UGT1A1 (TA)n PROMOTER GENOTYPE: DIAGNOSTIC AND POPULATION PHARMACOGENETIC MARKER IN SERBIA
Vukovic M, Radlovic N, Lekovic Z, Vucicevic K, Maric N, Kotur N, Gasic V, Ugrin M, Stojiljkovic M, Dokmanovic L, Zukic B, Pavlovic S
*Corresponding Author: Sonja Pavlovic, Ph.D., Laboratory for Molecular Biomedicine, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO BOX 23, 11010 Belgrade, Serbia. Tel: +38111-3976445. Fax: +38111-3975808. E-mail: sonya@sezampro.rs
page: 59

MATERIALS AND METHODS

Subjects. For this study, 51 blood samples were obtained from children who were previously diagnosed as positive for GS at the University Children’s Hospital, Belgrade, Serbia. Ethical approval was obtained from the Ethics Committee of University Children’s Hospital, University of Belgrade. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent for the molecular analyses was obtained from the participants’ parent or guardian before the collection of the specimens. A 5 mL Na-citrate tube of whole blood was obtained for each subject during routine clinical checkups. All personal identifiers were removed; isolated DNA samples were tested anonymously. Also, 100 unrelated healthy individuals (67 children and 32 adults) were enrolled in the study. Ethical approval was obtained from the Ethics committee of the Institute of Molecular Genetics and Genetics Engineering, University of Belgrade. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent for the molecular analyses was obtained from all subjects before the collection of the specimens. In case of minor participants, written informed consent was obtained from the participants’ parent or guardian and a blood sample was taken during routine pediatric check-ups. A 5 mL Na-citrate tube of whole blood was obtained for each subject. All personal identifiers were removed; isolated DNA samples were tested anonymously. All subjects enrolled in the study were unrelated healthy donors and self-declared as Serbs. Hypocaloric Diet and Phenobarbitone Testing. Levels of conjugated and total bilirubin was measured in the GS patients group at diagnosis and after a 3-day hypocaloric diet test (400 kcal per day) during standard laboratory examinations. Further, a 3-day phenobarbitone test (2 mg/kg/day) was performed in patients group and levels of conjugated and total bilirubin was measured also. Level of unconjugated bilirubin was calculated as a mathematical difference between the total and conjugated levels of bilirubin. UGT1A1 (TA)n Promoter Genotyping. DNA was extracted from blood samples using the QIAamp® DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). The number of TA repeats in the UGT1A1 promoter was genotyped as previously described [20] with slight modifications. The amplification reaction was performed in a total volume of 25 μL, and the reaction mix contained 20 pmol of each primer, 50-100 ng of genomic DNA, 200 μmol/L of each dNTP (Fermentas, Burlington, ON, Canada), 1 × PCR reaction buffer (Qiagen GmbH), 1 × Q solution (Qiagen GmbH), 2.75 mM MgCl2, 1 U HotStar DNA polymerase (Qiagen GmbH). The temperature profile of the PCR reactions was for the initial activation of DNA polymerase set at 95 °C for 15 min., followed by 35 cycles of 30 seconds denaturation at 95 °C, 30 seconds annealing at 63 °C, and 30 seconds elongation at 72 °C, ending with a final extension period of 7 min. at 72 °C. The PCR fragments were visualized on 2.0% agarose gel and finally analyzed using 15.0% acrylamide electrophoresis (19:1 acrylamide/bisacrylamide in 1 × TBE buffer, run at 300V and 60 mA for 5 hours) stained with Ag-nitrate [21]. Each acrylamide electrophoresis run had a positive (TA 7/7) and negative (TA 6/6) control sample, previously confirmed using the Sanger sequencing methodology. Twenty percent of samples were randomly chosen and results of UGT1A1 promoter genotyping by the PCR/ acrylamide electrophoresis methodology were checked and confirmed using fragment length analysis of fluorescent PCR products [22]. The 12 μL PCR reaction contained 50 ng of DNA, 1 × PCR buffer (Qiagen GmbH), 15.4 mM MgCl2, 0.4 mM dNTPs, 1 U of HotStar Polymerase (Qiagen GmbH) and 0.4 μM of both UGT1A1F (FAM dye-labeled, 5’-famTAC AGT CAC GTG ACA CAG-3’) and UGT1A1R (5’-TTT GCT CCT GCC AGA GGT TCG- 3’) primers. The temperature profile of the PCR reactions was for the initial activation of DNA polymerase set at 95 °C for 15 min., followed by 35 cycles of 30 seconds denaturation at 95 °C, 30 seconds annealing at 55 °C, and 30 seconds elongation at 72 °C, ending with a final extension period of 7 min. at 72 °C. The PCR products were separated on an ABI PRISM® 3130 DNA analyzer (Applied Biosystems, Foster City, CA, USA) and the collected data were analyzed with the GeneMapper version 4 software (Applied Biosystems). Ten percent of samples were randomly chosen and results of UGT1A1 (TA)n promoter genotyping by the PCR/ acrylamide electrophoresis methodology were checked and confirmed using Sanger sequencing methodology. Those samples were used as positive/negative controls on each acrylamide electrophoresis run [23]. UGT1A1 Gene Analysis. According to ENST00000 305208.9, primers were designed to analyze all five coding exons and nearby intronic sequences of the UGT1A1 gene. The amplification reaction was performed in a total volume of 30 μL, and the reaction mix contained 10 pmol of each primer, 50-100 ng of genomic DNA, 0.5 mM of each dNTP (Fermentas), 1 × PCR reaction buffer, 1.4 mM MgCl2, 1 U DNA polymerase (KAPA Biosystems, Wilmington, MA, USA). The temperature profile of the PCR reactions was for the initial activation of DNA polymerase set at 95 °C for 15 min., followed by 35 cycles of 30 seconds denaturation at 95 °C, 30 seconds annealing as given in Table 1, and 30 seconds elongation at 72 °C, ending with a final extension period of 10 min. at 72 °C. The PCR fragments were visualized on 2.0% agarose gel, separated on an ABI PRISM® 3130 DNA analyzer (Applied Biosystems) and the collected data were analyzed with the Gene Mapper version 4 software (Applied Biosystems). Statistical Analysis. All statistical analysis was performed using the SPSS® Statistics version 21 software (IBM). The difference in UGT1A1 (TA)n promoter genotype frequencies (risk and non-risk GS genotypes) between control group and GS patients was assessed using Fisher’s exact test. UGT1A1 (TA)n promoter genotype frequencies of the control group were checked for the Hardy-Weinberg equilibrium by exact test [24]. The distribution of unconjugated bilirubin in GS patients was checked for normality using Shapiro-Wilk and Kolmogorov-Smirnov tests. The difference in the level of unconjugated bilirubin between carriers of risk and non-risk GS genotypes was assessed using the Mann-Whitney test. Probability values of <0.05 were considered statistically significant. All tests were nondirectional (two-tailed).



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