Mutations Near Amino End of α1(I) Collagen Cause Combined Osteogenesis Imperfecta/Ehlers-Danlos Syndrome by Interference with N-propeptide Processing
2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês
10.1074/jbc.m414698200
ISSN1083-351X
AutoresWayne A. Cabral, Elena Makareeva, Alain Colige, Anne D. Letocha, Jennifer M. Ty, Heather N. Yeowell, Gerard Pals, Sergey Leikin, Joan C. Marini,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoPatients with OI/EDS form a distinct subset of osteogenesis imperfecta (OI) patients. In addition to skeletal fragility, they have characteristics of Ehlers-Danlos syndrome (EDS). We identified 7 children with types III or IV OI, plus severe large and small joint laxity and early progressive scoliosis. In each child with OI/EDS, we identified a mutation in the first 90 residues of the helical region of α1(I) collagen. These mutations prevent or delay removal of the procollagen N-propeptide by purified N-proteinase (ADAMTS-2) in vitro and in pericellular assays. The mutant pN-collagen which results is efficiently incorporated into matrix by cultured fibroblasts and osteoblasts and is prominently present in newly incorporated and immaturely cross-linked collagen. Dermal collagen fibrils have significantly reduced cross-sectional diameters, corroborating incorporation of pN-collagen into fibrils in vivo. Differential scanning calorimetry revealed that these mutant collagens are less stable than the corresponding procollagens, which is not seen with other type I collagen helical mutations. These mutations disrupt a distinct folding region of high thermal stability in the first 90 residues at the amino end of type I collagen and alter the secondary structure of the adjacent N-proteinase cleavage site. Thus, these OI/EDS collagen mutations are directly responsible for the bone fragility of OI and indirectly responsible for EDS symptoms, by interference with N-propeptide removal. Patients with OI/EDS form a distinct subset of osteogenesis imperfecta (OI) patients. In addition to skeletal fragility, they have characteristics of Ehlers-Danlos syndrome (EDS). We identified 7 children with types III or IV OI, plus severe large and small joint laxity and early progressive scoliosis. In each child with OI/EDS, we identified a mutation in the first 90 residues of the helical region of α1(I) collagen. These mutations prevent or delay removal of the procollagen N-propeptide by purified N-proteinase (ADAMTS-2) in vitro and in pericellular assays. The mutant pN-collagen which results is efficiently incorporated into matrix by cultured fibroblasts and osteoblasts and is prominently present in newly incorporated and immaturely cross-linked collagen. Dermal collagen fibrils have significantly reduced cross-sectional diameters, corroborating incorporation of pN-collagen into fibrils in vivo. Differential scanning calorimetry revealed that these mutant collagens are less stable than the corresponding procollagens, which is not seen with other type I collagen helical mutations. These mutations disrupt a distinct folding region of high thermal stability in the first 90 residues at the amino end of type I collagen and alter the secondary structure of the adjacent N-proteinase cleavage site. Thus, these OI/EDS collagen mutations are directly responsible for the bone fragility of OI and indirectly responsible for EDS symptoms, by interference with N-propeptide removal. Osteogenesis imperfecta (OI) 1The abbreviations used are: OI, osteogenesis imperfecta; EDS, Ehlers-Danlos syndrome; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcriptase. is a genetic disorder of connective tissue characterized by bone fragility, growth deficiency, and blue sclerae (1Sillence D.O. Senn A. Danks D.M. J. Med. Genet. 1979; 16: 101-116Crossref PubMed Scopus (1577) Google Scholar, 2Marini J.C. Behrman R.E. Kliegman R.M. Jenson H.B. Nelson Textbook of Pediatrics. 17th Ed. Saunders, Philadelphia2004: 2336-2338Google Scholar). Defects in type I collagen are well known to cause the full clinical range of OI (3Byers P.H. Cole W.G. Royce P.M. Steinmann B. Connective Tissue and Its Heritable Disorders. 2nd Ed. Wiley-Liss, Inc., New York2002: 385-430Crossref Google Scholar, 4Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1379) Google Scholar). Haploinsufficiency for type I collagen, caused by a null α1(I) allele, results in a very mild clinical phenotype (5Willing M.C. Deschenes S.P. Scott D.A. Byers P.H. Slayton R.L. Pitts S.H. Arikat H. Roberts E.J. Am. J. Hum. Genet. 1994; 55: 638-647PubMed Google Scholar). Collagen structural defects, which are usually glycine substitutions or exon skipping defects, have a dominant negative mechanism. They result in a phenotype that ranges from lethal to moderately severe depending on the chain in which the mutation occurs, its location in the chain, and the specific amino acid substituted (6Byers P.H. Wallis G.A. Willing M.C. J. Med. Genet. 1991; 28: 433-442Crossref PubMed Scopus (232) Google Scholar, 7Marini J.C. Lewis M.B. Wang Q. Chen K.J. Orrison B.M. J. Biol. Chem. 1993; 268: 2667-2673Abstract Full Text PDF PubMed Google Scholar). The great majority of mutations causing OI occur in the helical regions of either pro-α1(I) or pro-α2(I). Less than 5% of collagen structural mutations occur in the C-propeptides of the two chains; these mutations cause lethal to moderate OI by delaying chain association into heterotrimer (8Pace J.M. Kuslich C.D. Willing M.C. Byers P.H. J. Med. Genet. 2001; 38: 443-449Crossref PubMed Google Scholar). Ehlers-Danlos VII A and B are also caused by mutations in type I collagen (9Beighton P. De Paepe A. Steinmann B. Tsipouras P. Wenstrup R.J. Am. J. Med. Genet. 1998; 77: 31-37Crossref PubMed Scopus (1373) Google Scholar). These mutations have a well defined location and mechanism of action (10Giunta C. Superti-Furga A. Spranger S. Cole W.G. Steinmann B. J. Bone Joint Surg. Am. 1999; 81: 225-238Crossref PubMed Scopus (62) Google Scholar, 11Cole W.G. Chan D. Chambers G.W. Walker I.D. Bateman J.F. J. Biol. Chem. 1986; 261: 5496-5503Abstract Full Text PDF PubMed Google Scholar, 12D'Alessio M. Ramirez F. Blumberg B.D. Wirtz M.K. Rao V.H. Godfrey M.D. Hollister D.W. Am. J. Hum. Genet. 1991; 49: 400-406PubMed Google Scholar, 13Nicholls A.C. Sher J.L. Wright M.J. Oley C. Mueller R.F. Pope F.M. J. Med. Genet. 2000; 37: E33Crossref PubMed Scopus (19) Google Scholar, 14Byers P.H. Duvic M. Atkinson M. Robinow M. Smith L.T. Krane S.M. Greally M.T. Ludman M. Matalon R. Pauker S. Quanbeck D. Schwarze U. Am. J. Med. Genet. 1997; 72: 94-105Crossref PubMed Scopus (93) Google Scholar, 15Eyre D.R. Shapiro F.D. Aldridge J.F. J. Biol. Chem. 1985; 260: 11322-11329Abstract Full Text PDF PubMed Google Scholar, 16Wirtz M.K. Glanville R.W. Steinmann B. Rao V.H. Hollister D.W. J. Biol. Chem. 1987; 262: 16376-16385Abstract Full Text PDF PubMed Google Scholar, 17Weil D. D'Alessio M. Ramirez F. Steinmann B. Wirtz M.K. Glanville R.W. Hollister D.W. J. Biol. Chem. 1989; 264: 16804-16809Abstract Full Text PDF PubMed Google Scholar, 18Nicholls A.C. Oliver J. Renouf D.V. McPheat J. Palan A. Pope F.M. Hum. Genet. 1991; 87: 193-198Crossref PubMed Scopus (28) Google Scholar, 19Vasan N.S. Kuivaniemi H. Vogel B.E. Minor R.R. Wootton J.A. Tromp G. Weksberg R. Prockop D.J. Am. J. Hum. Genet. 1991; 48: 305-317PubMed Google Scholar, 20Chiodo A.A. Hockey A. Cole W.G. J. Biol. Chem. 1992; 267: 6361-6369Abstract Full Text PDF PubMed Google Scholar, 21Lehmann H.W. Mundlos S. Winterpacht A. Brenner R.E. Zabel B. Muller P.K. Arch. Dermatol. Res. 1994; 286: 425-428Crossref PubMed Scopus (12) Google Scholar, 22Ho K.K. Kong R.Y. Kuffner T. Hsu L.H. Ma L. Cheah K.S. Hum. Mutat. 1994; 3: 358-364Crossref PubMed Scopus (16) Google Scholar). All EDS VII mutations involve a complete or partial loss of exon 6 sequences from either α chain, with EDS VIIA due to mutations in pro-α1(I) and EDS VIIB due to similar mutations in pro-α2(I). Because exon 6 contains both the N-proteinase cleavage site and the interhelix cross-linking lysine, the N-propeptide cannot be cleaved from EDS VII mutant procollagen and cross-linking is defective. Patients with N-propeptide retention have severe generalized joint hypermobility and congenital bilateral hip dislocation. Many have scoliosis because of ligamentous laxity, dislocations of other joints and mild osteopenia, with a few fractures. In those patients in whom dermal fibrils were examined, cross-sectional diameters were normal or decreased and borders were irregular (13Nicholls A.C. Sher J.L. Wright M.J. Oley C. Mueller R.F. Pope F.M. J. Med. Genet. 2000; 37: E33Crossref PubMed Scopus (19) Google Scholar, 14Byers P.H. Duvic M. Atkinson M. Robinow M. Smith L.T. Krane S.M. Greally M.T. Ludman M. Matalon R. Pauker S. Quanbeck D. Schwarze U. Am. J. Med. Genet. 1997; 72: 94-105Crossref PubMed Scopus (93) Google Scholar, 15Eyre D.R. Shapiro F.D. Aldridge J.F. J. Biol. Chem. 1985; 260: 11322-11329Abstract Full Text PDF PubMed Google Scholar, 22Ho K.K. Kong R.Y. Kuffner T. Hsu L.H. Ma L. Cheah K.S. Hum. Mutat. 1994; 3: 358-364Crossref PubMed Scopus (16) Google Scholar, 23Carr A.J. Chiodo A.A. Hilton J.M. Chow C.W. Hockey A. Cole W.G. J. Med. Genet. 1994; 31: 306-311Crossref PubMed Scopus (22) Google Scholar). A small number of mutations at the amino end of the helical region of α2(I) collagen have been noted to cause a combination of EDS and OI symptoms. These mutations include two cases with exon 9 skipping, a case with exon 11 skipping, and a large duplication of E12–32 (24Raff M.L. Craigen W.J. Smith L.T. Keene D.R. Byers P.H. Hum. Genet. 2000; 106: 19-28Crossref PubMed Scopus (29) Google Scholar, 25Nicholls A.C. Oliver J. Renouf D.V. Heath D.A. Pope F.M. Hum. Genet. 1992; 88: 627-633Crossref PubMed Scopus (35) Google Scholar, 26Feshchenko S. Brinckmann J. Lehmann H.W. Koch H.-G. Muller P.K. Kugler S. Hum. Mutat. 1997; 12: 138Crossref Google Scholar, 27Sippola M. Kaffe S. Prockop D.J. J. Biol. Chem. 1984; 259: 14094-14100Abstract Full Text PDF PubMed Google Scholar, 28Dombrowski K.E. Vogel B.E. Prockop D.J. Biochemistry. 1989; 28: 7107-7112Crossref PubMed Scopus (21) Google Scholar). The symptoms of EDS predominate in the clinical presentation of these patients. All have bilateral hip dislocations and marked laxity of large joints. Two had shoulder joint dislocations and hernias (24Raff M.L. Craigen W.J. Smith L.T. Keene D.R. Byers P.H. Hum. Genet. 2000; 106: 19-28Crossref PubMed Scopus (29) Google Scholar, 26Feshchenko S. Brinckmann J. Lehmann H.W. Koch H.-G. Muller P.K. Kugler S. Hum. Mutat. 1997; 12: 138Crossref Google Scholar) and one had scoliosis (27Sippola M. Kaffe S. Prockop D.J. J. Biol. Chem. 1984; 259: 14094-14100Abstract Full Text PDF PubMed Google Scholar). In the case with duplication of E12–32, the dermal fibril diameter was ⅔ that of a matched control (24Raff M.L. Craigen W.J. Smith L.T. Keene D.R. Byers P.H. Hum. Genet. 2000; 106: 19-28Crossref PubMed Scopus (29) Google Scholar). OI symptoms in these cases were mild. All had blue sclerae, two had dentinogenesis imperfecta (24Raff M.L. Craigen W.J. Smith L.T. Keene D.R. Byers P.H. Hum. Genet. 2000; 106: 19-28Crossref PubMed Scopus (29) Google Scholar, 27Sippola M. Kaffe S. Prockop D.J. J. Biol. Chem. 1984; 259: 14094-14100Abstract Full Text PDF PubMed Google Scholar, 28Dombrowski K.E. Vogel B.E. Prockop D.J. Biochemistry. 1989; 28: 7107-7112Crossref PubMed Scopus (21) Google Scholar) and one had Wormian bones (27Sippola M. Kaffe S. Prockop D.J. J. Biol. Chem. 1984; 259: 14094-14100Abstract Full Text PDF PubMed Google Scholar, 28Dombrowski K.E. Vogel B.E. Prockop D.J. Biochemistry. 1989; 28: 7107-7112Crossref PubMed Scopus (21) Google Scholar). Only the child with duplication of E12–32 had a single tibial fracture. We describe here a group of seven patients with mutations in the α1(I) chain who have a distinct OI/EDS phenotype, in which the symptoms of OI are more prominent and the EDS less severe than in the OI/EDS mutations in α2(I). The α1(I) OI/EDS mutation cluster is located in a high stability folding region at the amino end of the type I collagen helix. These mutations interfere with removal of the N-propeptide, although the N-proteinase site in exon 6 is intact. Thus, OI/EDS and EDS VII are shown to have a common mechanism for EDS symptoms. Cell Culture—Skin fibroblast cultures were established from dermal punch biopsies. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 2 mm glutamine in the presence of 5% CO2. Osteoblast primary cultures were established from surgical bone chips using the method of Robey and Termine (29Robey P.G. Termine J.D. Calcif. Tissue Int. 1985; 37: 453-460Crossref PubMed Scopus (638) Google Scholar). In brief, osteoblasts were released from bone chips by digesting for 2 h at 37 °C with 0.3 units/ml collagenase P in serum-free medium, and grown in 45% low-calcium DMEM, 45% low-calcium Ham's F-12 Kinase medium (Biofluids, Rockville, MD), 25 μg/ml ascorbate, and 10% fetal bovine serum in the presence of 8% CO2. Steady State Collagen Synthesis—To label procollagens, confluent fibroblast cultures of probands and control cells (ATCC 2127, American Type Culture Collection, Manassas, VA) were incubated for 2 h in serum-free medium containing 50 μg/ml ascorbic acid, followed by incubation with 260 mCi/ml of 3.96 TBq/mmol l-[2,3,4,5-3H]proline in serum-free medium for 16 h. Procollagens were harvested from media and cell layer and precipitated with ammonium sulfate; collagens were prepared by pepsin digestion (50 μg/ml) of procollagen samples, as previously described (30Bonadio J. Holbrook K.A. Gelinas R.E. Jacob J. Byers P.H. J. Biol. Chem. 1985; 260: 1734-1742Abstract Full Text PDF PubMed Google Scholar). Mutation Identification—Total RNA was isolated from cultured fibroblasts of patient and control cell lines using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's directions (31Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63191) Google Scholar). The region of the α1(I) collagen mRNA corresponding to exons 5–12 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) (32Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13496) Google Scholar). Total RNA (1 μg) was reverse transcribed with 50 units of murine leukemia virus RT (Applied Biosystems, Foster City, CA) using an antisense primer complementary to nucleotides 813–842 of the cDNA sequence (GenBank™ AF017178) in exon 12 (5′-CCAGCAGGACCAGCATCTCCCTTGGCACCA-3′). The RT reaction was used as a template for PCR with a sense primer corresponding to nucleotides 377–406 of cDNA sequence and located in exon 5 (5′-CTGGCCGAGATGGCATCCCTGGACAGCCTG-3′). PCR used 0.1 mm dNTP, 2.5 units of Amplitaq, and 1× PCR Buffer II (Applied Biosystems). PCR cycling conditions were as follows: 94 °C for 5 min; then 35 cycles of 1 min at 94 °C, 1 min at 65 °C, and 1.5 min at 72 °C; and finally 7 min at 72 °C. RT-PCR products were sequenced directly on a Beckman Coulter CEQ2000 DNA Sequencer (Beckman Fullerton, CA) according to the manufacturer's protocol. Pericellular Processing—Processing of procollagens secreted by fibroblasts was examined by labeling confluent cells from probands and control with 260 mCi/ml of 3.96 TBq/mmol [3H]proline for 24 h and then replacing the media with DMEM containing 2 mm non-radioactive proline and 10% fetal bovine serum. Media from independent wells were harvested at 24-h intervals over a 5-day period as previously described (25Nicholls A.C. Oliver J. Renouf D.V. Heath D.A. Pope F.M. Hum. Genet. 1992; 88: 627-633Crossref PubMed Scopus (35) Google Scholar). Media procollagen samples from fibroblasts were precipitated with ammonium sulfate and electrophoresed on 6% polyacrylamide-urea-SDS gels. Matrix Deposition—Proband and control fibroblasts and osteoblasts were grown to confluence and stimulated every other day for 11 days (fibroblasts) or 9 days (osteoblasts) with fresh DMEM containing 10% fetal bovine serum and 100 μg/ml ascorbic acid. Cultures were then incubated for 24 h with 260 mCi/ml of [3H]proline in serum-free medium. Medium was collected and procollagens were precipitated with ammonium sulfate. Matrix collagens were serially extracted at 4 °C as previously described (34Bateman J.F. Golub S.B. Matrix Biol. 1994; 14: 251-262Crossref PubMed Scopus (46) Google Scholar). In brief, newly synthesized collagens were extracted for 24 h with neutral salt (50 mm Tris-HCl, pH 7.5, containing 0.15 m NaCl, 5 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 10 mm benzamidine, and 10 mmN-ethylmaleimide), separated from matrix by centrifugation, and precipitated with 2 m NaCl. Collagens with acid-labile cross-links were extracted from the matrix for 24 h with 0.5 m acetic acid and precipitated with 2 m NaCl. Collagens with mature cross-links were extracted by pepsin digestion (0.1 mg/ml) for 24 h and precipitated with 2 m NaCl. All fractions were electrophoresed on 6% polyacrylamide-urea-SDS gels. Transmission Electron Microscopy of Proband Dermal Fibrils—A dermal punch biopsy was obtained from each proband and from a control matched for age and race. The samples were fixed in 2.5% glutaraldehyde and then treated with 1% osmium tetroxide followed by en bloc staining with 2% uranyl acetate. After dehydration, the tissue was infiltrated with Spurr's plastic resin. 600–800-Å sections were obtained with an AO Reichert Ultracut ultramicrotome mounted on copper grids and stained with lead citrate. The stained grids were examined in a Zeiss EM10 CA transmission electron microscope and representative areas were photographed (JFE Enterprises, College Park, MD). Preparation of Proband Secreted Procollagens—Proband and control fibroblasts were grown to confluence at 37 °C. Culture medium was removed and fresh serum-free DMEM supplemented with 2 mm glutamine and 50 μg/ml ascorbate was added to the cell cultures. For procollagens used in calorimetry and N-propeptide processing studies, medium was harvested at 24-h intervals for 2 days and fresh medium containing 50 μg/ml ascorbate was replenished daily. Medium was buffered with 100 mm Tris-HCl, pH 7.4, and cooled to 4 °C. Protease inhibitors were added to obtain the following final concentrations: 250 mm EDTA, 0.2% NaN3, 1 mm phenylmethylsulfonylfluoride, 5 mm benzamidine, and 10 mmN-ethylmaleimide. Procollagen was precipitated by gradual addition of ammonium sulfate to a final concentration of 176 mg/ml and incubation at 4 °C overnight, followed by centrifugation at 12,000 × g for 2 h. Differential Scanning Calorimetry—DSC scans from 10 to 50 °C were performed at 0.125 and 1 °C/min heating rates in a Nano II DSC instrument (Calorimetry Sciences Corporation, Lindon, UT). To prevent fibrillogenesis of collagen, 0.1–0.3 mg/ml protein solutions in 0.2 m sodium phosphate, 0.5 m glycerol, pH 7.4, were used. The denaturation temperature (Tm) in phosphate/glycerol buffers depends linearly on the concentration of all buffer components and the corresponding proportionality coefficients do not depend on the scanning rate, allowing extrapolation of Tm to physiological conditions (35Leikina E. Mertts M.V. Kuznetsova N. Leikin S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1314-1318Crossref PubMed Scopus (450) Google Scholar). N-proteinase Cleavage—Ammonium sulfate procollagen precipitates were redissolved in 0.1 m sodium carbonate, 0.5 m NaCl, pH 9.3, labeled by covalent attachment of Cy2 and Cy5 fluorescent dyes (Amersham Biosciences, Piscataway, NJ) and transferred into 50 mm Tris, 0.5 m NaCl, 4 mm CaCl2, 0.5 mm phenylmethylsulfonylfluoride, 2.5 mmN-ethylmaleimide, 0.02% Brij 35, pH 8, as described (36Cabral W.A. Mertts M.V. Makareeva E. Colige A. Tekin M. Pandya A. Leikin S. Marini J.C. J. Biol. Chem. 2003; 278: 10006-10012Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Procollagen concentration was measured by Sircol assay (Biocolor Ltd., Belfast, Northern Ireland) and adjusted to 0.1 mg/ml. Binary mixtures of procollagens, one labeled by Cy5 and one labeled by Cy2, were prepared so that two different procollagens could be co-processed by an enzyme in the same sample tube and, therefore, under completely identical conditions. Procollagen N-proteinase (ADAMTS-2) was purified from fetal calf skin as described (37Colige A. Beschin A. Samyn B. Goebels Y. Van Beeumen J. Nusgens B.V. Lapiere C.M. J. Biol. Chem. 1995; 270: 16724-16730Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and stored in 5-μl aliquots in 50 mm Tris, 0.5 m NaCl, 2 mm CaCl2, 0.02% Brij 35, pH 7.6, at -80 °C. Enzyme was added to each collagen mixture on ice for an enzyme:substrate ratio of 15 units of enzyme/mg of substrate. Reactions were incubated at 34 °C to avoid partial denaturation of low-stability procollagens containing structural defects. Sample aliquots were collected at the indicated times after the start of the reaction, mixed with lithium dodecyl sulfate gel sample buffer (Invitrogen, Carlsbad, CA) with added EDTA (to stop enzymatic cleavage) and dithiothreitol. The samples were denatured and analyzed by gel electrophoresis on pre-cast 6% Tris/glycine minigels (Invitrogen, Carlsbad, CA). The gels were scanned on an FLA3000 fluorescence scanner (FUJI Medical Systems, Stamford, CT). Intensity profiles for each lane were extracted using ScienceLab software supplied with the scanner. Quantitative analysis of band intensities and deconvolution of overlapping bands were performed using PeakFit software (Systat, Point Richmond, CA). Phenotype of OI/EDS Patient Group—The seven probands in this study have a distinct OI/EDS phenotype (Table I). All seven first came to medical attention for symptoms of osteogenesis imperfecta. All have types III or IV OI, with multiple fractures of long bones; the children with G25V, G76E, and G88E had bone deformity sufficient to require osteotomy procedures. Their L1–L4 DEXA z-scores range from -3.0 to -5.2. All have the significant short stature of OI and a height age that ranges from 20 to 80% of the mean height for their chronological age. Also characteristic of the relatively shorter lower extremities in OI, most have arm span significantly greater than length. All probands have strikingly blue sclerae.Table IClinical characteristics of OI/EDS subjectsOI characteristicsEDS characteristicsSexCAaChronological age (years)OI typeHAbHeight age (years); age for which the height is at the 50th percentileProportionsDEXA ZeAge matched z-score (S.D. units) for bone density at the lumbar spine (L1–L4)ScleraefSclerae are scored on a scale from 1 (tinge blue) to 4 (dark blue)Beighton scoregBeighton score of joint hyperextensibility ranges from 1 to 9; (a) passive apposition of the thumb to the flexor aspect of the forearm (1 point each thumb), (b) passive dorsiflexion of the fifth finger beyond 90° (1 point each hand), (c) hyperextension of the elbow beyond 10° (1 point each elbow), (d) hyperextension of the knee beyond 10° (1 point each knee), (e) forward flexion of the trunk with knees fully extended so that palms of the hands rest on the floor (1 point)Scoliosis (degree)hDegree of thoracolumbar scoliosis as measured on AP radiographSpinal fixationUS: LScUpper segment (trunk, crown to pubis) to lower segment (legs, pubis to heel) ratioSpan:HtdArm span to height ratio1Exon 7 skippingM23IV12.34Large jointsSevereBraced2G13DF15.75IV101:1.341:0.91–4.94945,90iPatient has had 2 fusion/fixation surgeries with hardware placement. The first (9/2001) occurred after the curve reached 45°. Hardware failure occurred 4 months later; it was removed in 1/2002. A subsequent fusion with fixation and graft was completed in 11/2002, after the curve had progressed to 90°. At this time, the second fusion/fixation is holding143G25VF18IV4.251:1.81:0.69Rods475094G25VF2.9IV1.51:11:1–4.347553.55G34RM0.6III/IV0.670Fusion, 22m6G76EF16.2III31:0.791:0.73–5.23535–40None7G88EF23III/IV9.51:0.731:0.92–3.0375016a Chronological age (years)b Height age (years); age for which the height is at the 50th percentilec Upper segment (trunk, crown to pubis) to lower segment (legs, pubis to heel) ratiod Arm span to height ratioe Age matched z-score (S.D. units) for bone density at the lumbar spine (L1–L4)f Sclerae are scored on a scale from 1 (tinge blue) to 4 (dark blue)g Beighton score of joint hyperextensibility ranges from 1 to 9; (a) passive apposition of the thumb to the flexor aspect of the forearm (1 point each thumb), (b) passive dorsiflexion of the fifth finger beyond 90° (1 point each hand), (c) hyperextension of the elbow beyond 10° (1 point each elbow), (d) hyperextension of the knee beyond 10° (1 point each knee), (e) forward flexion of the trunk with knees fully extended so that palms of the hands rest on the floor (1 point)h Degree of thoracolumbar scoliosis as measured on AP radiographi Patient has had 2 fusion/fixation surgeries with hardware placement. The first (9/2001) occurred after the curve reached 45°. Hardware failure occurred 4 months later; it was removed in 1/2002. A subsequent fusion with fixation and graft was completed in 11/2002, after the curve had progressed to 90°. At this time, the second fusion/fixation is holding Open table in a new tab The symptoms of EDS are notably more severe than the mild to moderate joint hyperextensibility frequently found in OI. In addition to significant hyperextensibility of large and small joints (Fig. 1), these probands have laxity of paraspinal ligaments. This results in early scoliosis without vertebral compressions. The scoliosis is rapidly progressive and unresponsive to bracing. Spinal fixation has been required by the mid-teenage years. Collagen Biochemistry and Mutation Detection—Because the probands have clinically significant osteogenesis imperfecta, we examined the type I collagen synthesized by their dermal fibroblasts electrophoretically on SDS-urea-PAGE. Proband steady state media and cell layer collagen did not have delayed electrophoretic migration, as would be expected with the well known overmodification of type I collagen chains frequently seen in OI (Fig. 2A). Proband 1 had a leading edge in both media and cell layer α2(I) bands and proband 2 had a leading edge in the α2(I) band isolated from the cell layer. The normal collagen electrophoresis results prompted us to screen these patients for collagen mutations at the amino end of the helical region of either α1(I) or α2(I), which would not be expected to cause overmodification. Direct sequencing of RT-PCR products spanning exons 5–12 of both α chains revealed that all 7 probands had mutations located in exons 7–11 of COL1A1 (Fig. 2B), causing structural abnormalities in the 90 residues at the amino end of the α1(I) protein chain. Proband 1 has a mutation (IVS7 + 4 A > T; g.3756A>T) that causes skipping of exon 7 from the mutant transcript. The leading edge seen after pepsin digestion on SDS-PAGE is most likely the consequence of normal length α2(I) chain looping out of helices containing a shorter mutant α1(I) chain(s). The remaining 6 probands are heterozygous for glycine substitution mutations, located at Gly13, Gly25 (2 patients), Gly34, Gly76, and Gly88, respectively. The G13D substitution presumably disrupts the collagen helix sufficiently to expose the amino end of the α2(I) chain to pepsin digestion, generating the leading edge seen on the α2(I) band. Only RT-PCR products of the expected size were obtained from both α1(I) and α2(I) transcripts in the G13D case; sequencing ruled out a second mutation or use of a cryptic splice signal. Mutations Interfere with in Vitro and Pericelluar NH2-terminal Processing—The mutations causing OI/EDS interfere with in vitro processing of the N-propeptide of proband procollagen by purified N-proteinase (Fig. 3), although the sequence of the N-proteinase cleavage site in exon 6 is intact in all cases. Because of random association of pro-α chains during procollagen assembly, heterozygous patients generate 25, 50, and 25%, respectively, of procollagen molecules containing two, one, and no mutant pro-α1(I) chains. From probands heterozygous for mutations in exon 7 (ΔE7 and G13D), only about 25% of pro-α1(I) chains were processed in vitro. Apparently, these mutations prevent processing of the N-propeptide from all helices that contain one or two copies of the mutant pro-α1(I) chain, and only the 25% of helices with two normal pro-α1(I) chains could be processed. The procollagens with mutations in exons 8–11 (G25V, G34R, and G76E) underwent 70–88% cleavage, suggesting that helices with one or two normal pro-α1(I) chains were processed, whereas the 25% of helices containing two mutant chains could not be cleaved. The procollagen containing the G88E substitution was completely cleaved, but at a reduced rate compared with normal procollagen. Mutations located more distal to the cleavage site, at G121D and G136R, had complete in vitro cleavage with normal kinetics. We also examined the conversion of procollagen to collagen by the pericellular processing enzymes in a cell culture assay, by following the conversion of a pulse of [3H]procollagen to collagen over 5 days (data not shown). The procollagen secreted by all seven of the OI/EDS probands showed delayed processing to collagen by the pericellular enzymes, consistent with the in vitro processing results. In comparison with processing of normal control procollagen, amino propeptide processing is substantially delayed in probands 1 and 2 (exon 7 mutations), delayed to a lesser extent in probands 3 and 5 (exon 8 mutations), and only modestly delayed in probands 6 and 7 (exon 11 mutations). pN-collagen Deposited in Matrix in Fibroblast and Osteoblast Cultures—The pN-collagen present in the media of cultured fibroblasts and osteoblasts is incorporated into the matrix deposited by those cells (Fig. 4). In each proband, we see a substantial increase of pN-collagen in these fractions, as compared with control. For mutations in exon 7 (ΔE7 and G13D), the amount of pN-α1(I) is equivalent to the amount of fully proc
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