Results Generation of the Comp D469del Knockin Mouse Line Mice harboring the Comp D469del mutation in the TSP3 region of COMP were generated by homologous recombination in R1 ES cells using an appropriate targeting strategy (Supp. Fig. S1). The “long arm” of the targeting construct (8 kb) contained exons 1–16 of Comp and the “short arm” of the construct (2 kb) contained exons 17–19 of Comp (Supp. Fig. S1A). The 3-bp deletion required to produce the D469del mutation was introduced into exon 13 of Comp by site-directed mutagenesis. Three hundred and sixty ES cell clones that had been electroporated with the targeting construct and selected by antibiotic resistance were analyzed for homologous re- combination using Southern blotting with an external probe (Supp. Fig. S1B). The presence of the mutation was conﬁrmed by direct sequencing of DNA from ES clones that had successfully undergone homologous recombination. Positive clones resulting from the tar- geting procedure were microinjected into C57BL/6 blastocysts and then implanted into pseudopregnant foster mothers. The resultant chimeric mice were used for the foundation of a transgenic line and a breeding strategy was used to obtain wild-type mice and mice that were either heterozygous or homozygous for the mutation. The presence of the mutation was again conﬁrmed in mice that were homozygous for D469del (Supp. Fig. S1C). COMP mRNA and Protein Expression Levels are Comparable between Wild-Type and Mutant Mice Cartilage from 3-week-old mice was used for RNA extraction and for SDS-PAGE, and Western blot analysis of total protein to conﬁrm that the D469del mutation did not affect the expression of either Comp alleles. Prior to qRT-PCR, ﬁrst strand cDNA was analyzed to ensure that there was no contamination from genomic DNA. This was achieved by performing PCR using a primer pair span- ning intron 15 of the COMP gene, which conﬁrmed that there was no contamination from genomic DNA (Supp. Fig. S1D). There- after, qRT-PCR using Comp-speciﬁc primers was performed and this analysis conﬁrmed comparable Comp mRNA levels in chon- drocytes from wild-type and mutant mice (0.92 ± 0.09; wt vs. mut). Total COMP protein levels in cartilage were determined by SDS- PAGE analysis of DTT-reduced total protein samples followed by Western blotting using a COMP-speciﬁc antibody. Protein bands corresponding to wild-type and mutant COMP were present at ap- proximately 100 kDa, which corresponded to the COMP monomer (Supp. Fig. S1E). Wild-type and mutant cartilage protein samples showed comparable levels of COMP when normalized to Ponceau staining and quantiﬁed by densitometry (data not shown). Further- more, protein samples run under non-reducing conditions demon- strated that mutant COMP formed the correct pentameric structure (data not shown). Mutant Mice are Normal at Birth but Develop a Progressive Short-Limb Dwarﬁsm and Hip Dysplasia A normal Mendelian distribution of 1:2:1 (wt/wt:m/wt:m/m) was observed in litters produced from crosses of mice heterozygous for the Comp D469del mutation. There were no overt skeletal defects observed at birth in mice of any genotype (data not shown), an observation that is consistent with human PSACH patients when the clinical presentation only becomes apparent in early childhood. To quantify changes in growth that were caused by the Comp D469del mutation, littermates of all three genotypes were weighed at 3, 6, and 9 weeks of age and these measurements were used to prepare growth curves (Supp. Fig. S2). At 3 weeks of age, the body weights of all the mice were comparable, but by 6 weeks of age the body weights of male mice homozygous for the Comp D469del mutation were >7% (P < 0.05) less that their wild-type and heterozygous littermates. By 9 weeks of age, the differences in weight were statistically signiﬁcant for both male and female mice homozygous for the mutation (males ∼10%, P < 0.02 and females ∼8%, P < 0.05). Radiographs of individual mice of all three genotypes were col- lected at 3, 6, and 9 weeks of age and bone length measurements were performed (Fig. 1 and Supp. Fig. S3). To determine the effect of the mutation on intramembraneous ossiﬁcation, skull length and inner canthal distance (ICD) were measured; while the lengths of the pelvis, femur, and tibia were used to determine the effect of the mutation on endochondral ossiﬁcation. At 9 weeks of age, the mice homozygous for the mutation appeared shorter than their wild-type littermates and the ﬁnal reduction in adult male bone lengths, rel- ative to wild-type controls, was 6% (P < 0.05) for the femur, 7% (P < 0.05) for the pelvis, and 5% (P < 0.05) for the tibia (Fig. 1B). In female mice, the ﬁnal reduction in adult bone lengths was 6% (P < 0.05) for the femur, 3% (P < 0.001) for the pelvis, and 6% (P < 0.05) for the tibia (data not shown). In contrast, there were no differences in either the skull length or ICD for mice of all three genotypes and at all time points analyzed (Fig. 1B and data not shown). Patients with PSACH develop a waddling gait during childhood and we were interested to determine if Comp D469del mutant mice developed a hip dysplasia similar to that reported for Comp T585M mutant mice [Pirog-Garcia et al., 2007]. In order to determine the effect of the mutation on the development of the pelvis, the angle of deﬂection from the vertical of the tuberosity of the ischium was measured at 3, 6, and 9 weeks of age in mice of all three genotypes (Fig. 1C; only 9 weeks is shown). Interestingly, at 9 weeks of age the angle at the hip was greater in mice homozygous for the mutation when compared to their wild-type or heterozygous littermates. In males, the angle of deﬂection was increased by >57% (P < 0.01) (Fig. 1D; 10◦ in wild-type mice compared to 16◦ in mice homozy- gous for the mutation). In female mice, there was a >48% (P < 0.05) increase in the angle of deﬂection from 10.4◦ in wild-type mice to 15.4◦ in mice homozygous for the mutation (data not shown). Mutant Mice Exhibit Abnormal Growth Plate Morphology Characterized by Disorganized Chondrocyte Columns and Areas of Hypocellularity in the Proliferative Zone By 3 weeks of age, the growth plates of wild-type mice were well organized with ordered columns of proliferating chondro- cytes, while a distinct hypertrophic zone was visible that was ter- minated by a regular vascular invasion front (Fig. 2A). In con- trast, mice homozygous for the mutation had an abnormal growth plate characterized by disorganized chondrocytes within individual chondrons, while the chondrons themselves were also misaligned within each column and in some cases individual chondrocytes were seen to be aligned at right angles to their usual horizontal plane (Fig. 2B; red circles). Most striking however were the obvious areas of hypocellularity in the resting and upper proliferative zones of the mutant growth plates (Fig. 2B; asterisks). Furthermore, there ap- peared to be no clear distinction between the proliferative and hy- pertrophic zones of the growth plate and the vascular invasion front was also irregular. The growth plates of mice heterozygous for the mutation were comparable to those of wild-type mice in that they had an organized growth plate with clear proliferative and hyper- trophic zones and a regular vascular invasion front, however, some HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 221