Balkan Journal of Medical Genetics

ASSOCIATION BETWEEN THE POLYMORPHISM OF ANGIOTENSIN-CONVERTING ENZYME GENE AND INTERLEUKIN-1 BETA GENE AND THE RESPONSE TO ERYTHROPOIETIN THERAPY IN DIALYSIS PATIENTS WITH ANEMIA
Dzekova-Vidimliski P, Eftimovska-Otovikj N, Nikolov I G, Selim Gj, Rambabova-Bushljetik I, Pushevski V, Karanfilovski V, Matevska-Geshovska N, Dimovski A
*Corresponding Author: Assoc. Prof. Pavlina Dzekova-Vidimliski, MD, PhD, University Hospital for Nephrology, Mother Theresa str 17, 1000 Skopje, R. North Macedonia, Email address: pavlinadzekova@yahoo.com
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PATIENTS AND METHODS

The study included 69 patients with stage 5 chronic kidney disease on maintenance hemodialysis or perito- neal dialysis. All patients signed an informed consent for participation in the study. The study was approved by the ethical commission of the Faculty of Medicine, Ss. Cyril and Methodius University in Skopje, Skopje, RN Macedonia. The design of the study was a prospective, longitudinal study, with a duration of 12 months. Inclusive criteria for study patients: l older than 18 years, l treatment with dialysis for at least 3 months, l treatment of anemia with recombinant human erythropoietin. Exclusion criteria for study patients: l bleeding diagnosed before involvement in the study, l the persistence of malignant disease, l hemoglobinopathies and diseases of the bone mar- row. The patients were recruited from the hemodialysis unit and peritoneal dialysis unit at the University Hospital of Nephrology in Skopje and the Department of Nephrol- ogy and Dialysis at the General City Hospital “8 mi Septem- vri” in Skopje. The medical histories of the patients were used to determine demographic characteristics, etiology of kidney disease, dialysis vintage, and total weekly dose of erythropoietin. The laboratory data were obtained from the routine laboratory analyses of dialysis patients during the study period of 12 months. The total red blood cell count, hematocrit, hemoglobin (Hb), total protein, albumin, alka- line phosphatase, calcium, phosphorus, C-reactive protein (CRP), iron, and total iron binding capacity (TIBC) were analyzed monthly. The transferrin saturation index (TSAI) was calculated using the following equation: (serum Fe/ TIBC) x 100% (20). The serum concentration of ferritin was determined once in three months, with a target value of more than 500 ng/ml, but not exceeding 800 ng/ml (20). The serum concentration of intact parathyroid hor- mone (iPTH) and cholesterol was determined once in six months. The erythropoietin resistance index (ERI) was calculated monthly as the weekly rHuEPO dose per kg of body weight, divided by the hemoglobin concentration in g/dl (10). Genotyping of ACE and IL-1b polymorphism was done in all study patients at the initiation of the study in the Center for Biomolecular Pharmaceutical Analysis at the Institute of Pharmaceutical Chemistry at the Faculty of Pharmacy in Skopje. Genomic DNA from all study participants was isolated from peripheral blood using the MagCore Genomic DNA Whole Blood Kit (RBC Bioscience), following the manufacturer’s instructions. The ACE polymorphism (rs1799752) was genotyped by fluorescent PCR followed by fragment analysis on 3500 Automated Genetic Analyzer (Thermo Fisher Scientific), using the following primers: ACE_I/D_F:5’-CTGGAGA- CCACTCCCATCCTTTCT-3’ and ACE_I/D_R: 6FAM-5’- GATGTGGCCATCACATTCGTCAGAT-3’. A total of 100 ng of DNA was amplified in 25μL final volume including 2 mM Mg 2+ , 0.2 mM of each dNTPs, 0.5μM of each primer and 1U HOT FIREPol® DNA Polymerase (Solis Bio- Dyne), using the following program: initial denaturation at 95°C for 10 minutes; 35 cycles of 30 seconds at 95°C, 30 seconds at 58°C and 30 seconds at 72°C; and final elonga- tion at 72°C for 10 minutes. The IL-1b gene (rs1143627) polymorphism was genotyped by allele discrimination PCR on a Stratagene Mx3005P (Agilent Technologies) real-time PCR system using TaqMan® SNP genotyping assay (reference ID: C___1839944_10; Thermo Fisher Scientific). The genotypes were determined in a reaction mix containing 20 ng DNA in a total volume of 25 μL, according to the manufacturer’s recommended protocol. Positive and negative controls were included on each plate and reproducibility was checked by re-genotyping 10% of the cases.

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