GSE130088
Molecular Profiling of Failed Endochondral Ossification in Mucopolysaccharidosis VII
Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of ß-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification using whole transcriptome sequencing (RNA-Seq). Epiphyseal tissue was isolated from the vertebrae of control and MPS VII affected dogs at 9 and 14 days-of-age (n=5 for each group). Differences in global gene expression across this developmental window for both cohorts were established using RNA-Seq. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification, and a subset of gene expression changes were validated using qPCR. Principal Component Analysis revealed clustering of samples from each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expression between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. Targeted analysis revealed temporal changes in gene expression from 9 to 14 days-of-age in control samples consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/ß-catenin and BMP signaling were dysregulated. In conclusion, this study represents the first step towards identifying druggable therapeutic targets and putative biomarkers for bone disease in MPS VII patients during postnatal growth. Overall design: For this study, we used the naturally-occurring canine model of MPS VII. MPS VII dogs have a missense mutation (R166H) in the GUSB gene and exhibit a similar skeletal phenotype to human patients. Animals were raised at the University of Pennsylvania School of Veterinary Medicine under NIH and USDA guidelines for the care and use of animals in research, and all studies were carried out with IACUC approval. Litter-matched control (heterozygous) and MPS VII (homozygous) dogs were identified via gene sequencing analysis at birth. Animals were euthanized at 9 or 14 days-of-age, defined as number of postnatal days (n=5 for both control and MPS VII animals at each time point), using 80 mg/kg of sodium pentobarbital in accordance with the American Veterinary Medical Association guidelines. The thoracic spine was dissected out immediately following euthanasia, and epiphyseal tissue from both the cranial and caudal sides of the T12 vertebrae for each animal was excised adjacent to the growth plate, with adjoining intervertebral disc tissue carefully removed. Cranial and caudal tissue for each sample was combined and flash frozen in liquid nitrogen.