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The story of rickets leading to the discovery of vitamin D is an extraordinary tale, spread over many centuries and involving some remarkable characters with much speculation and a few mysteries, before reaching an exciting climax. It would be wrong to credit a single person as discovering rickets or being the first to describe its features, for reasons that will be set out here. Yet the emergence of the term 'rickets' is as important as the discovery of vitamin D itself and the possible causes of its deficiency. It permitted identification of a hitherto ill-defined disease entity, typically occurring in infants and children. It also provided a way for deciding if features of diseases that had been described earlier in the history of medicine could be seen as the symptoms and signs of related conditions.

Rickets was first documented as a cause of death in the Bills of Mortality for The City of London in 1634, but detailed descriptions were only published between 1645 and 1668. It was thought at the time that this was a new disease in England, but there was no indication as to the cause of the condition. However, air pollution from smoke produced by burning coal caused serious problems at that time, and so it can be suggested that vitamin D deficiency was responsible.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

Incubation of bovine parathyroid cells for 48 h in 0.4 mmol calcium/l had no significant effect on steady-state preproparathyroid hormone (preproPTH) mRNA levels when compared with cells incubated in 1.0 mmol calcium/l, but low calcium concentrations increased the membrane-bound polysomal content of preproPTH mRNA by 200 +/- 16% (mean +/- S.D.). No preproPTH mRNA was detected on free polysomes. Actinomycin D (5 and 10 micrograms/ml) had no effect on steady-state preproPTH mRNA levels measured in dot-blot assays after 24 h, but reduced levels in cells incubated in 1.0 mmol calcium/l to 54 +/- 16% and 39 +/- 12% of control values respectively after 48 h of incubation. Similarly, in cells incubated in 0.4 mmol calcium/l, actinomycin D (5 and 10 micrograms/ml) reduced steady-state preproPTH mRNA levels to 57 +/- 13% and 45 +/- 5% of control values respectively. Actinomycin D did not prevent the rise in polysomal content of preproPTH mRNA induced in cells by incubation in 0.4 mmol calcium/l, but increased polysomal content in cells incubated in 0.4 and 1.0 mmol calcium/l by 159 +/- 9% and 164 +/- 13% respectively after 48 h. These results demonstrate post-transcriptional regulation of PTH synthesis in cultured bovine parathyroid cells, and suggest that this control involves a protein which may be calcium-sensitive.

The active metabolite of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-DHCC), is a mononuclear phagocyte product with immunoregulatory properties which can influence not only surrounding T cells but also other mononuclear phagocytes; and which acts in an autocrine fashion. In this study we have used the U937 cell line as a starting point model to investigate further the comparative role of 1,25-DHCC and phorbol myristate acetate (PMA) upon growth, differentiation and phenotype in the mononuclear phagocyte system, and have correlated our findings with changes in 1,25-DHCC metabolism and receptor expression. Both 1,25-DHCC and PMA inhibit growth and differentiate U937 cells in a dose-dependent fashion. When used together, however, at low doses of PMA, 1,25-DHCC protects against the PMA-induced growth inhibition. At high concentrations of both compounds there is a decrease (1,25-DHCC) or an increase (PMA) in 1,25-DHCC receptor expression, with either 24-OHase (1,25-DHCC) or 1-OHase (PMA) synthesis. If the compounds are used in combination the receptor levels are equivalent to controls, and both enzymes are produced. The phenotype of the 1,25-DHCC-induced cells shows light adherence, class I+, increase in CD4 and CD14 and decrease in CD71. The PMA-induced cell is tightly adherent, class I+, and strongly positive for CD13 with a concomitant decrease in both CD4 and CD71. These findings suggest another role for 1,25-DHCC in the mononuclear phagocyte system, as a potential mitogenic agent. They also suggest that 1,25-DHCC may act at both membrane and nuclear levels within this model of the mononuclear phagocyte pathway and demonstrate one possible way in which physiological peripheral macrophage heterogeneity might be induced, i.e. due to the nature of the signals which are implicated during differentiation. The presence of increased CD4 and decreased CD13 on the surface of 1,25-DHCC-differentiated cells, and vice versa on PMA-differentiated cells, illustrates how this may then be reflected in functional mononuclear phagocyte heterogeneity, which may in turn be reflected in differential peripheral function.

This paper presents three markers, 16D/E, pHMAI (DXS208), and CRI-L1391 (DXS274), that show close linkage for X-linked hypophosphataemic rickets (HYP). DXS274 is closely linked to HYP (theta max = 0.00, Zmax = 4.20), and DXS41 (99.6), (theta max = 0.00, Zmax = 5.20). Marker 16D/E maps distal to the disease locus (theta max = 0.05, Zmax = 3.11). The pHMAI probe recognises the same restriction fragment length polymorphism (RFLP) as 99.6. Multipoint analysis suggests that the most probable order of loci is Xpter-(DXS43, 16D/E)-HYP-DXS274-(DXS208, DXS41)-Xcen. The location of DXS274 distal to HYP cannot be excluded, as no recombinants were observed between DXS274 and HYP, or between DXS274 and DXS41/DXS208. One of the families contains a large number of recombinants, four of which are double recombinants. This most probably means that the disease in this family maps elsewhere on the X chromosome or on an autosome, indicating locus heterogeneity.

The effects of 1,25(OH)2D3 on proliferation, c-myc mRNA levels and 1,25(OH)2D3 receptor expression in activated tonsillar T lymphocytes were studied. Activation of resting T cells with phytohaemagglutinin (PHA) for 72 hr led to an increase in proliferation, c-myc mRNA levels and to induction of 1,25(OH)2D3 receptor expression. However, when activation was carried out in the presence of 1,25(OH)2D3, there was inhibition of PHA-stimulated proliferation and c-myc mRNA levels. Increased cell proliferation, c-myc mRNA expression and 1,25(OH)2D3 receptor number were also observed, albeit to a lesser extent, when T cells were stimulated by phorbol myristate acetate (PMA), anti-CD3 antibody or A23187. However, in these cases 1,25(OH)2D3 was unable to prevent increased proliferation or c-myc mRNA expression. PMA and anti-CD3 used in combination produced similar or greater changes in proliferation, c-myc mRNA levels, 1,25(OH)2D3 receptor expression and responsiveness to the hormone when compared to PHA alone. Thus the inhibition of c-myc expression in activated T lymphocytes by 1,25(OH)2D3 can be related to its anti-proliferative effects. Moreover this inhibition seems to be dependent on the level of 1,25(OH)2D3 receptor expression, which in turn appears to be related to the degree of cell activation.

The human X-linked hypophosphatemic rickets gene locus (HYP, formerly HPDR) has been previously localized by linkage analysis to Xp22.31-Xp21.3 and the locus order Xpter-DXS43-HYP-DXS41-Xcen established. Recombination between HYP and these flanking markers is frequently observed and additional markers have been sought. The polymorphic loci DXS197 and DXS207 have been localized to Xpter-Xp11 and Xp22-Xp21, respectively. We have further localized DXS197 to Xpter-Xp21.3 by using a panel of rodent-human hybrid cells and have established the map positions of DXS197 and DXS207 in relation to HYP by linkage studies of hypophosphatemic rickets families. Linkage between DXS197 and the loci DXS43, DXS85, and DXS207 was established with peak lod score values of 6.19, 0 = 0.032; 4.14, 0 = 0.000; and 3.01, 0 = 0.000, respectively. Multilocus linkage analysis mapped the DXS197 and DXS207 loci distal to HYP and demonstrated the locus order Xpter-DXS85-(DXS207, DXS43, DXS197)-HYP-DXS41-Xcen. These additional genetic markers DXS197 and DXS207 will be useful as alternative markers in the genetic counseling of some families.

A patient with Hodgkin's disease spontaneously developed steroid-responsive hypercalcaemia during two consecutive summers. Administration of 3000 U/day of vitamin D, while he was normocalcaemic, caused a sharp increase in serum 1,25(OH)2D3 (from 59 pg/ml to 142 pg/ml) and subsequently hypercalcaemia while serum 25(OH)D3 rose moderately within the normal range (from 2.8 ng/ml to 10 ng/ml). During a spontaneous episode of hypercalcaemia which was accompanied by increased circulating 1,25(OH)2D3 concentrations, administration of hydrocortisone decreased serum 1,25(OH)2D3 rapidly (from 115 pg/ml to 62 pg/ml) and eventually led to normocalcaemia while serum 25(OH)D3 remained unchanged. Thus the disturbances of mineral metabolism found in this patient with Hodgkin's disease are very similar to those previously described in sarcoidosis.

Dichloromethylene bisphosphonate (C12MBP), a powerful inhibitor of bone resorption, was administered to 27 patients with primary hyperparathyroidism. It was given by either intravenous infusion (six patients, 500-100 mg day), or by intramuscular injection (six patients, 100-200 mg/day) or by mouth (15 patients, 1600-2400 mg/day) for 20-180 days. Sustained suppression of bone resorption was observed in all patients, as judged by a fall in the urinary hydroxyproline excretion. In contrast, the hypocalcaemic effect was inconsistent and short-lived, particularly in the patients without overt bone disease. The fall in serum calcium seemed largely to be due to a transient dissociation between bone resorption and bone formation and was associated with increases in circulating parathyroid hormone (PTH). In ten patients given the bisphosphonate orally for 6 months, serum calcium was unchanged but serum PTH was significantly raised. These results suggest that C12MBP may be of use for short-term correction of severe hypercalcaemia due to hyperparathyroidism, particularly in the patients with overt bone disease. However, its long-term use should not be recommended because of increased PTH secretion.

Receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) were prepared from bovine parathyroid glands and incubated with fragments of DNA of the 5'-flanking region of the bovine parathyroid hormone (PTH) gene covering 1700 base pairs (bp) upstream of the initiation site. In filter binding assays, incubation of the DNA fragment spanning -700 to +50 bp with 200 micrograms cytosolic protein gave 288 +/- 63% (mean +/- S.D.) of binding in the absence of protein. In contrast, there was no significant reaction with the -1350 to -700 bp fragment, nor was there binding of the receptor to a fragment of DNA covering the coding region of the PTH gene. Substitution of bovine serum albumin for the receptor preparation did not induce binding to the -700 to +50 bp fragment. The receptor-binding site was further defined to -700 to -100 bp as deletion of the -100 to +50 bp did not reduce receptor binding. Reaction of receptors further purified by sucrose density ultracentrifugation with a monoclonal antibody in immunoblots revealed a single species with a molecular mass of approximately 50,000 Da, which was absent in preparations of cos-1 cells. Autoradiography following incubation of receptors immobilized on nitrocellulose filters with the -700 to +50 bp fragment indicated a single reactive band coincident with the band in the immunoblot. The DNA fragment did not bind to filters containing preparations of cos-1 cells. Extraction of the receptors in the presence or absence of 1,25-(OH)2D3 (4 nmol/l) or the presence of KCl (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay. (ABSTRACT TRUNCATED AT 250 WORDS)

Idiopathic hypoparathyroidism has been reported to occur as an X-linked recessive disorder in two multigeneration kindreds. Affected individuals, who are males, suffer from infantile onset of epilepsy and hypocalcemia, which appears to be due to an isolated congenital defect of parathyroid gland development; females are not affected and are normocalcemic. We have performed linkage studies in these two kindreds (5 affected males, 11 obligate carrier females, and 44 unaffected members) and have used cloned human X chromosome sequences identifying restriction fragment length polymorphisms to localize the mutant gene causing this disorder. Our studies established linkage between the X-linked recessive idiopathic hypoparathyroid gene (HPT) and the DXS98 (4D.8) locus, peak LOD score = 3.82 (theta = 0.05), thereby mapping HPT to the distal long arm of the X chromosome (Xq26-Xq27). Multilocus analysis indicated that HPT is proximal to the DXS98 (4D.8) locus but distal to the F9 (Factor IX) locus, thereby revealing bridging markers for the disease. The results of this study will improve genetic counseling of affected families, and further characterization of this gene locus will open the way for elucidating the factors controlling the development and activity of the parathyroid glands.

In 10 patients with Paget's disease of bone and 2 patients with osteoporosis, we studied the effects of hypocalcemia and hypophosphatemia induced by disodium-(3-amino-1-hydroxypropylidene)-1,-bisphosphonate (APD) treatment on the serum concentration of PTH and 1,25-dihydroxy-vitamin D [1,25(OH)2D3] and on calcium absorption and balance. The fall in serum calcium and phosphate was associated with a rise in the serum concentration of PTH and 1,25(OH)2D3, coupled with increases in net calcium absorption and calcium balance. The concentration of 1,25(OH)2D3 was significantly related (P less than 0.001) to the serum calcium (r = 0.66), the serum phosphate (r = 0.78), and the serum PTH (r = 0.71), confirming the interrelated control of these parameters on 1,25(OH)2D3 production. Moreover the rise in 1,25(OH)2D3 caused an appropriate rise in calcium absorption (r = 0.74) and calcium balance (r = 0.86), showing that this vitamin D metabolite contributes as a hormone to calcium homeostasis.

The immunological properties of a synthetic peptide comprising the carboxyl-terminal 53--84 region of human parathyroid hormone (PTH) have been studied. The immunoreactivity of the synthetic human PTH-(53--84) peptide paralleled that of a 53--84 fragment of the native human hormone prepared by enzymic digestion, in both a standard radioimmunoassay, which was not region-specific, and also a radioimmunoassay specific for the carboxyl-terminal region of PTH. However, in both types of radioimmunoassay the synthetic human PTH-(53--84) peptide was four to five times more reactive than the native human PTH-(53--84) fragment.

The sequence of the amino-terminal portion of human parathyroid hormone, particularly the identity of residues 22, 28, and 30 (the subject of discrepancies in recent published reports), has been reexamined by two basic methods of structural analysis. A fresh lot of human parathyroid hormone isolated from pooled adenoma tissue was analyzed by Edman degradation with identification of critical residues by thin-layer chromatography and gas-liquid chromatography. In the second approach, -14C or tritiated amino acids were incorporated during biosynthesis of the human hormone in slices of parathyroid glands in vitro; the appropriate amino acid residues were then determined as the -14C or tritiated phenythiohydantoin derivatives of the amino acid after Edman degradation, or by peptide isolation after appropriate cleavage with endopeptidase, or both. The results confirm our previous findings that residue 22 is glutamic acid, residue 28 is leucine, and residue 30 is aspartic acid.

We have examined the effects of 1,25(OH)2D3 on T-cell populations isolated by buoyant density and E rosetting from human tonsils. Cell proliferation was assessed by measuring the incorporation of 125iododeoxyuridine; interleukin-2 (IL-2) production was measured using an IL-2-dependent cell line, and the number of 1,25(OH)2D3 receptors was measured by whole-cell nuclear association assay. At a concentration of 10(-7) M, 1,25(OH)2D3 inhibited mitogen-induced T-cell proliferation in all E+ T-cell populations. This effect was more pronounced in the cells from the intermediate and high density layers and was reflected both in cell proliferative responses and in relative IL-2 synthesis. By adding the 1,25(OH)2D3 during the course of the mitogen assay, we demonstrated that activation of the T cell precedes the 1,25(OH)2D3-mediated inhibition. Cells that had been preincubated with mitogen in the presence of the 1,25(OH)2D3 were refractory to further stimulation by mitogens. Receptors for 1,25(OH)2D3 could not be detected in unstimulated T cells. However, activation led to the expression of high-affinity receptors for 1,25(OH)2D3. Co-incubation of the cells with mitogen and 1,25(OH)2D3 increased the number of receptors compared with mitogen alone. The effects provide further evidence for the hypothesis that 1,25(OH)2D3 is an important potential modulator of the immune system through its action on T cells. Taking our observations in conjunction with the known capacity of monocytes to hydroxylate the precursor metabolite (and thus synthesize the active form of cholecalciferol), the results support the suggestion that 1,25(OH)2D3 plays a role as a local mediator of mononuclear phagocyte-T cell interaction in human lymphomedullary tissues.

Twenty eight hypocalcaemic patients were studied, 14 with primary hypoparathyroidism, nine with pseudohypoparathyroidism and two with hypo-hyperparathyroidism, to characterized the essential features of these disorders. Like tetany, which was present in 12 of the patients, epilepsy was a common symptom, occurring in 13, seven of whom had received anticonvulsants for two to eight years before hypocalcaemia was detected. Differentiation between primary and pseudohypoparathyroidism could not be made with certainty on clinical grounds but confident distinction could be made by measurement of endogenous parathyroid hormone concentrations and by testing for renal resistance to exogenous parathyroid hormone. This was achieved by measurement of the plasma and, in some patients, the urinary cyclic AMP response to an intravenous injection of highly purified bovine parathyroid hormone. These investigations were also valuable in the assessment of the other three hypocalcaemic patients in whom a diagnosis of parathyroid dysfunction would otherwise have been made. In 10 of the patients synthetic 1 alpha-hydroxylated forms of vitamin D were used to establish and maintain normocalcaemia, though their use required careful monitoring.

Highly sensitive assays have been developed that enable 25-hydroxycholecalciferol (25-hydroxyvitamin D3) and 25-hydroxyergocalciferol (25-hydroxyvitamin D2) to be measured in the same serum sample. With these assays it has been shown that endogenously produced cholecalciferol (vitamin D3) is important in man; the findings further emphasize the role of vitamin D metabolites as hormones rather than vitamins in the traditional sense. Dietary sources of vitamin D appear to be inadequate and vitamin D deficiency has been shown to the cause of rickets and osteomalacia in Asian immigrants to Britain. This condition may be readily treated with small doses of vitamin D. In addition, sub-clinical deficiency was found in the Asian community. In the elderly, also, vitamin D deficiency was established as an important cause of osteomalacia and again evidence for the existence of a sub-clinical deficiency state was found. It is therefore suggested that the present prophylactic practices should be reviewed. Secondary hyperparathyroidism (reflected by elevated concentrations of circulating immunoassayable parathyroid hormone) was shown to be the rule rather than the exception in vitamin D deficiency. Some patients, however, had failed to respond to a hypocalcaemic stimulus. In others, there were high concentrations of parathyroid hormone despite normal serum calcium concentrations. Thus the relationship between parathyroid hormone and metabolites of vitamin D may not be mediated through changes in serum calcium alone, and it is postulated that metabolites of vitamin D may directly affect the secretion of parathyroid hormone.

Preproparathyroid hormone (preproPTH) mRNA and PTH secretion were measured in human parathyroid adenomata (n = 8) cultured in 1.0 or 3.0 mmol calcium/l and compared with changes in bovine parathyroid glands (n = 3) as a control. Incubation of bovine glands in 3.0 mmol calcium/l for 24 h resulted in a fall in mRNA levels to 47.3 +/- 21.7% (mean +/- S.D.) compared with cells incubated in 1.0 mmol calcium/l, with a concomitant decrease in secretion to 62.6 +/- 10.8%. These values fell further to 30.1 +/- 15.5% and 42.1 +/- 18.7% respectively after 48-h incubation. One human adenoma responded to high levels of calcium in a similar manner with mRNA levels falling to 44.6 +/- 11.9% and secretion to 30.3 +/- 17.3% within 24 h. However, in the majority of adenomata (seven out of eight), after 24-h incubation in 3.0 mmol calcium/l, mRNA levels fell to 54.1 +/- 14.6% but there was no change in secretion. In two of these adenomata which were cultured for 48 h, there was no suppression of secretion despite mRNA levels having fallen to approximately 60% of control. Incorporation of [35S]methionine into PTH secreted from human adenomatous cells was quantified by densitometry. There was no difference in the amount of radiolabelled PTH secreted from cells incubated in high levels of calcium compared with those in normal levels of calcium. In similar experiments, the effects of high calcium on the synthesis of PTH in bovine cells was assessed by determination of radiolabelled intracellular PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Two regular hemodialysis patients were assessed before, during and after therapy for 8 1/2 months with 1alpha-hydroxycholecalciferol (1alphaOHD3). The first patient (F.S.), treated with 2 mug daily, improved considerably with complete resolution of histological osteomalacia (O.M.), reduction in osteitis fibrosa (O.F.) and healing of Looser zones. The second patient (T.Y.), who was treated at the same time with a combination of phenobarbitone and phenytoin, showed no improvement while taking 3 mug of 1alphaOHD3 daily. It is suggested that hepatic microsomal enzyme inducing drugs antagonize the action of 1alphaOHD3 by interfering with its subsequent hepatic 25-hydroxylation.

The changes of parathyroid hormone (PTH) and of vitamin D metabolites after intravenous administration of the bisphosphonate APD were studied in ten patients with Paget's disease of bone and in ten patients with tumour-induced hypercalcaemia. After APD all patients with Paget's disease became hypocalcaemic and showed an increase in both N-PTH and C-PTH values. Patients with malignancies had a nearly six-fold greater decrease in serum calcium but rises in N-PTH and C-PTH were observed only in those who developed hypocalcaemia. Overall, a clear rise in PTH was found when serum calcium fell below 2.20 mmol/l. Basal 25-hydroxy- and 24,25-dihydroxyvitamin D concentrations were similar in the two groups and showed no change after APD treatment. Circulating 1,25-dihydroxyvitamin D, however, increased in all patients with Paget's disease and in six of the hypercalcaemic patients. It is concluded that the main regulator of PTH secretion is the concentration of calcium per se rather than the magnitude or the rate of its change. The production of 24,25-dihydroxyvitamin D is not affected by wide variations in serum calcium while that of 1,25-dihydroxyvitamin D is sensitive to these changes.

1,24,25-trihydroxyvitamin D3 has been detected in human serum using a sensitive radioimmunoassay. Tritiated 1,24,25-trihydroxyvitamin D3 was synthesized biologically and used as tracer to monitor the recovery of endogenous metabolite during isolation from serum. Circulating 1,24,25(OH)3D3 in normal subjects ranged from 9.3 to 18.5 pmol/l but was not detectable (less than 2.3 pmol/l) in serum from nephrectomized subjects. The trihydroxymetabolite was elevated in three out of four vitamin D deficient subjects who were being treated with vitamin D3.