Subjects and sample collection: This case-control research study was performed between 2014 and 2016 and it included 163 subjects (Table 1). Fully informed consent was acquired from the control and diseased subjects, and this research was affirmed by the Umm Al Qura University’s IRB (Internal Review Board). Seventy eight unrelated type 2 diabetic patients were enlisted from health centers and hospitals in Makkah district, Saudi Arabia. The control subjects consisted of eighty five healthy subjects who either attended for a normal wellbeing check at a general practice or at work. Venous blood was collected from all subjects between 9:00 and 11:00 a.m. subsequent to fasting from 10:00-11:00 p.m. the previous day. Each specimen was divided into two halves, one half for the serum preparation and the other half was put in sterile K3EDTA (tri-potassium ethylene-diaminetetraacetic acid) coated tubes. Low speed centrifugation was used to isolate blood plasma; white cells were aspirated from the buffy coat for the isolation of DNA. Samples were kept at –20?C till the time of analysis.
Determination of fasting blood glucose and hemoglobin A1c (HbA1c):
We used glucose oxidase technique to determine the fasting glucose level, using a kit from Human Diagnostics, Wiesbaden, Germany. The amount of HbA1c was determined using enzymatic HbA1c assay kit according to the maker directions (Human Diagnostics, Wiesbaden, Germany).
Genomic DNA extraction:
Genomic DNA was separated from peripheral blood leukocytes utilizing/using Qiagen DNA extraction kit (QIAamp DNA Blood Mini Kits, Qiagen, Hilden, Germany). The extraction was performed according to the maker directions. For PCR, aliquots of genomic DNA were used.
VDR FokI and BsmI genotyping:
The FokI polymorphic region genotyping of exon 2 of VDR gene was done as described elsewhere with slight modification32. PCR was performed using the following oligonucleotide primers: 5′ -AGC TGG CCC TGG CAC TGA CTC TGC TCT-3′ (forward primer) and: 5′ -ATG GAA ACA CCT TGC TTC TTC TCC CTC- 3′ (used as the reverse primer). In 25 ml PCR mixture, 100 ng of genomic DNA were utilized for PCR amplification and 2 ´ PCR master mix was used (Thermo Fisher Scientific, Inc, MA USA). Following conditions were utilized for the PCR amplification: initial DNA denaturation at 96°C for 4 min, then 30 cycles of denaturation for 30 seconds at 94 °C, annealing for 30 seconds at 57 °C, and extension for 30 seconds at 72 °C. The reaction was terminated following 7 min elongation at 72 °C. PCR product was then digested with FokI restriction endonuclease. Restriction fragments were analyzed by electrophoresis through a 2 % agarose gel containing ethidium bromide, the bands were visualized under ultraviolet light and photographed.
For genotyping of VDR BsmI, the primers used were: the forward primer: 5¢-AAC CAG CGG GAA GAG GTC AAG GG- 3¢ -and the reverse primer: 5¢ -CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA- 3¢. The PCR reaction was done in 25 ml final volume and 2 ´ PCR master mix was utilized. The PCR program was as follows: At the start of PCR, we began by denaturation step at 95 °C for 3 min, then, 35 ´ (95 °C for 30 s, 65°C for 30s, 72°C for 30 s) and lastly, 72 °C for 10 min. The amplified PCR product was digested using BsmI restriction enzyme. DNA fragments were separated on 2 % agarose gel by electrophoresis and genotypes were identified as previously described 33.
Statistical Analysis: We used SPSS for Windows version 20.0 (SPSS Inc, Chicago, IL, USA) for data analysis. To compare mean values of continuous variable in cases and control, Student’s t test was utilized, whereas ?2 analysis was utilized to evaluate categorical data.
Table-1 demonstrates the demographic data in studied groups. Table 2 illustrates the genotype and allele frequencies in both T2DM and control groups. The genotype distribution of FokI polymorphism is in Hardy-Weinberg equilibrium in both the T2DM and control subjects. The genotypes FF, Ff and ff were 61.5 %, 24.3 % and 14.1% in subjects with T2DM respectively and were 55.2 %, 36.4 % and 8.2 % respectively in the control group.
We did not observe any statistically significant difference in the genotype and allele frequencies between patients with T2DM and healthy controls. The BsmI genotype and allele distribution, were observed to be in Hardy-Weinberg equilibrium in both T2DM and control groups. BsmI genotypes and allele frequencies in T2DM groups and in controls are shown in Table 2. In the T2DM patients, the genotypes BB, Bb and bb were 29.48 %, 33.33 % and 37.17 % respectively. However, these genotypes were 29.62 %, 32.1 % and 38.27 % in the control subjects respectively. There was no statistically significant difference in the distribution of genotypes and allele frequencies of BsmI between patients with T2DM and the matched healthy controls.