Association Between Vitamin D Receptor Polymorphism and Serum Vitamin D Levels in Children With Low-Energy Fractures

The study group consisted of 100 children aged 3 to 18 years (78% boys) hospitalized in the Department of Pediatric Orthopedics in 2011-2013 due to low-energy fractures. The control group (122 children, 68% boys) consisted of children aged 3 to 17 years, hospitalized for other reasons (injuries, diagnosis of knee ligament injuries and others) without fractures. Children with osteogenesis imperfecta (OI) were excluded from the study. From each patient and their parent/guardian a written informed consent to participate in the study was obtained. Low-energy fracture was defined as a fracture sustained from a fall from the patient's own height or a fall during team games. All fractures were radiologically confirmed.

During hospitalization the patients and their parents completed a questionnaire on demographic data, the place of residence (urban/rural), earlier fractures and physical activity. The degree of sexual maturity was assessed using a survey of puberty development on the basis of Puberty Development Scale [20-22]. The patients' body weights and heights were measured, and the Cole indicator was calculated to assess the nutritional status of the patients.

Serum concentrations of total vitamin D [25-hydroxy vitamin D3 ((25(OH)D3)) plus 25-hydroxy vitamin D2 ((25(OH)D2))] in the plasma of all the children were determined by electrochemiluminescence using paramagnetic particles coated with streptavidin and ruthenium compound on the Cobas e 411 apparatus by Roche. According to our laboratory, the reference value range for total vitamin D was 30.0 - 74.0 ng/ml.Whole blood samples were collected in tubes containing EDTA and stored at -20°C. DNA was isolated using the MasterPureTM DNA Purification Kit (Akor Laboratories) and quantified on a spectrophotometer (Nanodrop 2000, Thermo Scientific). The genotypes for four restriction fragment length polymorphisms of the vitamin D receptor (VDR) gene were determined by standard polymerase chain reaction (PCR) techniques and enzymatic digestion of the products with FokI, ApaI, TaqI and BsmI (Thermo Scientific). In short, PCR were performed in a final volume of 20 µl containing 50-100 ng DNA, 0.3 µM of each primer and JumpStartTM REDTaqTM ReadyMixTM (Sigma). After initial denaturation for 3 min at 94 °C, samples were subjected to 35 cycles of amplification, consisting of a 30-sec denaturing phase at 94 °C, a 30-sec annealing phase (FokI at 60 °C, ApaI and TaqI at 70 °C, BsmI at 62 °C), a 30-s extension phase at 72 °C, and 4-min at 72 °C on a Bio-Rad thermal cycler CFX96TM. The primers used for FokI polymorphism were: forward 5'-AGC TGG CCC TGG CAC TGA CTC TGC TCT-3', reverse 5'-ATG GAA ACA CCT TGC TTC TTC TCC CTC-3'; ApaI and TaqI: forward 5'-CAG AGC ATG GAC AGG GAG CAA-3', reverse 5'-GCA ACT CCT CAT GGC TGA GGT CTC-3' and BsmI: forward 5'-AGT GTG CAG GCG ATT CGT AG-3', reverse 5'-ATA GGC AGA ACC ATC TCT CAG-3' [25]. The PCR products were digested according to the manufacturer's instructions and separated on 2% agarose gel. The polymorphisms were documented by photographing under UV illumination using G:Box (Syngene). A random subset (20% of samples) was repeated to verify the results. Upper case letters "F", "A", "T" and "B" indicate the absence of the cut site for FokI, ApaI, TaqI and BsmI polymorphisms, respectively, whereas lower case letters "f", "a", "t", and "b" indicate its presence.To examine the relationships between quality attributes Chi-square test of independence and Fisher's exact test were used. The normality of distribution was verified using the Kolmogorov-Smirnov test with the Lilliefors significance correction and the Shapiro-Wilk test. There was no normal distribution of quantitative variables analyzed. To compare the quantitative variables without normal distribution a nonparametric Mann - Whitney U test was used. Models of uni-variate and multi-variate linear regression and logistic regression were established. Results were considered statistically significant at p <0.05. The calculations were performed using Statistica 10.0 by StatSoft, IBM SPSS Statistics 21.0 by Predictive Solutions Company and Stata / IC 13.1 packages by StataCorp LP