A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets.

Mutations in the vitamin D receptor (VDR) cause hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease resulting in target organ resistance to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. In this report, we describe the clinical case and molecular basis of HVDRR in an Asian boy exhibiting the typical clinical features of the disease including alopecia. Using cultured dermal fibroblasts from the patient, 1,25(OH)(2)D(3) resistance was demonstrated by a shift in the dose response required for 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) mRNA induction. Western blot showed that the cells express a normal size VDR but contained reduced levels of receptor compared to normal cells. At 24 degrees C, the affinity of the patient's VDR for [(3)H]1,25(OH)(2)D(3) was 50-fold lower than the VDR in normal fibroblasts. Sequence analysis identified a unique T to G missense mutation in exon 6 that changed phenylalanine to cysteine at amino acid 251 (F251C). The recreated F251C mutant VDR showed reduced transactivation activity using a 24-hydroxylase promoter-luciferase reporter. Maximal transactivation activity exhibited by the WT VDR was not achieved by the mutant VDR even when the cells were treated with up to 10(-6) M 1,25(OH)(2)D(3). However, the transactivation activity was partially rescued by addition of RXRalpha. In the yeast two-hybrid system and GST-pull-down assays, high concentrations of 1,25(OH)(2)D(3) were needed to promote F251C mutant VDR binding to RXRalpha, indicating defective heterodimerization. In conclusion, a novel mutation was identified in the VDR LBD that reduces VDR abundance and its affinity for 1,25(OH)(2)D(3) and interferes with RXRalpha heterodimerization resulting in the syndrome of HVDRR.

A rationale for treatment of hereditary vitamin D-resistant rickets with analogs of 1 alpha,25-dihydroxyvitamin D(3).

Hereditary vitamin D-resistant rickets (HVDRR) is caused by heterogeneous inactivating mutations in the vitamin D receptor (VDR). Treatment of HVDRR patients with high doses of oral calcium and supraphysiologic doses of 1 alpha,25-dihydroxyvitamin D(3) (1,25D(3)) has had limited success. In this study we explored the use of vitamin D analogs as a potential therapy for this disorder. The rationale for the use of vitamin D analogs is that they bind the VDR at different amino acid residues than 1,25D(3), and their ability to modulate VDR functions differs from that of the natural hormone. In this report, we examined the VDR from three HVDRR patients with mutations in the ligand-binding domain of the VDR (histidine 305 to glutamine, arginine 274 to leucine, and phenylalanine 251 to cysteine) for their responses to two vitamin D analogs, 20-epi-1,25D(3) and 1 beta-hydroxymethyl-3-epi-16-ene-26a,27a-bishomo-25D(3) (JK-1626-2). Our results reveal that vitamin D analogs partially or completely restore the responsiveness of the mutated VDR. Analog treatment seemed to be more successful when the mutation affects the amino acids directly involved in ligand binding rather than amino acids that contribute to a functional VDR interface with dimerization partners or coactivators of transcription.

Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor.

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

PPARgamma agonists enhance human vascular endothelial adhesiveness by increasing ICAM-1 expression.

Early atherosclerotic lesions are characterized by increased monocyte adhesion to the overlying endothelium. Oxidized LDL (oxLDL) stimulates the adhesion of human monocytes to endothelial cells, in part, by increasing expression of ICAM-1. However, the cellular role of oxLDL in endothelial adhesiveness is not well understood. The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is expressed in vascular endothelial cells. Whether it can be activated by a synthetic ligand, troglitazone, as well as by natural ligands, oxLDL and its lipid components (i.e., 9- and 13-HODE), has not yet been explored. This study was undertaken to determine whether PPARgamma is expressed in ECV304 human vascular endothelial cells and if so to define the biological effects of its activation by these agonists. Our results demonstrate that PPARgamma mRNA is expressed in ECV304 cells, and transfected cells with a PPARE luciferase construct respond to these agonists. In addition, ligand-dependent PPARgamma activation increased ICAM-1 protein expression and enhanced adherence of monocytes to ECV304 cells by two- to threefold. These findings suggest that the PPARgamma signaling pathway might contribute to the atherogenicity of oxLDL in vascular endothelial cells.

The vitamin D receptor gene start codon polymorphism: a functional analysis of FokI variants.

The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where "f" indicates the presence of the restriction site and the first ATG, while "F" indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]-1,25-dihydroxyvitamin D3 ([3H]-1,25(OH)2D3) (Kd = 131+/-78 pM, f-VDR; Kd = 237+/-190 pM, F-VDR; p = 0.24); however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose-response (0.1-10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF, Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets due to an opal mutation causing premature termination of the vitamin D receptor.

Mutations in the vitamin D receptor (VDR) gene have been shown to cause hereditary vitamin D-resistant rickets (HVDRR). The patient in this study is a young French-Canadian boy with no known consanguinity in his family. The child exhibited the clinical characteristics of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated 1,25-dihydroxyvitamin D (1,25(OH)2D) levels as well as total alopecia. Fibroblasts were cultured from a skin biopsy of the patient and used to assess the VDR. Northern blot analysis showed that a normal size VDR transcript was expressed; however, [3H]1,25(OH)2D3-binding levels were very low and Western blot analysis failed to detect any VDR protein. Total resistance to 1,25(OH)2D3 was demonstrated by the failure of the cultured fibroblasts to induce the transcription of the 25-hydroxyvitamin D-24-hydroxylase gene when treated with high concentrations of 1,25(OH)2D3. Analysis of the DNA sequence revealed a unique C to T base change corresponding to nucleotide 218 of the VDR cDNA. This single base substitution changes the codon for arginine (CGA) to an opal stop codon (TGA), resulting in the truncation of the VDR at amino acid 30. The Arg30stop mutation prematurely terminates translation and deletes 398 amino acids including most of the zinc fingers as well as the entire ligand-binding domain. Restriction fragment length polymorphism analysis of a DdeI restriction site created by the mutation showed that the parents were heterozygous for the mutant allele. In conclusion, the Arg30stop mutation truncates the VDR and leads to a hormone-resistant condition which is the molecular basis of HVDRR in this patient.

Vitamin D receptor gene polymorphisms: analysis of ligand binding and hormone responsiveness in cultured skin fibroblasts.

Recent reports have suggested that polymorphisms in the gene encoding the vitamin D receptor (VDR) determine a portion of the genetic contribution to bone mineral density (BMD). Individuals homozygous for the allele lacking the Bsm I restriction site in the intron between exons 8 and 9 (BB genotype) have been found to have lower BMD than individuals homozygous for the allele having the Bsm I site (bb genotype). Interestingly, this polymorphism has also been associated with prostate cancer risk. The observed changes in BMD and prostate cancer risk might be due to an alteration in the function or abundance of the VDR leading to differential responsiveness of target cells to the action of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. To test this hypothesis, we cultured dermal fibroblasts from donors with BB, Bb, and bb genotypes and determined the level of VDR expression and the cellular responsiveness to 1,25(OH)2D3 treatment. VDR abundance, affinity for [3H]1,25(OH)2D3, and VDR mRNA levels were not detectably different in BB cells compared to bb cells. Moreover, equal expression of both VDR gene alleles was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) on mRNA from Bb fibroblasts. Fibroblast responsiveness to 1,25(OH)2D3, assessed by induction of 24-hydroxylase mRNA, was similar between BB and bb cell types in dose-response experiments. Although there were individual variations in the parameters we measured, there were no detectable or consistent differences in mean values from our small sample of cultured dermal fibroblasts. In conclusion, we did not detect significant differences in VDR properties or cellular responsiveness to 1,25(OH)2D3 that correlated with VDR genotype. Our findings suggest that these polymorphisms do not affect VDR function, but rather may be a marker for a nearby gene that is responsible for the genotype-associated variation in osteoporosis and prostate cancer risk.

Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness.

Mutations in the vitamin D receptor (VDR) result in target organ resistance to 1alpha,25-dihydroxyvitamin D [1,25(OH)2D3], the active form of vitamin D, and cause hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR). We analyzed the VDR of a patient who exhibited three genetic diseases: HVDRR, congenital total lipodystrophy, and persistent mullerian duct syndrome. The patient was treated with extremely high dose calcitriol (12.5 microg/d) which normalized serum calcium and improved his rickets. Analysis of [3H]1,25(OH)2D3 binding in the patient's cultured fibroblasts showed normal abundance of VDR with only a slight decrease in binding affinity compared to normal fibroblasts when measured at 0 degrees C. The patient's fibroblasts demonstrated 1,25(OH)2D3-induction of 24-hydroxylase mRNA, but the effective dose was approximately fivefold higher than in control cells. Sequence analysis of the patient's VDR gene uncovered a single point mutation, H305Q. The recreated mutant VDR was transfected into COS-7 cells where it was 5 to 10-fold less responsive to 1,25(OH)2D3 in gene transactivation. The mutant VDR had an eightfold lower affinity for [3H]1,25(OH)2D3 than the normal VDR when measured at 24 degrees C. RFLP demonstrated that the patient was homozygous for the mutation while the parents were heterozygous. In conclusion, we describe a new ligand binding domain mutation in the VDR that causes HVDRR due to decreased affinity for 1,25(OH)2D3 which can be effectively treated with extremely high doses of hormone.

The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women.

We examined the association of bone mineral density (BMD) with a polymorphism in the gene encoding the vitamin D receptor (VDR) that causes a change in the predicted protein sequence. The polymorphism results from a C-to-T transition and creates an initiation codon (ATG) three codons proximal to a downstream start site. The polymorphism can be defined by a restriction fragment length polymorphism (RFLP) using the restriction endonuclease FokI. The presence of a FokI site, designated f, allows protein translation to initiate from the first ATG. The allele lacking the site (designated F), initiates from a second ATG site. Thus, translation products from these alleles are predicted to differ by three amino acids with the f variant elongated. In a group of 100 postmenopausal Mexican-American Caucasian women, subjects with the ff genotype (15% of the study population) had a 12.8% lower BMD at the lumbar spine than FF subjects (37% of the population) (p = 0.01). Heterozygote (Ff) subjects (48% of the population) had an intermediate BMD. This association between BMD and genotype was not apparent at the femoral neck or forearm. Over a 2-year follow-up period, a decrease in BMD at the femoral neck was greater in ff compared with FF subjects (-4.7% vs. -0.5%, p = 0.005). This trend was not apparent at the lumbar spine or forearm. There were no differences between genotype groups in measurements of 25-hydroxyvitamin D (25(OH)D), calcitriol, parathyroid hormone (PTH), osteocalcin, or urinary pyridinolines. We conclude that the FokI polymorphism of the VDR gene correlates significantly with decreased BMD at the lumbar spine and with an increased rate of bone loss at the hip in ff subjects. We emphasize that these initial data should be interpreted with caution but that the utility of this polymorphism as a genetic marker to determine BMD and osteoporosis risk warrants further study in larger populations with subjects of diverse ethnic backgrounds.

A novel mutation in the deoxyribonucleic acid-binding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets.

Mutations in the vitamin D receptor (VDR) result in hereditary 1,25-dihydroxyvitamin D3-resistant rickets (HVDRR), an autosomal recessive disease caused by target organ resistance to the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In this study, we investigated the molecular basis of HVDRR in a child from Saudi Arabia who was previously shown to be resistant to 1,25-(OH)2D3 action, but whose cultured skin fibroblasts exhibited normal [3H]1,25-(OH)2D3 binding. Using the PCR, exons 2 and 3 of the VDR gene that encode the DNA-binding region of the receptor were amplified and sequenced. A novel point mutation at nucleotide 252 in exon 2 of the VDR was identified. This missense mutation (GGC to GAC) resulted in the conversion of glycine to aspartic acid at amino acid position 46 (G46D), located at the base of the first zinc finger. This single base change was introduced into wild-type VDR complementary DNA by site-directed mutagenesis, and the mutant VDR was then expressed in COS-1 cells. The expressed mutant VDR displayed a normal binding affinity (Kd = 1.2 x 10(-10) mol/L) for [3H]1,25-(OH)2D3 as determined by Scatchard analysis. However, the mutant VDR was shown to have reduced binding affinity for DNA by DNA-cellulose chromatography. In COS-7 cells cotransfected with a vitamin D response element-chloramphenicol acetyltransferase reporter construct and the mutant VDR complementary DNA expression vector, the mutant VDR was unable to activate gene transcription in cells treated with up to 100 nmol/L 1,25-(OH)2D3. Restriction fragment length polymorphism analysis using MwoI restriction digests of exon 2 demonstrated that the affected child is homozygous for the mutation, whereas the child's father is heterozygous and a carrier of the defective allele. In conclusion, a new mutation was identified in exon 2 of the VDR gene. This mutation, which occurs in the first zinc finger of the DNA-binding domain of the receptor, blocks 1,25-(OH)2D3 action and leads to the syndrome of HVDRR.

Oestrogen-binding protein in Candida albicans: antibody development and cellular localization by electron immunocytochemistry.

Candida albicans, the most common fungal pathogen of humans, possesses an oestrogen (estrogen)-binding protein (EBP) that binds oestrogens with high affinity and specificity. The gene that encodes the EBP (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from Saccharomyces cerevisiae. Here, we report the purification and the subcellular localization of the EBP from C. albicans. Using ion-exchange chromatography and an oestradiol affinity column, the EBP was purified from a strain of C. albicans (strain 422) which was selected because it constitutively expressed elevated levels of the binding protein. The purified protein displayed a subunit molecular mass of approximately 46 kDa when examined by denaturing gel electrophoresis, which is consistent with the size estimated from the sequence of the cloned CaEBP1 gene. An immunoaffinity column, prepared using a polyclonal antisera generated against EBP, depleted the oestrogen-binding activity from C. albicans cell extracts. Western blot analysis showed that the antisera specifically recognized the EBP from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in S. cerevisiae and did not cross react with S. cerevisiae proteins. Using electron microscopy and antigen detection by immunogold staining, the EBP appeared to be primarily associated with vacuoles. However, when overexpressed in S. cerevisiae, the EBP was found diffusely throughout the cell. In conclusion, the EBP has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that EBP is found associated with vacuoles in C. albicans.

Hereditary 1 alpha,25-dihydroxyvitamin D-resistant rickets resulting from a mutation in the vitamin D receptor deoxyribonucleic acid-binding domain.

Hereditary 1 alpha,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a genetic disease that results from mutations in the gene encoding the vitamin D receptor (VDR). In this study of two siblings showing classical features of HVDRR, cultured dermal fibroblasts were used to characterize their VDR and assess responsiveness to 1,25-dihydroxyvitamin D3 treatment. The VDR displayed normal affinity and binding capacity for [3H]1,25-dihydroxyvitamin D3; however, the cells failed to exhibit induction of 25-hydroxyvitamin D 24-hydroxylase activity when treated with hormone. A decreased affinity of liganded VDR for DNA cellulose suggested that the defect was localized to the DNA-binding domain. Exons 2 and 3 of the VDR gene, which encode the two zinc fingers in the DNA-binding domain, were amplified and sequenced by polymerase chain reaction. Both siblings exhibited a G to A missense mutation (CGG to CAG) in exon 3, which results in the replacement of Arg77 by Gln at the base of the second zinc finger. This mutation has been described previously in two unrelated cases of HVDRR by Sone et al. It is unclear at this time whether these kindreds might be distantly related and, therefore, harbor the same mutation, or whether this represents a mutational hot spot in the VDR gene.

Candida albicans estrogen-binding protein gene encodes an oxidoreductase that is inhibited by estradiol.

Candida albicans, the most common fungal pathogen of humans, possesses an estrogen-binding protein (EBP) that binds mammalian estrogens with high affinity. We report here the cloning and complete nucleotide sequence of a gene encoding a C. albicans EBP. Amino acid sequences obtained from cyanogen bromide fragments of purified EBP were used to design oligonucleotide primers for PCR. An 800-bp product was amplified and used to screen a C. albicans genomic library. A clone was isolated containing an insert with an open reading frame of 1221 nt capable of encoding a protein with 407 amino acids and having a calculated molecular mass of 46,073 Da, the estimated size of EBP. The cloned gene, expressed in Escherichia coli as a lacZ fusion protein, demonstrated high-affinity binding for estradiol and a competition profile comparable to C. albicans wild-type EBP. Northern blots of C. albicans RNA revealed a single transcript of approximately 1600 nt, whereas Southern blots identified three hybridizing fragments. Computer searches of data bases showed that EBP shares a 46% amino acid identity with the old yellow enzyme, an oxidoreductase from Saccharomyces cerevisiae, but was unrelated to the human estrogen receptor as previously speculated. In addition, a 51-amino acid region of EBP is highly conserved among a group of flavoproteins including old yellow enzyme. Expressed EBP was shown to exhibit oxidoreductase activity that could be inhibited by 17 beta-estradiol in vitro. In conclusion, the EBP from C. albicans has no evident homology to the mammalian steroid receptor superfamily but appears to be a member of a recently identified family of flavoproteins.

Cloning and expression of the gene from Candida albicans that encodes a high-affinity corticosteroid-binding protein.

We have previously demonstrated the presence of a corticosteroid-binding protein (CBP) in Candida albicans and speculated on its homology to the glucocorticoid receptor. To explore this relationship further, we cloned the CBP gene. Our strategy employed sequencing enzymatically derived peptide fragments from purified CBP and using this information to synthesize degenerate oligonucleotide primers for use in the PCR. A 117-bp fragment amplified from C. albicans DNA was then used to screen a genomic library. Hybridizing clones were isolated, and DNA sequencing revealed an open reading frame of 1467 bp which encoded a protein with a molecular weight of 55,545. Southern analysis demonstrated that the gene was present at a unique locus within chromosome R of the Candida genome, while Northern analysis showed that the gene was expressed in C. albicans as a 1.8-kb transcript. CBP was over-expressed in Saccharomyces cerevisiae, and it exhibited an apparent dissociation constant (Kd) of 7 nM for [3H]corticosterone and displayed a steroid hormone binding profile comparable to that of the native protein. Searches of the data banks revealed little overall similarity to other cloned genes. However, the amino acid sequence contains a dinucleotide-binding fingerprint. In conclusion, we have cloned the gene encoding the CBP from C. albicans and have shown that the expressed protein has the properties of the native CBP. A comparison of the cloned gene to members of the steroid-thyroid-retinoic acid receptor gene superfamily showed that CBP is unrelated to these hormone receptors.

Cloning and characterization of the gene encoding the ADP-ribosylation factor in Candida albicans.

We have cloned and sequenced the gene (ARF) encoding the ADP-ribosylation factor (ARF) of Candida albicans. The gene contains an open reading frame of 537 nucleotides (nt) that codes for a protein with an Mr of 20,259. The C. albicans ARF gene is 67-70% identical at the nt level to other ARF sequences including those of humans; the deduced amino acid sequence of C. albicans ARF shows a 78-83% identity and 89-92% similarity to the other ARFs. Southern analysis of C. albicans genomic DNA suggested the presence of a second ARF gene. The presence of multiple ARF genes is a consistent finding among the other organisms previously shown to have ARFs.

Genetic defects of the 1,25-dihydroxyvitamin D3 receptor.

Target organ resistance to steroid hormone action is known to produce clinical disorders ranging from testicular feminization in the case of androgen resistance to hypocalcemic vitamin D-resistant ricets (HVDRR) in the case of 1,25-dihydroxyvitamin D3. The etiologic basis of these disorders is thought to be genetic mutations in the gene encoding receptors for these hormones. We investigated this possibility by analyzing the vitamin D receptor (VDR) protein, mRNA, and DNA from patients with HVDRR. This autosomal recessive disease of children is characterized by early onset rickets, hypocalcemia, hyperparathyroidism, and elevated levels of 1,25-(OH)2D3. Cells from patients fall into three general classes of molecular defects: (i) decreased or absent hormone binding; (ii) decreased affinity of VDR for DNA, or; (iii) defective nuclear translocation or retention. Analysis of the DNA and/or mRNA from these cells has identified missense mutations in the DNA binding (zinc finger) domain and a nonsense mutation in the steroid binding domain of VDR. The mutations were individually recreated in wild type VDR and the expressed mutant protein behaved biochemically identically to the patient receptor. Further studies have shown that the receptor is unable to interact with the specific hormone response element (HRE) of the osteocalcin gene and activate appropriate transcription. Rapid diagnostic genotyping of these mutations is possible with either restriction digestion or allele-specific oligonucleotide hybridization. Analysis of these naturally occurring, disease producing mutations of a gene regulatory protein should provide insight into the key amino acid residues of the protein and the mechanism by which steroids modulate gene transcription.

The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families.

Hereditary 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant rickets (HVDRR) is an autosomal recessive disease caused by target organ resistance to the action of 1,25(OH)2D3, the active form of the hormone. The defect in target cells is heterogenous and commonly appears to be a mutation in the gene encoding the vitamin D receptor (VDR). We have studied cultured skin fibroblasts and Epstein-Barr virus transformed lymphoblasts of seven family branches of an extended kindred having eight children affected with HVDRR. We have previously shown that cells from three affected children in this group contain an "ochre" nonsense mutation coding for a premature stop codon in exon 7 within the steroid-binding domain of the VDR gene. In the current studies, we found that cells from affected children failed to bind [3H]1,25(OH)2D3 and had undetectable levels of VDR as determined by immunoblots using an anti-VDR monoclonal antibody. Measurement of VDR mRNA by hybridization to a human VDR cDNA probe showed undetectable or decreased abundance of steady-state VDR mRNA. Parents, expected to be obligate heterozygotes, showed approximately half the normal levels of [3H]1,25(OH)2D3 binding, VDR protein, and mRNA. The mutation at nucleotide 970 (counting from the mRNA CAP site) results in the conversion of GTAC to GTAA, which eliminates an Rsa I restriction enzyme site and facilitates identification of the mutation. We found that polymerase chain reaction (PCR) amplification of exons 7 and 8 from family members and subsequent Rsa I digestion allows detection of the specific genotype of the individuals. When Rsa I digests of PCR-amplified DNA are subjected to polyacrylamide gel electrophoresis, children with HVDRR exhibit a homozygous banding pattern with loss of an Rsa I site. Parents exhibit a heterozygotic DNA pattern with detection of both normal and mutant alleles. In summary, our data show that the genetic abnormality is a point mutation within the steroid-binding domain of the VDR in all seven related families with HVDRR. Analysis of restriction fragment length polymorphism at the 970 locus of PCR-amplified DNA fragments can be used to diagnose this mutation in both affected children and parents carrying the disease.