immunoprecipitation followed by Western blotting of proteins from control chondrocytes demonstrated that CRT, Grp94, and Erp72 were directly associated with normal COMP, which was conﬁrmed by Flourescence Resonance Energy Transfer (FRET) [Hecht et al., 2001]. However, all of the previous studies on patient samples did not determine quantitatively the relative expression levels of these chaperone proteins and it is likely that their localization to the ER, and association with mutant COMP and other retained ECM pro- teins, is an indirect result of protein accumulation in the ER and the fact that they are all ER resident proteins. This observation is supported by IHC staining for PDI, BiP and Grp78, which show no differences in the localization and relative amounts of these three chaperones in mice that are wild-type or mutant for D469del COMP (Supp. Fig. S7). Finally, we also determined that there was no in- creased phosphorylation of eIF2α, a key mediator of the UPR, or any increase in the relative expression of CHOP, an important apoptosis- associated downstream target of prolonged UPR (Table 1). Taken together these data suggest that the expression of mutant COMP is inducing apoptosis that is independent of a conventional UPR. A consistent ﬁnding in all three knock-in mouse models of PSACH and MED is a signiﬁcant reduction in chondrocyte prolif- eration and the increased and/or spatially dysregulated apoptosis in the cartilage growth plate [Leighton et al., 2007; Pirog-Garcia et al., 2007]. The effects of the Comp D469del mutation on chondrocyte proliferation and apoptosis was more pronounced than in our pre- viously published models, which is consistent with the more severe phenotype caused by the COMP p.D469del mutation in PSACH patients. These detrimental alterations to chondrocyte proliferation and survival are therefore likely to be key factors in the initiation and progression of PSACH and MED. However, the speciﬁc genetic pathways leading to reduced proliferation and apoptosis are clearly different depending on the mutated gene and/or the speciﬁc muta- tion. For example, our previous studies have demonstrated that the expression of Matn3 V194D and Comp T585M cause ER stress that results in a conventional UPR characterized by the upregulation of various chaperone proteins [Leighton et al., 2007; Nundlall et al., 2010; Pirog-Garcia et al., 2007], which is clearly not the case for Comp D469del (Supp. Table S1 and S2). In the case of Comp T585M, the UPR appears sufﬁcient for the correct folding and secretion of mutant COMP, however, either the UPR itself or the antimorphic effect of mutant COMP in the ECM initiates CHOP-mediated apoptosis [Pirog-Garcia et al., 2007]. In contrast, the UPR is not capable of mediating the correct folding and secretion of the majority of mutant matrilin-3 (V194D), which appears to accumulate in the rER of chondrocytes and eventually leading to dysregulated apoptosis [Leighton et al., 2007]. However, unlike Comp T585M, this is not CHOP-mediated and the speciﬁc genetic pathways that result in apoptosis remain unresolved [Nund- lall et al., 2010]. Finally, we show in this study that the expression of Comp D469del results in signiﬁcantly increased apoptosis that is independent of both a conventional UPR and CHOP-mediated apoptosis. It is interesting to note that the COMP p. T585M muta- tion is generally accepted to result in a phenotype that is deﬁned as either mild PSACH or severe MED [Briggs et al., 1998; Song et al., 2003], in contrast to the p.D469del mutation that always causes the most severe form of PSACH. This difference in cellular mecha- nisms of COMP mutations provides insight into the broad clinical spectrum of the PSACH-MED disease group. Our microarray studies performed at newborn and 5 days of age have demonstrated that there are changes in the expression levels of a variety of different genes involved in oxidative stress, cell survival, attachment and/or migration, cell cycle arrest and apoptosis, and NF-κB signalling. These gene expression changes are consistent with the detrimental changes in chondrocyte phenotype that we have observed in the growth plate (i.e., reduced proliferation and increased apoptosis) and likely characterize a novel form of chondrocyte stress that is key to the pathogenesis of PSACH in this mouse model. Interestingly, several recent studies have shown that UPR- independent pathways, such as the ER overload response (EOR), are associated with the aggregation of mutant proteins in the ER, which induce a toxic gain of function. This is particularly the case with the serpinopathies in which the aggregation of insoluble mis- folded α1-antitrypsin triggers an alternative ER stress response that is independent of the UPR and involves the activation of NF-κB signalling [Ekeowa et al., 2010]. Recently, a role for Prdx2 in re- ducing apoptosis through NF-κB signalling has been demonstrated in mouse granulose cells [Yang et al., 2011], while the downreg- ulation of PRDX2 is implicated angiotensis II-mediated podocyte apoptosis [Hsu et al., 2011], thus providing further evidence that these pathways are important for cell survival. In addition, we have shown that the most highly upregulated gene in newborn chon- drocytes was Igfbp3, while other genes (i.e., Lyve1), known to be mediators of Igfbp3-dependant effects, were upregulated at 5 days of age. These data suggest that Igfbp3-mediated apoptosis may play a role in the initiation and progression of chondrocyte apoptosis due to the expression and accumulation of COMP D469del. In summary, we have generated a new mouse model of PSACH and demonstrated that expression of the most common form of mutant COMP causes a novel type of chondrocyte stress that is inde- pendent of a conventional UPR. This interesting ﬁnding highlights the emerging ﬁeld of UPR-independent pathways and supports al- ternative pathways such as the EOR in the pathogenesis of human genetic diseases. Acknowledgments This work was supported by grants from the Wellcome Trust.. We would like to thank Lynette Knowles for performing the microinjections, Leo Zeef for help with the statistical analysis of the microarray data, Roger Meadows for performing the EM analysis and David Thornton for helpful discussions. The research was undertaken in the Wellcome Trust Centre for Cell-Matrix Research and in the Histology, Bioimaging, Genomic Technologies, and Transgenic Core Facilities of the Faculty of Life Sciences at the University of Manchester. Disclosure Statement: There are no conﬂicts of interest to declare. References Boot-Handford RP, Briggs MD. 2009. The unfolded protein response and its relevance to connective tissue diseases. Cell Tissue Res 339:197–211. Briggs MD, Chapman KL. 2002. Pseudoachondroplasia and multiple epiphyseal dys- plasia: mutation review, molecular interactions, and genotype to phenotype cor- relations. Hum Mutat 19:465–478. Briggs MD, Hoffman SM, King LM, Olsen AS, Mohrenweiser H, Leroy JG, Mortier GR, Rimoin DL, Lachman RS, Gaines ES, Cekleniak JA, Knowlton RG, Cohn DH. 1995. Pseudoachondroplasia and multiple epiphyseal dysplasia due to mutations in the cartilage oligomeric matrix protein gene. Nat Genet 10:330–336. Briggs MD, Mortier GR, Cole WG, King LM, Golik SS, Bonaventure J, Nuytinck L, De Paepe A, Leroy JG, Biesecker L, Lipson M, Wilcox WR, Lachman RS, Rim- ion DL, Cohn DH. 1998. Diverse mutations in the gene for cartilage oligomeric matrix protein in the pseudoachondroplasia-multiple epiphyseal dysplasia disease spectrum. Am J Hum Genet 62:311–319. Chapman KL, Mortier GR, Chapman K, Loughlin J, Grant ME, Briggs MD. 2001. Mutations in the region encoding the von Willebrand factor A domain of matrilin- 3 are associated with multiple epiphyseal dysplasia. Nat Genet 28:393–396. Chen FH, Thomas AO, Hecht JT, Goldring MB, Lawler J. 2005. Cartilage oligomeric matrix protein/thrombospondin 5 supports chondrocyte attachment through in- teraction with integrins. J Biol Chem 280:32655–32661. HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 229