Progression of vertebral bone disease in mucopolysaccharidosis VII dogs from birth to skeletal maturity

https://doi.org/10.1016/j.ymgme.2021.06.005Get rights and content

Abstract

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, leading to accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans. Patients with MPS VII exhibit progressive spinal deformity, which decreases quality of life. Previously, we demonstrated that MPS VII dogs exhibit impaired initiation of secondary ossification in the vertebrae and long bones. The objective of this study was to build on these findings and comprehensively characterize how vertebral bone disease manifests progressively in MPS VII dogs throughout postnatal growth. Vertebrae were collected postmortem from MPS VII and healthy control dogs at seven ages ranging from 9 to 365 days. Microcomputed tomography and histology were used to characterize bone properties in primary and secondary ossification centers. Serum was analyzed for bone turnover biomarkers. Results demonstrated that not only was secondary ossification delayed in MPS VII vertebrae, but that it progressed aberrantly and was markedly diminished even at 365 days-of-age. Within primary ossification centers, bone volume fraction and bone mineral density were significantly lower in MPS VII at 180 and 365 days-of-age. MPS VII growth plates exhibited significantly lower proliferative and hypertrophic zone cellularity at 90 days-of-age, while serum bone-specific alkaline phosphatase (BAP) was significantly lower in MPS VII dogs at 180 days-of-age. Overall, these findings establish that vertebral bone formation is significantly diminished in MPS VII dogs in both primary and secondary ossification centers during postnatal growth.

Introduction

The mucopolysaccharidoses (MPS) are a family of inherited, lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs) due to mutations in associated genes [1]. MPS VII, also called Sly Syndrome, is characterized by deficient beta-glucuronidase (GUSB) activity, leading to incomplete degradation of heparan, dermatan and chondroitin sulfate GAGs [2]. These GAGs accumulate progressively in cells and tissues resulting in multi-organ system disease manifestations. MPS VII patients present with a spectrum of disease severities [3]. Progressive skeletal abnormalities, commonly present in patients with more severe disease and termed “dysostosis multiplex”, include short stature, spinal deformity and joint dysplasia, which result in pain, impaired mobility, and an overall diminished quality of life [[2], [3], [4]].

Abnormal bone growth contributes to the progression of skeletal disease in MPS VII due to failures of endochondral ossification during postnatal growth [5]. Endochondral ossification is the biological process underlying the development and growth of vertebrae and long bones, beginning with formation of a cartilaginous template that is then replaced by mineralized bone [6,7]. This processes occurs first in primary ossification centers (POCs) and subsequently in secondary ossification centers (SOCs), and also in growth plates, facilitating longitudinal bone growth [7,8]. Using the naturally-occurring canine model, we previously demonstrated that bone disease in MPS VII first manifests at the tissue level as delayed conversion of epiphyseal cartilage to mineralized bone in the SOCs of both vertebrae and long bones [9]. During later stages of postnatal growth, persistent cartilaginous lesions in vertebral SOCs resulted in reduced stiffness and increased range-of-motion of the intervertebral joints, implicating them as a contributing factor in the progression of spinal deformity [10,11]. These lesions were found to persist for the lifetime of the animals [12]. Impaired hypertrophic differentiation of epiphyseal and growth plate chondrocytes has been shown to contribute to failed cartilage-to-bone conversion in SOCs, and reduced longitudinal bone growth, respectively [9,13,14]. Recent work in MPS VII dogs showed that these cells exhibit significantly elevated lysosomal GAG storage from an early age, together with elevated apoptosis and impaired autophagy [15]. Additionally, studies have demonstrated impaired activation of key osteogenic signaling pathways required to initiate and sustain chondrocyte differentiation, and reduced expression of enzymes required for extracellular matrix remodeling and mineralization [16,17]. In the POCs of MPS VII dog vertebrae, despite bone cells (osteoblasts and osteocytes) exhibiting significantly elevated storage [15], bone content is normal during early postnatal development [9], and to date, no studies have examined how bone disease progresses in the POCs of these animals at later stages of growth. Studies in dogs with MPS I (α-L-iduronidase deficiency), however, have shown that POCs exhibit significantly lower bone volume fraction and mineral density from 6 months-of-age onwards [18]. A more comprehensive understanding of the temporal progression of abnormal ossification in both POCs and SOCs in MPS VII dogs throughout postnatal growth may provide new clinically-relevant insights into disease etiology and inform optimal timing for therapies specifically targeting skeletal disease manifestations in both canine and human patients.

With the advent of new, clinically-approved treatments for MPS VII such as enzyme replacement therapy (ERT), there is a pressing need for non-invasive biomarkers to monitor bone disease progression and response to treatment. There is evidence that serum levels of markers of bone turnover may be abnormal in MPS patients [19]. These biomarkers, when coupled with diagnostic imaging, can be used to monitor progression of other skeletal diseases characterized by altered bone turnover, including osteoporosis, osteoarthritis and rheumatoid arthritis [[20], [21], [22]]. To date, there have been no controlled, preclinical studies examining whether serum biomarkers can be used as surrogates for abnormal bone turnover in MPS VII.

The objectives of this study were to firstly characterize progression of vertebral bone disease in both POCs and SOCs from birth to skeletal maturity in MPS VII dogs, and secondly, to establish the efficacy of two serum biomarkers as non-invasive indicators of abnormal bone formation in this preclinical large animal model.

Section snippets

Animals and tissue collection

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 [23] and exhibit a similar skeletal phenotype to human patients [10,23,24]. 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. Control (heterozygous) and MPS VII (homozygous) dogs were identified via

Clinical findings

The mobility of MPS VII animals progressively declined beginning between 56 and 84 days-of-age, and these animals were unable to rise or walk by 180 days-of-age. Body weights in MPS VII and control animals were compared to each other within litters, because of the heterogeneity between litters. While weights within litters were similar at birth, MPS VII animals weighed around 25% less by 28 days-of-age, with the decreased trend in weight gain continuing until study termination. Weight gain in

Discussion

Bone disease is prevalent in MPS VII patients due to multiple failures of endochondral ossification during postnatal growth. Patients exhibit progressive kyphotic and scoliotic spinal deformity, due in part to abnormal development of the vertebrae. Until recently, treatment options for MPS VII were extremely limited. Enzyme replacement therapy (ERT) recently became FDA approved for clinical use [27,28]; however, as of yet there is little clinical data on its efficacy specifically for bone

Author contributions

SHP contributed to conceptual design, performed experiments and data analysis, and drafted the manuscript; YKL performed experiments and data analysis; JLK, ML, TA, DRM and JRB performed experiments; MEH, MLC and PO cared for animals, assisted with postmortem tissue collection and provided conceptual input on experimental design; LJS contributed to conceptual design and data analysis, and drafted the manuscript. All authors reviewed and approved the manuscript prior to submission.

Authors disclosures

The authors have no relevant conflicts of interest to disclose.

Acknowledgements

Funding for this work was received from the National Institutes of Health (R01AR071975, R01DK054481, R03AR065142, F32AR071298 and P40OD010939), the National MPS Society, and the Lisa Dean Moseley Foundation. Additional support was received from the Penn Center for Musculoskeletal Disorders (NIH P30AR069619). The authors thank animal care staff at the University of Pennsylvania School of Veterinary Medicine for their support.

References (41)

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