International Immunopharmacology 22 (2014) 200–203
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
The influence of osteoprotegerin genetic polymorphisms on bone mineral density and osteoporosis in Chinese postmenopausal women Tao Sun a,1, Mingqi Chen a,1, Xiaoyan Lin b, Ruixiang Yu a, Yong Zhao a, Jianhang Wang a,b,⁎ a b
Yantaishan Hospital, Yantai 264000, Shangdong Province, People's Republic of China Yantai Zhifu Hospital, Yantai 264000, Shangdong Province, People's Republic of China
a r t i c l e
i n f o
Article history: Received 23 May 2014 Received in revised form 10 June 2014 Accepted 11 June 2014 Available online 24 June 2014 Keywords: Osteoporosis Bone mineral density Osteoprotegerin gene Genetic polymorphisms Postmenopausal women
a b s t r a c t Previous studies suggest that the osteoprotegerin gene (OPG) plays an important role in the development of osteoporosis. This study aims to investigate the potential association between OPG genetic polymorphisms and bone mineral density (BMD) and osteoporosis in postmenopausal women. 938 Chinese postmenopausal women were enrolled. The lumbar spine (L2–4) BMD, neck BMD, and total hip BMD were measured by dual energy X-ray absorptiometry (DEXA). The genotypes of OPG genetic polymorphisms were evaluated by the created restriction site-polymerase chain reaction (CRS-PCR), PCR-restriction fragment length polymorphism (PCRRFLP), and DNA sequencing methods. Our data indicated that subjects with genotype TT of the g.26395TNC genetic polymorphism showed a significantly higher adjusted value of BMD when compared with those of genotypes TC and CC. Subjects with genotype AA of the g.27649ANG genetic polymorphism showed a significantly higher adjusted value of BMD than those of genotypes AG and GG. These findings suggest that the OPG genetic polymorphisms may affect BMD and osteoporosis in Chinese postmenopausal women. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Primary osteoporosis is a common complex health problem, particularly in postmenopausal women. It is a systemic skeletal disease characterized by a deterioration of bone microarchitecture with a consequent increase of fracture risk and a reduction in bone mineral density (BMD) [1–7]. Previous studies report that the osteoprotegerin gene (OPG) is one of the most important candidate genes for influencing the pathogenesis of BMD and osteoporosis [8–22]. The OPG genetic polymorphisms may affect the expression and function of the OPG protein, which contributes to genetic effects on BMD and osteoporosis [8–14,18,19,23]. Several studies have demonstrated that the OPG genetic polymorphisms are potentially associated with BMD and osteoporosis [6,7,9,13,18–29]. However, up to now, the exact molecular mechanism of osteoporosis remains uncertain. There are no similar studies on the potential association between the g.26395TNC and g.27649ANG genetic polymorphisms of the OPG gene and the risk of BMD and osteoporosis. Therefore, the objective of this study is to assess the influence of these two OPG genetic polymorphisms on BMD and osteoporosis in postmenopausal women.
2.1. Study subjects In the present study, we enrolled 938 postmenopausal women, including 468 primary postmenopausal osteoporosis patients (aged 47– 76 years) and 470 healthy controls (aged 48–78 years) from the Yantaishan Hospital (Yantai, China) between January 2009 and December 2013. All subjects were genetically unrelated and of Chinese Han nationality. Subjects suffering from diseases or taking drugs that could affect bone metabolism were excluded. This study was approved by the Ethics Committee of the Yantaishan Hospital, and the informed consents from all participants were obtained. 2.2. Measurement of bone mineral density The lumbar spine (L2–4) BMD, neck BMD, and total hip BMD were assessed by dual energy X-ray absorptiometry (DEXA) (Lunar Expert 1313, Lunar Corp., USA). The BMD values were calculated from bone mineral content (g) and bone area (cm2), and expressed as g/cm2. 2.3. PCR amplification
⁎ Corresponding author at: Yantaishan Hospital, No. 91 Jiefang Road, Zhifu District, Yantai 264000, Shangdong Province, People's Republic of China. Tel./fax: + 86 535 6602180. E-mail address: [email protected] (J. Wang). 1 Tao Sun and Mingqi Chen have contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.06.023 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Peripheral venous blood samples were collected from all participants. Following the supplier manual, the genomic DNA was isolated from blood samples using the Axygen DNA isolation kit (Axygen, CA), and stored at − 20 °C until analyzed. Based on the DNA sequences
T. Sun et al. / International Immunopharmacology 22 (2014) 200–203
201
Table 1 The detailed information of PCR, CRS-PCR and PCR-RFLP analyses used for OPG genetic variants. Genetic variants
Primer sequences
Annealing temperature (°C)
PCR amplification fragment (bp)
Region
Genotype method
Restriction enzyme
Genotype (bp)
g.26395TNC
5′-GAAAGAGTTCTGACTTCAGTAAGCT-3′ 5′-AGCAATCAGCTCTCAAAGACGT-3′
58.6
214
Intron 4
CRS-PCR
HindIII
g.27649ANG
5′-CGCACTAAAGCACTCAAAGACGT-3′ 5′-TTACTCATCCATGGGATCTCGC-3′
61.6
223
Exon 5
PCR-RFLP
Tsp45I
TT: 193, 21 TC: 214, 193, 21 CC: 193, 21 AA: 183, 40 AG: 223, 183, 40 GG: 214
Note: PCR, polymerase chain reaction; CRS-PCR, created restriction site-PCR; PCR-RFLP, PCR-restriction fragment length polymorphism. Underlined nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating genetic variations.
(GenBank ID: NG_012202.1) and mRNA sequences (GenBank ID: NM_ 002546.3) of the human OPG gene, the specific polymerase chain reaction (PCR) primers were designed by Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). The details of primer sequences, annealing temperature, region, and fragment sizes are shown in Table 1. The PCR amplifications were performed in a total volume of 20 μL of reaction mixture containing 50 ng mixed DNA template, 1× buffer (Tris–HCl 100 mmol/L, pH 8.3; KCl 500 mmol/L), 0.25 μmol/L primers, 2.0 mmol/L MgCl2, 0.25 mmol/L dNTPs, and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, China). Cycling conditions consisted of an initial 95 °C for 5 min followed by 30 cycles of 94 °C for 35 s, annealing at the corresponding temperature (shown in Table 1) for 35 s, and 72 °C for 35 s, with a final extension at 72 °C for 8 min. The PCR amplified products were separated by 2.5% agarose gel electrophoresis including ethidium bromide, and then observed under UV light.
Inc.; Chicago, IL, USA). All data are shown as the mean ± SD (standard deviation of the mean). The values of BMD were adjusted by age, weight and height. The Hardy–Weinberg equilibrium (HWE) in allele and genotype frequencies of different genetic mutations was analyzed by chisquared (χ2) test. The relationships between the variables were assessed by the stepwise multiple regression and logistic regression analyses. A P value less than 0.05 was defined as a statistically significant level. 3. Results 3.1. Genotyping of OPG genetic polymorphisms Through the CRS-PCR, PCR-RFLP and DNA sequencing methods, we detected two novel genetic polymorphisms (g.26395TNC and g.27649ANG) in the present study. DNA sequence analyses indicate that the g.26395TNC genetic polymorphisms cause from T to C mutations in intron 4 of the human OPG gene. The PCR amplified products of this genetic polymorphism were digested with the HindIII restriction enzyme, and divided into three genotypes: TT (193 and 21 bp), TC (214, 193 and 21 bp), and CC (214 bp, Table 1). As for the g.27649ANG genetic polymorphism, it is a non-synonymous mutation, which is caused by A to G mutations in exon 5 of the human OPG gene and resulting into the threonine (Thr) to alanine (Ala) amino acid replacement (p.Thr362Ala, reference sequences, GenBank IDs: NG_012202.1, NM_002546.3 and NP_002537.3). The PCR amplified products of these polymorphisms were digested with the Tsp45I restriction enzyme and divided into three genotypes, AA (223 bp), AG (223,183 and 40 bp) and GG (183 and 40 bp, Table 1).
2.4. OPG genetic polymorphism genotyping The genotypes of the OPG g.26395TNC genetic variant were evaluated by the created restriction site-PCR (CRS-PCR) method with one of the primers containing a nucleotide mismatch, which enables the use of restriction enzymes for discriminating sequence variations [30–34]. The PCR-restriction fragment length polymorphism (PCR-RFLP) method has been used to investigate the genotypes of the OPG g.27649ANG genetic variant. According to the manufacturer's instructions, the PCR amplified products (5 μL) were digested with 2 U of selected restriction enzymes (Table 1, MBI Fermentas, St. Leon-Rot, Germany) at 37 °C for 10 h. The digested products were separated by electrophoresis for 1 h at 100 V in 2.5% agarose gel containing ethidium bromide. In order to confirm the genotype results from the CRS-PCR and PCR-RFLP methods, 10% of the total samples were randomly selected to be re-analyzed by a DNA sequencing method (ABI3730xl DNA Analyzer, Applied Biosystems, Foster City, CA).
3.2. Allele and genotype frequencies Table 2 shows the allele and genotype frequencies of OPG g.26395TNC and g.27649ANG genetic polymorphisms. The frequencies of allele-T of g.26395TNC and allele-A g.27649ANG genetic polymorphisms were the maximums in the primary postmenopausal osteoporosis patients and healthy controls. As for g.26395TNC, the allele and genotype frequencies of patients (T: 68.70% and C: 31.30%; TT: 49.57%, TC: 38.25%, and CC: 12.18%) showed a significant difference with those of the healthy
2.5. Statistical analysis All statistical analyses were evaluated by the Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS
Table 2 The genotypic and allelic frequencies of OPG genetic variants in the studied subjects. Groups
g.26395TNC
g.27649ANG
Genotype frequencies (%)
Allele frequencies (%)
TT
TC
CC
T
Cases (n = 468)
232 (49.57)
179 (38.25)
57 (12.18)
Controls (n = 470)
247 (52.56)
193 (41.06)
30 (6.38)
Total (n = 938)
479 (51.06)
372 (39.66)
87 (9.28)
643 293 (68.70) (31.30) 687 253 (73.09) (26.91) 1330 546 (70.90) (29.10) 2 χ = 4.3775, P = 0.0364
2
χ = 9.3717, P = 0.0092
C
Genotype frequencies (%)
Allele frequencies (%)
GG
A
69 (14.74) 40 (8.51) 109 (11.62)
605 331 (64.64) (35.36) 650 290 (69.15) (30.85) 1255 621 (66.90) (33.10) 2 χ = 4.3120, P = 0.0378
χ2
P
AA
5.7347
0.0568
0.9007
0.6374
1.4254
0.4903
206 193 (44.02) (41.24) 220 210 (46.81) (44.68) 426 403 (45.42) (42.96) 2 χ = 8.8886, P = 0.0117
AG
G
χ2
P
4.4864
0.1061
1.0477
0.5922
0.8403
0.6569
202
T. Sun et al. / International Immunopharmacology 22 (2014) 200–203
Table 3 The characteristics of OPG genetic variants in the studied subjects. Genetic variants
g.26395TNC
Genotype
TT
TC
CC
*P
AA
g.27649ANG AG
GG
*P
Number (%) Age (years) Height (cm) Weight (kg) BMI Adjusted spine BMD (g/cm2) Adjusted neck hip BMD (g/cm2) Adjusted total hip BMD (g/cm2)
479 (51.06) 60.9 ± 7.2 161 ± 7.3 61.2 ± 7.6 23.2 ± 3.45 0.933 ± 0.122 0.751 ± 0.136 0.862 ± 0.145
372 (39.66) 61.8 ± 7.1 162 ± 6.9 62.3 ± 6.6 23.3 ± 3.67 0.867 ± 0.114 0.692 ± 0.222 0.821 ± 0.118
87 (9.28) 62.7 ± 7.6 163 ± 7.1 63.0 ± 5.2 23.7 ± 3.11 0.827 ± 0.132 0.668 ± 0.119 0.802 ± 0.109
– 0.427 0.326 0.234 0.226 0.038 0.016 0.038
426 (45.42) 60.8 ± 7.9 160 ± 7.8 61.5 ± 7.7 23.2 ± 3.54 0.941 ± 0.215 0.765 ± 0.205 0.872 ± 0.104
403 (42.96) 61.5 ± 7.4 162 ± 6.9 62.8 ± 6.9 23.4 ± 3.12 0.871 ± 0.121 0.686 ± 0.196 0.822 ± 0.205
109 (11.62) 62.6 ± 7.6 163 ± 6.5 63.2 ± 5.8 23.7 ± 3.29 0.833 ± 0.212 0.657 ± 0.133 0.807 ± 0.117
– 0.378 0.422 0.348 0.318 0.027 0.036 0.029
Note: BMI, body mass index; BMD, bone mineral density (BMD values adjusted by age, weight and height); data are shown as mean ± standard deviation (SD).
controls (T: 73.09% and C: 26.91%; TT: 52.56%, TC: 41.06%, and CC: 6.38%; for allele, χ2 = 4.3775, P = 0.0364; for genotype, χ2 = 9.3717, P = 0.0092, Table 2). As for g.27649ANG, the allele and genotype frequencies of patients (A: 64.64% and G: 35.36%; AA: 44.02%, AG: 41.24%, and GG: 14.74%) showed a significant difference with those of the healthy controls (A: 69.15% and G: 30.85%; AA: 46.81%, AG: 44.68%, and GG: 8.51%; for allele, χ2 = 4.3120, P = 0.0378; for genotype, χ2 = 8.8886, P = 0.0117, Table 2). The χ2 test suggested that the allele and genotype frequencies of these two genetic mutations were in accordance with the HWE in the studied populations (P N 0.05).
these two genetic polymorphisms may contribute to BMD and osteoporosis in Chinese postmenopausal women. It would be necessary to confirm our findings in further prospective investigations with large and different ethnic populations, and to explain the pathogenesis and molecular mechanism of osteoporosis. Acknowledgements None. References
3.3. Relationship of OPG genetic polymorphisms with bone mineral density Table 3 presents the age, weight, height, body mass index (BMI), adjusted neck hip BMD, adjusted spine BMD and adjusted total hip BMD in each genotype. All data are shown as mean ± SD (BMD values adjusted by age, height and weight). As for g.26395TNC, we detected significant differences of adjusted spine BMD, adjusted neck hip BMD, and adjusted total hip BMD among different genotypes in the subjects; subjects with genotype TT showed a significantly higher value of BMD when compared with those of genotypes TC and CC (P b 0.05, Table 3). The g.27649ANG was significantly associated with adjusted spine BMD, adjusted neck hip BMD and adjusted total hip BMD. Subjects with genotype AA had a significantly higher BMD value than those of genotypes AG and GG (P b 0.05, Table 3). 4. Discussion Osteoporosis is a multifactorial and polygenic disease, and is caused by the combined effects of genetic and environmental factors [24]. It is generally accepted that the genetic factors may play a key role in the development of osteoporosis [35–40]. In recent years, the OPG gene is considered to play an important role in the pathogenesis of osteoporosis [8–15,18], and several association studies have been conducted to assess the role of OPG genetic variants (such as A163G, T245G, T950C, G1181C, g.18861ANG, g.19190CNA, and g.27406CNT) on the risk of BMD and osteoporosis [6,7,9,13,18–21,23–29]. In this case–control study, we firstly investigated the influence of g.26395TNC and g.27649ANG genetic polymorphisms of the OPG gene with BMD and osteoporosis in Chinese postmenopausal women by association analyses. Our data suggested that there were significant differences in the distribution of allelic and genotypic frequencies between primary postmenopausal osteoporosis patients and healthy controls (Table 2). The allele-T/genotype-TT of g.26395TNC and allele-A/genotype-AA of g.27649ANG genetic polymorphisms of the OPG gene might be associated with a protection from osteoporosis (Table 3). Results from this study indicate that OPG genetic polymorphisms may affect BMD and osteoporosis in Chinese postmenopausal women. In summary, the current study is the first to report the potential association between g.26395TNC and g.27649ANG genetic polymorphisms of the OPG gene and BMD and osteoporosis. Our results indicate that
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
The influence of osteoprotegerin genetic polymorphisms on bone mineral density and osteoporosis in Chinese postmenopausal women Tao Sun a,1, Mingqi Chen a,1, Xiaoyan Lin b, Ruixiang Yu a, Yong Zhao a, Jianhang Wang a,b,⁎ a b
Yantaishan Hospital, Yantai 264000, Shangdong Province, People's Republic of China Yantai Zhifu Hospital, Yantai 264000, Shangdong Province, People's Republic of China
a r t i c l e
i n f o
Article history: Received 23 May 2014 Received in revised form 10 June 2014 Accepted 11 June 2014 Available online 24 June 2014 Keywords: Osteoporosis Bone mineral density Osteoprotegerin gene Genetic polymorphisms Postmenopausal women
a b s t r a c t Previous studies suggest that the osteoprotegerin gene (OPG) plays an important role in the development of osteoporosis. This study aims to investigate the potential association between OPG genetic polymorphisms and bone mineral density (BMD) and osteoporosis in postmenopausal women. 938 Chinese postmenopausal women were enrolled. The lumbar spine (L2–4) BMD, neck BMD, and total hip BMD were measured by dual energy X-ray absorptiometry (DEXA). The genotypes of OPG genetic polymorphisms were evaluated by the created restriction site-polymerase chain reaction (CRS-PCR), PCR-restriction fragment length polymorphism (PCRRFLP), and DNA sequencing methods. Our data indicated that subjects with genotype TT of the g.26395TNC genetic polymorphism showed a significantly higher adjusted value of BMD when compared with those of genotypes TC and CC. Subjects with genotype AA of the g.27649ANG genetic polymorphism showed a significantly higher adjusted value of BMD than those of genotypes AG and GG. These findings suggest that the OPG genetic polymorphisms may affect BMD and osteoporosis in Chinese postmenopausal women. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Primary osteoporosis is a common complex health problem, particularly in postmenopausal women. It is a systemic skeletal disease characterized by a deterioration of bone microarchitecture with a consequent increase of fracture risk and a reduction in bone mineral density (BMD) [1–7]. Previous studies report that the osteoprotegerin gene (OPG) is one of the most important candidate genes for influencing the pathogenesis of BMD and osteoporosis [8–22]. The OPG genetic polymorphisms may affect the expression and function of the OPG protein, which contributes to genetic effects on BMD and osteoporosis [8–14,18,19,23]. Several studies have demonstrated that the OPG genetic polymorphisms are potentially associated with BMD and osteoporosis [6,7,9,13,18–29]. However, up to now, the exact molecular mechanism of osteoporosis remains uncertain. There are no similar studies on the potential association between the g.26395TNC and g.27649ANG genetic polymorphisms of the OPG gene and the risk of BMD and osteoporosis. Therefore, the objective of this study is to assess the influence of these two OPG genetic polymorphisms on BMD and osteoporosis in postmenopausal women.
2.1. Study subjects In the present study, we enrolled 938 postmenopausal women, including 468 primary postmenopausal osteoporosis patients (aged 47– 76 years) and 470 healthy controls (aged 48–78 years) from the Yantaishan Hospital (Yantai, China) between January 2009 and December 2013. All subjects were genetically unrelated and of Chinese Han nationality. Subjects suffering from diseases or taking drugs that could affect bone metabolism were excluded. This study was approved by the Ethics Committee of the Yantaishan Hospital, and the informed consents from all participants were obtained. 2.2. Measurement of bone mineral density The lumbar spine (L2–4) BMD, neck BMD, and total hip BMD were assessed by dual energy X-ray absorptiometry (DEXA) (Lunar Expert 1313, Lunar Corp., USA). The BMD values were calculated from bone mineral content (g) and bone area (cm2), and expressed as g/cm2. 2.3. PCR amplification
⁎ Corresponding author at: Yantaishan Hospital, No. 91 Jiefang Road, Zhifu District, Yantai 264000, Shangdong Province, People's Republic of China. Tel./fax: + 86 535 6602180. E-mail address: [email protected] (J. Wang). 1 Tao Sun and Mingqi Chen have contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.06.023 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Peripheral venous blood samples were collected from all participants. Following the supplier manual, the genomic DNA was isolated from blood samples using the Axygen DNA isolation kit (Axygen, CA), and stored at − 20 °C until analyzed. Based on the DNA sequences
T. Sun et al. / International Immunopharmacology 22 (2014) 200–203
201
Table 1 The detailed information of PCR, CRS-PCR and PCR-RFLP analyses used for OPG genetic variants. Genetic variants
Primer sequences
Annealing temperature (°C)
PCR amplification fragment (bp)
Region
Genotype method
Restriction enzyme
Genotype (bp)
g.26395TNC
5′-GAAAGAGTTCTGACTTCAGTAAGCT-3′ 5′-AGCAATCAGCTCTCAAAGACGT-3′
58.6
214
Intron 4
CRS-PCR
HindIII
g.27649ANG
5′-CGCACTAAAGCACTCAAAGACGT-3′ 5′-TTACTCATCCATGGGATCTCGC-3′
61.6
223
Exon 5
PCR-RFLP
Tsp45I
TT: 193, 21 TC: 214, 193, 21 CC: 193, 21 AA: 183, 40 AG: 223, 183, 40 GG: 214
Note: PCR, polymerase chain reaction; CRS-PCR, created restriction site-PCR; PCR-RFLP, PCR-restriction fragment length polymorphism. Underlined nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating genetic variations.
(GenBank ID: NG_012202.1) and mRNA sequences (GenBank ID: NM_ 002546.3) of the human OPG gene, the specific polymerase chain reaction (PCR) primers were designed by Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). The details of primer sequences, annealing temperature, region, and fragment sizes are shown in Table 1. The PCR amplifications were performed in a total volume of 20 μL of reaction mixture containing 50 ng mixed DNA template, 1× buffer (Tris–HCl 100 mmol/L, pH 8.3; KCl 500 mmol/L), 0.25 μmol/L primers, 2.0 mmol/L MgCl2, 0.25 mmol/L dNTPs, and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, China). Cycling conditions consisted of an initial 95 °C for 5 min followed by 30 cycles of 94 °C for 35 s, annealing at the corresponding temperature (shown in Table 1) for 35 s, and 72 °C for 35 s, with a final extension at 72 °C for 8 min. The PCR amplified products were separated by 2.5% agarose gel electrophoresis including ethidium bromide, and then observed under UV light.
Inc.; Chicago, IL, USA). All data are shown as the mean ± SD (standard deviation of the mean). The values of BMD were adjusted by age, weight and height. The Hardy–Weinberg equilibrium (HWE) in allele and genotype frequencies of different genetic mutations was analyzed by chisquared (χ2) test. The relationships between the variables were assessed by the stepwise multiple regression and logistic regression analyses. A P value less than 0.05 was defined as a statistically significant level. 3. Results 3.1. Genotyping of OPG genetic polymorphisms Through the CRS-PCR, PCR-RFLP and DNA sequencing methods, we detected two novel genetic polymorphisms (g.26395TNC and g.27649ANG) in the present study. DNA sequence analyses indicate that the g.26395TNC genetic polymorphisms cause from T to C mutations in intron 4 of the human OPG gene. The PCR amplified products of this genetic polymorphism were digested with the HindIII restriction enzyme, and divided into three genotypes: TT (193 and 21 bp), TC (214, 193 and 21 bp), and CC (214 bp, Table 1). As for the g.27649ANG genetic polymorphism, it is a non-synonymous mutation, which is caused by A to G mutations in exon 5 of the human OPG gene and resulting into the threonine (Thr) to alanine (Ala) amino acid replacement (p.Thr362Ala, reference sequences, GenBank IDs: NG_012202.1, NM_002546.3 and NP_002537.3). The PCR amplified products of these polymorphisms were digested with the Tsp45I restriction enzyme and divided into three genotypes, AA (223 bp), AG (223,183 and 40 bp) and GG (183 and 40 bp, Table 1).
2.4. OPG genetic polymorphism genotyping The genotypes of the OPG g.26395TNC genetic variant were evaluated by the created restriction site-PCR (CRS-PCR) method with one of the primers containing a nucleotide mismatch, which enables the use of restriction enzymes for discriminating sequence variations [30–34]. The PCR-restriction fragment length polymorphism (PCR-RFLP) method has been used to investigate the genotypes of the OPG g.27649ANG genetic variant. According to the manufacturer's instructions, the PCR amplified products (5 μL) were digested with 2 U of selected restriction enzymes (Table 1, MBI Fermentas, St. Leon-Rot, Germany) at 37 °C for 10 h. The digested products were separated by electrophoresis for 1 h at 100 V in 2.5% agarose gel containing ethidium bromide. In order to confirm the genotype results from the CRS-PCR and PCR-RFLP methods, 10% of the total samples were randomly selected to be re-analyzed by a DNA sequencing method (ABI3730xl DNA Analyzer, Applied Biosystems, Foster City, CA).
3.2. Allele and genotype frequencies Table 2 shows the allele and genotype frequencies of OPG g.26395TNC and g.27649ANG genetic polymorphisms. The frequencies of allele-T of g.26395TNC and allele-A g.27649ANG genetic polymorphisms were the maximums in the primary postmenopausal osteoporosis patients and healthy controls. As for g.26395TNC, the allele and genotype frequencies of patients (T: 68.70% and C: 31.30%; TT: 49.57%, TC: 38.25%, and CC: 12.18%) showed a significant difference with those of the healthy
2.5. Statistical analysis All statistical analyses were evaluated by the Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS
Table 2 The genotypic and allelic frequencies of OPG genetic variants in the studied subjects. Groups
g.26395TNC
g.27649ANG
Genotype frequencies (%)
Allele frequencies (%)
TT
TC
CC
T
Cases (n = 468)
232 (49.57)
179 (38.25)
57 (12.18)
Controls (n = 470)
247 (52.56)
193 (41.06)
30 (6.38)
Total (n = 938)
479 (51.06)
372 (39.66)
87 (9.28)
643 293 (68.70) (31.30) 687 253 (73.09) (26.91) 1330 546 (70.90) (29.10) 2 χ = 4.3775, P = 0.0364
2
χ = 9.3717, P = 0.0092
C
Genotype frequencies (%)
Allele frequencies (%)
GG
A
69 (14.74) 40 (8.51) 109 (11.62)
605 331 (64.64) (35.36) 650 290 (69.15) (30.85) 1255 621 (66.90) (33.10) 2 χ = 4.3120, P = 0.0378
χ2
P
AA
5.7347
0.0568
0.9007
0.6374
1.4254
0.4903
206 193 (44.02) (41.24) 220 210 (46.81) (44.68) 426 403 (45.42) (42.96) 2 χ = 8.8886, P = 0.0117
AG
G
χ2
P
4.4864
0.1061
1.0477
0.5922
0.8403
0.6569
202
T. Sun et al. / International Immunopharmacology 22 (2014) 200–203
Table 3 The characteristics of OPG genetic variants in the studied subjects. Genetic variants
g.26395TNC
Genotype
TT
TC
CC
*P
AA
g.27649ANG AG
GG
*P
Number (%) Age (years) Height (cm) Weight (kg) BMI Adjusted spine BMD (g/cm2) Adjusted neck hip BMD (g/cm2) Adjusted total hip BMD (g/cm2)
479 (51.06) 60.9 ± 7.2 161 ± 7.3 61.2 ± 7.6 23.2 ± 3.45 0.933 ± 0.122 0.751 ± 0.136 0.862 ± 0.145
372 (39.66) 61.8 ± 7.1 162 ± 6.9 62.3 ± 6.6 23.3 ± 3.67 0.867 ± 0.114 0.692 ± 0.222 0.821 ± 0.118
87 (9.28) 62.7 ± 7.6 163 ± 7.1 63.0 ± 5.2 23.7 ± 3.11 0.827 ± 0.132 0.668 ± 0.119 0.802 ± 0.109
– 0.427 0.326 0.234 0.226 0.038 0.016 0.038
426 (45.42) 60.8 ± 7.9 160 ± 7.8 61.5 ± 7.7 23.2 ± 3.54 0.941 ± 0.215 0.765 ± 0.205 0.872 ± 0.104
403 (42.96) 61.5 ± 7.4 162 ± 6.9 62.8 ± 6.9 23.4 ± 3.12 0.871 ± 0.121 0.686 ± 0.196 0.822 ± 0.205
109 (11.62) 62.6 ± 7.6 163 ± 6.5 63.2 ± 5.8 23.7 ± 3.29 0.833 ± 0.212 0.657 ± 0.133 0.807 ± 0.117
– 0.378 0.422 0.348 0.318 0.027 0.036 0.029
Note: BMI, body mass index; BMD, bone mineral density (BMD values adjusted by age, weight and height); data are shown as mean ± standard deviation (SD).
controls (T: 73.09% and C: 26.91%; TT: 52.56%, TC: 41.06%, and CC: 6.38%; for allele, χ2 = 4.3775, P = 0.0364; for genotype, χ2 = 9.3717, P = 0.0092, Table 2). As for g.27649ANG, the allele and genotype frequencies of patients (A: 64.64% and G: 35.36%; AA: 44.02%, AG: 41.24%, and GG: 14.74%) showed a significant difference with those of the healthy controls (A: 69.15% and G: 30.85%; AA: 46.81%, AG: 44.68%, and GG: 8.51%; for allele, χ2 = 4.3120, P = 0.0378; for genotype, χ2 = 8.8886, P = 0.0117, Table 2). The χ2 test suggested that the allele and genotype frequencies of these two genetic mutations were in accordance with the HWE in the studied populations (P N 0.05).
these two genetic polymorphisms may contribute to BMD and osteoporosis in Chinese postmenopausal women. It would be necessary to confirm our findings in further prospective investigations with large and different ethnic populations, and to explain the pathogenesis and molecular mechanism of osteoporosis. Acknowledgements None. References
3.3. Relationship of OPG genetic polymorphisms with bone mineral density Table 3 presents the age, weight, height, body mass index (BMI), adjusted neck hip BMD, adjusted spine BMD and adjusted total hip BMD in each genotype. All data are shown as mean ± SD (BMD values adjusted by age, height and weight). As for g.26395TNC, we detected significant differences of adjusted spine BMD, adjusted neck hip BMD, and adjusted total hip BMD among different genotypes in the subjects; subjects with genotype TT showed a significantly higher value of BMD when compared with those of genotypes TC and CC (P b 0.05, Table 3). The g.27649ANG was significantly associated with adjusted spine BMD, adjusted neck hip BMD and adjusted total hip BMD. Subjects with genotype AA had a significantly higher BMD value than those of genotypes AG and GG (P b 0.05, Table 3). 4. Discussion Osteoporosis is a multifactorial and polygenic disease, and is caused by the combined effects of genetic and environmental factors [24]. It is generally accepted that the genetic factors may play a key role in the development of osteoporosis [35–40]. In recent years, the OPG gene is considered to play an important role in the pathogenesis of osteoporosis [8–15,18], and several association studies have been conducted to assess the role of OPG genetic variants (such as A163G, T245G, T950C, G1181C, g.18861ANG, g.19190CNA, and g.27406CNT) on the risk of BMD and osteoporosis [6,7,9,13,18–21,23–29]. In this case–control study, we firstly investigated the influence of g.26395TNC and g.27649ANG genetic polymorphisms of the OPG gene with BMD and osteoporosis in Chinese postmenopausal women by association analyses. Our data suggested that there were significant differences in the distribution of allelic and genotypic frequencies between primary postmenopausal osteoporosis patients and healthy controls (Table 2). The allele-T/genotype-TT of g.26395TNC and allele-A/genotype-AA of g.27649ANG genetic polymorphisms of the OPG gene might be associated with a protection from osteoporosis (Table 3). Results from this study indicate that OPG genetic polymorphisms may affect BMD and osteoporosis in Chinese postmenopausal women. In summary, the current study is the first to report the potential association between g.26395TNC and g.27649ANG genetic polymorphisms of the OPG gene and BMD and osteoporosis. Our results indicate that
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