©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A COL2A1 Mutation in Achondrogenesis Type II Results in the Replacement of Type II Collagen by Type I and III Collagens in Cartilage (*)

(Received for publication, September 19, 1994; and in revised form, November 9, 1994)

Danny Chan (1) William G. Cole (3) C. W. Chow (2) Stefan Mundlos (1) John F. Bateman (1)(§)

From the  (1)Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the (2)Department of Anatomical Pathology, Royal Children's Hospital, Parkville, Victoria 3052, Australia, and the (3)Division of Orthopaedics, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 alpha1(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.


INTRODUCTION

Type II collagen is the major fibril-forming collagen of cartilage. Each molecule contains three alpha1(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 alpha1(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 alpha1(II) chains. The cartilage lacked type II collagen but contained type I, III, and XI collagens, which were produced by the chondrocytes.


EXPERIMENTAL PROCEDURES

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.


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, times128).



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.

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-alpha1(II) chain, and Fig. 3shows their relative positions along the pro-alpha-chain. Each pair of PCR (^1)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) .




Figure 3: Location of the oligonucleotide primers. Primers used for the PCR of overlapping fragments covering the alpha1(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.

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 times 180 times 0.75 mm) containing 5% (v/v) glycerol in 1 times 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 times 10^6 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-^3H]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. (^2)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 alpha1(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 alpha1(X) cDNA. The type I collagen riboprobe was the insert from Hf667, an alpha1(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 10^5 dpm/µl in a hybridization buffer containing 25% (v/v/) deionized formamide, 10% (w/v) dextran sulfate, 0.3 M NaCl, 10 mM Na(2)HPO(4), 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 times SSC containing 50% (v/v) formamide at 48 °C and once in 2 times 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.


RESULTS

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.

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 alpha2(I) and dimeric beta12 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 alpha1(XI) chains relative to the alpha2(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 alpha1(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 alpha1(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 alpha1(II) cDNA for mutations. Low basal transcription of the COL2A1 gene by cultured dermal fibroblasts was used as the source of alpha1(II) mRNA and cDNA since only a limited amount of cartilage was available. Overlapping alpha1(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 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 alpha1(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 alpha1(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 alpha1(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).

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 alpha1(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).


Figure 7: Electrophoresis of pepsin-digested collagens produced by dedifferentiated and redifferentiated chondrocytes. The cultures were labeled with L-[2,3-^3H]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).


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, 10times) and B (magnification, 25times) are bright field images of a section hybridized to the type I collagen probe. Panels C (magnification, 10times) and D (magnification, 40times) 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 times magnification) is a bright field image of the control, and panel B (10 times 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.




DISCUSSION

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 alpha1(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 alpha1(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 alpha1(XI) chain then normal. It was not caused by comigrating type V collagen chains. The abnormal proportion of the alpha1(XI) chain may reflect anomalies in the composition of type XI collagen molecules, which usually contain an alpha3(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 alpha1(XI), alpha2(XI), and alpha3(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-alpha1(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 alpha2(I) transcript caused by the use of a cartilage-specific promoter within intron 2 of COL1A2(36) . This RNA does not encode alpha2(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 alpha1(II) chains or by mutations that produce premature stop codons in the alpha1(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 alpha1(II) chains are the usual cause of this phenotype.


FOOTNOTES

*
This work was supported by grants from the National Health and Medical Research Council of Australia and Royal Children's Hospital Research Foundation (to J. F. B.), the Medical Research Council of Canada (to W. G. C.), and Deutsche Forshungsgemeinscaft (to S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. Fax: 61-3-345-6668.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); PBS, phosphate buffered saline; SSC, sodium chloride-sodium citrate buffer; SSCP, single-stranded conformation polymorphism; TBE, Tris-borate-EDTA buffer.

(^2)
D. Chan, W. Cole, C. W. Chow, S. Mundlos, and J. F. Bateman, unpublished data.


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