MATRIX METALLOPROTEINASE-2 (MMP-2 ) AND-9 (MMP-9) GENE VARIANTS AND MICROVASCULAR COMPLICATIONS IN TYPE 2 DIABETES PATIENTS
Andjelic Jelic M٭ˡ, Radojkovic D², Nikolic A², Rakicevic Lj², Babic T², Jelic D³, Lalic NM⁴
*Corresponding Author: Marina Andjelic Jelic, Department of Endocrinology, Diabetes and Metabolic Diseases, Clinical Medical Centre Zvezdara, Dimitrija Tucovica 161, 11000 Belgrade, Serbia, Phone +381 64 122 2010 Fax +381 11 3088733, E mail: drmajelic@gmail.com
page: 6

MATERIAL AND METHODS

Study design, time and place This study contained 102 subjects with type 2 diabetes who were at the Zvezdara University Medical Center from February 2016 to April 2018. The diagnosis of DM was established using the criteria of the World Health Organization (WHO). The presence of microvascular diabetes complications was investigated in all patients on the basis of medical history, laboratory analysis and physical examination. Detailed ophthalmological examination and fundoscopy were done in order to confirm retinal changes due to diabetes. Classification was done to distinguish between nonproliferative retinopathy and proliferative retinopathy. The diagnosis of nephropathy was established based on urine albumin excretion in 24h urine. The cut off value was 30 mg/24h. A comprehensive diabetic foot examination comprising of visual inspection, monofilament examination, pinprick sensation, and ankle reflexes was performed in order to establish the diagnosis of diabetic polyneuropathy. The control group was comprised of 56 healthy subjects who were recruited during their regular annual health assessments. The local Ethics Committee gave permission to conduct the study and each participant signed informed consent. Variable Apart from demographic variables (gender and age), additional information regarding the duration of diabetes, antidiabetic medications, and smoking history was collected. Anthropometric variables included measurements of height, weight, and body mass index (BMI). The BMI was calculated according to the following formula: BMI (kg/m2) ꞊ body weight (kg)/ height (m2). Metabolic variables included measurements of fasting blood glucose (FBG), total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, total triglycerides, serum creatinine, and glycosylated hemoglobin (HbA1c). Peripheral blood samples were taken from the patients with type 2 diabetes and the control subjects after overnight fasting for at least 8 hours and metabolic parameters were analyzed using the biochemical analyzer Olimpus AU680. Detection of the MMP-2 and MMP-9 gene variants was performed using polymerase chain a reaction-restriction fragment length polymorphism (PCRRFLP) analysis [34]. The region containing the MMP-2 variant -1306C>T was amplified using a forward primer 5-CTTCCTAGGCTGGTCCTTACTGA-3 and a reverse primer 5-CTGAGACCTGAAGAGCTAAAGAGCT-3. The region containing the MMP-9 variant -1562C>T was amplified using a forward primer 5-GCCTGGCACAT-AGTAGGCCC-3 and a reverse primer 5-CTTCCTAGCCAGCCGGCATC- 3. Amplification of both regions was performed by PCR reaction directly from blood using the following program: 3 cycles at 98C for 5 min and 55C for 3 min; 95C for 5 min; 35 cycles at 95C for 30 sec; 58C for 45 sec; and 72C for 45 sec; and 72C for 10 min. The PCR products obtained with primers for MMP-2 (193bp) were incubated with the restriction endonuclease BfaI (Thermo Scientific, Waltham, MA, USA) at 37C overnight, resulting in either a 193bp fragment containing the -1306C allele or 164bp and 29bp fragments in the presence of the -1306T allele. The PCR products obtained with primers for MMP-9 (436bp) were incubated with the restriction endonuclease SphI (Thermo Scientific, Waltham, MA, USA) at 37C overnight, resulting in a 436bp fragment containing the -1562C allele or 194bp and 242bp fragments in the presence of the -1562T allele. Alleles were separated on agarose gel electrophoresis and visualized with ethidium-bromide staining and UV transillumination.



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