(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 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.
Type II collagen is the major fibril-forming collagen of
cartilage. Each molecule contains three 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.
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
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) .
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 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.
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,
128).
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
1(II) cDNA are shown.
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 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).
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 1(II)CB10.5 were not observed in
Coomassie blue (Fig. 5, lane 4) or silver stained gels (Fig. 5, lane 7).
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 1(II)CB10.5 was used as a
marker peptide for the presence of type II
collagen.
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 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).
Figure 6:
Sequences of 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 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 to A transition (data not shown).
Figure 7:
Electrophoresis of pepsin-digested
collagens produced by dedifferentiated and redifferentiated
chondrocytes. The cultures were labeled with L-[2,3-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.
Figure 9:
In situ hybridization. Frozen
sections of rib cartilage from the proband were hybridized to 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.
Figure 10:
In situ hybridization. Frozen
sections of rib cartilage from the proband were hybridized to 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.
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 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.
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 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) .
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 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.
Type XI collagen extracted from the proband's
cartilage had a higher ratio of the 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.
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 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) .
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
1(II) chains or by mutations that produce premature stop codons in
the
1(II) transcripts(39) .
Additional cases of
achondrogenesis type II need to be studied in order to determine
whether Gly substitutions near the mammalian collagenase cleavage site
of 1(II) chains are the usual cause of this phenotype.