Balkan Journal of Medical Genetics

DO GENE POLYMORPHISMS PLAY A ROLE IN NEWBORN HYPERBILIRUBINEMIA?
Hakan N, Aydin M, Ceylaner S, Di̇lli̇ D, Zenci̇roğlu A, Okumuş N
*Corresponding Author: Assoc. Prof. Nilay Hakan, MD, Division of Neonatology, Sitki Koçman University School of Medicine, Orhaniye Mah., Haluk Ozsoy Sk., 48000, Muğla / Türkiye, Phone: +90 (252) 214 13 26, Fax: +90 (252) 211 13 45, E-mail: nhakan@hotmail.com
page: 51

MATERIALS AND METHODS

Patients This study was conducted at Dr. Sami Ulus Maternity and Children’s Research and Training Hospital between April 2011 and May 2012. Of the 130 newborn babies included in the study, 89 jaundiced babies consisted of the study group (61 babies with idiopathic hyperbilirubinemia, 28 babies with prolonged jaundice of unexplained etiol- ogy), and 41 healthy babies without jaundice consisted of the control group. NH refers to newborn babies with serum total bilirubin levels above 17 mg/dl in the first 7 days of life. Prolonged jaundice is a condition in which serum total serum bilirubin (TSB) is above 10 mg/dl, which persists after the 14th day of life in newborns. The control group consisted of healthy newborns with peak serum TSB level ≤12.9 mg/dl in the first week of life. All newborns were born at 38-42 weeks of gestation and weighed over 2500 grams. Neonates with known risk factors such as major congenital malformations, sepsis, perinatal asphyxia, ma- ternal diabetes, polycythemia, glucose-6-phosphate dehy- drogenase (G6PD) activity deficiency, cephalic hematoma, dehydration, hypothyroidism, liver disease, Rh/subgroup and/or direct Coombs (DC) positive ABO incompatibility, or hemolysis for any reason were excluded from the study. Newborns in the control group were followed up for the development of jaundice, and in addition to determining the STB concentration, complete blood count, peripheral blood smear, blood group typing, DC and thyroid function tests were examined. In hyperbilirubinemia and prolonged jaundice groups, in addition to the above parameters, se- rum direct and indirect bilirubin levels, reticulocyte count, G6PD, liver function tests, thyroid function tests, urine culture and, if necessary, C-reactive protein (CRP) were examined. The study was approved by the Hacettepe University Faculty of Medicine Ethics Committee with Deci- sion number 08/II dated 02 May 2011. Genotyping procedure Blood samples were taken from all cases and then placed in EDTA vacuum containers. Genomic DNA was isolated from peripheral leukocytes using the standard phenol-chloroform procedure. DNA was isolated using the QIAamp DNA blood kit (Qiagen, Hilden, Germany). • Variants of the genes UGT1A1 (nucleotides 211), SLCO1B1 (nucleotides 388, 463, 521, 1463) and SLCO1B3 (nucleotides 334, 727+118, 1865+19721) The polymerase chain reaction-restriction frag- ment length polymorphism (PCR-RFLP) method was applied to detect variants of UGT1A1 at nucleotide 211, SLCO1B1 at nucleotides 388, 463, 521, 1463 and SLCO1B3 at nucleotides 334, 727+118, 1865+19721. PCR mixture (25 µL) consisted of 200 ng DNA, 0.2 mM of each dNTP, 120 nM of primer, 2.5 µL of 10 x buffer, and 1 µL of 50 mM MgCl2 solution. The final concentration of the PCR mixture was 1.5 mM MgCl2 in a volume of 100 μl of working solution. PCR amplification was performed in a DNA thermal cycler for 35 cycles of initial denaturation for 5 min, denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, primer extension for 1.5 min at 72 °C, and final extension for 7 min at 72 °C. The PCR product was digested with appropriate restriction enzymes and an- alyzed on 3% agarose gel (variants of the UGT1A153 gene were examined for 53 subjects with idiopathic hyperbilirubinemia, 27 subjects with prolonged hy- perbilirubinemia and 37 healthy subjects. Variants of the SLCO1B1 / SLCO1B3 genes were investigated in 61 subjects with idiopathic hyperbilirubinemia, 28 subjects with prolonged hyperbilirubinemia and 41 healthy subjects). • Variant of GST (GSTP1) gene PCR-RFLP method was applied to detect the GST variant (GSTP1). PCR mixture (25 µL) consisted of 200 ng DNA, 0.5 µL of 10 mM dNTP, 0.6 µL of 10 mM GSTP primer, 0.12 µL of 10 mM albumin, 2.5 µL of 10 x buffer, 1.25 µL of 50 mM MgCl2 solution. The final concentration of the PCR mixture was 1.5 mM MgCl2 in a volume of 100 μl of working solution. PCR amplification was performed in a DNA thermal cycler for 35 cycles of initial denaturation for 5 minutes, denaturation for 1 minute at 94°C, annealing for 1 minute at 64°C, primer extension for 1 minute at 72°C and final extension for 7 min- utes at 72°C. The PCR product was digested with appropriate restriction enzymes and analyzed on a 3% agarose gel as previously described (variants of the GST gene were examined for 55 subjects with idiopathic hyperbilirubinemia, 28 subjects with pro- longed hyperbilirubinemia and 40 healthy subjects. Statistical analysis All means are presented as means ± standard errors of the means. The weights and gestational ages of the groups were compared with one-way ANOVA. The Chi-square test was used to determine whether there were differences in gender distribution and genotype distribution between groups. Whether the quantitative data conformed to normal distribution was tested with the Shapiro-Wilk test or the Kol- mogorov Smirnov test. Since the STB values of the patients showed a normal distribution, the data are given as mean ± standard deviation values. Differences were considered statistically significant when the P value was less than 0.05.

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