association Between polymorphisms of VDR , Col 1 a 1 , and lCt genes and Bone Mineral Density in Belarusian women with severe postmenopausal osteoporosis

pavel Marozik1, Irma Mosse1, Vidmantas alekna2, 3, ema Rudenko4, Marija Tamulaitienė2, 5, Heorhi Ramanau6, Vaidilė Strazdienė3, 5, Volha samokhovec4, Maxim ameliyanovich1, nikita Byshnev1, alexander gonchar1, Liubov Kundas1, Krystsina Zhur1 1Institute of Genetics and Cytology NAS Belarus, Belarus, 2Faculty of Medicine, Vilnius University, Lithuania, 3State Research Institute Centre for Innovative Medicine, Lithuania, 4Belarusian Medical Academy of Postgraduate Education, Belarus, 5National Osteoporosis Centre, Lithuania, 6Gomel State Medical University, Belarus


Introduction
Osteoporosis is a common disease characterized by compromised bone strength predisposing a person to an increased risk of the osteoporotic fracture and leading to significant morbidity, mortality, and high social and economic burden (1).Age, sex, racial, and geographic disparities in the incidence and progression of osteoporosis also testify the multifactorial nature of this disease (2)(3)(4).The inheritance of bone mineral density (BMD) varies depending on the characteristics of analyzed population groups and reaches up to 80% (5,6).Variations of osteoporosis in the population are associated with an interaction between the genotype and the environment (7).Homeostasis of the bone tissue during lifetime is mainly maintained by the balanced processes of bone resorption and formation, resulting from the combined ac-tion of multiple genes and environmental factors (8).
Currently, a huge amount of information about the genetic determination of bone mineral density has been accumulated.More than 100 candidate genes associated with BMD and the risk of osteoporosis have already been identified (9)(10)(11)(12)(13).
Vitamin D as a part of the endocrine system has a pleiotropic effect on immune modulation, regulation of skeletal metabolism, and cellular proliferation and differentiation (14).The vitamin D receptor (VDR) gene is the most widely studied among other candidate genes determining the development of osteoporosis (15)(16)(17)(18)(19)(20)(21).The sequence of the VDR gene was found to be polymorphic in different individuals (18).BsmI, ApaI, and TaqI polymorphisms are located in the 3'-regulatory region of the VDR gene, so they are often marked as 1 haplotype; these polymorphisms increase the risk of osteoporosis (15)(16)(17)(18)(19). Cdx2 is a protective polymorphism that reduces the risk of osteoporosis (20,21).
The gene encoding type I collagen protein (COL1A1) is considered as a candidate gene in the pathogenesis of osteoporosis.The G>T polymorphism of the COL1A1 gene is associated with a decrease in bone mineral density and an increased incidence of the fragility fracture (22,23).
The lactase (LCT) gene encodes the amino acid sequence of the enzyme lactase.The T-13910C polymorphism in this gene leading to the intolerance of lactose may have an indirect impact on calcium supply, bone density, and osteoporotic fractures in the elderly (24,25).
The aim of this study was to analyze the association of polymorphisms of the VDR, COL1A1, and LCT genes with severe postmenopausal osteoporosis and BMD.

Study Population
A case-control study including Caucasian postmenopausal women who consecutively visited the City Center for Osteoporosis Prevention in Minsk, Belarus, was carried out.
Ambulatory women who were at least 2 years postmenopausal and who agreed to participate were included into in the study.Women with the conditions known to affect bone metabolism, i.e., diseases such as Paget's disease, osteogenesis imperfecta, rheumatoid arthritis, etc., or those using medications (glucocorticosteroids) were excluded from the study.
The women with the clinical diagnosis of postmenopausal osteoporosis (the BMD T-score of -2.5 or lower at the femoral neck or the lumbar spine) who sustained at least 1 fragility fracture confirmed by an x-ray examination were defined as the patients with severe osteoporosis.The control group comprised postmenopausal women with the BMD T-score of >-2.5 and without previous fragility fractures.The data of the medical history and the fracture history were obtained by a physician.
The local Research Ethics Committee at the belarusian Medical Academy of Postgraduate Education approved the study protocol.Written informed consent was obtained from all the participants.

Measurement of Bone Mineral Density
BMD was measured at the lumbar spine and both hips using dual-energy x-ray absorptiometry (Prodigy, GE Lunar, Madisson, WI, USA).The results of the lumbar spine L 1 -L 4 BMD and the right and left femoral neck BMD were analyzed.

Genotyping
For genetic analyses, venous blood samples were taken from the cubital vein using the Vacutainer system (Beckton-Dickinson, Franklin Lakes, NJ, USA).DNA was isolated from bloodspots dried on special NucleoSafe cards (Macherey-Nagel, Germany) using the standard proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation (26).The DNA solution was extracted with a phenol-chloroform-isoamyl alcohol mixture to remove protein contaminants and then was precipitated with 100% ethanol.The DNA was pelleted after the precipitation step, washed with 70% ethanol to remove salts and small organic molecules, and resuspended in a buffer at a concentration suitable for further investigation.
Polymorphic sites in osteoporosis predisposition genes (the ApaI, BsmI, TaqI, and Cdx2 polymorphisms of the VDR gene, the G2046T polymorphism of the COL1A1 gene, and the T-13910C polymorphism of the LCT gene) were determined using the polymerase chain reaction (PCR) analysis with specially designed primers (Table 1).
VDR ApaI, BsmI, TaqI.Polymorphic sites in VDR (BsmI B/b, rs1544410, ApaI A/a rs7975232, TaqI T/t, and rs731236) were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.PCR amplifications of the related regions were carried out in 20-μL volumes of reaction mixtures containing 2×PCR-buffer (with 3-mM MgCl 2 ), 200 μM of each dNTP, 0.5 μM of each set of specific primers, 1 unit of Tornado-Taq DNA polymerase (Primetech, Belarus), and 20-50 ng of the DNA sample.All the PCR reactions were run at 95°C for 15 minutes followed by 30 cycles at 99°C for 1 second, at 62°C for 10 seconds, and extension at 72°C for 20 seconds.The final extension was carried out at 72°C for 2 minutes in an automated thermal cycler (Applied biosystems 2720, USA).The amplified products were analyzed by electrophoresis on the 8% polyacrylamide gel.The BsmI, ApaI, and TaqI genotypes were analyzed by BsmI (37°C), ApaI (37°C), and TaqI (65°C) restriction enzyme digestion (Thermo Scientific, Lithuania), respectively.The B allele (191 bp) and the A and T alleles remained as a single 490-bp band; the b allele (115 and 76 bp) and the a (280 and 210 bp) and t (290 and 200 bp) alleles were observed as 2 bands.All of the digestion products were analyzed by electrophoresis on the 8% polyacrylamide gel.
VDR Cdx2.Two sets of primers were designed for the tetraprimer amplification refractory mutation system PCR (T-ARMS-PCR) (Table 1).G-Rev and A-For are allele-specific primers.These 4 primers generate 3 PCR fragments: the primer set G-For and G-Rev specifically amplifies the G allele with a size of 110 bp, A-For and A-Rev specifically amplify the A allele with a size of 235 bp, and the outprimer pair (G-For and A-Rev) amplifies the internal control PCR fragment with a size of 297 bp.The PCR amplification was carried out in an automated thermal cycler (Applied Biosystems 2720, USA).Ten microliters of the PCR reaction system consisted of 1.0 μL of 10× PCR buffer (1×buffer=10 mM Tris-Cl, pH 8.3; 50 mM KCl; 1.25 mM MgCl 2 ), 1.0 μL of 10×dNTPs (0.2 mM), 0.4 pmol of G-For, 0.6 pmol of G-Rev, 0.6 pmol of A-For, 0.4 pmol of A-Rev, 0.5 U of Super Taq (Thermo Scientific, Lithuania), and 10 ng of genomic DNA.The PCR was performed with an initial denaturation at 96°C for 5 minutes, followed by 28 cycles of denaturation at 94°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 45 seconds.The final extension was carried out at 72°C for 5 minutes.The PCR products were size-separated on the 8% polyacrylamide gel at 140 V for 45 minutes.The 100-bp DNA ladder (Thermo Scientific, Lithuania) was used to determine the size of the fragments.
COL1A1 G2046T.The G2046T polymorphism of the COL1A1 gene was detected by using the qPCR method.The amplification reactions were run in the CFX96 (BIO-RAD, USA) Real-Time PCR Detection System.The final reaction volume (20 μL) included 10 μL of 2×qPCR Master Mix (Thermo Scientific, Lithuania), 10 pmol of forward and reverse primers, 5 pmol of G2046 and 2046T probes, and 1 μL of the DNA sample (except the negative control ones).
LCT T-13910C.The position of the T-13910C polymorphism was based on the sequence analysis of the LCT gene.Two sets of primers were designed for the ASM-PCR test (Table 1).
The PCR amplification was carried out in an automated thermal cycler (Applied Biosystems 2720, USA).The PCR reaction system consisted of 10-μL 10×PCR buffer (1×buffer=10 mM Tris-Cl, pH 8.3; 50 mM KCl; 1.25 mM MgCl 2 ), 1.0 μL of 10×dNTPs (0.2 mM), 1.0 μL of each primer, 0.5 μL of poly-merase, 3.5 μL of mQ water, and 10 ng of genomic DNA.The PCR was performed with an initial denaturation at 95°C for 15 minutes, followed by 28 cycles of denaturation at 99°C for 1 second, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds.The final extension was performed at 72°C for 1 minute.The PCR products were size-separated on the 10% polyacrylamide gel at 125 V for 1 hour.The 100-bp DNA ladder (Thermo Scientific, Lithuania) was used to determine the size of the fragments.The amplified products were analyzed by electrophoresis on the 10% polyacrylamide gel.The C allele was observed as a 157-bp band; the T allele, as a 95-bp band; and the common fragment, as a 205 bp.

Statistical Analysis
The statistical analysis was performed using STA-TISTICA 10 for Windows (Statsoft Inc., USA).Based on the determined frequencies of genotypes and by using the χ 2 test, the Hardy-Weinberg equilibrium was tested.The association of the genotype alleles between the patients with postmenopausal osteoporosis and the control group was determined by the analysis of Pearson chi-square (χ 2 ).Crude odds ratios (ORs) were reported with 95% confidence intervals (CI).The differences between the groups were considered statistically significant at P<0.05.For BMD, the data are presented as mean (SD) (in g/cm 2 ).

Results
A total of 54 women with severe postmenopausal osteoporosis were included in this study.The control group consisted of 77 Caucasian women without osteoporosis.The clinical characteristics of the study population are summarized in Table 2. both groups were matched for age, duration of postmenopausal period, and body mass index, except for height and  The distribution of the analyzed gene polymorphisms in the controls and in the patients with osteoporosis were in correspondence with the one expected from the Hardy-Weinberg equilibrium (P>0.05 in all cases), showing that the analyzed groups were selected correctly.The genotype and allele frequencies of the analyzed polymorphisms are presented in Table 3.
There was a statistically significant difference in the genotype and allele frequencies of the ApaI polymorphism between the patients and the controls (χ 2 =13.74 and χ 2 =15.31, respectively, P<0.01).Furthermore, the patients with severe postmenopausal osteoporosis were 3.34 (95% CI, 1.51-7.37)and 2.73 (95% CI, 1.64-4.55)times more likely to carry the AA genotype and the A allele of the ApaI polymorphism, respectively, when compared with the controls.
There also was a statistically significant difference in the genotype and allele frequencies of the When the genotype frequency of other polymorphisms of the VDR gene -TaqI and Cdx2 -was compared between the patients and the controls, no statistically significant differences in the frequency of genotypes and alleles were found.The same picture was observed in the analysis of the G2046T polymorphism of the COL1A1 gene (Table 3).
The analysis of the data also revealed a statistically significant difference in the genotype and allele frequencies of the T-13910C polymorphism in the LCT gene.The patients with severe postmenopausal osteoporosis were 3.09 (95% CI, 1.40-6.84)and 2.24 (95% CI, 1.35-3.71)times more likely to carry the CC genotype and the C allele of the T-13910C polymorphism, respectively, when compared with the controls.
The relationship between the genotypes and BMD is shown in Table 4.The data showed that for all the gene polymorphisms, with the exception of the LCT gene polymorphism, the BMD was lower in the individuals carrying the osteoporosis predisposition genotypes as compared with the individuals with favorable genotypes.The statistically significant association was shown in the VDR ApaI polymorphism: the BMD of the left femoral neck (LF) in the carriers of the Aa genotype and the BMD of the right femoral neck (RF) in the carriers of the aa genotype were greater when compared with the carriers of the AA genotype.For VDR TaqI, a statistically significant difference in the BMD of the RF was observed between the bearers of the TT and tt genotypes.

Discussion
In the patients with severe postmenopausal osteoporosis, all the polymorphism genotype frequencies did not differ from the expected Hardy-Weinberg equilibrium.This allows us to use the multiplicative model of inheritance in the statistical analysis, performing the analysis of not only genotypes, but also alleles.
In the present study, a statistically significant association between postmenopausal osteoporosis and at least 3 analyzed gene polymorphisms was demonstrated.Interestingly, the statistically significant association was observed for 2 most studied polymorphisms of the VDR gene, i.e., ApaI and BsmI.Currently, the VDR gene is the best studied one in the set of the candidate genes that determine the The BsmI polymorphism contains a restriction site marked as the b allele.Up to 16% of the Caucasians are homozygotes for a functionally defective allele of this gene (BB) and are at risk of osteoporosis and osteoporotic fractures (16).In the present study, the frequency of the BB allele in the control group (14.3%) was very similar.The frequency of the Bb genotype was higher in the group of the patients with postmenopausal osteoporosis compared with the control subjects.The probability of having this genotype was more than 2.6 times greater for the patients compared with the control population.In total, the risk of osteoporosis for risk (B) allele bearers was 2.3 times higher compared with control subjects.
The LCT is the third gene, the polymorphism of which showed a statistically significant association with postmenopausal osteoporosis.The frequencies of both risk allele and homozygous genotypes were shown to be higher in the patients with postmenopausal osteoporosis as compared with the control group.
In the present study, we failed to show any association between TaqI and Cdx2 of the VDR gene and G2046T of the COL1A1 gene, and the risk of the development of postmenopausal osteoporosis.The relationship of these gene polymorphisms and osteoporosis has been well established in the European population (16,(20)(21)(22)(23).Moreover, for the COL1A1 gene, the haplotype analysis has demonstrated that the relationship with the risk of osteoporosis is specific for the G2046T polymorphism rather than for others (27).
For the VDR Cdx2 A allele, a statistically significant protective effect was not observed potentially reflecting a lack of statistical power, in particular for a very rare AA genotype group, which was absent in the present study.Therefore, further studies with a higher number of individuals have to be performed to determine its protective effect.The same possible explanation could be used for the absence of any association of the COL1A1 gene with postmenopausal studies, as no homozygous risk genotypes in the present study were found.
The absence of association between the TaqI polymorphism of the VDR gene and severe postmenopausal osteoporosis in the belarusian women was very surprising because this polymorphism is very well studied, and its association with osteoporosis has been shown in many European populations (13,28), including Russian (29).Its mechanisms mostly involve the hormonal regulation of osteogenesis, inactivating the vitamin D receptor.
Therefore, it is important to mention that the absence of a significant association of VDR TaqI and Cdx2 and the COL1A1 polymorphism with postmenopausal osteoporosis in the present study may be explained by the number of persons analyzed, which is insufficient for the investigation of rare alleles, and this does not reduce the interest in further studies.Further molecular and genetic analysis of these gene polymorphisms on the belarusian population may help obtain more information on their association with postmenopausal osteoporosis.
The analysis of the relationship between gene polymorphisms and bone mineral density in both investigated groups revealed a statistically significant association between the femoral neck BMD and the frequency of the VDR ApaI and TaqI polymorphisms.No statistically significant association was observed between the lumbar spine BMD and the gene polymorphism genotypes; however, for all the genes, except LCT, there was lower BMD in the individuals carrying osteoporosis-predisposing genotypes.
For VDR ApaI, we have found a statistically significant association between the femoral neck BMD and the genotypes: in the subjects with the Aa genotype, the RF BMD was higher by 14.5%; and in the women with the aa genotype, the LF BMD was higher by 11.2% than in those with the AA genotype.Thus, taking into consideration a significant association between VDR ApaI and severe postmenopausal osteoporosis in this case-control study, a conclusion might be drawn that this polymorphism may be a useful marker for osteoporosis screening at least in belarusian women.
The findings of this study suggest that the VDR ApaI gene polymorphism has a statistically significant association with the BMD level and severe postmenopausal osteoporosis in the analyzed group of the belarusian women.
Surprisingly, another polymorphism demonstrating a statistically significant association between the bone mass and the genotype was VDR TaqI, which did not reveal even a trend for osteoporosis predisposition in a genetic case-control study.In a comparative analysis, the carriers of the TT genotype had the RF BMD by 11.6% higher compared with the individuals with the tt genotype.
No statistically significant association between bone mineral density and the polymorphisms of the VDR BsmI and Cdx2, COL1A1 and LCT genes may be explained by an insufficient number of the subjects investigated.In another study, a relationship between the BMD level and osteoporosis predispo-sition genotypes is most evident among individuals with a relatively low calcium intake (30).This is especially important for the LCT gene T-13910C polymorphism, pathogenesis of which includes a reduced calcium intake as a result of lactose intolerance.
VDR Cdx2 was reported to have a significant association between the A allele and a higher BMD in postmenopausal women, predicting the association of this allele with a decreased risk of fracture (20,21).In the present study, we found no statistically significant association between BMD and the VDR Cdx2 polymorphism genotypes, possibly as a result of insufficient statistical power.
According to Uitterlinden et al. (17), the difference in BMD between women carrying the G or T alleles of the COL1A1 G2046T polymorphism increases with age.Interestingly, in their study, there was no significant difference in BMD between the GG and TT genotypes in the subjects aged 55-69 years; a significant difference in BMD was detected only in the subjects aged 70-80 years.Thus, it is possible that our study group was too young and represented a too narrow range of age to show any difference in the BMD level between the COL1A1 genotypes.
The findings of this study suggest that at least VDR ApaI and BsmI and LCT T-13910C polymorphisms are likely to be associated with the risk of postmenopausal osteoporosis and make the greatest contribution to its development in the Belarusian population.At the same time, the VDR Cdx2 and COL1A1 G2046T polymorphisms demonstrated a nonsignificant association with osteoporosis, and further molecular and genetic analysis is required.
Screening of these genetic markers may enable an early identification of risk groups to perform preventive measures in a timely manner and also to improve treatment effectiveness, avoid complications, reduce disability and mortality rates in these patients, as well as cut down the treatment costs.

Conclusions
The findings of this study suggest that the ApaI polymorphism of the VDR gene has the greatest association with the BMD level and severe postmenopausal osteoporosis in the analyzed group of the belarusian women.The present study prompts a further large-scale study of postmenopausal osteoporosis predisposition genes with larger sample sizes and in relation to fracture risk and environmental factors.

statement of Conflict of Interest
The authors state no conflict of interest.

acknowledgments
This work was funded by a grant (project Б11ЛИТ-017) from the Foundation for Basic Research of belarusian Republic and by a grant (No.TAP-21/2011) from the Research Council of Lithuania.

Table 1 .
Polymerase Chain Reaction Primers of VDR, COL1A1, and LCT Gene Polymorphisms Used for the Screening of Genetic Predisposition to Osteoporosis weight.The patients with severe osteoporosis had significantly lower BMD than the controls, in both absolute and relative values.

Table 2 .
Clinical Characteristics of Women Investigated

Table 3 .
Genotype and Allele Frequencies of Analyzed Osteoporosis Predisposition Gene Polymorphisms in Patients and ControlsPavel Marozik, Irma Mosse, Vidmantas Alekna, et al.

Table 4 .
-L4, lumbar spine; LF, left femoral neck; RF, right femoral neck; VDR, vitamin D receptor gene; COL1A1, type I collagen gene; LCT, lactase gene.Association Between Genotype and Bone Mineral Density at the Lumbar Spine, Left Femoral Neck, and Right Femoral Neck development of osteoporosis.The 3.3-times higher risk of low BMD in the presence of the VDR ApaI AA genotype shows the importance of the A allele in the development of postmenopausal osteoporosis.Moreover, there is a statistically significant decrease in the osteoporosis risk for the aa genotype carriers.