Vitamin D receptor mutations in patients with hereditary 1,25-dihydroxyvitamin D-resistant rickets

Abstract

Context: Hereditary vitamin D resistant rickets (HVDRR), also known as vitamin D-dependent rickets type II, is an autosomal recessive disorder characterized by the early onset of rickets with hypocalcemia, secondary hyperparathyroidism and hypophosphatemia and is caused by mutations in the vitamin D receptor (VDR) gene. The human gene encoding the VDR is located on chromosome 12 and comprises eight coding exons and seven introns.

Objectives, patients, and methods: We analyzed the VDR gene of 5 previously unreported patients, two from Singapore and one each from Macedonia (former Yugoslav Republic), Saudi Arabia and Turkey. Each patient had clinical and radiographic features of rickets, hypocalcemia, and the 4 cases that had the measurement showed elevated serum concentrations of 1,25-dihydroxyvitamin D (1,25(OH)(2)D). Mutations were re-created in the WT VDR cDNA and examined for 1,25(OH)(2)D(3)-mediated transactivation in COS-7 monkey kidney cells.

Results: Direct sequencing identified four novel mutations and two previously described mutations in the VDR gene. The novel mutations included a missense mutation in exon 3 causing the amino acid change C60W; a missense mutation in exon 4 causing the amino acid change D144N; a missense mutation in exon 7 causing the amino acid change N276Y; and a 2bp deletion in exon 3 5'-splice site (IVS3∆+4-5) leading to a premature stop.

Conclusions: These 4 unique mutations add to the previous 45 mutations identified in the VDR gene in patients with HVDRR.

Keywords: HVDRR; Hypocalcemia; Mutations; Rickets; Vitamin D; Vitamin D receptor.

Fig. 1. Mutations in the VDR causing HVDRR

A. Location of mutations in the DNA-binding domain (DBD). Conserved amino acids are shaded. B. Location of mutations in the ligand-binding domain (LBD). The α-helices are shown as black boxes and the β-turns as hatched box. Missense mutations are on top and nonsense mutations on bottom. E1 and AF-2 (activation function 2) represent helices important for transactivation. Adapted from Feldman et al [7]. Mutations from the current patients are indicated with an *.

Fig. 2. Transactivation of mutant VDRs by 1,25(OH)₂D₃ in COS-7 cells

Panels A–D, COS-7 cells were transfected with VDR expression vectors and CYP24A1 promoter luciferase reporter plasmids. Cells were treated with vehicle (0.1% ethanol) or graded concentrations of 1,25(OH)₂D₃ for 24 hrs then assayed for luciferase activity. Panel E, immunoblots of VDR mutant expression in COS-7 cells. Panel F, patient 2 fibroblasts and normal control fibroblasts were treated with vehicle (0.1% ethanol) or graded concentrations of 1,25(OH)₂D₃ for 24 hrs then assayed for CYP24A1 gene expression by real-time RT-PCR. Error bars represent standard deviation from the mean.