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Original Studies |
-Hydroxylase Gene Product in Pseudovitamin D Deficiency Rickets, Including That with Mild Clinical Manifestation
Institute of Molecular and Cellular Biosciences (S.Ki., A.M., K.T., S.Ka.), and the Department of Laboratory Medicine (S.F.), The University of Tokyo, 113-0032 Tokyo; the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University (T.S., K.I.), 606-8502 Kyoto; the Department of Pediatrics, Okayama University Medical School (Y.S.), 700-0914 Okayama; the Department of Pediatrics, Osaka University Medical School (M.S.), 565-0871 Osaka; the Department of Pediatrics and Child Health, Kurume University Medical School (S.Y.), 830-0011 Fukuoka; the Division of Metabolism, Chiba Children’s Hospital (M.T.), 266-0007 Chiba; the Department of Pediatrics, Chiba University School of Medicine (H.N.), 260-8677 Chiba; and Core Research for Evolutional Science and Technology, Japan Science and Technology Corp. (S.Ka.), 332-0012 Saitama, Japan
Address all correspondence and requests for reprints to: Shigeaki Kato, Ph.D., Institute of Molecular and Cellular Biosciences, University of Tokyo, 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. E-mail: uskato{at}hongo.ecc.u-tokyo.ac.jp
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-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 1-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
1-hydroxylase gene mutations that totally abolish the enzyme
activity. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D [1,25-(OH)2D]
with normal to elevated levels of 25-hydroxyvitamin D (25OHD) (4, 5).
As massive doses of vitamin D or 25OHD3 and
physiological doses of 1-(OH)D3 or
1,25-(OH)2D3 are
necessary for remission of the rickets (6), it was suspected that PDDR
is the consequence of a deficit in renal 25-hydroxyvitamin
D3 1-hydroxylase.
Although PDDR is a rare disease, patients with mild clinical symptoms
have been reported (7). Moreover, the serum
1,25-(OH)2D levels in the untreated state vary
with some patients reported to have normal serum
1,25-(OH)2D levels (2). Such mild clinical
manifestations of the disease are suspected to be caused by reduced
1-hydroxylase activity.
We recently isolated mouse 1-hydroxylase complementary DNA (cDNA)
(8) and subsequently isolated human cDNA and gene (cDNA, AB005989;
gene, AB005990) (9), which are independently cloned by other groups
(10, 11). By analysis of four unrelated Japanese patients with typical
PDDR, we identified four homozygous inactivating missense mutations in
this gene (9). Fourteen different inactivating mutations including
deletions and duplications in patients with PDDR from other ethnic
groups are also reported (10, 12, 13). However, no studies have been
performed on patients with mild phenotype to date, and it is unclear
whether such patients have residual activity of
1-hydroxylase.
In this study to investigate the molecular defects associated with the
phenotypic variation, we analyzed 1-hydroxylase gene in a patient
with a mild clinical manifestation together with five typical cases of
PDDR. Moreover, we analyzed the enzyme activity using a sensitive
assay, by overexpressing the enzyme in Escherichia coli
cells, to assess the subtle residual enzyme activity of the mutants. We
could find residual enzyme activity neither in the patient with mild
phenotype nor in the other five typical patients. We further discuss
the cause of clinical heterogeneity and the detectable serum
1,25-(OH)2D levels in these patients.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Six Japanese patients clinically diagnosed as having PDDR were
entered into this study. The patients were all unrelated to each other
as well as to those we analyzed previously (9). Some of their parents
and siblings were also studied. The study was approved by the
appropriate institutional review committees, and all subjects gave
informed consent. The clinical data for these patients are summarized
in Table 1
. Concentrations of serum
calcium, phosphate, alkaline phosphatase (AlP), 25OHD, and
1,25-(OH)2D were measured as previously
described (14). Serum PTH levels were measured by RIA using two-site
immunoradiometric assay kits (15).
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OHD3.
Patient 2 presented with failure to thrive and standing delay at 1 yr,
10 months. He had prominent rachitic rosary, kyphosis, and genu valgus.
He had hypocalcemia, a high serum AlP level, and typical
roentgenographic features of rickets with several psuedofractures. He
was first treated with 150,000 U/day vitamin D, then successfully with
3 µg/day 1OHD3.
Patient 3 (16) presented with failure to thrive at 1 yr, 5 months and
walking delay at 2 yr, 2 months. She had general convulsions when she
suffered from measles at age 1 yr, 9 months. Her height was -2.5
SD and she had craniotabes, rachitic rosary, bowing of
legs, and joint enlargements at 2 yr, 1 month. She had hypocalcemia and
a high serum AlP level, and showed rachitic changes on roentgenography.
She was treated with 600,000 U vitamin D3, three
times per week, im. Her serum 1,25-(OH)2D
level was 11.3 pg/mL without therapy at age 10 yr.
Patient 4 (17) was first found to have high serum alkaline phosphatase
activity by a laboratory test before immunization at age 1 yr, 1 month.
She had hypocalcemia, hypophosphatemia, high serum levels of PTH, and
generalized aminoaciduria. She had bowing of the legs, and rachitic
change was evident on roentgenography. She was successfully treated
with 0.1 µg/kg·day 1OHD3. Her serum
1,25-(OH)2D level during 3 weeks cessation of
therapy was 14 pg/mL and did not increase with stimulation by PTH or by
low calcium diet.
Patient 5 presented with failure to thrive at 1 yr, 1 month. His
laboratory data were typical for PDDR. He was successfully treated with
1.6 µg/day 1OHD3.
Patient 6 (18) presented with delay of walking at age 1 yr, 6 months.
She had hypocalcemia and a high serum AlP level, and rachitic change
was evident on roentgenography. She was successfully treated with
20,000 U vitamin D2/day at maximum. However, she
no longer presented symptoms on cessation of treatment from 3 yr, 8
months of age. At age 13 yr, she presented with knee pain and was found
to have rachitic changes on roentgenography. Her height was within the
normal range (-1 SD), and she had neither bowing of legs
nor muscle weakness. Her serum calcium level was 5.8 mg/dL, and AlP and
PTH levels were high. Her serum level of 25OHD was high, whereas that
of 1,25-(OH)2D was low. Normalization of serum
calcium level and bone roentgenograms were obtained with 3 µg/day and
maintained with 1 µg/day 1(OH)D3.
The parents of patient 2 were first cousins, and the parents of patient 3 were second cousins, whereas the other four parents denied consanguinity.
PCR amplification and sequence analysis of 1-hydroxylase gene in
PDDR patients
The genomic DNA of the patients and all family members were
extracted from peripheral white blood cells. Nine exon segments of the
1-hydroxylase gene were amplified from genomic DNA using specific
primers derived from the intronic sequences, and the products were
sequenced as previously described (9). In the case of compound
heterozygous mutations, the mutations were further verified by
subcloning the PCR products using a TA cloning kit
(Invitrogen) and sequencing more than eight clones
(19).
Plasmid construction
For 1-hydroxylase activity assay using vitamin D receptor
(VDR)-mediated transcriptional activation method in mammalian cells,
1-hydroxylase expression plasmid for mammalian cells, expression
plasmid for VDR ligand-binding domain fused to GAL4 DNA-binding domain,
and a chloramphenicol acetyltransferase (CAT) reporter plasmid
containing GAL4 upstream activating sequence and ß-globin promoter
(17M2-G-CAT) were used. Wild-type human 1-hydroxylase cDNA in the
mammalian expression vector pcDNA3 (Invitrogen, San Diego,
CA), the expression plasmid for the ligand-binding domain of VDR
(VDR-DEF) fused to the GAL4 DNA-binding domain [GAL4-VDR(DEF)], the
reporter plasmid 17M2-G-CAT, and adrenodoxin and adrenodoxin reductase
expression plasmids were prepared as previously described (9). The
desired mutations were introduced into the wild-type human
1-hydroxylase expression plasmid as previously described (9). The
wild-type minigene was constructed by inserting
SpeI-digested genomic fragment (corresponding to introns
1–6) of subcloned 1-hydroxylase genome (20) into the
XbaI site of expression vector pcDNA3. Minigene containing
the splice donor site mutation was constructed using the PCR products
amplified from DNA of patient 6. For 1-hydroxylase activity analysis
using the E. coli expression system, 1-hydroxylase
expression plasmid for E. coli cells was used. The
expression plasmid for the putative mature form of human
1-hydroxylase, pKMat-h1, was constructed by inserting human
1-hydroxylase cDNA fragment corresponding to codons 33–508 into
E. coli expression vector pkk233–3 (21). The mutant (P143L,
D164N, T321R, R389C, W433X) plasmids in pkk233–3 were constructed by
inserting the corresponding fragments of mutant 1-hydroxylase cDNA
created in pcDNA3. The complete sequence of each construct was verified
to ensure that no extraneous mutations were introduced during PCR.
Enzyme activity analyzed by the VDR-mediated transcriptional activation method
1-Hydroxylase activity assay was first analyzed by
overexpressing mutant 1-hydroxylase in mammalian cells and detecting
the conversion of 25OHD3 to
1,25-(OH)2D3 by the
trans-activation function of VDR, as previously described
(9). Briefly, COS-1 cells were transiently transfected with 0.5 µg
GAL4-VDR(DEF), 1 µg 17M2-G-CAT reporter plasmid, 0.2 µg each of the
adrenodoxin and adrenodoxin reductase expression vectors, 3 µg of
either wild-type or mutant 1-hydroxylase expression vector, and 1
µg ß-galactosidase expression plasmid pCH110 (Pharmacia Biotech, Madison, WI) using lipofection reagent (Life Technologies, Inc., Gaithersburg, MD). Twelve hours after
transfection, 25OHD3 (10-7
mol/L) was added to the medium, and after an additional 36 h, cell
extracts were prepared and used for CAT assays.
Enzyme activity analyzed by E. coli expression system
1-Hydroxylase activity was analyzed by overexpressing mutant
1-hydroxylase in E. coli cells and detecting the
converted 1,25-(OH)2D3
by high performance liquid chromatography (HPLC), as previously
described (21). Briefly, each of the wild type pKMat-h1 or the
mutants were transformed to E. coli JM109. The recombinant
cells were gently shaken at 26 C for 24 h. 1-Hydroxylase
activity was measured in the reconstituted system containing the
membrane fraction of the cells containing 2 mg protein, 5.0 µmol/L
25OHD3, 0.5 µmol/L NADPH-adrenodoxin reductase,
and 5 µmol/L adrenodoxin at 30 C. The reaction was initiated by the
addition of NADPH, and aliquots of the reaction mixture were collected
after 3 h and extracted with chloroform-methanol. The samples were
subjected to HPLC under the conditions described. The concentrations of
25OHD3 and 1,25-(OH)2D3
were estimated from their molar extinction coefficient (1.80 x
104 mol/L·cm at 264 nm).
Transfection of minigene and RT-PCR
To determine the transcription product of a detected splice site mutation, minigene constructs were transfected into mammalian cells, and the transcripts were analyzed by RT-PCR. COS-1 cells maintained in DMEM supplemented with 10% calf serum in 100-mm dishes were transiently transfected with 5 µg of the minigene plasmid constructed from either normal control or patient 6 DNA, using the calcium phosphate method. Twenty-four hours after transfection, the medium was washed with PBS and renewed. Forty-eight hours after transfection, the cells were washed twice with PBS and collected. Polyadenylated ribonucleic acid (RNA) was extracted from these cells using QuickPrep Micro messenger RNA (mRNA) purification kit (Pharmacia Biotech), followed by DNase digestion. mRNA (0.1 µg) was reverse transcribed using the Superscript preamplification system (Life Technologies, Inc.) according to the instruction manual using oligo(deoxythymidine)12–18 primer (15). PCR was performed in a 25-µL reaction medium with 0.5 µL cDNA, using sense primer (5'-GCTTCTCGCCCTGGACGGAG-3') located in exon 2, antisense primer (5'-CATCGCCATGGTCAACAGCGTGGACAC-3') located in exon 4, and AmpliTaq Gold under the following conditions: initial denaturation at 95 C for 9 min, and 23 cycles of 95 C for 30 s, 60 C for 30 s, and 72 C for 1 min. The RT-PCR products were run on an 1.5% agarose gel and visualized. The corresponding PCR products were purified and sequenced directly.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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All six patients were diagnosed as having PDDR from the clinical
features. To determine whether these patients with PDDR have mutations
in the 1-hydroxylase gene, we analyzed genomic DNA obtained from the
leukocytes of these patients. Each of the nine exons including the
exon-intron boundaries of the 1-hydroxylase gene were PCR amplified
and directly sequenced in both directions. As shown in Fig. 1A, all of the patients were revealed to
have mutations in the 1-hydroxylase gene in both alleles. Patients
1, 2, and 3 were homozygous, and patients 4, 5, and 6 were compound
heterozygous for the mutations. Four novel missense mutations, P143L
(CCG to CTG) and D164N (GAC to AAC) in exon 3, T321R (ACG to AGG) in
exon 5, and R389C (CGT to TGT) in exon 7, were identified. A homozygous
nonsense mutation, W433X (TGG to TGA) in exon 8, was detected in
patient 3. This residue lies on the 5'-side of the conserved
heme-binding domain and is considered to code a truncated protein that
lacks the functional domain. The R107H mutation, previously identified
as homozygous in an unrelated patient, was found to be heterozygous in
patients 4 and 5.
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). The mother of patient 5 was heterozygous for R107H. The father of
patient 6 carried the T321R heterozygous mutation; however, the mother
was completely normal for the 1-hydroxylase gene. In addition, the
mother of patient 1 did not have any mutation. The possibility of a
large deletion of this gene in patient 1 and the mother was
excluded by Southern blotting analysis of their genomic DNA. These
findings suggest that although most of the mutations were inherited
recessively, the intron mutation in patient 6 and the D164N mutation in
patient 1 might be de novo mutations. A splice site mutation in intron 3 results in intron retention
A splice site mutation, guanine to adenine transition in
the first nucleotide of intron 3 (IVS3 + 1 g
a), was detected in
patients 5 and 6, both of whom were heterozygous for it. To determine
the transcription product of the donor splice site mutation, we
conducted an expression study using a minigene construct. Wild-type and
mutant minigenes that span exons 2–6 were constructed in an expression
vector. These plasmids were transiently transfected to COS-1 cells, and
the transcripts were analyzed by RT-PCR using primer pairs located in
exons 2 and 4. As shown in Fig. 2A, the
RT-PCR product of wild-type plasmid was 209 bp, as expected. However,
that of mutant plasmid was 294 bp, 85 bp longer than the wild-type
product. Similar results were obtained using other primer pairs located
in exons 2 and 6 and in exons 3 and 6. We confirmed by increasing the
number of cycles of RT-PCR and Southern blotting analysis that none of
the mutant genes was spliced correctly (data not shown). To determine
the structure of the abnormally spliced mRNA, we performed direct
sequencing analysis of the RT-PCR products. The results indicated that
the wild-type product consisted of exons 2–6, whereas the mutant
product contained intron 3 (Fig. 2B).
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-hydroxylase gene causes altered translation and results in a
premature stop codon at 63 bp downstream from the end of exon 3. The
putative C-terminal-truncated 1-hydroxylase, which has only 197 of
508 amino acids is considered to have no enzyme activity.
No 1-hydroxylase activity in the missense and the nonsense
mutants in the typical cases of PDDR
1-Hydroxylase activity of the newly identified missense and
nonsense mutants was analyzed by VDR-mediated CAT assay (9). Mutations
P143L, D164N, R389C, and W433X were introduced into the
1-hydroxylase expression plasmid by site-directed mutagenesis. In
the absence of 1-hydroxylase expression plasmid,
25OHD3 did not show transcriptional activation of
VDR (Fig. 3, lane 1). Addition of
wild-type 1-hydroxylase, however, efficiently induced the activation
(Fig. 3, lane 2). In contrast to wild-type 1-hydroxylase, none of
the mutants induced CAT activity (Fig. 3, lanes 3–6). This result
indicates that the three novel missense mutations and a nonsense
mutation found in the typical patients abolish 1-hydroxylase
activity.
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That patient 6 was asymptomatic for 10 yr without any treatment
and had a near-normal level of serum
1,25-(OH)2D suggests she had a relatively mild
form of the disease. Usually patients with PDDR have recurrence of
rickets after cessation of therapy within several months (16, 17).
Patient 6 was compound heterozygous for the splicing mutation and a
unique missense mutation T321R (Fig. 1). As the splicing mutation,
which was also found in a typical patient (patient 5), results in
intron retention and a premature stop codon, we suspected that T321R
mutant has some residual enzyme activity. We could not detect any
enzyme activity in T321R by the VDR-mediated enzyme assay (Fig. 3, lane
7), and considered that the small amount of converted
1,25-(OH)2D3 may not be
able to induce transactivation function of VDR. Therefore, we employed
another assay overproducing 1-hydroxylase in E. coli
cells. This method has been used for multiple P450 enzymes, including
CYP24 (23) and CYP27 (24), and has been revealed to be very useful for
kinetic analysis of mitochondrial P450 enzymes. We previously showed
that mouse 1-hydroxylase expressed in E. coli catalyzes
25OHD3 to
1,25-(OH)2D3 with a
Km of 2.7 µmol/L (21), which is similar to that
in another report (10), and it seems to induce a more efficient
conversion than that expressed in mammalian cells. Using this
reconstituted system containing the membrane fraction of the
recombinant E. coli cells, we further examined the enzyme
activity of T321R mutant together with those of the other mutants.
However, even by this assay method, we could not detect enzyme activity
in T321R (<1% of the wild type) or in any other mutant (Fig. 4; data not shown). This result indicates
that all of the mutations described in this study abolish the enzyme
activity.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylase gene mutations
and found all six to have either homozygous or compound heterozygous
mutation in the 1-hydroxylase gene. The mutations varied, and we
identified a splicing mutation of the 1-hydroxylase gene for the
first time.
Patient 6, who was considered to have a relatively mild form of the
disease, was compound heterozygous for T321R and a splicing mutation,
and as the splicing mutation results in premature stop codon, we
speculated that residual enzyme activity lies in the T321R mutation.
However, analysis of the enzyme activity of T321R mutant revealed that
this mutation totally abrogates the activity to the same level as the
other missense and nonsense mutations. This result is supported by the
fact that the corresponding residue, Thr252, in
P450cam plays a crucial role in the catalysis (25), and that same
threonine to arginine transition in the corresponding residue of
CYP11B1 detected in a patient with typical 11ß-hydroxylase deficiency
also abolishes the enzyme activity (26). Accordingly, although it was
proved that the mild type patient also had 1-hydroxylase gene
mutation, there seems to be no residual activity in the mutant
1-hydroxylase.
Serum 1,25-(OH)2D levels in most of the
patients analyzed, including those in our previous report, were
detectable and were near normal in patient 6 (9). However, each of the
mutations found in these patients totally abolished the enzyme
activity. We cannot exclude the possibility that these mutants have
minimal residual enzyme activity that cannot be detected by our assay
system and exert the activity when markedly overexpressed in
vivo. However, as patients with a nonsense mutation in the 5'-site
of the heme-binding domain (patient 3) and with frameshift mutations
(10, 12) also had detectable serum 1,25-(OH)2D
levels, this may not be the case. There may be several reasons for the
discrepancy between serum 1,25-(OH)2D levels
and 1-hydroxylase activity of the mutants. First, 1-hydroxylase
activity may be also exerted in either renal or extrarenal tissues by
enzymes other than the cloned mitochondrial 1-hydroxylase. It has
been reported that enzyme for 25-hydroxylation also has activity for
1-hydroxylation (27). Another report suggests the presence of
1-hydroxylase activity in microsomes of the kidney and in the liver
(28, 29). Although it may be weak in the normal state, the activity of
such compensatory enzymes may be potentiated in the patients and may
produce 1,25-(OH)2D. Second,
1,25-(OH)2D may be detectable by exogenous
factors, such as oral intake of foods that contain 1-hydroxylated
vitamin D metabolites. It is known that human and cow milk and fish
contain 1,25-(OH)2D (30, 31). Third, some
other vitamin D metabolites may be measured as
1,25-(OH)2D by the RRA. In an abnormal state
of vitamin D metabolism in patients with PDDR, some metabolites,
unknown or known, may be produced and detected by the assay as
1,25-(OH)2D. It is suspected that the clinical
variations may also be due to the presence of such compensatory enzymes
or to the exogenous factors.
In this and our previous study (9), we have identified a total of 10 types of mutations in 10 families of Japanese decent. We also found de novo mutations for the first time in this study. These results suggested that there is no founder effect in Japanese patients.
We have identified eight missense mutations that totally abolish the
enzyme activity widely distributed from exons 2–6. Naturally occurring
missense mutation in the 1-hydroxylase gene seems to be more
variable than that in other mitochondrial P450 enzymes (32). There are
conserved functional domains, presumably for ferredoxin binding and
heme binding in human 1-hydroxylase (9, 10). However, most of the
missense residues are not located in these domains. Each of these eight
residues is highly conserved among several mitochondrial P450 enzymes
(8, 32, 33, 34). This fact suggests that these residues are important for
the function of P450 enzymes in general. Thus, analysis of the function
of the missense mutants found in the 1-hydroxylase gene may lead to
the elucidation of other functional regions important for P450 enzyme
activity.
In conclusion, we have analyzed six, in addition to four previously
reported, patients with PDDR and found that this disease is
indiscriminately caused by mutations in the 1-hydroxylase gene.
Furthermore, we found that a patient with mild symptoms also had
mutations that totally abolish the enzyme activity. We suspect that the
phenotype of PDDR may be modified by endogenous factors with
1-hydroxylase activity or by exogenous factors.
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Acknowledgments |
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Received April 5, 1999.
Revised June 17, 1999.
Accepted July 9, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylase and vitamin D synthesis. Science. 277:1827–1830.-hydroxylase gene in patients with
pseudovitamin D-deficiency rickets. N Engl J Med. 338:653–661.-hydroxylase and mutations causing
vitamin D-dependent rickets type I. Mol Endocrinol. 11:1961–1970.-hydroxylase,
P450c1. DNA Cell Biol. 16:1499–1507.[Medline]
-hydroxylase deficiency in 17 families. Am J Hum
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-hydroxylase mutations in French-Canadians with vitamin D
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-hydroxylase gene by parathyroid hormone,
calcitonin and 1,25-(OH)2D3 in intact animals. Endocrinology. 140:2224–2231.-hydroxylase gene confers positive and
negative responsiveness to PTH, calcitonin, and
1,25-(OH)2D3. Biochem Biophys Res Commun. 249:11–16.[CrossRef][Medline]
-hydroxylase expressed in
Escherichia coli. Eur J Biochem. 259:731–738.[Medline]
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D3. Proc Natl Acad Sci USA. 91:10014–10018.-hydroxylation of 25-hydroxyvitamin D3 in
piglets suffering from pseudo vitamin D-deficiency rickets, type I.
Biochim Biophys Acta 1407:234–242.
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