Collagen XXVII Function in Growth Plate Figure 7. Appearance of mice carrying a cartilage-specific deletion in type XXVII collagen. Mice homozygous for a cartilage specific deletion of the collagen XXVII gene (MDskel/MDskel) were viable but were severely dwarfed. Left of figure – littermates at 4 weeks of age (+/MDskel is the phenotypically normal control). Right of figure - X-rays of 3 week old mice. MDskel/MDskel mice exhibited a slight distortion of the thoracic cage, a mild thoracic kyphosis and a distortion of the pelvis (indicated by red dashed lines) compared to control littermates. Scale bar = 10 mm. doi:10.1371/journal.pone.0029422.g007 Through the application of a conditional knock-in strategy, we have been able to determine the effects of two distinct mutations in collagen XXVII on endochondral bone growth. The Gly to Cys mutation within the C-terminal region of the collagenous domain produced a very mild phenotype whereas the much larger 87 amino acid residue in-frame deletion in the collagenous domain not only disrupted endochondral ossification but also caused defects in other tissues including lung. Interesting, neither collagen XXVII mutation caused any overt phenotype in articular cartilage. It was anticipated that the del87 allele would produce a hypomorph or functional for collagen XXVII due to misfolding of the triple helical domain and subsequent intracellular protein degradation by way of ER associated degradation or autophagy [16]. In some ways, the most surprising finding was that the mice heterozygote for either the Gly to Cys or the del87 mutation had no phenotype and appeared to secrete collagen XXVII efficiently based on immunolocalisation. In a heterozy- gote, the trimerisation of mutant and wild type proa1(XXVII) chains at the carboxyl- terminal non-collagenous domain should result in 87.5% of trimers containing at least one mutant chain. In the classical fibrillar collagens, this is sufficient to cause significant consequences either in terms of protein misfolding and reduced secretion or if secreted, in the disruption of the resulting fibril structure. Type XXVII collagen forms very thin fibrils that are distinct from the thicker fibrils formed by types II and XI collagen in cartilage [7,8]. The lack of phenotype in terms of growth plate function and immunostaining in heterozygotes for the collagen XXVII missense or deletion mutation suggests that this collagen is far more tolerant of sequence change compared to its classical counterparts. Furthermore, the phenotypic effects of the Gly to Cys mutation were extremely mild with significant pathology only becoming apparent with a much larger deletion in the collagenous domain. Unfortunately, we have been unable to detect quantifiable levels of collagen XXVII in tissue extracts using western blotting and so have been unable to confirm the levels of secretion and molecular form of the molecule secreted in control and mutant cartilage by biochemical means. Nevertheless, these results suggest disease associated mutations in collagen XXVII may be less common than one would predict based on extensive studies of classical fibrillar collagens [e.g. 11,12]. Human disease resulting from mutations in collagen XXVII are likely to result more commonly from larger deletions in the collagen XXVII gene rather than missense mutations and may prove perinatal lethal involving not only chondrodysplasia but also problems in other tissues such as lung and midline defects. In summary, collagen XXVII plays an important role in organizing the pericellular ECM of proliferative zone chondro- cytes. Without the correct organisation of this matrix, the pattern of cell division and the ability of the proliferative chondrocyte to correctly organise into columns are disrupted resulting in a significant decrease in the rate of endochondral bone growth. Materials and Methods Ethics statement The mouse work described here was approved by the University Animal Ethical Review Group and conducted under a project licence (Animals [Scientific Procedures] Act 1986) granted by the UK Home Office. Generation of Collagen XXVII mutant mice Gene targeting was performed as described [17]. Briefly, a PAC clone was identified and ordered from HGMP (Hinxton, UK: at http://www.hgmp.mrc.ac.uk) using a 129/sv DNA library (RPCI- 21) supplied as filters and screened with a cDNA encoding the C- PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29422