Subjects
Two hundred subjects referred to Peymanieh hospital (Jahrom city, Iran) including 100 healthy individuals and 100 type 2 diabetes mellitus patients were recruited in the study. Patients had a fasting blood glucose (FBG) > 125 mg/dl. However, subjects with underlying disease including cancer, liver and kidney disease, gastrointestinal tract disease were excluded from the study. Low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triacylglycerol (TG) and FBG were measured by routine biochemical assays.
DNA extraction and PCR-RFLP
Venous blood samples were drawn in an EDTA-containing tube and stored in -80 C until the DNA extraction. Salting out technique was used to extract the genomic DNA (21) and stored rapidly in -20. Three single nucleotide polymorphisms were investigated in the study. On the other hand, rs4380143 T > C with minor allele frequency (MAF) 0.3 was located on upstream promoter region, rs34125392 T>- with MAF 0.26 was located on 5ʹUTR region, and rs1800437 G > C (Glu to Gln) with MAF 0.2 was located on coding region. RFLP-PCR was applied to genotyping and allele identification. The sequences of primers used in the study are summarized in Table 1.
Table 1
Sequence of primers used for detection of rs34125392, rs4380143 and rs1800437 in GIPR gene
Primers
|
Sequence
|
GIPR5392. R
|
5'-GGTGGGACAGCATGAGAGATTGTA − 3'
|
GIPR5392.F
|
5'-GTTATCTAGCAGCTAACCAGAGATGGA-3'
|
GIPR0143. R
|
5'-CCAAGAGTTGGAGACCAGCATGG -3'
|
GIPR0143.F
|
5'-CAGTTCCAACAACACTGTCAATCACC-3'
|
GIPR0143.Nes.R
|
5'-GTTCCAGTGCACTCCACTCTCAT − 3'
|
GIPR0143.Nes.F
|
5'-CAGGCTGGTCTCAAACTCCTG-3'
|
GIPR00437. R
|
5'-GCATTCTTGGCATTCTCCTGTCC − 3'
|
GIPR00437.F
|
5'-GAAGGAGCTGAGGAAGATCTCAAAGC-3'
|
F: forward primer, R: Reverse primer, Nes: Nested-PCR |
Reactions were performed in a micro-tube with final volume of 25 µl containing 0.2 µg genomic DNA, 0.8 U Hot start Taq DNA polymerase and 1.5 mM MgCl2. The temperature cycles for rs34125392 were 940C for 30 seconds, 630C for 45 seconds and 720C for 35 seconds for 30 cycles. The temperature cycles for rs4380143 were 940C for 30 seconds, 650C for 45 seconds and 720C for 50 seconds for 30 cycles. The product of this step was the template for Nested-PCR with new pair of primers (Table 1) to detect rs43800143. The temperature cycles for Nested-PCR were 940C for 30 seconds, 620C for 25 seconds and 720C for 35 seconds for 25 cycles. The temperature cycles for rs1800437 were 940C for 30 seconds, 650C for 45 seconds and 720C for 20 seconds for 30 cycles. Initial incubation period was 5 minutes at 940C and a final extension incubation step was set at 720C for 5 minutes for all reactions.
Then, the PCR products of rs34125392, rs4380143 and rs1800437 were subjected to digestion with BtsI, FatI and BssSI, respectively (Newengland Biolab; 10U, overnight). The digested PCR products were run on 3% agarose gel and visualized by UV transillumination after DNA green viewer staining.
Statistical analysis
SPSS v.18 (Chicago) was used for statistical analysis. Normality of the data was checked by Kolmogorov-Smirnov test. Hardy-Weinberg equilibrium was performed to survey allele distribution. Logistic regression was used to survey odds ratios. The numeric data were reported as mean ± Standard Error (SE). Student t and Chi square tests were applied to investigate the differences between groups, genotype and allele variations. P value less than 0.05 was considered to be significant.