4, Bedford, MA, USA) for lumbar spine (LS) (L1–L4), total hip and whole body (WB). All measured values were transformed into Z-scores using equipment-specific age- and sex-adjusted reference data for US Caucasian children; all subjects were of normal height. Body composition was analyzed with DXA to distinguish between lean and fat mass. Calibration

of the measurements was performed with a spine phantom; inter-CV% for the phantom BMC, BA, and BMD was 0.35%, 0.21%, and 0.41%, respectively. The reproducibility of the DXA measurement for bone, fat, and lean mass is 1.2%, 1.9% and 0.7%, respectively, in children between 10 and 18 years of age [18]. Age- and gender-specific reference values were utilized to derive Z-scores for fat percentage selleck products [19] and [20]. Volumetric BMD and bone geometry

were measured from nondominant radius with pQCT (XCT-2000; Stratec, Pforzheim,Germany, software version 5.50) as described previously [21] and [22]. The scans were analyzed using contour mode 2 (45%) and peel mode 1 to assess total bone (TB) and trabecular bone (Trab) parameters at the 4% site. At the 66% site, cortical bone (Cort) was detected with separation mode 1 and a threshold of 710 mg/cm3. In addition, we calculated age- and sex-specific Z-scores for total cross-sectional area (CSA), bone mineral content (BMC), Cort mineral density, TB mineral density and stress and strain index (SSI) using the published Cole’s formula, which is based on mostly Caucasian reference data [23] and [24]. Patient DNA was extracted from peripheral blood by standard methods. Primers for FGF23 http://www.selleckchem.com/screening/stem-cell-compound-library.html (hg18/uc001qmq1) were designed with Primer3

v.0.4.0 (http://frodo.wi.mit.edu/primer3/) for all three Loperamide exons, UTRs and a minimum of 30 bases of flanking introns. Due to the length of exon 3 and the 3′UTR, this segment was sequenced with four overlapping primer pairs. PCR amplification was performed with AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA). The DNA fragments were then visualized with ethidium bromide on a 1.2% agarose gel, purified with ExoSAP (USB, Cleveland, OH, USA) and labeled with BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). After sequencing with an ABI3730 sequencer (Applied Biosystems), chromatograms were analyzed with Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI, USA). Primer sequences and detailed PCR protocols are available upon request from the authors. The haplotype analysis was performed with Haploview 4.2. The statistical analysis was performed on diplotypes due to the relatively small study population. Descriptive data are reported as medians and ranges or as means ± SD. Association of variables was tested with Pearson or Spearman correlation, as appropriate. Partial correlation was used to describe the association after controlling for confounding factor(s).