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Original Studies |
Department of Orthopedic Surgery, Osaka University Medical School (Y.F., K.N., N.Y., E.K., T.O.), Suita City, Osaka 565-0871; the Department of Orthopedic Surgery, Osaka Medical Center for Maternal and Child Health (Y.M.), Izumi-City, Osaka 594-1101; the Department of Orthopedic Surgery, Osaka National Hospital (K.H.), Osaka City, Osaka 540-0006, and the Department of Orthopedic Surgery, Hyogo Nojigiku Medical Center for Disabled Children (R.S.), Kobe-City, Hyogo 651-2215, Japan
Address all correspondence and requests for reprints to: Dr. Yoshi Fujita, Department of Orthopedic Surgery, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: yfujita{at}ort.med.osaka-u.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In this study we analyzed the sequence of the coding region of the CK gene in four patients from unrelated families and identified three different mutations of the CK gene. Three patients had mutations altering the amino acid sequence of the mature peptide, and one patient had an amino acid substitution in the signal peptide region of this protein. We examined the expression of this mutant CK protein in COS-7 cells using a transient expression assay. The signal peptide mutation detected in our patient was a new type of mutation disrupting the function of CK protein.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Four Japanese patients were studied. Patients 1, 3, and 4 were
men, aged 32, 36, and 37 yr, respectively. Patient 2 was a 54-yr-old
woman. They were all unrelated. Patients 1 and 4 were the offspring of
the nonconsanguinous parents and were unaware of any congenital
anomalies in their families. Parents of patients 2 and 3 were the first
cousins, respectively. All four patients showed characteristic clinical
findings, such as short stature, frequent fractures, osteosclerosis,
skull deformities, and acroosteolysis of the distal phalanges. There
was no phenotypical difference among these cases, and they all showed
similar radiographic features (Fig. 1
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Whole blood was obtained from each patient as well as from the parents and sister of patient 4. Blood samples from 80 normal individuals were used as a control. Genomic DNA was extracted from whole blood samples using the DNA Extraction Kit (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations. CK genomic DNA were amplified in 6 overlapping fragments by genomic DNA PCR. A 1.6-kbp product, corresponding to nucleotides (nt) 29–225 interrupted by intron 1, was amplified using the forward primer (PR-1) 5'-TAA ATC TAG CAC CCC TGA TGG-3' and the reverse primer (PR-2) 5'-CTT GTT GTT ATA TTG CTT CCT GTG GG-3'. A 0.9-kbp product, corresponding to nt 139–471 interrupted by introns 2 and 3, was amplified using the forward primer (PR-3) 5'-GTG AGC TTT GCT CTG TAC CCT-3' and the reverse primer (PR-4) 5'-TCG ATA GTC GAC AGA GTC TGG-3'. A 2.0-kbp product, corresponding to nt 362–697 interrupted by intron 4, was amplified using the forward primer (PR-5) 5'-TGG TTC AGA AGA TGA CTG GAC-3' and the reverse primer (PR-6) 5'-CAG AGT CAA TAC CCC GGT TCT-3'. A 4.6-kbp product, corresponding to nt 580–881 interrupted by intron 5, was amplified using the forward primer (PR-7) 5'-CTC TTA AAT CTG AGT CCC CAG-3' and the reverse primer (PR-8) 5'TAA AAC TGG AAG GAG GTC AGG-3'. A 0.5-kbp product, corresponding to nt 756–980 interrupted by intron 6, was amplified using the forward primer (PR-9) 5'-GGC AGC TAA ATG CAG AGG GTA-3' and the reverse primer (PR-10) 5'-CAG TGC TTG TTT CCC TTC TGG-3'. A 2.5-kbp product, corresponding to nt 917-1155 interrupted by intron 7, was amplified using the forward primer (PR-11) 5'-GCG ATA ATC TGA ACC ATG CGG-3' and the reverse primer (PR-12) 5'-TCG TTA CAC TGC ACC ATC GTG-3'. The PCR conditions to amplify the 4.6-kbp product were 30 cycles of 96 C for 1 min, 65 C for 1 min, and 72 C for 3 min. The other products were amplified for 30 cycles at 96 C for 1 min, 63 C for 1 min, and 72 C for 2 min. Each product was used for direct sequencing or subcloned into the pCR 2.1 vector using an Original TA Cloning Kit (Invitrogen, San Diego, CA). Positive clones were selected, and isolated plasmids were used for sequencing on an automated fluorescence-based sequencer (ABI, Perkin Elmer Corp., Foster City, CA).
Ribonucleic acid (RNA) extraction, RT-PCR, and PCR amplification of CK complementary DNA (cDNA)
RNA was prepared from fracture callus and cultured bone marrow
cells of patient 4. Callus was harvested at the time of surgery for a
fractured right femur. Total RNA was extracted with guanidinium
thiocyanate followed by centrifugation in CsCl. Bone marrow blood
samples was also obtained from patient 4, and bone marrow cells were
cultured for 14 days in 
MEM (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10-8 mol/L
1,25-hydroxyvitamin D3, 10% FBS, and 10%
autologous bone marrow supernatant to induce the osteoclastic phenotype
(19, 20). Total RNA was extracted using the phenol/guanidine
isothiocyanate method (Trizol reagent, Life Technologies, Inc.). cDNA was synthesized using oligo-(deoxythymidine) primers
and the Superscript II preamplification system (Life Technologies, Inc.). CK cDNA was amplified in two overlapping
fragments by cDNA PCR. A 569-bp product, corresponding to nt 71–639 of
exons 1–5, was amplified using the forward primer (PR-13) 5'-GAA ACG
AAG CCA GAC AAC AG-3' and the reverse primer (PR-14) 5'-TCC ACA GCC ATC
ATT CTC AG-3'. A 576-bp product, corresponding to nt 563-1138 of exons
5–8, was amplified using the forward primer (PR-15) 5'-AGA AGA AAA CTG
GCA AAC TC-3' and the reverse primer (PR-16) 5'-CGT GGA AGA AAT GGA AGA
GC-3'. In addition, a 1068-bp product, corresponding to nt 71–1138 of
exons 1–8, was amplified using forward primer PR-13 and reverse primer
PR-16. The PCR conditions to amplify the 569- and 576-bp products were
30 cycles of 96 C for 1 min, 60 C for 1 min, and 72 C for 1 min. The
PCR conditions for the 1068-bp product were 30 cycles of 96 C for 1
min, 60 C for 1 min, and 72 C for 2 min. The amplified products were
subjected to direct sequencing or were subcloned into pCR
2.1 vector. Positive clones were selected, and
the isolated plasmids were used for sequencing.
Transient expression of wild-type and mutant CK genes tagged with FLAG in COS-7 cells
The entire cDNA of the nt 131C mutant and wild-type CK genes tagged at the COOH-terminus with a FLAG epitope was constructed as follows. First, a PCR fragment containing the full coding sequence of the nt 131C mutated CK was amplified from the cDNA of patient 3 with a sense primer containing an EcoRI site (PR-17, 5'-CCG GAA TTC GAA ACG AAG CCA GAC AAC AGA TTT CC-3'), and an antisense primer containing a FLAG sequence, termination codon, and EcoRI site (PR-18, 5'-CCG GAA TTC GAG TCA CTT GTC ATC GTC GTC CTT GTA GTC CAT CTT GGG GAA GCT GGC CAG GTT GGC-3'). A single point mutation (C to T) was introduced at nt 131 of the mutant CK cDNA using oligonucleotide-directed mutagenesis (21). Consequently, wild-type CK cDNA was obtained. The wild-type and mutant CK cDNAs tagged with FLAG were subcloned in pcDNA3.1 (Invitrogen) and were designated pCK-FLAG and pL9PCK-FLAG. All DNA constructs were verified by sequencing.
COS-7 cells were maintained and propagated in DMEM containing 10% FBS (Life Technologies, Inc.) at 37 C in 95% room air and 5% CO2 with a humidity of 100%. One day before transfection the cells were trypsinized and plated into six-well cell culture dishes. When the cells reached a density of 70–80%, they were transfected with 2 µg plasmid DNA using 3 µl FuGene 6 Transfection Reagent (Roche Molecular Biochemicals, Mannheim, Germany).
Immunohistochemical staining and Western blotting
At 48 h after transfection, COS-7 cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.04% Triton X-100, and incubated with anti-FLAG Bio-M2 antibody (10 µg/ml; Sigma, St. Louis, MO) at room temperature for 1 h. This was followed by incubation with avidin labeled with fluorescein isothiocyanate (10 µg/ml; EY Laboratories, Inc., San Mateo, CA) at room temperature for 1 h. The cells were washed in PBS, mounted in glycerol, and examined with a fluorescence microscope. In addition, after incubation with the primary antibody, the immunoreaction was visualized by incubation with avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA) for 30 min and with diaminobenzidine solution containing 0.01% H2O2 for 10 min. The cells were examined under a light microscope.
For Western blotting, the cells were harvested at 48 h after transfection, and COS-7 cell homogenates were resolved on 4–20% gradient polyacrylamide gel and transferred to nitrocellulose filters. FLAG-tagged CK protein was detected using anti-FLAG Bio-M2 antibody. The approximate molecular size of the protein was determined by comparison with ECL molecular weight standards (Amersham Pharmacia Biotech, Aylesbuy, UK).
In all cases, mock-transfected COS-7 cells were used as the control.
mRNA extraction and RT-PCR
At 48 h after transfection, cells were harvested, and mRNA was extracted using the Micro-FastTrack kit (Invitrogen). RNA was reverse transcribed, and the cDNA was immediately amplified using forward primer PR-17 and reverse primer PR-19 (5'-CTT GTC ATC GTC GTC CTT GTA GTC-3'), a primer pair specific for FLAG-tagged CK. The PCR conditions were 30 cycles of 94 C for 1 min, 63 C for 1 min, and 72 C for 2 min. PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase gene was also performed.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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CK genomic DNA was amplified as six overlapping fragments by the genomic DNA PCR. After cloning into the pCR 2.1 vector, the entire coding region of CK genomic DNA was sequenced.
Patient 1 revealed the presence of a C to T transition of nt 935 in
exon 7. Direct sequencing of genomic DNA showed that the patient was
homozygous for this mutation. This point mutation resulted in the
substitution of an Ala277 by a Val (A277V) in the
mature CK polypeptide (Fig. 2a).
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). Direct genomic sequencing showed that the patient was also
homozygous for this mutation, which resulted ina frame shift from codon
142 and a premature stop at codon 160.
Direct sequencing of genomic DNA indicated that patient 4 was
homozygous for a T to C transition at nt 131 in exon 2, which replaced
the normal Leu9 (CTA) with a Pro (CCA) (L9P) in
the signal peptide of CK (Fig. 3). No
mutations was found in the other amplified DNA fragments. The
patient’s parents were heterozygous for this mutation, and the
unaffected sister was homozygous for the normal gene (Fig. 3). This
base substitution was not detected in any of the 80 normal controls by
direct sequencing of this region of the CK gene, suggesting that it was
unlikely to be a neutral polymorphism. Sequencing of the cDNA of
patient 4 transcribed from fracture callus and from cultured bone
marrow cells showed the same mutation. No other mutations were found in
the amplified cDNA fragments.
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Immunohistochemical staining using a monoclonal FLAG antibody
revealed immunoreactivity in the cytoplasm of the cells transfected
with pCK-FLAG (Fig. 4a). No immunoreactivity
was detected in cells transfected with pL9PCK-FLAG and mock-transfected
COS-7 cells (Fig. 4, b and c). The same results were obtained by
avidin-biotin-peroxidase immunohistochemistry (data not shown).
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). In homogenates of COS-7 cells transfected
with pCK-FLAG, a protein band of 37 kDa, the approximate molecular mass
for the pro-CK, was detected. In homogenates of COS-7 cells transfected
with pL9PCK-FLAG, only a trace of this protein was detected, and no
immunoreactivity was detected in homogenates of mock-transfected COS-7
cells. To determine whether the reduced expression of CK was due to a
defect at the transcriptional or translational level, we performed
RT-PCR analysis of RNA isolated from COS-7 cells transfected with
normal CK or the L9P mutant (Fig. 6). The
expected 1061-bp product was amplified from both wild-type and mutant
mRNA. No difference in the density of the amplified PCR products was
observed between the pCK-FLAG and pL9PCK-FLAG transfectants.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) (8). This was the third mutation
reported at nt 935, after two reports of an Ala to Gln substitution by
C to A transition at the same site, suggesting that nt 935 is a
mutational hot spot for pycnodysostosis (9). No A277V mutation was
detected in analysis of more than 50 normal individuals by Hou et
al. They also performed the expression analysis and showed that
the A277V mutation resulted in a precursor protein lacking
autoactivating function and unstable in the presence of pepsin (9).
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) (10). Therefore, the truncated
protein was considered to be biologically inactive, because it lacked
two of the three amino acids that are essential for catalytic activity.
It also lacked four of the six cysteines that are predicted to be
important in the formation of intramolecular disulfide bonds.
In patient 4, we characterized a novel missense mutation of CK, a T to
C transition, at nt 131 in exon 2, which replaced Leu (CTA) in codon 9
with Pro (CCA; Fig. 7). This mutation was not located in the coding
region of the mature protein, but in the signal peptide of prepro-CK.
Signal peptides play a critical role in the targeting of proteins to
the endoplasmic reticulum and translocation of proteins across the
membrane (22, 23). They typically have three distinct domains: a
positively charged amino-terminal region (N-region), a central
hydrophobic core (H-region), and a more polar carboxyl-terminal domain
(C-region) (23). The signal peptide sequence of CK contains a
positively charged amino acid (Lys5) close to the
initial methionine and a subsequent stretch of hydrophobic amino acids
terminated by a consensus alanine (Ala15) (10).
Substitution of amino acid 9 may disrupt the hydrophobic core of the
signal peptide of the CK.
The functional properties of the L9P mutant of CK were examined by transient expression in COS-7 cells. The L9P mutant and wild-type CK genes were tagged with FLAG in the mammalian expression vector and transfected into COS-7 cells. Both genes were transcribed at an approximately equal level, as confirmed by RT-PCR of the m RNA of each transfectant, consistent with the fact that these two genes were driven by the strong cytomegalovirus promoter. In the immunohistochemical study, however, cells transfected with FLAG-tagged mutant CK cDNA did not show any positive staining by the monoclonal FLAG antibody, whereas cells transfected with the wild-type fusion gene showed positive staining. On Western blotting, positive bands were detected corresponding to the size of pro-CK, although the density of the mutant fusion gene product was significantly reduced compared to that of the wild-type product. Thus, expression of the mutant protein was markedly reduced, and it was probably below the detection limit of immunohistochemistry.
Some experiments have suggested that disruption of the hydrophobic core by either charged or helix-breaking residues mostly leads to a more or less severe kinetic defect in translocation, but rarely blocks export altogether (23, 24). In the present case, the replacement of Leu9 by a helical-breaking residue, Pro, is expected to impair translocation of the nascent preproenzyme across the endoplasmic reticulum membrane, which would be followed by its degradation.
Some mutations in human signal sequences have been reported to have a
direct correlation with defective protein synthesis and pathological
status (25, 26, 27). Signal peptide mutations have been described for
prepro-PTH and bilirubin-UDP-glucuronosyltransferase, which were
similar to the mutation found in patient 4, and these are reported to
cause disruption of the hydrophobic core of the signal peptide (25, 26). Such mutations have mainly been found in secretory proteins or
membrane proteins, and the effect of a mutation in the signal peptide
sequence on the intracellular transport of a lysosomal enzyme is not
well understood. In the case of lysosomal disease, there have only been
two reports on mutations in the signal peptide sequence. A missense
mutation, resulting in Pro to Arg substitution at amino acid 5 in the
hydrophobic core of the signal peptide of -L-fucosidase,
has been reported in a patient with fucosidosis, an autosomal recessive
lysosomal storage disease (28). The other report was of a missense
mutation in an atypical variant of Fabry’s disease, resulting in
substitution at amino acid 20 in the signal peptide of
-galactosidase (29).
In conclusion, we have analyzed the structure of the CK gene in four Japanese patients with pycnodysostosis and identified mutations that explained the pathogenesis of the disease. Although one had a mutation identical to that previously reported (8, 9), the others had novel mutation sites. One of them had a missense mutation at the CK signal peptide that disrupted the function of the signal peptide. This is the first report of signal peptide dysfunction due to an amino acid alteration as one of the genotypic subtypes of pycnodysostosis.
Received July 16, 1999.
Revised August 31, 1999.
Accepted September 9, 1999.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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