Family pedigree and DNA sequences.

<p>(A) Family pedigree showing <i>VDR</i> mutation status. Small solid circle indicates p.H229Q carrier, while small open circle indicates p.M4I/FOKI-F carrier. Index case is large solid circle (B) Heterozygous variants in c. 2T>C (p.M1T), c. 12G>A (p.M4I), and c.687C>G (p.H229Q) were found in patient’s genomic DNA. Full-length cDNA derived from patient’s peripheral lymphocyte showed amino acids M1, M4, and H229Q are located on the same allele.</p

Patient’s biochemical profile.

<p>* This data was collected during oral calcium and active Vitamin D supplement.</p><p>Patient’s biochemical profile.</p

The VDR transactivation activity is markedly reduced in p.H229Q mutant.

<p>COS-7 cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-WT (WT) or pcDNA3.1-H229Q (H229Q) mutant VDR expression vectors and a VDRE-luciferase reporter. Cells were treated with different concentrations of 1,25(OH)₂D₃ for 24 h and luciferase activity measured. VDR protein levels in transfected cells at the five different 1,25(OH)₂D₃ concentration are shown. Results represent the average of at least 3 independent experiments performed in triplicates and are annotated as means±SE. *<i>p</i><0.05 compared with control tested using one-way ANOVA.</p

Exon-flanking primers used for PCR in the human VDR <i>gene</i>.

<p>Exon-flanking primers used for PCR in the human VDR <i>gene</i>.</p

The conservation of amino acid H229 of <i>VDR</i> gene in different species.

<p>Clustal Omega (<a href="http://www.ebi.ac.uk/Tools/msa/clustalo/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalo/</a>) was used for sequence alignment of VDR from different species. Hs, <i>Homo sapiens</i>, Mm, <i>Mus musculus</i>, Gg, <i>Gallus gallus</i>, Xt, <i>Xenopus tropicalis</i>, Dr, <i>Denio rario</i>, Dm, <i>Drosophila melanogaster</i>, Ci, <i>Ciona intestinalis</i>.</p

Heterodimerization of wild-type and mutant p.H229Q VDR with RXR.

<p>HEK293 cells were transfected with wild-type or p.H229Q mutant HA-VDR with FLAG-RAR expression plasmid. Cells were harvested, extracts prepared and coimmunoprecipitated with anti-FLAG antibody. The immunopellets were subjected to Western blotting with anti-HA and anti-FLAG antibodies. WCE, whole cell extract.</p

Restricted Cubic Spline Regression Plot of the U-shape Association between sK and the Risk for End Stage Renal Disease.

<p>Covariates included in the model were the same as the Cox regression in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067140#pone-0067140-t004" target="_blank">Table 4</a>. Serum potassium (sK), body mass index, mean blood pressure and C-reactive protein were treated as restricted cubic spline functions. The solid line represents the log transformed multivariable-adjusted hazard ratio of ESRD. The dashed lines indicate the 95% confidence intervals. sK below 4.03 and above 5.11 mEq/L were associated with higher hazard (log hazard ratio >0). Tick marks on the x-axis indicate individual observations at corresponding levels of sK.</p

Pedigree analysis of <i>CYP17A1</i> gene.

<p>The patient is found to be a compound heterozygous for mutations of <i>CYP17A1</i>. The first mutation was identified as IVS1 +2T>C and the second mutation p.R358X (CGA→TGA). The first intron mutation is originated from her mother and p.R358X from her father, based on the analysis of her uncle's genomic DNA. Proband's brother is also heterozygous for the affected maternal allele.</p

Heterologous expression and mRNA analysis using minigene and full-length <i>CYP17A1</i> gene, wild-type and mutation IVS1 +2T>C.

<p>A) RT-PCR analysis shows only the correct 311 bp amplicon when wild-type <i>CYP17A1</i> minigene or full-length gene is expressed in COS-7 or HEK293 cells. In contrast, the IVS1 +2T>C mutant <i>CYP17A1</i> gene affords primarily a ∼400 bp amplicon, best demonstrated in COS-7 cells, as well as lesser amounts of the normal 311 bp amplicon and larger molecular species. B) Cartoon demonstrating size and sequence of aberrantly spliced transcripts obtained from transfected HEK293 cells expressing the IVS1 +2T>C <i>CYP17A1</i> mutation, obtained after TOPO TA subcloning and sequencing. The asterisks indicate position of in-frame termination codons. C) Cartoon demonstrating splice sites used to generate transcripts obtained from transfected HEK293 cells expressing the IVS1 +2T>C <i>CYP17A1</i> mutation. Figure length is not drawn to scale. D) Western blot analysis of protein obtained from transiently transfected HEK293 cells expressing the wild type (W) and IVS1 +2T>C mutation (Mu) full-length <i>CYP17A1</i> gene. Decreased CYP17A1 protein expression was noted for the IVS1 +2T>C mutant full-length <i>CYP17A1</i> gene compared to the wild-type gene.</p

Baseline Demographic and Clinical Characteristics.

<p>Data expressed as mean ± standard deviation, median (interquartile range) or percentage.</p><p>BMI, body mass index; MBP, mean blood pressure; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; CRP, C-reactive protein; HbA1c, glycated hemoglobin; UPCR, Urine protein-to-creatinine ratio; ESRD, End Stage Renal Disease.</p><p>Comparisons are made by ANOVA or the chi-square test.</p>*<p>eGFR slope less than −6.88 mL/min/1.73 m²/yr (%).</p>#<p>Urine protein-to-creatinine (mg/g).</p