Association between g.19163A>G and g.23298T>C genetic variants of the osteoprotegerin gene and bone mineral density in Chinese women

Address for correspondence:
Zhi Yi, Department of Orthopaedics, Shaanxi Provincial People’s Hospital, NO.256 Youyi West Road, Xi’an 710068, Shaanxi province, People’s Republic of China, Tel.: +86-29-85236987, Fax: +86-29-85236987, E-mail: zhiyi_12@sina.com

Received 06-03-2013, Accepted 29-04-2013

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INTRODUCTION

Osteoporosis is a common polygenic health problem of considerable complexity and importance, particularly in postmenopausal women. It is characterized by a reduction in bone mineral density (BMD) and a microarchitectural deterioration of bone tissue with a consequent increase of fracture risk.^1-6 Evidence from previous studies suggested that BMD is a complex trait that is caused by multiple genes and environmental factors.⁷ A large number of studies have demonstrated that genetic factors may play a significant role in the pathogenesis of osteoporosis.^8-13 To date, several genes have been verified as being involved in bone remodeling, bone mineral homeostasis, and bone matrix composition, including osteoprotegerin (OPG),^14-23 vitamin D receptor (VDR),^21,24-28 estrogen receptor (ESR),^29-31 collagen type 1a1 (COL1A1),³² and transforming growth factor b1 (TGFB1).³³ Previous studies indicated that the OPG gene is considered to be one of the most important candidate genes in the pathogenesis of osteoporosis.^14-23 The single nucleotide polymorphisms (SNPs) in the OPG gene may contribute to genetic effects on osteoporosis and BMD.^14-20,22,23 The correlation analyses between the OPG genetic variants and BMD have been analyzed in osteoporosis, such as A163G, T245G, T950C and G1181C.^{6,7,15,19,22,23,34-39} However, the association of g.19163A>G and g.23298T>C SNPs of the OPG gene with osteoporosis and BMD have not previously been published. Therefore, the aim of this study was to evaluate the association between these two SNPs and osteoporosis and BMD in postmenopausal women.

MATERIALS AND METHODS

Subjects

A total of 739 postmenopausal women were enrolled in this study, including 367 primary postmenopausal osteoporosis patients (aged 48-79 years) and 372 healthy controls (aged 47-79 years). All subjects were genetically unrelated and of the Chinese Han population. Subjects were excluded when suffering from diseases or taking drugs that could affect bone metabolism. The present study was approved by the local ethics committee and all participants gave their informed consent.

Bone mineral density measurement

The BMD of the lumbar spine (L_2-4), neck, and total hip were evaluated through dual energy X-ray absorptiometry (DEXA) (Lunar Expert 1313, Lunar Corp., USA). The value of BMD was automatically calculated from bone mineral content (g) and bone area (cm²) and then expressed as g/cm.²

Genotyping of genetic variants

Genomic DNA was isolated from peripheral venous blood samples using the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Germany), in accordance with the supplier manual, and then stored at -20°C until analyzed. Polymerase chain reaction (PCR) primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA) based on the DNA sequences (GenBank ID: NG_012202.1) and mRNA sequences (GenBank ID: NM_002546.3) of the human OPG gene. Primer sequences, annealing temperature, region, and fragment sizes are presented in Table 1. PCR amplifications were carried out in a 20 μL reaction mixture containing 50 ng mixed DNA template, 10 pM each primer, 0.20 mM dNTPs, 2.5 mM MgCl₂ and 0.5 U Taq DNA polymerase (TaKaRa, Dalian, China). The protocol of PCR was 95°C for 5 minutes followed by 32 cycles of 94°C for 30 seconds, annealing at the corresponding temperature (shown in Table 1) for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. The PCR amplified products were separated on a 2.5% agarose gel including ethidium bromide. The g.19163A>G variant was genotyped by the PCR-restriction fragment length polymorphism (PCR-RFLP) method. The g.23298T>C variant was detected 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.^40-44 Aliquots of 5 μL PCR amplified products were digested with 2 Uselected restriction enzymes (MBI Fermentas, St. Leon-Rot, Germany) at 37°C for 10 h following the manufacturer’s instructions (Table 1), and then were separated by electrophoresis for 1 h at 100 V in a 1.5% agarose gel. Random samples (10% of the total samples) were analyzed by DNA sequencing methods with an ABI 3730 sequencer (Bioasia Biotechnology Co., Ltd. Shanghai, China) to verify the genotype results of allelic variation, which were based on the electrophoretic pattern of the restriction enzyme-treated PCR products.

Statistical analyses

Allele and genotype frequencies were calculated in the studied subjects. The chi-squared test (χ²) was used to assess the Hardy-Weinberg equilibrium of the genotypes of different genetic mutations. The BMD values were adjusted by age, height, and weight through stepwise multiple regression and logistic regression analyses. The one-way analysis of variance (ANOVA) and least-significant difference (LSD) post hoc test were utilized to compare the quantitative data. All statistical analyses were performed with the Statistical Package for Social Sciences software (SPSS, Windows version release 15.0; SPSS Inc.; Chicago, IL, USA). All data were presented as the mean ± SD (standard deviation of the mean). A P value of <0.05 was considered statistically significant.

RESULTS

SNPs genotyping

In the present study, two novel genetic variants (g.19163A>G and g.23298T>C) were detected by PCR-RFLP, CRS-PCR, and DNA sequencing methods, including A→G mutation (resulting in phenylalanine (Phe) to leucine (Leu) amino acid replacement, p.Phe117Leu) at position 19163 and T→C mutation (not resulting in amino acid replacement, p.Arginine (Arg)161Arg) at position 23298 of the human OPG gene, respectively in exon2 and exon3 (reference sequences, GenBank IDs: NG_012202.1, NM_002546.3 and NP_002537.3). The PCR amplified products of g.19163A>G was digested with SacI restriction enzyme and divided into three genotypes: AA (256 bp), AG (256, 183 and 73 bp) and GG (183 and 73 bp) (Table 1). The PCR amplified products of g.23298T>C was digested with the TaqI restriction enzyme and divided into three genotypes: TT (190 and 20 bp), TC (210, 190 and 20 bp), and CC (210 bp) (Table 1).

Genotype and allele frequencies

The result of the χ² test for g.19163A>G and g.23298T>C variants in the studied populations indicated that these genetic variants were in the Hardy-Weinberg equilibrium (P >0.05). Table 2 presents the genotype and allele frequencies of the g.19163A>G and g.23298T>C variants. As for the g.19163A>G variant, the allele frequencies of the cases (A: 55.59% and G: 44.41%) were significantly different from those of the controls (A: 64.65% and G: 35.35%, P < 0.001, χ² test, Table 2). Additionally, the genotype frequencies in cases were not similar to those of controls, the differences being statistically significant (P = 0.003, χ² test, Table 2). As for the g.23298T>C variant, the allele and genotype frequencies of cases (for allele, T: 71.25% and C: 28.75%; for genotype, TT: 52.04%, TC: 38.42%, and CC: 9.54%) showed differences from those of the controls (for allele, T: 74.33% and C: 25.67%; for genotype, TT: 54.03%, TC: 40.59%, and CC: 5.38%), the differences, however, not being statistically significant (for allele, P = 0.184; for genotype, P = 0.098, χ² test, Table 2).

Association between OPG genetic variants and bone mineral density

Age, height, weight, body mass index (BMI), spine BMD, neck hip BMD, and total hip BMD in each genotype group were presented as the mean ± SD (Table 3). The results of this study suggest that g.19163A>G SNP is significantly associated with adjusted spine BMD, neck hip BMD, and total hip BMD. Subjects with genotype AA had significantly higher BMD value than thoseof genotypes AG and GG (P <0.05, Table 3). However, we failed to detect any significant association between g.23298T>C SNP and adjusted spine BMD, neck hip BMD, and total hip BMD in the present study (P >0.05, Table 3).

DISCUSSION

The OPG gene is an important candidate gene in the pathogenesis of osteoporosis.^14-22 Several studies have reported the association between genetic variants and osteoporosis and BMD.^{6,7,15,19,22,23,34-39} However, the results from these observations still remain poorly understood. In this study, we firstly detected the association of g.19163A>G and g.23298T>C SNPs of the OPG gene with osteoporosis and BMD. Our data indicated that g.19163A>G was significantly associated with osteoporosis and BMD. Allele-A could be a risk factor for osteoporosis in Chinese postmenopausal women. As for g.23298T>C, we failed to detect any statistically significant association between this variant and adjusted spine BMD, neck hip BMD, and total hip BMD (P >0.05). It seems that the allele-C might have potential effects on osteoporosis and BMD. These findings suggest that the OPG gene is related to osteoporosis and BMD in Chinese postmenopausal women. Previously published studies have demonstrated the association between SNPs (e.g., A163G, T245G, T950C and G1181C) osteoporosis and BMD in different ethnicities, which is consistent with our findings, and that genetic variants in the OPG gene may contribute to genetic effects on osteoporosis and BMD.^{6,7,15,19,22,23,34-39} Further studies in larger populations to investigate the association of g.19163A>G, g.23298T>C, or other genetic variants, with osteoporosis and BMD are necessary to verify these results and to further elucidate the pathogenesis and molecular mechanism of osteoporosis.

In conclusion, our data suggest that the OPG g.19163A>G variant could be associated with osteoporosis and BMD. The results of this study could form the basis for further analysis of the implication of OPG genetic variants in osteoporosis risk factors.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

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