RESEARCH ARTICLE A Novel Form of Chondrocyte Stress is Triggered by a COMP Mutation Causing Pseudoachondroplasia Farhana Suleman,1 Benedetta Gualeni,1 Hannah J. Gregson,1 Matthew P. Leighton,1 Katarzyna A. Piro´ g,1 Sarah Edwards,1 Paul Holden,2 Raymond P. Boot-Handford,1 and Michael D. Briggs1∗ 1Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom; 2Shriner’s Hospitals for Children, Research Center, Portland, Oregon Communicated by Iain McIntosh Received 21 July 2011; accepted revised manuscript 16 September 2011. Published online 17 October 2011 in Wiley Online Library (www.wiley.com/humanmutation).DOI: 10.1002/humu.21631 OFFICIAL JOURNAL www.hgvs.org ABSTRACT: Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarﬁsm. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte prolif- eration was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mu- tations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH- MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). How- ever, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identiﬁed ex- pression changes in groups of genes implicated in oxida- tive stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress trig- gered by the expression of mutant COMP is central to the pathogenesis of PSACH. Hum Mutat 33:218–231, 2012. C 2011 Wiley Periodicals, Inc. KEY WORDS: pseudoachondroplasia; cartilage oligomeric matrix protein; chondrocyte stress Introduction Pseudoachondroplasia (PSACH; MIM# 177170) is an autoso- mal dominant disease that affects skeletal development and has a Additional Supporting Information may be found in the online version of this article. ∗Correspondence to: Michael D. Briggs, Wellcome Trust Centre for Cell Ma- trix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, United Kingdom. E-mail: mike.briggs@ manchester.ac.uk Contract grant sponsor: Wellcome Trust (Grants 071161/Z/03/Z, 084353/Z/07/Z, and 078456/Z/05/Z). clinical spectrum ranging from mild to severe [Maroteaux et al., 1980; Rimoin et al., 1994; Wynne-Davies et al., 1986]. The dis- ease results almost exclusively from mutations in the gene encod- ing cartilage oligomeric matrix protein (COMP; MIM# 600310) [Briggs and Chapman, 2002; Briggs et al., 1995; Hecht et al., 1995], a 550-kDa extracellular matrix (ECM) glycoprotein found in car- tilage, tendon, and ligament [DiCesare et al., 1994; Hecht et al., 1998; Oldberg et al., 1992]. Mutations in COMP can also result in a related chondrodysplasia, multiple epiphyseal dysplasia (MED; MIM# 132400), which is a clinically variable and genetically het- erogeneous disease that can also result from mutations in the genes encoding matrilin-3 (MATN3; MIM# 602109) and type IX collagen (COL9A1; MIM# 120210, COL9A2; MIM# 120260, and COL9A3; MIM# 120270) [Briggs and Chapman, 2002; Chapman et al., 2001; Muragaki et al., 1996; Paassilta et al., 1999]. COMP is thought to be important for ECM assembly within the cartilage growth plate by acting as a catalyst in the regulation of collagen ﬁbril assembly [Halasz et al., 2007; Rosenberg et al., 1998], as well as interacting with other ECM proteins such as members of the matrilin protein family [Fresquet et al., 2007; Mann et al., 2004] and type IX collagen [Holden et al., 2001; Thur et al., 2001]. COMP has also been shown to be central to the integrity of chondrocytes within the ECM. In the ﬁrst instance, it is important in the regulation of chondrocyte attachment to the ECM through integrin receptors [Chen et al., 2005; Rock et al., 2010]. Second, COMP plays an important regulatory role in chondrocyte proliferation [Xu et al., 2007] and has recently been proposed to have a cell survival role by promoting the upregulation of inhibitors of apoptosis proteins (IAPs), particularly XIAP, cIAP1, cIAP2, and survivin [Gagarina et al., 2008]. Overall, these studies have suggested an important function for COMP in the regulation and maintenance of cell– matrix integrity within the cartilage growth plate. It was therefore surprising that a knockout mouse model of COMP displayed no overt skeletal phenotype [Svensson et al., 2002], which supports the hypothesis that the compromised skeletal devel- opment in PSACH and MED results from antimorphic (dominant negative) COMP mutations [Briggs and Chapman, 2002]. COMP is a modular protein and comprises an N-terminal oligomerization domain, four epidermal growth factor-like (TSP2) repeats, eight calcium binding type 3 (TSP3) repeats, and a C- terminal globular domain. An increasing number of mutations (∼15%) have been reported in the C-terminal domain of COMP [Kennedy et al., 2005b]; however, the majority of PSACH and MED mutations (85%) are clustered in the TSP3 repeats [Kennedy et al., 2005a]. The most common disease-causing mutation is the in-frame deletion of an aspartic acid residue (p.D469del) from the seventh T3 repeat (T37), which accounts for approximately 30% of all PSACH C 2011 WILEY PERIODICALS, INC.