This was a cross-sectional study, which included 97 Egyptian women. Their ages ranged between 25 and 65 years with a mean age of 48.85 ± 9.88 years. They were recruited and randomly chosen, from all employees and workers, of all categories, of the “National Research Centre”, Egypt. All participated women were free from any chronic disease or under long-term medications. A written informed consent was obtained from all participants after being informed about the purpose of the study. This research paper was derived from a cross-sectional survey of a project funded by “National Research Centre”, Egypt, 2016–2019 entitled “Bone mass among Overweight and Obese Women: Mechanism and Intervention.” “National Research Centre”, with an approval obtained from the Ethics Committee of “National Research Centre” (registration number is 16/127).
For each participant woman, anthropometric measurements, dual-energy X-ray absorptiometry (DEXA) measurements, and molecular genetic analysis were done.
Anthropometric measurements
Body weight and height were measured, following the recommendations of the “International Biological Program” [16]. Body weight (Wt) was determined to the nearest 0.01 kg using a Seca Scale Balance, with the woman wearing minimal clothes and with no shoes. Body height (Ht) was measured to the nearest 0.1 cm using a Holtain portable anthropometer. Body mass index (BMI) was calculated: [BMI: weight (in kilograms) divided by height (in meters squared)]. The participant women were classified according to their BMI into 2 groups: 31 women with normal BMI (<25 kg/m2) and 66 overweight/obese women (> 25 kg/m2).
DEXA measurements
Both bone mineral density “BMD” (gm/cm2) and BMD T score at the neck of the femur were measured using dual-energy DEXA (DEXA Norland XR-46 version 3.9.6/2.3.1, USA). A full body DEXA scan, based on the woman’s age, weight, and height, was performed with the participant keeping the precise distance between her arms and legs according to the machine instruction manual. A well-qualified operator executed and evaluated all analyses using the same protocol for all assessments. According to the WHO diagnostic criteria [17] depending on BMD T score at any of the recommended sites (lumbar spine or femoral neck), the women were classified into 3 groups: women with healthy bone ( > −1), osteopenia (between −1 and > −2.5), and osteoporosis (<−2.5) [18]. After that, BMD T score −1 was taken to group the participating women to a non-osteoporotic or osteoporotic group.
Molecular genetic analysis
Genomic DNA extraction
Genomic DNA was extracted using Qiagen QIAamp DNA Blood Mini Kit from whole blood samples according to the manufacturer’s protocol. The concentration of genomic DNA was determined by quantitative method, based on optical density measurement using NanoDrop UV/V (Thermo Scientific, UK). The purity of DNA was determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm (A260/A280). Pure DNA should have an A260/A280 ratio of 1.7–1.9, respectively.
VDR gene polymorphism genotyping by PCR-restriction fragment length polymorphism (RFLP)
The ApaI and TaqI polymorphic sites of VDR were considered. The targeted SNP was amplified by conventional polymerase chain reaction (PCR) and followed by restriction digestion. The VDR genotype of each subject was identified according to the digestion pattern and alleles.
TaqI polymorphism
PCR amplification: PCR reaction was carried out in 25-μL reaction mixture containing 1.5 mM MgCl2, 0.2 mM dNTP, and 10 pmoles of each primer sequences F: 5′-CAG AGC ATG GAC AGG GAG CAA-3′ and R: 5′-CAC TTC GAG CAC AAG GGG CGT TAG C-3′ as described previously by Mohamed and El-Askary [19], 0.5 U of Taq DNA polymerase, and 200ng of genomic DNA. PCR conditioning was as follows: initial denaturation for 5 min at 95°C, 30 cycles of 30s at 94°C, annealing for 45s at 57°C, extension for 60s at 72°C, and a final extension for 5 min at 72 °C.
ApaI polymorphism
PCR amplification: PCR reaction was carried out in 25-μL reaction mixture containing 1.5 mM MgCl2, 0.2 mM dNTP, and 10 pmoles of each primer sequences F: 5′-CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA-3′ and R: 5′-CAC TTC GAG CAC AAG GGG CGT TAG C-3′ as described previously by Mohamed and El-Askary [19], 0.5 U of Taq DNA polymerase, and 200ng of genomic DNA. PCR conditioning was as follows: initial denaturation for 5 min at 95°C, 30 cycles of 30s at 94°C, annealing for 45s at 55°C, extension for 60s at 72°C, and a final extension for 10 min at 72 °C.
Post-PCR-RFLP: The resulting DNA fragments were subjected to restriction digestion using respective enzymes ApaI and Taq-I (Promega, Madison, USA, 10 U/ml). The Eppendorf tubes for RFLP were prepared as follows: 10 μl of PCR product; 16.3 μl of sterile, deionized water; 0.2 μl of 100X BSA; and 2 μl of 10X RE Buffer, and mixed by pipetting. Finally, 10 units of each of the respective restriction enzymes were added. The tubes were incubated (2 h at 37°C for ApaI and Taq I polymorphisms) and heat inactivated for 15 min at 80°C. The genotypes were resolved on 2% (w/v) agarose gels.
3-Genotyping: The genotypes were resolved on 2% (w/v) agarose gels. ApaI polymorphism genotyping for wild type homozygosis fragment (AA) at 740-bp mutant homozygosis fragments (aa) at 530 and 210 bp and heterozygosis produces fragment (Aa) of 740, 530, and 210 bp. In the presence of A-allele, there was no restriction enzyme cleavage site and a product of 740 bp was obtained. In subjects carrying “a-allele,” the cleavage products of 530 and 210 bp were detected. Alleles “A” and “a” were assigned based on the presence of a 740-bp (uncleaved) fragment and the 530-bp and 210-bp (cleaved) fragments, respectively (Fig. 1). TaqI polymorphism genotyping for wild type homozygosis produces fragment (TT) at 495 bp while heterozygosis produces fragments (Tt) at 495, 290, and 205 bp. The allele “T” was associated with the presence of a 495-bp fragment, while allele “C” was assigned in the presence of 290-bp and 205-bp fragments (Fig. 2).
Statistical analysis
Data were analyzed using the Statistical Package for Social Sciences (SPSS/Windows version 16, SPSS Inc., Chicago, IL, USA). The normality of data was tested using the Kolmogorov-Smirnov test. The data of DEXA, weight, and BMI were not normally distributed. So, non-parametric tests were used.
The 97 participant women were classified twice into 2 groups: first according to their BMI (31 normal weight and 66 overweight/obese) and second according to their BMD T score (23 osteoporotic and 74 non-osteoporotic). The parametric data were expressed as mean ± SD, where the qualitative ones were expressed as number and percentage (%). The various parametric variables of the different groups were analyzed and compared using the Mann-Whitney test for independent groups, while the frequency distribution of the vitamin D receptors among different groups (non-parametric data) were compared using the chi-square test. P < 0.05 was regarded as statistically significant for all tests.
