Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures.
OBJECTIVE
Hereditary vitamin D resistant rickets (HVDRR) has been shown to be due to mutations in the gene encoding the vitamin D receptor (VDR). In two patients with the characteristic phenotype we have investigated the functional defect and sequenced the VDR cDNA. We report two new mutations in the DNA binding domain of the VDR gene and we have used the crystallographic structure of the glucocorticoid and oestrogen receptors (GR and ER respectively) as models to explain the stereochemical consequences of these mutations.


METHODS
Patient and control cell lines prepared from skin fibroblasts were used to measure binding of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and functional responses to this hormone. These cells were also used to isolate VDR mRNA from which cDNA was prepared and sequenced. VDR cDNA from affected and control patients was also transfected into receptor defective cells to analyse further functional responses to 1,25(OH)2D3. Computer analysis of mutations in the VDR gene was carried out using the glucocorticoid and oestrogen receptors as model systems.


METHODS
Two patients with HVDRR from unrelated families.


METHODS
Cytosolic binding and nuclear association of 1,25(OH)2D3 were determined in control and affected patients, and functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamin D-24-hydroxylase activity (24-hydroxylase). VDR cDNA was sequenced and transfected into VDR-deficient CV-1 cells for further analysis of functional response to 1,25(OH)2D3 following cotransfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid.


RESULTS
Cells from HVDRR patients I and II showed detectable numbers of VDR with normal hormone binding. However, unlike controls, the HVDRR cells did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)2D3. Sequencing of cDNA revealed single mutations, in patient I (Phe44-->IIe) and in patient II (Lys42-->Glu). Both these residues are conserved in the steroid/thyroid hormone receptor superfamily and stereochemical analysis has been used to deduce the importance of these amino acids and the deleterious effect of these and other mutations in the DNA-binding domain of the VDR.


CONCLUSIONS
Two new mutations in the vitamin D receptor which cause hereditary vitamin D resistant rickets have been described and using molecular modelling we have been able to analyse the genesis of this inherited disease at the level of stereochemistry.
Rut
A R
AR
Department of Medicine, University College London Medical School, Jules Thorn Institute, Middlesex Hospital, UK.
Hewison
M
M
Kristjansson
K
K
Luisi
B
B
Hughes
M R
MR
O'Riordan
J L
JL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002117:Calcitriol / Q000235:genetics; D002648:Child; D003198:Computer Simulation; D017931:DNA Primers; D004270:DNA, Circular / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D006801:Humans; D007015:Hypophosphatemia, Familial / Q000235:genetics*; D007223:Infant; D008297:Male; D008957:Models, Genetic; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D018167:Receptors, Calcitriol / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics
D017931:DNA Primers; D004270:DNA, Circular; D018167:Receptors, Calcitriol; D002117:Calcitriol