Association Between Vitamin D Receptor Polymorphism and Serum Vitamin D Levels in Children With Low-Energy Fractures
Condition: Vitamin D Receptor Defect
MeSH terms: Fractures, Bone
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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
    

Observational model: Case-Control
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Study Group: Healthy children with low energy fractures Control group: Healthy children
        without fractures
      
Biospecimen description: 
      Whole blood samples
    
Keywords: 25-Hydroxy-vitamin D, children, bone fractures, vitamin D receptor polymorphism