Association Between Vitamin D Receptor Polymorphism and Serum Vitamin D Levels in Children With Low-Energy Fractures
Association Between Vitamin D Receptor Polymorphism and Serum Vitamin D Levels in Children With Low-Energy Fractures
This study was designed to determine the impact of vitamin D serum levels and vitamin D
 receptor (VDR) polymorphisms on the occurrence of low energy fractures in children.
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
Inclusion Criteria:

 -  low-energy fractures.

 -  healthy children hospitalized for other reasons than fractures (injuries, diagnosis of
 knee ligament injuries and others) without fractures

 Exclusion Criteria:

 -  Children with osteogenesis imperfecta (OI) were excluded from the study.