Nutritional rickets among children admitted with severe pneumonia at Mulago hospital, Uganda: a cross-sectional study.

BACKGROUND: There's abundant sunshine in the tropics but severe rickets is still observed. Nutritional rickets is associated with an increased risk of acute lower respiratory infections. Pneumonia is the leading cause of death in the under 5 -year old children with the highest burden in developing countries. Both Pneumonia and rickets are common in the developing countries and may affect clinical presentation and outcome. This study aimed to determine the prevalence and associated factors of nutritional rickets in children admitted with severe pneumonia. METHODS: This was a cross-sectional study of children aged 2-59 months presenting with severe pneumonia at an emergency unit. We enrolled 221 children between February and June 2012 after consent. A pre-coded questionnaire was used to collect data on socio-demographic, nutritional and past medical history. Physical exam was done for signs of rickets and anthropometric measurements. Serum calcium, phosphorus, and alkaline phosphatase (ALP) were assessed. Children with any physical signs of rickets or biochemical rickets (ALP > 400 IU); had a wrist x-ray done. Nutritional rickets was defined as the presence of radiological changes of cupping or fraying and/ or metaphyseal thickening. Severe pneumonia was defined using the WHO criteria. Statistical analysis was performed using the Stata 10 statistical package. P- value < 0.05 was significant. RESULTS: The prevalence of nutritional rickets among children with severe pneumonia is 9.5%. However, 14.5% had raised ALP (biochemical rickets). The factors independently associated with rickets was an elevated alkaline phosphatase; p-value < 0.001, or 32.95 95% CI (10.54-102.93). Other factors like breastfeeding, big family size, birth order were not significantly associated with rickets. Low serum calcium was detected in 22 (9.9%) of the 221 participants. Overall few children with rickets had typical clinical features of rickets on physical examination. CONCLUSION: Rickets is a common problem in our setting despite ample sunshine. Clinicians should actively assess children for rickets in this setting and screen for rickets in those children at high risk even without clinical features.

CYP3A4 mutation causes vitamin D-dependent rickets type 3.

Genetic forms of vitamin D-dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR; however, using whole exome sequencing analysis, we identified a recurrent de novo missense mutation, c.902T>C (p.I301T), in CYP3A4 in both subjects that alters the conformation of substrate recognition site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than WT CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide insight into vitamin D metabolism and demonstrate that accelerated inactivation of vitamin D metabolites represents a mechanism for vitamin D deficiency.

Vitamin D-Dependent Rickets Type 1B (25-Hydroxylase Deficiency): A Rare Condition or a Misdiagnosed Condition?

Vitamin D requires a two-step activation by hydroxylation: The first step is catalyzed by hepatic 25-hydroxylase (CYP2R1, 11p15.2) and the second one is catalyzed by renal 1α-hydroxylase (CYP27B1, 12q13.1), which produces the active hormonal form of 1,25-(OH)2 D. Mutations of CYP2R1 have been associated with vitamin D-dependent rickets type 1B (VDDR1B), a very rare condition that has only been reported to affect 4 families to date. We describe 7 patients from 2 unrelated families who presented with homozygous loss-of-function mutations of CYP2R1. Heterozygous mutations were present in their normal parents. We identified a new c.124_138delinsCGG (p.Gly42_Leu46delinsArg) variation and the previously published c.296T>C (p.Leu99Pro) mutation. Functional in vitro studies confirmed loss-of-function enzymatic activity in both cases. We discuss the difficulties in establishing the correct diagnosis and the specific biochemical pattern, namely, very low 25-OH-D suggestive of classical vitamin D deficiency, in the face of normal/high concentrations of 1,25-(OH)2 D. Siblings exhibited the three stages of rickets based on biochemical and radiographic findings. Interestingly, adult patients were able to maintain normal mineral metabolism without vitamin D supplementation. One index case presented with a partial improvement with 1alfa-hydroxyvitamin D3 or alfacalcidol (1α-OH-D3 ) treatment, and we observed a dramatic increase in the 1,25-(OH)2 D serum concentration, which indicated the role of accessory 25-hydroxylase enzymes. Lastly, in patients who received calcifediol (25-OH-D3 ), we documented normal 24-hydroxylase activity (CYP24A1). For the first time, and according to the concept of personalized medicine, we demonstrate dramatic improvements in patients who were given 25-OH-D therapy (clinical symptoms, biochemical data, and bone densitometry). In conclusion, the current study further expands the CYP2R1 mutation spectrum. We note that VDDR1B could be easily mistaken for classical vitamin D deficiency. © 2017 American Society for Bone and Mineral Research.

Increased tartrate-resistant Acid phosphatase expression in osteoblasts and osteocytes in experimental osteoporosis in rats.

Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were analyzed for TRAP gene expression by in situ hybridization, TRAP protein by immunogold labeling, and TRAP enzyme activity using the fluorescent substrate ELF97. Osteoblasts and osteocytes close to intracortical remodeling sites and bone surfaces demonstrated TRAP, most prominently in cancellous bone and osteocytes. Intracellular TRAP was located to electron-dense vesicles with similar morphology in both cell types. Ovx-D increased osteoclast activity (p < 0.001) and ELF97⁺ osteocytes (p < 0.05) in cancellous bone, but no corresponding increase was observed in the osteocyte lacunar area. The level of TRAP⁺ vesicles in cortical osteoblasts (p < 0.01) in Ovx-D rats was also increased. Enhanced osteoclast activity was noted in healing rickets after 72 h (p < 0.05), but no differences in TRAP expression were detected in osteoblasts or osteocytes. Thus, increased osteoclast activity does not affect TRAP expression in osteoblasts and osteocytes, favoring the notion that increased TRAP in these cells is rather due to increased synthesis. Although the role of TRAP in osteoblasts and osteocytes remains elusive, we speculate that the function is related to the capability of the enzyme to regulate the phosphorylation of proteins known to be expressed by these cells.

High incidence of rickets in extremely low birth weight infants with severe parenteral nutrition-associated cholestasis and bronchopulmonary dysplasia.

Risk factors for rickets of prematurity have not been re-examined since introduction of high mineral formula, particularly in ELBW infants. We analyzed the incidence and the risk factors of rickets in extremely low birth weight (ELBW) infants. As a retrospective case-control study from 2004 to 2008, risk factors were analyzed in 24 patients with rickets versus 31 patients without. The frequency of rickets in ELBW infants was 24/55 (44%). Infants with rickets were diagnosed at 48.2 ± 16.1 days of age, and improved by 85.3 ± 25.3 days. By radiologic evaluation, 29% were grade 1 rickets, 58% grade 2 and 13% grade 3. In univariate analysis, infants with rickets had significantly higher incidence of patent ductus arteriosus, parenteral nutrition associated cholestasis (PNAC), severe PNAC and moderate/severe bronchopulmonary dysplasia (BPD). In multiple regression analysis, after adjustment for gestation and birth weight, rickets significantly correlated with severe PNAC and with moderate/severe BPD. Serum peak alkaline phosphatase levels were significantly elevated in rickets (P < 0.001). In ELBW infants, the incidence of rickets of prematurity remains high and the incidence of severe PNAC and moderate/severe BPD was significantly increased 18 and 3 times, respectively.

Novel mutations of CYP27B1 gene lead to reduced activity of 1α-hydroxylase in Chinese patients.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder resulting from a defect in renal 25-hydroxyvitamin D 1α-hydroxylase, the key enzyme in the pathway of vitamin D metabolism. We identified ten different mutations in the 1α-hydroxylase gene (CYP27B1) in eight Chinese families with PDDR by DNA-sequence analysis. Six of them are novel missense mutations: G57V, G73W, L333F, R432C, R459C, and R492W; three are novel deletion mutations: c48-60del, c1310delG, and c1446delA; and an insertion mutation c1325-1332insCCCACCC reported previously. Functional assay revealed that the missense mutants identified in this study retain 5.5-12.1% 1α-hydroxylase activity of the wild type. The study describes nine novel mutations in addition to 37 known mutations of CYP27B1 gene and shows the correlation between these mutations and the clinical findings of 1α-hydroxylase deficiency.

Clinical and genetic analysis of patients with vitamin D-dependent rickets type 1A.

CONTEXT: Vitamin D-dependent rickets type 1A (VDDR-IA, OMIM 264700) is a rare autosomal recessive disorder and is caused by mutations in the CYP27B1 gene. OBJECTIVES: We aim to investigate CYP27B1 mutation in seven patients from four separate families and characterize the genotype-phenotype correlation. METHODS: The entire coding region of the CYP27B1 gene was sequenced, and genotype-phenotype correlation among patients was assessed. RESULTS: Sequencing analysis identified biallelic CYP27B1 mutations in all patients and monoallelic mutations in their parents. One patient from the first family was compound heterozygous for c.1166G>A (p.Arg389His) and a novel nonsense mutation c.1079 C>A (p.Ser360*). Two patients from the second family were homozygous for a novel splice donor site mutation in intron 1 (c.195 + 2 T>G), causing partial retention of the intron and a shift in the reading frame. Both novel mutations lead to the complete loss of vitamin D1α-hydroxylase activity. Four patients from families 3 and 4 were homozygous for a previously reported duplication mutation in exon 8 (1319-1325dupCCCACCC, Phe443Profs*24). Interestingly, one patient who was presented with severe hypocalcaemia and seizures at 4 months of age as a result of Phe443Profs*24 has improved spontaneously since 11 years of age and does not need regular treatment. Her laboratory tests showed normal serum calcium and 1,25(OH)(2) D after refusing to take medication for 12 months. CONCLUSIONS: There is a good genotype-phenotype correlation in VDDR-IA. However, some patients may recover from the loss of CYP27B1 function, probably due to 1α-hydroxylase activity exerted by a non-CYP27B1 enzyme.

High Incidence of Rickets in Extremely Low Birth Weight Infants with Severe Parenteral Nutrition-Associated Cholestasis and Bronchopulmonary Dysplasia

Risk factors for rickets of prematurity have not been re-examined since introduction of high mineral formula, particularly in ELBW infants. We analyzed the incidence and the risk factors of rickets in extremely low birth weight (ELBW) infants. As a retrospective case-control study from 2004 to 2008, risk factors were analyzed in 24 patients with rickets versus 31 patients without. The frequency of rickets in ELBW infants was 24/55 (44%). Infants with rickets were diagnosed at 48.2 +/- 16.1 days of age, and improved by 85.3 +/- 25.3 days. By radiologic evaluation, 29% were grade 1 rickets, 58% grade 2 and 13% grade 3. In univariate analysis, infants with rickets had significantly higher incidence of patent ductus arteriosus, parenteral nutrition associated cholestasis (PNAC), severe PNAC and moderate/severe bronchopulmonary dysplasia (BPD). In multiple regression analysis, after adjustment for gestation and birth weight, rickets significantly correlated with severe PNAC and with moderate/severe BPD. Serum peak alkaline phosphatase levels were significantly elevated in rickets (P < 0.001). In ELBW infants, the incidence of rickets of prematurity remains high and the incidence of severe PNAC and moderate/severe BPD was significantly increased 18 and 3 times, respectively.

High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support.

BACKGROUND: Osteopenia and rickets are common among extremely low birth weight infants (ELBW, <1000 g birth weight) despite current practices of vitamin and mineral supplementation. Few data are available evaluating the usual course of markers of mineral status in this population. Our objectives in this study were to determine the relationship between birth weight (BW) and peak serum alkaline phosphatase activity (P-APA) in ELBW infants and evaluate our experience with the diagnosis of rickets in these infants. METHODS: We evaluated all ELBW infants admitted to Texas Children's Hospital NICU in 2006 and 2007. Of 211 admissions, we excluded 98 patients who were admitted at >30 days of age or did not survive/stay for >6 weeks. Bone radiographs obtained in 32 infants were reviewed by a radiologist masked to laboratory values. RESULTS: In this cohort of 113 infants, P-APA was found to have a significant inverse relationship with BW, gestational age and serum phosphorus. In paired comparisons, P-APA of infants <600 g (957 +/- 346 IU/L, n = 20) and infants 600-800 g (808 +/- 323 IU/L, n = 43) were both significantly higher than P-APA of infants 800-1000 g (615 +/- 252 IU/L, n = 50), p < 0.01. Thirty-two patients had radiographic evaluation for evidence of rickets, based on P-APA greater than 800 IU/L, parenteral nutrition greater than 3 to 4 weeks, or clinical suspicion. Of these, 18 showed radiologic rickets and 14 showed osteopenia without rickets. Infants with BW <600 g were more likely to have radiologic rickets (10/20 infants) compared to those with BW 600-800 g (6/43 infants) and BW 800-1000 g (2/50 infants), p < 0.01 for each. P-APA was not significantly higher in infants with radiologic rickets (1078 +/- 356 IU/L) compared to those without radiologic evidence of rickets (943 +/- 346, p = 0.18). CONCLUSION: Elevation of P-APA >600 IU/L was very common in ELBW infants. BW was significantly inversely related to both P-APA and radiologic rickets. No single value of P-APA was related to radiological findings of rickets. Given the very high risk of osteopenia and rickets among ELBW infants, we recommend consideration of early screening and early mineral supplementation, especially among infants <600 g BW.

Expression of matrix metalloproteinases during vascularization and ossification of normal and impaired avian growth plate.

Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. In this paper, we induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. These results suggest that MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate.

[Vitamin D1alpha-hydroxylase deficiency as the cause of severe rickets in a 1-year-old-old boy]. / D-vitamin-1 alpha-hydroxylase-mangel som årsag til svaer rakitis hos en etårig dreng.

We describe a 15-month-old boy referred to the hospital because of delayed development of motor skills and growth retardation. Blood samples and X-rays of the wrists and knees revealed rickets. He was treated with oral calcium and vitamin D with modest clinical and biochemical effect. 1,25-dihydroxyvitamin D was undetectable in laboratory tests. Vitamin D1alpha-hydroxylase deficiency was suspected and confirmed by DNA analysis, which revealed a 7 bp duplication in exon 8 of the CYP27B1 gene. The treatment was changed to an activated formula of vitamin D, alphacalcidol, whereupon the clinical and biochemical symptoms rapidly improved.

A novel splicing defect (IVS6+1G>T) in a patient with pseudovitamin D deficiency rickets.

A 15-month-old boy with severe rickets, that by clinical analysis was diagnosed as affected by hereditary pseudovitamin D deficiency rickets (PDDR), was evaluated for mutations in the 25OHD3 1alpha-hydroxylase gene. Molecular analysis showed a double heterozygous state for a novel splicing mutation in the invariant dinucleotide of the donor site of IVS6 and a 7 nucleotide insertion in the exon 8, which is common in different ethnical backgrounds.

Structure-function analysis of CYP27B1 and CYP27A1. Studies on mutants from patients with vitamin D-dependent rickets type I (VDDR-I) and cerebrotendinous xanthomatosis (CTX).

We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111-4117]. None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3. Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.

Genetic mutation in the human 25-hydroxyvitamin D3 1alpha-hydroxylase gene causes vitamin D-dependent rickets type I.

Vitamin D is deeply involved in a wide variety of biological events such as calcium homeostasis, bone formation and cellular differentiation. An active form of vitamin D, 1alpha,25(OH)2D3, serves as a vitamin D receptor (VDR)-specific ligand to activate the expression of a particular set of target genes. 1Alpha,25(OH)2D3, is biosynthesized from cholesterol, and at the final biosynthesis step, 25-hydroxyvitamin D3 1alpha-hydroxylase [1alpha(OH)ase] in kidney conducts 1alpha-hydroxylation of 25(OH)2D3. This enzymatic activity is under multihormonal regulation and critical for the biosynthesis. Molecular cloning of 1alpha(OH)ase from several species has revealed that this enzyme belongs to a member of the cytochrome P450 enzyme superfamily, with highest homologies to the P450 hydroxylases for vitamin D derivatives. The renal gene expression is strictly regulated at the transcriptional level through its gene promoter by PTH and calcitonine (positive) and 1alpha,25(OH)2D3 (negative). Most importantly in clinical aspects, genetic mutations in this gene to abolish the enzymatic activity are now shown to cause the one of three kinds of hereditary rickets, vitamin D-dependent rickets type I.

No enzyme activity of 25-hydroxyvitamin D3 1alpha-hydroxylase gene product in pseudovitamin D deficiency rickets, including that with mild clinical manifestation.

Pseudovitamin D deficiency rickets (PDDR) is an autosomal recessive disorder caused by defect in the activation of vitamin D. We recently isolated 25-hydroxyvitamin D3 1alpha-hydroxylase gene and identified four homozygous inactivating missense mutations in this gene by analysis of four typical cases of PDDR. This disease shows some phenotypic variation, and it has been suspected that patients with mild phenotypes have mutations that do not totally abolish the enzyme activity. To investigate the molecular defects associated with the phenotypic variation, we analyzed six additional unrelated PDDR patients: one with mild and five with typical clinical manifestation. By sequence analysis, all six patients were proven to have mutations in both alleles. The mutations varied, and we identified four novel missense mutations, a nonsense mutation, and a splicing mutation for the first time. The patient with mild clinical symptoms was compound heterozygous for T321R and a splicing mutation. The splice site mutation caused intron retention. Enzyme activity of the T321R mutant was analyzed by overexpressing the mutant 1alpha-hydroxylase in Escherichia coli cells to detect the subtle residual enzyme activity. No residual enzyme activity was detected in T321R mutant or in the other mutants. These results indicate that all of the patients, including those of mild phenotype, are caused by 1alpha-hydroxylase gene mutations that totally abolish the enzyme activity.

Novel mutations in the 1alpha-hydroxylase (P450c1) gene in three families with pseudovitamin D-deficiency rickets resulting in loss of functional enzyme activity in blood-derived macrophages.

Pseudovitamin D-defiency rickets (PDDR) is an autosomal recessive disorder characterized by hypocalcemia, rickets (which are resistant to treatment with vitamin D), and low or undetectable serum levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). The symptoms are corrected with 1,25(OH)2D treatment, and the disease is now believed to result from a defect in the cytochrome P450 component (P450c1; CYP27B1) of the renal 25-hydroxyvitamin D-1alpha-hydroxylase (1-OHase). We have studied genomic DNA from three families with PDDR and have identified the same homozygous mutation in the P450c1 gene in two of the index cases, causing a frameshift in exon 8, resulting in a premature stop codon in the heme-binding domain. The two cases in the third kindred were compound heterozygotes with missense mutations in exons 6 and 9. We have also identified a C/T polymorphism in intron 6 of the P450c1 genomic DNA. Interferon gamma-inducible 1-OHase activity in blood-derived macrophages was shown by 1,25(OH)2D synthesis in all control cells tested (37-184 fmol/h/106 cells) and those from the PDDR family parents (34-116 fmol/h/106 cells) but was totally absent from the patients' cells, indicating a defect in their macrophage 1-OHase, similar to the presumed renal defect. The assumption of similarity between the renal and macrophage P450c1 was supported by our ability to clone a 514 bp sequence, including the heme-binding region of the macrophage P450c1 cDNA from controls, which was identical to that published for both the renal and keratinocyte P450c1 cDNAs.

Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alpha-hydroxylase gene by parathyroid hormone, calcitonin, and 1alpha,25(OH)2D3 in intact animals.

Reflecting the prime role of 1alpha,25(OH)2D3 in calcium homeostasis, the activity of 25-hydroxyvitamin D3 1alpha-hydroxylase, a key enzyme for 1alpha,25(OH)2D3 biosynthesis, is tightly regulated by 1alpha,25(OH)2D3, PTH and calcitonin. Its significant activity is found in kidney, though the enzymatic activity is also reported in extra-renal tissues. In the present study, we found that the 1alpha-hydroxylase gene abundantly expresses in kidney, and at low levels in other tissues and in some cell lines. Positive and negative regulations of 1alpha-hydroxylase gene by PTH, calcitonin, or 1alpha,25(OH)2D3 were observed at transcriptional levels in kidneys of animals and in a mouse proximal tubule cell line. Moreover, the protein kinase A inhibitor abrogated the PTH-mediated positive regulation. In mice lacking the vitamin D receptor, the 1alpha-hydroxylase gene expression was overinduced, and the inducible effect of either PTH or calcitonin, but not the repression by 1alpha,25(OH)2D3, was evident. Thus, vitamin D receptor is essential for the negative regulation by 1alpha,25(OH)2D3. Moreover, we demonstrate that renal 1alpha-hydroxylase gene expression in chronic renal failure model rats was decreased and the positive effect by PTH and calcitonin was diminished. The present study demonstrates that PTH and calcitonin positively regulate renal 1alpha-hydroxylase gene expression via PKA-dependent and independent pathway, respectively, and that 1alpha,25(OH)2D3 negatively regulates it mediated by vitamin D receptor. Furthermore, in a moderate state of chronic renal failure, renal cells expressing the 1alpha-hydroxylase gene appear to have diminished potential in response to PTH and calcitonin.

Genetics of vitamin D 1alpha-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 1 alpha-hydroxylation of 25-hydroxyvitamin D3 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 1 alpha, 25-dihydroxyvitamin D3 considerably lower than in healthy control piglets. It has been suggested that the rachitic piglets have a defective renal 1 alpha,25-dihydroxyvitamin D3 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 1 alpha-hydroxylation of 25-hydroxyvitamin D3. The renal mitochondrial 1 alpha-hydroxylase activity was higher in the rachitic piglets whereas the renal microsomal 1 alpha-hydroxylase activity was decreased. The immunodetectable levels in kidney of a mitochondrial 1 alpha-hydroxylase (CYP27) and a microsomal 1 alpha-hydroxylase (vitamin D3 25-hydroxylase) were correlated with the 1 alpha-hydroxylase activities. The results suggest that the renal microsomal 1 alpha-hydroxylase is affected by the rachitic condition. It is concluded that the primary genetic defect of systemic 1 alpha,25-dihydroxyvitamin D3 deficiency in the rachitic PVDRI piglets does not reside in a defective function or absence of renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase. From this, it may also be concluded that PVDRI in man and pig appear to be two different forms of the disease.