Original Article
Inactivating Mutations in the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene in Patients with Pseudovitamin D–Deficiency Rickets
N Engl J Med 1998; 338:653-662March 5, 1998
- Abstract
Background
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 1α-hydroxylase, the key enzyme for the synthesis of 1α,25-dihydroxyvitamin D3.
Methods
We cloned human 25-hydroxyvitamin D3 1α-hydroxylase complementary DNA (cDNA) using a mouse 1α-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 1α-hydroxylase gene in four unrelated patients with pseudovitamin D–deficiency rickets by DNA-sequence analysis. Both the normal and the mutant 1α-hydroxylase proteins were expressed in COS-1 cells and were assayed for 1α-hydroxylase activity.
Results
The gene for 25-hydroxyvitamin D3 1α-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 1α-hydroxylase protein revealed that all four mutations abolished 1α-hydroxylase activity.
Conclusions
Inactivating mutations in the 25-hydroxyvitamin D3 1α-hydroxylase gene are a cause of pseudovitamin D–deficiency rickets.
Media in This Article
Figure 2
Northern Blot Analysis of 1α-Hydroxylase Gene Expression in Human Tissues.Figure 1
Chromosomal Localization of the 1α-Hydroxylase Gene According to Fluorescence in Situ Hybridization.Article Activity
- Article
Hereditary pseudovitamin D–deficiency rickets, also known as vitamin D–dependent rickets type I, is characterized clinically by hypotonia, weakness, growth failure, and hypocalcemic seizures in early infancy.1 The patients also have hypocalcemia, radiologic findings typical of rickets, elevated serum parathyroid hormone concentrations, and generalized aminoaciduria.2,3 It is inherited as an autosomal recessive trait.
Two other inherited types of rickets are known,4 and the mutated genes in patients with these diseases have been identified. The diseases are hypocalcemic vitamin D–resistant rickets, also known as type II vitamin D–dependent rickets, in which the gene for the vitamin D receptor is mutated,5-7 and X-linked hypophosphatemic vitamin D–resistant rickets, in which the PEX gene (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is mutated.8 In contrast, the molecular basis of pseudovitamin D–deficiency rickets has remained unclear, although the disease locus has been mapped to chromosome 12q14 by linkage analysis.9
Pseudovitamin D–deficiency rickets is distinguished from other types of hereditary rickets in that affected patients have low serum concentrations of 1α,25-dihydroxyvitamin D and normal or high concentrations of 25-hydroxyvitamin D.10,11 In these patients, physiologic doses of 1α,25-dihydroxyvitamin D3 but massive doses of vitamin D or 25-hydroxyvitamin D3 are required to cure the rickets.12 These findings suggest that the activity of renal 25-hydroxyvitamin D3 1α-hydroxylase,13,14 which converts 25-hydroxyvitamin D3 to 1α,25-dihydroxyvitamin D3, is defective in patients with pseudovitamin D–deficiency rickets. Whether this defect is due to abnormalities in the 25-hydroxyvitamin D3 1α-hydroxylase gene or to genetic abnormalities in other factors required for the full activity of this enzyme is unknown.13,15 Since direct enzymatic measurement of 1α-hydroxylase activity in these patients is difficult, the human 1α-hydroxylase gene was cloned in the present study to investigate genetic mutations in patients with pseudovitamin D–deficiency rickets.
Using a novel vitamin D receptor–mediated expression cloning method,16 we recently cloned 1α-hydroxylase complementary DNA (cDNA) from the kidneys of vitamin D receptor–deficient knockout mice in which the 1α-hydroxylase gene was overexpressed.17 In the present study, we cloned human 1α-hydroxylase cDNA, determined the structure of the gene and the chromosomal locus, and analyzed whether this gene is mutated in patients with pseudovitamin D–deficiency rickets.
Methods
Patients
We studied four unrelated Japanese patients with pseudovitamin D–deficiency rickets and four of their unaffected parents, one sibling, and one nephew. The diagnosis of pseudovitamin D–deficiency rickets in these patients was based on the early onset of hypocalcemia, radiologic findings characteristic of rickets, and the requirement of high doses of vitamin D and physiologic doses of 1α-hydroxyvitamin D3.2,3 The clinical data on these patients are summarized in Table 1Table 1
Clinical and Biochemical Features in Four Patients with Pseudovitamin D–Deficiency Rickets.. The diagnosis was confirmed by low serum concentrations of 1α,25-dihydroxyvitamin D determined when the patients were 10 to 20 years old, after the cessation of therapy with vitamin D2 or 1α-hydroxyvitamin D3. The parents of Patient 1 were second cousins; those of Patient 2 were first cousins; those of Patient 3 were unrelated and came from the same remote area; those of Patient 4 were unrelated and came from different areas.The studies were approved by the appropriate institutional review committees, and all subjects gave informed consent.
Isolation of 1α-Hydroxylase cDNA and Gene
A human kidney cDNA library was prepared from poly(A)+RNA purified from normal kidney tissue.19 A total of 1×106 plaques were screened by hybridization with a mouse 1α-hydroxylase cDNA N-terminal fragment (0.7 kb).16 Two positive plaques were subcloned and sequenced automatically in a Prism 377 DNA sequencer (Applied Biosystems, Foster City, Calif.), with AmpliTaq DNA polymerase FS (Perkin–Elmer, Norwalk, Conn.) and dye terminator.
The genomic sequence of human 1α-hydroxylase was determined by sequencing the polymerase-chain-reaction (PCR) products amplified with three sets of primers specific to the exon and the untranslated region, with normal human leukocyte DNA as a template.
Fluorescence in Situ Hybridization
Three human genomic fragments covering all areas of the 1α-hydroxylase gene were used as probes. Fluorescence in situ hybridization was carried out on more than 20 chromosomes in prometaphase.20
PCR Amplification and Sequence Analysis of the 1α-Hydroxylase Gene
Genomic DNA of all patients and family members was extracted from peripheral white cells. Exons of the 1α-hydroxylase gene were amplified by PCR with specific primers derived from intronic sequences. Information about the sequence of the primers is available elsewhere (NAPS). AmpliTaq Gold (Perkin–Elmer) and its standard buffer were used in all reactions. All exons were amplified in a PCR thermocycler (Perkin–Elmer) by initial denaturation at 95°C for nine minutes, followed by 30 cycles at 95°C for one minute, 60°C for one minute, and 72°C for one minute. The corresponding PCR products were purified and sequenced directly in both directions.
Plasmid Construction
The wild-type human 1α-hydroxylase cDNA was introduced into the expression vector pcDNA3 (Invitrogen, San Diego, Calif.), and the patients' mutations were introduced with a site-directed mutagenesis kit (Quick Change, Stratagene, La Jolla, Calif.). The expression plasmid for the ligand-binding domain of the vitamin D receptor (VDR) fused to the GAL4 DNA binding domain [GAL4-VDR(DEF)] was prepared as described elsewhere.21
Assay of 1α-Hydroxylase Activity
1α-Hydroxylase activity was assayed both by vitamin D receptor–mediated transactivation assay and by high-performance liquid chromatography.16 Briefly, for the vitamin D receptor–mediated transactivation assay, COS-1 cells were transiently transfected with 0.5 μg of GAL4-VDR(DEF), 1 μg of 17M2-G-CAT reporter plasmid,22 0.2 μg each of the adrenodoxin and adrenodoxin reductase expression plasmids, 1 to 2 μg of either wild-type or mutant 1α-hydroxylase expression plasmid, and 1 μg of β-galactosidase expression plasmid pCH110 (Pharmacia Biotech, Uppsala, Sweden). Twelve hours after transfection, the ligands were added to the medium. After incubation for an additional 36 hours, cell extracts were prepared and used for chloramphenicol acetyltransferase assays.23
For analysis by high-performance liquid chromatography, COS-1 cells were transfected with 0.5 μg each of adrenodoxin and adrenodoxin reductase expression plasmids and 3 μg of either wild-type or mutant 1α-hydroxylase expression plasmid and were then incubated with tritiated 25-hydroxyvitamin D3 for six hours. The incubation medium and cells were extracted and analyzed by normal-phase and reverse-phase high-performance liquid chromatography. The eluent fractions were collected, and the radioactivity was estimated by liquid-scintillation counting.16,24,25 Authentic vitamin D derivatives were chromatographed, and the retention times were determined by ultraviolet absorption at 264 nm.
Results
Isolation of 25-Hydroxyvitamin D3 1α-Hydroxylase cDNA and Gene
A 2.4-kb cDNA clone was isolated from a normal human kidney cDNA library by using the mouse 1α-hydroxylase cDNA fragment as a probe. This cDNA contains a 1527-bp open reading frame predicted to encode a protein of 508 amino acids. The sequence of this cDNA was verified by determination of the genomic sequence.
The sequence of the coding region was 82 percent identical to that of mouse 1α-hydroxylase at both the nucleotide and the amino acid levels.16 The deduced amino acid sequence of this cDNA has substantial homology with members of the mitochondrial P450 family,26 particularly human vitamin D3 25-hydroxylase (CYP27, 40 percent),27 25-hydroxyvitamin D3 24-hydroxylase (CYP24, 32 percent),28 P450scc (CYP11A, 33 percent),29 and 11β-hydroxylase (CYP11B1, 30 percent).30
The structure of the human 1α-hydroxylase gene was determined by PCR amplification of normal human leukocyte DNA, with use of exon-specific primers designed from the cDNA sequence, on the basis of the finding that the known members of the mitochondrial P450 family usually have short introns.31 The 1α-hydroxylase gene consisted of nine exons spanning a region of approximately 4.8 kb.
Chromosomal Localization of the 1α-Hydroxylase Gene
Fluorescence in situ hybridization revealed that the 1α-hydroxylase gene was located on chromosome band 12q13.3 (Figure 1AFigure 1
Chromosomal Localization of the 1α-Hydroxylase Gene According to Fluorescence in Situ Hybridization. and Figure 1B). Since the gene responsible for pseudovitamin D–deficiency rickets has been mapped to this locus,9 this finding suggested that pseudovitamin D–deficiency rickets is caused by a defect in the structural gene encoding 1α-hydroxylase.Tissue Expression of the 1α-Hydroxylase Gene
Using the human 1α-hydroxylase cDNA clone as a probe, we examined its expression in various human tissues by Northern blot analysis. The 1α-hydroxylase transcript (approximately 2.4 kb) was detected only in renal tissue (Figure 2Figure 2
Northern Blot Analysis of 1α-Hydroxylase Gene Expression in Human Tissues.).Homozygous Mutations in the 1α-Hydroxylase Gene in Patients with Pseudovitamin D–Deficiency Rickets
To determine whether the 1α-hydroxylase gene was mutated in patients with pseudovitamin D–deficiency rickets, we analyzed genomic DNA from the four patients. Four different missense mutations in four different positions were detected in these patients (Figure 3AFigure 3
Mutation Analysis of the 1α-Hydroxylase Gene in Four Families with Pseudovitamin D–Deficiency Rickets. and Figure 3B). Patient 1 had a mutation in exon 2, Arg107His (CGC to CAC), and Patient 2 had a mutation in exon 2, Gly125Glu (GGA to GAA). Patient 3 had a mutation in exon 6, Arg335Pro (CGG to CCG), that eliminates a restriction site for HpaII. The presence of the mutation was further confirmed by digesting the PCR product of exon 6 with the enzyme (data not shown). Patient 4 had a mutation in exon 7, Pro382Ser (CCT to TCT). All these patients were homozygous for their mutations, and the parents and one sibling who were studied were all heterozygous carriers (Figure 3B). A nephew of Patient 4 was a normal homozygote.Functional Analysis of Wild-Type and Mutant 1α-Hydroxylase Proteins
The 1α-hydroxylase activities of the wild-type and the mutant enzymes in the four patients were assessed by examining the ligand-induced transactivation function of the vitamin D receptor. The vitamin D receptor was activated only when the 25-hydroxyvitamin D3 added to the culture medium was converted to an active ligand, possibly 1α,25-dihydroxyvitamin D3 (Figure 4AFigure 4
Functional Analysis of Wild-Type and Mutant 1α-Hydroxylase Proteins.). The expression of wild-type 1α-hydroxylase activated the reporter gene (Figure 4B, lanes 3 and 4), as in mouse 1α-hydroxylase16 (lane 5). In contrast, none of the mutants (Figure 4B, lanes 8, 9, 10, and 11) increased the activity of the reporter gene. In vitro translation analysis indicated that the wild-type and the mutant plasmids were translated with similar efficiency at 55 kd in size (data not shown).The 1α-hydroxylase activity of these mutants was assayed to determine the ability of the enzyme to catalyze the conversion of 25-hydroxyvitamin D3 to 1α,25-dihydroxyvitamin D3. When tritiated 25-hydroxyvitamin D3 was added to cells transfected with the wild-type 1α-hydroxylase expression plasmid, a metabolite was detected (Figure 4C, lower panels) at a retention time identical to that of authentic 1α,25-dihydroxyvitamin D3 (Figure 4C, upper panels). However, none of the mutants converted 25-hydroxyvitamin D3 into 1α,25-dihydroxyvitamin D3 (Figure 4D). These results indicate that the protein products of the four mutations found in the patients with pseudovitamin D–deficiency rickets had no 1α-hydroxylase activity.
Discussion
The responsible genes and their mutations have been identified for two types of hereditary rickets besides pseudovitamin D–deficiency rickets.5-8 We isolated a human cDNA encoding 25-hydroxyvitamin D3 1α-hydroxylase and identified different homozygous missense mutations in this gene in four patients with pseudovitamin D–deficiency rickets. Each of the four mutations abolished 1α-hydroxylase activity. The asymptomatic parents and sibling were heterozygous for the mutations, demonstrating the autosomal recessive inheritance of this disease. We conclude that pseudovitamin D–deficiency rickets is caused by inactivating mutations in the 1α-hydroxylase gene.
We found no correlation between phenotype and genotype; all four mutations abolished 1α-hydroxylase activity, and the clinical features in all four patients were similar. Clinical heterogeneity has been reported in pseudovitamin D–deficiency rickets,2,34 and there may be other mutations that only partially reduce enzyme activity, as is the case with other P450 enzymes.35,36 In a preliminary study of a patient who had pseudovitamin D–deficiency rickets with mild clinical features, we detected no mutations in the coding region (unpublished data), a fact that suggests that gene expression may be impaired by a mutation in the promoter region. Alternatively, there may be genetic heterogeneity in pseudovitamin D–deficiency rickets, and further analysis may reveal mutations in another gene. We found only homozygous mutations in the four patients, a result in agreement with the finding that in rare autosomal recessive diseases, homozygous mutations are frequent and compound heterozygotes are uncommon.37
The mutated residue (Pro382Ser) in one of our patients was located in a conserved region among various P450 enzymes that is assumed to be required for substrate binding,38,39 and another mutated residue (Gly125Glu) was located in the region corresponding to the proposed substrate-recognition site in the P450 family (CYP2).40 These mutations probably abolish the activity of 1α-hydroxylase by reducing its affinity for 25-hydroxyvitamin D3. We do not know how the other two mutations abolish 1α-hydroxylase activity, but because these four residues are highly conserved among several mitochondrial P450 enzymes, they are likely to be important for the enzymatic function of this protein.
With the data obtained in this study, we now know the molecular basis of all three major types of hereditary rickets. The phenotypes of pseudovitamin D–deficiency rickets and hypocalcemic vitamin D–resistant rickets are similar, except for alopecia in the latter and differences in serum concentrations of vitamin D metabolites.4 There are many endogenous vitamin D derivatives with biologic activity, 41 and therefore altered serum concentrations of vitamin D derivatives in these types of hereditary rickets may cause different changes.
Supported by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to Dr. Kato).
NAPS See NAPS document no. 05444 for one page of supplementary material. To order, contact NAPS c/o Microfiche Publications, 248 Hempstead Tpk., West Hempstead, NY 11552.
We are indebted to Mr. Yoshikazu Masuhiro, Dr. Yasuo Yanagi, Dr. Seiji Fukumoto, Dr. Masato Kobori, Dr. Takehiko Yokomizo, Mr. Fumihiko Ichikawa, and Dr. Naoki Kubota for technical assistance; to Dr. Toshiyuki Yasuda and Dr. Ayako Tanae for providing data on patients; to Mr. Tatsuya Yoshizawa for helpful discussions; to Dr. Chifumi Kitanaka for valuable comments; to Dr. Yoshiyasu Yabusaki for the kind gift of adrenodoxin and adrenodoxin reductase cDNA plasmids; and to Chugai Pharmaceuticals for vitamin D–related compounds.
Source Information
From the Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo (S. Kitanaka, K.T., A.M., T.S., J.Y., S. Kato); Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama (S. Kato); the Faculty of Bioresources, Mie University, Mie (K.O., M.N.); Tokyo Metropolitan Kiyose Children's Hospital, Tokyo (Y.H.); the Department of Pediatrics, Chiba University School of Medicine, Chiba (H.N.); and the Department of Endocrinology and Metabolism, National Children's Medical Research Center, Tokyo (T.T.) — all in Japan.
Address reprint requests to Dr. Kato at the Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
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