MED is genetically heterogeneous and results from mutations in the genes encoding matrilin-3, type IX collagen and COMP. However, extensive genetic analysis has consistently demonstrated that mutations in other, as yet unidentified genes, can also result in MED. The proportion of MED that does not have a genetic basis ascribed varies between 30-70% depending on diagnostic rigour. Our recent comprehensive study also identified cohorts of patients that were phenotypic outliers of the MED ‘disease spectrum’, but which shared distinct clinical and/or radiographic features. Consensus diagnoses for these patients focused around specific forms of familial hip dysplasia (FHD) and it is clear that these diseases are genetically distinct from ‘classical’ forms of MED. Similarly, this study also highlighted gaps in our understanding of the genetic basis of PSACH and identified patients/families that have one or more of the following features; a distinct severe-PSACH phenotype, consanguineous parents (i.e. recessive inheritance) and are COMP mutation negative.
Next generation sequencing (NGS) offers the technical innovation to determine the full genetic basis of PSACH-MED-FHD that has not been possible to date. DNA from mutation negative families and cohorts of patients with a similar clinical/radiographic presentation has been collected and is available for study. Exome sequencing will be performed at the Institute of Genetic Medicine, which has state-of-the-art high-throughput DNA sequencing facilities and bioinformatics pipeline.
What role to do ARMET and CRELD2 play in genetic skeletal diseases?
Armet/Manf (arginine-rich, mutated in early stage tumors) and Creld2 (cysteine-rich with EGF- like domains 2) are two recently identified ER-stress responsive proteins and despite being induced by a variety of physiological and pathological triggers and reported to function as cytoprotective factors against oxidative and ER stresses, very little is known about the function of these two proteins. ARMET and CRELD2 are upregulated and secreted in cell and mouse models of Matn3-V194D andCol10a1-N617K; but not Comp mutations. Preliminary studies using cell models confirm that ARMET and CRELD2 form complexes with mutant matrilin-3, but not COMP, whilst substrate-trapping experiments indicate that CRELD2 processes PDI-like activity. To advance this line-of-enquiry we have generated cartilage-specific knockouts of Creld2 and Armet and then crossed these onto Matn3-V194D mice. These novel mice lines along with validated cell models will be used for investigating the role of ARMET and CRELD2 in protein folding, UPR and growth plate dysplasia.
Determining the function of ARMET and CRELD2 and understanding their potential cytoprotective role in the pathobiology of GSDs will provide novel insight into disease mechanisms.
This line-of-enquiry will be underpinned by our well-established phenotypic analyses that comprise qualitative and quantitative assays of skeletal dysplasia, cell-matrix pathology and genetic mechanisms. Histology and immunohistochemistry for visualising growth plate dysplasia and changes in protein localisation.Cell proliferation and apoptosis assays provide quantitative markers of chondrocyte phenotype. Omics technologies (gene expression and proteomics) will be used to gain a global overview of mRNA and protein changes that occur as a result of the different gene deletions. The zone-specific changes in pathology may require precise analysis and micro-dissected growth plate cartilage will be used for these studies.
Investigating the role of XBP1 mediated UPR in pathogenesis of MATN3-MED
This line of enquiry will characterise the role of Xbp1-mediated UPR in the pathogenesis of MED caused by Matn3-V194D and to compliment these studies we will compare findings to the Col10a1-N617K model of MCDS.
Xbp1 was specifically chosen due to its key transcriptional role in UPR and Xbp1 splicing was confirmed in both models. Moreover, ARMET and CRELD2 are putative downstream targets of spliced-Xbp1.
Conditional Xbp1 mice with a chondrocyte-specific Col2–cre allele were bred to homozygosity with Matn3-V194D or Col10a1-N617K alleles and preliminary analysis has confirmed very different phenotypic consequences.
1) Col2–Xbp1-/-Matn3m/m mice are significantly shorter than Matn3m/m mice with extensive disruption to growth plate architecture and chondrocyte proliferation.
2) In contrast, chondrocyte-deletion of Xbp1 appears to have no detrimental effect on growth plate pathology or bone-lengths in Col10a1-N617K mice.
This unexpected dichotomy between the phenotypic consequences of Xbp1-deletion in the two models, in view of their similar ER-stress disease signatures, highlights differences in ER-stress pathways in related cell-types (proliferative vs. hypertrophic chondrocytes). Finally, although previously reported to have no overt skeletal phenotype, our preliminary analysis has demonstrated reduced adult bone-lengths in Col2–Xbp1-/- mice, which will constitute another relevant line-of-enquiry. Defining these phenotypic differences and cellular responses at the genetic and molecular level will form a major objective of these lines-of-enquiry.