ROLE OF THE APOB GENE POLYMORPHISM (c.12669G>A, p.Gln4154Lys) IN CORONARY ARTERY DISEASE IN THE INDIAN PUNJABI POPULATION
Sharma R1,*, Mahajan M2, Singh B1, Singh G3, Singh P3
*Corresponding Author: Ritu Sharma, Department of Biochemistry, Government Medical College, Circular Road, Amritsar-143001, Punjab, India; Tel.: +91-183-257-3637; Fax: +91-183-242-6506: E-mail: ritu_gmc@ rediffmail.com
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MATERIAL AND METHODS

We recruited 87 CAD patients from the Out Patient Departments and Medical Wards of the Employee’s State Insurance (ESI) and Guru Nanak Dev hospitals, Amritsar, India, diagnosed with CAD by the clinician on the basis of clinical symptoms, echocardiogram (ECG), changes, stress test and angiography (if required). Seventy-four healthy individuals without any evident symptoms of CAD and any past history of the disease, were taken as controls from the general population. Subjects were segregated into males and females and divided into three age groups: 35-45, 46- 55 and 56-65 years. A written informed consent was signed by each individual. A detailed questionnaire was prepared for all the necessary details of both patients and control subjects to answer. Coronary artery disease patients suffering from diabetes mellitus, renal or thyroid disease, rheumatoid arthritis, acute infections, on lipid lowering medications or women on oral contraceptives or who had undergone hysterectomy were excluded. Venous blood samples were taken after 12-hour overnight fasting. Serum was used for various biochemical investigations. High-density lipoprotein (HDL) was isolated from serum by precipitating LDL and very lowdensity lipoprotein (VLDL) with magnesium chloride and sodium phosphotungstate reagent [14]. Total cholesterol and HDL cholesterol levels were estimated by extracting it quantitatively into acetone-ethanol mixture. The cholesterol residue was dissolved in glacial acetic acid and allowed to react with concentrated sulphuric acid and ferric chloride reagent [15]. Serum triglycerides were estimated using a commercially available kit from Biotech, Baroda, Gujrat, India [16]. The LDL cholesterol levels were calculated from the formula given by Friedwald et al. [17]. Serum apoB levels were estimated using an immunoturbidimetric kit (Diasys, Holzheim, Germany). The LDL was isolated from serum. Briefly, the precipitation buffer consisted of 0.064 M trisodium citrate adjusted to pH 5.05 and contained 50,000 IU/L heparin. The insoluble lipoproteins were sedimented by centrifugation at 1,000g for 10 min. The pellet was resuspended in 1 mL of 0.1 M sodium phosphate buffer at pH 7.4 containing 0.9% normal saline [18]. The LDL protein oxidation as apoB carbonyl content was assessed. Briefly, 0.2 mL of dinitrophenylhydrazine (10 mM DNPH in 2 M HCl) was added to the isolated LDL solution in a centrifuge tube. The contents were incubated for 1 hour at room temperature. Then 0.6 mL of denaturing buffer (0.15 M sodium phosphate buffer, pH 6.8, containing 3% sodium dodecyl sulphate) was added and the tubes were allowed to vortex for 1min. After mixing, an equal volume of ethanol and heptane mixture was added and the contents were mixed again. The tubes were centrifuged at 1,000 g for 5 min. The LDL was recovered from the interface and washed three times with ethanol ethyl acetate (1:1 v/v) mixture. Each DNPH sample was dissolved in denaturing buffer and was scanned from 320 to 410 nm. The peak absorbance was used to calculate protein carbonyls with extinction coefficient 22,000 M–1 cm–1 [19]. Results were expressed as nmoles carbonyl/mg LDL protein. The LDL lipid peroxidation as malondialdehyde (MDA) levels was estimated by the method of Beuge and Aust [20]. The protein content of the LDL sample was estimated according to the protocol of Lowry et al. [21]. DNA was isolated from the blood samples by the phenol chloroform method [22] and the quality was checked on 0.8% agarose gel electrophoresis. DNA amplification kits and primers were procured from Genei, Bangalore, India. A 480 bp sequence of APOB gene containing the EcoR1 restriction site was amplified by polymerase chain reaction (PCR) on a thermal cycler (Bio-Rad India Pvt Ltd., Gurgaon, India). The following primers were used to amplify the desired sequence in APOB gene: forward primer (F) 5’-CTG AGA GAA GTG TCT TCG AAG-3’ and reverse primer (R) 5’- CTC GAA AGG AAG TGT AAT CAC-3’. The reaction mixture was prepared to a final volume of 50 μL; 2 μL of DNA (1 μg/μL) was added to the assay mixture. The amplified sequence was isolated after 0.8% agarose gel electrophoresis and digested overnight at 37°C with 20 U of EcoR1. The digested products were resolved on 2.5% agarose gel electrophoresis in 1X Tris-acetate ethylenediaminetetraacetic (TAE) buffer, pH 8.0, at a constant voltage of 50 V. A 480 bp sequence containing the EcoR1 restriction site was digested into fragments of 253 and 227 bp, respectively. In the absence of the EcoR1 restriction site, the 480 bp sequence produced only a single band on the agarose gel. The respective alleles were designated as R+ and R–. Stastistical Analysis. Results were expressed as mean ± standard deviation (SD). Chi-Square was applied to determine the significance in allele frequency. The Student’s t test was applied to assess the significance in other parameters. Statistical significance was determined at p <0.05.



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