Study subjects
The study design is a case–control, consisting of 400 T2DM patients in addition to 400 individuals as control. The specimen collections were done from Oct. 2019 till Jan. 2020. The analysis of phenotyping and genotyping were done in the postgraduate laboratory of department of biochemistry in faculty of medicine in Kufa University. The selection of sample size was done according to online software (osse.bii.a-star.edu.sg/calculation1.php).
A total of 400 type 2 diabetes individuals were enrolled in present study (205 female and 195 male). Patients varied in age from 30 to 69 years old, the mean of age equal to 52.93 \(\pm\) 7.00 years as SD. Specialist physicians have examined the patients. They were randomly selected from AL-Sader Teaching Hospital in al Najaf Province. For the patients to be included in this study, the diagnosis of T2DM according to WHO criteria. Exclusion criteria were adopted to deport patients who have the following:
A second group consist of 400 normal individuals were randomly selected from the general population (205 males and 195 female) as control group.
From all participants in this study a 5 ml of blood were taken by venipuncture and collected in two tubes, 3.0 ml of 5 placed in plain tube (without anticoagulant) and the remaining quantity (2 ml) has been placed in tube with EDTA. The first tube (plain tube) was centrifuged at 2000×g for 10 min after being allowed to coagulate at room temperature for 10–15 min. The serum that obtained from previous step was stored at − 20 °C until analysis for phenotyping estimation. Extraction of DNA was done from other tube that contains EDTA the samples of DNA were maintained frozen at − 20 °C until gene polymorphisms were examined.
Informed consent has been taken from patients and control group before sampling, the protocol of this study was approved by the Kufa Medical Faculty Ethical Committee.
Measurements
Anthropometric data as BMI and biochemical parameters such as FPG, serum total cholesterol, serum triglyceride, VLDL, LDL, HDL insulin levels and HOMA-IR were measured for all individuals participating in the study. Serum total cholesterol, triglyceride, HDL in addition to fasting blood glucose estimated according to "standard enzymatic colorimetric assay" method in contrast LDL and VLDL were calculated by using mathematical formulas. Enzyme-linked immunosorbent assay ELISA was used to estimate insulin concentration whereas HOMA-IR was used for Insulin Resistance assessment.
Genotyping
DNA purification kit (G-spin) was used for DNA extraction from blood samples for all individuals participating in the study. Polymerase chain reaction-Restriction Fragment Length Polymorphism (PCR–RFLP) technique were used to determined CDKN2A/B rs10811661 T>C and rs2383208 A>G gene polymorphisms.
The primers of SNPs that used in current study were designed by Primer3plus software as the following:
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For rs10811661 SNP:
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For rs2383208 SNP:
The conditions of PCR–RFLP technique
A total volume of DNA amplification reaction was 25 µl which consist of GoTaq® Green Master Mix from Promega (12.5 µl), forward and reverse primer (1.5 µl) from both of them, genome DNA (5 µl) then Complete the final volume by nuclease-free water (4.5 µl). The conditions of thermo-cycle for rs10811661 SNP amplification was initial denaturation (5 min at 95 °C) for one cycle then for 30 cycles included denaturation step (1 min at 95 °C), annealing step (1 min at 62 °C) and extension step (1 min at 72 °C) then final extension step (10 min. at 72 °C). The product of PCR was 232 bp detected by 2% agarose gel electrophoresis. The same programed protocol of PCR reaction done for rs2383208 SNP, while the amplification protocol was as following: initial denaturation (5 min at 95 °C) then 35 cycles consisting of denaturation step (30 s at 95 °C), annealing step (30 s at 59.3 °C) and extension step (30 s at 72 °C) then the final extension step (5 min. at 72 °C). Also the size of PCR product also detected by 2% agarose gel electrophoresis which was 246 bp. PCR product for both SNPs were digested by specific restriction enzymes. For rs10811661 SNP the restriction enzymes was BspHI, the recognition sequence T^CATGA, the digestion temp. was 37 °C and the time of digestion was 8 h. while for rs2383208 SNP the restriction enzymes was Hpy166II, the recognition sequence GTN^NAC.
The digestion temp. was 37 °C and the digestion time was 15 min. Digested fragments electrophoresed in 3% agarose gel agarose (75 V and 120 min) with diamond stain and visualized under UV light.
Statistical analysis
For statistical analysis, SPSS version 23 was used. Phenotypic data were expressed as mean ± SD, T test used to determine the significance between the two groups. ANOVA test was used to comparing mean levels for continuous characteristics across genotypes Chi-square test was used to determine genotype frequencies among T2DM patients and healthy controls. The Hardy Weinberg Equilibrium equation was used to look at allelic frequencies. The associations between CDKN2A/B (rs10811661 and rs2383208) genotype with T2DM risk were estimated by calculating odds ratio with 95% CI using multinominal logistic regression analysis. P values of less than 0.05 were regarded significant.
