Generation and Characterization of a Mouse Model to Investigate Tissue-Specific Pathologies in Spondyloepiphyseal Dysplasia, MGP Type

Abstract

Skeletal dysplasia is a group of clinically and genetically heterogeneous disorders associated with abnormal bone and cartilage development. We recently reported a dominant mutation in the matrix Gla protein gene (c.56G>T:p.C19F) and named this spondyloepiphyseal dysplasia (SED), MGP type. Because of the early lethality of mice carrying this dominant mutation, Mgp+/56G>T mice, our lab previously employed CRISPR/Cas-9 and in vitro fertilization (IVF)-based mouse models to examine the pathology of SED in vivo. This mutation converts the cysteine residue at the end of the signal peptide to phenylalanine, preventing the protein's transport out of the cell. As a result, the protein accumulates in the endoplasmic reticulum (ER), leading to ER stress and the death of prehypertrophic and hypertrophic chondrocytes, thereby disrupting normal skeletal development. Currently, a comprehensive understanding of the craniofacial abnormalities linked to SED, MGP type, remains lacking. Our first aim was to generate a mouse model with tissue-specific expression of the mutant protein to study its impact on skeletal and craniofacial development. We employed CRISPR/Cas-9 technology to insert a ‘floxed’, transcription-blocking sequence upstream of the Mgp+/56G>T mutation to inhibit the expression of the mutant protein. This Mgp+/IND56G>T mouse was bred with Mgp-Cre and Col2a1-Cre mice, resulting in the expression of the mutant protein in all chondrocytes of the compound mutant mice. Our second aim was to characterize the skeletal phenotype resulting from chondrocyte-specific C19F MGP expression in the newly developed model. Mgp+/IND56G>T; Col2a1-Cre mice were significantly shorter than wild-type controls, with reduced long bone lengths and body weights. Three-dimensional (3D) reconstruction of the micro-CT for the vertebrae showed a shortened 13 vertebra, premature closure of the growth plate, and a low bone mass in 6-week-old Mgp+/IND56G>T; Col2a1-Cre mice compared to control mice. These phenotypes are consistent with those seen in the IVF-generated mouse model previously used to study SED pathophysiology. 3D reconstructions of the heads revealed midface hypoplasia and a class III malocclusion in mutant mice compared with controls. Cephalometric analyses of whole heads show a decrease in basicranial length in these mice compared with WT mice. Further analysis revealed abnormal mineralization and premature fusion of cranial base synchondroses, leading to deficient anterior-posterior growth of the skull and resulting in class III malocclusion. No calcification was observed in the nasal septum. However, the nasal septum was thicker, with increased cell apoptosis. Conclusion We successfully generated and characterized a preclinical mouse model with chondrocyte-specific expression of the C19F mutant MGP, which replicates the original whole-body expression model generated by IVF. The findings of this study confirm that the pathologies associated with SED, MGP type, result from the expression of the mutant protein in chondrocytes. Additionally, we demonstrate that midface hypoplasia in SED, MGP type, arises from distinct underlying mechanisms not seen in MGP-deficient mice. This model provides a valuable tool for understanding the pathophysiology of SED, MGP type, exploring the effects of the mutation on other cell types, and developing therapeutic strategies to prevent or halt disease progression.