Vitamin D-Resistant Rickets and Type 1 Diabetes in a Child With Compound Heterozygous Mutations of the Vitamin D Receptor (L263R and R391S): Dissociated Responses of the CYP-24 and rel-B Promoters to 1,25-Dihydroxyvitamin D3

Vitamin D-Resistant Rickets and Type 1 Diabetes in a Child With Compound Heterozygous Mutations of the Vitamin D Receptor (L263R and R391S): Dissociated Responses of the CYP-24 and rel-B Promoters to 1,25-Dihydroxyvitamin D3
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  Vitamin D–Resistant Rickets and Type 1 Diabetes in a Child WithCompound Heterozygous Mutations of the Vitamin D Receptor(L263R and R391S): Dissociated Responses of the CYP-24 and  rel  - B Promoters to 1,25-Dihydroxyvitamin D 3 Minh Nguyen, 1 Arnold d’Alesio, 1 Jean Marc Pascussi, 2 Rajiv Kumar, 3 Matthew D Griffin, 3 Xiangyang Dong, 3 Huguette Guillozo, 1 Marthe Rizk-Rabin, 4 Christiane Sinding, 5 Pierre Bougnères, 1,5 Frédéric Jehan, 1 andMichèle Garabédian 1 ABSTRACT: We report here the first association between vitamin D–resistant rickets, alopecia, and type 1diabetes in a child with compound heterozygous mutations in the  VDR  gene. Transfection studies suggestdissociated effects of   VDR  gene mutations on the regulation of genes involved in vitamin D metabolism anddendritic cell maturation.Introduction:  Whereas vitamin D may play a role in the immune tolerance process, no patient has beenreported to associate hereditary vitamin D–resistant rickets (HVDRR) and an autoimmune disease, and noattempt has been made to delineate the outcome of mutations of the vitamin D receptor (VDR) on thetranscription of genes controlling immune tolerance. Materials and Methods:  The VDR gene was analyzed in a child with vitamin D–resistant rickets, total alopecia,and early childhood–onset type 1 diabetes. Patient’s fibroblasts and COS-7 cells transfected with wildtype ormutant VDRs were studied for ligand-binding capacity, transactivation activity using two gene promoters[CYP-24, a classical 1,25(OH) 2 D 3 -responsive gene, and  relB , a critical NF-  B component for regulation of dendritic cell differentiation], VDR-RXR heterodimers association to CYP 24 VDREs by gel mobility shiftassays, and co-activator binding by Glutathione- S -transferase pull-down assays. Results:  Two novel compound heterozygous mutations (L263R and R391S) were identified in the VDRligand-binding domain in this child. Both mutations significantly impaired VDR ligand-binding capacity buthad dissociated effects on CYP-24 and  RelB  promoter responses to vitamin D. CYP 24 response binding toSRC-1 and RXR-heterodimer binding to CYP24 VDREs were abolished in L263R mutants but normal orpartially altered in R391S mutants. In the opposite,  RelB  responses to vitamin D were close to normal inL263R mutants but abolished in R391S mutants. Conclusions:  We report the first clinical association between HVDRR, total alopecia, and early childhood–onset type 1 diabetes. Mutations in the VDR ligand-binding domain may hamper the 1,25(OH) 2 D 3 –mediated relB  responses, an effect that depends on the site of the VDR mutation and cannot be anticipated from VDRligand-binding ability or CYP-24 response. Based on these results, we propose to survey the immune functionin patients with HVDRR, including those with moderate features of rickets. J Bone Miner Res 2006;21:886–894. Published online on May 8, 2006; doi: 10.1359/JBMR.060307Key words: hereditary 1,25-dihydroxyvitamin D–resistant rickets, vitamin D receptor mutations, CYP24,relB, type 1 diabetesINTRODUCTION I N ADDITION TO  its central role in bone and mineral me-tabolism, a rapidly increasing number of observationssuggest that 1,25-dihydroxyvitamin D 3  [1,25(OH) 2 D 3 ] mayplay a part in the complex processes leading to immunetolerance. (1–4) In vitro studies support this hypothesis byshowing inhibitory effects of 1,25(OH) 2 D 3  on the matura-tion of antigen presenting dendritic cells (DC), which arecritical in the induction of T cell–mediated immune re-sponses. (3,5,6) This vitamin also decreases the transcrip-tional activity of a number of genes involved in initiation of  The authors state that they have no conflicts of interest. 1 INSERM Unité 561, Hôpital St Vincent de Paul, Paris, France;  2 INSERM Unité 632, Université Montpellier I, Montpellier, France; 3 Department of Internal Medicine, Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota, USA;  4 INSERMU567-CNRS UMR 8104, Faculté Cochin, Paris, France;  5 Department of Pediatric Endocrinology, Hôpital St Vincent de Paul, Paris,France. JOURNAL OF BONE AND MINERAL RESEARCHVolume 21, Number 6, 2006Published online on May 8, 2006; doi: 10.1359/JBMR.060307© 2006 American Society for Bone and Mineral Research 886  cognate cellular (T cell) immune responses. (2,7) One of these genes is  relB , (7) a gene in the NF-  B pathway that ispivotal in regulating dendritic cell immunogenicities. (8) Animal studies bring further evidence, because mice withtargeted germline deletions of their  vitamin D receptor  ( VDR)  gene show increased maturity of DC in the subcu-taneous lymph nodes. (3) These mice also show immune de-fects such as impaired macrophage chemotaxis and lowersplenocyte response to anti-CD3 stimulation, although bothabnormalities can be prevented by normalization of serumcalcium. (9) In addition, 1,25(OH) 2 D 3  has been reported todelay the onset of various autoimmune diseases, includingtype 1 diabetes in the nonobese diabetic (NOD) mouse (10) and to prevent streptozotocin-induced diabetes in wildtypemice but not in VDR-knockout (KO) mice. (9) Finally, inhumans, negative associations have been observed in case-control and birth-cohort studies between vitamin D supple-mentation during the first year of life and the risk of child-hood onset type 1 diabetes. (11–13) The nuclear actions of 1,25(OH) 2 D 3  involve binding tothe ligand-binding domain of the VDR, interaction of theVDR heterodimerization sites with the retinoic acid X re-ceptor (RXR), and binding of its DNA-binding domain tovitamin D response elements (VDREs) in promoter re-gions of target genes. (14) The structure/function analysis of the VDR has been facilitated greatly by the characteriza-tion of a variety of mutations in the VDR of patients withhereditary 1,25(OH) 2 D 3 –resistant rickets (HVDRR), alsoknown as vitamin D–dependent rickets, type II. Mutationscausing premature termination of the VDR protein andmutations in its DNA-binding domain result in completehormone resistance and alopecia. (15,16) In contrast, muta-tions in the ligand binding domain have diversely impairedVDR functions, such as ligand binding, heterodimerizationwith RXR, or co-activator binding. They have been associ-ated with variable degrees of vitamin D resistance and havenot been consistently associated with alopecia, (15,17–20) sug-gesting the structural complexity of this domain. Interest-ingly, and despite a putative role of vitamin D on immunefunctions, none of the reported case, thus far, presentedwith type 1 diabetes.To date, the transactivation consequences of   VDR  mu-tations have been looked for in genes regulating vitamin Dmetabolism, especially CYP24, a classical marker of 1,25(OH) 2 D 3  responsiveness in the evaluation of HVDRR, (15) and in genes involved in bone metabolism,such as osteocalcin. (17,21–23) However, no attempt has beenmade to delineate the outcome of   VDR  mutations on thetranscription of genes controlling immune tolerance. Wereport here the first association in a patient of HVDRR,alopecia, and early childhood–onset type 1 diabetes. Thedetection in this child of two novel heterozygous mutationsin the VDR ligand binding domain (L263R and R391S)offered a unique opportunity to analyze the consequencesof   VDR  mutations on the transcription of genes specificallyexpressed in immune cells. Two genes were studied: the relB  gene because it bears functional VDREs and is nega-tively regulated by 1,25(OH) 2 D 3(7) and the classical  CYP- 24  gene for comparison. MATERIALS AND METHODS Patients Blood samples were obtained from the patient and hisfamily, and a skin biopsy was obtained from the patient. Alocal human subjects ethics committee approved the fibro-blast cultures and genomic studies, and the parents gavewritten informed consent. Culture conditions Skin fibroblasts were obtained from the patient at age 48months and grown in DMEM (Gibco, Invitrogen, CergyPontoise, France) supplemented with 10% FCS, penicillin,streptomycin, and fungizone until confluent as de-scribed. (19) Control fibroblasts from two age-matched subjects withno known alteration of calcium metabolism were culturedin identical conditions. VDR binding, VDR protein immunoblot, and 25(OH)D  3  24-hydroxylase assays Cytosol samples were prepared from the patient’s andcontrol fibroblasts and from transfected COS-7 cells. Li-gand-binding assays were performed by incubating 0.2 mlcytosol with 0.15–1.5 nM  3 H 1,25(OH) 2 D 3  for either 1 h at25°C or 18 h at 4°C in the absence or presence of an excess(1200 nM) of unlabeled 1,25(OH) 2 D 3 . Bound and free li-gands were separated by hydroxyapatite. (19) The presenceof the VDR protein was ascertained in transfected cells byWestern blot as described. (19) The 25(OH)D 3  24-hydroxylase assays were performed inthe patient’s and control fibroblasts after culture in serum-free DMEM, with 1,25(OH) 2 D 3  or its solvent (ethanol) for18 h. Cells were rinsed with serum-free medium and incu-bated for 120 minutes in the presence of 1.6 nM  3 H25(OH)D 3 . The  3 H vitamin D 3  derivatives present in themedium were extracted and chromatographed using astraight phase high-performance liquid chromatography(HPLC) system as described. (24) DNA isolation, PCR, and genomicDNA sequencing Genomic DNA was prepared from blood lymphocytes of the patient, parents, and unaffected brother. (19) Exons 7–9were amplified from genomic DNA (1  g) using 0.16  M of each of the following sets of primers: 5  -GCGAATTCC-GTTACTGGTAACCTGACCTCTTC-3   and 5  -TGTC-TAGAATACACCCCGCTCCCCAGTCCCTGAG-3   forexons 7–8 and 5  -CAGAGCATGGACAGGGAGCAAG-3   and 5  -CAACTCCTCATGGCTGAGGTCTC-3   forexon 9. PCR amplification was performed using  Taq  poly-merase (Appligene Oncor, Illkirch, France) in buffer con-taining either 2 (exons 7–8) or 2.5 mM (exon 9) of MgCl 2 and 2.5% dimethylsufoxide (DMSO) for 30 cycles. Eachcycle consisted of denaturation at 95°C for 1 minute, an-nealing at either 58°C (exons 7–8) or 65°C (exon 9) for 1minute, and primer extension at 72°C for 10 minutes.PCR products were purified and sequencing was carriedout using the  Taq  Dye-Deoxy Terminator cycle sequencing CYP24 AND  RELB  RESPONSES IN A HVDRR CHILD 887  kit and an ABI PRISM 377 DNA sequencer (Perkin-Elmer, Biosystems, CourtaBoeuf, France). cDNA synthesis and sequencing cDNA was synthesized from RNA (5   g) of immortal-ized patient leukocytes using Superscript II-RNase H re-verse transcriptase (Gibco BRL). (19) The PCR product wassubcloned into the pGEM-T Easy Vector (Promega).Transformant  Escherichia coli  clones (strain JM 109) wereprinted on nylon membrane and screened using a humanVDR probe (a gift from JW Pike) [ 32 P]labeled by randompriming. Positive clones were sequenced as describedabove. Site-directed mutagenesis and construction of VDRexpression vectors Site-directed mutagenesis of the wildtype VDR cDNAwas performed using the Gene Editor TM system (Pro-mega, Madison, WI, USA). The mutagenic oligonucleo-tides used were 5  -CAGATCGTACTGCGGAAGT-CAAGTGCCATTG-3   (for Leu263Arg mutant), 5  -AGCTAGCCGACCTGAGCAGCCTCAATGA-3   (forArg391Ser mutant), and 5  -AGCTAGCCGACCTGTG-CAGCCTCAATGA-3   (for Arg391Cys mutant; the mu-tated base is underlined).The mutated VDR cDNA wasinserted into a pTracer-CMV expression vector (InvitrogenLife Technologies, Cergy-Pontoise, France) by the  Eco RIsites. Each site-directed mutant was confirmed by sequenc-ing. Transient transfection experiments COS-7 cells were grown in monolayers in DMEM con-taining 10% FCS, penicillin, streptomycin, and fungizone.One day before transfection, cells were plated in 6-wellplates (2 × 10 5 cells/well; Costar, Cambridge, MA, USA)with 2 ml of medium. Then, 2   l of Fugene-6 (Roche) anda mixture of 1   g of plasmid DNA, containing 150 ng of wildtype or mutated, pTracer-CMV VDR, 50 ng of    -ga-lactosidase, and 800 ng of either luciferase reporter CYP-24promoter, luciferase reporter human  relB  promoter, or lu-ciferase reporter human  relB  promoter bearing mutationson its two VDRE motifs, (7) were added to the cells.Twenty-four hours after transfection, cells were incubatedwith ethanol or 1,25(OH) 2 D 3  for 16 h and harvested with200   l of 1× reporter lysis buffer (Promega). Luciferaseactivities were measured in 10   l of cell extracts with a LGBerthold Lumat LB 9507.   -Galactosidase activity was de-termined in the same cell extracts using  o -nitrophenyl-  - D -galactopyranoside (ONPG) as substrate. GST pull-down assays [ 35 S]methionine-labeled wildtype and mutant VDRswere prepared by in vitro translation using the transcriptionand translation (TNT)-coupled transcriptional translationsystem (Promega).Glutathione- S -transferase-steroid receptor co-activator 1(GST-SRC-1) fusion proteins were expressed in the  Esche-richia coli  BL21 strain (4 h with 0.1 mM isopropyl-  - D -thiogalactoside) and purified using glutathione sepharose-4B bead affinity chromatography (Pharmacia, Uppsala,Sweden). The beads were subsequently washed and resus-pended in 20 mM Tris (pH 8.0), 100 mM NaCl, 1 mMdithioerythritol (DTT), and 0.1% Nonidet P-40 buffer(NETN) containing anti-proteases cocktail (Roche). GSTproteins bound to glutathione-sepharose (40  l slurry) wereincubated with 5   l of [ 35 S]methionine-labeled proteins inthe presence of NETN buffer and with 1,25(OH) 2 D 3  or 1%DMSO, in a final volume of 600   l. After overnight incu-bation at 4°C with gentle shaking, agarose beads were ex-tensively washed with NETN buffer and bound proteinswere eluted in sample buffer and analyzed by SDS-PAGE.Gels were incubated in an autoradiography enhancer (Du-pont NEN), dried, and analyzed using a PhosphoImagerapparatus (Molecular Dynamics). Electrophoretic mobility shift assays Wildtype VDR, the two VDR mutants, and RXR   werein vitro synthesized using 500 ng of plasmid and transcrip-tion and translation-coupled (TNT-coupled) transcriptionaltranslation (Promega). The following double-stranded syn-thetic oligonucleotides containing human VDREs wereused. For human 24-hydroxylase VDREs (25) : VDRE proxi-mal: 5  -ATGGAGTCAGCGAGGTGAGCGAGGGC-GTCC-3  ; VDRE distal: 5  - ACGCCGGAGTTCACC-GGGTGTGCTTCGA-3  ; and for human  relB  VDREs (7) : relB  A: 5  -CACCATGTCGGTCAGGCTGGTCTC-GAAATCC-3   and  relB  B: 5  -AAACGGCAGGTTCAA-GTCCCACTGGGAGACC-3  .They were [  - 32 P]-ATP end-labeled by T4 polynucleo-tide kinase (Invitrogen Life Technologies) and purified ona nondenaturing 10% polyacrylamide gel before gel shiftstudies.For the binding reaction, 0.1 ng of labeled oligonucleo-tide was incubated for 20 minutes on ice with 1   l of VDRprotein, 1   l of RXR protein (undiluted or diluted), and 1  g of poly(dI–dC) (Amersham Biosciences) in a buffercontaining 10% glycerol, 5 mM Tris, pH 7.5, and 150 mMKCl. In some experiments, a monoclonal antibody 9A7   ora monoclonal antibody against the carboxy terminus of theVDR (Santa Cruz Biotechnology, Santa Cruz, CA, USA)was added and incubated for 30 minutes before the additionof the probe. The samples were electrophoresed on a 4%nondenaturing polyacrylamide gel. Gels were transferredand dried, followed by autoradiography. The developedfilm was subjected to densitometric quantification (Gel Doc2000; BioRad). RESULTS Clinical  This white French patient, with no known consanguinity,was first seen at the age of 27 months with florid rickets,total alopecia, and postnatal growth retardation (height: 72[+1.5 SD], 73 [−0.5 SD] and 81 [−2 SD] cm at 6, 12, and 27months, respectively). Blood tests revealed hypocalcemia,hypophosphatemia, elevated alkaline phosphatase activity,elevated PTH levels, subnormal 25-hydroxyvitamin D (7ng/ml), and low normal 1,25-dihydroxyvitamin D (28 pg/ml)concentrations (Fig. 1). Oral administration of elementalcalcium (1 g/day) and 1  (OH)D 3  (2–8  g/day) for 3 months NGUYEN ET AL.888  did not substantially improve the abnormal biochemical pa-rameters. Treatment with calcium (1 g/day) and higher1  (OH)D 3  doses (15  g/day) for 4 months had only a slightbeneficial effect on serum calcium and alkaline phospha-tase activity. The vitamin D treatment was switched to highdoses of 25(OH)D 3  to further increase 1,25-dihydroxy-vitamin D levels, as previously reported. (26) After a5-month treatment with 25(OH)D 3  (200–250   g/day) andcalcium (1 g/day), complete healing of clinical and radio-logical signs of rickets and normalization of all biologicalabnormalities, including secondary hyperparathyroidism,were obtained. During this treatment, serum 25(OH)D and1,25(OH) 2 D levels were 10–20 fold above normal (200–450ng/ml and 200–1800 pg/ml, respectively). Thereafter, treat-ment with 100–75   g/day 25(OH)D 3  with no calciumsupplements was sufficient to maintain a normal clinical,radiological, and biological status, with the exception of alopecia, and to correct growth retardation (height: 158 cm[+0.8 SD] at 13.5 years).At the age of 5 years, the child presented with insulin-dependent diabetes. Retrospective analysis of collectedblood samples showed the presence of antiislet autoanti-bodies, detected by immunofluorescence (27) before vitaminD treatment (i.e., 3 years before the onset of diabetes; 160Juvenile Diabetes Foundation [JDF] units at 27 and 44months and 40 JDF units at 5 years; reference value < 10JDF units). Insulin autoantibodies were in the normal range(1% binding at 27 and 44 months; reference value < 2.3%).His HLA DRB1 genotype is HLA DRB1*03.His parents and an older half brother are phenotypicallyunaffected regarding bone and calcium metabolism anddiabetes.  Amplification of cDNA, genomic DNA, andnucleotides sequence analysis Sequencing of the full-length VDR cDNA from the pa-tient’s leukocytes revealed two heterozygous missense mu-tations on separate cDNA clones. The first one, in exon 7,causes the substitution of leucine by arginine at amino acidposition 263 (L263R), and the second one, in exon 9, causesthe substitution of arginine by serine at amino acid position391 (R391S) (Fig. 2). The absence of a  Fok 1 cleavage site atthe first VDR translation start codon (F/F genotype) sug-gests the expression in this child of a three amino acidshorter VDR isoform with normal or increased transcrip-tional potency. (14) The remainder of the VDR coding re-gion was normal.Sequence analysis of DNA samples from the parentsshowed that the mother is a heterozygous carrier of the FIG. 1.  Effects of vitamin D therapy on biological parameters inpatient. Normal ranges for calcium, phosphate, alkaline phospha-tase activities, PTH, and vitamin D metabolites are given as grayareas. The daily dose of 1  (OH)D 3  is indicated as dark areas andthat of 25(OH)D 3  as hatched areas. FIG. 2.  Family pedigree with VDR nucleotide sequence analy-sis. Roman numerals represent generations. Partial nucleotide se-quences of exons 7 and 9 in the normal and mutant VDR arerepresented. The altered nucleotides are indicated in boldfacetype. Direct sequencing of the VDR gene revealed two com-pounds heterozygous mutations for the patient (L263R andR391S). Analysis of the VDR cDNA synthesized from total RNAof patient’s leukocytes confirmed the presence of these mutationson separate cDNA clones. Direct sequencing of the  VDR  generevealed the presence of the heterozygous mutation L263R forthe father and his son by the first marriage and that of the het-erozygous mutation R391S for the mother. These three subjectsare phenotypically unaffected. CYP24 AND  RELB  RESPONSES IN A HVDRR CHILD 889  mutant  R391S  allele, whereas the father and the half brother are heterozygous carriers of the mutant  L263R  al-lele. VDR binding assay and 24-hydroxylase activity in patient fibroblasts The VDR present in the patient’s fibroblasts specificallybound  3 H 1,25(OH) 2 D 3  with a normal affinity ( K  d : 1.2 ×10 −11 M), regarding the results obtained in two age-matched control fibroblasts ( K  d : 1 × 10 −11 M). The numberof binding sites, however, was reduced to a level that was10–30% that found in control cells (Table1). The patient’sfibroblasts produced detectable amounts of   3 H24,25(OH) 2 D 3  under basal conditions. After treatment with10 nM 1,25(OH) 2 D 3 , 24-hydroxylase activity showed a low2-fold increase compared with the 18- to 24-fold stimulationobserved in control cells (Table1). No further 24-hydroxylase activity stimulation was observed after cell in-cubations with higher concentrations of 1,25(OH) 2 D 3 , up to1000 nM (data not shown). VDR binding assay and 24-hydroxylase geneactivation in transfected COS-7 cells To test the effects of each mutation on VDR function,COS-7 cells were transiently transfected with wildtype(WT) or mutant VDR cDNA (R391S and L263R) and a24-hydroxylase promoter VDRE-luciferase reporter plas-mid. No VDR protein expression and no activation of 24-hydroxylase gene transcription were observed in untrans-fected COS-7 cells or COS-7 cells transfected with theempty plasmid p-Tracer CMV (data not shown).All WT and mutant cells expressed an immunoreactiveprotein at  ∼ 50 kDa, corresponding to the predicted size of hVDR, when using the 9A7    anti-VDR antibody (Figs. 3and 4, insets). WT and VDR mutants had a similar VDR/actin ratio in basal conditions (0.6, 0.8, and 0.6 in WT,R391S, and L263R cells, respectively). R391S  mutants bound  3 H 1,25(OH) 2 D 3  with an affinitysimilar to that found in WT cells, but with a 2- to 3-foldlower binding capacity (Fig. 3; Table 2). They showed cleardose-dependent 24-hydroxylase responses to 1,25(OH) 2 D 3 ,albeit the maximal response only reached one half that ob-served with WT cells and required 10- to 100-fold higher1,25(OH) 2 D 3  concentrations (Fig. 4). L263R  mutants, like  R391  mutants, bound  3 H1,25(OH) 2 D 3  with an affinity similar to that found in WTcells, but with a 3-fold lower binding capacity, after cytosolincubation at 4°C (Fig. 3; Table 2). To be noticed, this bind-ing capacity was almost abolished after cytosol incubationat 25°C, suggesting thermal lability of the binding (Table 2). L263R  mutants showed no 24-hydroxylase response to1,25(OH) 2 D 3  despite the 1,25(OH) 2 D 3  concentrations used(Fig. 4). RXR-VDR heterodimer associations with the CYP  24 promoter  To explore the mechanism underlying the different ef-fects of the two mutations on the 24-hydroxylase responsesto 1,25(OH) 2 D 3 , we performed gel mobility shift assays at4°C using in vitro–synthesized VDR (wildtype or mutants),RXR, and oligonucleotides containing the proximal anddistal VDREs of the  CYP 24  gene. These experimentsshowed a specific association between wildtype VDR,RXR  , and CYP 24 VDREs that was eliminated by pre-incubation with the 9A7   monoclonal antibody and super-shifted after incubation with the monoclonal antibody di-rected against the C-terminal region of the VDR (Fig. 5A).Increasing dilutions of RXR   above 1:4 decreased the sig-nal intensity of the complex formation with both CYP 24VDREs: distal (Fig. 5B) and proximal (Fig. 5C). T ABLE  1. S PECIFIC  1,25(OH) 2 D 3  B INDING AND 24-H YDROXYLASE  A CTIVITY IN  P ATIENT AND C ONTROL  F IBROBLASTS 1,25(OH)  2 D  3 binding (fmol/mg protein) 25(OH)D  3  –24-aseactivity (fmol/500.000cells/120 minutes)Basal Stimulation fold Controls (10–72months old) 8–24 0.9–2.1 18–24Patient (48months old) 2.5 0.9 2.1 Quantitation of specific binding capacity was performed with 2 nM of  3 H1,25(OH) 2 D 3  in the absence or presence of a 50-fold excess of unlabeled1,25(OH) 2 D 3 . For 24-hydroxylase activity, cells were grown for 3 days inDMEM plus 10% FCS and then for 18 h in serum-free medium with etha-nol or 1,25(OH) 2 D 3  (10 -8 M). They were incubated in fresh serum-freemedium for 2 h with 1.6 nM  3 H 25(OH)D 3 . Media were collected, and  3 H24,25(OH) 2 D 3  production was determined by HPLC. FIG. 3.  Analysis of specific 3H 1,25(OH) 2 D 3  binding in cytosolsfrom COS-7 cells transfected with WT or L263R or R391S VDRmutants. Cells extracts were incubated at 4°C with increasing con-centrations of 3H 1,25(OH) 2 D 3  in the absence or presence of anexcess of unlabeled 1,25(OH) 2 D 3 . Bound and free ligand wereseparated by hydroxyapatite. Scatchard analysis was performed inthree separate experiments. One of these Scatchard analysis plotsis shown. The presence of the VDR protein was ascertained in cellextracts by Western blot (inset). NGUYEN ET AL.890
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