Arterial pathology in canine mucopolysaccharidosis-I and response to therapy.

Mucopolysaccharidosis-I (MPS-I) is an inherited deficiency of α-L-iduronidase (IdU) that causes lysosomal accumulation of glycosaminoglycans (GAG) in a variety of parenchymal cell types and connective tissues. The fundamental link between genetic mutation and tissue GAG accumulation is clear, but relatively little attention has been given to the morphology or pathogenesis of associated lesions, particularly those affecting the vascular system. The terminal parietal branches of the abdominal aorta were examined from a colony of dogs homozygous (MPS-I affected) or heterozygous (unaffected carrier) for an IdU mutation that eliminated all enzyme activity, and in affected animals treated with human recombinant IdU. High-resolution computed tomography showed that vascular wall thickenings occurred in affected animals near branch points, and associated with low endothelial shear stress. Histologically these asymmetric 'plaques' entailed extensive intimal thickening with disruption of the internal elastic lamina, occluding more than 50% of the vascular lumen in some cases. Immunohistochemistry was used to show that areas of sclerosis contained foamy (GAG laden) macrophages, fibroblasts and smooth muscle cells, with loss of overlying endothelial basement membrane and claudin-5 expression. Lesions contained scattered cells expressing nuclear factor-κβ (p65), increased fibronectin and transforming growth factor β-1 signaling (with nuclear Smad3 accumulation) in comparison to unaffected vessels. Intimal lesion development and morphology was improved by intravenous recombinant enzyme treatment, particularly with immune tolerance to this exogenous protein. The progressive sclerotic vasculopathy of MPS-I shares some morphological and molecular similarities to atherosclerosis, including formation in areas of low shear stress near branch points, and can be reduced or inhibited by intravenous administration of recombinant IdU

Immune-Mediated Inflammation May Contribute to the Pathogenesis of Cardiovascular Disease in Mucopolysaccharidosis Type I.

BackgroundCardiovascular disease, a progressive manifestation of α-L-iduronidase deficiency or mucopolysaccharidosis type I, continues in patients both untreated and treated with hematopoietic stem cell transplantation or intravenous enzyme replacement. Few studies have examined the effects of α-L-iduronidase deficiency and subsequent glycosaminoglycan storage upon arterial gene expression to understand the pathogenesis of cardiovascular disease.MethodsGene expression in carotid artery, ascending, and descending aortas from four non-tolerized, non-enzyme treated 19 month-old mucopolysaccharidosis type I dogs was compared with expression in corresponding vascular segments from three normal, age-matched dogs. Data were analyzed using R and whole genome network correlation analysis, a bias-free method of categorizing expression level and significance into discrete modules. Genes were further categorized based on module-trait relationships. Expression of clusterin, a protein implicated in other etiologies of cardiovascular disease, was assessed in canine and murine mucopolysaccharidosis type I aortas via Western blot and in situ immunohistochemistry.ResultsGene families with more than two-fold, significant increased expression involved lysosomal function, proteasome function, and immune regulation. Significantly downregulated genes were related to cellular adhesion, cytoskeletal elements, and calcium regulation. Clusterin gene overexpression (9-fold) and protein overexpression (1.3 to 1.62-fold) was confirmed and located specifically in arterial plaques of mucopolysaccharidosis-affected dogs and mice.ConclusionsOverexpression of lysosomal and proteasomal-related genes are expected responses to cellular stress induced by lysosomal storage in mucopolysaccharidosis type I. Upregulation of immunity-related genes implicates the potential involvement of glycosaminoglycan-induced inflammation in the pathogenesis of mucopolysaccharidosis-related arterial disease, for which clusterin represents a potential biomarker

The RPGRIP1-deficient dog, a promising canine model for gene therapy

PURPOSE: To evaluate the RPGRIP1-deficient miniature longhaired dachshund (MLHD) dog as a potential candidate for gene therapy. METHODS: Six RPGRIP1-deficient MLHD dogs from our dog colony have been observed for two years using a variety of noninvasive procedures. These included bilateral full-field electroretinograms (ERG) to evaluate retinal function, fundus photographs to evaluate retinal vascularization, and optical coherence tomographs (OCT) to evaluate retinal thickness. We also performed histological examination of hematoxylin- and eosin-stained retinal sections as well as sections labeled in situ by the terminal dUTP nick end labeling (TUNEL) method. RESULTS: ERG findings showed that as early as 2 months of age, cone function was lost while rod function was preserved. However, by 9 months of age, both cone and rod functions could not be detected. Functional visual assessment based on the ability to avoid obstacles showed that vision was retained up to the age of 11 months. Both OCT and histopathology studies revealed a progressive thinning of the outer nuclear layer (ONL) over the first 2 years of age. TUNEL labeling identified apoptotic photoreceptor cell death as the cause of this thinning of the ONL. CONCLUSIONS: A treatment strategy should consist in initiating gene therapy as early as possible after birth to prevent or delay the loss of rod function. In the MLHD, successful subretinal delivery of a therapeutic vector is feasible at 2 months of age and may prevent or delay the loss of rod function

A Mutation in LTBP2 Causes Congenital Glaucoma in Domestic Cats (Felis catus)

The glaucomas are a group of diseases characterized by optic nerve damage that together represent a leading cause of blindness in the human population and in domestic animals. Here we report a mutation in LTBP2 that causes primary congenital glaucoma (PCG) in domestic cats. We identified a spontaneous form of PCG in cats and established a breeding colony segregating for PCG consistent with fully penetrant, autosomal recessive inheritance of the trait. Elevated intraocular pressure, globe enlargement and elongated ciliary processes were consistently observed in all affected cats by 8 weeks of age. Varying degrees of optic nerve damage resulted by 6 months of age. Although subtle lens zonular instability was a common feature in this cohort, pronounced ectopia lentis was identified in less than 10% of cats examined. Thus, glaucoma in this pedigree is attributed to histologically confirmed arrest in the early post-natal development of the aqueous humor outflow pathways in the anterior segment of the eyes of affected animals. Using a candidate gene approach, significant linkage was established on cat chromosome B3 (LOD 18.38, θ = 0.00) using tightly linked short tandem repeat (STR) loci to the candidate gene, LTBP2. A 4 base-pair insertion was identified in exon 8 of LTBP2 in affected individuals that generates a frame shift that completely alters the downstream open reading frame and eliminates functional domains. Thus, we describe the first spontaneous and highly penetrant non-rodent model of PCG identifying a valuable animal model for primary glaucoma that closely resembles the human disease, providing valuable insights into mechanisms underlying the disease and a valuable animal model for testing therapies

Discovery and Use of a Natural Mutation that Results in Severe Combined Immuno Deficiency in Pigs

Piglets from the low residual feed intake (RFI) line at ISU were found to be affected with a lethal autosomal recessive mutation that causes Severe Combined Immunodeficiency (SCID). Bone marrow allotransplantation rescued the immune deficiency in four of nine attempted transfers; the other five exhibited signs of severe graft versus host disease and were euthanized. A genome wide association study identified a 5.6 Mb region that contained the causative mutation. Affected haplotypes were traced back to the founders of the RFI population, who were sourced from the purebred Yorkshire population. The SCID pigs will be useful as a biomedical model, as pigs are anatomically and genetically more similar to humans than SCID mice, which are now widely used. Development of a genetic test for the causative mutation will be valuable to the swine industry, allowing breeders to identify carriers

Central nervous system pathology in preclinical MPS IIIB dogs reveals progressive changes in clinically relevant brain regions

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24-30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model

Early-Onset Progressive Retinal Atrophy Associated with an IQCB1 Variant in African Black-Footed Cats (Felis nigripes)

African black-footed cats (Felis nigripes) are endangered wild felids. One male and full-sibling female African black-footed cat developed vision deficits and mydriasis as early as 3 months of age. The diagnosis of early-onset progressive retinal atrophy (PRA) was supported by reduced direct and consensual pupillary light reflexes, phenotypic presence of retinal degeneration, and a non-recordable electroretinogram with negligible amplitudes in both eyes. Whole genome sequencing, conducted on two unaffected parents and one affected offspring was compared to a variant database from 51 domestic cats and a Pallas cat, revealed 50 candidate variants that segregated concordantly with the PRA phenotype. Testing in additional affected cats confirmed that cats homozygous for a 2 base pair (bp) deletion within IQ calmodulin-binding motif-containing protein-1 (IQCB1), the gene that encodes for nephrocystin-5 (NPHP5), had vision loss. The variant segregated concordantly in other related individuals within the pedigree supporting the identification of a recessively inherited early-onset feline PRA. Analysis of the black-footed cat studbook suggests additional captive cats are at risk. Genetic testing for IQCB1 and avoidance of matings between carriers should be added to the species survival plan for captive management

Therapeutic Neonatal Hepatic Gene Therapy in Mucopolysaccharidosis VII Dogs

Dogs with mucopolysaccharidosis VII (MPS VII) were injected intravenously at 2–3 days of age with a retroviral vector (RV) expressing canine β-glucuronidase (cGUSB). Five animals received RV alone, and two dogs received hepatocyte growth factor (HGF) before RV in an attempt to increase transduction efficiency. Transduced hepatocytes expanded clonally during normal liver growth and secreted enzyme with mannose 6-phosphate. Serum GUSB activity was stable for up to 14 months at normal levels for the RV-treated dogs, and for 17 months at 67-fold normal for the HGF/RV-treated dog. GUSB activity in other organs was 1.5–60% of normal at 6 months for two RV-treated dogs, which was likely because of uptake of enzyme from blood by the mannose 6-phosphate receptor. The body weights of untreated MPS VII dogs are 50% of normal at 6 months. MPS VII dogs cannot walk or stand after 6 months, and progressively develop eye and heart disease. RV- and HGF/RV-treated MPS VII dogs achieved 87% and 84% of normal body weight, respectively. Treated animals could run at all times of evaluation for 6–17 months because of improvements in bone and joint abnormalities, and had little or no corneal clouding and no mitral valve thickening. Despite higher GUSB expression, the clinical improvements in the HGF/RV-treated dog were similar to those in the RV-treated animals. This is the first successful application of gene therapy in preventing the clinical manifestations of a lysosomal storage disease in a large animal

Early-onset progressive retinal atrophy associated with an IQCB1 variant in African black-footed cats (Felis nigripes)

African black-footed cats (Felis nigripes) are endangered wild felids. One male and full-sibling female African black-footed cat developed vision deficits and mydriasis as early as 3 months of age. The diagnosis of early-onset progressive retinal atrophy (PRA) was supported by reduced direct and consensual pupillary light reflexes, phenotypic presence of retinal degeneration, and a non-recordable electroretinogram with negligible amplitudes in both eyes. Whole genome sequencing, conducted on two unaffected parents and one affected offspring was compared to a variant database from 51 domestic cats and a Pallas cat, revealed 50 candidate variants that segregated concordantly with the PRA phenotype. Testing in additional affected cats confirmed that cats homozygous for a 2 base pair (bp) deletion within IQ calmodulin-binding motif-containing protein-1 (IQCB1), the gene that encodes for nephrocystin-5 (NPHP5), had vision loss. The variant segregated concordantly in other related individuals within the pedigree supporting the identification of a recessively inherited early-onset feline PRA. Analysis of the black-footed cat studbook suggests additional captive cats are at risk. Genetic testing for IQCB1 and avoidance of matings between carriers should be added to the species survival plan for captive management