From the Orthopaedic Research Laboratories,
University of Washington, Seattle, Washington 98195-6500, § Ahmanson Department of Pediatrics, Steven Spielberg
Pediatric Research Center, Burns and Allen Cedars-Sinai Research
Institute, Cedars-Sinai Medical Center, Los Angeles, California
90048-1869, Departments of ** Pediatrics and
Radiology, UCLA School of Medicine, Los
Angeles, California 90048-1869, and
Department of Medical
Genetics, Ajou University College of Medicine, Suwon, South Korea
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ABSTRACT |
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Type II collagen mutations have been identified
in a phenotypic continuum of chondrodysplasias that range widely in
clinical severity. They include achondrogenesis type II,
hypochondrogenesis, spondyloepiphyseal dysplasia congenita,
spondyloepimetaphyseal dysplasia, Kniest dysplasia, and Stickler
syndrome. We report here results that define the underlying genetic
defect and consequent altered structure of assembled type II collagen
in a neonatal lethal form of Kniest dysplasia. Electrophoresis of a
cyanogen bromide (CNBr) (CB) digest of sternal cartilage revealed an
1(II)CB11 peptide doublet and a slightly retarded mobility for all
major CB peptides, which implied post-translational overmodification. Further peptide mapping and sequence analysis of CB11 revealed equal
amounts of a normal
1(II) sequence and a chain lacking the 18 residues (361-378 of the triple helical domain) corresponding to exon
24. Sequence analysis of an amplified genomic DNA fragment identified a
G to A transition in the +5 position of the splice donor consensus
sequence of intron 24 in one allele. Cartilage matrix analysis showed
that the short
1(II) chain was present in collagen molecules that
had become cross-linked into fibrils. Trypsin digestion of the
pepsin-extracted native type II collagen selectively cleaved the normal
length
1(II) chains within the exon 24 domain. These findings
support a hypothesis that normal and short
-chains had combined to
form heterotrimeric molecules in which the chains were in register in
both directions from the deletion site, accommodated effectively by a
loop out of the normal chain exon 24 domain. Such an accommodation,
with potential overall shortening of the helical domain and hence
misalignment of intermolecular relationships within fibrils, offers a
common molecular mechanism by which a group of different mutations
might act to produce the Kniest phenotype.
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INTRODUCTION |
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The chondrodysplasias are a clinically and genetically heterogeneous group of skeletal disorders characterized by abnormal endochondral ossification (1, 2). Phenotypes within the group of chondrodysplasias caused by mutations in the gene for type II collagen, COL2A1, range in severity from the neonatal lethal dwarfing conditions achondrogenesis type II and hypochondrogenesis to mild phenotypes, such as Stickler syndrome, in which there is little observed skeletal growth abnormality (3). Together with the spondyloepiphyseal dysplasias and spondyloepimetaphyseal dysplasias, Kniest dysplasia is a moderately severe disorder within the clinical spectrum of type II collagenopathies (1-4). The phenotype is characterized by disproportionate dwarfism, a short trunk and small pelvis, kyphoscoliosis, and short limbs with prominent joints that can have restricted mobility. Craniofacial anomalies may include a flat face, myopia and retinal detachment, cleft palate, and hearing loss. Radiographic features include narrowed joint spaces, platyspondyly, vertical clefts of the vertebral bodies, short tubular bones, large epiphyses, and flared metaphyses.
Cartilage from Kniest dysplasia patients displays a distinct morphology, termed "Swiss cheese" cartilage (5), which is diagnostic for the phenotype. The matrix has a foamy appearance with sparse, thin collagen fibrils surrounding the chondrocytes and thickened fibrils in the periphery. Abnormal type II collagen can be isolated in high yield from Kniest dysplasia cartilage (6, 7), suggesting that the incorporation of mutant type II collagen into the matrix produces the characteristic histologic appearance.
Of about 10 distinct mutations known to cause Kniest dysplasia, most result in whole or partial exon deletions that cluster in the amino-terminal half of the triple-helical domain of the type II collagen molecule (8). Previously we suggested two models for the assembly of type II collagen trimers composed of normal chains and chains with a deletion: 1) maintenance of the triple-helix along the length of the molecule from the carboxyl terminus, resulting in a misregister of short chains relative to normal chains from the deletion site to the amino terminus and 2) "loop out" of the normal chain(s) with maintenance of chain registration and a normal amino-terminal structure (6). Here, we describe a new dominant mutation that results in the skipping of exon 24 and produces a severe form of Kniest dysplasia. Analysis of the abnormal type II collagen from the tissue provides evidence for the second model, that the normal chain sequences in effect loop out at the site of the deletion.
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MATERIALS AND METHODS |
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Clinical Summary
The proband, the female product of a 37-week gestation, died of respiratory distress at 10 days of age. The infant had short limbs, club feet, cleft palate, midface hypoplasia, and a narrow chest. X-rays revealed flattened vertebral bodies with coronal clefts, slight shortening of the ribs, and dumbbell-shaped femurs. These findings were compatible with a diagnosis of Kniest dysplasia (9). Cartilage and other tissues were obtained for analysis.
Protein Analysis
Pepsin Extraction of Collagen-- Sternal cartilage was extracted in 4 M guanidine HCl, 50 mM Tris/HCl, pH 7.0, at 4 °C for 48 h. The washed residue was digested with pepsin (10). Cartilage from a 4-day-old infant was similarly treated as a control.
Collagen Peptide Analysis--
Sternal cartilage was digested
with CNBr in 70% (w/v) formic acid for 24 h at room temperature
(11), and the resulting peptides were fractionated by sequential
cation-exchange and reverse-phase HPLC1 (12, 13). Fractions
containing 1(II)CB11 (identified by SDS-PAGE) were pooled, dried,
and digested further with either trypsin or endoproteinase Asp-N.
Resulting peptides were fractionated by reverse-phase HPLC (13).
Peptide yields were estimated by integration of peak areas of 220-nm
absorbance and by the recoveries of phenylthiohydantoin-derivatives on
subsequent sequence analysis.
Trypsin Susceptibility Assay-- Pepsin-solubilized native collagens from patient and control tissues were dissolved in 0.1 M NH4HCO3, 0.01% (w/v) SDS, pH 7.8, at 24 °C. Trypsin was added at a 1:100 (w/w) enzyme:substrate ratio, and aliquots were incubated at various temperatures ranging from 24 to 37 °C for 18 h. The digests were then run on 6% gels (SDS-PAGE) and electroblotted to polyvinylidene difluoride membrane for sequence analysis.
SDS-PAGE--
The method of Laemmli (14) was used with 6% and
12.5% gels, respectively, for whole -chains and CNBr peptides.
Protein Microsequencing-- Amino-terminal sequence analysis of individual peptides was carried out by Edman chemistry on a Porton 2090E machine equipped with on-line HPLC analysis of the phenylthiohydantoin-derivatives. The standard program was modified to resolve 4-hydroxyproline, hydroxylysine, and the two hydroxylysine glycosides.
Collagen Cross-linking Analysis-- Cartilage was acid-hydrolyzed (6N HCl, 110 °C, 24 h) for analysis by reverse-phase HPLC (15) of the pyridinoline cross-links of collagen. Hydroxyproline was quantified in the same hydrolysate by colorimetric assay (16). Pyridinoline concentration was expressed in mol/mol of collagen for comparison of tissues.
The CNBr digests of pepsin-extracted type II collagen were also analyzed by molecular sieve HPLC (Toso-Haas G3000SW, 7.5 mm x 60 cm, two columns in series (6); eluent, 0.1 M sodium phosphate, pH 6.8, 30% (v/v) acetonitrile) monitoring for pyridinoline cross-link fluorescence in the resolved peptides.Histology
Cartilage from the proband was prepared for light and electron microscopy as described previously (17).
COL2A1 Analysis
Total RNA was isolated from cartilage by the method of Chomczynski and Sacchi (18). RT-PCR was carried out essentially as described previously (19). Briefly, COL2A1 cDNA was synthesized by reverse transcription using 1 µg of total RNA and a gene-specific primer complementary to sequences in exon 25 (5'-CCAGGACGACCATCTTCACCA-3'). Amplification by PCR used the reverse transcription primer and a second gene-specific primer that annealed within exon 23 (5'-CAAGGGAGCCAACGGTGACC-3'). DNA was denatured at 94 °C for 2 min followed by 35 cycles of 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C in a DNA Thermal Cycler (Perkin-Elmer). The final 72 °C step was extended to 10 min. PCR products were analyzed by electrophoresis through 6% polyacrylamide gels.
Genomic DNA was isolated from fibroblasts or lymphoblastoid cell lines by standard methods. A genomic DNA fragment was amplified by PCR using a COL2A1 intron 23 forward primer (5'-CAGCCCTGCACTGCCAGGAT-3') and the exon 25 reverse primer described above. Cycling conditions were as described above. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Inc.) and cloned using the TA cloning kit (Invitrogen Corp.). Colonies were grown and plasmid DNA was isolated by standard methods. Sequencing was with the fluorescent dideoxy terminator method of cycle sequencing on a Perkin-Elmer/ABI 373a automated DNA sequencer following ABI protocols.
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RESULTS |
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The collagen peptides from CNBr-digested Kniest cartilage migrated
more slowly than their control counterparts on SDS-PAGE (Fig.
1). Peptide CB11 appeared to run as a
doublet of bands, one migrating more slowly and the other faster than
the control CB11 (Fig. 1, left lane). The pepsin-solubilized
1(II) chains from Kniest cartilage migrated as a broad band,
slightly slower than control
1(II). These observations suggested a
deletion in peptide CB11 in about half the
1(II) chains of the
tissue. The faster CB11 band was the deletion candidate, and the slower
CB11 band behaved as a post-translationally overmodified normal
peptide. There was no obvious enrichment or abnormality of collagen
1(II) chains in the guanidine HCl extract of Kniest cartilage
compared with control cartilage (not shown).
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Enrichment from the cartilage CNBr digest by cation-exchange HPLC and
analysis by SDS-PAGE confirmed that CB11 was the source of the doublet
(Fig. 2). Pooled fractions of CB11 were
purified by reverse-phase HPLC (not shown), and aliquots were digested with trypsin or endoproteinase Asp-N for peptide mapping in comparison with control CB11. Fig. 3 shows the
resulting tryptic peptide profiles. (The endoproteinase Asp-N results
are not shown.) Peptides that appeared to be unique to the patient were
selected for amino-terminal sequence analysis. The observed peptide
sequence shown underlined in Fig. 3a would result
from trypsin cleavage if the exon 24 domain (residues 361-378 of the
triple helix; Ref. 20) were deleted to create a new tryptic cleavage
site between the terminal Arg-360 of exon 23 and the initial Gly-379 of
exon 25. Deletion of the exon 24 sequence was confirmed by sequence
analysis of an endoproteinase Asp-N peptide, which spanned the deletion
site. It was estimated from densitometry of the two CB11 bands on
SDS-PAGE and the yields of the various peptides on sequence analysis
that the proportion of deletion-bearing -chains in the Kniest tissue
was 40-50% that of total
1(II).
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The pepsin-solubilized molecule was probed with trypsin in an attempt
to distinguish between two hypotheses concerning the structural
consequences of forming trimers from a mixture of normal and deleted
chains (6). The first possibility was that the -chains were in
correct register with respect to their X- and Y-position amino acids,
both amino-terminal and carboxyl-terminal to the disrupted region at
the deletion site. This hypothesis requires that the normal chains in
effect loop out to preserve the chain registration. The second
possibility is that the molecules fold into a continuous triple helix
without regard to chain registration. This means that the X- and
Y-position residues would be out of register amino-terminal to the
deletion and that the chain(s) with the deletion would stop short of
the amino terminus of the molecule.
The pepsin-solubilized collagen used as the substrate contained a ratio
of short to normal chains of about 2:3 based on the CB11 doublet on
SDS-PAGE of a cyanogen bromide digest. On digestion with trypsin at
24 °C, this collagen yielded two cleavage products when analyzed by
SDS-PAGE. Microsequence analysis of the larger product (2/3 fragment,
Fig. 4A) gave two sequences;
one beginning at residue 361 of the triple helical domain
(GLTGRPG ... ) and the other at residue 375 (VGPSGAP ... ).
Both originate in the exon 24-coded sequence (Fig. 4B) and
so must have derived from normal 1(II) chains that presumably had
been incorporated into heterotrimers that also included one or two
deletion-bearing chains. Amino-terminal sequence analysis of the
smaller product (1/3 fragment, Fig. 4A) revealed
the NH2 terminus of the
1(II) chain beginning in the NH2-telopeptide and running into the triple-helical domain
(GVMQGPM ... ). Trypsin digestion of the collagen at 34 °C also
yielded two cleavage products when analyzed by SDS-PAGE. The larger
product (2/3 fragment, Fig. 4A) yielded the same two
amino-terminal sequences found at 24 °C plus a third sequence
starting at residue 379 (GAPGEDG ... ) in the domain encoded by
exon 25. This trypsin cleavage product can only arise from the exon
24-deleted product of the mutant allele. At 37 °C, trypsin digestion
produced little or no observable 2/3 and 1/3 fragments, although at
least half of the starting collagen had been degraded, as seen by the
decrease in
1(II). Presumably, this reflects rapid melting of the
two triple helical fragments and further proteolysis of their denatured
chains at the higher temperature. The faint band in the 37 °C Kniest
lane (Fig. 4A) that appears to be the 1/3 fragment may in
fact be a degradation product of the 2/3 fragment. (The minor band seen running midway between the 2/3 and 1/3 fragments in the 34 °C Kniest
lane proved by sequence analysis to be a COOH-terminal piece of the 2/3
fragment.)
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To explore the apparent melting properties of the collagen in more detail, the trypsin digestion experiment was repeated with brief digestion (2 min) and a higher enzyme:substrate ratio (1:4). Electrophoresis of the products formed at 31-43 °C in 2 °C increments revealed the same 2/3 and 1/3 fragments at 31-35 °C (results not shown). No other discrete cleavage products were seen, and the remaining full-length molecules melted and were degraded to small fragments with an apparent Tm above 39 °C for both Kniest and control collagens.
Analysis of the CNBr digest of type II collagen from the Kniest
cartilage by molecular sieve HPLC gave a similar peptide profile to
that of control cartilage (Fig. 5). The
fluorescence profiles each had two peaks that contain the cross-linked
peptides from the two sites of pyridinoline cross-linking, two
NH2-telopeptides linked to 1(II)CB9,7 and two
COOH-telopeptides linked to
1(II)CB12. For both control and Kniest
tissue, the ratio of fluorescence between these two peptide pools was
similar. This indicates that for the total pool of collagen in the
Kniest tissue there is no obvious imbalance in relative occupancy
of the two cross-linking sites, which lie at opposite ends of the
triple-helix (13). The concentration of hydroxylysyl pyridinoline
cross-links in the Kniest cartilage collagen was 0.87 residues/collagen
molecule, compared with a range of 0.5-1.5 residues/collagen molecule
for control infant cartilage. The collagen content of the Kniest
tissue, however, was 19% that of the dry weight, compared with
50-60% for neonatal control cartilage.
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Light microscopy of the Kniest cartilage showed the typical Swiss cheese and a perilacunar foamy appearance characteristic of the phenotype (Fig. 6A; Refs. 1 and 5). Transmission electron microscopy showed sparse, thin collagen fibrils immediately surrounding the chondrocytes and thickened fibrils in the periphery (not shown). The chondrocytes contained large inclusion bodies of rough endoplasmic reticulum swollen with a granular material that is presumably abnormal type II collagen (Fig. 6B; Ref. 21).
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To identify the underlying mutation, a cDNA fragment designed to contain the exon 24 coding domain was amplified from total RNA prepared from the Kniest cartilage. This analysis revealed the expected 189-bp fragment, also amplified from control cartilage RNA, plus a shorter fragment of approximately 135 bp (Fig. 7). This smaller fragment would result from mRNA in which exon 24 was deleted, compatible with an exon skipping mutation and consistent with the protein data. Direct sequence analysis of an amplified genomic DNA fragment containing the consensus splice sequences flanking exon 24 demonstrated that the patient was heterozygous for a G to A transition at position +5 of the intron 24 splice donor (Fig. 8).
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DISCUSSION |
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The results define the effects at the protein level of a new mutation in the type II collagen gene that causes Kniest dysplasia. This extends the number of distinct COL2A1 mutations known to cause this phenotype to 10. They include splice-site mutations that cause or predict skipping of exons 12 (22), 15 (23), 18 (8), 20 (Kniest-like; Ref. 24), and now exon 24. Short deletions of seven amino acids in exon 12 (6), six amino acids in exon 21 (25), 34 (8), and 49 (26) also cause Kniest dysplasia. Two single amino acid substitutions, G103D (7) and G127D2 have also been found in Kniest-like cases. It is notable that the exon 20 skipping mutation manifested as a spondyloepiphyseal dysplasia phenotype in the father and a Kniest-like radiographic phenotype in an affected fetus (24). Similarly, the G103D substitution resulted in a Kniest diagnosis from birth to 2 years (7), but the child went on to develop features of an spondyloepiphyseal dysplasia phenotype.3
The present molecular defect, a G to A transition at the +5 position of
the donor splice site of intron 24, results in skipping of exon 24 in
the spliced product of the mutant allele. The other allele and its
spliced transcript are normal. This gene defect explains the recovery
of both normal 1(II) protein sequences and exon 24-deleted
1(II)
sequences from the structural collagen of the Kniest cartilage. Our
detection of mRNA lacking exon 24 was nonquantitative and based on
RT-PCR using primers for exons 23 and 25. It is possible that other
mRNA splice forms had resulted from the mutation that were not
detected by this strategy and also that some of the abnormal
transcripts were degraded in the nucleus.
Yields of cyanogen bromide peptides on SDS-PAGE and of sequenced
peptides indicate a ratio of short 1(II)/normal
1(II) of about
two-thirds in the tissue. The inextractability of the short
1(II)
chains in 4 M guanidine HCl and their recovery in
triple-helical collagen molecules after pepsin digestion of the tissue
indicate that they had become incorporated into the structural fabric
of the extracellular matrix as components of cross-linked fibrils. The
pyridinoline content/mol of tissue collagen and the distribution of
these cross-links between the two intermolecular cross-linking sites in
type II collagen (27) were in the normal range for neonatal cartilage.
The content of collagen/tissue dry weight (19%) was low, however,
consistent with the sparse distribution of collagen fibrils in Kniest
cartilage seen by electron microscopy (28). Matrix deposition of
collagen-containing mutant
1(II) chains appears to be a common
feature in Kniest dysplasia. Including the present results, we and
others have observed this for at least five different COL2A1 mutations
that showed a Kniest-like phenotype and from which cartilage was
available for analysis (6, 7, 23-25, 29).2 The phenotype
may be owing, therefore, to the dominant negative effect of a
particular form of mutant
1(II) chain being incorporated into
molecules and fibrils of the extracellular matrix of cartilage. The
deficiency of extracellular collagen and effects on chondrocytes of
retaining abnormal protein presumably also must contribute greatly to
the pathology. However, deficiency alone is unlikely to explain the
unique phenotype since a matrix deficiency of type II collagen and
chondrocyte accumulation of inclusion bodies is seen in the other type
II collagenopathies (achondrogenesis II, hypochondrogenesis,
spondyloepiphyseal dysplasia congenita, spondyloepimetaphyseal dysplasia), but their clinical presentation and cartilage
histopathology are different from Kniest dysplasia.
An extensive body of published data on similar but not identical
mutations that affect type I procollagen genes and cause osteogenesis
imperfecta provides the basis for interpreting the results (30, 31).
These include glycine substitutions and several multi-exon (32) and
single exon (33) deletions in COL1A1 and COL1A2 that cause in-frame
shortening of the triple-helical domain of the expressed collagen
chains. Cultured skin fibroblasts were routinely studied to define the
consequences of such mutations on the molecular assembly of procollagen
type I. There are relatively few data, however, on the structural
collagen of osteogenesis imperfecta patient tissues, particularly from
cases of single exon deletion. There is one report on a patient with
osteogenesis imperfecta type IV caused by a mutation in COL1A2 that
resulted in skipping of exon 12 (33). Both skin fibroblasts and
osteoblasts in culture incorporated the product of the mutant allele
into secreted procollagen (33). On pepsin treatment, some of the molecules were shortened at the amino terminus of the helix in both
1(I) and
2(I) chains, consistent with a helix disruption at the
deletion site. The present study, therefore, is novel in providing data
on structural collagen, in this case type II collagen, extracted from
the extracellular matrix of a tissue assembled in vivo.
The trypsin digestion results reveal how exon 24-deleted 1(II)
chains affect the structure of molecules into which they become incorporated. In theory, from equal pools of normal and exon 24-deleted chains, three kinds of molecule could assemble. One-eighth would be
normal homotrimers,
would be short-chain homotrimers and
would be heterotrimers of normal and short chains (34).
Whether all these forms assemble into stable molecules and get secreted
from the chondrocytes is unknown. What is clear is that a significant
fraction of molecules containing short chains do get out of the cell
and become cross-linked into extracellular fibrils. The best
explanation for the trypsin cleavage results is that heterotrimers
present a locally disrupted domain at the deletion site (35) in which
the normal chain exon 24 domains lack a triple-helical conformation and
so can be cleaved after arginine and lysine (Fig. 4B). At
24-31 °C, the normal
1(II) chains were clipped but only in their
exon 24 domain, indicating a local disruption within the triple helix.
At 34 °C, the local structure further unfolded to allow cleavage
after the arginine residue in the exon 24-deleted chain(s). The
resulting
and
segments retained a native triple
helix, so explaining the products of trypsin digestion identified on SDS-PAGE (Fig. 4A). Molecules assembled from three normal
1(II) chains are not cleaved at 24 or 34 °C, as seen for the
control type II collagen. If heterotrimers of short and normal chains had folded as an uninterrupted zipper into a continuous triple helix
from the COOH to the NH2 terminus through the deletion site (36-38), then trypsin would not be expected to have cleaved at the
bonds observed. The data, therefore, strongly support the loop-out
explanation and a mechanism of triple-helix folding that can
accommodate a relatively large deletion of 18 residues. This is
consistent with the concept that rather than being a uniform rod, the
triple helix consists of a series of alternating domains of high and
low stability that can fold cooperatively but somewhat independently as
helix formation proceeds from initial registration and nucleation at
the COOH terminus (39). Indeed, regions of micro-unfolding can be
observed in preparations of normal types I and II collagens as cleavage
products when pepsin is used at acid pH above 4 °C (40). But such
products were not a relevant feature of our starting collagen
preparation made at 4 °C.
The fibrillar matrix of hyaline cartilage is a cross-linked copolymer
of collagen types II, IX, and XI (41, 42). Type II collagen is the main
component. Types IX and XI together represent about 20% of the total
collagen in fetal cartilage and decrease to <5% in mature articular
cartilage (43). In mature cartilage collagen, the main intermolecular
cross-links are trivalent pyridinoline residues, which link type II
collagen molecules in fibrils at two head-to-tail sites (27). Two
COOH-telopeptides link to helix Lys-87, and two
NH2-telopeptides link to helix Lys-930. The present analyses for cross-links in Kniest cartilage and the occupancy of
cross-linking sites in the isolated type II collagen show no gross
effect on intermolecular cross-linking of the short 1(II) chains.
There may be subtle effects, for example in restricting the potential
number of cross-linking interactions between nearest neighbor molecules
packed in the polymer. Thus, if molecules containing short
1(II)
chains are shorter than normal molecules and become part of the fibril
polymer, then not all telopeptide-to-helix interaction sites may be
aligned correctly, so that the normal spatial pattern of intermolecular
cross-linking may be disturbed. It may be relevant in this respect that
the known mutations causing Kniest dysplasia appear to be mostly
clustered about one region of the NH2-terminal half of the
molecule. One exception is a deletion of the COOH-terminal six residues
of the triple-helix (26). Conceivably, shortening here could affect the
positioning of the nearby COOH-telopeptides relative to their normal
interaction sites in the helical domains of adjacent molecules in a
fibril. The initial physical interactions responsible for the assembly of monomers into a type II collagen fibril, which may require specific
binding domains along the triple helix (44), could also be
affected.
The susceptibility of collagen molecules from Kniest tissue to helical
domain cleavage by trypsin raises another potential source of cartilage
morbidity. Matrix proteases that are involved in extracellular
remodeling that do not normally attack collagen might damage Kniest
fibrils. Thus, the collagen network might be prone to progressive
proteolytic damage at the sites of the helical domain imperfections.
This could explain the degenerate, vacular appearance of the matrix.
The presence of abnormal type II collagen molecules might also disturb
the copolymeric assembly of types IX and XI collagens into the fibril
architecture. Finally, since the collagen type XI molecule normally
includes one 1(II) chain (34), incorporating a short
1(II) chain
could negatively affect collagen type XI function.
The exceptions to the emerging pattern of full or partial exon-skipping mutations underlying Kniest dysplasia are two G to D substitutions (Ref. 7).2 They are close to each other (G103D, G127D), and one of them (G103D) is within the 7 amino acid sequence at the COOH terminus of exon 12 that is deleted in two other, unrelated Kniest cases (6). Therefore, if a G to D substitution is particularly disruptive, as collagen type I mutations in osteogenesis imperfecta suggest (36, 45), it might also result in a looping out of normal chains in this domain of the heterotrimer so that the net effect on the molecule and fibril may be the same as a short deletion. We suggest this unifying hypothesis to explain in part how different mutations might act through a common molecular mechanism to produce characteristic features of the Kniest phenotype.
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ACKNOWLEDGEMENTS |
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We thank Mary Ann Priore and Sheila Levin of the International Skeletal Dysplasia Registry at Cedars-Sinai Medical Center for assisting with sample collection and Kae Pierce for manuscript preparation.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HD22657, AR37318, and GM16219 (to D. W.). Additional support was from the Division of Intramural Research, National Human Genome Research Institute, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-1267.
§§ To whom correspondence should be addressed: Dept. of Orthopaedics, University of Washington, P. O. Box 356500, Seattle, WA 98195-6500. Tel.: 206-543-4700; Fax: 206-685-4700.
1 The abbreviations used are: HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pair(s); RT, reverse transcription; CB, cyanogen bromide.
2 D. R. Eyre, M. A. Weis, R. S. Lachman, and D. L. Rimoin, unpublished results.
3 D. L. Rimoin, unpublished observations.
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REFERENCES |
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