Novel Vitamin D Receptor Mutations in Hereditary Vitamin D Resistant Rickets in Chinese

Abstract

Hereditary 1, 25-dihydroxyvitamin D-resistant rickets (HVDRR), a rare recessive disease, is caused by mutation in the VDR gene encoding the vitamin D receptor leading to the resistance to vitamin D. We described a female toddler with initial presentation of leg tenderness and clinical features of HVDRR including severe rickets, hypocalcemia and hypophosphatemia without alopecia. Genetic analysis revealed novel compound heterozygous mutations of p.M4I and p.H229Q in patient's VDR gene. In cis p.M4I with FOKI-F eliminated both translation start sites of the VDR protein. The p.H229Q VDR exhibited significantly reduced VDR transactivation activity with intact dimerization with RXR. Our report expanded the mutation spectrum of HVDRR, and provided the first case of a benign variant p.M4I plus a common p.M1T polymorphism leading to a pathogenic allele.

Fig 1. (A) Antero-posterior radiograph of the patient’s hand with rickets demonstrates cupping and fraying of the metaphyseal region. (B) Antero-posterior radiograph of the patient’s chest showing scoliosis of the thoracolumbar spine with Cobb’s angle of 31degrees. (C) Antero-posterior radiograph of lower extremities revealed widening between the distance of the end shaft and epiphyses. (D) The curvatures of legs revealed genu valgum with intermalleolar distance (IMD) of 5.0cm.

Fig 2. Family pedigree and DNA sequences.

(A) Family pedigree showing VDR 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.

Fig 3. The conservation of amino acid H229 of VDR gene in different species.

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

Fig 4. The VDR transactivation activity is markedly reduced in p.H229Q mutant.

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. *p<0.05 compared with control tested using one-way ANOVA.

Fig 5. Heterodimerization of wild-type and mutant p.H229Q VDR with RXR.

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.