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-Hydroxylase Deficiency That Confer Partial Enzyme Activity in Vitro
Department of Pediatrics, University of California, San Francisco, California 94143
Address all correspondence and requests for reprints to: Anthony A. Portale, M.D., University of California, 533 Parnassus Avenue, Room U-585, Box 0748, San Francisco, California 94143-0748. E-mail: . aportale{at}peds.ucsf.edu
Abstract
The rate-limiting, hormonally regulated step in the biological activation of vitamin D is its 1
-hydroxylation to 1,25-dihydroxyvitamin D [1,25-(OH)2D] in the kidney, catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1. We previously cloned the human P450c1 cDNA and gene, and identified 14 different mutations, including 7 missense, in 19 patients with 1-hydroxylase deficiency, also known as vitamin D-dependent rickets type 1. None of the missense mutations encoded a protein with detectable enzymatic activity in vitro. Although there is phenotypic variation among such patients, the molecular basis of this variation is unknown. We analyzed 6 additional patients with clinical and radiographic features of rickets; in 4 patients the laboratory abnormalities were typical of 1-hydroxylase deficiency, but in 2 they were unusually mild [mild hypocalcemia and normal serum 1,25-(OH)2D concentration]. Direct sequencing revealed that all patients had P450c1 mutations on both alleles. Five new and 2 known mutations were identified. The new mutations included a 5-bp deletion with a 6-bp novel insertion causing a frameshift in exon 2, and a G to A change at +1 of intron 2; a minigene experiment proved that this intronic mutation prevented proper splicing. Three new missense mutations were found and tested by expressing the mutant cDNA in mouse Leydig MA-10 cells. The R389G mutant was totally inactive, but mutant L343F retained 2.3% of wild-type activity, and mutant E189G retained 22% of wild-type activity. The two mutations that confer partial enzyme activity in vitro were found in the 2 patents with mild laboratory abnormalities, suggesting that such mutations contribute to the phenotypic variation observed in patients with 1-hydroxylase deficiency.
VITAMIN D IS a biologically inactive prehormone that must be converted to its hormonally active form before it can bind to and activate the VDR and thus exert its biological effects. Vitamin D first undergoes 25-hydroxylation in the liver, catalyzed by P450c25 (1, 2), resulting in 25-hydroxyvitamin D (25OHD) which has minimal activity. 25OHD then undergoes 1-hydroxylation in the kidney, resulting in 1,25-dihydroxyvitamin D [1,25-(OH)2D], the hormonally active form of vitamin D. This reaction is hormonally regulated and rate-limiting, and is catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1 (3, 4). Circulating concentrations of 1,25-(OH)2D primarily reflect its synthesis in the kidney, although 1-hydroxylase activity is present in keratinocytes (5), macrophages (6, 7, 8, 9, 10), and osteoblasts (11), and its mRNA is also expressed in testis and brain (4).
1-Hydroxylase deficiency, also known as vitamin D- dependent rickets type I or pseudo-vitamin D-deficiency rickets, is an autosomal recessive disease characterized clinically by failure to thrive, muscle weakness, hypocalcemia, secondary hyperparathyroidism, and clinical and radiological findings of rickets (3, 12, 13). The hallmarks of this disease are reduced serum concentrations of 1,25-(OH)2D despite normal or increased serum concentrations of 25OHD, and reversal of clinical and laboratory abnormalities by physiological replacement doses of 1,25-(OH)2D3 (12, 13). These findings suggest that the disease is caused by defective renal conversion of 25OHD to 1,25-(OH)2D. Indeed, in 1997 we (4, 14) and others (15, 16, 17, 18) cloned the cDNA and gene for P450c1, and we showed that 1-hydroxylase deficiency is caused by mutations in this gene (4). We subsequently reported 14 different mutations in 19 patients from widely divergent ethnic backgrounds, and identified 2 common mutations, a 7-bp duplication and a deletion of guanine at nucleotide 958 (958
G), the latter found commonly among French Canadian patients from the Charlevoix region of Quebec (19). Currently, many mutations have been identified in multiple ethnic groups, including missense mutations, deletions, duplications, and splice site mutations (19, 20, 21, 22, 23). All of the missense mutations identified to date, including 1 in a Japanese patient with mild clinical manifestations (22), were found to be totally inactive when expressed in vitro (19, 20, 22, 23).
We now have analyzed the P450c1 gene in six additional patients with 1-hydroxylase deficiency, including two with mild laboratory abnormalities. Mutations were identified in all patients, including five previously undescribed mutations, of which three were missense. These novel missense mutations were tested by expressing the mutant cDNA in mouse Leydig MA-10 cells, which revealed significant residual enzyme activity in the two patients whose laboratory abnormalities were mild. These findings suggest that such mutations contribute to the phenotypic variation observed in patients with 1-hydroxylase deficiency and provide information about the structure and function of the enzyme.
Subjects and Methods
Patients
The clinical data for each of six unrelated patients with clinical and laboratory features of 1-hydroxylase deficiency are summarized in Table 1
. DNA samples were obtained from patients and their parents; parental DNA was not available for patient 5, who was adopted. This study was approved by the committee on human research, University of California-San Francisco.
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Patient 2 developed hypocalcemic seizures at the age of 5 months. Physical examination of the limbs was unremarkable, but radiographs of the wrist revealed mild rachitic changes. Laboratory data were typical for 1-hydroxylase deficiency, and he required treatment with 1,25-(OH)2D3 to maintain clinical remission.
Patient 3 presented with stridor at the age of 7 months. She had radiographic evidence of rickets, hypocalcemia, hypophosphatemia, and high serum ALP activity. The serum PTH concentration was increased when measured at 24 months of age. She has been treated with 1,25-(OH)2D3.
Patient 4 presented with failure to thrive at the age of 13 months. She had muscle wasting, rachitic rosary, enlargement of the wrists, and radiographic findings of rickets. Laboratory tests showed hypophosphatemia and high serum concentrations of ALP and PTH, but normal concentrations of 25OHD and 1,25-(OH)2D. Administration of 1 µg/d 1,25-(OH)2D3 resulted in normalization of clinical, radiographic, and laboratory abnormalities.
Patient 5 had failure to thrive and bowed legs at the age of 24 months. She had severe rickets and typical laboratory findings of 1-hydroxylase deficiency. She was treated with dihydrotachysterol, but complete normalization of clinical and laboratory abnormalities was achieved only with the addition of oral phosphorus supplements.
Patient 6 presented with hypotonia and leg deformity at the age of 21 months. She had rachitic rosary, enlargement of the wrists and ankles, genu varus, and radiographic findings of severe rickets. The serum concentration of PTH was increased, but concentrations of calcium, phosphorus, and 1,25-(OH)2D were within the normal range. Treatment with high dose vitamin D3 for 1 month induced no clinical or laboratory changes, whereas treatment with 0.25 µg/d 1,25-(OH)2D3 induced rapid normalization of hyperparathyroidism and the clinical and radiographic signs of rickets.
Mutation analysis
Genomic DNA was prepared from whole blood using the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI), and the entire human P450c1 gene was amplified in a single PCR reaction as previously described (19). The PCR product was either sequenced directly without cloning or was subcloned into vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA), and plasmid DNA from single colonies was sequenced on both strands. Putative mutations were checked again by PCR amplification and sequencing of the affected exon directly from genomic DNA of the patients and their parents as previously described (19).
Microsatellite haplotype analysis
PCR primers for the microsatellite markers D12S90, D12S305, and D12S104 were synthesized and end-labeled with [
-32P]ATP (Amersham Pharmacia Biotech, Arlington Heights, IL) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA) as previously described (19). The PCR reactions included 40 ng genomic DNA, 20 ng of each primer, 0.2 mM of each dNTP, and 1 U of a 20:1 mixture of Taq and Pfu DNA polymerases (Promega Corp.) in 20 µl 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl2, and 2% dimethylsulfoxide. PCR was performed using a PT-100 thermal cycler (MJ Research, Inc., Watertown, MA) under the following conditions: 95 C for 3 min, followed by 30 cycles at 95 C for 30 sec, 57 C (for D12S90 and D12S305) or 52 C (for D12S104) for 30 sec, and 72 C for 30 sec. The PCR products were then separated by electrophoresis on a 6% polyacrylamide sequencing gel, and the data were analyzed on a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Minigene construction, transfection, and RT-PCR
To determine the effect of a mutation at a splice junction, we built two minigenes containing exons 2–6 and their intervening introns, with and without the splice donor mutation in intron 2. Genomic DNA was PCR-amplified from patient 2 and a normal control, using the sense primer 5'-CCTCACCCAAAGGTTAAATAG-3' and the antisense primer 5'-AGAGTGTTTGAGAACAGGGTT-3' as previously described (19). The PCR products were digested with SpeI, yielding exons 2–6; this fragment was subcloned into the XbaI site of pcDNA3, and the presence of the mutation was confirmed by direct sequencing.
COS-1 cells maintained in DMEM-H21 with 10% FBS at 60–70% confluence were transfected in 10-cm dishes with 20 µg DNA using the calcium phosphate method. Sixteen hours after transfection, the cells were washed with PBS, and the medium was changed; 48 h after transfection, the cells were washed and collected, and total RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). The RNA was then reverse transcribed using the SuperScript II system (Life Technologies, Inc.), using the primer 5'-AAATGCAAACATCTGGTC-3'. PCR was performed using the sense primer (in exon 2) 5'-TAGCCAGCTTTGGGACAGTG-3' and the antisense primer (in exon 4) 5'-ATCGCCATGGTCAACAGCG-3' with Taq DNA polymerase (Promega Corp.) under the following conditions: 95 C for 3 min, followed by 30 cycles of 95 C for 30 sec, 55 C for 30 sec, and 72 C for 1 min. The PCR products were separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide.
Site-directed mutagenesis and cDNA expression
The patients’ mutations were recreated by PCR-based oligonucleotide-mediated mutagenesis of our human P450c1 cDNA in the expression vector pcDNA3 (4), using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The PCR reaction included 200 ng template DNA, 250 ng of each primer, 0.4 mM of each dNTP, and 5 U Pfu-Turbo DNA polymerase (Stratagene) and was performed under the following conditions: 95 C for 30 sec, followed by 15 cycles at 95 C for 30 sec, 55 C for 1 min, and 65 C for 20 min. Wild-type template plasmids were selectively digested from the PCR product with 20 U DpnI (Promega Corp.). The mutations were confirmed by sequencing, and 2 µg plasmid DNA were transfected into MA-10 cells at 50–60% confluence using adenovirus-mediated transfection as previously described (4). Forty-eight hours after transfection, cells were transferred to serum-free medium and incubated with 0.1 µM 25OHD for 1 h; cells and medium were extracted with acetonitrile, and after C18 and silica Sep-Pak chromatography, 1,25-(OH)2D was determined in duplicate by RRA as previously described (4). The activity of the mutants is expressed as a percentage of wild-type activity.
Results
The diagnosis of 1-hydroxylase deficiency was made in all six patients based on their clinical manifestations; their parents were asymptomatic. We amplified the entire 4.2-kb P450c1 gene from genomic DNA obtained from patients and their parents and directly sequenced all nine exons, including exon-intron boundaries. Mutations were found on both alleles in all patients (Fig. 1). Patients 1, 2, and 6 were homozygous, and patients 3, 4, and 5 were compound heterozygous. Each of their asymptomatic parents was heterozygous, carrying a mutation on only one allele, consistent with the autosomal recessive nature of the disease. Of the seven mutations identified in the six patients, two are known and five are novel, the latter including one 6-bp substitution insertion, one donor splice site mutation, and three missense mutations.
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-hydroxylase deficiency (24), and the numbers of alleles described by these three markers are 7, 5, and 3, respectively; thus, these markers can describe 105 unique haplotypes. We found that the microsatellite haplotype of the Chilean family is quite different from that of our previously studied Filipino family (Fig. 2), indicating that the same mutation occurred in these 2 families independently.
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a). This disrupts the splice donor site, which we predicted would result in retention of intron 2. The retained intron would create a translational termination signal at codon 130, resulting in a severely truncated peptide that cannot have enzymatic activity. To assess this prediction, we conducted a minigene experiment (Fig. 3). Minigenes that included wild-type or mutant exons 2–6 were subcloned into the expression vector pcDNA3 and transiently transfected into COS-1 cells. The transcripts produced from the minigenes were reverse transcribed and amplified by PCR using primer pairs located in exons 2 and 4. The transcript from the wild-type cDNA yielded a band of 484 bp, representing exons 2, 3, and 4, whereas the transcript from the mutant yielded a band of 995 bp, representing exons 2, 3, and 4 plus retention of the 511-bp intron 2 (Fig. 3). Retention of intron 2 caused a shift in the reading frame and premature termination of translation, yielding a peptide devoid of enzyme activity.
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). This substitution caused a shift in the reading frame, resulting in a premature stop at codon 332 and thus a peptide devoid of enzymatic activity. The deletion of GGGCG results in a loss of eight restriction endonuclease cleavage sites, and the insertion of CTTCGG results in the creation of seven novel restriction sites, including creation of a unique ApaLI site (GTGCAC); no other ApaLI sites were found in the wild-type gene. A 3062-bp fragment containing either the GGGCG to CTTCGG substitution or the wild-type sequence was amplified from genomic DNA and digested with ApaLI. Digestion of the mutant fragment from the heterozygous patient and his father yielded three bands of 3062, 2160, and 902 bp, whereas digestion of the wild-type fragment yielded only one band of 3062 bp (Fig. 4). The mutant sequence contained a duplication of the sequence GCACTTCGG separated by only four bases, suggesting a slipped strand mispairing mechanism for this mutation.
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). Patients 4 and 6 had normal serum calcium and 1,25-(OH)2D concentrations, suggesting that their mutant P450c1 retained enzymatic activity. To test this possibility, we recreated each mutation in our P450c1 cDNA expression vector and transfected the mutants into mouse Leydig MA-10 cells. These steroidogenic cells contain large amounts of the ferredoxin reductase and ferredoxin required by mitochondrial (type 1) P450 enzymes such as P450c1, and thus expression of mitochondrial P450 enzymes in MA-10 cells yields 1000-fold more activity than expression in monkey kidney COS-1 cells (25). The mutation R389G (patient 5) had no 1-hydroxylase activity (0.1% of the wild-type); however, mutation L343F (patient 4) had 2.3% of the wild-type activity, and mutation E189G (patient 6) had 22% of the wild-type activity (Fig. 6). Thus, some P450 mutations are associated with partial enzymatic activity in vitro, suggesting that such mutations contribute to the variable phenotype observed in patients with 1-hydroxylase deficiency.
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In the present study we identified two known and five novel mutations in the P450c1 gene in six unrelated patients with 1-hydroxylase deficiency. The five novel mutations included a 5-bp deletion with a 6-bp insertion in exon 2, and a guanine to adenine substitution in the first nucleotide of intron 2 that prevented proper splicing. Both mutations caused a shift in the reading frame and premature termination of translation, thus yielding a truncated protein that cannot have activity. The other three mutations identified in three patients were missense: E189G, L343F, and R389G.
Although vitamin D 1-hydroxylase deficiency is a rare disease, a total of 31 different mutations have been found on 88 distinct chromosomes since our first description of mutations in this gene in 1997 (4) (Table 2). The mutations observed most frequently are G958, commonly found in French Canadian patients (19) due to a founder effect (24), and a 7-bp duplication that arose independently in several populations (19). The large number of amino acid replacement (missense) mutations observed combined with our computational predictions of the structure of P450c1 (19) permit an analysis of the mechanism by which each mutation disrupts activity of the enzyme. All of the missense mutations reported to date, including the mutation T321R in a Japanese patient with mild clinical manifestations (22), were reported to be totally inactive when assayed for enzymatic activity in vitro. A variety of in vitro assays for P450c1 activity have been used, including promoter trans-activation by 1,25-(OH)2D (20, 22), cDNA expression in mammalian cells (19) and bacteria (22), and determination of activity in patient-derived macrophages (23); hence, it is not possible at present to compare the activity data from different investigators. Thus, we can only compare the activity data of the 10 missense mutations analyzed in this laboratory previously (19) and in the present work.
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The mutation L343F changes leucine, a small uncharged residue to phenylalanine, a bulky uncharged residue. L343 lies in the J helix, which is a structurally conserved region that is important structurally, but not catalytically. The mutation L343F could disrupt activity by creating a conformational mutant.
The most remarkable mutation found in our study is E189G, which retained 22% of the wild-type activity and hence is the first missense mutation described that retains partial activity in vitro. Residue E189 lies in the E helix, which is part of the four-helix bundle (D, E, I, and L helixes) that is a hallmark of all cytochrome P450 enzymes (26). A change from glutamic acid (E) to glycine (G) removes a three-carbon side-chain and replaces an acidic residue with a neutral one; such a change could cause a conformational disturbance that still permits substrate binding and interaction with ferredoxin, albeit at decreased efficiency. We previously reported a mutation at this same residue (19); due to a typographical error in that report, the mutation was identified as E189L (glutamic acid to leucine), whereas the actual mutation was E189K (glutamic acid to lysine). Of the seven mutants analyzed in our previous report, only the E189K mutant had activity above that of the vector control (1% of wild-type), although this was thought to be an insignificant level (19). Because E189G retained 22% of activity, we reexamined the E189K mutant and found that it retained 11% of the wild-type activity.
Patient 6 was homozygous for the E189G mutation that retained 22% of the wild-type activity. This patient presented clinically with hypotonia and leg deformity and was found to have hyperparathyroidism, but the serum concentrations of calcium, phosphorus, and 1,25-(OH)2D were not reduced. The diagnosis of 1-hydroxylase deficiency was made when the patient failed to respond to large doses of vitamin D3, but showed rapid improvement with administration of 0.25 µg/d 1,25-(OH)2D3. Similarly in patient 4, whose mutation retained 2.3% of the wild-type activity, serum concentrations of 25OHD and 1,25-(OH)2D were not reduced, but the diagnosis was more readily considered because of hypophosphatemia and increased serum concentrations of ALP and PTH. Patients 4 and 6 demonstrate that the classical laboratory criteria for the diagnosis of 1-hydroxylase deficiency may fail to identify patients with partial, but significant, defects in this enzyme, and hence, 1-hydroxylase deficiency syndromes may be more common than previously appreciated.
Acknowledgments
We thank the following individuals for providing DNA specimens and clinical information on their patients: Drs. Veronica Mericq (patient 1), Adnal Alshaikh and Sangwy Kooh (patient 2), Lisa Guay-Woodford (patient 3), Willem Proesmans (patient 4), Clifford Kashtan (patient 5), and Eric Girardin and Petra Genet Donati (patient 6).
Footnotes
This work was supported by the NIH Grants DK-54433 (to A.A.P.), DK-37922 and DK-42154 (to W.L.M.), grants from the March of Dimes Birth Defects Foundation (to A.A.P.), and gifts from the David Carmel Trust (to A.A.P.).
Abbreviations: ALP, Alkaline phosphatase; 1,25-(OH)2D, 1,25-dihydroxyvitamin D; 25OHD, 25-hydroxyvitamin D.
Received September 20, 2001.
Accepted January 3, 2002.
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  C. J. Kim, L. E. Kaplan, F. Perwad, N. Huang, A. Sharma, Y. Choi, W. L. Miller, and A. A. Portale Vitamin D 1{alpha}-Hydroxylase Gene Mutations in Patients with 1{alpha}-Hydroxylase Deficiency J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3177 - 3182. [Abstract] [Full Text] [PDF]
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 H. Chen, M. Hewison, B. Hu, and J. S. Adams Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: A cause of vitamin D resistance PNAS, May 13, 2003; 100(10): 6109 - 6114. [Abstract] [Full Text] [PDF]
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T. O. Carpenter Variable Degrees of 1-{alpha} Hydroxylase Activity--Fine Tuning the Rachitic Rheostat J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2421 - 2423. [Full Text] [PDF]
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