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(Received for publication, September 19,
1994; and in revised form, November 9, 1994) From the
An autosomal dominant mutation in the COL2A1 gene was
identified in a fetus with achondrogenesis type II. A transition of
G Type II collagen is the major fibril-forming collagen of
cartilage. Each molecule contains three Four cases of
hypochondrogenesis have been shown to be caused by heterozygous
mutations of the COL2A1 gene that result in the substitution
of glycine residues in Gly-X-Y triplets that form the
mandatory repetitive structure of the triple helical domain of the
In
contrast, the cartilage of patients with achondrogenesis type II lacks
type II collagen(9, 10, 11) . It contains
type I collagen and small amounts of normal type IX and XI collagens.
The molecular defects that account for the lack of type II collagen in
such cases have not previously been described. We report a case of
achondrogenesis type II that was caused by a heterozygous mutation of
the COL2A1 gene that resulted in the substitution of
Gly
Figure 1:
Lateral and anteroposterior radiographs
of the proband.
The epiphyses of the long
bones were gelatinous. Light microscopy of a rib showed that the
columnar structure of the normal growth plate and the hyaline cartilage
structure of the normal epiphysis were lacking. Both the growth plate
and epiphysis were traversed by abnormal bands of fibrovascular tissue.
The cartilage matrix was markedly decreased and the chondrocytes were
lying in dilated lacunae (Fig. 2). The cytoplasm of the
chondrocytes contained occasional vacuoles and moderate amounts of
glycogen. Inclusion bodies were not seen. The clinical, radiological,
and pathological features were typical of achondrogenesis type
II(12, 13, 14) .
Figure 2:
Light micrograph of rib epiphyseal
cartilage. The cartilage is traversed by abnormal fibrovascular septa (arrows). The chondrocytes, which are contained within dilated
lacunae, are surrounded by a markedly reduced amount of matrix. The
section was stained with haematoxylin and eosin (magnification,
The proband's
parents were clinically normal and unrelated. Dermal fibroblast and
femoral epiphyseal chondrocyte cultures were established from the
proband with parental consent and the approval of the Ethics Committee
of this hospital.
Figure 3:
Location of the oligonucleotide primers.
Primers used for the PCR of overlapping fragments covering the
For
sequencing, the amplification products of the predicted sizes were
purified and cloned into a SmaI-cut and dephosphorylated
M13mp18 vector(17) . Single-stranded DNA preparations from the
individual clones were sequenced using a Sequenase kit (U. S.
Biochemical Corp.). In all cases, multiple products of at least two
independent amplification reactions were cloned and sequenced.
The age-matched control cartilage contained type II collagen and a
small amount of type XI collagen (Fig. 4). In contrast, the
proband's cartilage produced a dermal profile of type I and III
collagen chains together with some type XI collagen chains (Fig. 4). The prominent
Figure 4:
Electrophoresis of pepsin-digested
collagen from cartilage. Pepsin-digested collagens were analyzed by
SDS-polyacrylamide (5%, w/v) gel electrophoresis (see
``Experimental Procedures'' for details). Lane 1,
pepsin-digested dermal type I and III collagen standard; lane
2, pepsin-digested cartilage type II collagen standard; lane
3, age-matched control cartilage collagen; lane 4,
proband's pepsin-digested cartilage collagen. The gel was stained
with Coomassie Brilliant blue. Lanes 5 and 6, western
blot of proband and control cartilage collagens, respectively, probed
with an antibody to bovine type XI collagen; lane 7, 3 µg
of normal human type XI collagen probed with the same antibody. The
identities of the various collagen chains are
indicated.
To
further characterize the collagen chains, the pepsin digest was
subjected to CNBr cleavage. Electrophoresis of the control samples
showed the expected CNBr peptides of type II collagen (Fig. 5).
However, the proband's sample contained mainly type I collagen
and a small amount of type III collagen peptides. Type II collagen
marker peptides such as the
Figure 5:
Electrophoresis of CNBr peptides from
pepsin-digested collagens of cartilage. CNBr peptides were resolved by
SDS-polyacrylamide (12.5%, w/v) gel electrophoresis as described under
``Experimental Procedures.'' Lanes 1 and 6,
type II collagen CNBr peptide standard; lanes 2 and 5, type I and III collagen CNBr peptide standard; lane
3, CNBr peptides from control cartilage collagen; lanes 4 and 7, CNBr peptides from proband's cartilage
collagens. Lanes 1-4 were stained with Coomassie
Brilliant blue, and lanes 5-7 were stained with silver
to increase detection sensitivity. The identities of the various CNBr
peptides are indicated. The peptide
To identify the potential mutation, the PCR
product amplified using primers 11 and 12 was cloned into the M13mp18
vector for sequencing. The mutation was identified to be a transition
of G
Figure 6:
Sequences of
To confirm this finding, primers 12 and 13 were used to amplify a
290-bp genomic DNA fragment from the proband's fibroblasts. These
primers spanned the mutation that was predicted to be in exon
41(20) . The PCR fragment was also cloned into M13mp18 for
sequencing, which confirmed that the proband was heterozygous for the
G
Figure 7:
Electrophoresis of pepsin-digested
collagens produced by dedifferentiated and redifferentiated
chondrocytes. The cultures were labeled with L-[2,3-
Figure 9:
In situ hybridization. Frozen
sections of rib cartilage from the proband were hybridized to
Figure 10:
In situ hybridization. Frozen
sections of rib cartilage from the proband were hybridized to
The typical achondrogenesis type II phenotype in the proband
was shown to be caused by a heterozygous point mutation in the COL2A1 gene. A transition of G The epiphyseal cartilage was gelatinous and
contained a reduced amount of extracellular matrix, which completely
lacked type II collagen(8, 9) . Although autosomal
recessive inheritance has been proposed for this lack of type II
collagen in achondrogenesis type II(28) , our findings show
that it is caused by an autosomal dominant mutation of COL2A1.
We did not determine if the proband had a new mutation or if it had
been inherited from a mosaic parent. The cartilage matrix in the
proband consisted of predominantly type I and type III collagens, which
are normally not produced by cartilage cells and are characteristic
markers of a fibroblastic cell phenotype. However, chondrocytes were
present throughout the hyaline cartilage of the proband and were shown
by in situ hybridization to produce type II collagen mRNA and
by culture to produce type XI collagen. Other studies have also shown
that achondrogenesis type II cartilage contains normal type IX and XI
collagens and normal cartilage-specific proteoglycans(9) .
These chondrocytic markers indicate that the chondrocytes were
differentiated despite the lack of type II collagen in the matrix.
Likewise, the expression of type X collagen mRNA by the hypertrophic
chondrocytes in the growth plate cartilage demonstrated that not only
were the chondrocytes differentiated, but they were able to undergo
maturation and hypertrophy within this anomalous type I collagen
matrix. These data are consistent with in vitro culture
experiments demonstrating that hypertrophic chondrocytes express type X
collagen when grown within type I collagen
gels(29, 30) . The abnormal collagen phenotype of
the proband's chondrocytes was stable in vitro. Cultures
of the proband's chondrocytes in alginate beads produced a
collagen profile that was similar to that of the abnormal matrix in the
cartilage tissue. However, in these biosynthetic labeling experiments,
a trace of overmodified type II collagen was detected within the cell
fraction, but no type II collagen was detected in the secreted
fraction. The lack of type II collagen in the cartilage matrix was not
caused by the absence of type II collagen mRNA since the in situ hybridization studies showed that most chondrocytes produced type
II collagen mRNA. We did not quantify the steady state levels of the
normal and mutant The
finding of small amounts of mutant type II collagen within the cell
that migrated slowly on electrophoresis because of excess
post-translational modifications demonstrated that the Gly to Ser
mutation perturbed helix folding and prevented collagen secretion. By
analogy with other glycine substitution
mutations(1, 31, 32, 33) , this type
II collagen mutation would be expected to compromise collagen helix
stability, and it is likely that the chondrocytes produced and then
degraded the mutant-containing collagen molecules. However, the
complete degradation of type II collagen is not typical of
substitutions of Gly by Ser at other sites(4, 7) . For
example, substitutions of Gly Type XI collagen extracted from the proband's
cartilage had a higher ratio of the In situ hybridization of cartilage showed that about half of the
chondrocytes produced both type I and II collagen mRNAs. The
chondrocytes producing type I mRNA were widely dispersed throughout the
cartilage and were not confined to the fibrovascular septa. We did not
determine which cells were producing the type III collagen. The in
vivo production of type I collagen by chondrocytes was confirmed
by the in vitro production of type I collagen by chondrocyte
cultures. The production of type XI collagen by the cultured
chondrocytes indicated that the cells had redifferentiated in the
alginate beads(19, 21) . Our findings are similar
to those observed in a fetus with hypochondrogenesis caused by the
heterozygous substitution of Gly Our results suggest that
achondrogenesis type II, the severest phenotype produced by mutations
of COL2A1, is caused by the absence of type II collagen in the
cartilage matrix. The type I and III collagens that replace it appear
to be unable to compensate for the lack of type II collagen.
Hypochondrogenesis, a slightly less severe phenotype, shares many of
the same abnormalities except that the cartilage also contains abnormal
type II collagen. Spondyloepiphyseal dysplasia congenita,
spondyloepimetaphyseal dysplasia, and Kniest syndromes are also caused
by dominant-negative mutations of COL2A1 in which the
cartilage contains abnormal type II collagen but no detectable type I
or III collagens(17, 19, 23, 38) .
Stickler and Wagner syndromes are caused either by mutations that alter
the primary structure of the aminoterminal region of the helix of
Additional cases of
achondrogenesis type II need to be studied in order to determine
whether Gly substitutions near the mammalian collagenase cleavage site
of
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1747-1753
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
to A in exon 41 produced a substitution of Gly
by Ser within the triple helical domain of the
1(II) chain
of type II collagen, interrupting the mandatory Gly-X-Y triplet sequence required for the normal formation of stable
triple helical type II collagen molecules, resulting in the complete
absence of type II collagen in the cartilage, which had a gelatinous
composition. Type I and III collagens were the major species found in
cartilage tissue and synthesized by cultured chondrocytes along with
cartilage type XI collagen. However, cultured chondrocytes produced a
trace amount of type II collagen, which was retained within the cells
and not secreted. In situ hybridization of cartilage sections
showed that the chondrocytes produced both type II and type I collagen
mRNA. As a result, it is likely that the chondrocytes produced type II
collagen molecules, which were then degraded. The close proximity of
the Gly substitution by Ser to the mammalian collagenase
cleavage site at Gly
-Leu
may have produced
an unstable domain that was highly susceptible to proteolysis. The type
I and III collagens that replaced type II collagen were unable to
maintain the normal structure of the hyaline cartilage but did support
chondrocyte maturation, evidenced by the expression of type X collagen
in the hypertrophic zone of the growth plate cartilage.
1(II) chains that are
encoded by the COL2A1 gene(1) . Mutations of this gene
produce a family of spondyloepiphyseal dysplasias that include
achondrogenesis type II, hypochondrogenesis, spondyloepiphyseal
dysplasia congenita, and the Kniest, Stickler, and Wagner
syndromes(2, 3) . Achondrogenesis type II and
hypochondrogenesis are perinatal lethal phenotypes, with
achondrogenesis type II being the more severe form.1(II) chains. The mutations include Gly to
Ser(4) , Gly
to Glu(5) , Gly
to Ala(6) , and Gly
to Ser(7) . The
cartilage matrix in these patients contained normal type IX and XI
collagens but a reduced amount of type II collagen, which was
overmodified(5, 6, 8) . Type I collagen,
which is not found in normal hyaline cartilage, was also present in the
cartilage matrix of two of these cases(6, 8) .
by Ser in the triple helical domain of
1(II)
chains. The cartilage lacked type II collagen but contained type I,
III, and XI collagens, which were produced by the chondrocytes.
Clinical Summary
The proband was shown by
ultrasonography at 19 weeks of gestation to have severe shortening of
the limbs and trunk and marked oedema around the neck. The pregnancy
was terminated at 20 weeks of gestation. External examination showed
extremely short limbs, a large head, short trunk, bulging abdomen, and
edema of the head and neck. Radiographs (Fig. 1) showed very
short tubular bones with metaphyseal expansion and cupping, absent
ossification of the vertebrae and sacrum, small iliac wings with absent
ossification of the pubis and ischium, and short ribs but relatively
normal ossification of the calvarium.
128).
Preparation of Cartilage Collagens
The abnormal
gelatinous center of the distal femoral epiphysis and from hyaline
cartilage of an age-matched control were freeze-milled and extracted
with 50 mM Tris/HCl buffer, pH 7.5, containing 0.15 M NaCl, 5 mM EDTA, 0.1 mM phenylmethysulfonyl
fluoride, 10 mMN-ethylmaleimide, and 4 M guanidine HCl for 48 h at 4 °C to remove proteoglycans and
other noncollagenous proteins. The extract was desalted by dialysis and
freeze-dried. The cartilage residue was washed thoroughly with water
and freeze-dried. Portions of the dried residue were digested with
pepsin (Sigma) for 24 h at 4 °C using an enzyme:substrate ratio of
1:10 and a final pepsin concentration of 100 µg/ml in 0.5 M acetic acid.Amplification, Cloning, and Sequencing of
cDNA
Total cytoplasmic RNA was extracted from fibroblast
cultures(15) . First-strand cDNA was synthesized from total RNA
using an oligo(dT)primer and a cDNA synthesis kit (Amersham Corp.). Table 1lists the primers used to amplify overlapping cDNA
fragments covering the pro-1(II) chain, and Fig. 3shows
their relative positions along the pro--chain. Each pair of PCR (
)primers contained sequences from different exons, which
ensured that amplification products from the cDNA template could be
distinguished from those amplified from contaminating genomic
DNA(16) . Negative control reactions were also included to
detect contamination from previously amplified cDNAs, and the
conditions for PCR were as previously described(17) .
1(II) cDNA are shown.
Single-stranded Conformation Polymorphism
Analysis
Purified amplification products were digested with one
or more restriction endonucleases (Table 1) to produce fragments
of optimal size for SSCP analysis(18) . The fragments were
recovered by ethanol precipitation; dissolved in a buffer containing
45% (v/v) formamide, 10 mM EDTA, 0.025% (w/v) bromophenol
blue, and 0.025% xylene cyanol FF; denatured at 95 °C for 5 min;
and analyzed by electrophoresis on a 7.5% (w/v) nondenaturing
polyacrylamide gel (180 180 0.75 mm) containing 5%
(v/v) glycerol in 1 TBE buffer. Electrophoresis was carried out
at a constant temperature of 15 °C using a Bio-Rad PAC 3000 power
supply with a temperature probe. The voltage did not exceed 500 V. The
normal running time was approximately 4 h. The gel was stained with
silver nitrate(19) .Amplification, Cloning, and Sequencing of Genomic
DNA
Genomic DNA was prepared from confluent fibroblast cultures
from the proband(19) . Approximately 50 ng of DNA was amplified
over 35 cycles using primers 12 and 13 (Table 1). The 290-bp PCR
product extended from exon 40 to exon 41 of the COL2A1 gene
and included the mutation, which was predicted to be in exon
41(20) .Chondrocyte Cultures
Chondrocyte cultures were
established from the gelatinous center of the distal femoral epiphysis
using previously described methods(19) . The cells were grown
as monolayer cultures, and a sufficient number of cells for collagen
analysis were obtained by the fourth passage. The dedifferentiated
chondrocytes were released from the monolayer cultures by trypsin
digestion and redifferentiated within alginate beads(21) . The
beads were prepared from a suspension of 2 10
cells/ml of alginate(19, 21) . The alginate
beads were suspended in Dulbecco's modified Eagle's basal
medium containing 10% (v/v) fetal calf serum and 0.25 mM sodium ascorbate. At least 4 weeks was allowed for
redifferentiation of the chondrocytes(19) .Preparation of Collagen from Chondrocyte
Cultures
Collagens produced by redifferentiated chondrocytes
grown in alginate beads were analyzed by biosynthetic labeling of the
collagen with L-[2,3-
H]proline in fresh
Dulbecco's modified Eagle's basal medium containing 10%
(v/v) dialyzed fetal calf serum for 24 h(19) . The medium was
removed, and the beads were gently washed with 0.15 M NaCl.
The medium and NaCl wash were not analyzed since previous experiments
showed that the collagen synthesized during the labeling period was
retained within the alginate beads. (
)The chondrocytes were
released by depolymerization of the beads in 5 ml of 0.15 M sodium citrate buffer, pH 7.5, at 37 °C(19) . The
procollagens in the cell-associated and secreted fractions were
precipitated by ammonium sulfate at 25% saturation and converted to
collagen by limited pepsin digestion(22) . Portions of these
samples were also cleaved with CNBr.SDS-Polyacrylamide Gel Electrophoresis
Collagen
chains were analyzed on 5% (w/v) polyacrylamide gels, and the CNBr
peptides were analyzed on 12.5% (w/v) polyacrylamide gels. The methods
of sample preparation, fluorography, western blotting, Coomassie
Brilliant Blue, and silver staining have been described
elsewhere(19, 22) .Preparation of mRNA Hybridization Probes
The type
II collagen riboprobe was a subclone of HC22(23) . A 1232-bp
fragment from an EcoRI and SacI digest of HC22 was
subcloned into pGEM7Zf(+) from Promega. It included nucleotides
3142-4373 of the 1(II) cDNA(24) . The type X
collagen riboprobe was a subclone of pSAh10f(25) . A 710-bp
fragment from a HindIII and SacI digest was subcloned
into pGEM7Zf(+). It included nucleotides 1694-2403 of the
1(X) cDNA. The type I collagen riboprobe was the insert from
Hf667, an 1(I) clone(26) , recloned into SP64 and SP65
vectors (Promega). The cDNAs were transcribed with greater than 90%
efficiency from the T7 or SP6 promoters to generate sense and antisense
cRNAs. Linearized plasmids were transcribed in the presence of
[S]CTP (1000 Ci/mM, DuPont NEN) using a
Riboprobe gemini transcription system (Promega). The resultant
S-labeled cRNAs were hydrolyzed to generate fragments of
approximately 200 bp prior to hybridization. The probes were selected
to contain the carboxyl-terminal noncollagenous domains to ensure
specificity and to minimize cross-reactivity between collagen types.
In Situ Hybridization
In situ hybridization was performed as previously described(27) .
8-µm frozen sections were cut on a Leitz cryostat and were mounted
onto aminoalkysilane-treated slides by baking at 60 °C on a heating
block for 10 min. The slides were immersed in 4% (w/v) paraformaldehyde
in PBS for 20 min, washed twice in PBS for 5 min, rinsed briefly in
deionized water, and rebaked at 60 °C until dry. The sections were
rehydrated in deionized water before digestion with pronase at a
concentration of 0.3 mg/ml of 50 mM Tris/HCl buffer, pH 7.5,
containing 5 mM EDTA for 8 min at room temperature. They were
rinsed in water and fixed in 4% (w/v) paraformaldehyde in PBS for 10
min. The slides were then washed for 3 min in PBS and dehydrated in a
graded series of ethanol and air-dried. The riboprobes were diluted to
specific activities of 10
dpm/µl in a hybridization
buffer containing 25% (v/v/) deionized formamide, 10% (w/v) dextran
sulfate, 0.3 M NaCl, 10 mM NaHPO
, 10 mM Tris/HCl (pH 7.5), 5
mM EDTA, 0.02% (w/v) bovine serum albumin, 0.02% (w/v) Ficoll
400, 0.02% (w/v) polyvinylpyrrolidone, 10 mM dithiothreitol,
and 0.8 mg/ml of yeast tRNA. The sense strand of each probe was used as
a negative control. Approximately 60 µl was used per slide.
Hybridization was performed overnight at 48 °C in a humidified
chamber. Slides were washed three times in 2 SSC containing 50%
(v/v) formamide at 48 °C and once in 2 SSC at room
temperature for 30 min followed by digestion with RNAse (Sigma) for 10
min at 37 °C and a wash with PBS. After dehydration, the slides
were dipped in emulsion (Kodak NTB-2 diluted 1:1 in water), air-dried
for 2 h, and autoradiographed in a dry chamber at 4 °C for 20 days.
The slides were developed, fixed, and stained with a progressive
Mayer's hematoxylin stain.
Cartilage Collagens
Collagen chains were not
detected in the guanidine HCl extracts of control or proband cartilage
(data not shown). However, the collagens in the pepsin extracts were
representative of the collagens in the tissue since the proband and
control cartilages were completely solubilized by pepsin digestion. 2(I) and dimeric 
12 chains
indicated that type I collagen was the major collagen in the
proband's cartilage. The chains of type XI collagen migrated
normally but with an abnormally high ratio of the 1(XI) chains
relative to the 2(XI) chains. This observation was verified by
western blotting with an antibody specific to type XI collagen (a
generous gift from Dr. Garry Gibson, Henry Ford Hospital, Detroit). The
abnormal ratio of the type XI collagen chains was shown not to be
caused by contaminating type V collagen by western blotting using an
antibody specific for human type V collagen (data not shown).
1(II)CB10.5 were not observed in
Coomassie blue (Fig. 5, lane 4) or silver stained gels (Fig. 5, lane 7).
1(II)CB10.5 was used as a
marker peptide for the presence of type II
collagen.
Characterization of the COL2A1 Mutation
The
molecular basis of the type II collagen deficiency was studied by
screening of 1(II) cDNA for mutations. Low basal transcription of
the COL2A1 gene by cultured dermal fibroblasts was used as the
source of 1(II) mRNA and cDNA since only a limited amount of
cartilage was available. Overlapping 1(II) cDNA PCR products were
digested with restriction endonucleases to yield fragments of suitable
size for mutation screening by SSCP ( Table 1and Fig. 3).
An additional single strand was observed on SSCP analysis of the XhoI digest of the PCR fragment amplified using primers 11 and
12 (data not shown). to A, which changed the codon GGT for Gly
to AGT for Ser in the helical domain of the
1(II) chain. Of
the 18 clones sequenced, 10 were mutant and 8 were normal, indicating
that the proband was heterozygous for the mutation (Fig. 6).
1(II) cDNA clones from
the proband's dermal fibroblasts. The 563-bp cDNA PCR product
that produced a band shift on SSCP analysis (data not shown) was cloned
into M13mp18 and sequenced. Normal and mutant sequences were obtained
as shown. The circles and the arrow indicate the site
of the point mutation. The corresponding coding strand sequences and
the deduced amino acid sequences are shown below. The box encloses the abnormal codon resulting in the substitution of
Gly-769 by Ser in the carboxyl-terminal region of the CB10.5 peptide of
the mutant 1(II) chain.
to A transition (data not shown).
Collagen Metabolism by Redifferentiated Cultured
Chondrocytes
Chondrocytes isolated from a small sample of
cartilage were grown in monolayer cultures to gain sufficient cell
numbers for collagen analysis. The dedifferentiated chondrocytes
obtained from both control and proband cartilage produced both type I
and III collagens similar to dermal fibroblasts (Fig. 7). The
chondrocyte phenotype was re-established after 4 weeks of cell culture
in alginate beads in the presence of ascorbic acid. The control cells
re-expressed type II and type XI collagens (Fig. 7). The
proband's cells re-expressed type XI collagen, but type I and III
collagens were the major collagens produced and secreted by these
cells. Trace amounts of slowly migrating 1(II) chains and CNBr
peptides were observed in the proband's cell fraction ( Fig. 7and 8). Type II collagen CNBr peptides were not detected
in the secreted fraction even after prolonged exposure of the
fluorograms. Similar results were obtained after a further 2 weeks of
culture in alginate beads (data not shown).
H]proline, and the collagen from
the cell and medium fractions were subjected to limited pepsin
digestion. The resultant collagen chains were analyzed by
SDS-polyacrylamide (5%, w/v) gels. Collagens produced by
redifferentiated chondrocytes are shown in lane 1 (proband
cell collagens), lane 2 (proband secreted collagens), lane
3 (control cell collagens), and lane 4 (control secreted
collagens). Collagens produced by dedifferentiated chondrocytes are
shown in lane 5 (control secreted collagens) and lane 6 (proband secreted collagens). Samples were analyzed without
reduction of disulfide bonds, and the protein bands were detected by
fluorography. The identities of the various collagen chains are
indicated.
In Situ Hybridization
The source of type I
collagen in the proband's cartilage was further studied in
vivo using in situ hybridization of frozen sections from
rib cartilage of the proband and control. Control samples showed
specific hybridization of the type II collagen probe to chondrocytes
throughout the cartilage and specific hybridization of the type X
collagen probe to the hypertrophic chondrocytes. There was no
detectable hybridization of the type I collagen probe to the control
chondrocytes (results not shown). The proband sample also showed
similar specific hybridizations of the type II (Fig. 9, C and D). At least 90% of the chondrocytes hybridized to
the type II collagen probe, and approximately 50% of the chondrocytes
also hybridized to the type I collagen probe (Fig. 9, A and B). These percentages were estimated from the mean of
five randomly selected regions of the cartilage. A total of
approximately 1000 cells were counted. Hybridization of the type I
collagen probe was uniformly distributed in the proband's
cartilage and was not localized to the abnormal fibrovascular strands.
Hybridization with a type X collagen probe demonstrated the localized
expression of type X collagen mRNA in the hypertrophic chondrocytes of
the growth plate cartilage of the control (Fig. 10A)
and the proband (Fig. 10B).
S-labeled antisense cRNAs that were specific for human
type I and II collagens. Panels A (magnification, 10
)
and B (magnification, 25) are bright field images of a
section hybridized to the type I collagen probe. Panels C (magnification, 10) and D (magnification,
40) are bright field images of a section hybridized to the type
II collagen probe. p, perichondrium; c, cartilage.
Examples of positive cells are indicated by closed arrowheads,
and negative cells are indicated by open
arrows.
S-labeled antisense cRNAs that were specific for human
type X collagen. Panel A (10
magnification) is a bright field
image of the control, and panel B (10 magnification)
is a dark field image of the proband cartilage. Panel C is the
bright field histology corresponding to panel B. Regions of
the aligned sections corresponding to bone, the adjacent growth plate
cartilage, and hyaline cartilage are
indicated.
to A in exon 41
produced a substitution of Gly
by Ser within the triple
helical domain of the
1(II) chain of type II collagen. It
interrupted the mandatory Gly-X-Y triplet sequence
required for the normal formation of stable triple helical type II
collagen molecules.1(II) mRNAs in the cartilage or in the cultured
chondrocytes. However, the steady state levels were probably similar
since approximately equal numbers of mutant and normal cDNA clones were
obtained from the transcripts produced by low basal transcription of COL2A1 by cultured dermal fibroblasts(17) . and Gly
result in the production of overmodified type II collagen by
chondrocytes(4, 7) . In our proband, the close
proximity of the Gly
substitution by Ser to the mammalian
collagenase cleavage site at Gly
-Leu
may have produced an unstable domain that was highly susceptible
to proteolysis.
1(XI) chain then normal. It
was not caused by comigrating type V collagen chains. The abnormal
proportion of the 1(XI) chain may reflect anomalies in the
composition of type XI collagen molecules, which usually contain an
3(XI) chain encoded by COL2A1(9) . Since type XI
collagen co-polymerizes with type II collagen fibrils within the
cartilage tissue(34) , the absence of type II collagen fibrils
in the mutant cartilage may result in abnormal regulation of type XI
collagen expression. In contrast, the 1(XI), 2(XI), and
3(XI) chain ratios of type XI collagen produced in alginate
cultures were similar to the control ratios. by Ala in the triple
helical domain of type II collagen(6) . In both cases, the COL2A1 mutations resulted in the abnormal production of type I
collagen by chondrocytes of hyaline cartilage. Normal human hyaline
cartilage lacks type I collagen and pro-
1(I) mRNA(35) .
These findings suggest that the COL1A1 and COL1A2 genes of type I collagen are not transcribed by normal human
chondrocytes. Chick chondrocytes produce an alternative 2(I)
transcript caused by the use of a cartilage-specific promoter within
intron 2 of COL1A2(36) . This RNA does not encode
2(I) chains but may encode a noncollagenous protein. We did not
determine the mechanism of stimulation of transcription of the COL1A1 and COL1A2 genes by the proband's
chondrocytes. It may be a response to the abnormal pericellular
environment (7) or to an abnormal concentration of transforming
growth factors(37) .1(II) chains or by mutations that produce premature stop codons in
the 1(II) transcripts(39) .1(II) chains are the usual cause of this phenotype.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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