the mutation were comparable to those of the wild-type mice (data not shown). To determine the cytotoxic effect of mutant Comp expression on chondrocyte viability, we performed the TUNEL assay on tibia growth plate sections from 3-week-old mice. In wild-type animals, TUNEL positive chondrocytes were observed primarily at the vas- cular invasion front and typically 1–2 positive cells were counted per section, whereas there were very few TUNEL positive cells in the hypertrophic zone and none at all observed in the prolifer- ative zone of the growth plate (Fig. 4B; left panel). In contrast, there was a > ﬁvefold increase (P < 0.0001) in TUNEL positive cells in the hypertrophic zone of mice homozygous for the muta- tion (Fig. 4C) and we also observed numerous clusters of TUNEL positive chondrocytes in the proliferative zone (Fig. 4B; right panel). In order to identify the initial time of onset and subsequent pro- gression of chondrocyte apoptosis, we also performed the TUNEL assay on the tibia growth plates of 5-day-old and 2-week-old mice that were wild-type and homozygous for Comp D469del (Supp. Fig. S5A). There was a ∼threefold increase in apoptosis in the pro- liferative zone of 5-day-old mice and a ∼ﬁvefold increase in 2-week- old mice compared to wild-type littermates (Supp. Fig. S5B). There was no increase in apoptosis in the hypertrophic zone of 5-day- old mice, but a ∼twofold increase in 2-week-old mice compared to wild-type littermates (data not shown). Overall these data conﬁrm that increased and spatially dysregulated apoptosis occurs shortly after birth and gets progressively worse, which is consistent with the gradual accumulation of mutant COMP and progressive shortening of the limbs. The Expression of Comp D469del Does Not Induce a Conventional Unfolded Protein Response (UPR) Studies on COMP mutations to date have shown that mutant COMP is co-localized with chaperone proteins in the ER of chondro- cytes from PSACH patients [Hecht et al., 2001; Vranka et al., 2001]. Furthermore, we have recently reported a UPR in mouse models of PSACH-MED harboring mutations in Comp (T585M) and Matn3 (V194D) [Boot-Handford and Briggs, 2009; Leighton et al., 2007; Nundlall et al., 2010; Pirog-Garcia et al., 2007]. Therefore, in order to determine if the expression of Comp D469del caused a UPR, we looked at the relative expression of key chaperone proteins using a combination of real time PCR and SDS-PAGE Western blotting. qRT-PCR analysis showed no relative increase in the expression of Grp94 and Grp78/BiP at 5 days of age and Grp94, Grp78/BiP, Erp72, Cnx, and Crt at 3 weeks of age (Table 1). The corresponding protein levels of these chaperones was determined by Western blotting of intracellular proteins, which conﬁrmed that there was no increase in the relative intracellular levels of Grp94, Grp78/BiP, and Erp72 in mutant chondrocytes at 3 weeks of age (Table 1). Furthermore, we could not detect any increase in the levels of phosphorylated eIF2α, a key downstream mediator of the UPR and there was also no increase in the relative expression of Chop at 5 days and 3 weeks of age (Table 1). We also performed microarray analysis on RNA isolated from the rib chondrocytes of newborn and 5-day-old mice that were either heterozygous (5 day) or homozygous new born (newborn and 5 day) for Comp D469Del relative to the wild-type controls. These data conﬁrmed that there was no increase in the relative expression of more than 250 genes associated with a conventional UPR (Supp. Table S1 and S2), some of which have previously been shown to be upregulated in chondrocytes from mice harboring the Matn3 226 HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 Table 1. The Expression Levels of Selected ER Stress and UPR-Associated Genes was Determined Using qRT-PCR Analysis of mRNA Isolated from Chondrocytes at 5 Days and 3 Weeks of Age and Western Blotting of Total Cartilage Protein Isolated from the Cartilage of 3-Week-Old Mice ER stress/UPR marker Grp94 Grp78/BiP Erp72/Pdia4 Calnexin Calreticulin CHOP eIF2α eIF2αP 5 Days (wt vs. mut) 0.57 (±0.17) 0.93 (±0.28) - - - 0.71 (±0.03) - - mRNA 3 Week (wt vs. mut) 1.21 (± 0.32) 1.20 (±0.18) 1.11 (±0.31) 0.71 (±0.23) 0.76 (±0.27) 0.91 (±0.35) - - Protein 3 Week (wt vs. mut) 1.27 (±0.19) 1.04 (±0.12) 1.31 (±0.18) - - 1.06 (±0.31) 0.98 (±0.09) The table shows the relative levels of mRNA or protein in mutant samples compared to wild-type controls (wt vs. mut ± SEM). n ≥ 3 litters per genotype (∼5–8 mice per litter) and ≥3 separate experiments were performed in duplicate. V194D mutation [Nundlall et al., 2010]. The only exception was ﬁbroblast growth factor 18 (Fgfr18), which was upregulated twofold in newborn homozygous mutant mice. Finally, human HT1080 ﬁbrosarcoma cells expressing the COMP p.D469del mutation, which showed the retention of mutant COMP protein (Supp. Fig. S6) also failed to demonstrate a conventional UPR (Supp. Table S2), thereby conﬁrming consistency between relevant in vivo and in vitro models. Gene Expression Studies Show a Complex Disease Proﬁle Implicating Oxidative Stress, Apoptosis, and Cell Cycle Arrest in the Pathogenesis of PSACH Following the unequivocal exclusion of a classical UPR as a po- tential disease mechanism in Comp D469del mice, the Robust Mul- tiarray Averaging (RMA) method was used to ascertain the top 20 signiﬁcantly up- and downregulated genes in the chondrocytes from newborn and 5-day-old mice in order to identify potential genetic pathways involved in the pathogenesis of PSACH (Supp. Table S3–S6). The genes upregulated in chondrocytes from newborn mice were clustered into the following groupings; oxidative stress (Abcc9, Rgs5, Cdh5), cell proliferation and/or apoptosis (Igfbp3, Kera, Cilp, Lamb1-1, Sdpr, Sox17), cell attachment and/or migration (Ibsp, Chodl, Cd93, Cd34, Thbs4, Cdh5, Egﬂ6), cell survival (Ednrb), and NF-κB signalling (Meox2) (Supp. Table S3). The most highly up- regulated gene was Igfbp3 (↑12.19), which has been shown to in- duce rapid apoptosis in a non-IGF-dependant manner and has an- tiproliferative effects via the transforming growth factor β (TGF-β) signalling pathway [Han et al., 2011; Lee et al., 2005]. The down- regulated genes included those linked with NF-κB signalling (Ccl5, Prdx2, T2bp, Hivep3, Tac1, Srxn1), cell proliferation (Pfkfb3), cell survival (Bdkrb1), cell signalling (Gng4, Lif, Otos, A2m, Rspo3), and cell cycling (Rnd1) (Supp. Table S4). Interestingly, we found the downregulation of three members of the chemokine ligand protein family, namely Ccl5, Cxcl2, and Cxcl16, and also other genes that were involved in the same pathways such as Hivep3 and T2bp. By 5 days of age, the upregulated genes included those in- volved in oxidative stress and degradative pathways (Amacr, Hmox1, Car3, Arsj, Ctsc, Heph), cell survival and/or proliferation (Iﬁ202b, Camk4, Sox11, Snca, Agtr2), regulators of insulin receptor and IGFBP-3-dependent effects (Enpp1, Lyve1), and NF-κB signalling