Medical Oncology

, 30:389

Association analyses of osteoprotegerin gene polymorphisms with bone mineral density in Chinese postmenopausal women

Authors

  • Feng Zhang
    • Orthopedics DepartmentThe Second Affiliated Hospital Zhejiang University College of Medicine
  • Chunlei He
    • Orthopedics DepartmentThe Second Affiliated Hospital Zhejiang University College of Medicine
  • Gang Chen
    • Orthopedics DepartmentThe Second Affiliated Hospital Zhejiang University College of Medicine
  • Fangcai Li
    • Orthopedics DepartmentThe Second Affiliated Hospital Zhejiang University College of Medicine
    • Orthopedics DepartmentThe Second Affiliated Hospital Zhejiang University College of Medicine
    • Department of OrthopedicsThe First Affiliated Hospital of Gannan Medical University
Original Paper

DOI: 10.1007/s12032-012-0389-3

Cite this article as:
Zhang, F., He, C., Chen, G. et al. Med Oncol (2013) 30: 389. doi:10.1007/s12032-012-0389-3

Abstract

Osteoprotegerin gene (OPG) is an important candidate gene of osteoporosis. The objective of this study was to evaluate the association between OPG gene polymorphisms and bone mineral density (BMD). A total of 336 Chinese postmenopausal women were included in this study. OPG gene polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP), created restriction site–PCR (CRS–PCR) and DNA sequencing methods. BMD was evaluated at the lumbar spine (L2–4), total hip and femoral neck. Two single-nucleotide polymorphisms (SNPs) (g.21775C>T and g.23367T>C) were identified and the association analysis showed significant difference of spine BMD among different g.23367T>C genotype, subjects with the genotype TT was significantly higher than those of genotype TC and CC. Such a significant difference was not observed at the neck hip BMD and total hip BMD. The g.21775C>T polymorphism was not significantly associated with spine BMD, total hip BMD and neck hip BMD in the studied subjects. These findings suggested that OPG gene polymorphisms were associated with BMD in Chinese postmenopausal women. Results from this study will be helpful in further studies to determine the role of OPG gene in osteoporosis.

Keywords

Association analysisBone mineral densityOsteoporosisOsteoprotegerin genePostmenopausal womenSingle-nucleotide polymorphisms

Introduction

Osteoporosis is a common disease in the postmenopausal women, which is characterized by a reduction in bone mineral density (BMD) and a consequent increase in bone fracture risk [16]. Low BMD is considerate to be a major risk factor for osteoporosis, meanwhile it has high heritability [7, 8]. Several studies proved that genetic factors play an important role in the pathogenesis of osteoporosis [913]. A number of candidate genes have been investigated to be associated with BMD and osteoporosis, such as osteoprotegerin (OPG) [1420], vitamin D receptor (VDR) [21, 22], collagen type 1a1 (COL1A1) [23] and transforming growth factor b1 (TGFB1) [24]. OPG gene is an important candidate gene for BMD and osteoporosis, as polymorphisms in OPG could contribute to the genetic influence on BMD and osteoporosis [1420]. Evidence from previous studies suggested that common polymorphisms in OPG gene are likely candidates to confer risk for BMD and osteoporosis [1420]. Several OPG gene single-nucleotide polymorphisms (SNPs), such as A163G, T245G, T950C and G1181C, have been approved to be associated with BMD and osteoporosis [6, 8, 15, 19, 2530]. However, the association of g.21775C>T and g.23367T>C polymorphisms in OPG gene with BMD and osteoporosis have not been analyzed. The present study aimed to detect the OPG gene g.21775C>T and g.23367T>C polymorphisms and investigated the effect of polymorphisms on BMD and osteoporosis in Chinese postmenopausal women.

Materials and methods

Subjects

This study was performed on 204 women (aged 46–79 years) with primary postmenopausal osteoporosis. A second group consisting of 132 healthy age-matched women donors (aged 47–77 years) was collected as controls. Those suffering diseases or taking drugs which could affect bone metabolism were excluded. All subjects were genetically unrelated ethnic Han Chinese postmenopausal women. This study was approved by the local ethics committee, and all the subjects had completed written informed consent forms before any measurements.

Bone mineral density measurement

BMD of the lumbar spine (L2–4), femoral neck and total hip were measured by dual-energy X-ray absorptiometry (DEXA; Lunar Expert 1313, Lunar Corp., USA).

PCR–RFLP and CRSPCR analysis

Genomic DNA was extracted from peripheral venous blood samples according to the standard protocol. According to the DNA sequences (GenBank ID: NG_012202.1) and mRNA sequences (GenBank ID: NM_002546.3) of human OPG gene, specific PCR primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) to amplify and verify the SNPs (Table 1). Primers, annealing temperature, region, fragment sizes and selected restriction enzymes (MBI Fermentas, St. Leon-Rot, Germany) were given in Table 1. Polymerase chain reaction (PCR) amplifications were performed in 20 μl reaction mixture containing 50 ng mixed DNA template, 10 pM of each primer, 0.20 mM dNTP, 2.5 mM MgCl2 and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, China). The polymerase chain reaction (PCR) protocol was 95 °C for 5 min followed by 32 cycles of 94 °C for 30 s, annealing at the corresponding temperature (shown in Table 1) for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR products were separated on 1.5 % agarose gel. The PCR amplified products were purified using a Wizard Prep PCR purification kit and sent to the Bioasia Biotechnology Co., Ltd. (Shanghai, China) for sequencing in both directions by ABI 3730 sequencer. The g.21775C>T was genotyped by employing 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 [3135]. The g.23367T>C was investigated by PCR–restriction fragment length polymorphism (PCR–RFLP). Following the supplier’s manual, aliquots of 5 μl PCR amplified products were digested with 2 U restriction enzyme at 37 °C for 10 h. The digested products were separated by electrophoresis in 2.5 % agarose gel.
Table 1

PCR, PCR–RFLP and CRS–PCR analysis used for genotyping SNPs in OPG gene

SNPs

Primer sequences

Annealing temperature (°C)

PCR amplification fragment (bp)

Region

Restriction enzyme

Genotype (bp)

g.21775C>T

5′-GGACCCAGGTCTCCCTTTGTTAA-3′

63.8

205

Intron2

HpaI

CC:184,21

5′-CAACCCTAGCTACTCAGGAGGCTG-3′

    

CT:205,184,21

     

TT: 205

g.23367T>C

5′-GCTGGTTAAGATTCAAGAAAGGG-3′

60.8

249

Exon3

NdeI

TT: 169,80

5′-CACCCTGTAGAAAACACACAAATTG-3′

    

TC:249,169,80

     

CC:249

Underlined nucleotides mark nucleotide mismatches enabling the use of the selected restriction enzymes for discriminating sequence polymorphisms

PCR polymerase chain reaction, PCRRFLP PCR–restriction fragment length polymorphism, CRSPCR created restriction site–PCR, SNPs single-nucleotide polymorphisms

Statistical analysis

The Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS Inc.; Chicago, IL, USA) was used to analyze all statistical analyses. All data were expressed as mean ± SD (standard deviation of the mean). Hardy–Weinberg equilibrium was tested for the SNPs in the group of participants using the Chi-square (χ2) test. Allelic and genotypic frequencies were calculated by gene-counting method. Quantitative data were compared by one-way analysis of variance (ANOVA). Multiple regression and logistic regression analyses were investigated to evaluate the association between the variables. A P value of <0.05 was considered statistically significant.

Results

Identification of SNPs in OPG gene

In the present study, the g.21775C>T and g.23367T>C SNPs were detected by CRS–PCR, PCR–RFLP and DNA sequencing methods, including C → T mutation at position 21775 and T → C mutation (resulting in Isoleucine (Ile) to Methionine (Met) amino acid replacement, p.Ile184Met) at position 23367 of human OPG gene, respectively, in intron2 and exon3 (reference sequence NG_012202.1, NM_002546.3 and NP_002537.3). The PCR product of g.21775C>T was digested with HpaI enzyme and divided into three genotypes: CC (184 and 21 bp), CT (205, 184 and 21 bp) and TT (205 bp; Table 1). As for the PCR product of g.23367T>C was digested with NdeI enzyme and divided into three genotypes: TT (169 and 80 bp), TC (249, 169 and 80 bp) and CC (249 bp; Table 1).

Allelic and genotypic frequencies

The allelic and genotypic frequencies of the g.21775C>T and g.23367T>C polymorphisms were showed in Table 2. Allele C and T were predominant alleles and the value of genotype CC and TT frequencies was the maximum in the studied populations, respectively, in g.21775C>T and g.23367T>C SNPs (Table 2). The Chi-square (χ2) test of g.21775C>T and g.23367T>C in the studied subjects suggested that the polymorphism sites were corresponded to Hardy–Weinberg equilibrium (P > 0.05).
Table 2

Frequencies of genotype and allele of g.21775C>T and g.23367T>C polymorphism in the studied subjects

Groups

g.21775C>T

g.23367T>C

Genotypic frequencies

Allelic frequencies

Genotypic frequencies

Allelic frequencies

CC

CT

TT

C

T

TT

TC

CC

T

C

Case group (n = 204)

106 (0.5196)

78 (0.3824)

20 (0.0980)

290 (0.7108)

118 (0.2892)

118 (0.5784)

70 (0.3431)

16 (0.0784)

306 (0.7500)

102 (0.2500)

Control group (n = 132)

65 (0.4924)

51 (0.3864)

16 (0.1212)

181 (0.6856)

83 (0.3144)

70 (0.5303)

48 (0.3636)

14 (0.1061)

188 (0.7172)

76 (0.2879)

 

χ2 = 0.5214, P = 0.7705

χ2 = 0.4847, P = 0.4863

χ2 = 1.1129, P = 0.5733

χ2 = 1.1811, P = 0.2771

Association analysis of SNPs in OPG gene with bone mineral density

Age, weight, height, body mass index (BMI), spine BMD, neck hip BMD and total hip BMD in each genotype group were performed as mean ± SD in Table 3. Association analysis showed significant difference of spine BMD among different g.23367T>C genotype, subjects with the genotype TT was significantly higher than those of genotype TC and CC (Table 3, P < 0.05). Such a significant difference was not observed at the neck hip BMD and total hip BMD. The g.21775C>T polymorphism were not significantly associated with spine BMD, neck hip BMD and total hip BMD in the studied subjects (Table 3, P > 0.05).
Table 3

Characteristics of g.21775C>T and g.23367T>C polymorphism in the total group of subjects

SNPs

g.21775C>T

g.23367T>C

Genotype

CC

CT

TT

P

TT

TC

CC

P

Number

171 (0.5089)

129 (0.3839)

36 (0.1071)

188 (0.5595)

118 (0.3512)

30 (0.0893)

Age (years)

61.8 ± 7.8

62.6 ± 7.7

62.8 ± 6.5

0.454

61.9 ± 7.7

62.3 ± 7.5

62.6 ± 6.6

0.321

Weight (kg)

61.9 ± 7.1

62.2 ± 6.1

62.5 ± 5.3

0.339

61.7 ± 7.4

62.3 ± 6.8

62.5 ± 5.8

0.338

Height (cm)

159 ± 7.2

160 ± 6.8

161 ± 6.6

0.442

159 ± 7.7

160 ± 6.8

161 ± 6.5

0.431

BMI

23.6 ± 3.54

23.7 ± 3.19

23.9 ± 3.12

0.447

23.1 ± 3.56

23.4 ± 3.43

23.7 ± 3.22

0.324

Spine BMD (g/cm2)

0.855 ± 0.128

0.843 ± 0.121

0.838 ± 0.111

0.314

0.929 ± 0.135

0.849 ± 0.127

0.839 ± 0.125

0.037

Neck hip BMD (g/cm2)

0.723 ± 0.114

0.695 ± 0.111

0.686 ± 0.112

0.245

0.733 ± 0.102

0.695 ± 0.103

0.681 ± 0.101

0.075

Total hip BMD (g/cm2)

0.836 ± 0.109

0.821 ± 0.104

0.816 ± 0.108

0.062

0.839 ± 0.112

0.826 ± 0.105

0.822 ± 0.106

0.066

Data are shown as mean ± SD (BMD values adjusted by age and weight)

SNPs single-nucleotide polymorphisms, BMI body mass index, BMD bone mineral density

Discussion

Osteoporosis is a common and polygenic disease resulting from complex interactions between genetic and environmental factors. Previous studies have evaluated that OPG gene is an important candidate gene for mediating the genetic influence on BMD and osteoporosis [1420]. In the present study, two novel SNPs (g.21775C>T and g.23367T>C) were identified and association analysis suggested that the g.23367T>C variant in exon3 was significantly associated with spine BMD in Chinese postmenopausal women, individuals with the genotype TT was significantly higher than those of genotype TC and CC (Table 3, P = 0.037) and the TT genotype had higher total hip BMD and neck hip BMD than those of genotype TC and CC, with almost reached at the significant level (Table 3, P = 0.066 and P = 0.075). The C allele could be an increased risk for BMD and osteoporosis in Chinese postmenopausal women. As for g.21775C>T in intron2, our findings revealed that the OPG g.21775C>T polymorphism was not significantly associated with spine BMD, neck hip BMD and total hip BMD. Subjects with CC genotype had higher spine BMD, total hip BMD and neck hip BMD than those of genotype CT and TT, without reaching at the significant level (Table 3, P = 0.314, P = 0.062 and P = 0.245, respectively). Although introns were not coding sequences, the evidences were provided constantly to prove that introns played an important role on regulating mRNA splicing, transcription and gene expression and regulation [33, 36, 37]. Whether the detected g.21775C>T locus in OPG gene intron2 affected the gene expression and regulation needs to be further verified. Results from the present study provided more evidences to reveal the role of OPG gene in BMD and osteoporosis.

In conclusion, our findings suggested that the OPG g.23367T>C polymorphism was associated with BMD and osteoporosis, but not shown in g.21775C>T variant. The results from this study may be providing a framework for further analysis of the importance of OPG polymorphisms in the osteoporosis risk. Further studies investigating the association between g.21775C>T, g.23367T>C or other polymorphisms and osteoporosis are necessary to gain more reliable results on larger populations and to clarify the underlying molecular mechanism.

Conflict of interest

The authors declare that they have no conflict of interest.

Copyright information

© Springer Science+Business Media New York 2013