The Influence of g.19124G>A Genetic Polymorphism in the OPG Gene on Bone Mineral Density in Chinese Women

Abstract

Objective: The aim of this study was to detect the influence of g.19124G>A genetic polymorphism in the osteoprotegerin (OPG) gene on bone mineral density (BMD) and osteoporosis in Chinese postmenopausal women. Methods: A total of 403 primary osteoporosis subjects and 409 healthy controls were enrolled in this study. The BMD value of the femoral neck hip, lumbar spine (L_2-4), and total hip was analyzed by Norland XR-46 dual energy X-ray absorptiometry. The polymerase chain reaction-restriction fragment length polymorphism method was utilized to investigate the genotype of g.19124G>A genetic polymorphism. Results: We found significant differences of the femoral neck hip, lumbar spine (L_2-4), and total hip of BMD among different genotypes of g.19124G>A genetic polymorphism, individuals with the genotype GG had significantly higher BMD than those of genotype GA and AA (p<0.05). Conclusion: These findings indicate that the g.19124G>A genetic polymorphism in the OPG gene is potentially related to BMD and osteoporosis in Chinese postmenopausal women, and the allele A could be associated with a lower BMD and an increased risk factor for osteoporosis.

Introduction

Primary osteoporosis is a common polygenic and multifactorial disease in the postmenopausal women. It is characterized by a deterioration of bone microarchitecture with a consequent increase of fracture risk and a reduction in bone mineral density (BMD) (Cummings et al., 1985; Riggs and Melton, 1986; Peck, 1993; Kanis et al., 1994; Geng et al., 2007; Garcia-Unzueta et al., 2008; Li et al., 2012; Woo et al., 2012). BMD is a complex trait that is determined by genetic, metabolic, and environmental factors. Low BMD has high heritability, and is a major risk factor for osteoporosis (Nguyen et al., 2000; Lee et al., 2010; Ozbas et al., 2012). Genetic factors play an important function in the pathogenesis of osteoporosis (Nguyen et al., 2000; Ohmori et al., 2002; Albagha and Ralston, 2006; Ferrari, 2008; Cheung et al., 2010; Hosoi, 2010; Lee et al., 2010; Ralston, 2010; Feng et al., 2012; Ozbas et al., 2012; Woo et al., 2012; Zhang et al., 2013). A number of candidate genes have been investigated for the correlation with BMD and osteoporosis, for example, the osteoprotegerin (OPG) gene (Pocock et al., 1987; Arko et al., 2002, 2005; Hofbauer and Schoppet, 2002; Langdahl et al., 2002; Yamada et al., 2003; Vidal et al., 2011; Feng et al., 2012; Hussien et al., 2013; Zhang et al., 2013), the collagen type 1a1 (COL1A1) gene (Mann and Ralston, 2003; Falcon-Ramirez et al., 2011), the transforming growth factor b1 (TGFB1) gene (Yamada, 2001), the vitamin D receptor (VDR) gene (Fang et al., 2005; Jakubowska-Pietkiewicz et al., 2012; Kurt et al., 2012; Li et al., 2012; Horst-Sikorska et al., 2013; Hussien et al., 2013), and the estrogen receptor alpha (ERalpha) gene (Kurt et al., 2012). The OPG gene is one of the most important candidate genes for mediating genetic influence on osteoporosis. There were several studies, which indicated that the common genetic polymorphisms of OPG gene had an effect on BMD and osteoporosis (Pocock et al., 1987; Arko et al., 2002, 2005; Hofbauer and Schoppet, 2002; Langdahl et al., 2002; Yamada et al., 2003; Vidal et al., 2011; Feng et al., 2012; Zhang et al., 2013). Many genetic polymorphisms, such as A163G, T245G, T950C and G1181C, G23276A, C21775T and T23367C, have been investigated to evaluate the potential association with BMD and osteoporosis (Arko et al., 2002; Langdahl et al., 2002; Ohmori et al., 2002; Jorgensen et al., 2004; Zhao et al., 2005; Kim et al., 2007; Ueland et al., 2007; Garcia-Unzueta et al., 2008; Moffett et al., 2008; Lee et al., 2010; Feng et al., 2012; Zhang et al., 2013). Up to date, however, no similar studies report the association between g.19124G>A genetic polymorphism in the OPG gene and BMD and osteoporosis. Therefore, this study focused on evaluating the potential correlation of this genetic polymorphism with BMD and osteoporosis.

Materials and Methods

Subjects

In total, 812 subjects were enrolled from the Second Affiliated Hospital, Sun Yat-sen University (Guangzhou, China), including 403 postmenopausal women subjects with primary osteoporosis and 409 healthy age-matched subjects. All individuals were of Chinese Han nationality. Those suffering from diseases or taking drugs that are known to interfere with bone metabolism were excluded. The study was approved by the ethics committee of the Second Affiliated Hospital, Sun Yat-sen University. All participants had completed written informed consent forms.

BMD measurement

The BMD at the lumbar spine (L_2-4), femoral neck hip, and total hip were assessed using Norland XR-46 dual energy X-ray absorptiometry (DEXA; Norland Coopersurgical Corp., Fort Atkinson, WI) (Tothill et al., 1999). The value of BMD was automatically calculated from the bone mineral content (g) and bone area (cm²), and then expressed as g/cm².

Genotyping

Peripheral venous blood was collected from all subjects. Genomic DNA was isolated using the Qiagen method and stored at −80°C until analyzed. Using the Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA), we constructed the polymerase chain reaction (PCR) primers. The information of primers sequences, annealing temperature, fragment region, and genotype size are given in Table 1. The PCRs were performed in a total volume of 20 μL 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 (Qiagen, Hilden, Germany). The PCR cycling conditions were carried out as followed: an initial 5 min at 94°C followed by 32 cycles of 30 s at 94°C, 30 s at 58.5°C, and 30 s at 72°C, and a final extension of 5 min at 72°C. The g.19124G>A genetic polymorphism was genotyped by the PCR-restriction fragment length polymorphism (RFLP) method. Five microliters of each amplified PCR product was digested with 2 units of FnuDII restriction enzymes (MBI Fermentas, St. Leon-Rot, Germany) at 37°C for 10 h, electrophoresed, and observed under UV light. To verify the genotype accuracy of the PCR-RFLP method, random samples (10% of the total samples) were characterized by DNA sequencing (ABI3730xl DNA Analyzer; Applied Biosystems, Foster City, CA).

Table 1.

The PCR-RFLP Analysis Used for Detecting the g.19124G>A Genetic Polymorphism in OPG Gene

Primer sequences	Annealing temperature (°C)	PCR amplification fragment (bp)	Region	Restriction enzyme	Genotype (bp)
5′-TGACAAATGTCCTCCTGGTACC-3′	58.5	248	Exon2	FnuDII	GG:186, 62
5′-AGCTCCTATGTTTCAAGCAGAAC-3′					GA:248, 186, 62
					AA:248

PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; OPG, osteoprotegerin.

Statistical analyses

The chi-square (χ²) test was utilized to assess the Hardy-Weinberg equilibrium (HWE) for allele and genotype distributions. The one-way analysis of variance (ANOVA) and unpaired t-tests evaluated the quantitative data. All data are shown as mean±standard deviation (SD). The multiple regression analyses were used to investigate the association between variables. p-Value <0.05 was considered statistically significant. All statistical analyses were performed using the Statistical Package for Social Sciences software (SPSS 15.0; SPSS, Inc., Chicago, IL).

Results

OPG genetic polymorphism identification

In the current study, we found a novel genetic polymorphism (g.19124G>A) using PCR-RFLP, and verified by DNA sequencing methods. The sequence analyses based on the OPG gene reference sequences (GenBank IDs: NG_012202.1, NM_002546.3, NP_002537.3) suggested that this genetic polymorphism was a nonsynonymous mutation in exon2 at 19124 position of the OPG gene, causing a G→A mutation and resulting in valine (Val) to methionine (Met) amino acid replacement (p.Val104Met). The PCR products were digested with the FnuDII restriction enzyme and divided into three genotypes: GG (186 and 62 bp), GA (248, 186, and 62 bp), and AA (248 bp, Table 1).

Allelic and genotypic distribution

The allelic and genotypic distribution of the g.19124G>A genetic polymorphism in cases of osteoporosis and healthy controls are shown in Table 2. The allele-G and genotype GG were predominant in the studied populations. The allelic frequencies of osteoporosis subjects (G, 63.65%; A, 36.35%) were significantly different from those of healthy controls (G, 69.32%; A, 30.68%; χ²=5.8543, p=0.0155). In addition, genotypic frequencies in osteoporosis subjects were significantly different from those in healthy controls, the differences being statistically significant (χ²=7.1500, p=0.0280). The χ² test suggested that the distributions of genotypes corresponded to the HWE for the studied populations (p>0.05).

Table 2.

The Allelic and Genotypic Frequencies of g.19124G>A Genetic Polymorphism in OPG Gene in the Studied Subjects

	Allelic frequencies (%)		Genotypic frequencies (%)
Groups	G	A	GG	GA	AA	p	χ²
Case group (n=403)	513 (63.65)	293 (36.35)	174 (43.18)	165 (40.94)	64 (15.88)	0.0689	5.3503
Control group (n=409)	567 (69.32)	251 (30.68)	198 (48.41)	171 (41.81)	40 (9.78)	0.9417	0.1201
Total (n=812)	1080 (66.50)	544 (33.50)	372 (45.81)	336 (41.38)	104 (12.81)	0.1274	4.1212
	χ²=5.8543, p=0.0155				χ²=7.1500, p=0.0280

Association analyses

The age, height, weight, body mass index, femoral neck hip BMD, spine BMD, and total hip BMD in each genotype are shown in Table 3. All data are performed in mean±SD (BMD values adjusted by age and weight). Significant differences of femoral neck hip BMD, spine BMD, and total hip BMD among different genotypes were found in the subjects, and subjects with the genotype GG showed significantly higher values of BMD when compared with those of genotype GA and AA (p<0.05, Table 3).

Table 3.

The Characteristics of g.19124G>A Genetic Polymorphism in the Total Group of Subjects

Genotype	GG	GA	AA	p
Number (%)	372 (45.81)	336 (41.38)	104 (12.81)	—
Age (years)	62.3±8.3	63.1±7.8	63.6±7.2	0.342
Height (cm)	162±6.7	163±7.1	166±6.4	0.521
Weight (kg)	61.6±6.4	61.8±6.7	62.9±6.8	0.252
BMI	23.2±3.34	23.3±3.27	23.8±3.12	0.429
Femoral neck hip BMD (g/cm²)	0.747±0.111	0.696±0.153	0.678±0.135	0.041
Spine BMD (g/cm²)	0.943±0.122	0.851±0.129	0.831±0.213	0.035
Total hip BMD (g/cm²)	0.880±0.102	0.826±0.115	0.801±0.314	0.021

Data are shown as mean±standard deviation (BMD values adjusted by age, height, and weight).

BMD, bone mineral density; BMI, body mass index.

Discussion

Primary osteoporosis is a systemic skeletal disease caused by the combined effects of environmental and genetic factors (Ohmori et al., 2002; Zhao et al., 2005). Genetic factors have been considered to play key roles in the development of osteoporosis (Nguyen et al., 2000; Ohmori et al., 2002; Albagha and Ralston, 2006; Ferrari, 2008; Cheung et al., 2010; Hosoi, 2010; Lee et al., 2010; Ralston, 2010; Feng et al., 2012; Ozbas et al., 2012; Woo et al., 2012; Zhang et al., 2013). The OPG gene is one of the most potentially likely genes to affect BMD and osteoporosis (Pocock et al., 1987; Arko et al., 2002, 2005; Hofbauer and Schoppet, 2002; Langdahl et al., 2002; Yamada et al., 2003; Vidal et al., 2011; Feng et al., 2012; Hussien et al., 2013; Zhang et al., 2013). Previous studies indicated that genetic polymorphisms of the OPG gene could affect BMD and cause osteoporosis (Pocock et al., 1987; Arko et al., 2002, 2005; Hofbauer and Schoppet, 2002; Langdahl et al., 2002; Yamada et al., 2003; Vidal et al., 2011; Feng et al., 2012; Hussien et al., 2013; Zhang et al., 2013). However, the results from these observations are still inconsistent. In the current study, we evaluated the relevance of OPG genetic polymorphisms in relation to BMD and osteoporosis using association analysis. We first detected the g.19124G>A genetic polymorphism in exon2 of the OPG gene using PCR-RFLP, and examined the potential association with BMD and osteoporosis. Our data indicated that there were statistically significant associations between this genetic polymorphism and BMD and osteoporosis in Chinese postmenopausal women; individuals with the genotype GG had significantly higher BMD than those of GA and AA genotypes (p<0.05, Table 3). The allele G could be a decreased risk for BMD and osteoporosis. Previous studies have reported that many genetic polymorphisms, for example, A163G, T245G, T950C and G1181C, G23276A, C21775T and T23367C, have been found to have correlations with BMD and osteoporosis, which were consistent with our findings that genetic polymorphisms in the OPG gene may have an important genetic influence on BMD and osteoporosis (Arko et al., 2002; Langdahl et al., 2002; Ohmori et al., 2002; Jorgensen et al., 2004; Zhao et al., 2005; Kim et al., 2007; Ueland et al., 2007; Garcia-Unzueta et al., 2008; Moffett et al., 2008; Lee et al., 2010; Feng et al., 2012; Zhang et al., 2013). The g.19124G>A genetic polymorphism is a nonsynonymous mutation and causes Val to Met amino acid replacement, and it might alter the function of the OPG protein. This genetic polymorphism may be linked to other known nonsynonymous genetic polymorphisms, such as lysine (Lys)3 asparagine (Asn), isoleucine (Ile)184Met, and threonine (Thr)154Met. These genetic polymorphisms were significantly associated with the risk of BMD and osteoporosis (Zhao et al., 2005; Feng et al., 2012; Zhang et al., 2013). Our findings could provide more evidence to investigate the role of the OPG gene in BMD and osteoporosis. Further studies are necessary to evaluate the association between this genetic polymorphism, or other genetic polymorphisms and osteoporosis in larger different populations, and to elucidate the underlying molecular mechanism.

Footnotes

Author Disclosure Statement

The authors declare that they have no conflict of interests.

References

Albagha

, Ralston

. 2006. Genetics and osteoporosis. Rheum Dis Clin North Am, 32:659-680.

Arko

, Prezelj

, Kocijancic

et al. 2005. Association of the osteoprotegerin gene polymorphisms with bone mineral density in postmenopausal women. Maturitas, 51:270-279.

Arko

, Prezelj

, Komel

et al. 2002. Sequence variations in the osteoprotegerin gene promoter in patients with postmenopausal osteoporosis. J Clin Endocrinol Metab, 87:4080-4084.

Cheung

, Xiao

, Kung

. 2010. Genetic epidemiology of age-related osteoporosis and its clinical applications. Nat Rev Rheumatol, 6:507-517.

Cummings

, Kelsey

, Nevitt

et al. 1985. Epidemiology of osteoporosis and osteoporotic fractures. Epidemiol Rev, 7:178-208.

Falcon-Ramirez

, Casas-Avila

, Miranda

et al. 2011. Sp1 polymorphism in collagen I alpha1 gene is associated with osteoporosis in lumbar spine of Mexican women. Mol Biol Rep, 38:2987-2992.

Fang

, van Meurs

, d'Alesio

et al. 2005. Promoter and 3′-untranslated-region haplotypes in the vitamin D receptor gene predispose to osteoporotic fracture: the rotterdam study. Am J Hum Genet, 77:807-823.

Feng

, Meng

, Wang

et al. 2012. Single-nucleotide polymorphism of the osteoprotegerin gene and its association with bone mineral density in Chinese postmenopausal women. J Pediatr Endocrinol Metab, 25:1141-1144.

Ferrari

. 2008. Human genetics of osteoporosis. Best Pract Res Clin Endocrinol Metab, 22:723-735.

10.

Garcia-Unzueta

, Riancho

, Zarrabeitia

et al. 2008. Association of the 163A/G and 1181G/C osteoprotegerin polymorphism with bone mineral density. Horm Metab Res, 40:219-224.

11.

Geng

, Yao

, Yang

et al. 2007. Association of CA repeat polymorphism in estrogen receptor beta gene with postmenopausal osteoporosis in Chinese. J Genet Genomics, 34:868-876.

12.

Hofbauer

, Schoppet

. 2002. Osteoprotegerin gene polymorphism and the risk of osteoporosis and vascular disease. J Clin Endocrinol Metab, 87:4078-4079.

13.

Horst-Sikorska

, Dytfeld

, Wawrzyniak

et al. 2013. Vitamin D receptor gene polymorphisms, bone mineral density and fractures in postmenopausal women with osteoporosis. Mol Biol Rep, 40:383-390.

14.

Hosoi

. 2010. Genetic aspects of osteoporosis. J Bone Miner Metab, 28:601-607.

15.

Hussien

, Shehata

, Karam

et al. 2013. Polymorphism in vitamin D receptor and osteoprotegerin genes in Egyptian rheumatoid arthritis patients with and without osteoporosis. Mol Biol Rep, 40:3675-3680.

16.

Jakubowska-Pietkiewicz

, Mlynarski

, Klich

et al. 2012. Vitamin D receptor gene variability as a factor influencing bone mineral density in pediatric patients. Mol Biol Rep, 39:6243-6250.

17.

Jorgensen

, Kusk

, Madsen

et al. 2004. Serum osteoprotegerin (OPG) and the A163G polymorphism in the OPG promoter region are related to peripheral measures of bone mass and fracture odds ratios. J Bone Miner Metab, 22:132-138.

18.

Kanis

, Melton

3rd , Christiansen

et al. 1994. The diagnosis of osteoporosis. J Bone Miner Res, 9:1137-1141.

19.

Kim

, Kim

et al. 2007. Association between osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB (RANK), and RANK ligand (RANKL) gene polymorphisms and circulating OPG, soluble RANKL levels, and bone mineral density in Korean postmenopausal women. Menopause, 14:913-918.

20.

Kurt

, Yilmaz-Aydogan

, Uyar

et al. 2012. Evaluation of ERalpha and VDR gene polymorphisms in relation to bone mineral density in Turkish postmenopausal women. Mol Biol Rep, 39:6723-6730.

21.

Langdahl

, Carstens

, Stenkjaer

et al. 2002. Polymorphisms in the osteoprotegerin gene are associated with osteoporotic fractures. J Bone Miner Res, 17:1245-1255.

22.

Lee

, Woo

, Choi

et al. 2010. Associations between osteoprotegerin polymorphisms and bone mineral density: a meta-analysis. Mol Biol Rep, 37:227-234.

23.

, Xi

, Li

et al. 2012. Association between vitamin D receptor gene polymorphisms and bone mineral density in Chinese women. Mol Biol Rep, 39:5709-5717.

24.

Mann

, Ralston

. 2003. Meta-analysis of COL1A1 Sp1 polymorphism in relation to bone mineral density and osteoporotic fracture. Bone, 32:711-717.

25.

Moffett

, Oakley

, Cauley

et al. 2008. Osteoprotegerin Lys3Asn polymorphism and the risk of fracture in older women. J Clin Endocrinol Metab, 93:2002-2008.

26.

Nguyen

, Blangero

, Eisman

. 2000. Genetic epidemiological approaches to the search for osteoporosis genes. J Bone Miner Res, 15:392-401.

27.

Ohmori

, Makita

, Funamizu

et al. 2002. Linkage and association analyses of the osteoprotegerin gene locus with human osteoporosis. J Hum Genet, 47:400-406.

28.

Ozbas

, Tutgun Onrat

, Ozdamar

. 2012. Genetic and environmental factors in human osteoporosis. Mol Biol Rep, 39:11289-11296.

29.

Peck

. 1993. Consensus development conference: diagnosis, prophylaxis, and treatment of osteoporosis. Am J Med, 94:646-650.

30.

Pocock

, Eisman

, Hopper

et al. 1987. Genetic determinants of bone mass in adults. A twin study. J Clin Invest, 80:706-710.

31.

Ralston

. 2010. Genetics of osteoporosis. Ann N Y Acad Sci, 1192:181-189.

32.

Riggs

, Melton

3rd.

1986. Involutional osteoporosis. N Engl J Med, 314:1676-1686.

33.

Tothill

, Laskey

, Orphanidou

et al. 1999. Anomalies in dual energy X-ray absorptiometry measurements of total-body bone mineral during weight change using Lunar, Hologic and Norland instruments. Br J Radiol, 72:661-669.

34.

Ueland

, Bollerslev

, Wilson

et al. 2007. No associations between OPG gene polymorphisms or serum levels and measures of osteoporosis in elderly Australian women. Bone, 40:175-181.

35.

Vidal

, Formosa

, Xuereb-Anastasi

. 2011. Functional polymorphisms within the TNFRSF11B (osteoprotegerin) gene increase the risk for low bone mineral density. J Mol Endocrinol, 47:327-333.

36.

Woo

, Kim

, Lee

. 2012. Heterogeneous genetic associations of nucleotide sequence variants with bone mineral density by gender. Mol Biol Rep, 39:2259-2265.

37.

Yamada

. 2001. Association of polymorphisms of the transforming growth factor-beta1 gene with genetic susceptibility to osteoporosis. Pharmacogenetics, 11:765-771.

38.

Yamada

, Ando

, Niino

et al. 2003. Association of polymorphisms of the osteoprotegerin gene with bone mineral density in Japanese women but not men. Mol Genet Metab, 80:344-349.

39.

Zhang

, He

, Chen

et al. 2013. Association analyses of osteoprotegerin gene polymorphisms with bone mineral density in Chinese postmenopausal women. Med Oncol, 30:389.

40.

Zhao

, Liu

, Ning

et al. 2005. The influence of Lys3Asn polymorphism in the osteoprotegerin gene on bone mineral density in Chinese postmenopausal women. Osteoporos Int, 16:1519-1524.