Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4583-4587, May 1992 Medical Sciences Characterization of a type II collagen gene (COL2AI) mutation identified in cultured chondrocytes from human hypochondrogenesis WILLIAM A. HORTON*t, MIRTA A. MACHADO*, JEFFREY ELLARD*, DIANNA CAMPBELL*, JAMES BARTLEYt, FRANCESCO RAMIREZ§, EMILIA VITALE§, AND BRENDAN LEE§¶ *Division of Medical Genetics, Department of Pediatrics, University of Texas Medical School, Houston, TX 77225; *Loma Linda Medical Center, Loma Linda, CA 92350; and §Brookdale Center for Molecular Biology, Mount Sinai Medical Center, New York, NY 10029 Communicated by Victor A. McKusick, January 6, 1992 ABSTRACT A subtle mutation in the type H collagen gene The detection of mutations in genes expressed during COL2AI was detected in a case of human hypochondrogenesis chondrogenesis remains difficult in humans. Cartilage is not by using a chondrocyte culture system and PCR-cDNA scan- easily accessible, and it is relatively acellular. Consequently, ning analysis. Chondrocytes obtained from cartilage biopsies it is a poor source of RNA for cDNA synthesis and analysis. were dedifferentiated and expanded in monolayer culture and Even more problematic has been investigation of the conse- then redifferentiated by culture over agarose. Single-strand quences of mutations on chondrogenesis. Despite detailed conformation polymorphism and direct sequencing analysis knowledge ofgrowth plate morphology (17), there is a dearth identified a G -- A transition, resulting in a glycine substitution of biologic information about endochondral ossification in at amino acid 574 of the proal(ll) collagen triple-helical humans. Furthermore, experimental models for studying domain. Morphologic assessment of cartilage-like structures chondrogenesis in humans are generally not available. Chon- produced in culture and electrophoretic analysis of collagens drocytes cultured under usual conditions lose their pheno- synthesized by the cultured chondrocytes suggested that the typic characteristics and cease to express many of the genes glycine substitution interferes with conversion oftype II procol- normally expressed during chondrogenesis (18). Thus, even lagen to collagen, impairs intracellular transport and secretion when a mutation is identified, its biologic significance is of the molecule, and disrupts collagen fibril assembly. This difficult to determine. experimental approach has broad implications for the investi- To circumvent these technical problems and facilitate gation of human chondrodysplasias as well as human chon- characterization ofchondrogenesis mutations, we developed drocyte biology. an experimental approach based on analysis of the products of cultured chondrocytes from affected patients. The plas- Chondrogenesis is responsible for the formation of the car- ticity of the chondrocyte phenotype was exploited to expand tilage templates that serve as temporary models for the the population of cultured chondrocytes and to promote vertebrate skeleton. The process is crucial for the develop- chondrogenesis in vitro. The mutation was detected by ment of most embryonic bones and their subsequent linear scanning analysis of COL2A1 cDNA derived from reverse- growth-i.e., endochondral ossification. In both instances, transcribed RNA extracted from cultured chondrocytes, this process involves the biosynthesis, secretion, and assem- rather than by direct analysis of genomic DNA. We used the bly of a highly ordered extracellular matrix by differentiated approach to identify a COL2AJ mutation and examine its chondrocytes (1). The ability of cartilage matrix to serve its effects on chondrogenesis in a case of human hypochondro- template functions depends on its integrity, which in turn genesis. depends on the proper interactions between a three- dimensional network of collagen fibrils comprised of type II METHODS collagen and lesser amounts of types IX and XI collagen and a host ofother matrix constituents, such as proteoglycans (2). Cell Culture. Epiphyseal chondrocytes were obtained from Abnormalities of chondrogenesis and of the genes ex- costal cartilage at autopsy from an infant with radiographic pressed during chondrogenesis, especially type II collagen, characteristics of hypochondrogenesis. Control epiphyseal have long been suspected of causing certain of the human chondrocytes were obtained similarly from infants who chondrodysplasias. Patients with chondrodysplasias typi- showed no evidence of skeletal abnormalities. cally have short, deformed bones that grow slowly (3). Their The methods for chondrocyte isolation and monolayer clinical features vary widely and reflect the degree to which culture have been described in detail (19). Briefly, the car- endochondral ossification and other normal cartilage func- tilage was trimmed to remove perichondrium, minced, and tions are disturbed. The candidate disorders for mutations of digested overnight with collagenase. The cells were sus- COL2AJ, the gene encoding type II collagen, are the spondy- pended in Dulbecco's modified Eagle's medium/10% fetal loepiphyseal dysplasias (SEDs) (4, 5). In these disorders the calf serum/gentamycin, and primary cultures were estab- distribution of clinical disease parallels the distribution of lished. The cultures were fed twice weekly and passaged two type II collagen, defective chondrogenesis has been inferred to four times. from microscopic studies ofgrowth plate and other cartilages (3, 6-8), and electrophoretic abnormalities oftype II collagen Abbreviations: SED, spondyloepiphyseal dysplasia; TEM, transmis- have been found (5, 9-11). Recently, reports of COL2AJ sion electron microscopy; rER, rough endoplasmic reticulum; SSCP, mutations in patients with SED have begun to emerge, single-strand conformation polymorphism. validating the earlier suspicions (12-16). tTo whom reprint requests should be addressed at: Division of Medical Genetics, Department of Pediatrics, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225. The publication costs of this article were defrayed in part by page charge Present address: Department of Microbiology and Immunology, payment. This article must therefore be hereby marked "advertisement" State University of New York, Health Science Center at Brooklyn, in accordance with 18 U.S.C. §1734 solely to indicate this fact. New York, NY 11203. 4583' Downloaded by guest on October 3, 2021 4584 Medical Sciences: Horton et al. Proc. Natl. Acad Sci. USA 89 (1992) The agarose culture scheme was adapted from that de- recommendations (Amersham). Three overlapping cDNA scribed by Tacchetti et al. (20) for culture of chicken chon- fragments (exon 1-22, 22-38, and 38-50) spanning the trans- drocytes. The cells were trypsinized, and 2 x 106 cells were lated domain ofCOL2AJ were PCR amplified. These primary suspended in 1 ml of medium (Dulbecco's modified Eagle's amplification products were used for amplification of medium/10o fetal calf serum/gentamycin). The cell suspen- [32P]dATP internally labeled subfragments for SSCP analy- sion was layered over culture dishes precoated with high- sis. For SSCP analysis, 200- to 400-base-pair (bp) fragments temperature agarose. Fresh medium was added every third were PCR amplified from the primary amplification cDNAs day. Ascorbic acid at 50 jug/ml was added daily, except for and internally labeled with the addition of 10 ,uCi of days 1 and 2, when 10 and 25 ,ug/ml were added, respectively; [32P]dATP (800 Ci/mmol) per 100 ,1d ofPCR reaction volume. ascorbic acid was omitted from some cultures. The agarose One microliter of the resulting products was denatured and cultures were monitored daily and maintained for 7-10 days. electrophoresed on a 4% polyacrylamide/[with or without Microscopic Analysis. Specimens of costochondral carti- 10%1 (vol/vol) glycerol] nondenaturing gel at a constant 30 W lage were dissected and processed into Spurr epoxy resin and autoradiographed overnight at -700C. (Ted Pella) by standard techniques for both light microscopy Cloning and Sequencing. Direct sequencing of cDNA and and transmission electron microscopy (TEM) (19). The tis- genomic PCR amplification products were done as described sue-like structures formed in agarose culture were also ex- (25). cDNA and genomic PCR amplification products were amined by both light microscopy and TEM. The structures cloned into the Sma I site of pUC18 and sequenced by the were fixed in 1.5% (wt/vol) paraformaldehyde/1.5% glutar- dideoxynucleotide chain-termination method. aldehyde/0.05% ruthenium hexamine trichloride/0.1 M so- dium cocadylate buffer, pH 7.2. Cultures used for TEM were postfixed in 1.-0% (wt/vol) OSO4. Both were embedded in RESULTS Spurr epoxy resin. Light microscopy sections were stained Tissue Studies. The costochondral cartilage histology was with toluidine blue, whereas TEM ultrathin sections were typical ofhypochondrogenesis. It showed a poorly organized stained with 4% (wt/vol) uranyl acetate/2% (wt/vol) Rey- growth plate, unevenly distributed epiphyseal chondrocytes nolds lead citrate (Ted Pella). that occupied prominent lacunae, sparse matrix, and exten- Collagen Biosynthetic Analysis. Seven- to ten-day cultures with vascular canals were labeled with [3H]proline for 24 hr (20). After being sive fibrous tissue associated cartilage washed, the cartilage-like structures were homogenized and (data not shown). TEM revealed distended rough endoplas- extracted for 24 hr in 1.0 M NaCl containing protease mic reticulum (rER) in most epiphyseal chondrocytes and inhibitors.