Anthropol. Anz. 72/3 (2015), pp.

311–319 Article
J. Biol. Clinic. Anthropol.
published online 25 June 2015; published in print September 2015

The effect of A163G polymorphism in the osteoprotegerin

gene on osteoporosis related traits in Slovak postmeno-
pausal women

Vladimira Krajcovicova1, Radoslav Omelka1, Jana Durisová1,

Monika Martiniakova2, Drahomir Galbavy3, and Maria Bauerova1
1
Department of Botany and Genetics, Constantine the Philosopher University in Nitra, Nitra,
Slovak Republic
vkrajcovicova@ukf.sk
2
Department of Zoology and Anthropology, Constantine the Philosopher University in Nitra,
Nitra, Slovak Republic
3
Private Orthopedic Ambulance, Nitra, Slovak Republic

With 1 figure and 5 tables

Summary: Osteoprotegerin (OPG) plays an important role in the osteoclast differentiation as

an effective inhibitor of osteoclast maturation and activation. We examined a potential effect
of A163G single nucleotide polymorphism in the promoter region of the OPG gene on femo-
ral neck (FN-BMD) and lumbar spine BMD (LS-BMD), as well as circulating alkaline phos-
phatase, osteocalcin (ALP, OC; formation markers), beta-CrossLaps (CTx; resorption
marker) in Slovak postmenopausal women. In addition, fractures of spine, radius and femur
were examined. Altogether 284 women (62.28 ± 8.40 years) were selected according to strict
inclusion criteria. The polymorphism was detected by PCR-RFLP method. Genotype fre-
quencies were tested using the chi-square test. The differences of quantitative variables
between the genotypes were analyzed by covariance analysis (GLM procedure) after correc-
tion of the measurements for age and BMI. Fracture incidence in association with OPG geno-
type was evaluated by Binary Logistic Regression with the genotype, age, and BMI as covari-
ates. The frequencies of genotypes were 76.8 %, 21.1 %, and 2.1 % for AA, AG, and GG,
respectively. Statistically significant associations of OPG genotypes with FN-BMD (p <
0.01) and LS-BMD (p < 0.05) were observed. The GG genotype was associated with higher
BMD values likewise decreased CTx concentration (p < 0.05) in compared with the other
genotypes, which indicates that the allele G has a protective effect on bone. These associa-
tions were not followed by the effect of OPG on fracture incidence. Our results suggest that
OPG/A163G polymorphism could contribute to the genetic regulation of BMD or bone turn-
over markers in Slovak population and thus could increase or decreased osteoporosis risk.

Key words: Osteoprotegerin gene, A163G polymorphism, osteoporosis, bone mineral den-
sity, bone turnover markers, fracture incidence.

Introduction
Osteoporosis is a skeletal disorder characterized by a low bone mass and microarchi-
tectural deterioration of bone tissue connected with increased fracture risk (Pocock et

쏘 2015 E. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart, Germany www.schweizerbart.de

DOI: 10.1127/anthranz/2015/0494 0003-5548/15/0494 $ 2.25
eschweizerbart_XXX
312 Vladimira Krajcovicova et al.

al. 1987). The main reason for disease manifestation is an imbalance between bone
formation and bone resorption caused by changes in several systemic hormones and
local factors (Manolagas 2000). Osteoclastogenesis is a tightly regulated process in
bone remodeling, in which osteoprotegerin (OPG, also known as TNFRS11B), a sol-
uble decoy receptor, modulates the RANK/RANKL signaling. OPG is secreted by
stromal cells and other cell types including osteoblast cells (Simonet et al. 1997,
Bezerra et al. 2005). RANKL (Receptor Activator of Nuclear Factor-κB Ligand) is
produced by osteoblasts/stromal cells and binds to its receptor RANK (Receptor
Activator of Nuclear Factor-κB), expressed by osteoclast precursors. OPG recognizes
RANKL and blocks interactions between RANK and RANKL. This OPG activity
leads to a modulation of osteoclast function and bone metabolism (Boyce & Xing
2007, Reid & Holen 2008). Crucial role lies in the inhibition of the osteoclast differ-
entiation, maturation and activation (Burgess et al. 1999).
It is well-known that bone mineral density (BMD) and other osteoporosis related
traits are under the strong genetic control. Genetic variability in responsible genes could
therefore affect the bone remodeling process and the variability of BMD, bone mass and
bone quality. Over the last decades a number of genes and alleles involved in the patho-
genesis of osteoporosis were identified (Huang et al. 2003). Candidate-gene association
studies play a key role in genetic of complex diseases. Osteoprotegerin gene belongs
to important osteoporosis candidate genes as evidenced by numbers of studies where
several polymorphisms were identified but with inconsistent results (Arko et al. 2002,
Langdahl et al. 2002, Casado-Dı́as et al. 2007, Ueland et al. 2007).
The aim of our study was to evaluate the genotype frequency and the influence of
individual genotypes of OPG/A163G (rs310 2735) polymorphism to femoral (FN-
BMD) and spinal BMD (LS-BMD), bone turnover markers and fracture incidence in
the Slovak population of postmenopausal women.

Material and methods

Study population
A total of 284 postmenopausal women (62.28 ± 8.40 years) were finally included in the study.
Women were selected according to the strict inclusion criteria. We excluded women with seri-
ous internal, endocrine, chronic and hereditary diseases, patients treated with certain medica-
tions (glucorcorticoids, hormones), obese women (body mass index more than 30), women
with significant abuse (alcoholism, excessive smoking and excessive caffeine intake) and
serious disturbances in the menstrual cycle. The baseline assessment was focused on the
medical and familiar history (anamnesis) of tested women, age, body mass index (BMI) and
BMD measurements, bone turnover markers, biochemical analysis and fracture incidence
evaluation.

Anamnesis and anthropometric data collection

Personal and family history, age, life style habits and diet (mainly calcium intake) were exam-
ined using a questionnaire that was completed by the subjects and reviewed by the qualified
practitioner. According to the calcium intake the subjects were divided into three groups – A,
B, and C, for average daily calcium intake of 250 mg, 500 mg, and 750 mg, respectively. BMI
was calculated from weight and height according to the World Health Organization (1995). In
addition, the information about fractures incidence was statistically evaluated in the study.
For this purpose we utilized information about presence (P) or absence (A) of spinal (also

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 Effect of A163G polymorphism in the osteoprotegerin gene on osteoporosis 313

included compression fractures), radius and femoral fractures detected on radiographs. The
fractures were identified and evaluated by qualified orthopedist.

BMD and biochemical measurements

Using dual-energy X-ray absorptiometry (DXA, HOLOGIC) the LS-BMD at the lumbar
spine (L2–4) and FN-BMD at the femoral neck were evaluated. The value of BMD was auto-
matically calculated from bone mineral content (g) and bone area (cm2) and then expressed
as T-score. Finally, the BMDs were categorized according to an accepted classification
(World Health Organization, 1994) as osteoporosis (T score 울 2.5 SD), osteopenia (–1 울 T-
score 욷 –2.5 SD), or normal BMD (T-score 욷 –1). Biochemical markers of bone remodeling
– bone isoenzyme of alkaline phosphatase (ALP; μkat/l), serum osteocalcin (OC; μg/l) as
osteoformation markers and serum beta CrossLaps (CTx; ng/l) as an osteoresorption marker
– were measured by electrophoretic and ELISA tests within the diagnostic screening.

Genotypic analysis of OPG/A163G polymorphism

Genomic DNA was purified from whole blood using a kit Simax Genomic DNA Extraction
kit (SBS Genetech, China). PCR was carried out in a total volume of 20 μl and amplification
was performed in thermocycler Primus 25/96 (MWG-BIOTECH). The PCR product was
generated using 5’-CTGGAGACATATAACTTGAACA-3’ as the forward primer and 5’-
CCATCATCAAAAGGCCTATTGGT-3’ as the reverse primer (Langdahl et al. 2002) to pro-
duce a 300 bp fragment. PCR was performed in 45 cycles with the following steps: 94 °C for
5 minutes and then 94 °C for 30 seconds, 62 °C for 45 seconds, and 72°C for 45 seconds. The
PCR was completed by a final extension cycle at 72 °C for 7 minutes. The PCR products
were digested overnight with restriction enzyme VspI (Fermentas) at 37°C. The products
were separated on 4 % agarose gel containing 0.5 μg ml–1 ethidium bromide. Documentation
of the gel was provided by DNR Bio-Imaging Systems (MiniBIS Pro, Israel).

Statistical analysis
Genotype distribution was tested for Hardy-Weinberg equilibrium using the chi-square test.
The differences of quantitative variables among the genotypes were analyzed by covariance
analysis (General Linear Model procedure) after correction of the measurements for age and
BMI. For an evaluation of categorical dependent variables (e.g. fracture incidence) we used
Binary Logistic Regression with the genotype, age, and BMI as covariates. Age and BMI
have been selected since they dispose significant effect on analyzed traits according to used
statistical methods (Table 1). Statistical analysis was realized using SPSS software version
17.0 (SPSS Inc.; Chicago, IL, USA). The p-value of less than 0.05 was considered to be sta-
tistically significant.
All information related to the study was obtained from subjects with their informed con-
sent in an anonymous form by the qualified practitioner. All procedures in our study were
approved by the Ethical Committee of the Specialized Hospital of St. Svorad in Nitra.

Results
The baseline characteristics of the study population are given in Tables 2 and 3. OPG
genotypes of each subject were identified according to the digestion pattern and
alleles according to the absence (“A”) or presence (“G”) of the VspI restriction site.
The restriction endonuclease digests 300 bp PCR product into two fragments – 162
bp and 138 bp (Fig. 1).

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314 Vladimira Krajcovicova et al.

Table 1. Effects of age, BMI and diet (calcium intake) as covariates on analyzed traits.
Trait Covariates (p-value)
age BMI diet (calcium intake)
FN-BMD 0.001 0.196 0.445
LS-BMD 0.001 0.002 0.238
ALP 0.003 0.012 0.477
OC 0.113 0.044 0.759
CTx 0.011 0.040 0.813
Fracture incidence 0.001 0.134 0.241

Table 2. Baseline characteristics of the total study population (n = 284).

Baseline characteristics Mean ± SD
Age (years) 62.28 ± 8.40
Body mass index 27.67 ± 1.43
Femur-BMD (T-score) –1.78 ± 0.56
Spinal BMD (T-score) –2.33 ± 0.75
Isoenzyme of alkaline phosphatase (μkat l–1) 0.57 ± 0.71
Osteocalcin (μg l–1) 3.80 ± 1.02
BetaCrosslaps (ng l–1) 697.94 ± 245.17
SD – standard deviation

Table 3. Incidences of fractures in studied population.

Fractures Presence (P) Absence (A)
Spinal fractures 69 215
Radius fractures 43 241
Femoral fractures 3 281

M AG AA AA AG AA AA AA AA GG

300 bp

162 bp
138 bp

Fig. 1. Representative results of PCR-RFLP; M – marker (50 bp), AA, AG, GG – genotypes.

The distribution of OPG/A163G genotypes and allele frequencies in the Slovak

population was in Hardy-Weinberg equilibrium (χ2 = 0.361, p = 0.835). In the ana-
lyzed population AA genotype (76.8 %) was the most frequent; the least frequent was

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 Effect of A163G polymorphism in the osteoprotegerin gene on osteoporosis 315

GG genotype (2.1 %). The frequency of the heterozygous AG genotype was 21.1 %.
The allelic frequencies of “A” and “G” alleles were 0.87 and 0.13, respectively.
Table 4 shows an association of A163G polymorphism with analyzed osteoporotic
traits. We found a statistically significant effect of OPG genotypes on FN-BMD (p <
0.01) and L-BMD (p < 0.05). The T-score for BMD parameters were significantly
higher in the subjects with GG genotype as compared with AA and AG genotypes.
Moreover, GG genotype was significantly associated with bone turnover markers.
Significantly decreased CTx concentrations (p < 0.05) were observed in this group.
We did not find ALP and OC concentrations to be different across the genotypes;
however, the differences in ALP and OC levels between GG and the other genotypes
were close to the significance level (p = 0.054). Finally, the fracture incidence was
not associated with A163G genotypes in total, as well as in any skeletal site (Table 5).
We did not include femoral fractures in this evaluation because of small number of
femoral fracture carriers.

Table 4. Associations of OPG/A163G genotypes with analyzed traits.

Genotypes p-value
AA AG GG
n = 218 n = 60 n=6
FN-BMD –1.77 ± 0.04 –1.88 ± 0.07 –1.24 ± 0.21 AGvsGG:0.004
(T-score) AAvsGG:0.013
LS-BMD –2.30 ± 0.05 –2.48 ± 0.09 –1.76 ± 0.29 AGvsGG:0.016
(T-score)
Isoenzyme of alkaline 0.54 ± 0.05 0.74 ± 0.09 0.25 ± 0.29 NS
phosphatase (μkat l–1)
Osteocalcin (μg l–1) 3.76 ± 0.07 3.82 ± 0.13 3.16 ± 0.46 NS
Beta-Crosslaps (ng l–1) 704.88 ± 16.10 693.05 ± 30.81 494.66 ± 97.45 AAvsGG:0.034
Data are presented as Mean ± SE (SE – standard error of the mean), values are adjusted
for age and BMI; NS – not significant

Table 5. Distribution of fracture incidence and effect of OPG/A163G genotypes on fracture

incidence (femoral fractures not evaluated).
Fractures Distribution B SE p-value Exp(B) 95 % CI for
of genotypes Exp(B)
AA AG GG AA-AG
Spinal P 55 14 0 0.257 0.396 0.515 1.293 0.595 2.811
A 163 46 6
Radius P 33 10 0 0.015 0.422 0.971 1.407 1.044 1.897
A 185 50 6
Total P 64 16 0 0.314 0.369 0.393 1.369 0.664 2.822
A 154 44 6
P– presence of fractures; A – Absence of fractures; B – parameter estimate; SE – standard
error of the estimate; Exp(B) – exponentiated B; CI – confidence interval; tested by binary
logistic regression with adjusting for age and BMI

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316 Vladimira Krajcovicova et al.

Discussion
Since OPG has an important role as an inhibitor of osteoclast differentiation, poly-
morphisms in the gene coding for OPG (located on chromosome 8q24.13; GenBank
accession number U94 332) might influence the bone remodeling process, and OPG
could thus be a candidate gene for identifying individuals at risk of developing low
bone mass or osteoporosis (Jørgensen et al. 2004). The A163G polymorphism in the
osteoprotegerin gene located in the promoter region was identified by Kusk (2000).
Although it does not affect any recognition sites of the known transcription factors
there is a possibility that the OPG polymorphism is in a linkage disequilibrium (LD)
with nearby genetic variations that are the actual causes of observed associations
with BMD (Lee et al. 2010).
Our results of genotype and allele frequencies are similar to those observed from
the other Caucasian populations (Langdahl et al. 2002, Jørgensen et al. 2004, Ueland
et al. 2007, Zajı́čková et al. 2008, Piedra et al. 2011).
In our work we revealed an association of A163G polymorphism in the OPG gene
with BMD and CTx concentration, which indicates reduced bone resorption in the
GG group. CTx is released into circulation as a result of the osteoclast mediated deg-
radation of type 1 collagen during the bone resorption process and it is considered to
be highly bone specific (Christenson 1997). According to WHO, the main criteria for
diagnosis of osteoporosis are bone density measurements. Biochemical markers are
able to complete the static measurement of BMD and indicate a dynamic assessment
of the skeleton (Camacho & Kleerokoper 2006).
The data in our study was statistically analyzed by General Linear Model where
we used the OPG genotype as fixed factor and age and BMI as covariates. Similarly,
BMI and age were included also in Binary Logistic Regression. Age and BMI are
known to have significant effect on analyzed traits in many studies. Within the statis-
tical analysis age showed the significant effect on all tested traits excluding OC; BMI
was significant for LS-BMD and all biochemical markers. On the other hand we did
not confirm the effect of diet on any analyzed trait (Table 1); therefore this factor was
not included in any statistical analysis in our study.
Several studies have tested the association between OPG variants and BMD or
osteoporotic fractures but yielded inconclusive findings. Langdahl et al. (2002)
revealed that A163G polymorphism predicts bone mass and tends to predict osteopo-
rotic fractures independently of BMD. However, together with T245G polymor-
phism, it was associated with increased risk of vertebral fractures. Jørgensen et al.
(2004) found a significant effect on bone mass and fracture status, where the G allele
of the A163G was associated with lower T-scores of forearm BMD. The A163G
polymorphism was not associated with BMD in 650 Korean postmenopausal women.
However, haplotype analysis of A163G and C1181G polymorphisms revealed that
BMDs differ significantly according to OPG combination of haplotypes where sub-
jects with the A allele of A163G and C allele of G1181C had higher values of the dis-
tal radius and calcaneus BMD (Choi et al. 2005). In consistent with our findings,
Hsu et al. (2006) recorded that subjects with the GG genotype had a 70 % reduced
risk of having extremely low hip BMD and higher whole body BMD. In contrast,
Ueland et al. (2007), Garcia-Unzueta et al. (2008) and Vidal et al. (2006) found no
relationships between OPG sequence variation and bone mass, bone turnover or frac-
ture incidence. Polymorphisms in the OPG gene were analyzed in relation to quanti-

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 Effect of A163G polymorphism in the osteoprotegerin gene on osteoporosis 317

tative ultrasound variables of the heel in a cohort of postmenopausal women by Zaj-

ı́čková et al. (2008) where women with the presence of G allele (AG+GG genotypes)
had significantly lower velocity of sounds (VOS) than women with AA genotype.
Because of conflicting results of the association studies between osteoprotegerin
polymorphisms and BMD, a meta-analysis was published by Lee et al. (2010). The
study demonstrated that G1181C polymorphism is associated with lumbar BMD in
European and Asian and in Europeans with femoral neck and total hip BMD. Differ-
ences in total hip BMD was found between the AG and GG genotypes in Europeans.
Furthermore, a recent meta-analysis revealed that the G allele of A163G polymor-
phism may be related to an increased risk of osteoporosis. Stratified analyses showed
that this effect was evident in Caucasians and in the postmenopausal women sub-
groups (Luo et al. 2014). These results contrast with our findings. BMD regulation
and osteoporotic traits have obviously a multifactorial aetiology and polygenic type
of inheritance, which is determined by the effect of several genes each with relatively
small effect, whose interaction results in a clinical manifestation of the disease. Pre-
vious studies showed that effects of genes are different in various populations, which
is a consequence of different genetic and environmental interactions.
Inconsistent conclusions of many studies can be ascribed to gene combinations,
e.g. interactions between OPG gene and other candidate gene polymorphisms. Chro-
mosome position of OPG gene on the locus 8q23–24 represents a region closely
linked to the gene involved in hereditary multiple exocytoses (HME), a skeletal dis-
order resulting in bone malformation (Ahn et al. 1995). This same locus harbours the
gene for the bone morphogenetic protein BMP-1 (Tabas et al. 1991), a member of the
TGF-beta super family which has potent bone-forming activity. Due to this fact, there
raises the possibility that region of chromosome 8 may harbour a gene cluster
involved in the regulation of bone development and metabolism (Simonet et al.
1997). From this point of view, it is important to mention the findings from GWASs.
Two SNPs, rs644 69804 and rs699 3813, showed a genome-wide significant associa-
tion with both spine and hip bone BMD. Moreover they are in LD with the G1181C
and A163G polymorphisms in the OPG gene (Styrkarsdottir et al. 2008).
Although the main limitation of our study is the low frequency of the GG genotype,
the effect of OPG on analyzed traits is sufficient to achieve the statistical significance.
The present study demonstrates that the OPG A163G polymorphism could contribute
to the genetic regulation of bone mass. Results obtained from the study might contribute
to a personalized genetics and screening for the individuals with higher risk of low
BMD. Knowing the risk status can help to minimize a risk of the disease and give the
opportunity to take control of individual’s health. Moreover, the analysis of associations
between candidate genes and BMD or bone turnover markers in specific populations
can extend our knowledge about molecular background of bone remodeling and loss.

Conclusions
This study demonstrates a significant effect of the OPG/A163G polymorphism on oste-
oporosis-related traits in analyzed population of Slovak postmenopausal women. We
identified an association of the polymorphism with femoral and spinal BMD, and CTx
concentration, which indicates reduced bone resorption in the GG group. These associ-
ations were not followed by the effect of OPG on fracture incidence. The results could

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318 Vladimira Krajcovicova et al.

be applicable in predictive genetics, osteoporosis research and they can contribute to

the knowledge about molecular background of bone remodeling and loss.

Acknowledgements
This study was supported by the grants KEGA 035UKF-4/2013, KEGA 025UKF-4/2012.
This work was supported by European Community under project No. 262 20220 180: Build-
ing Research Centre “AgroBioTech”.

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osteoprotegerin gene associated with heel velocity of sound in postmenopausal women? –
Physiol. Res. 57 (Suppl. 1): 153–157.

Submitted: 09 June 2014;

accepted: 20 November 2014.

Address for correspondence: Vladimı́ra Krajčovičová, Department of Botany and Genetics,

Faculty of Natural Sciences, Constantine the Philosopher University in Nitra, Nábrežie
mládeže 91, 949 74 Nitra, Slovak Republic
vkrajcovicova@ukf.sk

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