98195, the 'Ahmanson Department of Pediatrics, Steven Spielberg Pediatric Research Center, Cedars-Sinai Medical Center, Los. Angeles, California 90048 ...
THEJOURNAL
OF
Vol. 267, No. 31, Issue of November 5, pp. 22522-22526,1992 Printed in U.S.A.
BIOLOGICAL CHEMISTRY
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
An Amino Acid Substitution (Glys53+ Glu) in theCollagen al(I1) Chain Produces Hypochondrogenesis* (Received for publication, April 27, 1992)
Raymond Bogaert,"9bGeorge E. Mary Ann Weis," Helen E. Gruber,c*d,fDavidL. Rimoin:dpf Daniel H. Cohn:dsf and David R. Eyre"~g~n FromtheDepartments of "Orthopaedics, Biology, andPBiochemistry,University of Washington,Seattle,Washington 98195, the 'Ahmanson Departmentof Pediatrics, Steven Spielberg Pediatric Research Center, Cedars-Sinai Medical Center,Los Angeles, California 90048, and the dDepartmentof Pediatrics, UCLA School of Medicine, Los Angeles, California 90024
The spondyloepiphyseal dysplasia subclassification characterized clinically by abnormal epiphyses, flattened verof bone dysplasiasincludesachondrogenesis, hypo- tebral bodies, varyingdegrees of metaphyseal irregularity, chondrogenesis, and spondyloepiphyseal dysplasia myopia, and vitreous degeneration (2-4). At one end of the congenita. The phenotypic expression of these disor- spectrum,theperinatallethal formachondrogenesis I1 is ders ranges from mild to perinatal lethal forms. We usually characterized by an absence of type I1 collagen in report the detection and partial characterizationof a cartilage matrix and thepresence of type I collagen (5,6). In defect in type I1 collagen in a perinatal lethal formof the radiographically less severe but still lethal form, hypohypochondrogenesis. Electrophoresis in sodium dode- chondrogenesis, the matrix typically contains post-translacy1 sulfate-polyacrylamide of CB peptides (where CB tionally overmodified type I1 collagen, with variable amounts represents cyanogen bromide) from typeI1 collagen of of type I collagen (7, 8). Cartilage from moderately severe the diseased cartilage showed a doublet bandfor pep- spondyloepiphysealdysplasia congenitapatientscontains tide al(I1)CBlO and evidence for post-translational mostly type I1 collagen, which can be structurally abnormal overmodification of the major peptides (CB8, CB10, (9). and CB11) seen as a retarded electrophoreticmobility. Recently, dominant mutations in the typeI1 collagen gene, Peptide CBlO wasdigested by endoproteinase Asp-N; COL2A1, have been identified within the spondyloepiphyseal and on reverse-phase high pressure liquid chromatog-dysplasia congenita spectrum of disease (10-12). These findraphy, fragments of abnormal mobility were noted. ings provide genetic support for the concept of a family of Sequence analysis of a unique peptide D l 2 revealed a type I1 collagenopathies with a phenotypic continuum ranging single amino acid substitution(Gly + Glu) at position from mild toperinatallethal,similartothe osteogenesis 853 of the triple helical domain. This was confirmed imperfecta spectrum of clinical phenotypes that results from by sequenceanalysis of amplified COLZA1 cDNA, a diversity of structural mutations in the genes for type I which revealed a single nucleotide substitution (GGA collagen (13-16). For the type I1 collagen mutations, it is not + GAA) in 5 of 10 clones. Electron micrographsof the yet clear how different molecular defects relate to theclinical diseased cartilage showed a sparse extracellular ma- phenotype or whether the abnormal protein chains are incortrix and chondrocytes containing dilated rough endo- porated into the extracellular matrix and so directly affect its plasmic reticulum, which suggested impaired assembly structure and function. and secretion of the mutant protein. This case further This study identifies a moleculardefect in the COLZA1 documents the molecular basis of the spondyloepiphyseal dysplasia spectrum of chondrodysplasias as mu- gene and extracellular type I1 collagen of cartilage in a case of hypochondrogenesis. tations in COLZA1. MATERIALSANDMETHODS
Clinical Summary
The chondrodysplasias are a heterogeneous group of disorderscharacterized by abnormalformationand growth of cartilage (1). The spondyloepiphysealdysplasia congenita subclassification includes a spectrum of heritable disorders
* This work was supported in part by National Institutes of Health Grants PO1 HD22657 and AR37318. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s)reported in this paper has been submitted totheGenBankTM/EMBL Data Bankwith accession numbeds) L00977. e Supported by an Arthritis Foundation postdoctoral fellowship. Present address: Div. of Genetics,VanderbiltUniversity Medical Center, Nashville, T N 37240. Supported by the Ahmanson Department of Pediatrics and the Steven Spielberg Pediatric Research Center. To whom correspondence should be addressed Dept. of Orthopaedics, RK-10, University of Washington, Seattle, WA 98195. Tel.: 206-543-4700; Fax: 206-685-3139.
'
The proband was born at 33 weeks of gestation to clinically normal parents, with a birth length of 34.5 cm (50th percentile for a 26-week fetus). Short limbs and a small chest were noted on prenatal ultrasound examination at 19 weeks of gestation. In addition to these findings, unilateral polydactyly was noted at birth.Early radiographs were consistent with a severe form of spondyloepiphyseal dysplasia congenita, whereas skeletal films at 3 months of age more closely resembled hypochondrogenesis (17). The infant required continuous respiratory support until his death at 3 months of age.
Protein Analysis Pepsin-solubilized Collagen-Rib cartilage was extracted in 4 M guanidine HC1,0.05 M Tris/HCl, pH 7.0, at 4 "C for 48 h. The washed residue was digested withpepsin(18). Humanarticular cartilage collagen from a 25-year-old normal male was used as acontrol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE)' was carried out by the method of Laemmli (19) in 6 and 12.5% gels. The abbreviations used are: SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; CB, cyanogen bromide; HPLC, high pressure liquid chromatography.
22522
22523
Type 11Collagen Mutation in Hypochondrogenesis Peptide Analysis-Rib cartilage was digested with cyanogen bromide in 70% (w/v) formic acid for 24 h a t room temperature (20). T h e CB peptides were fractionated by sequential cation-exchange HPLC (Mono-S HR 5/5, Pharmacia LKB Biotechnology Inc.) and reverse-phase HPLC (CRRP-300, 25 cm X 4.6 mm, Brownlee Labs) (21,22). Pooled fractions containing nl(1I)CBlO (identifiedby SDSPAGE) were dried, and the peptidewas digested withendoproteinase Asp-N (sequencinggrade,Boehringer Mannheim).Theresulting peptides were fractionated by reverse-phase HPLC using a gradient (0-30% in 60 min) of acetonitrile/l-propanol (3:1, v/v) in 0.1% (v/v) trifluoroacetic acid a t 1 ml/min (22). Peptide yields were estimated by areas under absorbance peaks in the chromatography profiles and by recoveries of phenylthiohydantoin-derivativeson sequence analysis. Protein Microseguencing-Edman amino-terminal sequencing of individualpeptides was carriedouton a Porton 2090E machine equipped with on-line HPLC analysis of phenylthiohydantoin-derivatives using the manufacturer's standardprogram. Collagen Cross-link Analysis-An acid hydrolysate (6 M HCI, 24 h, 110 "C)of cartilage was analyzed for the hydroxypyridinium (pyridinoline) cross-links of collagen by reverse-phase HPLC as previously described (23). Hydroxyproline was measured in the hydrolysate by colorimetricanalysis (32). Thecross-linkconcentration was expressed asmoles/mole of collagen relative to normal type I1 collagen. Nucleic Acid Analysis Total cellular RNA was extracted from fresh-frozen cartilage by the method of Chomczynski and Sacchi (24). First-strandcDNA was synthesized using avian myeloblastosis virus reverse transcriptase and reagents from a kit (Promega Biotec) with the COL2Al exon 44 specific primer 5'-GGCCGAATTCAGCACCAGTCTCACCACGATC-3'. Double-stranded cDNA was generated using the polymerase chain reaction (25) with the reverse transcription primer and a primer that anneals the at exon 31/32 junction, 5'-CGGCCGGATCCCAAAGGTGCATCTGGCCCAGCA-3'. The amplification conditions were 40 cycles a t 94 "C for 1 min, 55 "C for 1 min, and 72 "C for 2 min. After purification through a low melting point agarose gel (26), the1021-base pair polymerase chain reaction product was digested with BamHI and EcoRI and ligated into bacteriophage M13mp18. DNA sequence analysis was performed using Sequenase 2.0 (United States Biochemical Corp.). The primer 5"AACGTGGTGAGAGAGGATTC-3' was used to generate the sequence shown in Fig. 5. GenomicDNAwaspurified fromparentalspermandpatient cartilage (27) and used to amplify a 917-basepair fragment containing the mutation. The primersused were the sequencing primer and the first-strand cDNA primer described above. The conditions were 40 cycles a t 94 "C for 1 min, 55 "C for 1 min, and 72 "C for 1.5 min. The polymerase chain reaction product wasdigested with HinfI (New England BioLabs, Inc.), and the resulting fragments were separated by electrophoresis through a 6% polyacrylamide gel.
y.
--
Bal(II)- . I )
>B8 ;B 9,7
Pepsin-solubilized type II collagen
CNBr digest
FIG. 1. SDS-PAGE of pepsin-solubilized collagen and CB peptides from hypochondrogenesis tissue. The pepsin-solubilized nl(I1)chains (6% gel; S E D ) showa doubletandretarded mobility in comparison with normal human al(I1) (control). The CB digestshowsa doublet for nl(1I)CBlO and retarded mobilities of peptides CB10, CB11, and CB8 compared with CB peptides from control human cartilage.
t
l.o
20
310
410
40
ELUTION TIME(min)
P CB 10 CB 11. al(I) CB 7 .al(l) CB 8
Electron Microscopy Cartilage specimens from rib, femur, iliac crest, and vertebrawere fixed in 2.5% glutaraldehyde, post-fixed in osmium, and dehydrated and embedded in Spurr resin. Thin sectionswere cut with a diamond knife using Pharmacia microtome, collected on one-hole Formvarcoated grids, stained with uranyl acetate andlead citrate, andviewed with a Zeiss 902A transmission electron microscope.
CB 8
31 32 33 34 35
RESULTS
FIG. 2. Upper, elution profile from cation-exchange HPLC of CB
The al(I1) chain of pepsin-solubilized collagen extracted peptides of type I1 collagen from the hypochondrogenesis cartilage. The fractions marked by the barwere pooled for reverse-phase HPLC. from the hypochondrogenesis cartilage migrated as a doublet Lower, SDS-12.5% PAGE of fractions sampledacross the nl(I1)CBlO on SDS-PAGE, with most of the protein in the more slowly peak as indicated by the bar (upper). PeptideCBlO ran as a doublet migrating component (Fig. 1).The CB peptides of the abnor- on the gel. The upper band eluted slightly earlier in the chromatomal type I1 collagen also migrated moreslowly than their gram. counterparts from control human cartilage, with al(I1)CBlO running asa doublet (Fig. 1). individual Asp-N peptides unique to thehypochondrogenesis Analysis of the CB digest by cation-exchange HPLC and patient cartilage were selected for amino-terminal microseSDS-PAGE showed that theupper (slower) component of the quence analysis (Fig. 3). The sequence of a novelform of CBlO doublet was more acidic (Fig. 2). Fractions containing peptide Dl2 (residues 849-856 of the al(I1)chain triple helix) al(1I)CBlOwere pooled. The peptide was further purified by identified glutamate at cycle 5 (corresponding to position 853 reverse-phase HPLC and digested with endoproteinase Asp- of the triple helical domain) insteadof the glycine seen in the N. On the basis of the elution profile on reverse-phase HPLC, normal version of peptide Dl2 at thisposition. The ratio of
Type 11 Collagen Mutation in
22524
20
30
SO
40
60
ELUTION TIME (MIN) C
D3
552
P G E R G A A G I A G P K G 4 0 R G 1 D V G E
573
K
G
P
E
594
P
G
P
A
615
A
G G
A
P
A
G
K
N
G
'
D
E
K
G
G
R
G
L
T
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V
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P
P
G G
P P
I
G A
572
P
593
G
S
614
G A R G A P G E R G E T G P P G P A G F 05 A G P P G A ' D G O P G A K G E O G E A G O
635
636 657
K G ' O A G A P G P O G P S G A P G P O G P
677
678
T
G
V
T
G
P
K
G
A
R
G
A
O
G
P
P
G
A
T
G
F
698
699
P
G
A
A
G
R
V
G
P
P
G
S
N
G
N
P
G
P
P
G
P
719
720
P G P S G K ' O G P K G A R G ' D S G P P G R
741
A
G
E
762
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G
P
S
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A
E
G
P
P
G
P
783
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G
L
P
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R
G
E
R
G
F
804
P G K O G A P G A S G ' O R G P P G P V G P
815
P
846
P G R I 012 D G A A E V K G P O R G E T G A V G A
866
867
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G
887
888
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G
G
L
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S
G
A
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P
P
G
G
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P
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O
A
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740
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656
761 I
782
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803
P
O
Hypochondrogenesis
distinctive peak of a phenylthiohydantoin-derivativefor galactosylhydroxylysine.Hence, theearlier-eluting version of peptide D5 gave a blank a t cycle 7. Peptide D5 from control tissue gave a mixture of hydroxylysine and lysine at cycle 7, but no galactosylhydroxylysine. The concentration of hydroxylysylpyridinoline cross-links in the hypochondrogenesis cartilage collagen was 0.55 residues/mol of collagen, compared with a mean of -0.5 residues/ mol for control neonatal cartilage (28). Ultrastructural examination of thecartilage revealed a normal cell density within a sparse extracellular matrix (Fig. 4). Chondrocytes contained inclusion bodies of dilated rough endoplasmic reticulum filled with a granular material. Sequence analysis of cDNA clones derived from RNA isolated from the patient's cartilage showed that the glutamate for glycine substitution at residue 853 of the helix was the result of a point mutationin the second positionof the glycine codon (GGA to GAA) (Fig. 5A). The mutation obliterated a HinfI restriction endonuclease cleavage site,and cleavage affected individual with HinfI was used to determine that the was heterozygous for the absence of the site in polymerase chain reaction-amplified genomic DNA (Fig. 5B). During the cDNA sequence analysis, we determined that the affected individual was also heterozygous for a C or T in the third position of the codon for the glycine a t residue 565 of the triple helical domain. This presumably neutral polymorphism can be detected as a restriction fragment length polymorphism by cleavage with MueIII.Restriction analysis of the cDNAclones demonstrated that the mutation was carried by the MueIII (+)-allele. The unaffected father was homozygous for the presence of the site and therefore must havecontributedtheabnormal allele (datanot shown). Within the limitsof the sensitivityof the assay, there was no fragment thatcorresponded to anallele lackingthe HinfI site
045
M
FIG.3. Reverse-phase HPLC separation of peptides generated by endoproteinase Asp-N digestion of purified CB fragment al(1I)CBlO from hypochondrogenesis cartilage (a)and ( b )and amino acid sequence of human human control cartilage al(I1)CBlO from published cDNA data (31)( c ) . Endoproteinase Asp-N cleavage sites are indicated by arrows. The Asp-N peptides subjected to microsequence analysis are indicated. The sequence of the mutation-containing peptide, D12, is boxed.
glutamate to glycine peptide forms was -19. The galactosylhydroxy1ysine:hydroxylysine:lysine ratio a t cycle 7 was the same in the glutamate-containing peptide as in the normal peptide from the hypochondrogenesis tissue (data not shown). Two versions of peptide D3 (residues 569-581 of al(I1)) were derived from the hypochondrogenesis tissue in equal yield, an earlier-eluting peak that yielded hydroxylysine at cycle 5 (residue 573 of the triplehelix) and a secondpeak that gave lysine a t cycle 5. Control D3 gave only lysine at this position. Similarly, peptide D5 (residues 642-658) was also resolved as a doublet, withthe glucosylgalactosylhydroxylysyl form a t cycle 7 elutingfirst.Thelater-eluting version of peptide D5 gave two peaks a t cycle 7, indicating a mixture of galactosylhydroxylysine and hydroxylysine. Our experience with glycosylated hydroxylysines on microsequence analysis is a blank cycle for glucosylgalactosylhydroxylysine and a
FIG.4. Ultrastructure of cartilage from patient. The low magnification transmission electron micrograph shows a representative chondrocyte from the resting zone. Note thesparse collagen matrix surrounding the cell and the large inclusion bodies (magnification X 13,600).
Type 11Collagen Mutation in Hypochondrogenesis A
equally in both normal and mutant chains of molecules that contain a mutant chain. The observations thatonly a small Val Lp proportion of protein corresponding to normally migrating 1 Glu al(I1) chainswas present in a pepsin digest, and similarly for NORMAL Patlent Father the CBpeptides, support these theoreticalconsiderations. The lysine a t residue 573 in peptide D3 appears not to be hydroxylated in normal cartilage since the peptide elutesa as single peak on reverse-phase HPLC (Fig. 3) and gave only lysine on sequence analysis. From the abnormaltissue, about equal amounts of hydroxylysine-containing and lysine-containing peptides were resolved (Fig. 3). Our interpretation is that the lysine a t residue 573 normally resists hydroxylation (probably because it follows glutamate), and the extent of overmodification in thehypochondrogenesis case is therefore limited a t this residue to partialhydroxylation. It may be that this lysine residue escapes hydroxylation even insome chains of molecules that contain one ormore mutant (ul(I1) chains. FIG. 5. Identification of mutation. A, sequence of the normal At other lysine sites (the lysine a t residue 648 of peptide D5 and abnormalalleles in theregion of the cDNA carrying the mutation. allele. for example), each residue may normally be largely hydroxT h e arrow marks thelocation of the mutation in the abnormal A partial sequence is shown above. The HinfI site altered by the ylated, and so the overmodification is in the degree of glycomutation is underlined. B, HinfI cleavage of amplified genomic DNA sylation. In short, thedegree of post-translational modificafrom the patient and from sperm DNA of the father. The 562-base tion of lysine residues clearly varies dependent on the local pair fragment diagnostic of the mutation is marked with an arrow. Shown above is a line diagram of the region of the COL2A1 genethat sequence in normal a(I1) chains, thus resulting in site-dependent variability in theovermodification of molecules bearcarries the mutation, the region amplified by the primers, and the by HinfI (H)digestion. ing mutant al(I1) chains. These results and the electropholocations andsizes of the fragments generated T h e HinfI site disruptedby the mutation is markedwith an asterisk. retic evidenceshow post-translational overmodificationin peptides amino-terminal to the mutation site, consistent with destroyed by the mutation in amplified DNAfrom the father's theory. How does expressionof this mutant proteinmolecule affect sperm (Fig. 5). Therefore, the fatherwas not detectably mothe organization of the collagen fibril and associated matrix? saic for the mutation in hisgerm line. The measured content of mature cross-links in the cartilage collagen implies that there is no dramaticeffect on intermoDISCUSSION lecular cross-linking of the mutant protein. The ultrastrucT o date, specific mutations in typeI1 collagen reported for tural observation of grossly dilated endoplasmic reticulum, patients with clinical phenotypes within thespondyloepiphyseal dysplasia family of chondrodysplasia have been identified which presumably contains abnormal type I1 collagen moleby analysis of genomic DNA or cDNA clones (10-12). This cules (29), suggests that there isa reduced amount of type I1 study has detected and defined a new mutation at the protein collagen in the extracellular matrix and therefore a reduced level. The mutation was a glutamate for glycine substitution ratio of type I1 collagen to other matrix molecules. It is also at residue 853 of the al(I1) triple helix, which produced a possible that the intracellular retention secondarily affected the function of chondrocytes at the growth plate. The overhypochondrogenesis phenotype. We demonstrated that the mutation arose in the paternally inherited allele, but that modification and/or the directeffect on proteinconformation there was no evidence of the abnormal allele in sperm from of the amino acid substitution may interfere with specific the father. We conclude that this hypochondrogenesis phe- associations between the type I1 collagen fibrils and other notype resulted from a new dominant mutation in the type I1 matrix molecules, such as type IX and XIcollagens, decorin, collagen gene, COL2A1. biglycan, fibromodulin, or aggrecan (30). Finally, fibrils conThe amino acid substitution produced a more slowly mi- taining the mutant molecules may not be turned over norgrating population of collagen al(I1) chains, suggesting an mally within thematrix. increased degree of lysyl hydroxylation and hydroxylysyl glyThis case of hypochondrogenesis adds support to the concosylation. Judging by the g1utamate:glycine ratio in peptide cept that dominant mutations in the COLZA1 gene characD12, which contains the mutatedresidue, there were roughly terize at the molecular level those disorders that have been equal amountsof the mutantal(I1) and normal al(I1) chains clinically grouped into thespondyloepiphyseal dysplasia faminthe cartilagematrix. Thus,bothnormalandmutant ily of chondrodysplasias. Analysis of additional cases of type COLZA1 alleles had contributed equally to matrixprotein. I1 collagenopathies at thehistologic, protein, andnucleic acid The aminoacid sequencing results on peptides D3 and D5 levels will likely lead to the identification of functional dofrom the hypochondrogenesis tissue at cycles where lysine mains within the type I1 collagen molecule and provide a occurs at theY-position of the Gly-X- Yrepeat are consistent better understandingof how a molecular defect translates into with post-translational overmodification, but this appears to a clinical phenotype and of the relationships between the be variable in extent, dependent on the actual sequence. In various macromolecules of the cartilage matrix. theory, for the homotrimeric type I1 collagen molecule, threequarters of the molecules would contain one or two mutant Acknowledgments-We thank Dr. Susan Winter for bringing the chains, one-eighth would contain three normal chains, and patient to our attention and arranging procurement of tissue samples, one-eighth three mutant chains. If all molecules with a t least Dr. Ralph Lachman for radiologic interpretation of films, Maryann one abnormal chain became post-translationally overmodi- Priore and Shielah Levin for coordination of the International Skelfied, seven-eighths of the chains should have an increased etal Dysplasia Registry, and Betty Mekikian and Loyda Nolasco for hydroxylysine content, assuming that overmodification occurs technical assistance with light and electron microscopy. @
GGC GCT GCT GGGly Ala Ala Gly
MurAur
B
22525
M G
22526
Type II Collagen Mutation in
REFERENCES 1. Rimoin, D. L., and Lachman, R. S. (1990) in Principles and Practice of Medical Genetics (Emery, A. E. N., and Rimoin, D. L., eds) pp. 895-932, Churchill-Livingstone, Inc., New York 2. Spranger, J. W., and Langer, L. O., Jr. (1970) Radiology 94,313-322 3. Spranger, J. (1975) Birth Defects Orig. Artic. Ser. 1 1 , 177-182 4. Lachman, R. S., Rimoin, D. L., Hall, J. G., Kozlowski, K., Langer, L. O., Scott, C. I., and Spranger, J. (1975) Birth Defects Or&. Artic. Ser. 1 1 , 231-248 5. Eyre, D. R., Upton, M. P., Shapiro, F. D., Wilkinson, R. H., and Vawter, R. H. (1986) Am. J. Hum. Genet. 39,52-67 6. Eyre, D. R. (1988) Pathol. Immunopathol. Res. 7,90-94 7. Murray, L. W., and Rimoin, D. L. (1988) Pathol. Immunopathol. Res. 7 , 99-103 8. Godfrey, M., and Hollister, D. W. (1988) Am. J. Hum. Genet. 43, 904-913 9. Murray, L. W., Baustista, J., James, P. L., and Rimoin, D. L. (1989) Am. J. Hum. Genet. 45,5-15 10. Lee, B., Vissing, J. H., Ramirez, F., Rogers, D., and Rimoin, D. (1989) Science 244,978-980 11. Vissing, H., D'Alessio, M., Lee, B., Ramirez, F., Godfrey, M., and Hollister, D. W. (1989) J. Biol. Chem. 2 6 4 , 18265-18267 12. Tiller, G. E., Rimoin, D. L., Murray, L. W., and Cohn, D. H. (1990) Proc. Natl. Acad. Sci. LI. S. A . 8 7 , 3889-3893 13. Kuivaniemi, H., Tromp, G., and Prockop, J. (1991) FASEB J. 5 , 20522060 14. Byers, P. H., Tsipouras, P., Bonadio, J. F., Starman, B. J., and Schwartz, R. C. (1988) Am. J.Hum. Genet. 4 2 , 237-248 15. Cohn, D. H., Apone, S., Eyre, D. R., Starman, B. J., Andreassen,P.,
Hypochondrogenesis
Charbonneau, H., Nicholls, A. C., Pope, F. M., and Byers, P. H. (1988) J. Biol. Chem. 2 6 3 , 14605-14607 16. Starman B. J., Eyre, D., Charbonneau, H., Harrylock, M., Weis, M. A., Weiss,'L., Graham, J. M., and Byers, P. H. (1989) J. Clin. Invest. 84, 1206-1 21 4 ~~~~
""
17. Taybi, H., and Lachman, R. S. (1990) Radiology of Syndromes Metabolic Disorders, and Skeletal Dysplasias, 3rd Ed., pp. 733-735 and 798-800, Year Book Medical Publishers, Inc., Chicago 18. Miller, E. J. (1972) Biochemcst 1 1 , 4903-4909 19. Laemmli, U. K. (1970) Nature~27,680-685 20. Eyre, D. R., and Muir, H. (1975) Biochem. J. 161,595-602 21. Bateman, J. F., Mascara,T.,Chan, D., and Cole, W. G. (1986) A d . Biochem. 54,338-344 22. Eyre, D. R. (1987) Methods Enzymol. 4,115-139 23. Eyre, D. R., Koob, T. J., and Van Ness, K. P. (1984) Anal. Biochem. 1 3 7 ,
m n --"m __-
24. Chomczynski P. and Sacchi N. (1987) Anal. Biochem. 162 156-159 25. Saiki, R. K belkand D. H.btoffel, S., Scharf, S. J. Hi ch; R Horn, G. T., Mull& K. B., i n d Ehhich, H. A. (1987) Scienle 2%, $872491 26. Benson S. A. (1984) BzoTechn ues 2 66 67 27. Sambrdok J Fritsch E. F., an3 Maniati; T. (1989) Molecular Clonin A &boratA$Manual,'pp. 9.16-9.17, Cold Spring Harbor Laboratory,told Spring Harbor, NY 28. Eyre, D. R., Dickson, I. R., and Van Ness, K.(1988) Biochem. J. 252,4955nn
29. Godfrey M. Keene, D. R., Blank, E., Hori H. Sakai L. Y., Sherwin, L. A., a i d Hbllister, D. W. (1988) Am. J. HLm. &net. i3,894-903 30. Heinegird, D., and Oldber , A. (1989) FASEB J. 3, 2042-2051 M., Smith, C., Jimmez, S. A., and Prockop, D. 31. Baldwln C. T. Regmato J. (19h9) Bi6chem. J. 262.521-528 32. Stegemann, H. (1958) Z . Phys. Chem. 311,41-45
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