pathways (Il1rl2, Phex) (Supp. Table S5). The downregulated genes included those implicated in oxidative stress (Npas4, Prdx2), apop- tosis (Camk2d, Adi1), cell cycle control (Ddx3y, Eif2s3y), cell pro- liferation/migration and cell–cell interactions (Lypd3, Gjb4, Bop1, Cdh1, Hbegf, Trio), and NF-κB signalling pathways (Prdx2, Snno2) (Supp. Table S6). The Downregulation of Peroxiredoxin-2 is a Consistent Finding in Both Mutant Mice and the Cell Culture Model Interestingly, the only gene whose expression was consistently changed at both time points was Prdx2, which was downregulated at both newborn (↓3.89) and 5 days of age (↓2.91) (Supp. Table S4 and S6). Furthermore, PRDX2 expression was also signiﬁcantly down- regulated in the HT1080 cell culture model of COMP p.D469del (↓2.60) and at 5 days in mice heterozygous for the mutation (↓3.32) (data not shown). In view of these data and given the known as- sociation between the downregulation of PRDX2 and apoptosis in human diseases such as Down syndrome and Fuchs Endothelial Dystrophy (FED) [Jurkunas et al., 2008; Sanchez-Font et al., 2003], we used a combination of Western blotting and IHC to validate the downregulation of peroxiredoxin-2 both in vivo and in vitro. Total protein was extracted from the cartilage of 3-week-old mice and used for SDS-PAGE and Western blotting (Fig. 5A). Following normalization by Ponceau staining for total protein, densitometry conﬁrmed that the protein levels of peroxiredoxin-2 were signiﬁ- cantly reduced by 13% (P < 0.0003) in mutant chondrocytes relative to wild-type (Fig. 5B). In addition, total cell lysate samples were collected from HT1080 cells at 48 and 72 hr after conﬂuence and the relative levels of peroxiredoxin-2 were quantiﬁed by densitometry after normalizing to a β actin loading control (Fig. 5C). There was a >95% (P < 0.0001) decrease in the levels of peroxiredoxin-2 in the COMP p.D469del mutant cell line compared to wild-type (Fig. 5D). Finally, IHC was performed on the tibia growth plates of 3-week- old mice homozygous for the Comp D469del mutation and their wild-type littermates. This analysis demonstrated the intracellular staining of peroxiredoxin-2 in growth plate chondrocytes from mu- tant mice was much reduced in chondrocytes from wild-type mice growth plates (Fig. 5E). Discussion A single mutation in COMP, the in-frame deletion of an aspartic acid codon (p.D469del) from the T37 calcium-binding repeat of COMP, is responsible for approximately 30% of all PSACH [Hecht et al., 1995; Ikegawa et al., 1998; Kennedy et al., 2005a]. To deter- mine the disease mechanisms that underpin the pathophysiology of PSACH in vivo, we generated a knock-in mouse model of PSACH re- sulting from this p.D469del mutation. This is the ﬁrst physiologically relevant mouse model of PSACH to closely model the phenotypic and cellular consequences of Comp D469del. However, in contrast to the human PSACH phenotype, which is a dominant disease, both copies of the Comp D469del allele were required for the mice to develop a quantiﬁable chondrodysplasia phenotype. We have pre- viously noted similar differences in other mice models of PSACH and MED, such as Comp Thr585Met [Pirog-Garcia et al., 2007] and Matn3 Val194Asp [Leighton et al., 2007], and metaphyseal chon- drodysplasia type Schmid Col10a1 Asn617Lys [Rajpar et al., 2009]. Furthermore, a mouse model of achondroplasia showed a similar inconsistency [Li et al., 1999] and these discrepancies in phenotypic presentation are suggestive of dominance modiﬁcation [Dixon and Dixon, 2004; Nadeau, 2001] Mutant mice were normal at birth but developed signiﬁcant short-limb dwarﬁsm by 9 weeks of age (Fig. 1). This is consistent with the clinical progression of the PSACH phenotype in which patients are normal at birth but during childhood they exhibit progres- sive short-limbed dwarﬁsm [Maroteaux and Lamy, 1959; Rimoin et al., 1994]. Mutant mice also developed a progressive hip dyspla- sia, which was characterized radiographically by a deﬂection from the vertical of the tuberosity of the ischium (Fig. 1). This observation was consistent with, although more severe than that reported in the Comp T585M [Pirog-Garcia et al., 2007], Col10a1 N617K [Rajpar et al., 2009] and Dtdst A386V [Forlino et al., 2005] mutant mice. In contrast, neither a transgenic Comp D469Del mouse [Schmitz et al., 2008] nor the Matn3 V194D mice [Leighton et al., 2007] had a recognizable hip dysplasia, even though the former displayed ster- nal malformations. This disparity in radiographic presentation in the transgenic Comp D469Del mouse may be due to differences in the level of transgene expression, while the Matn3 V194D mutation may not have as pronounced an effect on the development of the pelvis. The chondrocyte columns in the growth plates of mutant mice were disorganized and there were distinct changes to the morphol- ogy of individual chondrocytes. Immunohistochemical analysis also showed the retention of COMP, matrilin-3, and to a lesser extent type collagen IX, within the chondrocytes of mutant mice (Fig. 2). Numerous studies to date, both in vitro and in vivo, have con- sistently shown the accumulation of mutant COMP and since it interacts with both matrilin-3 and type IX collagen in vitro these interactions are possibly the cause of their co-retention. The effect that the retained mutant COMP has on the trafﬁcking of type II collagen has not been fully resolved and there are conﬂicting reports as to whether type II collagen and other cartilage ECM molecules (such as aggrecan) are also retained in the rER of chondrocytes [Hecht et al., 2001; Vranka et al., 2001]. Our own IHC studies sug- gest that type II collagen is not co-retained with mutant COMP in the rER, however, there were clear differences in the localization of type II collagen in the ECM, which may be a direct consequence of the defect in trafﬁcking of COMP, matrilin-3, and type IX collagen and/or the reduced levels of these proteins in the cartilage ECM (Supp. Fig. S4). Alternatively, the presence of mutant COMP in the ECM may disrupt speciﬁc interactions, thereby inﬂuencing in turn the localization of its binding partners. This hypothesis is consistent with the pericellular localization of mutant COMP and wild-type matrilin-3 in the resting zone of mutant growth plates (Fig. 2D and F). Quite striking were the regions of hypocellularity within the pro- liferative zone (Fig. 2), which were consistent with the signiﬁcantly increased and dysregulated apoptosis within this zone of the growth plate. This ﬁnding is in agreement with studies on cultured PSACH chondrocytes, which show an increase in cell death following pro- longed culture in vitro [Duke et al., 2003], and also a transgenic model of Comp D469del in which there was a signiﬁcant increase in cell death when it was crossed onto a Comp background [Schmitz et al., 2008]. However, in this transgenic model the in- crease in apoptotic cells was primarily detected at the interface with the secondary center of ossiﬁcation, which is in direct contrast to our knock-in model in which increased apoptosis is seen in the prolifer- ative and hypertrophic zones. It is difﬁcult to explain this difference in the localization of apoptosis, but it is possible that differences in the relative levels of mutant Comp expression between these two models might inﬂuence the location of dysregulated apoptosis. Based on our recent ﬁndings in the PSACH and MED mouse models of Comp T585M [Pirog-Garcia et al., 2007] and Matn3 V194D [Leighton et al., 2007; Nundlall et al., 2010] mutations, we HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 227