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View of Vitamin-D Receptor (VDR) Gene Polymorphisms (Apai&Bsmi) In Iraqi Osteoporosis Patients

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Vitamin-D Receptor (VDR) Gene Polymorphisms (Apai&Bsmi) In Iraqi Osteoporosis Patients

Suroor Mohammed Ali 1, Mohammed Abdullah Jebor2*

1,2Department of Biology, College of Sciences, Babylon University, Iraq

* Corresponding author: [email protected]

ABSTRACT

Osteoporosis is a polygenic disorder and has been demonstrated to be associated with ~30 candidate genes, the majority of which have also been implicated in the regulation of bone mineral density (BMD). VitaminD receptor (VDR) mediates Vit-D activity, thus VDR gene polymorphismsmay correlate with different diseases. This study aimed todetermine the distribution of VDR gene (ApaI&BsmI) polymorphismsusing a RFLP in in Iraqi osteoporosis patients. The present results revealed There were no significant differences in ApaI genotype(rs7975232) frequencies between the cases and control. the genotypic frequency of heterozygous C/A with homozygous C/C (OR=0.1365, 95% CI=0.0072 - 2.5895, P= 0.1847). and the genotypic frequency of homozygous variant A/A with the homozygous C/C(OR=0.1127,95% CI= 0.0060 - 2.1070, P=0.1440). There were a significant differences in A allele frequencies in patients as compared to the controls inthe VDR ApaI (rs7975232) polymorphism (OR=0.9252, 95% CI =0.4556 - 1.8784, P=0.8295).Also, there were no significant differences in BsmIgenotype (rs15444410 ) frequencies between the cases and control. the genotypic frequency of heterozygous A/G with homozygous G/G (OR=0.3652, 95% CI=0.0402 - 3.3174, P= 0.3709), and the genotypic frequency of homozygous variant A/A with the homozygous G/G (OR=0.2833, 95% CI= 0.0293 - 2.7444, P= 0.2763). There were a significant difference in A allele frequencies in patients as compared to the controls in the CAT C-262T polymorphism (OR= 0.7463, 95%

CI=0.4075 - 1.3669, P=0.3433).

Keywords:Osteoporosis; VDRpolymorphism;rs7975232;rs15444410; VDR ApaI;VDRBsmI; PCR-RFLP.

Introduction

Osteoporosis is a systemic bone disease mostly occurring in elderly individuals. In this disease, disturbance in bone remodeling (bone resorption and formation) leads to a bone mass reduction, bone fragility, and eventually, to fracture. Osteoporotic fracture may cause disability, decreased qual¬ity of life, and ultimately, mortality – it affects all aspects of the patient’s life [1]. There have also been studies reporting the rate of this disease in a local region; for example, in 2009, an Iranian multi- center study indicated that 70% of women and 50% of men aged ≥50 years suffered from osteoporosis or osteopenia[2].Peak bone mineral density (BMD) as a major determi¬nant of bone strength achieved in early adulthood plays an important role in the prediction of osteoporotic fracture in later life.1 In addition to many confirmed factors, such as race, sex, age, nutrition, hormonal status, menopausal state, smoking, alcohol intake, and physical activity, there are many studies that support the remarkable influence of genetic factors on bone strength. Studies show that up to 80% of BMD variation is attributable to genetic factors [3,4]. The VDR gene located on the long arm of chromosome 12 (12q13.11) is a member of the nuclear receptor superfamily, and many studies have shown that VDR gene polymorphisms play an important role in the pathogenesis of osteoporosis [5].

VDR polymorphisms include VDR TaqI (rs17880019), VDR BsmI (rs1544410), VDR FokI (rs17881966), and VDR ApaI (rs17879735) [6,7] . Other studies indicate a relationship between VDR gene polymorphisms and poor bone health including Osteoporosis [8].A meta-analysis of BsmI, TaqI, ApaI and FokI VDR polymorphisms concluded that no clear association exists with OP in females [9].

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Material and Methods Ethical statement

Every volunteer has informed written consent. The ethics committee of the MOH and MOHSER in Iraq's ethical approval for scientific research has accepted this research.

Study Population

The study subjects comprised from 64 patients selected from Imam Al-Hussein Medical-City all were female as patients’ group with age range (20-82 years). The control group study included 36 people apparentlyhealthy that also were females with age range (20–71) years. All subjects in this study were taken written consent before participation in this study.

DNA extraction and genotyping

DNA of blood was extracted and purified using extraction and purification kit from Geneaid company (UK). The genotyping of the study groups was performed using the PCR-RFLP technique after DNA extraction from blood samples.

The targeted sites of DNA were amplified using design specific primers which used to identify VDR (rs7975232) , obtained from Macrogen company/south Korea . Forward primer: 5’-

CAGAGCATGGACAGGGAGCAA -3’, and reverse primer: 5’-

GCAACTCCTCATGGCTGAGGTCTC-3’.PCR was carried out in 20 μl reaction volumes containing 1 μl of each forward and reverse primer, 12.5 μl of Green Master Mix, 3 μl of Genomic DNA, and 2.5 μl of nuclease-free water to bring the reaction volume up to 20 μl . Amplification was conducted in a thermocycler (Biometra, Germany) with the following settings: 5 min pre- denaturation at 95°C; 30 cycles with denaturation for 20 seconds at 95°C, annealing for 20 seconds at 62°C, extending for 20 seconds at 72°C; and a final extension of 5 min [10]. For VDR (rs1544410) specific primers which used, obtained from Macrogen company/south Korea . Forward primer: 5’- AGTGTGCAGGCGATTCGTAG -3’, and reverse primer: 5’- ATAGGCAGAACCATCTCTCAG - 3’.Amplification was conducted in a thermocycler (Biometra, Germany) with the following settings:

5 min pre-denaturation at 95°C; 30 cycles with denaturation for 30 seconds at 95°C, annealing for 30 seconds at 62°C, extending for 30 seconds at 72°C; and a final extension of 5 min [11].

PCR products were electrophoresed in 1 percent agarose at 75 V using gel electrophoresis (cleaver science – UK) and visualized with ethidium bromide. A gel documentation system (Cleaver Scientific –UK) was used to take photos.

polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP).

For VDR (rs7975232) the PCR product was digested with 2 units of one of the specific endonucleases (ApaI ) for 1-4 h at 37˚C , according to manufacturer’sinstructions(Promega company) . For VDR (rs1544410) the PCR product was digested with 2 units of one of the specific endonucleases (BsmI ) for 5-15min at 65˚C , according to manufacturer’sinstructions (Biolabs company) .PCR and digestion products were analyzed by electrophoresis in 1 percent agarose at 75 V using gel electrophoresis (cleaver science – UK) and visualized with ethidium bromide.

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Statistical analysis

All the statistical analyses were done with the SPSS statistical software (version 23; SPSS Inc., Chicago, IL), p<0.05 was considered statistically significant

Results and Discussion

The genomic DNA (Fig.1) was extracted from the blood samples as a first step to amplify the target region of VDR (rs15444410and rs7975232) gene.

Figure 1. The electrophoresis pattern of gnomic DNA extracted from blood samples of osteoporosis patients and healthy control groups.

(Lane 1 -lane 10 refers to genomic DNA from blood samples; Electrophoresis conditions, 1% agarose, 75 V, 20 mA for 1h, stained with ethidium bromide).

Genotyping of VDR (rs7975232) Gene Polymorphisms

For VDR (rs7975232) genotyping, the genomic DNA was amplified using specific primers and accomplished by the Thermo-cycler apparatus under the optimal conditions. The results revealed that the presence a single band (745bp) of the target sequence of VDR (rs7975232) gene in agarose gel (Fig. 2).

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Figure 2. Agarose gel electrophoresis of an amplified product patterns of vitamin D receptor (rs7975232) with specific primer.

(M: refers to DNA size marker; lanes 1 - 9 refer to PCR products of vitamin D receptor (rs7975232) (745bp) of osteoporosis patients and healthy control groups. Electrophoresis conditions: 1% agarose

concentration 1%; 75 V, 20 mA for 120 min. Staining method; precast ethidium bromide).

After that, the PCR products of the VDR (rs7975232) target sequences were digested withApaI (5' GGGCCꜜC3')restriction enzyme (Fig. 3) to detect the rs7975232SNP in VDR gene. The results of PCR-RFLP showed that the presence of three different genotypes as in figure (3). the first A1/A1 (AA) homozygous, presents the expected 745bp fragment, the second A1/A2 (AG) demonstrated 745, 528 & 217 fragment. While the third A2/A2 (CC) 528 &217 bp fragment.

M 1 2 3 4 5 6 7 8 9

745 bpbp

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Figure 3. Electrophoresis patterns of allelotyping of VDR (rs7975232) gene of osteoporosis patients and healthy control groups using ApaI enzyme by PCR-RFLP method

(M: DNA ladder (100 bp); Lanes 5,7,8&10 refer to a homozygous allele (AA) had a single band with 745 bp molecular size; Lanes 1,2&9 refer to a heterozygous allele (CA) had 3 bands with 745, 528 &217 bp; Lanes

3,4&6 refer to a homozygous allele (CC) had a two bands with 528 &217 bp molecular size).

The Genotypes Distribution of (rs7975232) Polymorphisms with Allele Frequency in Control and Case Groups.

The distribution observed in VDR (rs7975232) gene polymorphism in cases group and control group are showed in Table (1). The highest genotype in control group was mutant homozygote AA (58%) followed by CA heterozygote genotype (42%) and homozygote genotype CC (0%).In osteoporosiscases, the highest genotype mutant homozygote AA (48.4%) followed by CA heterozygote genotype (42.1%) and homozygote genotype CC (9.5%). analyses showed significant differences between cases and controls in the VDR ApaI polymorphism (CAvs CC: OR=0.1365, 95%

CI =0.0072 - 2.5895, P= 0.1847). analyses showed no significant differences between cases and controls in the VDR ApaIpolymorphism (AAvs CC: OR=0.1127, 95% CI =0.0060 - 2.1070, P=

0.1440).

M 1 2 3 4 5 6 7 8 9 10 10

528bp 148 bp 217bp 148 bp 745bp

148 bp

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Table 1. Genotype distribution and odd ratio of rs7975232 polymorphisms between the patient’s vs healthy control

Genotype of rs7975232

Patients No.(%)

Control No.(%)

Significance

level O.R CI (95%)

CCa 6 (9.5%) 0 (0%)

CA 27 (42.1%) 15 (42%) 0.1847

0.1365

0.0072 - 2.5895

AA 31 (48.4%) 21 (58%) 0.1440 0.1127 0.0060 - 2.1070

Total No. 64 36

Allele Frequency Frequency

C 0.31 0. 21

A 0.69 0.79 0.8295

0.9252 0.4556 - 1.8784

P ≤ 0.05 ; OR=(95%CI); a reference

In 2003, Zhang et al.[12] reported a significant relationship between the (AA) genotype of ApaI and the incidence of osteoporosis in Chinese menopausal women. In 2009, Dundaret al.[13]. studied the genotype distribution of ApaI in Turkish menopausal women and showed that individuals with the (aa) genotype had a lower bone density and higher serum calcium level than those with the (AA) genotype.

Other study found that ApaI rs7975232 (dominant comparison: OR = 0.77, P = 0.007; allele comparison: OR = 0.81, P = 0.04), polymorphisms was significantly associated with the risk of postmenopausal osteoporosis in Caucasians, this meta-analysis shows that ApaI rs7975232 polymorphisms may affect the risk of postmenopausal osteoporosis in Caucasians, while BsmI rs1544410 polymorphism may affect the ris k of postmenopausalosteoporosis in Asians [14].

Furthermore, comparison of frequency distribution of genotype and allele for VDR ApaI between osteoporotic patients and controls did not show any significant difference [15]. A significant increase in the genotype frequencies of the ApaI (Aa) and (aa) was observed among osteoporotic patients compared to controls (P = 0.002 and P < 0.0001, respectively). The minor “a” allele of ApaI was significantly more common in the patients as compared to controls (P < 0.0001) [16].

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In the VDR gene, several common polymorphic sequence variations have been reported. VDR genotypes were found to be associated with a wide variety of bone diseases as well as multiple sclerosis, osteoporosis, vitamin D-dependent rickets type II, and other complex maladies [17,18].

Genotyping of VDR (rs15444410) Gene Polymorphisms

For VDR (rs15444410) genotyping, the genomic DNA was amplified using specific primers and accomplished by the Thermo-cycler apparatus under the optimal conditions .The results revealed that the presence a single band (191bp) of the target sequence of VDR (rs15444410)gene in agarose gel (Fig. 4).

Figure 4. Agarose gel electrophoresis of an amplified product patterns of vitamin D receptor (rs15444410) with specific primer.

(M: refers to DNA size marker; lanes 1 - 9 refer to PCR products of vitamin D receptor ( rs15444410 ) (191bp) of osteoporosis patients and healthy control groups. Electrophoresis conditions: 1% agarose concentration 1%; 75

V, 20 mA for 120 min. Staining method; precast ethidium bromide).

After that, the PCR products of the VDR (rs15444410) target sequences were digested withBsmI (5' GAATGCN ꜜ 3')restriction enzyme (Fig.5) to detect the rs15444410 SNP in VDR gene. The genotypes of the studied subjected has been distributed into three groups based on the presence or absence of the Polymorphisms: A/A homozygous 191 bp, A/G heterozygote demonstrated 191, 115

& 76 bp and G/G homozygous 115 & 76 bp (Fig.5).

M 1 2 3 4 5 6 7 8 9

191bp

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Figure 5. Electrophoresis patterns of allelotyping of VDR (rs15444410) gene of osteoporosis patients and healthy control groups using BsmI enzyme by PCR-RFLP method.

(M: DNA ladder (50 bp); Lanes 5 refer to a homozygous allele (AA) had a single band with 191 bp molecular size; Lanes 1,2,4, 6,7,9&10 refer to a heterozygous allele (AG) had 3 bands with 191, 115 &76 bp; Lanes 3&8

refer to a homozygous allele (GG) had two bands with 115 &76 bp molecular size).

The Genotypes Distribution of VDR (rs15444410) Polymorphisms with Allele Frequency in Control and Case Groups.

The distribution observed in VDR (rs15444410) gene polymorphism in cases group and control group are showed in Table (2). The highest genotype in control group was AG heterozygote genotype (64%) followed by homozygote genotype AA (33.3%) and GG homozygote (2.7%) .In breast cancer disease, the highest genotype was AG heterozygote genotype (65.6%) followed by homozygote genotype AA (26.5%) and GG homozygote (7.9%). analyses showed significant differences between cases and controls in the VDR BsmI polymorphism (AGvsGG: OR=0.3652, 95% CI =0.0402 - 3.3174, P=0.3709). analyses showed no significant differences between cases and controls in the VDR ApaIpolymorphism (AAvsGG: OR=0.2833, 95% CI =0.0293 - 2.7444, P=

0.2763).

M 1 2 3 4 5 6 7 8 9 10

76bp

115bp 148 bp 191bp

100bp bbpbp 200 bp 500bp

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Table 2. Genotype distribution and odd ratio of rs15444410 polymorphisms between the patient’s vs healthy control.

Genotype of rs15444410

Patients No.(%)

Control No.(%)

Significance

level O.R CI (95%)

GGa 5 (7.9%) 1 (2.7%)

AG 42

(65.6%) 23 (64%) 0.3709 0.3652 0.0402 - 3.3174

AA 17

(26.5%) 12

(33.3%) 0.2763 0.2833 0.0293 - 2.7444

Total No. 64 36

Allele Frequency Frequency

G 0.41 0. 35

A 0.59 0.65 0.3433 0.7463 0.4075 -

1.3669 P ≤ 0.05 ; OR=(95%CI); a reference

Overall, statistically significantly increased osteoporosis risk was found in Indians and women for VDR gene polymorphism. Statistically significantly decreased osteoporosis risk was found in West Asians for VDR BsmI polymorphism. However, when we performed a sensitivity analysis after excluding low quality and Hardy-Weinberg Disequilibrium (HWD) studies, significantly decreased osteoporosis risk was only found in overall population for VDR BsmI polymorphism. Further, less- credible positive results were identified when we evaluated the credibility of positive results [19].Other study concluded that the VDR BsmI genetic polymorphism was correlated with LS BMD level in pediatric patients: compared with those with the B allele, children with the bb genotype were less likely to have lower BMD levels. No significant difference was identified in the pediatric FN BMD levels [20].

Further study founded that BsmI rs1544410 (OR = 0.69, P = 0.002 ) polymorphisms was significantly associated with the risk of postmenopausal osteoporosis in Caucasians, whereas FokI rs10735810 polymorphism was significantly associated with the risk of postmenopausal osteoporosis in Asians (dominant comparison: OR = 0.61, P = 0.0001; recessive comparison: OR = 2.02, P = 0.001; allele comparison: OR = 0.68, P = 0.002). ,this meta-analysis shows that ApaI rs7975232, BsmI rs1544410 a ndTaqI rs731236 polymorphisms may affect the risk of postmenopausal osteoporosis in Asians[21].

A meta-analysis of BsmI, TaqI, ApaI and FokI VDR polymorphisms concluded that no clear association exists with osteoporosis in females [22]. In addition, Kow et al. [23], found that,there were no significant differences between the case and control groups in the VDR A/G genotype (p=0.

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5, OR[95% CI] =1.45 (0.65–3.24), similarly no significant differences between the case and control groups in the VDR A/A genotype (p=0. 5, OR[95% CI] =1.74 (0.66–4.63) .

Conclusion

Based on the present statistical analysis, our studyshows no significant association of VDR ApaI(rs7975232) and VDR BsmI (rs15444410) geneswith the development of osteoporosis.

References

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[2] Rahnavard Z, Zolfaghari M, Hossein-Nezad A, VahidDastgerdi M.(2009). The incidence of osteoporotic Hip fracture: Iranian Multicenter Osteoporosis Study (IMOS). Res J Biol Sci.;4:171–173.

[3] Harsløf T, Husted LB, Nyegaard M.(2011). Polymorphisms in the ALOX12 gene and osteoporosis. Osteoporos Int.;22:2249–2259.

[4] Xiao WJ, Ke YH, He JW.(2012). Polymorphisms in the human ALOX12 and ALOX15 genes are associated with peak bone mineral density in Chinese nuclear families. Osteoporos Int.;23:1889–1897.

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[6] Deng H, Liu F, Pan Y.(2011). BsmI, TaqI, ApaI, and FokI polymorphisms in the vitamin D receptor gene and periodontitis:a meta-analysis of 15 studies including 1338 cases and 1302 controls. J ClinPeriodontol; 38: 199-207.

[7]Ozel L, Ata P, Ozel MS.(2011). Risk factors for osteoporosis after renal transplantation and effect of vitamin D receptor Bsm I polymorphism. Transplant Proc; 43: 858-62.

[8] Mencej-Bedrac S, Prezelj J, Kocjan T.(2009). The combinations of polymorphisms in vitamin D receptor, osteoprotegerin and tumour necrosis factor superfamily member 11 genes are associated with bone mineral density. J Mol Endocrinol.;42(3):239–47.

[9] Zintzaras E, Rodopoulou P, Koukoulis GN. (2006). BsmI, TaqI, ApaI and FokI polymorphisms in the vitamin D receptor (VDR) gene and therisk of osteoporosis: a meta- analysis. Dis Markers. 22(5–6):317–326.

[10] Al-Darraji SZ.;, Al-AzzawieHF.;and Al-Kharsani AR(2018). Vitamin D Status and its Receptor Genes BsmI, FokI, ApaI, TaqIPolymorphism in Relation to Glucose Metabolism in Obese Iraqi Type 2Diabetes Mellitus Patients.J Mol Genet Med 2017, 11:2,1-10.

[11] Marozik P, Mosse I, Alekna V, Rudenko E, Tamulaitienė M, Ramanau H, Strazdienė V, Samokhovec V, Ameliyanovich M, Byshnev N, Gonchar A, Kundas L, Zhur K. (2013).

Association Between Polymorphisms of VDR, COL1A1, and LCT genes and bone mineral density in Belarusian women with severe postmenopausal osteoporosis. Medicina (Kaunas).;49(4):177-84.

[12] Zhang YY, Long JR, Liu PY, Liu YJ, Shen H, Zhao LJ, (2003). Estrogen receptor alpha and vitamin D receptor gene polymorphisms and bone mineral density:Association study of healthy pre-and postmenopausal Chinese women. BiochemBiophys Res Commun.;308:777–

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[13] Dundar U, Solak M, Kavuncu V, Ozdemir M, Cakir T, Yildiz H.(2009). Evidence of association of vitamin D receptor Apa I gene polymorphism with bone mineral density in postmenopausal women with osteoporosis. Clin Rheumatol.;28:1187–91.

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[14] Lijuan F.; Jinhuan M.;Sumei Y.; and Qijun.(2020). A meta-analysis of VDR polymorphisms and postmenopausal osteoporosis. Endocrine Connections 9: 9, 882–889.

[15]Ahmad I, Jafar T, Mahdi F, Ameta K, Arshad M, Das SK, Waliullah S, Rizvi I, Mahdi AA.(2018). Association of vitamin D receptor gene polymorphism (TaqI and Apa1) with bone mineral density in North Indian postmenopausal women. Gene. 15;659:123-127.

[16] Banjabi AA, Al-Ghafari AB, Kumosani TA, Kannan K, Fallatah SM. (2020). Genetic influence of vitamin D receptor gene polymorphisms on osteoporosis risk. International journal of health sciences, 14(4), 22–28.

[17] Cantorna MT, Mahon BD. (2004). Mounting evidence for vitamin D as an environmental factor affecting autoimmune disease prevalence. Exp Biol Med (Maywood);229:1136–42.

[18].Valdivielso JM, Fernandez E. (2006).Vitamin D receptor polymorphisms and diseases.

ClinChim Acta.;371:1–12.

[19] Chen B, Zhu WF, Mu YY, Liu B, Li HZ, He XF.(2020). Association between vitamin D receptor BsmI, FokI, and Cdx2 polymorphisms and osteoporosis risk: an updated meta- analysis. Biosci Rep. Jul 31;40(7) .

[20] Bao L, Chen M, Lei Y, Zhou Z, Shen H, Le F.(2017). Association between vitamin D receptor BsmI polymorphism and bone mineral density in pediatric patients: A meta-analysis and systematic review of observational studies. Medicine (Baltimore). Apr;96(17):e6718

[21]. Lijuan F.; Jinhuan M.;Sumei Y.; and Qijun.(2020). A meta-analysis of VDR polymorphisms and postmenopausal osteoporosis. Endocrine Connections 9: 9, 882–889.

[22] Zintzaras E, Rodopoulou P, Koukoulis GN. (2006). BsmI, TaqI, ApaI and FokI polymorphisms in the vitamin D receptor (VDR) gene and the risk of osteoporosis: a meta- analysis. Dis Markers. 22(5–6):317–326

[23] Kow M, Akam E, Singh P, Singh M, Cox N, Bhatti JS, Tuck SP, Francis RM, Datta H, Mastana S. (2019). Vitamin D receptor (VDR) gene polymorphism and osteoporosis risk in White British men. Ann Hum Biol. Aug;46(5):430-433.

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