RESUMO
Skeletal alterations in the head and neck region, such as midfacial hypoplasia, foramen magnum stenosis and spinal canal stenosis, are commonly observed in patients with mucopolysaccharidosis (MPS). However, enzyme replacement therapy (ERT), one of the major treatment approaches for MPS, shows limited efficacy for skeletal conditions. In this study, we analysed the craniofacial morphology of mice with MPS type VII, and investigated the underlying mechanisms promoting jaw deformities in these animals. Furthermore, we investigated the effects of C-type natriuretic peptide (CNP), a potent endochondral ossification promoter, on growth impairment of the craniofacial region in MPS VII mice when administered alone or in combination with ERT. MPS VII mice exhibited midfacial hypoplasia caused by impaired endochondral ossification, and histological analysis revealed increased number of swelling cells in the resting zone of the spheno-occipital synchondrosis (SOS), an important growth centre for craniomaxillofacial skeletogenesis. We crossed MPS VII mice with transgenic mice in which CNP was expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP. The maxillofacial morphological abnormalities associated with MPS VII were ameliorated by CNP expression, and further prevented by a combination of CNP and ERT. Histological analysis showed that ERT decreased the swelling cell number, and CNP treatment increased the width of the proliferative and hypertrophic zones of the SOS. Furthermore, the foramen magnum and spinal stenoses observed in MPS VII mice were significantly alleviated by CNP and ERT combination. These results demonstrate the therapeutic potential of CNP, which can be used to enhance ERT outcome for MPS VII-associated head and neck abnormalities.
Assuntos
Mucopolissacaridose VII , Peptídeo Natriurético Tipo C , Humanos , Camundongos , Animais , Peptídeo Natriurético Tipo C/farmacologia , Constrição Patológica/complicações , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/tratamento farmacológico , Osteogênese , Camundongos TransgênicosRESUMO
Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-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.
Assuntos
Doenças Ósseas/fisiopatologia , Progressão da Doença , Mucopolissacaridose VII/complicações , Coluna Vertebral/patologia , Animais , Animais Recém-Nascidos , Doenças Ósseas/genética , Osso e Ossos/patologia , Cães , Feminino , Crescimento e Desenvolvimento , Masculino , Mucopolissacaridose VII/genética , OsteogêneseRESUMO
Mucopolysaccharidosis type VII (MPS VII) is a rare autosomal recessive lysosomal storage disorder. MPS VII is caused by mutations in the GUSB gene that encodes ß-glucuronidase. Adult MPS VII patients present with musculoskeletal abnormalities, coarse features, and corneal clouding. Cardiac and valvular impairment are common; however, severe valvular disease necessitating surgery has not yet been reported. We present a 32-year-old male MPS VII patient admitted to our hospital with decompensated heart failure. We identified aortic valve disease with severe stenosis (valve area 0.69 cm2) and moderate regurgitation. Severe mitral valve stenosis (valve area 1 cm2) with moderate to severe regurgitation was also found in the patient. In addition, an occlusion of the right coronary artery (RCA) was documented. The patient underwent surgical replacement of the mitral and aortic valves with mechanical prostheses and implantation of a venous bypass graft to his RCA. The surgery led to a significant improvement of his clinical symptoms. Six months after the procedure, both mechanical valves function normally. Histopathological assessment identified chronic inflammatory infiltrates, fibrosis and calcifications in both resected valves. Foamy cytoplasmic transformation was most evident in the valvular interstitial cells. The ultrastructural vacuolar abnormality seen in these cells corresponded to storage changes observed in other MPSs. In conclusion, we describe clinical findings and valvular pathology in an MPS VII patient with the first-reported successful combined surgical valve replacement and myocardial revascularization. The histological and ultrastructural analyses revealed that the lysosomal storage predominantly affected the valvular interstitial cells.
Assuntos
Insuficiência da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/cirurgia , Ponte de Artéria Coronária , Oclusão Coronária/cirurgia , Implante de Prótese de Valva Cardíaca , Insuficiência da Valva Mitral/cirurgia , Estenose da Valva Mitral/cirurgia , Mucopolissacaridose VII/complicações , Adulto , Insuficiência da Valva Aórtica/diagnóstico por imagem , Insuficiência da Valva Aórtica/etiologia , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/etiologia , Oclusão Coronária/diagnóstico por imagem , Oclusão Coronária/etiologia , Humanos , Masculino , Insuficiência da Valva Mitral/diagnóstico por imagem , Insuficiência da Valva Mitral/etiologia , Estenose da Valva Mitral/diagnóstico por imagem , Estenose da Valva Mitral/etiologia , Mucopolissacaridose VII/diagnóstico , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Purpose: Mucopolysaccharidosis (MPS) VII is a genetic, lysosomal storage disease characterized by abnormal accumulation of glycosaminoglycans in cells and tissues. MPS VII patients exhibit multiple failures of endochondral ossification during postnatal growth, including markedly delayed cartilage-to-bone conversion in the vertebrae and long bones. Cartilage canals provide the template for vascularization at the onset of secondary ossification. The objective of this study was to investigate whether abnormal cartilage canal architecture and enzyme-mediated extracellular matrix (ECM) remodeling contribute to delayed cartilage-to-bone conversion in MPS VII.Materials and Methods: The epiphyseal cartilage canal networks of 9-day-old healthy control and MPS VII-affected dog vertebrae were characterized using high-resolution, contrast-free quantitative susceptibility mapping magnetic resonance imaging. Relative expression levels of matrix metalloproteinases (MMPs) 9, 13 and 14 were examined using immunohistochemistry, while tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were examined using in situ enzyme staining.Results: Interestingly, the density, number, connectivity and thickness of cartilage canals was not significantly different between MPS VII and control vertebrae. Immunohistochemistry revealed diminished MMP-9, but normal MMP-13 and 14 expression by epiphyseal cartilage chondrocytes, while ALP and TRAP enzyme expression by chondrocytes and chondroclasts, respectively, were both diminished in MPS VII.Conclusions: Our findings suggest that while the epiphyseal cartilage canal network in MPS VII is normal at the onset of secondary ossification, expression of enzymes required for cartilage resorption and replacement with mineralized ECM, and initiation of angiogenesis, is impaired.
Assuntos
Doenças Ósseas , Mucopolissacaridose VII , Animais , Cães , Matriz Extracelular/patologia , Lâmina de Crescimento , Humanos , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/patologia , OsteogêneseRESUMO
OBJECTIVES: Nonimmune hydrops fetalis (NIHF) accounts for 90% of hydrops fetalis cases. About 15% to 29% of unexplained NIHF cases are caused by lysosomal storage diseases (LSD). We review the spectrum of LSD and associated clinical findings in NIHF in a cohort of patients referred to our institution. METHODS: We present a retrospective case-control study of cases with NIHF referred for LSD biochemical testing at a single center. Cases diagnosed with LSD were matched to controls with NIHF and negative LSD testing and analyzed according to the STROBE criteria to the extent the retrospective nature of this study allowed. RESULTS: Between January 2006 and December 2018, 28 patients with NIHF were diagnosed with a LSD. Eight types of LSD were diagnosed: galactosialidosis 8/28 (28.6%), sialic acid storage disease (SASD) 5/28 (17.9%), mucopolysaccharidosis VII 5/28 (17.9%), Gaucher 4/28 (14.3%), sialidosis 2/28 (7.1%), GM1 gangliosidosis 2/28 (7.1%), Niemann-Pick disease type C 1/28 (3.6%), and mucolipidosis II/III 1/28 (3.6%). Associated clinical features were hepatomegaly 16/21 (76.2%) vs 22/65 (33.8%), P < .05, splenomegaly 12/20 (60.0%) vs 14/58 (24.1%), P < .05, and hepatosplenomegaly 10/20 (50.0%) vs 13/58 (22.4%) P < .05. CONCLUSION: The most common LSD in NIHF were galactosialidosis, SASD, mucopolysaccharidosis VII, and Gaucher disease. LSD should be considered in unexplained NIHF cases, particularly if hepatomegaly, splenomegaly, or hepatosplenomegaly is visualized on prenatal ultrasound.
Assuntos
Hidropisia Fetal/etiologia , Doenças por Armazenamento dos Lisossomos/complicações , Adulto , Ascite/diagnóstico por imagem , Estudos de Casos e Controles , Edema/diagnóstico por imagem , Feminino , Doença de Gaucher/complicações , Doença de Gaucher/diagnóstico , Idade Gestacional , Hepatomegalia/diagnóstico por imagem , Humanos , Hidropisia Fetal/diagnóstico por imagem , Recém-Nascido , Doenças por Armazenamento dos Lisossomos/diagnóstico , Masculino , Mucolipidoses/complicações , Mucolipidoses/diagnóstico , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/diagnóstico , Doença de Niemann-Pick Tipo C/complicações , Doença de Niemann-Pick Tipo C/diagnóstico , Derrame Pericárdico/diagnóstico por imagem , Derrame Pleural/diagnóstico por imagem , Poli-Hidrâmnios/diagnóstico por imagem , Gravidez , Diagnóstico Pré-Natal , Estudos Retrospectivos , Doença do Armazenamento de Ácido Siálico/complicações , Doença do Armazenamento de Ácido Siálico/diagnóstico , Pele/diagnóstico por imagem , Esplenomegalia/diagnóstico por imagem , Adulto JovemRESUMO
Growth impairment in mucopolysaccharidoses (MPSs) is an unresolved issue as it is resistant to enzyme replacement therapy (ERT) and growth hormone therapy. C-type natriuretic peptide (CNP) is a promising agent that has growth-promoting effects. Here we investigate the effects of CNP on growth impairment of MPSs using Gusbmps-2J mice, a model for MPS type VII, with combination therapy of CNP and ERT by hydrodynamic gene delivery. Although monotherapies were not sufficient to restore short statures of treated mice, combination therapy resulted in successful restoration. The synergistic effects of CNP and ERT were not only observed in skeletal growth but also in growth plates. ERT reduced cell swelling in the resting zone and increased cell number by accelerating proliferation or inhibiting apoptosis. CNP thickened the proliferative and hypertrophic zones. Regarding changes in the bone, ERT restored bone sclerosis through decreased bone formation and increased bone resorption, and CNP did not adversely affect this process. In addition, improvement of joint deformation by ERT was suggested by analyses of joint spaces and articular cartilage. CNP additively provided restoration of the short stature of MPS VII mice in combination with ERT, which improved abnormalities of growth plates and bone metabolism.
Assuntos
Terapia Genética/métodos , Transtornos do Crescimento/terapia , Mucopolissacaridose VII/terapia , Peptídeo Natriurético Tipo C/uso terapêutico , Animais , Cartilagem Articular/anatomia & histologia , Terapia de Reposição de Enzimas , Glucuronidase/genética , Transtornos do Crescimento/etiologia , Lâmina de Crescimento/anatomia & histologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucopolissacaridose VII/complicações , Peptídeo Natriurético Tipo C/genéticaRESUMO
Short stature is a characteristic feature of most of the mucopolysaccharidoses, a group of inherited lysosomal storage disorders caused by a single enzyme deficiency. MPS patients present with progressive skeletal defects from an early age, including short stature due to impaired cartilage-to-bone conversion (endochondral ossification). The aim of this study was to determine which murine MPS model best reproduces the bone length reduction phenotype of human MPS and use this model to determine the earliest developmental stage when disrupted endochondral ossification first appears. Gusmps/mps mice representing severe MPS VII displayed the greatest reduction in bone elongation and were chosen for histopathological analysis. Tibial development was assessed from E12.5 to 6â¯months of age. Chondrocytes in the region of the future primary ossification center became hypertrophic at a similar age to normal in the MPS VII mouse fetus, but a delay in bone deposition was observed with an approximate 1â¯day delay in the formation of the primary ossification centre. Likewise, chondrocytes in the region of the future secondary ossification center also became hypertrophic at the same age as normal in the MPS VII early postnatal mouse. Bone deposition in the secondary ossification centre was delayed by two days in the MPS VII proximal tibia (observed at postnatal day 14 (P14) compared to P12 in normal). The thickness of the tibial growth plate was larger in MPS VII mice from P9 onwards. Abnormal endochondral ossification starts in utero in MPS VII and worsens with age. It is characterized by a normal timeframe for chondrocyte hypertrophy but a delay in the subsequent deposition of bone in both the primary and secondary ossification centres, accompanied by an increase in growth plate thickness. This suggests that the signals for vascular invasion and bone deposition, some of which are derived from hypertrophic chondrocytes, are altered in MPS VII.
Assuntos
Doenças Ósseas/etiologia , Doenças Ósseas/patologia , Modelos Animais de Doenças , Mucopolissacaridose VII/complicações , Osteogênese , Tíbia/patologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Feminino , Masculino , Camundongos , GravidezRESUMO
Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in ß-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII.
Assuntos
Doenças do Sistema Nervoso Central/terapia , Terapia Genética/métodos , Glucuronidase/genética , Mucopolissacaridose VII/terapia , Animais , Animais Recém-Nascidos , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Cães , Vetores Genéticos/administração & dosagem , Glucuronidase/líquido cefalorraquidiano , Glicosaminoglicanos/metabolismo , Injeções Intravenosas , Injeções Espinhais , Masculino , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/genética , Mucopolissacaridose VII/metabolismoRESUMO
Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.
Assuntos
Condrócitos/citologia , Epífises/citologia , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/fisiopatologia , Osteogênese , Animais , Doenças Ósseas/etiologia , Doenças Ósseas/fisiopatologia , Diferenciação Celular , Cães , Glicosaminoglicanos/metabolismo , Humanos , Hipertrofia , Coluna Vertebral/fisiologia , Microtomografia por Raio-XRESUMO
We present the cardiac findings from the autopsy of a 28-year-old male with mucopolysaccharidosis VII (MPS VII), also known as Sly Syndrome, whose diagnosis was confirmed by biochemical testing. The patient died a sudden cardiac death. Autopsy showed thickened and stenotic aortic valve leaflets as well as marked concentric intimal thickening of the aorta and muscular arteries. There was left ventricular hypertrophy as well as mild papillary muscle thickening and fusion. Increased colloid iron staining was seen in the small- and medium-sized arteries of the heart and at the intercalated discs. We discuss the patient's premortem echocardiographic and electrocardiographic studies. In addition, we discuss the pathogenesis of MPS VII and review previous literature on its anatomic and pathologic features.
Assuntos
Aorta/patologia , Valvas Cardíacas/patologia , Mucopolissacaridose VII/patologia , Miocárdio/patologia , Adulto , Autopsia , Morte Súbita Cardíaca/etiologia , Morte Súbita Cardíaca/patologia , Humanos , Masculino , Mucopolissacaridose VII/complicaçõesAssuntos
Histiócitos/patologia , Hidropisia Fetal/etiologia , Linfócitos/patologia , Doenças por Armazenamento dos Lisossomos/complicações , Mucopolissacaridose VII/complicações , Vacúolos/patologia , Adulto , Feminino , Humanos , Recém-Nascido , Doenças por Armazenamento dos Lisossomos/patologia , Masculino , Mucopolissacaridose VII/patologia , GravidezRESUMO
BACKGROUND: Mucopolysaccharidoses (MPSs) are rare genetic diseases caused by a deficient activity of one of the lysosomal enzymes involved in the glycosaminoglycan (GAG) breakdown pathway. These metabolic blocks lead to the accumulation of GAGs in various organs and tissues, resulting in a multisystemic clinical picture. The pathological GAG accumulation begins a cascade of interrelated responses: metabolic, inflammatory and immunological with systemic effects. Metabolic inflammation, secondary to GAG storage, is a significant cause of osteoarticular symptoms in MPS disorders. OBJECTIVE AND METHOD: The aim of this review is to present recent progress in the understanding of the role of inflammatory and immune processes in the pathophysiology of osteoarticular symptoms in MPS disorders and potential therapeutic interventions based on published reports in MPS patients and studies in animal models. RESULTS AND CONCLUSIONS: The immune and skeletal systems have a number of shared regulatory molecules and many relationships between bone disorders and aberrant immune responses in MPS can be explained by osteoimmunology. The treatment options currently available are not sufficiently effective in the prevention, inhibition and treatment of osteoarticular symptoms in MPS disease. A lot can be learnt from interactions between skeletal and immune systems in autoimmune diseases such as rheumatoid arthritis (RA) and similarities between RA and MPS point to the possibility of using the experience with RA in the treatment of MPS in the future. The use of different anti-inflammatory drugs requires further study, but it seems to be an important direction for new therapeutic options for MPS patients.
Assuntos
Doenças Ósseas/imunologia , Artropatias/imunologia , Mucopolissacaridoses/imunologia , Doenças Ósseas/etiologia , Doenças Ósseas/metabolismo , Cartilagem Articular/imunologia , Cartilagem Articular/metabolismo , Disostoses/etiologia , Disostoses/imunologia , Disostoses/metabolismo , Glicosaminoglicanos/imunologia , Glicosaminoglicanos/metabolismo , Humanos , Artropatias/etiologia , Artropatias/metabolismo , Mucopolissacaridoses/complicações , Mucopolissacaridoses/metabolismo , Mucopolissacaridose I/complicações , Mucopolissacaridose I/imunologia , Mucopolissacaridose I/metabolismo , Mucopolissacaridose II/complicações , Mucopolissacaridose II/imunologia , Mucopolissacaridose II/metabolismo , Mucopolissacaridose VI/complicações , Mucopolissacaridose VI/imunologia , Mucopolissacaridose VI/metabolismo , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/imunologia , Mucopolissacaridose VII/metabolismo , Sinovite/etiologia , Sinovite/imunologia , Sinovite/metabolismoRESUMO
Mucopolysaccharidosis VII (MPS VII) is due to the deficient activity of ß-glucuronidase (GUSB) and results in the accumulation of glycosaminoglycans (GAGs) in lysosomes and multisystemic disease with cardiovascular manifestations. The goal here was to determine the pathogenesis of mitral valve (MV) disease in MPS VII dogs. Untreated MPS VII dogs had a marked reduction in the histochemical signal for structurally-intact collagen in the MV at 6 months of age, when mitral regurgitation had developed. Electron microscopy demonstrated that collagen fibrils were of normal diameter, but failed to align into large parallel arrays. mRNA analysis demonstrated a modest reduction in the expression of genes that encode collagen or collagen-associated proteins such as the proteoglycan decorin which helps collagen fibrils assemble, and a marked increase for genes that encode proteases such as cathepsins. Indeed, enzyme activity for cathepsin B (CtsB) was 19-fold normal. MPS VII dogs that received neonatal intravenous injection of a gamma retroviral vector had an improved signal for structurally-intact collagen, and reduced CtsB activity relative to that seen in untreated MPS VII dogs. We conclude that MR in untreated MPS VII dogs was likely due to abnormalities in MV collagen structure. This could be due to upregulation of enzymes that degrade collagen or collagen-associated proteins, to the accumulation of GAGs that compete with proteoglycans such as decorin for binding to collagen, or to other causes. Further delineation of the etiology of abnormal collagen structure may lead to treatments that improve biomechanical properties of the MV and other tissues.
Assuntos
Doenças das Valvas Cardíacas/etiologia , Valva Mitral/patologia , Mucopolissacaridose VII/complicações , Animais , Cordas Tendinosas/metabolismo , Colágeno/metabolismo , Cães , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Doenças das Valvas Cardíacas/metabolismo , Masculino , Valva Mitral/metabolismo , Mucopolissacaridose VII/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Transdução de SinaisRESUMO
Mucopolysaccharidosis VII (MPS VII) is due to deficient activity of the lysosomal enzyme ß-glucuronidase (GUSB) and results in the accumulation of glycosaminoglycans (GAGs). This study determined the long-term effect of neonatal intravenous injection of a gamma retroviral vector (RV) on cardiac valve disease in MPS VII dogs. Transduced hepatocytes secreted GUSB into the blood for up to 11 years at levels similar to or greater than those achieved with enzyme replacement therapy (ERT). Valve regurgitation and thickening were scored from 0 (normal) to +4 (severely abnormal). At 1 year, untreated MPS VII dogs had mitral regurgitation, mitral valve thickening, aortic regurgitation, and aortic valve thickening scores of 2.3 ± 0.7, 2.3 ± 0.6, 1.8 ± 0.5, and 1.6 ± 0.7, respectively, which were higher than the values of 0.6 ± 0.1, 0.1 ± 0.4, 0.3 ± 0.8, and 0.1 ± 0.4, respectively, in treated MPS VII dogs. Treated MPS VII dogs maintained low aortic regurgitation and aortic valve thickening scores in their lifetime. Although mitral regurgitation and mitral valve thickening scores increased to 2.0 at ≥ 8 years of age in the treated MPS VII dogs, older normal dogs from the colony had similar scores, making it difficult to assess mitral valve disease. Older treated dogs had calcification within the mitral and the aortic valve annulus, while GUSB staining demonstrated enzyme activity within the mitral valve. We conclude that neonatal RV-mediated gene therapy reduced cardiac valve disease in MPS VII dogs for up to 11 years, and propose that neonatal initiation of ERT should have a similar effect.
Assuntos
Gammaretrovirus/genética , Terapia Genética , Vetores Genéticos/genética , Doenças das Valvas Cardíacas/etiologia , Doenças das Valvas Cardíacas/terapia , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/genética , Animais , Animais Recém-Nascidos , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/patologia , Modelos Animais de Doenças , Cães , Ecocardiografia , Feminino , Glucuronidase/genética , Glucuronidase/metabolismo , Doenças das Valvas Cardíacas/diagnóstico por imagem , Masculino , Valva Mitral/diagnóstico por imagem , Valva Mitral/patologiaRESUMO
Recombinant adeno-associated viruses (rAAVs) have been tested in humans and other large mammals without adverse events. However, one study of mucopolysaccharidosis VII correction in mice showed repeated integration of rAAV in cells from hepatocellular carcinoma (HCC) in the Dlk1-Dio3 locus, suggesting possible insertional mutagenesis. In contrast, another study found no association of rAAV integration with HCC, raising questions about the generality of associations between liver transformation and integration at Dlk1-Dio3. Here we report that in rAAV-treated ornithine transcarbamylase (Otc)-deficient mice, four examples of integration sites in Dlk1-Dio3 could be detected in specimens from liver nodule/tumors, confirming previous studies of rAAV integration in the Dlk1-Dio3 locus in the setting of another murine model of metabolic disease. In one case, the integrated vector was verified to be present at about one copy per cell, consistent with clonal expansion. Another verified integration site in liver nodule/tumor tissue near the Tax1bp1 gene was also detected at about one copy per cell. The Dlk1-Dio3 region has also been implicated in human HCC and so warrants careful monitoring in ongoing human clinical trials with rAAV vectors.
Assuntos
Carcinoma Hepatocelular/genética , Dependovirus/genética , Vetores Genéticos/efeitos adversos , Mucopolissacaridose VII/terapia , Ornitina Carbamoiltransferase/uso terapêutico , Proteínas de Ligação ao Cálcio , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/virologia , Ensaios Clínicos como Assunto , Terapia Genética/efeitos adversos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Iodeto Peroxidase/genética , Fígado/patologia , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/genética , Ornitina Carbamoiltransferase/efeitos dos fármacos , Ornitina Carbamoiltransferase/genética , Integração Viral/genéticaRESUMO
Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme ß-glucuronidase (GUS), leading to accumulation of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) effectively clears GAG storage in the viscera. Recent studies showed that a chemically modified form of GUS (PerT-GUS), which escaped clearance by mannose 6-phosphate and mannose receptors and showed prolonged circulation, reduced CNS storage more effectively than native GUS. Clearance of storage in bone has been limited due to the avascularity of the growth plate. To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treated mice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement.
Assuntos
Doenças Ósseas/etiologia , Doenças Ósseas/terapia , Terapia de Reposição de Enzimas , Glucuronidase/uso terapêutico , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/terapia , Proteínas Recombinantes/uso terapêutico , Animais , Doenças Ósseas/diagnóstico , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Vértebras Cervicais/diagnóstico por imagem , Vértebras Cervicais/patologia , Glucuronidase/administração & dosagem , Glucuronidase/química , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/patologia , Camundongos , Mucopolissacaridose VII/diagnóstico , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Tomografia Computadorizada por Raios XRESUMO
Hundreds of different human skeletal disorders have been characterized at molecular level and a growing number of resembling dysplasias with orthologous genetic defects are being reported in dogs. This study describes a novel genetic defect in the Brazilian Terrier breed causing a congenital skeletal dysplasia. Affected puppies presented severe skeletal deformities observable within the first month of life. Clinical characterization using radiographic and histological methods identified delayed ossification and spondyloepiphyseal dysplasia. Pedigree analysis suggested an autosomal recessive disorder, and we performed a genome-wide association study to map the disease locus using Illumina's 22K SNP chip arrays in seven cases and eleven controls. A single association was observed near the centromeric end of chromosome 6 with a genome-wide significance after permutation (p(genome)=â0.033). The affected dogs shared a 13-Mb homozygous region including over 200 genes. A targeted next-generation sequencing of the entire locus revealed a fully segregating missense mutation (c.866C>T) causing a pathogenic p.P289L change in a conserved functional domain of ß-glucuronidase (GUSB). The mutation was confirmed in a population of 202 Brazilian terriers (pâ=â7,71×10(-29)). GUSB defects cause mucopolysaccharidosis VII (MPS VII) in several species and define the skeletal syndrome in Brazilian Terriers. Our results provide new information about the correlation of the GUSB genotype to phenotype and establish a novel canine model for MPS VII. Currently, MPS VII lacks an efficient treatment and this model could be utilized for the development and validation of therapeutic methods for better treatment of MPS VII patients. Finally, since almost one third of the Brazilian terrier population carries the mutation, breeders will benefit from a genetic test to eradicate the detrimental disease from the breed.
Assuntos
Osso e Ossos/anormalidades , Glucuronidase/genética , Mucopolissacaridose VII/enzimologia , Mucopolissacaridose VII/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos de Mamíferos/genética , Cães , Nanismo/complicações , Feminino , Testes Genéticos , Estudo de Associação Genômica Ampla , Glucuronidase/química , Glucuronidase/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Dados de Sequência Molecular , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/patologia , Osteocondrodisplasias/complicações , Osteogênese/genéticaRESUMO
Mucopolysaccharidosis type VII (MPS VII) is characterized by deficient ß-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan and dermatan sulfate glycosaminoglycans (GAGs), and multisystemic disease. MPS VII patients can develop kypho-scoliotic deformity and spinal cord compression due to disease of intervertebral disks, vertebral bodies, and associated tissues. We have previously demonstrated in MPS VII dogs that intervertebral disks degenerate, vertebral bodies have irregular surfaces, and vertebral body epiphyses have reduced calcification, but the pathophysiological mechanisms underlying these changes are unclear. We hypothesized that some of these manifestations could be due to upregulation of destructive proteases, possibly via the binding of GAGs to Toll-like receptor 4 (TLR4), as has been proposed for other tissues in MPS models. In this study, the annulus fibrosus of the intervertebral disk of 6-month-old MPS VII dogs had cathepsin B and K activities that were 117- and 2-fold normal, respectively, which were associated with elevations in mRNA levels for these cathepsins as well as TLR4. The epiphyses of MPS VII dogs had a marked elevation in mRNA for the cartilage-associated gene collagen II, consistent with a developmental delay in the conversion of the cartilage to bone in this region. The spine obtained at autopsy from a young man with MPS VII exhibited similar increased cartilage in the vertebral bodies adjacent to the end plates, disorganization of the intervertebral disks, and irregular vertebral end plate morphology. These data suggest that the pathogenesis of destructive changes in the spine in MPS VII may involve upregulation of cathepsins. Inhibition of destructive proteases, such as cathepsins, might reduce spine disease in patients with MPS VII or related disorders.
Assuntos
Vértebras Lombares , Mucopolissacaridose VII/complicações , Doenças da Coluna Vertebral/etiologia , Animais , Animais Geneticamente Modificados , Catepsinas/genética , Catepsinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Masculino , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radiografia , Doenças da Coluna Vertebral/diagnóstico por imagem , Doenças da Coluna Vertebral/patologiaRESUMO
Mucopolysaccharidosis VII (MPS VII) is due to deficient ß-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan, and dermatan sulfate glycosaminoglycans in various tissues including those of the spine. Associated spine disease can be due to abnormalities in the vertebrae, the intervertebral disks, or other spine tissues. The goal of this study was to determine if neonatal gene therapy could prevent lumbar spine disease in MPS VII dogs. MPS VII dogs were injected intravenously with a retroviral vector (RV) expressing canine GUSB at 2 to 3 days after birth, which resulted in transduction of hepatocytes that secreted GUSB into blood. Expression was stable for up to 11 years, and mean survival was increased from 0.4 years in untreated dogs to 6.1 years in treated dogs. Despite a profound positive clinical effect, 6-month-old RV-treated MPS VII dogs still had hypoplastic ventral epiphyses with reduced calcification in the lumbar spine, which resulted in a reduced stiffness and increased range of motion that were not improved relative to untreated MPS VII dogs. At six to 11 years of age, ventral vertebrae remained hypoplastic in RV-treated MPS VII dogs, and there was desiccation of the nucleus pulposus in some disks. Histochemical staining demonstrated that disks did not have detectable GUSB activity despite high serum GUSB activity, which is likely due to poor diffusion into this relatively avascular structure. Thus, neonatal gene therapy cannot prevent lumbar spine disease in MPS VII dogs, which predicts that enzyme replacement therapy (ERT) will similarly be relatively ineffective even if started at birth.
Assuntos
Terapia Genética , Vértebras Lombares , Mucopolissacaridose VII/complicações , Mucopolissacaridose VII/terapia , Doenças da Coluna Vertebral/etiologia , Doenças da Coluna Vertebral/terapia , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Fenômenos Biomecânicos , Cálcio/metabolismo , Cães , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glucuronidase/sangue , Glicosaminoglicanos/urina , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/metabolismo , Vértebras Lombares/patologia , Masculino , Mucopolissacaridose VII/mortalidade , Radiografia , Retroviridae/genética , Doenças da Coluna Vertebral/diagnósticoRESUMO
Mucopolysaccharidosis VII (MPS VII) is due to mutations within the gene encoding the lysosomal enzyme ß-glucuronidase, and results in the accumulation of glycosaminoglycans. MPS VII causes aortic dilatation and elastin fragmentation, which is associated with upregulation of the elastases cathepsin S (CtsS) and matrix metalloproteinase 12 (MMP12). To test the role of these enzymes, MPS VII mice were crossed with mice deficient in CtsS or MMP12, and the effect upon aortic dilatation was determined. CtsS deficiency did not protect against aortic dilatation in MPS VII mice, but also failed to prevent an upregulation of cathepsin enzyme activity. Further analysis with substrates and inhibitors specific for particular cathepsins suggests that this enzyme activity was due to CtsB, which could contribute to elastin fragmentation. Similarly, MMP12 deficiency and deficiency of both MMP12 and CtsS could not prevent aortic dilatation in MPS VII mice. Microarray and reverse-transcriptase real-time PCR were performed to look for upregulation of other elastases. This demonstrated that mRNA for complement component D was elevated in MPS VII mice, while immunostaining demonstrated high levels of complement component C3 on surfaces within the aortic media. Finally, we demonstrate that neonatal intravenous injection of a retroviral vector encoding ß-glucuronidase reduced aortic dilatation. We conclude that neither CtsS nor MMP12 are necessary for elastin fragmentation in MPS VII mouse aorta, and propose that CtsB and/or complement component D may be involved. Complement may be activated by the GAGs that accumulate, and may play a role in signal transduction pathways that upregulate elastases.