Mutations in the vitamin D receptor gene in three kindreds associated with hereditary vitamin D resistant rickets.

Hereditary vitamin D resistant rickets has been associated with a number of mutations within the DNA and ligand binding domains of vitamin D receptors (VDR). The aim of our study was to identify and characterize the causative mutations in three kindreds with this condition. Resistance of 1,25(OH)2D3 was confirmed in cultured skin fibroblasts in which there was no induction of 24-hydroxylase activity; binding of 1,25(OH)2D3 to VDR was undetectable in patients 1 and 2, but normal in patients 3 and 4. The coding region of the VDR gene was sequenced to seek mutations. A mutation in the VDR gene of patient 1 resulted in a STOP codon, patient 2 showed a 56 bp deletion leading to frameshift and premature termination of VDR; a point mutation of A to C lying within the hormone-binding domain was shown for patients 3 and 4, who were siblings. Transactivation studies confirmed that these were functional mutations. Gel shift assays using nuclear extract from patient 3 demonstrated that the mutation that altered a conserved amino acid (glutamine-259) known to be involved in heterodimerization with other nuclear receptors affected protein: protein interactions.

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP).

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.

Functional analysis of vitamin D response elements in the parathyroid hormone gene and a comparison with the osteocalcin gene.

We have previously localised two putative vitamin D response elements (VDRE) in the bovine parathyroid hormone (PTH) gene to within -485 to -452 and -451 to -348 base pairs (bp). To confirm the functional significance of these elements, a series of reporter gene constructs were generated and the ability of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to suppress gene transcription was assessed. These data confirmed the presence of two regions within -485 to -348 bp which confer responsiveness to 1,25(OH)2D3 and mediate its down-regulatory effect on PTH gene transcription. Second, we investigated whether the putative bovine PTH VDRE and the osteocalcin VDRE require the same nuclear proteins in their interaction with VDR. In gel shift assays, three specific DNA-protein complexes were formed using CV-1 nuclear extract and PTH VDRE. However, unlabelled osteocalcin VDRE (50-fold) failed to inhibit the formation of these complexes. These results suggest that interactions of VDR with the PTH and osteocalcin genes require different accessory factors.

Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping.

OBJECTIVE: Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disorder resulting in target organ resistance to the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In many cases, this disorder has been shown to be due to mutations in the gene encoding vitamin D receptors (VDR). In a patient with characteristic features of this disorder, we investigated the functional defect and sequenced the coding region of the gene for mutations. DESIGN: Skin fibroblasts from patient and control were used to measure binding of 1,25(OH)2D3 and functional responses to the hormone. These cells were also used to prepare RNA from which cDNA was prepared and sequenced. Furthermore, genomic DNA was prepared from the fibroblasts and the intron/exon boundaries sequenced. PATIENT: A child with classic features of HVDRR with alopecia diagnosed as having rickets due to resistance to 1,25(OH)2D3. MEASUREMENTS: Nuclear association of 1,25(OH)2D3 was determined in patient and control cells and the functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamin D-24-hydroxylase(24-hydroxylase) activity. VDR cDNA and genomic DNA prepared from patient and control cells were sequenced. RESULTS: Cells from the patient with HVDRR had undetectable amounts of VDR compared to control cells and did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)2D3. Sequencing of the VDR coding region after RT-PCR of RNA revealed an absence of exon 4 in patient RNA which was not due to a deletion in genomic DNA but was caused by exon skipping during RNA processing. In addition, the deletion of exon 4 sequences from RNA leads to a frameshift in translation resulting in a premature stop codon. Amplification of genomic DNA around the intron/exon boundary of exon 4 revealed a point mutation in the 5' donor splice site of intron 4. CONCLUSION: In this study, we have identified a novel mutation in the gene for vitamin D receptors in a patient with the characteristic phenotype of hereditary vitamin D resistant rickets. The mutation at the +5 position in intron 4 is most likely to cause skipping of exon 4 in this patient.

Translational regulation of parathyroid hormone gene expression and RNA: protein interactions.

The aim of this study was to investigate the mechanism by which translation of parathyroid hormone (PTH) mRNA is regulated with regard to the subcellular distribution of PTH mRNA and RNA:protein interactions. Sucrose density ultracentrifugation of RNA from bovine parathyroid cells indicated that there was no evidence for a pool of nonribosomal PTH mRNA, and the extracellular calcium concentration had no effect on polysome size. UV cross-linking studies revealed two proteins in parathyroid cell cytosol which bound specifically to the 5'-untranslated region (UTR) of PTH mRNA with molecular masses of 66 and 68 kD while proteins with apparent molecular masses of 48 and 70 kD bound to the 3'-UTR. In vitro translation assays indicated that parathyroid cell cytosol contains factors that inhibit translation of PTH mRNA. Fractionation of cytosol revealed that this effect was associated with proteins within the molecular mass range 30-90 kD. To determine which sequences in PTH mRNA mediate translational regulation, RNA was synthesized from luciferase gene constructs containing the 5'- and/or 3'-UTR of PTH mRNA, and translated in vitro. Addition of parathyroid cell cytosol reduced the translation of RNA containing the 5'- and 3'-UTR of PTH mRNA by 44 +/- 7% but had no effect on the translation of RNA containing only the luciferase coding region. Translation of RNA containing only the 5'-UTR of PTH mRNA was unchanged; however, cytosol reduced the translation of RNA containing the 3'-UTR by 31 +/- 9%. These data demonstrate a role for RNA:protein interactions in the regulation of PTH synthesis and that translational control is mediated primarily through interactions with the 3'-UTR of PTH mRNA.

Antisense inhibition of vitamin D receptor expression induces apoptosis in monoblastoid U937 cells.

The active vitamin D3 metabolite 1,25-dihydroxycholecalciferol (1,25(OH)2D3) acts as an antiproliferative and differentiating agent for the monoblastoid cell line U937 and as an important immunologic mediator implicated particularly in the function of cells belonging to the monocyte/macrophage lineage. These effects are controlled by the vitamin D receptor (VDR), a member of the steroid hormone receptor family. The objective of this study was to develop U937 transfectants expressing antisense VDR mRNA, and to use these to examine the role of 1,25(OH)2D3-VDR interaction in this lineage. A 2-kb VDR cDNA insert (including the complete VDR coding region) was cloned in an antisense orientation into the EBV episomal vector pMEP4 under the control of an inducible promoter and transfected into U937. The resultant cell line, DH42, was hygromycin resistant, contained VDR cDNA, expressed fewer VDRs than controls, and showed a substantial decrease in antiproliferative response to 1,25(OH)2D3. However, 1,25(OH)2D3 increased the number of cells expressing macrophage cell surface Ags, including CD14 and CD11b. A subpopulation of smaller cells did not express the differentiation markers after cadmium stimulation. Cell cycle analysis showed shifts in the distribution of cells from G1 to S phase, which were more pronounced after cadmium treatment. A considerable proportion of cells were outside the cycle and DNA fragmentation confirmed apoptosis. Thus, the functional outcome of the VDR antisense transfection suggests that in the myelomonocytic lineage, VDR expression may act as a protective mechanism against programmed cell death.

The gene for X-linked hypophosphataemic rickets maps to a 200-300kb region in Xp22.1, and is located on a single YAC containing a putative vitamin D response element (VDRE).

The location of the HYP gene, which determines X-linked hypophosphataemic rickets, has been refined considerably by linkage analysis, and three new microsatellite primers isolated, Cap32 (DXS7473), Cap29 (DXS7474) and 7v2 (DXS7475). The locations of four other markers have also been determined (DXS1226, AFMa176zb1, AFMa152wc5, and AFM346azc1). Markers Cap29 and Cap32 are the closest distal markers to the gene with zetamax=11.93, thetamax= 0.018 and zetamax=12.03, thetamax = 0.015 respectively. Both Cap29 and Cap32 are proximal to DXS365 and AFMa176zb1, as deduced by screening non-chimaeric yeast artificial chromosomes (YACs) from a contig spanning the HYP gene. A single crossover places AFMa176zbl distal to the disease gene. There are no recombinations between 7v2 and HYP (zetamax=12.9, thetamax=0.0), or between 7v2 and adjacent markers Cap32, Cap29, AFMa176zb1, DXS1683 and DXS365. However screening of YAC clones encompassing the HYP gene and also P1 clones localises 7v2 distal to Cap29 and Cap32, and proximal to DXS443. Marker DXS1226 is placed outside the region containing the gene, and is located proximal to DXS274 as confirmed by a crossover for this marker and DXS41 against HYP and its presence on YAC 83B05. Genetic mapping of CEPH pedigrees, and screening of YACs places AFMa152wc5 and AFMa346zcl between DXS1683 and DXS1052. The following gene marker map presents the best order for the HYP region: Xptel-DXS43-DXS999-DXS443-(DXS365/DXS74 75/AFMa176zb1)-(DXS7474/DXS7473)-HYP- DXS1683-(AFMa152wc5/AFMa346zc1)-DXS1052-DXS 274 -(DXS41/DXS1226)-Xcen. The distance between the cluster of distal flanking markers Cap29 (DXS7474), Cap32 (DXS7473), and DXS1683 is approximately 300 kb, as deduced from physical map data from a YAC contig spanning the gene. Thus the gene for HYP is contained within a single YAC (900AO472). Of further interest, is the location of a putative vitamin D response element (VDRE) on this YAC.

Mutations in the RET proto-oncogene and the von Hippel-Lindau disease tumour suppressor gene in sporadic and syndromic phaeochromocytomas.

Phaeochromocytomas may occur sporadically, or as part of the inherited cancer syndromes multiple endocrine neoplasia (MEN) type 2, von Hippel-Lindau disease (VHL), and, rarely, in type 1 neurofibromatosis. In MEN 2, germline missense mutations have been found in one of eight codons within exons 10, 11, 13, 14, and 16 of the RET proto-oncogene. In VHL, germline mutations within one of the three exons are responsible for the majority of cases. To determine if somatic mutations similar to those seen in the germline in MEN 2 or VHL disease play a role in the pathogenesis of sporadic or familial phaeochromocytomas, we analysed 48 sporadic tumours and tumours from 17 MEN 2 and five VHL patients for mutations in RET exons 9, 10, 11, 13, 14, 15, and 16, and the entire coding sequence of VHL. Five of 48 sporadic phaeochromocytomas had RET mutations within exons 10, 11, and 16. Of these, one was proven to be germline and two were proven to be somatic mutations. Four of 48 had VHL mutations; these included both the bilateral cases in the series (one was proven to be a germline mutation) and two others, of which one was proven somatic.

Transfection of vitamin D receptor cDNA into the monoblastoid cell line U937. The role of vitamin D3 in homotypic macrophage adhesion.

A 2-kB cDNA for the vitamin D receptor (VDR) was cloned in sense orientation into the plasmid pMEP4 (containing a cadmium-inducible metallothionein II promoter and a hygromycin-resistance selection gene) and transfected into monoblastoid U937 cells. The resultant cell line, DH39, expressed two species of VDR mRNA: 4.6-kb wild-type mRNA (present in native U937 cells or cells transfected with pMEP4 alone) and 2-kb transfected mRNA, which increased with cadmium treatment. Binding studies (using the active vitamin D metabolite, 1,25-dihydroxycholecalciferol (1,25-DHCC)) showed that DH39 cells contained five times more VDR per cell than controls, and ten times more after cadmium treatment. DH39 were sensitive to 1,25-DHCC: adding cadmium with 100 nM 1,25-DHCC for 72 h completely inhibited proliferation and induced concomitant differentiation. Unlike control cells, differentiation of DH39 by 1,25-DHCC led to homotypic cell-cell adhesion and formation of macrophage clusters. FACS analysis showed that 1,25-DHCC increased the number of cells expressing CD11b in both DH39 and controls, and the number of cells expressing CD11c in DH39. There was a quantitative increase in mean fluorescence intensity of expression of CD11a and CD18 in DH39. Northern blotting showed increased CD11a and CD18 mRNA in DH39. Ab inhibition of 1,25-DHCC-induced homotypic adhesion showed that CD11a/18 mediated the cell-cell clustering. CD50 expression was decreased on DH39, but the CD11a/18 ligand implicated was CD54. DH39 provides a model system not only for investigating the VDR role in 1,25-DHCC anti-proliferative effects, but also for regulation of homotypic macrophage adhesion mechanisms that are important in disease pathogenesis.

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. DESIGN: 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. PATIENTS: Two patients with HVDRR from unrelated families. MEASUREMENTS: 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.

Construction of a high-resolution linkage map for Xp22.1-p22.2 and refinement of the genetic localization of the Coffin-Lowry syndrome gene.

The genes responsible for two X-linked diseases, the Coffin-Lowry syndrome (CLS) and juvenile retinoschisis (RS), have been previously mapped, through linkage studies, to an 8-cM region, in Xp22.1-p22.2, flanked distally by two tightly linked markers, DXS207 and DXS43, and proximally by DXS274. In the present study, five Genethon markers have been assigned to the (DXS207, DXS43)-DXS274 interval using somatic cell hybrids and a meiotic breakpoint panel and ordered together with three markers previously mapped to this region. A genetic map, which includes 13 loci and spans a distance of approximately 13 cM, was derived from linkage analysis using the CEPH families. The most likely locus order and map distances (in centimorgans) are Xpter-DXS16-(3.4)-(DXS207, DXS43, DXS1053)-(2.0)-(DXS999, DXS257)-(1.7)-AFM291 wf5-(1.4) - DXS443 - (2.0) - (DXS1229, DXS365) - (2.1) - (DXS1052, DXS274, DXS41)-Xcen. Analysis of multiply informative crossovers established AFM291 wf5 and DXS1052 as new flanking markers for CLS, which significantly reduces the candidate region for this disease gene to a 4- to 5-cM interval. Three markers, DXS443, DXS1229, and DXS365, mapping within this interval showed complete cosegregation with the disease phenotype, giving a multipoint lod score of 14.2. The present map provides the framework for constructing a YAC contig for the CLS and RS region and should be useful for refining the localization of other disease genes mapping to this region. The panel of somatic cell hybrids characterized for the present study has also allowed us to refine the localization of five genes (CALB3, GRPR, PDHA1, GLRA2, and PHKA2) and two expressed sequence tags (DXS1118E and DXS1006E) previously assigned to the Xp22 region.

Binding of 1,25-dihydroxyvitamin D3 receptors to the 5'-flanking region of the bovine parathyroid hormone gene.

To further define the binding site for receptors for 1,25(OH)2D3 (VDR) in the bovine PTH gene and to study the interactions of transcription factors with VDR, Southwestern and gel shift assays were used. Data from the former indicated binding of VDR to DNA fragments spanning the regions -451 to -348 bp and -668 to -452 bp. Studies using gel shift assays confirmed binding to the -451 to -348 bp fragment and specificity was shown by using excess concentrations of unlabelled -451 to -348 bp fragment to compete for binding, whereas excess unlabelled -347 to +50 bp did not compete. Binding was also observed with the -668 to -452 bp fragment but excess concentrations of unlabelled -668 to -452 or -451 to -348 bp fragments did not compete for binding to radiolabelled fragments. These data indicate the presence of two binding domains within this region; the upstream element having a lower affinity for VDR than the downstream element. In addition, there was no interaction between VDR and consensus sequences for AP1, AP2, AP3 and SP1. The putative vitamin D3 response element (VDRE) contains two similar hexameric steroid response element-like half-sites placed as AGGTCA-related direct repeats. The upstream repeat is at -461 to -456 bp and the downstream element is at -449 to -444 bp. The presence of these half-sites is consistent with our experimental data in which cleavage with SspI at -452 bp resulted in two DNA fragments which bound VDR.

A YAC contig spanning the hypophosphatemic rickets disease gene (HYP) candidate region.

Dominant X-linked hypophosphatemic rickets (HYP) is the most common form of familial rickets. Linkage studies have localized the gene for this disorder to Xp22.1 between the markers DXS365 and DXS274, a region estimated to be approximately 3.5 cM. We have constructed a 1.5-Mb YAC contig encompassing this region by hybridization screening of high-density YAC clone filters. Rapid chromosome walking was achieved by direct hybridization of a pool of Alu-PCR products derived from a YAC containing DXS365 to the filter grids. Overlaps between YACs in the contig were estimated by hybridization of end probes to YAC digest blots and by analysis of cosmid fingerprints obtained by hybridization of YAC inserts to a flow-sorted chromosome X cosmid library. All YACs in the contig have been verified by fluorescence in situ hybridization. Several YACs spanning the HYP gene candidate region were selected for further analysis by rare-cutter enzyme digestion and pulsed-field gel electrophoresis. We estimate that the markers flanking the disease region, DXS365 and DXS274, are less than 1 Mb apart. This clone contig map provides an essential resource for the isolation of the HYP gene.

Hormone-nuclear receptor interactions in health and disease. Vitamin D resistance.

Tissue resistance to vitamin D, or vitamin D-dependent rickets (VDDR), can be classified as two separate conditions--VDDR type I and VDDR type II--both of which present with the classical clinical, radiological and biochemical features of rickets despite adequate vitamin D intake. VDDR II can also be associated with alopecia, for reasons that are not clear. The two syndromes result from distinct disorders of vitamin D metabolism or action. Both are inherited in an autosomal recessive fashion. VDDR I is caused by decreased production of the active form of vitamin D, 1,25-dihydroxycholecalciferol, with the proposed defect being in the gene encoding the enzyme 1 alpha-hydroxylase. VDDR II results from mutations in the gene for the intracellular receptor for 1,25-dihydroxycholecalciferol (vitamin D receptor), resulting in changes in hormone or DNA binding, depending on the mutation. These mutations are analogous to those affecting receptors for other steroid-thyroid hormones, which have also been shown to cause resistance to hormone action.

Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene.

OBJECTIVE: Hereditary vitamin D resistant rickets (HVDRR) is characterized by severe rickets and is often accompanied by alopecia. Mutations in the gene encoding the vitamin D receptor have been found in this condition. In a patient with the characteristic phenotype we have investigated the functional defect and sequenced the gene to seek a mutation. DESIGN: Patient and control cell lines prepared from skin fibroblasts and peripheral blood lymphocytes were used to measure binding of 1,25(OH)2D3 and to isolate vitamin D receptor mRNA. VDR cDNA was sequenced and transfected into receptor defective cells. PATIENT: A child with alopecia diagnosed as having rickets due to resistance to 1,25(OH)2D3. MEASUREMENTS: Cytosolic binding and nuclear association of 1,25(OH)2D3 were determined in patient and control cells, and functional response to 1,25(OH)2D3 assessed by measurement of 24-hydroxylase activity. VDR mRNA was prepared, reverse transcribed, and cDNA sequenced. VDR cDNA was also transfected into VDR-deficient CV-1 cells and functional response to 1,25(OH)2D3 assessed by co-transfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS: VDR from the patient were able to bind 1,25(OH)2D3 but showed no nuclear localization resulting in an absence of functional response to 1,25(OH)2D3. Sequencing revealed that the VDR coding region was normal. Expression studies of the patient's VDR showed functionally normal VDR as evidenced by normal transactivation in the presence of 1,25(OH)2D3. CONCLUSION: These data indicate a new cause of tissue resistance to 1,25(OH)2D3 which occurs in the absence of mutations in the coding region of VDR gene and which is characterized by defective nuclear localization of this receptor.

Scintillation proximity assay for calcitriol in serum without high pressure liquid chromatography.

A rapid isolation step for 1,25-dihydroxyvitamin D3 without high pressure liquid chromatography (HPLC) and a sensitive radioimmunoassay (RIA) have been developed. The time required for extraction and isolation with a combination of Extrelut-1-minicolumns and Sep-Pak silica cartridges from as little as 0.5 ml serum is only 2 h. The assay can be counted after 8 h of incubation. It is performed in the vial that collects the eluate, thus eliminating transfer losses and errors. No separation of bound and free hormone is necessary before beta-counting in the scintillation proximity assay. The detection limit of the assay is 2.7 ng/l. The intra-assay coefficients of variation are 7.3% and 5.2% for samples with calcitriol concentrations of 31 and 148 ng/l, respectively. The inter-assay coefficients of variation are 11.3%, 13.3% and 16.1% for low (16 ng/l), medium (30 ng/l) and high (148 ng/l) control pool samples, respectively. Normal values for calcitriol range from 32 to 80 ng/l. Elderly subjects, patients with reduced kidney function and pregnant women were also evaluated for their calcitriol levels. This assay correlates well with a RIA employing HPLC prepurification and charcoal separation of bound/free calcitriol (r = 0.94).

Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3.

We have identified and characterized two mutations in the hormone binding domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a premature stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC) resulting in the substitution of arginine 271 by leucine (R271L) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cells showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, however, be elicited by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the wild-type receptor. This shows that Arg 271 directly affects the affinity of the VDR for its ligand and its conversion to leucine decreases its affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located immediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270) that is conserved among members of the steroid/thyroid/retinoid hormone receptor superfamily. These results represent the first missense mutation identified in the hormone binding domain of VDR and further define the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor.