VDR gene polymorphism and trace elements in Thai postmenopausal women with risk of osteoporosis: Cross -sectional study

Osteoporosis progresses without any symptoms until a bone fracture occurs. Osteoporotic fractures are a common problem in public health control and intervention, which relate to increasing immobility and mortality, affecting quality of life, and high economic impact due to extended hospital stay (Black and Rosen, 2016; Kawalkar, 2015). Intervention policies are necessary to diagnose and reduce the risk of fractures (Si et al., 2015; Williams et al., 2021). There are various associated factors, such as female gender (Black and Rosen, 2016; Pietschmann et al., 2009), old age (Aspray and Hill, 2019; Giusti and Bianchi, 2014), calcium intake (Cano et al., 2018), vitamin D level (Cano et al., 2018; Weaver et al., 2016), and genetic variations (Conti et al., 2015; He et al., 2015; Kubota et al., 2001). There are various genes involved in osteoclast proliferation, which are associated with bone mass reduction or osteoporotic fractures, including the vitamin D receptor (VDR), estrogen receptor, and collagen type I alpha-I genes (Stewart and Ralston, 2000). Vitamin D and its active metabolites participate in the processes of bone tissue mineralization, maintaining calcium homeostasis, and bone remodeling, which are mediated through the VDR (Ahn et al., 2009). The variations VDR gene polymorphisms have been reported, and VDR genotypes are associated with bone diseases, including multiple sclerosis, osteoporosis, vitamin D-dependent rickets type II (Cantorna and Mahon, 2004; Jiang et al., 2020; Valdivielso and Fernandez, 2006), and essential hypertension (Banjabi et al., 2020). VDR VDR gene polymorphism and trace elements in Thai postmenopausal women with risk of osteoporosis: Cross -sectional study


INTRODUCTION
Osteoporosis progresses without any symptoms until a bone fracture occurs. Osteoporotic fractures are a common problem in public health control and intervention, which relate to increasing immobility and mortality, affecting quality of life, and high economic impact due to extended hospital stay (Black and Rosen, 2016;Kawalkar, 2015). Intervention policies are necessary to diagnose and reduce the risk of fractures (Si et al., 2015;Williams et al., 2021). There are various associated factors, such as female gender (Black and Rosen, 2016;Pietschmann et al., 2009), old age (Aspray and Hill, 2019; Giusti and Bianchi, 2014), calcium intake (Cano et al., 2018), vitamin D level (Cano et al., 2018;Weaver et al., 2016), and genetic variations (Conti et al., 2015;He et al., 2015;Kubota et al., 2001). There are various genes involved in osteoclast proliferation, which are associated with bone mass reduction or osteoporotic fractures, including the vitamin D receptor (VDR), estrogen receptor, and collagen type I alpha-I genes (Stewart and Ralston, 2000).
Vitamin D and its active metabolites participate in the processes of bone tissue mineralization, maintaining calcium homeostasis, and bone remodeling, which are mediated through the VDR (Ahn et al., 2009). The variations VDR gene polymorphisms have been reported, and VDR genotypes are associated with bone diseases, including multiple sclerosis, osteoporosis, vitamin D-dependent rickets type II (Cantorna and Mahon, 2004;Jiang et al., 2020;Valdivielso and Fernandez, 2006), and essential hypertension (Banjabi et al., 2020). VDR VDR gene polymorphism and trace elements in Thai postmenopausal women with risk of osteoporosis: Cross -sectional study gene polymorphisms occur in coding or noncoding regions, which can be changed in an amino acid sequence and also affect VDR expression (Uitterlinden et al., 2004). Single nucleotide polymorphisms (SNPs) of the VDR gene can be identified with the proper restriction endonucleases such as FokI, ApaI, TaqI, and BsmI by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Recently, VDR gene variants and the risk of osteoporosis have been associated with ethnic differences (Abbasi et al., 2012;Eisman, 1995;Jiang et al., 2020;Zintzaras et al., 2006). The FokI polymorphism (C>T; rs2228570) of the VDR gene was significantly correlated with bone mineral density (BMD) reduction at the lumbar spine and elevation of hip bone loss in osteoporotic Mexican-American women (Gross et al., 1996). The association of the haplotype of TaqI, FokI, and BsmI SNPs of the VDR gene had been reported where the T allele was a risk factor for osteoporosis in an Indian population (Singh et al., 2013). FokI SNPs could be a link between calcium and vitamin D intake, BMD level, and osteoporosis in obese Iranian women (Moradi et al., 2017). FokI SNPs were also a significant risk factor for osteoporosis in Thai postmenopausal women with osteoporosis, and the TT genotype was susceptible to osteoporosis [odds ratio (OR) = 2.30] (Techapatiphandee et al., 2018).
Nutritional disturbances, especially deficiencies of trace elements and vitamins, pose increased risk for osteoporosis during menopause. Elements maintain skeleton development and function in the bone matrix (Howard et al., 1992). Abnormal metabolism of trace elements may play a role in osteoporosis development (Liu et al., 2009;Nieves, 2005;Odabasi et al., 2008). In addition, trace element supplementation with or without calcium (Ca) can increase BMD in postmenopausal women (Saltman and Strause, 1991). Early BMD screening for osteoporosis in Thai postmenopausal women is needed to focus on the community level (Sudjaroen and Thongmuang, 2019;Sudjaroen and Thongmuang, 2020), and calcium and vitamin D supplementation in a pilot study improved bone biochemical parameters (Sudjaroen and Thongmuang, 2018). This study aimed to compare and correlate bone-related parameters (i.e., BMD, Ca and alkaline phosphatase, and ALP) and trace elements [magnesium (Mg), zinc (Zn), and selenium (Se)] and to identify the genotypes of FokI SNPs and estimate its osteoporosis risk between normal and risk groups of postmenopausal women.

Subject recruitment
This research was a cross-sectional study during the period from December 2020 to March 2021. Approximately 1,500 participants were in the Public Health Service Program in the Osteoporosis Prevention Program, which was under the responsibility of the Samut Songkhram Provincial Government Office and Samut Songkhram Education Centre, Suan Sunandha Rajabhat University. Seventy subjects were randomly recruited in this study. Inclusion criteria were as follows: postmenopausal women who 1) were aged ≥ 55-65 years, 2) were conscious and interactive, and 3) had no serious symptom conditions. Exclusion criteria were as follows: women who had 1) complications with metabolic bone diseases, 2) a previous accident with a fracture, and 3) a pathological fracture . Anthropometric data were recorded by interviews during health service provision. The Ethics Committee of Suan Sunandha Rajabhat University approved this research protocol (COA.1-050-2020). The Director of the Osteoporosis Prevention Program permitted work on this research. All subjects were informed and consented.

BMD measurements
BMD was determined by a calcaneal quantitative ultrasound bone densitometer (SONOST-2000, OsteoSys, Korea), and the instrument operation and data interpretations were made according to manufacturer instruction. Interpretation was made according to BMD status, which was categorized as osteoporotic (T score at or below −2.5), osteopenic (T score between −1.0 and −2.5), and normal (T score at above −1.0) postmenopausal women. The normal postmenopausal women group (N = 24) had a normal BMD status, and the low-BMD postmenopausal women group (N = 46) included the osteoporotic and osteopenic status.

Blood collection and biochemical measurement
Each 5 ml blood sample was obtained by venipuncture from the median cubital vein during the morning (7-9 a.m.) and drawn into clotting blood and Ethylenediaminetetraacetic acid (EDTA) tubes for 3 and 2 ml, respectively. The clotting blood tube was further centrifuged, and serum was separated within 2 hours (Young and Bermes, 1999). Serum ALP, Ca, and Mg were measured by an automatic analyzer, COBAS c501 (Roche-Diagnostics, Rotkreuz, Switzerland). Zn and Se from the serum in the clotting blood tube were extracted and analyzed (Roychowdhury et al., 2002;Wach et al., 2018) by inductively coupled plasma-optical emission spectrometry (Avio200, Perkin Elmer, Thailand).

DNA extraction and genotype analysis
Whole blood contained in EDTA tubes was prepared for genomic DNA extraction by using the QIAamp Blood DNA Mini Kit (QIAGEN Thailand, Bangkok, Thailand), and genomic DNA was stored at −20°C. Genomic DNA was amplified along with the genotype of the FokI (rs2228570) SNPs of the VDR gene by PCR-RFLP. The primers were reverse primer (3¢′-TTGTACCCTGCCCGCAAGAAA-5′¢) and forward primer (5′¢-ACCAAGGATGCCAGCTGG-3′¢). The PCR mixture used was OnePCR Ultra Supermix with a fluorescent dye (Bio-Helix, Gibthai, Thailand). The reaction conditions were optimized for initial denaturation at 94°C for 5 minutes followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds and extension at 72°C for 60 seconds, and one step of final extension at 72°C for 10 minutes in a thermal cycler (Prasad et al., 2021;Techapatiphandee et al., 2018). The amplified PCR product was 266 bp, which was confirmed by agarose gel electrophoresis. The PCR amplified product (20 ml) was conducted with RFLP by using 0.4 ml of the FokI restriction endonuclease enzyme (R0109S, NEB), 2.5 ml of reaction buffer, and 2.1 ml of autoclaved water (total volume of reaction mixture = 25 ml). The reaction mixture was incubated at 37°C for 60 minutes in a water bath for making DNA fragments (Prasad et al., 2021) and genotyped in each sample by DNA fragment separation on 2.5% agarose gel electrophoresis along with DNA ladder (VC 100 bp DNA Ladder, Vivantis Technologies, Malaysia). DNA electropherogram was performed by Bio-Rad Universal Hood II-GelDoc System (Bio-Rad Laboratories-Segrate, Italy). FokI SNPs were 266 bp (uncut) for the homozygous dominant (CC) genotype; 184 and 63 bp (cut) for the TT genotype; and 266, 184, and 63 bp (cut/uncut) for the heterozygous (CT) genotype (Techapatiphandee et al., 2018). Direct sequencing confirmed genotypes in 15% of randomized samples.

Statistical analysis
Descriptive data was represented by using mean, standard deviation, and frequency. The independent t-test was used for the comparison of individual and biochemical parameters between the normal and risk (low-BMD) groups. Pearson's correlation was used to analyze the relationship between individual and biochemical parameters. Chi-square was used to calculate odd risk estimation on different FokI genotypes. Statistical analysis was conducted by the SPSS 21.0 software.

Bone-related parameters and trace elements in postmenopausal women
Age between the normal and low-BMD (risk) groups was not significantly different. ALP and serum Ca of the risk group were significantly higher than in the normal group (p = 0.035 and < 0.0001); therefore, they were within reference ranges. Serum Mg, Zn, and Se of the risk group were significantly lower than in the normal group (p = 0.012, 0.04, and 0.028, respectively). Serum Zn in the risk group and Se in both groups were lower than reference ranges (Table 1). BMD was significantly negatively correlated with ALP and Ca (r = −0.239 and −0.673) and positively correlated with Mg and Zn (r = 0.327 and 0.383). Serum calcium was significantly negatively correlated with all trace elements. The correlation of serum Zn and Se was the strongest (r = 0.824) due to cofactors of antioxidant enzymes (Table 2).
Previous studies reported higher Ca and ALP (within reference ranges) in women with osteoporosis (Ali, 2018;Sasmita et al., 2015). Serum Ca was controlled and maintained homeostasis between the serum and bone compartment. Increasing of serum Ca and BMD reduction in osteoporotic women were implied to have a negative correlation between serum Ca and BMD status (Ali, 2018;Rana, 2013). ALP is a clinical marker of bone metabolism, and its activity arises from the bone and liver. The ALP level of the risk and normal groups was significantly different; therefore, it was still within the reference range. A significant finding was represented in the relationship of the BMD status or osteoporosis and ALP (Narula et al., 2013). Mg is an important mineral for bone cell function especially osteoblast mitogenicity. Thus, when Mg has a deficit, the growth of osteoblasts will be inhibited (Rude and Gruber, 2004). A significant difference of Mg between the osteoporotic and healthy postmenopausal women was also observed in red blood cells, which was described by the Mg transport mechanism into the cell in osteoporosis (Odabasi et al., 2008). Zn is an essential element for over 200 enzymes, collagen production, and bone mineralization (Hyun et al., 2004). Se is also important for human health, and depletion of Se will affect bone tissue (Chariot and Bignani, 2003). Oxidative stress is implicated in postmenopausal osteoporosis by loss of balance between antioxidative and oxidative markers. Monitoring of oxidative stress-related markers is useful for the diagnosis and prognosis of osteoporosis (Zhao et al., 2021). Hence, reduction of serum Mg, Zn, and Se in osteoporosis may affect the antioxidant status, especially antioxidant enzymes. Our finding was summarized in that serum Ca and ALP in the risk group were higher than in normal postmenopausal women, and serum Mg, Zn, and Se will reduce along with BMD during osteoporosis progress. High serum Ca in postmenopausal women is a risk for bone loss, and estrogen deficit increases bone turnover and bone resorption. Hence, the hormonal and BMD status in females are important factors, which Normal BMD was defined as postmenopausal women who had T-Score were above −1.0. b Low BMD was included osteopenia and osteoporosis women; and T-Score were within −1.0 to −2.5 and lower than −2.5, respectively. *Statistically significant at p < 0.05; ** Statistically significant at p < 0.001. should be addressed in osteoporosis studies (Dalemo et al., 2018;Lerner, 2006;Liu et al., 2019).

FokI genotypes of VDR gene polymorphisms in postmenopausal women
The DNA electrophoresis of the FokI genotypes identified a homozygous dominant (CC) for the 266 bp fragment, heterozygous (CT) for the 266, 184, and 63 bp fragments, and homozygous recessive (TT) for the 184 and 63 bp fragments (Fig. 1). The distribution of the T allele is more frequent in the risk group, and the TT genotype was high risk (OR = 2.69, 0.508-4.882) and susceptible to osteoporosis (Table 3). Our significant finding corresponded to a previous study, where it was reported that the FokI genotypes of the VDR gene were related to osteoporosis and the TT genotype was high risk and most susceptible in Thai osteoporotic patients (Techapatiphandee et al., 2018). We confirmed that these SNPs were useful in osteopenia and osteoporotic postmenopausal women with asymptomatic appearances and no history of fracture for at least the past 3 years. There was supportive evidence for osteoporosis occurrence in tropical countries, which have enough sunlight for vitamin D production. The FokI genotypes of the VDR gene were also related to primary hypertension (Prasad et al., 2021), periodontitis , and tumorigenesis (Rai et al., 2017). Identification of the FokI SNPs was used to predict osteoporotic progression in postmenopausal women without symptoms or fractures.

CONCLUSION
We concluded that serum Ca and ALP were negatively correlated with BMD. However, serum Mg, Zn, and Se were positively correlated with BMD in postmenopausal women. It was implied that osteoporosis was related to antioxidant reduction by trace element depletion. Identification of the FokI SNPs was used to predict osteoporotic progression in postmenopausal women without symptoms or fractures.