Pseudovitamin D-deficiency Rickets Research Articles

Genetics of Vitamin D 1α-Hydroxylase Deficiency in 17 Families

Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency."

Renal and hepatic 1α-hydroxylation of 25-hydroxyvitamin D 3 in piglets suffering from pseudo vitamin D-deficiency rickets, type I

The piglets examined suffer from rickets and have symptoms similar to those of classic pseudo vitamin D-deficiency rickets, type I (PVDRI), including plasma concentrations of 1α,25-dihydroxyvitamin D 3 considerably lower than in healthy control piglets. It has been suggested that the rachitic piglets have a defective renal 1α,25-dihydroxyvitamin D 3 production. The present study shows that partially purified mitochondrial and microsomal cytochrome P450 from kidney and liver of both rachitic and control animals is able to catalyze 1α-hydroxylation of 25-hydroxyvitamin D 3. The renal mitochondrial 1α-hydroxylase activity was higher in the rachitic piglets whereas the renal microsomal 1α-hydroxylase activity was decreased. The immunodetectable levels in kidney of a mitochondrial 1α-hydroxylase (CYP27) and a microsomal 1α-hydroxylase (vitamin D 3 25-hydroxylase) were correlated with the 1α-hydroxylase activities. The results suggest that the renal microsomal 1α-hydroxylase is affected by the rachitic condition. It is concluded that the primary genetic defect of systemic 1α,25-dihydroxyvitamin D 3 deficiency in the rachitic PVDRI piglets does not reside in a defective function or absence of renal mitochondrial 25-hydroxyvitamin D 3 1α-hydroxylase. From this, it may also be concluded that PVDRI in man and pig appear to be two different forms of the disease.

Inactivating mutations in 25-hydroxyvitamin D3 1α-hydroxylase gene in pseudovitamin D-deficiency rickets patients

Inactivating mutations in 25-hydroxyvitamin D3 1α-hydroxylase gene in pseudovitamin D-deficiency rickets patients

Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets.

Pseudovitamin D-deficiency rickets is characterized by the early onset of rickets with hypocalcemia and is thought to be caused by a deficit in renal 25-hydroxyvitamin D3 1alpha-hydroxylase, the key enzyme for the synthesis of 1alpha,25-dihydroxyvitamin D3. We cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase complementary DNA (cDNA) using a mouse 1alpha-hydroxylase cDNA fragment as a probe. Its genomic structure was determined, and its chromosomal location was mapped by fluorescence in situ hybridization. We then identified mutations in the 1alpha-hydroxylase gene in four unrelated patients with pseudovitamin D-deficiency rickets by DNA-sequence analysis. Both the normal and the mutant 1alpha-hydroxylase proteins were expressed in COS-1 cells and were assayed for 1alpha-hydroxylase activity. The gene for 25-hydroxyvitamin D3 1alpha-hydroxylase was mapped to chromosome 12q13.3, which had previously been reported to be the locus for pseudovitamin D-deficiency rickets by linkage analysis. Four different homozygous missense mutations were detected in this gene in the four patients with pseudovitamin D-deficiency rickets. The unaffected parents and one sibling tested were heterozygous for the mutations. Functional analysis of the mutant 1alpha-hydroxylase protein revealed that all four mutations abolished 1alpha-hydroxylase activity. Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene are a cause of pseudovitamin D-deficiency rickets.

Stronger bonds between 25-hydroxyvitamin D 1alpha-hydroxylase and pseudovitamin D-deficiency rickets.

Stronger bonds between 25-hydroxyvitamin D 1alpha-hydroxylase and pseudovitamin D-deficiency rickets.

The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25-hydroxyvitamin D-1alpha-hydroxylase, a renal cytochrome P450 enzyme (P450[1alpha]) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13-q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P450(1alpha) gene (hereafter referred to as 1alpha-OHase). We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase (CYP24; 24-OHase) sequence to identify and clone the 1alpha-OHase cDNA. The full-length 1alpha-OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time-of-flight mass spectrometry. The expression of the gene was analyzed in vitamin D-replete animals; treatment with 1alpha,25(OH)2D3 reduced 1alpha-OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2-fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1alpha-OHase gene mapped to 12q13.1-q13.3, providing strong evidence that a mutation in the 1alpha-OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1alpha-OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.

Evidence for vitamin D-independent active calcium absorption in newborn piglets.

The role of 1,25-dihydroxycholecalciferol (calcitriol) for intestinal calcium (Ca2+) absorption was studied in newborn (< 1 week old) and weaned piglets (> 6 weeks old). In both groups, normal piglets and piglets suffering from inherited pseudo vitamin D-deficiency rickets, type I (PVDRI) were used. In this inherited disorder, renal production of calcitriol is absent. Plasma samples were assayed for calcitriol and total Ca, and dissociation constants (Kd) and maximum binding capacities (Bmax) of intestinal calcitriol receptors were determined under equilibrium conditions at 4 degrees C. Unidirectional Ca(2+)-flux rates were measured across stripped duodenal mucosae in Ussing chambers in the absence of electrochemical gradients. The plasma calcitriol concentrations of neonatal (26.5 +/- 7.1 pg/ml, n = 11; mean +/- SEM) and weaned PVDRI piglets (18.8 +/- 5.7 pg/ml, n = 8) were unphysiologically low and differed significantly from control animals (83.6 +/- 14.8 pg/ml, n = 8, and 86.9 +/- 9.6 pg/ml, n = 11, respectively). However, newborn PVDRI piglets had normal plasma Ca levels at least during the first days of life. They became hypocalcemic and developed clinical symptoms of rickets during the following weeks. In newborn PVDRI and control piglets, Bmax was significantly lower (84 +/- 28 fmol/mg protein and 127 +/- 55 fmol/mg protein, n = 9, respectively) than in weaned piglets (741 +/- 82 fmol/mg protein, n = 9, and 778 +/- 121 fmol/mg protein, n = 8, respectively). Significant net Ca(2+)-fluxes were found in both newborn PVDRI and control piglets (88.8 +/- 25.1 nmol.cm-2 x h-1, n = 6, and 86.5 +/- 10.5 nmol.cm-2 x h-1, n = 9, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Linkage mapping by simultaneous screening of multiple polymorphic loci using Alu oligonucleotide-directed PCR.

We present the use of our recently described multiple-loci polymorphic DNA markers ("alumorphs") for linkage mapping of the human genome. By using the polymerase chain reaction (PCR) with an Alu-specific primer we could reveal, in a single experiment, up to 20 genomic polymorphisms seen as the presence or absence of amplified DNA fragments originating from genomic segments flanked by Alu repeats. Using this approach we examined genomic DNA samples from two families with a history of pseudovitamin D-deficiency rickets (PDDR), an autosomal recessive disorder. An indication of linkage with the PDDR phenotype was found for one of the polymorphic bands, denoted 30A. A significant linkage [logarithm-of-odds (lod) score greater than 3.0] was obtained between this polymorphism and a number of chromosome 12q markers tightly linked to PDDR. The 30A band specifically hybridized to DNA digests from hybrid cell lines carrying a human chromosome 12, thus independently assigning the 30A marker to this chromosome. Since Alu elements are ubiquitous in human DNA, the use of alternative Alu-specific primers, which reveal different sets of Alu-flanked loci, should provide an efficient and rapid approach to human genetic mapping.

The effects of 1,25-dihydroxyvitamin D-3 deficiency on Ca 2+-transport and Ca 2+-uptake into brush-border membrane vesicles from pig small intestine

BBMV were prepared from duodenal segments of untreated, 1,25-(OH) 2D 3- or vitamin D-3-treated rachitic piglets and from non-rachitic controls by the Mg 2+ precipitation method. The rachitic piglets were offspring from the ‘Hannover Pig Strain’ which suffer from pseudo vitamin D-deficiency rickets, type I (no renal 1-hydroxylase activity). Initial uptake of Ca 2+ (up to 35 s) at low [Ca 2+] (between 0.02–0.25 mmol/l) into isolated BBMV consisted of a saturable and non-saturable component. The apparent V max of the saturable component was significantly lower in rachitic piglets than in control piglets. In the presence of an inside negative potassium diffusion potential, the difference in uptake extended over at least 15 min. The apparent K m of Ca 2+-uptake was not influenced by the rachitic condition. Treatment of rachitic piglets with sequential doses of 1,25-(OH) 2D 3 for three days 91 μg/day) or with a single dose (2.5 mg) of vitamin D-3 elevated the saturable Ca 2+-uptake component to values similar to those of control piglets. Addition of 1 mmol/l verapamil to the vesicular suspension inhibited Ca 2+-uptake in BBMV of control piglets by 40–60% but was without effect on preparations from rachitic piglets. It was concluded from the study that 1,25-(OH) 2D 3 dependent Ca 2+-uptake unto BBMV constituted a saturable process which can be inhibited by a known Ca 2+-channel blocking agent. It appears that 1,25-(OH) 2D 3 increases the number of verapamil sensitive Ca 2+-transport components in brush-border membranes. The vitamin D-dependent changes in vesicular Ca 2+-uptake were paralleled by the expression of an active transmural Ca 2+-transport across the mucosa.

Binding properties of plasma vitamin D-binding protein and intestinal 1,25-dihydroxyvitamin D 3 receptor in piglets with pseudo-vitamin D-deficiency rickets, type I: Treatment effects with pharmacological doses of vitamin D 3

The effective treatment of the rachitic symptoms of pseudo-vitamin D-deficiency rickets, type I (PVDRI) by massive doses of vitamin D 3 was examined. For this purpose, the affinities and the maximum binding capacities ( B max) of the plasma vitamin D-binding protein (DBP) and of the intestinal 1,25-dihydroxyvitamin D 3 (1,25- (OH) 2D 3) receptor for vitamin D 3, 25-hydroxyvitamin D 3 (25-OHD 3) and 1,25-(OH) 2D 3, were investigated in normal piglets and in rachitic piglets that suffered from PVDRI. The piglets were 5 to 10 weeks old and of both sexes. The B max of plasma DBP for 25-OHD 3 was 6.77 ± 0.45 μ m for PVDRI piglets and 7.30 ± 0.41 μ m for control piglets and showed no differences between the two groups. Equilibrium association constants ( K a ) of DBP for 25-OHD 3 were 4.3 × 10 8 m −1 for PVDRI piglets and 4.0 × 10 8 m −1 for controls and showed also no differences between the two groups. Similarly the K a of DBP for 1,25-(OH) 2D 3 was also the same for rachitic and control piglets (1.45 × 10 7 and 1.54 × 10 7 m −1, respectively). Due to the lower circulating concentration of 1,25-(OH) 2D 3 in the plasma of rachitic piglets compared to that of controls its free metabolite index was significantly lower in rachitic (0.42 ± 0.05 × 10 −5) than in control piglets (3.63 ± 0.30 × 10 −5). The K d and B max of the intestinal nuclear receptor for 1,25-(OH) 2D 3 of rachitic and control piglets were 0.31 ± 0.05 and 0.33 ± 0.05 n m and 674 ± 103 and 719 ± 123 fmol/mg protein, respectively, and were also not different between the two groups of piglets. It was concluded from these observations that the rachitic symptoms of PVDRI piglets resulted solely from the lower free 1,25-(OH) 2D 3 concentration in plasma compared to that of normal piglets. The relative affinities of the intestinal 1,25-(OH) 2D 3 receptor for vitamin D 3 and 25-OHD 3 were also measured. It was found that 50% displacement of 1,25-(OH) 2D 3 from the intestinal receptor of PVDRI and control piglets required a 220,000- and 245,000-fold excess of the free concentration of vitamin D 3, respectively, and a 20- to 42- and 23- to 71-fold excess of the free concentration of 25-OHD 3, respectively. Treatment of PVDRI piglets with an effective dose of 7.5 × 10 4 IU of vitamin D 3 led to a transient 1073-fold excess of the free concentration of vitamin D 3 and a 59-fold excess of the free concentration of 25-OHD 3 over free 1,25-(OH) 2D 3 in plasma. Since a 59-fold excess of free 25-OHD 3 in plasma of treated piglets could cause at least 50% displacement of 1,25-(OH) 2D 3 from the intestinal 1,25-(OH) 2D 3 receptor it is suggested that after administration of pharmacological amounts of vitamin D 3 to PVDRI piglets both 25-OHD 3 and 1,25-(OH) 2D 3 acted upon the intestinal 1,25-(OH) 2D 3 receptor. The two metabolites probably exerted a coordinate effect in healing the rachitic symptoms.

Role of 1,25-(OH)2D3 during pregnancy; studies with pigs suffering from pseudo-vitamin D-deficiency rickets, type I.

Biochemical parameters of Ca homeostasis in fetal and maternal plasma and placental transfer of Ca, Pi and vitamin D metabolites were measured during pregnancy and at parturition. Control pigs and pigs with inherited pseudo-vitamin D-deficiency rickets, type I (PVDR), which are devoid of renal calcitriol production, were used. Although sows with PVDR normally require treatment with massive doses of vitamin D at intervals of 4 weeks to stabilize Ca and Pi concentrations in plasma, these plasma parameters tended to normalize during the first 3 months of pregnancy when the vitamin D treatment was discontinued about 4 weeks before conception. In homozygote sows plasma calcitriol concentrations remained unphysiologically low and the 25OHD3 concentrations increased steadily during pregnancy reaching higher levels than those found in control sows. Despite hypocalcaemia and hypophosphataemia of the homozygote sows at term, fetal Ca and Pi concentrations were normal. This demonstrated a higher feto-maternal concentration gradient in PVDR pigs than in control pigs. It is concluded that maintenance of Ca and Pi homeostasis of the feto-maternal system, including active transplacental transport in pigs, is at least partly independant from calcitriol.

Placental transport of calcium and phosphorus in pigs.

The role of vitamin D in regulating placental transport of calcium and phosphate in pigs was studied using the "Hannover Pig Strain" which suffers from pseudo-vitamin D-deficiency rickets, type I. Sows and fetuses of normal phenotype (heterozygotes) and of homozygote phenotype which suffered from clinical symptoms of rickets were used. The homozygote animals are devoid of renal 25-cholecalciferol-1-hydroxylase. In the rachitic sows which normally depend on treatment with pharmacological doses of vitamin D3 at intervals of four to six weeks, vitamin D-treatment was discontinued two months before conception. Ca, P and 1.25-(OH)2D3 concentrations in plasma of sows and fetuses (Aa. umb., V. umb.) were measured in samples obtained at term during cesarian section. Mean concentrations of Ca and P in heterozygote sows at parturition were 2.34 mmol/l and 2.33 mmol/l, respectively, and were significantly higher than in homozygote sows, 1.58 mmol/l and 1.26 mmol/l, respectively. Ca concentrations in plasma of the umbilical vein of fetuses from both homozygote and heterozygote sows were normal, however, (3.23 mmol/l and 2.96 mmol/l, respectively) and statistically not different from each other. No significantly different P concentrations in arterial plasma of fetuses from heterozygote or homozygote sows were seen, either. The concentrations of Ca and P in the arterial umbilical plasma were significantly higher (p less than 0.001) than in venous plasma of both homozygote and heterozygote fetuses, indicating net placental transfer of these elements in both genotypes. The concentration of 1.25-(OH)2D3 in hypocalcemic sows at term (25.5 +/- 8.25 pg/ml) was significantly lower than that in the normocalcemic (heterozygote) sows (84.1 +/- 25.6 pg/ml). The 1.25-(OH)2D3 concentration in arterial plasma of fetuses from homozygote sows was only 46% that of fetuses from normocalcemic sows which showed transfer of calcitriol from mother to fetus. It is concluded from these studies that in pigs, renal production and physiological concentrations of 1.25-(OH)2D3 in either mother or fetus is not essential for the maintainance of the Ca and P homeostasis in the fetal-maternal system.

Vitamin D3 metabolism in a pig strain with pseudo vitamin D-deficiency rickets, type I.

Vitamin D metabolism was studied in the 'Hannover Pig', a strain which suffers from pseudo vitamin D-deficiency rickets, type I. Animals of this strain are known to be devoid of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase and -24-hydroxylase activities. Pigs with florid rickets and hypocalcaemia were treated with single im injections of 0.25 to 1.25 mg of vitamin D3, doses that have been shown in previous studies to be effective in producing transient healing of rachitic symptoms. The levels of vitamin D3 and its most relevant physiological metabolites in plasma were estimated at intervals before and after this vitamin D3 treatment. Vitamin D3 rose from 14.8 +/- 8.1 to 364 +/- 190 nmol/l (mean +/- SD) 2 to 3 days post injectionem, 25-hydroxyvitamin D3 from 131.0 +/- 46.2 to 1068 +/- 160 nmol/l within 7 days post injectionem. The 1 alpha, 25-dihydroxyvitamin D3 concentration in plasma was elevated from 73.9 +/- 25.0 to 281 +/- 168 pmol/l 2 to 3 days post injectionem and declined continually from that time. 24R,25-dihydroxyvitamin D3 and 25S,26-dihydroxyvitamin D3 levels after treatment showed different responses in different animals being either elevated or unchanged. Clinical healing of the pigs with these doses of vitamin D3 was attributed to the transient rise of 1 alpha,25-dihydroxyvitamin D3 in plasma. It was assumed that 1 alpha,25-dihydroxyvitamin D3 synthesis takes place under these circumstances in extrarenal tissues.

Pseudo-vitamin D deficiency rickets (PDR) and relative hypoparathyroidism: a report of a family.

Three siblings with hypocalcemia, variable serum phosphate and elevated serum alkaline phosphatase levels, who are considered to have pseudo-vitamin D deficiency rickets (PDR), are described. In one patient metabolic balance studies were performed during acid loading with ammonium chloride (18 g in 4 days) and dihydrotachysterol (5 mg per day for 4 days) administration. During the acid loading period, fecal calcium excretion increased and was carried over into the dihydrotachysterol administration period resulting in a negative calcium balance, thus indicating defective calcium absorption. Acid loading with ammonium chloride in this patient and in one of his siblings demonstrated an impairment in the acidification of the urine. In one patient the increase in urinary titrable acids (T.A.) was mainly by increased excretion of organic acid. Serum parathyroid levels in two of the subjects were within normal limits in the presence of hypocalcemia, a finding which may indicate relative hypoparathyroidism. The relationship between the role of kidney, parathyroid glands and vitamin D metabolism is discussed and an attempt is made to implicate these factors in the causation of this condition.