Cloning of Human 25-Hydroxyvitamin D-1{alpha}-Hydroxylase and Mutations Causing Vitamin D-Dependent Rickets Type 1

Glenn K. Fu, Dong Lin, Martin Y. H. Zhang, Daniel D. Bikle, Cedric H. L. Shackleton, Walter L. Miller and Anthony A. Portale

Departments of Pediatrics (G.K.F., D.L., M.Y.H.Z., W.L.M., A.A.P), Medicine (D.D.B., A.A.P.), and Dermatology (D.D.B.) Child Health Research Center Veterans Affairs Medical Center (D.D.B.) San Francisco, California 94121
Children’s Hospital Research Institute (C.H.L.S.) Oakland, California 94609


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The secosteroid hormone, 1,25-dihydroxyvitamin D [1,25(OH)2D], plays a crucial role in normal bone growth, calcium metabolism, and tissue differentiation. The key step in the biosynthesis of 1,25-(OH)2D is its 1{alpha}-hydroxylation from 25-hydroxyvitamin D (25-OHD) in the kidney. Because its expression in the kidney is very low, we cloned and sequenced cDNA for 25-OHD-1{alpha}-hydroxylase (P450c1{alpha}) from human keratinocytes, in which 1{alpha}-hydroxylase activity and mRNA expression can be induced to be much greater. P450c1{alpha} mRNA was expressed at much lower levels in human kidney, brain, and testis. Mammalian cells transfected with the cloned P450c1{alpha} cDNA exhibit robust 1{alpha}-hydroxylase activity. The identity of the 1,25(OH)2D3 product synthesized in transfected cells was confirmed by HPLC and gas chromatography-mass spectrometry. The gene encoding P450c1{alpha} was localized to chromosome 12, where the 1{alpha}-hydroxylase deficiency syndrome, vitamin D-dependent rickets type 1 (VDDR-1), has been localized. Primary cultures of human adult and neonatal keratinocytes exhibit abundant 1{alpha}-hydroxylase activity, whereas those from a patient with VDDR-1 lacked detectable activity. Keratinocyte P450c1{alpha} cDNA from the patient with VDDR-1 contained deletion/frameshift mutations either at codon 211 or at codon 231, indicating that the patient was a compound heterozygote for two null mutations. These findings establish the molecular genetic basis of VDDR-1, establish a novel means for its study in keratinocytes, and provide the sequence of the key enzyme in the biological activation of vitamin D.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hormone 1,25-dihydroxyvitamin D [1,25(OH)2D] plays an essential role in calcium metabolism, bone growth, and cellular differentiation. Synthesis of 1,25(OH)2D from its endogenous precursor, 25-hy-droxyvitamin D (25-OHD), is catalyzed by 25-OHD-1{alpha}-hydroxylase (1{alpha}-hydroxylase), a mitochondrial cytochrome P450 enzyme (1, 2, 3) that is subject to complex regulation by PTH, calcium, 1,25(OH)2D, and phosphorus (4, 5, 6, 7). The principal site of 1,25(OH)2D synthesis is the proximal renal tubule (1, 8, 9, 10, 11). Serum concentrations of 1,25(OH)2D are decreased in patients with chronic renal insufficiency and are greatly reduced yet detectable in anephric patients (12, 13, 14, 15, 16). 25-OHD also is converted to 24,25-dihydroxyvitamin D, a hormone whose physiological role is uncertain, by 25-OHD-24-hydroxylase (24-hydroxylase). The cDNAs for both the renal 24-hydroxylase (P450c24) and hepatic 25-hydroxylase (P450c25) have been cloned, demonstrating that each is a unique mitochondrial cytochrome P450 enzyme (17, 18, 19, 20).

Synthesis of 1,25(OH)2D is impaired in numerous disorders including chronic renal insufficiency, renal tubular diseases, and autosomal recessive vitamin D-dependent rickets type 1 (VDDR-1). VDDR-1 (or pseudo-vitamin D-deficiency rickets) is characterized by failure to thrive, muscle weakness, skeletal deformities, hypocalcemia, secondary hyperparathyroidism, and greatly reduced serum concentrations of 1,25(OH)2D despite normal concentrations of 25-OHD (21, 22). These abnormalities are reversed by administration of physiological amounts of 1,25-(OH)2D3. The genetic defect is unknown but is presumed to result in defective renal 1{alpha}-hydroxylation of 25-OHD (23). Although such a defect in renal tissue has not been demonstrated directly, cells isolated from human placental decidua of patients with VDDR-1 fail to convert 25-OHD to 1,25(OH)2D, which suggests that 1{alpha}-hydroxylase in decidua and kidney, or a regulator of its activity, are encoded by the same gene (24). The mutation causing VDDR-1 has been mapped to chromosome 12q14 by linkage analysis (25, 26).

Because the activity and presumably mRNA content of renal 1{alpha}-hydroxylase are very low, we sought an alternative source of RNA for its cloning. Keratinocytes, which synthesize vitamin D3 from endogenous 7-dehydrocholesterol upon exposure to UV light, also synthesize 1,25(OH)2D from exogenous 25-OHD with high activity in vitro (27, 28, 29). Such 1{alpha}-hydroxylase activity is tightly regulated and coupled to the differentiation of these cells (27, 28, 29). We now report the cloning of the cDNA for 25-OHD-1{alpha}-hydroxylase, designated P450c1{alpha}, from human keratinocytes, its expression in transfected mammalian cells, and that mutations in its gene cause VDDR-1.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of the Human 1{alpha}-Hydroxylase cDNA
Our initial efforts to clone the P450c1{alpha} cDNA from vitamin D-deficient rat kidney by cross-hybridization with an oligonucleotide, based on the presumably conserved heme-binding site of mitochondrial P450c24, were unsuccessful. As an alternative source of RNA, we used human keratinocytes, which exhibit high 1{alpha}-hydroxylase activity in vitro (27, 28). Efforts to screen a human keratinocyte cDNA library, by probe hybridization and by expression cloning, were unsuccessful. We then performed RT-PCR using multiple sets of degenerate-sequence primers corresponding to the region of the ferredoxin-binding and heme-binding sites of P450c24 and P450c25. One set of primers (see Materials and Methods for details) yielded a predominant cDNA fragment of ~300 bp with a unique DNA sequence homologous to both P450c24 and P450c25 (17, 18, 19, 20). This PCR product was used to screen a human keratinocyte cDNA library, yielding a partial-length, 1.9-kb clone, whose complete sequence was then obtained by rapid amplification of cDNA ends (5'-RACE). The full-length, 2.4-kb 1{alpha}-hydroxylase cDNA, P450c1{alpha}, encodes a protein of 508 amino acids with predicted molecular mass of 56 kDa (Fig. 1AGo). The predicted topology is similar to that of known mitochondrial cytochrome P450 enzymes, with the NH2 terminus having characteristics of a mitochondrial signal sequence (30). Within the heme-binding region, its amino acid sequence identity is 73% and 65% to that of human P450c24 and P450c25, respectively (17, 18, 19, 20). However, the overall sequence identity of P450c1{alpha} to that of other mitochondrial P450 enzymes is limited: P450c25 (39%), P450c24 (30%), P450scc (32%), and P450c11ß (33%) (Fig. 1BGo).



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Figure 1. Human P450c1{alpha} cDNA

A, The full-length nucleotide and predicted amino acid sequences are shown; the ferredoxin-binding site (beginning at base 1116) and heme-binding site (beginning at base 1374) are underlined. Human keratinocyte poly(A)+ RNA was reverse transcribed into cDNA, which was PCR-amplified using degenerate sequence primers, yielding a predominant ~300-bp fragment. This PCR product was cloned, sequenced, and used to screen a human keratinocyte cDNA library, yielding a partial-length 1.9-kb clone containing the 3'-sequence shown, 5'-RACE was then performed to yield a 650-bp nested PCR product, which was cloned, sequenced, and ligated to the 5'-end of clone pDL1.9 containing the 3'-end of the cDNA. The full-length, 2.4-kb 1{alpha}-hydroxylase cDNA (Genbank accession number AF020192) encodes a protein of 508 amino acids with predicted molecular mass of 56 kDa. B, Sequence comparison of P450c1{alpha} with other human mitochondrial P450 sequences. The alignments were performed with the ClustalW program (52). The top line depicts the sequence of P450c1{alpha}, followed by P450c25 (53), P450c24 (20), P450scc (54), and P450c11ß (55). The 62 residues identical in all five sequences are designated by an asterisk (*), the 68 highly conservative substitutions by a colon (:), and the 32 moderately conservative substitutions by a period (.). The conserved ferredoxin-binding and heme-binding sites are underlined, as in panel A.

 
Tissue Distribution of P450c1{alpha} mRNA Expression
Northern blotting of keratinocyte RNA revealed a single P450c1{alpha} band of ~2.5 kb (Fig. 2AGo), but ribonuclease protection assays did not detect the mRNA in other tissues. Therefore, we used RT-PCR to amplify a specific 553-bp fragment of the cDNA, which was then detected by Southern blotting. This approach demonstrated expression of P450c1{alpha} mRNA in human adult kidney (not shown) and fetal kidney, brain, and testis, all at less than 1/25 of the level of its expression in cultured neonatal keratinocytes (Fig. 2BGo). Expression was not detectable in human placenta or fetal liver, heart, adrenal, or lung, whereas the ß-actin RT-PCR product was obtained from all tissues examined. The absence of detectable P450c1{alpha} mRNA in placental tissue is surprising, since 1{alpha}-hydroxylase activity has been observed in human placental decidual cells (24). However, it has been suggested that 1{alpha}-hydroxylase activity in placental trophoblastic tissue is nonenzymatic, since it can be abolished by antioxidants and is unaffected by ketoconazole, unlike that in kidney (31). Southern blotting of human genomic DNA probed with a small fragment of cDNA under low-stringency conditions revealed a single band in each restriction digest, which suggests that there is only one copy of the gene for P450c1{alpha} (not shown).



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Figure 2. Expression of 1{alpha}-Hydroxylase mRNA

A, Northern blot of 30 µg of human keratinocyte total RNA probed with a 340-bp KpnI fragment of the cDNA reveals a single band of ~2.5 kb; nonradioactive molecular size markers were run in an adjacent lane not shown. B, Tissue distribution of human 1{alpha}-hydroxylase mRNA. Random primers were used to prepare cDNA from 1 µg of total RNA from each human fetal tissue shown, and this cDNA was then amplified for 30 cycles to yield a 553-bp 1{alpha}-hydroxylase cDNA fragment; a similar procedure was used to amplify a 439 bp actin cDNA fragment as control. The PCR products were resolved on agarose gels, blotted, and probed with the P450c1{alpha} cDNA (above) or actin cDNA (below). Only 1/25 of the keratinocyte cDNA product was loaded.

 
While the mRNA abundance in the cultured keratinocytes is relatively great, it should be noted that these cells were grown in serum-free calcium-deficient medium, which maximizes their 1{alpha}-hydroxylase activity (27) and presumably their P450c1{alpha} mRNA. Similarly, the relatively low abundance of P450c1{alpha} mRNA in kidney tissue presumably reflects the small contribution of P450c1{alpha} mRNA from proximal tubule cells, the site of 1{alpha}-hydroxylase activity, relative to that of remaining renal tissue, where 1{alpha}-hydroxylase activity is absent.

Expression of P450c1{alpha} cDNA in Mammalian Cells
To determine whether the cloned P450c1{alpha} indeed catalyzes 1{alpha}-hydroxylase activity, we expressed the cDNA in mammalian cells. The enzymatic activity of all mitochondrial P450 enzymes requires the expression of the electron transport proteins, ferredoxin and ferredoxin reductase (32). Therefore, we transfected a plasmid expressing the full-length P450c1{alpha} cDNA into cultured mouse Leydig MA-10 cells (33), which contain both of these electron transfer proteins and which exhibit marked enzymatic activity when expressing P450c11AS, another mitochondrial P450 found in low abundance (34). Minimal 1{alpha}-hydroxylase activity was detected both in untransfected MA-10 cells and in those transfected with empty vector. MA-10 cells expressing the P450c1{alpha} cDNA converted 25-OHD3 to putative 1,25(OH)2D3 at levels ~200 times above control transfections (Fig. 3AGo). The 1{alpha}-hydroxylated product coeluted with authentic 1,25(OH)2D3 on HPLC using two solvent systems that distinguish it from other metabolites of vitamin D (35). Thus, the cloned cDNA encodes an enzyme, P450c1{alpha}, with robust 1{alpha}-hydroxylase activity. Lineweaver-Burke analysis of the cloned enzyme’s kinetics yielded an apparent Michaelis-Menten constant (Km) of 2.6 x 10-7 M (Fig. 3BGo). This value, obtained in whole cells, may not represent the true kinetic constant, as substrate access to the inner mitochondrial membrane may be rate limiting (36, 37). However, this value is comparable to that obtained previously in chick and pig kidney and human placenta and skin (27, 38, 39, 40).



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Figure 3. Characterization of 1{alpha}-Hydroxylase Activity

A, 1{alpha}-hydroxylase activity, expressed as picograms of 1,25(OH)2D3 produced per 10-cm plate of cultured cells. The y-axis (logarithmic scale) begins at the detection limit of 1,25(OH)2D per plate. The three left bars depict MA-10 cells transfected with: 1{alpha}, vector expressing 1{alpha}-hydroxylase cDNA; V, empty vector; or Un, untransfected cells. The three right bars depict keratinocytes from: N, neonatal foreskin; Ad, healthy adult subject; or Pt, patient with VDDR-1. MA-10 cells expressing P450c1{alpha} converted 25-OHD3 to 1,25(OH)2D3 at levels ~200 times above control transfections. Minimal 1{alpha}-hydroxylase activity was detected in untransfected MA-10 cells, but no activity was detected (ND) in keratinocytes from the patient with VDDR-1. MA-10 cells were transfected at 50% confluence with 2 µg/ml DNA using adenovirus-mediated transfection. Keratinocytes were cultured in KGM containing 0.07 mM calcium. Cells were incubated with 0.1 µM 25-OHD3 for 1 h in serum-free medium, and the 1{alpha}-hydroxylated product was determined by RRA after extraction and C-18 and silica SepPak chromatography (49). Each bar represents the mean and range of activity determined in two separate transfections of MA-10 cells or two separate cultures of keratinocytes, each assayed in duplicate. Identity and quantification of the assayed product as authentic 1,25(OH)2D3 was confirmed by HPLC. B, Lineweaver-Burke plot of 1{alpha}-hydroxylase activity in MA-10 cells expressing the cloned P450c1{alpha}. C, GC/MS selected-ion recording of five ions diagnostic for the trimethylsilyl ether of 1,25(OH)2D3 (41). The left panel represents authentic 1,25(OH)2D3, and the right panel represents that biosynthesized in MA-10 cells expressing P450c1{alpha}. To conserve space, only the central portions of the recordings showing the major pyro- form at 19.5 min is depicted. The ions found and their intensities and retention times are identical.

 
To prove the identity of the assayed product as authentic 1,25(OH)2D3, a 1,25(OH)2D3 reference standard and the material biosynthesized (230 ng) in transfected MA-10 cells were trimethylsilylated and analyzed by gas chromatography/mass spectrometry (GC/MS). Due to limited amount of material, both samples were analyzed by selected ion monitoring of five representative ions of 1,25(OH)2D3 (see Ref. (41) for published spectrum): m/z 632 (molecular ion), m/z 542 (M -90, loss of trimethylsilanol), m/z 501 (M -131, loss of terminal three carbons of side chain with trimethylsilylated 25-hydroxyl), m/z 452 (loss of two trimethylsilanols), and m/z 131 (base peak, the fragment representing the terminal part of the side chain). Analysis of both authentic and biosynthesized 1,25-(OH)2D3 gave rise to two chromatographic peaks of retention time 19.5 min (major) and 21.3 min (minor), respectively, which represent the pyro- and isopyro- forms of the ring B recyclized hormone (41). Proof of identify of biosynthesized 1,25(OH)2D3 was established by the presence of the appropriate ions, with intensities and retention time identical to those of authentic hormone (Fig. 3CGo). Further confirmation of identity was achieved by obtaining a full mass spectrum of the hormone.

Since the surface area of skin is large and its in vitro 1{alpha}-hydroxylase activity high relative to that in kidney, the question arises as to whether 1,25(OH)2D produced in skin contributes to the maintenance of its serum concentration (27). The 1,25(OH)2D produced in skin is rapidly degraded, and its export from the cell is very inefficient (27). Furthermore, 1{alpha}-hydroxylase activity in skin is much more sensitive to suppression by exogenous 1,25(OH)2D3 than that in kidney (27), which suggests that at physiological concentrations of 1,25-(OH)2D, its production by skin is inhibited. However, the skin can contribute significant amounts of 1,25-(OH)2D to the circulation when renal function is absent or greatly decreased, provided that sufficient amounts of 25-OHD are provided (42, 43).

Mutations in P450c1{alpha} Causing VDDR-1
Keratinocytes from patients with VDDR-1 have not been studied previously. Primary cultures of keratinocytes from a patient with clinical and laboratory features characteristic of VDDR-1 (see Materials and Methods and Table 1Go), failed to convert 25-OHD3 to 1,25-(OH)2D3 in amounts above assay background, whereas keratinocytes from adult and neonatal tissue, examined in an identical fashion, had 1{alpha}-hydroxylase activity ~1000 times and ~3000 times, respectively, above assay background (Fig. 3AGo). Since VDDR-1 is presumed to result from a defect in renal 1{alpha}-hydroxylase, the failure to detect its activity in the patient’s keratinocytes strongly suggests that renal and keratinocyte 1{alpha}-hydroxylase activities are encoded by the same gene, as suggested by our finding of one size of mRNA and expression of the same cDNA in kidney, keratinocytes, and other tissues.


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Table 1. Serum Findings in a Patient with VDDR-1

 
To prove that the P450c1{alpha} gene expressed in keratinocytes is the gene mutated in VDDR-1, we sought inactivating mutations in P450c1{alpha} cDNA prepared from the patient’s keratinocyte mRNA. Using primers based on our human P450c1{alpha} sequence (Fig. 1Go), RT-PCR of mRNA from the patient’s keratinocytes yielded cDNA of ~2.4 kb, demonstrating that the mutation was not a gene deletion or a severe promoter mutation. Sequencing of multiple cDNA clones revealed that all clones had a deletion/frameshift mutation either at codon 211 or at codon 231 (Fig. 4Go), but no clones had both mutations. Each of these mutations leads to the creation of a premature TGA transitional stop codon after reading 233 amino acids. Although the RT-PCR of the patient’s keratinocyte cDNA was performed in two fragments, both mutations lie on the same fragment. Each frameshift created a novel restriction endonuclease cleavage site; digestion of multiple cDNA clones confirmed that all clones carried either one mutation or the other, but no clones carried both, indicating that the patient was a compound heterozygote, having inherited a different frame-shift mutation from each parent (Fig. 4Go). Unfortunately, neither parent was available for study. Thus, the gene encoding the cloned keratinocyte vitamin D 1{alpha}-hydroxylase cDNA is homozygously disrupted in VDDR-1, giving rise to a severely truncated protein that cannot bind heme and thus cannot have P450 catalytic activity. Screening of a panel of rodent/human somatic cell hybrid cell lines demonstrated that the gene for P450c1{alpha} lies on chromosome 12, where the mutation causing VDDR-1 has previously been mapped (25, 26). Thus, our findings that cloned keratinocyte P450c1{alpha} confers authentic 1{alpha}-hydroxylase activity, and that its disruption causes VDDR-1, demonstrate that the keratinocyte and renal 1{alpha}-hydroxylase enzymes are encoded by the same gene and establish the molecular genetic basis of VDDR-1. Furthermore, our studies demonstrate that P450c1{alpha} can be characterized both enzymatically and genetically in cultured keratinocytes.



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Figure 4. Mutations Causing VDDR-1

A, Sequence of cDNA from normal (left) and patient (right) keratinocytes in the region of codon 211. Deletion of a G (arrow) changes the normal sequence TGCCTGGAGGCT to TGCCTGAGGCT, destroying an EcoRII/Bst NI site (CCNGG) and creating the Bsu 36I recognition sequence CCTNAGG. B, Normal (left) and patient (right) sequence in the region of codon 231. Deletion of a C (arrow) changes the normal sequence GTGTCCACG to GTGTCACG, creating the Tsp 45I site GTNAC. C, Confirmation of compound heterozygosity. A 796-bp fragment containing both codon 211 and codon 231 was amplified from patient keratinocyte RNA using primers GF41 and GF21. The four left hand lanes show digestion with Bsu 36I (top panel) and Tsp 45I (bottom panel); the right hand lanes are the same samples undigested and DNA size markers. Two independently prepared samples of patient keratinocyte DNA could be half-digested by extensive treatment with each enzyme, but keratinocyte cDNA from both a normal control and the cDNA clone remained uncut.

 
VDDR-1 is a rare disorder; however, defective 1{alpha}-hydroxylation of vitamin D is characteristic of chronic renal insufficiency and other renal tubular disorders. The cloning of the cDNA for 1{alpha}-hydroxylase, the key enzyme in the metabolic activation of vitamin D, will permit the detailed study of the molecular genetic basis of its regulation in health and disease.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Keratinocytes were isolated from human neonatal foreskins by incubation with 25 U/ml Dispase (Collaborative Biomedical Products, Bedford, MA) in Hank’s basal salt solution, calcium- and magnesium free, containing 0.05 mg/ml gentamycin, at 4 C overnight. The epidermis was separated from the remaining tissue and incubated in 0.05% trypsin for 15 min. Cells were centrifuged, suspended in keratinocyte growth medium (KGM; Clonetics, San Diego, CA) containing 0.07 mM calcium, plated into T-185 plastic flasks, and grown at 37 C, under 5% CO2/95% air. Keratinocytes were isolated from human skin obtained by biopsy by incubation with Dispase and trypsin as above. Cells were suspended in keratinocyte serum-free medium (GIBCO-BRL, Grand Island, NY) containing 5 ng/ml epidermal growth factor (GIBCO-BRL) and 0.09 mM calcium, plated into T-25 plastic flasks, and grown as above. Keratinocytes were passaged at ~80% confluence, and preconfluent cultures between the second to fourth passage used for subsequent procedures. Mouse Leydig MA-10 cells (33) (American Type Culture Collection, Rockville, MD) were maintained in Waymouth medium containing 2.24 g/liter NaHCO3, 0.35 g/liter glutamine, and 10% fortified bovine calf serum (Hyclone, Logan, UT) as previously described (44). Cells between the fourth to tenth passage were plated at a density of 1.5 x 106 per 10-cm plate and incubated for 36 h before transfection.

Construction and Screening of cDNA Library
Using poly(A)+ RNA obtained from preconfluent neonatal keratinocytes, a unidirectional, oligo(dT)-primed, size-selected (>1000 bp) cDNA library was prepared in the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). The unamplified library size was 1.2 x 106 recombinants and was amplified for use in subsequent procedures.

Poly(A)+ RNA (0.5 µg) isolated from neonatal keratinocytes was reverse transcribed into cDNA using 20 µl 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM each of deoxynucleotide triphosphate, 2.5 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (Perkin-Elmer, Foster City, CA), and 2.5 mM of random primers. PCR amplification of the single-stranded cDNA product was performed using Taq polymerase (Perkin-Elmer) and multiple sets of degenerate-sequence oligonucleotide primers based on the relatively conserved regions of ferredoxin-binding and heme-binding regions of P450c24 and P450c25 (17, 18, 19, 20). The primers, 5'-CTSCTSAARGCYGTSATYAARGA-3' and 5'-TGYMTBGGYCGCCGCMTGGCYGAR-3' (where R = G or A, Y = C or T, S = G or C, M = A or C, and B = C, T, or G), yielded a predominant PCR product of ~300 bp. Degeneracy of these primers was decreased slightly based on a partial amino acid sequence of rat 1{alpha}-hydroxylase reported in preliminary form (45). PCR was performed in a Perkin-Elmer DNA Thermal Cycler under the following program: 94 C, 45 sec; 60 C, 45 sec; 72 C, 30 sec for 30 cycles. The PCR product was extracted from gels, purified through a Centricon-100 filter (Amicon, Beverly, MA), subcloned into vector pCR2.1 (Invitrogen), and sequenced by the dideoxy chain-termination technique using Sequenase (United States Biochemical, Cleveland, OH) (46). This clone was used to screen the human keratinocyte cDNA library, yielding 18 positive colonies. After rescreening, representative clones were subcloned into pcDNA3 and sequenced.

5'-RACE
Poly(A)+ mRNA (1 µg) isolated from neonatal keratinocytes was reverse transcribed using 100 ng of primer DR2, 5'-GTGACACAGAGTGACCAGCATAT- 3' (bases 1226–1248 in Fig. 1Go), and Superscript reverse transcriptase (GIBCO-BRL), Escherichia coli DNA ligase (20 U), E. coli DNA polymerase (80 U), and E. coli RNase H (4 U). The resulting double-stranded cDNA was ligated to 10 µg of the double-stranded adaptor GF6, 5'- CCTCACGCTGCAGAAATTCCAGACTGAACCTTGAT-3', corresponding to bases -160/-126 of the human P450scc promoter (47), using 10 U of T4 DNA ligase (Boeringer-Mannheim, Indianapolis, IN) at 4 C for 24 h. This material was PCR amplified using primers GF6 and GF21, 5'-GCAAACATCTGGTCCCAGTCT- 3' (bases 777–797 in Fig. 1Go), with the program: 94 C, 40 sec; 60 C 40 sec; 72 C, 50 sec; for 30 cycles. A second nested PCR amplification was performed using GF6 and GF29, 5'-CAGCCCAAGCGCGAGCCGAG-3' (bases 619–638 in Fig. 1Go) under the same conditions. The resulting 650-bp PCR product was subcloned into pCR2.1 and sequenced. The full-length cDNA was assembled by ligating the RACE clone containing the 5'-end of the cDNA into clone pDL1.9 containing the 3'-end of the cDNA, through the EcoRI and SacII sites.

Characterization of the P450c1{alpha} mRNA
For Northern blotting, 30 µg of total RNA from keratinocytes was size-fractionated on a 0.7% formaldehyde-agarose gel, blotted to a nylon membrane (Amersham, Arlington Heights, IL), and probed with a 340-bp KpnI fragment of the P450c1{alpha} cDNA labeled with [32P]dCTP by random priming. Hybridization was performed for 18 h at 60 C. For analysis of tissue distribution, 1 µg total RNA from various human tissues was used to synthesize cDNA using MMLV-RT and random primers. PCR amplification of the single-stranded cDNA product was performed using primers DF2, 5'-ACGCTGTTGACCATGGC-3', and DR2 for 30 cycles at 94 C, 45 sec; 60 C, 45 sec; 72 C, 30 sec; to yield a 553-bp 1{alpha}-hydroxylase cDNA fragment. PCR also was performed using ß-actin primers to yield a 439-bp actin cDNA fragment as control. The amplified products were separated on 1% agarose gel, blotted to nylon membrane, and probed with [32P]P450c1{alpha} or actin cDNA probes.

Expression of Human P450c1{alpha} cDNA and Characterization of the 1,25(OH)2D3 Product
The full-length P450c1{alpha} cDNA was subcloned into pcDNA3, which contains the cytomegalovirus promoter for expression of DNA in mammalian cells. MA-10 cells at 50–60% confluence were transfected with 2 µg/ml plasmid DNA using adenovirus-mediated transfection modified from that described (48). Seventy-two hours after transfection, cells were transferred to serum-free medium and incubated with 0.1 µM chromatographically purified 25-OHD3 for 1 h. Cells and medium were extracted with acetonitrile, and the 1{alpha}-hydroxylated product was determined in duplicate by RRA after C-18 and silica SepPak chromatography (49). To confirm quantification of the assayed product, after SepPak chromatography a fraction of selected samples was subjected to sequential HPLC using a Zorbax Sil column (0.46 x 25 cm) (Dupont Instruments, Wilmington, DE) at a flow rate of 2.0 ml/min, equilibrated first in isopropanol-hexane (11:89) and then in isopropanol-dichloromethane (5:95) (35). The paired values for each sample, quantitated by RRA, did not differ significantly from one another. The identity of the assayed product as authentic 1,25(OH)2D3 was determined by two methods: MA-10 cells expressing P450c1{alpha} were incubated with 200,000 dpm of chromatographically purified [3H]25-OHD for 1 h. After extraction and SepPak chromatography, the radiolabeled 1{alpha}-hydroxylated product was subjected to sequential HPLC in the solvent systems described. GC/MS of the assayed product also was performed using a Hewlett-Packard 5970 instrument housing a DB 17 fused silica capillary column as described (41).

Patient with VDDR-1
A caucasian female first came to medical attention at the age of 9 months because of respiratory distress. Radiographic examinations revealed osteopenia and metaphyseal abnormalities consistent with rickets, and treatment was initiated with Vitamin D2, 4000 U/day. At age 13 months, the patient’s weight and height were below the fifth percentile for age. The skin was normal but she had developed no deciduous teeth; she had enlargement of the costochondral junctions of the ribs, moderate genu varus, enlargement of the wrists and ankles, and inability to sit, crawl, or stand. These clinical findings and the data in Table 1Go were characteristic of VDDR-1 (21, 22). At age 14 months, therapy with 1,25-(OH)2D3 and calcium carbonate was initiated with doses adjusted to maintain normocalcemia. The patient was able to stand at age 18 months and to walk at 23 months. At 54 months of age, genu varus is minimal, but the patient’s height and weight remain below the 5th percentile for age although growth velocity has been normal.

Characterization of P450c1{alpha} cDNA in VDDR-1
Total RNA was isolated from keratinocytes from the patient with VDDR-1 and from a healthy adult as control. After RT with random primers, cDNA from the patient with VDDR-1 was PCR amplified in two overlapping pieces with primers GF41, 5'-CTGACCCAGACCATGACCCAGACCCTCAA-3', and GF21 for the 5'-half (bases 1–797) and DF2 and GF42, 5'-GGTCAGATAGGCATTAGGGGAAGATGT-3', for the 3'-half (bases 705-1619). The PCR products were cloned into pCR2.1 and several clones were picked for sequencing. Complementary cDNA from the healthy control was PCR amplified using primers GF41 and GF42, the PCR product was cloned into pCR2.1, and two clones were picked for sequencing. To confirm the mutations detected by DNA sequencing, the RT-PCR products from the patient and normal control were digested with either Tsp 45I (New England Biolabs, Beverly, MA) or Bsu 36I (Promega, Madison, WI), and the products were separated on a 1% agarose gel. The chromosomal location of the P450c1{alpha} gene was determined by PCR amplification of genomic DNA samples in NIGMS rodent/human somatic cell hybrid mapping panel 2 (50, 51) (Coriell Institute, Camden, NJ). PCR, performed with primers GF41 and GF21, yielded a 2-kb genomic fragment.


    ACKNOWLEDGMENTS
 
We thank John Forsayeth for help with adenovirus-mediated transfection and Sally Pennypacker for help with keratinocyte culture.


    FOOTNOTES
 
Address requests for reprints to: Anthony A. Portale, M.D., University of California San Francisco, 533 Parnassus Avenue, Room U-585, San Francisco, California 94143.

Dr. Portale was supported by the Department of Pediatrics, grants from the University of California, San Francisco Academic Senate and Research Evaluation and Allocation Committee, and generous gifts from the Carmel David Trust. Dr. Miller was supported by NIH Grants DK-37922 and DK-42154 and a grant from the March of Dimes. Dr. Fu was supported by Pediatric Endocrinology Training Grant DK-07161 and Dr. Lin by the UCSF Child Health Research Center Grant HD-28825. Dr. Bikle was supported by NIH Grants RO1 AR-38386 and PO1 AR-39448.

Received for publication August 20, 1997. Revision received September 16, 1997. Accepted for publication September 17, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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