a risk factor for osteoporosis. As seen in our combined analysis result, TT genotype not alone but together with BGLAP gene CC genotype was a risk factor for osteoporosis. If our study population was larger this CC genotype or C allele might be significant for osteoporosis. Calcitonin receptor gene is one of the most important candidate genes for predisposition to osteoporosis. Masi and colleagues first identified an association between CALCR genotype and BMD in a study of Italian women in 1998 and Tsai et al. (2003) reported that women with genotype TT had a greater risk for developing osteoporosis at the lumbar spine and at the femoral neck and this polymorphism is associated with reduced bone mineral density and predisposes women to osteoporosis. Zofkova et al. (2003) suggested that this polymorphism
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is associated with bone mass at the femoral neck in a cohort of postmenopausal women, the lower values being found in carriers of C allele compared to subjects with the TT genotype. Similarly Bandres et al. (2005) studied 177 postmenopausal Spanish women and revealed that women with the CC genotype had a lower adjusted BMD at the femoral neck. Drews et al. (2005) studied 139 postmenopausal Polish women and they reported T-score was higher in CT genotype carriers. In the present study, CALCR CC genotype compared with TT + TC genotypes was found to be protective against osteoporosis. In the combined genotype analysis, ER1/CALCR TCCC combined genotype was found to have a protective effect against osteoporosis. However, BGLAP/Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were risk factors for osteoporosis. In Turkish population TT genotype might be significant for osteoporosis but our sample size might not be big enough to detect the difference. The effect of an individual SNP is generally small, and the genetic effect of combinations of functionally relevant SNPs may additively or synergistically contribute to increased OP risk. For this reason the analysis of combined genotypes of different SNPs is more helpful in the identification of predisposing of complex diseases. To the best of our knowledge, this is the first study to simultaneously examine the possible relationship between polymorphism of BGLAP − 298 C>T, ER1 397 T>C, Col1A1 2046 G>T and CALCR 1340 T>C gene polymorphisms and osteoporosis and also to investigate the effect of combined genotype in the same subjects. The limitation of our study is the sample size that is relatively small because none of the women had a history of taking medicines for the treatment of osteoporosis. In conclusion, the polymorphic genotypes of BGLAP, ER1, Col1A1 and CALCR are not found to be associated with osteoporosis in a single form but found to be associated in combined forms. By enlarging the study we hope that more explanatory and definitive results can be obtained. And further studies are also necessary in different populations. Conflicts of interest None. Acknowledgments This study was presented as poster in European Human Genetics Conference Amsterdam RAI, The Netherlands, May 28–31, 2011. Additionally, this study was completely supported by Ondokuz Mayis University (Project No. T-593). References Bandres, E., Pombo, I., Gonzalez-Huarriz, M., Rebollo, A., Lopez, G., Garcia, Foncillas J., 2005. Association between bone mineral density and polymorphism of the VDR, ERalpha, COL1A1 and CTR genes in Spanish postmenopausal women. J. Endocrinol. Invest. 28 (4), 312–321. Bustamante, M., et al., 2007. COL1A1, ESR1, VDR and TGFB1 polymorphisms and haplotypes in relation to BMD in Spanish postmenopausal women. Osteoporos. Int. 18, 235–243. Chen, H., Tsai, H., Chen, W., Wu, J., Tsai, F., Tsai, C., 2001. Relation of polymorphism in the promoter region for the human osteocalcin gene to bone mineral density occurrence of osteoporosis in postmenopausal Chinese women in Taiwan. J. Clin. Lab. Anal. 15, 251–255. Choi, Y.J., Oh, H.J., Kim, D.J., Lee, Y., Chung, Y.S., 2012. The prevalence of osteoporosis in Korean adults aged 50 years or older and the higher diagnosis rates in women who were beneficiaries of a national screening program: The Korea National Health and Nutrition Examination Survey 2008–2009. J. Bone Miner. Res. http://dx.doi.org/ 10.1002/jbmr.1635 (Epub ahead of print). Dean, A.G., Sullivan, K.M., Soe, M.M., OpenEpi, . Open Source Epidemiologic Statistics for Public Health, Version 2.3.1www.OpenEpi.com (updated 2010/19/09, accessed 2011/02/21). Dohi, Y., et al., 1998. A novel polymorphism in the promoter region for human osteocalcin gene: the possibility of a correlation with bone mineral density in postmenopausal Japanese women. J. Bone Miner. Res. 13, 1633–1639.
Drews, K., et al., 2005. Genetic polymorphism of the calcitonin receptor gene and bone mineral density in Polish population of postmenopausal women. Ginekol. Pol. 76 (8), 612–618. Erdogan, M.O., et al., 2010. Association of estrogen receptor alpha and collagen type I alpha 1 gene polymorphisms with bone mineral density in postmenopausal women. Osteoporos. Int. http://dx.doi.org/10.1007/s00198-010-1312-4. Gennari, L., et al., 2005. Estrogen receptor gene polymorphisms and the genetics of osteoporosis. A HuGE review. Am. J. Epidemiol. 161 (4), 307–320. Grant, S.F.A., et al., 1996. Reduced bone density and osteoporosis associated with a polymorphic sp1 site in the collagen type I alpha 1 gene. Nat. Genet. 14, 203–205. Hubacek, J.A., Weichetova, M., Bohuslavova, R., Skodova, Z., Adamkova, V., 2006. Genetic polymorphisms of TGF-beta, PAI-1, and COL1A-1, and determination of bone mineral density in Caucasian females. J.Endocrinol. Regul. 40 (3), 77–81. Ioannidis, J.P., et al., 2002. Association of polymorphisms of the estrogen receptor alpha gene with bone mineral density and fracture risk in women: a meta-analysis. J. Bone Miner. Res. 17, 2048–2060. Ivaska, S., Kaisa, A., 2005. Novel insights into the use of osteocalcin as a determinant of bone metabolism. Osteocalcin . (Turku, Finland). Jeedigunta, Y., et al., 2010. Association of estrogen receptor α gene polymorphisms with BMD and their affect on estradiol levels in pre- and postmenopausal women in south Indian population from Andhra Pradesh. Clin. Chim. Acta 411, 597–600. Jiang, D.K., et al., 2007. No evidence of association of the osteocalcin gene HindIII polymorphism with bone mineral density in Chinese women. J. Musculoskelet. Neuronal Interact. 7 (2), 149–154 (37). Kobayashi, S., et al., 1996. Association of bone mineral density with polymorphism of the estrogen receptor gene in post-menopausal women. J. Bone Miner. Res. 11, 306–311. Lai, I.C., et al., 2002. Association study of the estrogen receptor polymorphisms with tardive dyskinesia in schizophrenia. Neuropsychobiology 46 (4), 173–175. Lee, H.J., et al., 2010. Fracture, bone mineral density, and the effects of calcitonin receptor gene in postmenopausal Koreans. Osteoporos. Int. 21, 1351–1360. Long, J.R., et al., 2004. Association between COL1A1 gene polymorphisms and bone size in Caucasians. Eur. J. Hum. Genet. 12, 383–388. Mann, V., et al., 2001. COL1A1Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J. Clin. Invest. 107 (pp.). Masi, L., Brandi, M.L., 2007. Calcitonin and calcitonin receptors. Clin. Cases Miner. Bone Metab. 4 (2), 117–122. McGuigan, F., Kumar, J., Ivaska, K., Obrant, K., 2010. Osteocalcin gene polymorphisms influence concentration of serum osteocalcin and enhance fracture identification. J. Bone Miner. Res. 25, 1392–1399. Miller, S.A., Dykes, D.D., Polesky, H.H., 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16 (3), 1215. Mitra, S., Desai, M., Khatkhatay, M.I., 2006. Association of estrogen receptor a gene polymorphisms with bone mineral density in postmenopausal Indian women. Mol. Genet. Metab. 87 (1), 80–87 (899–907). Mo, X.Y., et al., 2004. Lack of association between the HindIII RFLP of the osteocalcin (BGP) gene and bone mineral density (BMD) in healthy pre- and postmenopausal Chinese women. J. Bone Miner. Metab. 22, 264–269. Navarro, M.C., Sosa, M., Pino-Montes, J., Torres, A., Salido, E., Saavedra, P., Corral-Gudino, L, Montilla, CA, 2007. Collagen type 1 (COL1A1) Sp1 binding site polymorphism is associated with osteoporotic fractures but not with bone density in post-menopausal women from the Canary Islands: a preliminary study. Aging Clin. Exp. Res. 19 (1), 4–9. Nguyen, T.V., et al., 2005. Contribution of the collagen I 1 and vitamin D receptor genes to the risk of hip fracture in elderly women. J. Clin. Endocrinol. Metab. 90 (12), 6575–6579. Ralston, S.H., 2010. Genetics of osteoporosis. Ann. N. Y. Acad. Sci. 1192, 181–189. Ralston, S.H., Uitterlinden, A.G., 2010. Genetics of osteoporosis. Endocrinol. Rev. 31 (5), 629–662. Speer, G., et al., 2006. Myocardial infarction is associated with Spl binding site polymorphism of collagen type 1A1 gene. Acta Cardiol. 61 (3), 321–325. Sroga, G.E., Vashishth, D., 2012. Effects of bone matrix proteins on fracture and fragility in osteoporosis. Curr. Osteoporos. Rep. (Epub ahead of print). Todhunter, C.E., et al., 2005. Influence of IL-6, COL1A1, and VDR gene polymorphisms on bone mineral density in Crohn's disease. Gut 54 (11), 1579–1584. Tsai, F.J., Chen, H.Y., Tsai, C.H., 2003. The AluI calcitonin receptor gene polymorphism (TT) is associated with low bone mineral density and susceptibility to osteoporosis postmenopausal women. Gynecol. Obstet. Invest. 5, 82–87. Vidal, C., Brincat, M., Xuereb-Anastasi, A., 2007. Effects of SNPs in the COL1A1 and methylenetetrahyhdrofolate reductase genes on BMD in postmenopausal women in Malta. Balkan J. Med. Genet. 10 (1), 9–18. Wolfe III, L.A., et al., 2003. In vitro characterization of a human calcitonin receptor gene polymorphism. Mutat. Res. 522, 93–105. Wu, H.C., Lin, C.C., Chen, W.C., Chen, H.Y., Tsai, F.J., 2003. Osteocalcin gene HindIII C/T polymorphism is a biomarker for prostate cancer and responsiveness to hormone therapy. Eur. Urol. 43 (2), 197–200. Yamada, Y., Ando, F., Niino, N., Shimokata, H., 2002. Association of polymorphism of interleukin-6, osteocalcin, and vitamin D receptor genes, alone or in combination, with bone mineral density in community dwelling Japanese women and men. J. Clin. Endocrinol. Metab. 88 (7), 3372–3378. Zhao, H.Y., et al., 2005. Study of the impact of candidate genes on bone mineral density in postmenopausal women. Zhonghua Fu Chan Ke Za Zhi 40 (12), 803–807. Zofkova, I., Zajıckova, K., Hill, M., Krepelova, A., 2003. Does polymorphism C1377T of the calcitonin receptor gene determine bone mineral density in postmenopausal women. Exp. Clin. Endocrinol. Diabetes 111, 447–449.
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Association between osteoporosis and polymorphisms of the bone Gla protein, estrogen receptor 1, collagen 1-A1 and calcitonin receptor genes in Turkish postmenopausal women Sengul Tural a,⁎, Nurten Kara a, Gamze Alayli b, Leman Tomak c a b c
Ondokuz Mayis University, Faculty of Medicine, Dept. of Medical Biology and Genetics, Samsun, Turkey Ondokuz Mayis University, Faculty of Medicine, Dept. of Physical Medicine and Rehabilitation, Samsun, Turkey Ondokuz Mayis University, Faculty of Medicine, Dept. of Biostatistics, Samsun, Turkey
a r t i c l e
i n f o
Article history: Accepted 10 October 2012 Available online 5 November 2012 Keywords: BGLAP ER1 COL1A1 CALCR Polymorphism Osteoporosis
a b s t r a c t In this study, we have investigated the association between osteoporosis and osteocalcin (BGLAP) −298 C>T, estrogen receptor 1 (ER1) 397 T>C, collagen type1 alpha 1 (Col1A1) 2046 G>T and calcitonin receptor (CALCR) 1340 T>C polymorphisms. Genomic DNA was obtained from 266 persons (158 osteoporotic and 108 healthy controls). Genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method and analyzed by PCR-RFLP. As a result, there was no statistically significant difference in the genotype and allele frequencies of patients and controls for BGLAP −298 C>T, Col1A1 2046 G>T, ER1 397 T>C and CALCR 1340 T>C polymorphisms. However, ER1 CC genotype compared with TT+ TC genotypes was found to increase the two fold the risk of osteoporosis [p=0.039, OR=2.156, 95% CI (1.083–4.293)] and CALCR CC genotype compared with TT+TC genotypes was found to have protective effect against osteoporosis [p=0.045, OR=0.471, 95% CI (0.237–0.9372)]. In the combined genotype analysis, ER1/CALCR TCCC combined genotype was estimated to have protective effect against osteoporosis [p=0.0125, OR=0.323, 95% CI (0.1383–0.755)] whereas BGLAP/Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were estimated as risk factors for osteoporosis in Turkish population (p=0.027, p =0.009 respectively). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis (OMIM166710) is a common skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue with increased susceptibility to fracture (Ralston and Uitterlinden, 2010). According to the International Osteoporosis Foundation (IOF), 30–50 % of women and 15–30 % of men will be afflicted in the course of their lives. It affects one in three postmenopausal women and the majority of the elderly. The incidence of osteoporotic fractures increases with age and it is higher in whites than blacks (Choi et al., 2012; Gennari et al., 2005; Lee et al., 2010; Ralston, 2010; Ralston and Uitterlinden, 2010).
Abbreviations: BGLAP, osteocalcin bone Gla protein; ER1, estrogen receptor 1; Col1A1, collagen type1 alpha 1; CALCR: CTR, calcitonin receptor; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; IOF, Osteoporosis Foundation; DEXA, dual energy X-ray absorptiometry; SNP, single nucleotide polymorphism; BMI, body mass index; WHO, World Health Organization; SD, standard deviation; Cis, confidence intervals; OP, osteoporosis. ⁎ Corresponding author at: Ondokuz Mayis University, Faculty of Medicine, Dept. of Medical Biology and Genetics, Turkey. Tel.: +90 5334925282/0362 3121919 3245. E-mail addresses: [email protected] (S. Tural), [email protected] (N. Kara), [email protected] (G. Alayli), [email protected] (L. Tomak). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.10.041
The major determinant of bone strength and osteoporotic fracture risk is bone mineral density (BMD), as assessed by dual photon absorptiometry or dual energy X-ray absorptiometry (DEXA). Osteoporosis is a multifactorial disorder with a strong genetic component. Twin and family studies suggest that about 50–85% of the variance in BMD is genetically determined (Ralston, 2010). There are several genes that play a role in the genetic determination of osteoporosis. In this regard, a large number of polymorphisms in multiple candidate genes have been investigated (Ralston, 2010). Osteocalcin (BGLAP) (also known as bone Gla protein, BGP) is one of the major noncollagenous proteins of bone (Chen et al., 2001). Noncollagenous proteins together with collagen, contribute to structural and mechanical properties of bone (Sroga and Vashishth, 2012). BGLAP also plays a role in bone resorption and remodeling (Wu et al., 2003). BGLAP gene −298 C>T polymorphic promoter region is important for the controlling expression of the BGLAP gene (containing osteocalcin box) (Wu et al., 2003). Various promoter elements lying less than a kilobase 5′ to the transcription initiation site contribute to basal expression and osteoblast specificity (Ivaska and Kaisa, 2005). −298 C>T polymorphism in the promoter region has been associated with osteoporosis in postmenopausal women (Chen et al., 2001). The other important and widely studied candidate gene for osteoporosis is estrogen receptor 1 (ER1). ER1 gene encodes ligand-activated
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transcription factor estrogen receptor alpha which belongs to nuclear receptor superfamily. Estrogen deficiency plays a major role in the pathogenesis of postmenopausal osteoporosis (Ivaska and Kaisa, 2005; Jeedigunta et al., 2010). The skeletal effects of estrogens are mediated by its binding to specific estrogen receptors. While many polymorphisms have been described in ESR1, the most widely studied are T397C and C351G polymorphisms located in the first intron. It is noted that the T>C transition associated with loss of the PvuII site results in a potential binding site for myb transcription factors. Thus, in some settings, the presence of the T allele might amplify ESR1 transcription (Gennari et al., 2005). Another important candidate gene for predisposition to osteoporosis is the collagen type1 alpha 1 (COL1A1) gene, which encodes the 1(I) protein chain of type I collagen. It makes up 90% of the organic matrix that has a role in bone mineralization and gives flexibility to the bone (Erdogan et al., 2010). It is a heterotrimer protein consisting of two α1 chains and one α2 chain of type 1 collagen. The Sp1 transcription factor binding site is located in the first intron of the COL1A1 gene, an important region for the regulation of collagen transcription (Hubacek et al., 2006). This is a single nucleotide polymorphism affecting the recognition site of the transcription factor Sp1. Presence of the T allele leads to abnormal production of the α-1collagen chain in comparison to the α-2 collagen chain, which has an adverse effect on bone composition and mechanical strength (Hubacek et al., 2006). The 1340 T>C polymorphism among human calcitonin receptor (CALCR) (also named CTR) gene polymorphism has generated interest precisely because of this single nucleotide polymorphism (SNP) in the coding region. It has been suggested that this locus modulates the susceptibility of postmenopausal women to osteoporotic phenotypes (Lee et al., 2010). CALCR is a member of the transmembrane receptor family and a point mutation polymorphism (1340 T>C) (codon 447) has been identified in the 3-region of the calcitonin receptor gene which included a Pro → Leu shift in the third intracellular domain of the protein (Masi and Brandi, 2007). This change may play a role in G-protein coupling and signal transduction (Wolfe et al., 2003). The absence of the proline residue could alter the secondary structure of the calcitonin receptor (Wolfe et al., 2003). The aim of this study is to investigate if there is an association between BGLAP − 298 C>T, ESR1 397 T>C, Col1A1 2046 G>T and CALCR 1340 T>C gene polymorphisms and osteoporosis risk in a Turkish population. 2. Materials and methods 2.1. Subjects 266 postmenopausal women with a mean age of 61.60 ±8.51 years were included in the study. Among them, a total of 158 had osteoporosis (T score b −2.5, mean age 63.65± 8.62 years) and 108 had normal BMD (T score>−1, mean age 58.81± 7.97 years). Subjects with a history of bone disease, metabolic or endocrine disorders such as hyperthyroidism and hyperparathyroidism, diabetes mellitus, liver disease,
renal disease, and medications known to affect bone metabolism (e.g., corticosteroids, anticonvulsants, and heparin sodium) were excluded. None of the women had a history of taking medicines for the treatment of osteoporosis, such as active vitamin D3, bisphosphonates, SERM, or calcium. All subjects were from the Black Sea Coastal region from Turkey. The subjects were interviewed using a standard questionnaire including demographics, body mass index (BMI), years since menopause, menarche age, menopause age and history. All the patients gave informed consent and the study was approved by the ethical committee of Ondokuz Mayıs University. 2.2. Bone mineral density measurements Dual-energy X-ray absorptiometry (Norland EXCELL, USA) was used to assess BMD. Left hip and posterior-anterior lumbar spine (L2–L3–L4) scans were performed with the patient lying supine on the imaging table using the protocols recommended by the manufacturer. Osteoporosis was defined according to the conventional World Health Organization (WHO) definition. 2.3. DNA extraction and determination of BGLAP, ER1, Col1A1 and CALCR genotypes Peripheral venous blood samples were obtained from all subjects, and genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method (Miller et al., 1988). Genotyping of BGLAP, ER1, Col1A1 and CALCR polymorphisms were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. PCR amplification was performed in thermal cycler (Techne Gradient, Cambridge, UK) and digestion of the PCR products was carried out with restriction enzymes. The primer sequences, restriction enzymes, and fragment lengths are given in Table 1. Osteocalcin and Col1A1 genes polymorphism identification were conducted by a slight modification based on the method of Chen et al. (2001) and Todhunter et al. (2005) respectively. ER1 and CALCR polymorphisms were determined by a slight modification of the polymerase chain reaction (PCR) described by Bandres et al. (2005). Amplification of the 253 bp fragment encompassing the BGLAP − 298 C>T polymorphic site was performed in 25 μl, 1 × PCR buffer containing 10 pmol of each primer (GmbH Biotech, Deutschland), 2 mM MgCl2, 200 μM of each dNTP (MBI, Fermentas, Lithuania), 200 ng DNA, and 1.5 U Taq polymerase (Promega, Madison, WI, USA). After initial denaturation at 94 °C for 8 min, amplification was performed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 60 s. Final extension was allowed to proceed at 72 °C for 10 min (Chen et al., 2001). Amplification of the 1361 bp fragment encompassing the ER1 397 T>C polymorphic site was performed in 25 μl, 1 × PCR buffer containing 20 pmol of each primer, 2 mM MgCl2, 200 μM of each dNTP, 200 ng DNA, and 1.5 U Taq polymerase (Promega). Following
Table 1 Single nucleotide polymorphisms (SNPs) investigated in this study and PCR primers for the genotyping of BGLAP, ER1, COL1A1 and CALCR. Gene and polymorphism
Region
BGLAP (−298C>T) ER1 (+397 T>C) Col1A1 (+2046 G>T) CALCR (+1377 T>C)
Promoter
rs1800247
Intron 1
rs2234693
Intron 1
rs1800012
Exon 13
rs1801197
a
F: forward primer, R: reverse primer.
rs number
Primer pairs
(F)5′CCGCAGCTCCCAACCACAATAAGCT-3′a (R)5′CAATAGGGCGAGGAGT-3′ (F)5′CTGCCACCCTATCTGTATCTTT-3 (R)5-ACCCTGGCGTCGATTATCTGA-3′ (F)5′CTGGACTATTTGCGGACTTTTTGG-3′ (R)5′GTCCAGCCCTCATCCTGGCC-3′ (F)5′AGGTCCAAACCACCGTGAAG-3′ (R)5′GCAGTGGGAGACTCCATTCC-3′
Method
Hind III based PCR-RFLP Pvu II based PCR-RFLP MscI/Bal I based PCR-RFLP Alu I based PCR-RFLP
Genotype Wild Ref
Heterozygote
Variant
CC
CT
TT
[5]
TT
TC
CC
[16]
GG
GT
TT
[15]
TT
TC
CC
[16]
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Fig. 1. Determination of BGLAP, ESR1, COL1A1 and CALCR gene polymorphisms by PCR-RFLP. (a) BGLAP genotypes (b) ER1 genotypes (c) COL1A1 genotypes (d) CALCR genotypes (M; puC19 DNA/MspI (HpaII). Marker for (a), (c) and (d), ΦX174 DNA/BsuRI (HaeIII) marker for (b).
initial denaturation at 94 °C for 10 min, amplification was performed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 64 °C for 60 s, and extension at 72 °C for 1.5 min. The reaction was terminated by final extension at 72 °C for 10 min (Bandres et al., 2005). Amplification of the 255 bp fragment encompassing the Col1A1 2046 G>T polymorphic site was performed in 25 μl, 1 × PCR buffer containing 20 pmol of each primer, 2 mM MgCl2, 200 μM of each dNTP, 200 ng DNA, and 1.25 U Taq polymerase (Promega) (Bandres et al., 2005). Following initial denaturation at 94 °C for 5 min, amplification was performed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 64 °C for 60 s, and extension at 72 °C for 1 min. The reaction was terminated by final extension at 72 °C for 7 min. Amplification of the 443 bp fragment encompassing the CALCR 2046 1340 T>C polymorphic site was performed in 25 μl, 1 × PCR buffer containing 20 pmol of each primer, 2 mM MgCl2, 200 μM of each dNTP, 200 ng DNA, and 1.25 U Taq polymerase (Promega) (Dean and Sullivan, 2010). Following initial denaturation at 94 °C for 10 min, amplification was performed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 45 s. The reaction was terminated by final extension at 72 °C for 7 min. The BGLAP, ER1 Col1A1 and CALCR gene PCR products were digested with HindIII, PvuII, and MscI/BalI restriction enzymes respectively at 37 °C for 16 h. PCR reaction products were separated on a 2–3% agarose gel and RFLP digested products were separated on 3% nu-micropore agarose gel stained with ethidium bromide (Fig. 1 ). In order to validate the accuracy and reproducibility of this method, each PCR reaction included internal controls for each
genotype. Second PCR was performed to confirm samples which results are not clear. Also, to confirm the accuracy of the genotyping, repeated analysis was performed on randomly selected samples. No discrepancies were found. 2.4. Statistical analysis Analysis of the data was performed using the computer software SPSS 13.0 (SPSS, Chicago, IL, USA) and OpenEpi Info software package program (Dean and Sullivan, 2010). Continuous data were given as mean ± SD (standard deviation) and median (min-max), categorical data were given as frequency (percent). The frequencies of the alleles and genotypes in patients and controls were compared with X 2 analysis. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated. The comparisons of two groups were performed by t test and Mann–Whitney test. P value smaller than 0.05 (two-tailed) was regarded as statistically significant. Power analysis was made by using Minitab 15.0 package program. 3. Results Clinical and laboratory findings of the patients and controls were given in Table 2. In power analysis, the power of ER1/CALCR CCTT genotype comparison between patients and controls was 82% in 95% confidence interval. The results of the genotype and allele frequencies of BGLAP − 298 C>T, ER1 397 T>C, Col1A1 2046 G>T and CALCR 1340 T>C for osteoporosis patients and control groups are presented in Table 3. The genotype frequencies of the BGLAP, Col1A1 and CALCR
Table 2 Clinical and laboratory findings. Characteristics
Patients (n = 158) (mean ± SD)
Median (min–max)
Controls (n = 108)
Median (min–max)
P value
BMI (kg/m2) Age at menopause (years) Years since menopause (years) Age at menarche (years) Number of birth (n) Serum calcium (mg/dl) Serum phosphorus (mg/dl) Serum PTH (pg/ml) Serum ALP (U/l)
28.04 ± 3.837 47.28 ± 5.527 15.37 ± 7.640 13.64 ± 1.529 3.74 ± 2.087 9.54 ± 0.550 3.99 ± 2.67 79.07 ± 53.91 189.02 ± 61.91
28.00 47.50 14.0 13.0 3.0 9.5 3.73 76.0 188.35
29.48 ± 4.622 46.76 ± 4.7243 9.67 ± 7.460 13.59 ± 1.309 2.63 ± 1.624 9.72 ± 0.4992 3.90 ± 0.69835 74.42 ± 28.583 188.43 ± 46.146
29 (22–39) 47 (33–59) 7 (1–26) 13 (11–17) 3 (0–6) 9.8 (8.5–11) 4 (2.07–5.51) 73.2 (31–178) 187.5 (97–313)
0.002 0.011 0.0002 0.856 0.003 0.022 0.047 0.560 0.991
The results that are statistically significant are typed in bold.
(19–39) (34–60) (2–35) (9–17) (0–10) (8.01–11.6) (2.2–26.20) (11.90–446) (2.34–410)
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Table 3 Comparison of genotype and allele frequencies of BGLAP, ER1, Col1A1 and CALCR gene polymorphisms between osteoporotic patients and controls. SNP
Genotype/allele
Patients (n = 158) (%)
Controls (n = 108) (%)
χ2
P value
BGLAP −298 (C>T) rs1800247
CC CT TT CC + CT:TT CC:TC + TT C T TT TC CC TT + TC:CC TT:TC + CC T C GG GT TT GG + GT:TT GG:GT + TT G T TT TC CC TT:TC + CC TT + TC:CC T C
87 (55.1) 51 (32.3) 20 (12.7) 138:20 87:71 225 (71.2) 91 (28.8) 30 (19) 92 (58.2) 36 (22.8) 122:36 30:128 152 (48.1) 164 (51.8) 71 (44.9) 61 (38.6) 26 (16.5) 132:26 71:87 203 (64.2) 113 (35.8) 53 (33.5) 88 (55.7) 17 (10.8) 53:105 141:17 194 (61.4) 122 (38.6)
51 (47.2) 45 (41.7) 12 (11.1) 96:12 51:57 147 (75) 69 (25) 24 (22.3) 71 (65.7) 13 (12) 95:13 24:84 119 (55) 97 (45) 48 (44.4) 49 (45.4) 11 (10.2) 97:11 48 :60 145 (67.1) 71 (32.9) 31 (28.7) 55 (50.9) 22 (20.4) 31:77 86:22 117 (54.2) 99 (45.8)
2.455
0.293
0.036 1.580 0.6042
0.850 0.209 0.437
4.944
0.084
4.242 0.239 2.59
0.039a 0.625 0.113
2.526
0.283
1.615 0.006 0.473
0.204 0.936 0.491
4.789
0.091
0.695 3.999 2.750
0.404 0.045 0.097
ER1 +397 (T>C) rs2234693
Col1A1 +2046 (G>T) rs1800012
CALCR +1340 (T>C) rs1801197
OR (95% CI)
1.159 (0.541–2.483) 0.730 (0.447–1.193) 0.8616 (0.5918–1.254)
2.156 (1.083–4.293) 1.219 (0.666–2.228) 0.755 (0.534–1.07)
1.737 (0.818–3.685) 0.980 (0.599–1.604) 1.137 (0.789–1.638)
1.254 (0.736–2.134) 0.471 (0.237–0.9372) 1.346 (0.948–1.910)
The results that are statistically significant are typed in bold. a Fisher exact test.
polymorphisms in the control group were in Hardy–Weinberg equilibrium. Only ER1 gene polymorphism was not in Hardy–Weinberg equilibrium. Our small sample size might be the reason of disequilibrium. Combined genotype analysis results were presented in Table 4. ER1 CC genotype compared with TT +TC genotypes was found to increase two fold the risk of osteoporosis [p=0.039, OR =2.156, 95% CI (1.083–4.293)]. CALCR CC genotype compared with TT +TC genotypes was found to have protective effect against osteoporosis [p =0,045, OR=0.471, 95% CI (0.237–0.9372)]. There was no statistically significant difference in the genotype and allele frequencies of patients and controls
for BGLAP −298 C>T (p=0.293 and p= 0.437, respectively) and Col1A1 2046 G>T polymorphisms (p= 0.283, p =0.491, respectively). In the combined genotype analysis, ER1/CALCR TCCC combined genotype was found to have protective effect against osteoporosis [p = 0.0125, OR = 0.323, 95% CI (0.1383–0.755)]. However, BGLAP/ Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were risk factors for osteoporosis (p = 0.027 and p = 0.009). ER1/CALCR TCCC combined genotype was estimated to have protective effect against osteoporosis (p = 0.0125). However, BGLAP/Col1A1 CCTT, ER1/CALCR TTTT combined genotypes were risk factors for osteoporosis (p =
Table 4 Comparison of combined genotypes of BGLAP, ER1, Col1A1 and CALCR gene polymorphisms between osteoporotic patients and controls. Genes
Combined genotypes
Patients (%)
Controls (%)
χ2
P value
OR (95% CI)
BGLAP /COL1A1 rs1800247/rs1800012
CCGG CCGT CCTT CTGG CTGT CTTT TTGG TTGT TTTT CCTT CCTC CCTT TCTT TCTC TCCC TTTT TTTC TTCC
40 (25.3) 33 (20.9) 14 (8.9) 22 (13.9) 22 (13.9) 7 (4.4) 9 (5.7) 6 (3.8) 5 (3.2) 19 (12) 12 (7.6) 5 (3.2) 26 (16.5) 57 (36.1) 9 (5.7) 8 (5.1) 19 (12) 3 (1.8) 158
27 (25.0) 22 (20.4) 2 (1.9) 15 (13.9) 24 (22.0) 6 (5.6) 6 (5.6) 3 (2.8) 3 (2.8) 3 (2.8) 8 (7.4) 2 (1.9) 21 (19.4) 33 (30.6) 17 (15.7) 7 (6.4) 14 (13)) 3 (2.8) 108
0.003 0.010
0.9350 0.9188 0.027a 0.8632 0.1110 0.8979 0.8245 0.4664a 0.8804b 0.009a 0.8573 0.0810 0.6427 0.3501 0.0125 0.8245 0.9365 0.9693
1.017 (0.578–1.788) 1.032 (0.5634–1.911)
BGLAP /CALCR rs2234693/rs1801197
Total The results that are statistically significant are typed in bold. a Fisher exact test. b Mid-P exact test.
0.029 2.536 0.016 0.049
0.0320 0.2152 0.8733 6.2440 0.0491 0.0014
1.003 0.566 0.788 1.027
(0.494–2.074) (2.988–1.073) (0.2486–2.566) (0.3504–3.187)
1.027 (0.405–2.604) 0.816 (0.4322–1.541) 1.283 (0.7606–2.163) 0.323 (0.1383–0.755) 0.769 (0.2706–2.189) 0.917 (0.4387–1.920)
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0.0139 and p = 0.009). There were no statistically significant differences in terms of BMI, years since menopause, tobacco smoking, menarche age or menopause age between the genotypes for each RFLP (data not shown). Additionally effect of combinations of three and four SNPs were analyzed and an association was not found (data not shown). 4. Discussion The results of the present study showed that polymorphic genotypes of BGLAP, ER1, Col1A1 and CALCR genes are not associated with osteoporosis in single form but associated in combined forms. In the present study, there was no statistically significant difference in the genotype and allele frequencies of patients and controls for BGLAP − 298 C>T, Col1A1 2046 G>T, ER1 397 T>C and CALCR 1340 T>C polymorphisms. However, ER1 CC genotype compared with TT + TC genotypes was found to increase two fold the risk of osteoporosis and CALCR CC genotype compared with TT + TC genotypes was found to have protective effect against osteoporosis. Osteocalcin is well known as a marker for differentiated mature osteoblasts and a determinant of the bone calcification process (Chen et al., 2001). BGLAP gene − 298C>T polymorphism in the promoter region has been found to be associated with osteoporosis in postmenopausal women (Dohi et al., 1998). In the present study, when we compared the genotype distribution and allele frequencies of patients and controls, we did not find a statistically significant difference, however in the combined genotype analysis, BGLAP/Col1A1 CCTT combined genotype was a risk factor for osteoporosis. The first research of BGLAP gene − 298C>T polymorphism and osteoporosis performed by Dohi et al. (1998) reported that C allele was associated with lower BMD in Japanese postmenopausal women. The following study in Taiwanese postmenopausal women supported these results (Chen et al., 2001). Another study was carried out in the Japanese women and significant association between lumbar spine, hip BMD and the BGLAP polymorphic genotypes was found only in postmenopausal women but not in pre-menopausal women (Yamada et al., 2002). In a recent study, McGuigan et al. (2010) (Yamada et al., 2002) did not find an association with 298C>T polymorphism and BMD in a large Swedish postmenopausal women population, but they observed a significant association with BMI. However a number of studies failed to detect a significant association (Jiang et al., 2007; Mo et al., 2004; Zhao et al., 2005). As seen in our combined analysis result, CC genotype not alone but together with COL1A1 TT genotype was a risk factor for osteoporosis. Our findings suggest that when certain genotypes of some genes act together, they influence each other and affect the person's osteoporosis risk. Estrogen functions as a potent regulator of peak bone mass and bone remodeling (Ivaska and Kaisa, 2005). ER1 gene 397 T>C (also named Pvu II polymorphism) is also associated with preeclampsia, endometriosis uterine fibroids and tardive dyskinesia in schizophrenia besides osteoporosis (Lai et al., 2002). The present study revealed that ER1 CC genotype was found to increase the risk of osteoporosis two fold when compared with TT + TC genotypes (p= 0.039). In the combined genotype analysis, ER1/CALCR TCCC combined genotype was estimated to have protective effects against osteoporosis and ER1/CALCR CCTT combined genotype was a risk factor for osteoporosis. As seen in our combined analysis result, ER1 CC genotype not alone but together with CALCR TT genotype was a risk factor for osteoporosis. Kobayashi et al. (1996) first demonstrated the association between 397 T>C polymorphism and BMD in Japanese women. A study conducted in Indian women indicated that average lumbar vertebra BMD value of TT genotypes was found to be higher than those of the CC genotype (Mitra et al., 2006). These findings are in agreement with our results. Our results are also in agreement with the findings of Erdogan et al.'s (2010) study which was carried out in 126 postmenopausal Turkish women and which revealed that average lumbar vertebra BMD value of women
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with TT genotype was significantly higher than that with CC genotype. Similarly, a recent study performed in a south Indian population from Andhra Pradesh declared that ER1 gene C allele was significantly higher in pre- and postmenopausal osteoporotics when compared to controls (Jeedigunta et al., 2010). A meta-analysis pooled the association between the ER1 polymorphisms and BMD in sixteen eligible studies involving 4,297 Chinese women and the data suggested that TT homozygotes had lower BMD than TC/CC genotype (Ralston, 2010). However, another meta-analysis (Ioannidis et al., 2002) including eight European centers did not find a significant association between ESR1 gene polymorphisms and BMD. Also there are several studies which revealed no association between 397 T>C polymorphism and osteoporosis (Bandres et al., 2005; Mitra et al., 2006). Col1A1 gene 2046 G>T (also named Sp1 polymorphism) has been associated with low BMD and an increased risk of osteoporotic fracture in several studies (Bandres et al., 2005; Hubacek et al., 2006). Col1A1 gene 2046 G>T polymorphism is also associated with osteosclerosis, Chron's disease and myocardial infarction (Speer et al., 2006). In our study, we have demonstrated that there were not statistically significant differences between the allele frequencies and genotype frequencies for Col1A1 gene 2046 G>T polymorphism. However BGLAP/Col1A1 CCTT combined genotype was a risk factor for osteoporosis. Grant et al. (1996) have first documented that unfavorable GT and TT genotypes are associated with low BMD and prevail in an osteoporotic fracture group compared to normal controls. In several studies, an association between osteoporosis and Col1A1 gene 2046 G>T polymorphism was not found (Long et al., 2004; Vidal et al., 2007). Our study was also similar to Hubacek et al.'s (2006) study in which there was no significant difference in terms of 2046 G>T polymorphism genotype frequency among women with normal, osteopenic, and osteoporotic bone mass in Caucasian women. Recently, a meta-analysis of 26 studies confirmed the association of the T allele of the 2046 G>T polymorphism with a modest reduction in BMD and a significant risk of osteoporotic fracture (Mann et al., 2001). COLIA1 TT genotype was associated with increased hip fracture risk in Caucasian women, and this association was independent of BMD and age (Nguyen et al., 2005). Bandres et al. (2005) described that the TT genotype was found to be overrepresented in Spanish women who had a personal history of fractures. Bustamante et al. (2007) reported that COL1A1-1997 G>T and 2046 G>T polymorphisms are associated with increased BMD for lumbar spine. Our results are in agreement with these results by our combined analysis. In contrast, a study carried out in post-menopausal Caucasian women from Canary Island showed that all patients with osteoporotic fractures carried the GG allele more frequently than TT homozygotic women (Navarro et al., 2007). Our results are also in agreement with Erdogan et al. (2010) who carried out a study in 126 postmenopausal Turkish women and revealed that there was no association between 2046 G>T polymorphism and osteoporosis. Ramirez reported that the frequency of T allele was significantly higher in Mexican women with OP than in women without OP (Drews et al., 2005). In a recent meta-analysis, for the 2046 G>T polymorphism, BMD values in TT homozygotes were lower at the spine and lower at the hip than GG homozygotes (Navarro et al., 2007). In the present study, BGLAP/Col1A1 CCTT combined genotype was a risk factor for osteoporosis. As seen in our combined analysis result, TT genotype not alone but together with BGLAP gene CC genotype was a risk factor for osteoporosis. If our study population was larger this CC genotype or C allele might be significant for osteoporosis. Calcitonin receptor gene is one of the most important candidate genes for predisposition to osteoporosis. Masi and colleagues first identified an association between CALCR genotype and BMD in a study of Italian women in 1998 and Tsai et al. (2003) reported that women with genotype TT had a greater risk for developing osteoporosis at the lumbar spine and at the femoral neck and this polymorphism is associated with reduced bone mineral density and predisposes women to osteoporosis. Zofkova et al. (2003) suggested that this polymorphism
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is associated with bone mass at the femoral neck in a cohort of postmenopausal women, the lower values being found in carriers of C allele compared to subjects with the TT genotype. Similarly Bandres et al. (2005) studied 177 postmenopausal Spanish women and revealed that women with the CC genotype had a lower adjusted BMD at the femoral neck. Drews et al. (2005) studied 139 postmenopausal Polish women and they reported T-score was higher in CT genotype carriers. In the present study, CALCR CC genotype compared with TT + TC genotypes was found to be protective against osteoporosis. In the combined genotype analysis, ER1/CALCR TCCC combined genotype was found to have a protective effect against osteoporosis. However, BGLAP/Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were risk factors for osteoporosis. In Turkish population TT genotype might be significant for osteoporosis but our sample size might not be big enough to detect the difference. The effect of an individual SNP is generally small, and the genetic effect of combinations of functionally relevant SNPs may additively or synergistically contribute to increased OP risk. For this reason the analysis of combined genotypes of different SNPs is more helpful in the identification of predisposing of complex diseases. To the best of our knowledge, this is the first study to simultaneously examine the possible relationship between polymorphism of BGLAP − 298 C>T, ER1 397 T>C, Col1A1 2046 G>T and CALCR 1340 T>C gene polymorphisms and osteoporosis and also to investigate the effect of combined genotype in the same subjects. The limitation of our study is the sample size that is relatively small because none of the women had a history of taking medicines for the treatment of osteoporosis. In conclusion, the polymorphic genotypes of BGLAP, ER1, Col1A1 and CALCR are not found to be associated with osteoporosis in a single form but found to be associated in combined forms. By enlarging the study we hope that more explanatory and definitive results can be obtained. And further studies are also necessary in different populations. Conflicts of interest None. Acknowledgments This study was presented as poster in European Human Genetics Conference Amsterdam RAI, The Netherlands, May 28–31, 2011. Additionally, this study was completely supported by Ondokuz Mayis University (Project No. T-593). References Bandres, E., Pombo, I., Gonzalez-Huarriz, M., Rebollo, A., Lopez, G., Garcia, Foncillas J., 2005. Association between bone mineral density and polymorphism of the VDR, ERalpha, COL1A1 and CTR genes in Spanish postmenopausal women. J. Endocrinol. Invest. 28 (4), 312–321. Bustamante, M., et al., 2007. COL1A1, ESR1, VDR and TGFB1 polymorphisms and haplotypes in relation to BMD in Spanish postmenopausal women. Osteoporos. Int. 18, 235–243. Chen, H., Tsai, H., Chen, W., Wu, J., Tsai, F., Tsai, C., 2001. Relation of polymorphism in the promoter region for the human osteocalcin gene to bone mineral density occurrence of osteoporosis in postmenopausal Chinese women in Taiwan. J. Clin. Lab. Anal. 15, 251–255. Choi, Y.J., Oh, H.J., Kim, D.J., Lee, Y., Chung, Y.S., 2012. The prevalence of osteoporosis in Korean adults aged 50 years or older and the higher diagnosis rates in women who were beneficiaries of a national screening program: The Korea National Health and Nutrition Examination Survey 2008–2009. J. Bone Miner. Res. http://dx.doi.org/ 10.1002/jbmr.1635 (Epub ahead of print). Dean, A.G., Sullivan, K.M., Soe, M.M., OpenEpi, . Open Source Epidemiologic Statistics for Public Health, Version 2.3.1www.OpenEpi.com (updated 2010/19/09, accessed 2011/02/21). Dohi, Y., et al., 1998. A novel polymorphism in the promoter region for human osteocalcin gene: the possibility of a correlation with bone mineral density in postmenopausal Japanese women. J. Bone Miner. Res. 13, 1633–1639.
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