Novel Homozygous ADAMTS2 Variants and Associated Disease Phenotypes in Dogs with Dermatosparactic Ehlers-Danlos Syndrome.

Tissue fragility, skin hyperextensibility and joint hypermobility are defining characteristics of Ehlers-Danlos syndrome (EDS). Human EDS is subclassified into fourteen types including dermatosparactic EDS, characterized by extreme skin fragility and caused by biallelic ADAMTS2 mutations. We report two novel, ADAMTS2 variants in DNA from EDS-affected dogs. Separate whole-genome sequences from a Pit Bull Terrier and an Alapaha Blue Blood Bulldog each contained a rare, homozygous variant (11:2280117delC, CanFam3.1), predicted to produce a frameshift in the transcript from the first coding ADAMTS2 exon (c.10delC) and a severely truncated protein product, p.(Pro4ArgfsTer175). The clinical features of these dogs and 4 others with the same homozygous deletion included multifocal wounds, atrophic scars, joint hypermobility, narrowed palpebral fissures, skin hyperextensibility, and joint-associated swellings. Due to severe skin fragility, the owners of all 6 dogs elected euthanasia before the dogs reached 13 weeks of age. Cross sections of collagen fibrils in post-mortem dermal tissues from 2 of these dogs showed hieroglyphic-like figures similar to those from cases of severe dermatosparaxis in other species. The whole-genome sequence from an adult Catahoula Leopard Dog contained a homozygous ADAMTS2 missense mutation, [11:2491238G>A; p.(Arg966His)]. This dog exhibited multifocal wounds, atrophic scars, and joint hypermobility, but has survived for at least 9 years. This report expands the spectrum of clinical features of the canine dermatosparactic subtype of EDS and illustrates the potential utility of subclassifying canine EDS by the identity of gene harboring the causal variant.

Diagnosis and surgical management of a retrobulbar abscess causing unilateral exophthalmos in a Boer goat.

A 10-year-old Boer goat wether presented for unilateral exophthalmos of 2- to 3-week duration. Ocular ultrasonography and computed tomography (CT) were utilized in the diagnosis of the patient's orbital disease and surgical planning. Exenteration was performed under the same general anesthetic event as CT. Cytology, culture, and histopathology were performed after exenteration. Cytology was consistent with a mixed bacterial infection. Culture confirmed the presence of Streptococcus ovis. Histopathology on the enucleated globe and mass revealed no evidence of tumor and confirmed intraocular extension of retrobulbar inflammation. Histopathologic diagnosis was consistent with severe chronic orbital pyogranuloma and fibrinosuppurative endophthalmitis confined to the subretinal space. The abscess recurred in the orbital space 2 weeks postoperatively; the orbit was explored. Repeat culture was consistent with S. ovis, Staphylococcus schleigeri subspecies coagulans, and Fusobacterium necrophorum. Complete resolution was obtained after drainage and lavage of the orbit. Abscess is cited as a cause of exophthalmos in small ruminants, but no individual case reports exist. Advanced imaging allowed presumptive diagnosis and surgical planning. Histopathology confirmed intraocular extension of retrobulbar disease.

Surgical correction of congenital entropion in related Boer goat kids using a combination Hotz-Celsus and lateral eyelid wedge resection procedure.

Five related Boer goat kids (≤4 months of age) were presented to the University of Missouri, Veterinary Teaching Hospital (MU-VMTH) with epiphora and blepharospasm of several weeks duration and commencing prior to 1 month of age in all animals. Clinical examination confirmed euryblepharon and entropion bilaterally in two females and one male and unilaterally in two female kids. Deep stromal corneal ulceration was present in two eyes, and corneal granulation tissue and fibrosis were present in half (5/10) the affected eyes. A combination Hotz-Celsus and lateral eyelid wedge resection procedure was performed on all affected eyelids. Recheck examinations and long-term follow-up confirmed resolution of the entropion, preservation of normal eyelid conformation, and restoration of ocular comfort. Pedigree analysis ruled out sex-linked and autosomal dominant inheritance patterns; a specific mode of inheritance could not be determined. The Boer goat breed may be at increased risk for the development of entropion. This cases series represents the first report of entropion in the caprine species.

Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity.

OBJECTIVE: To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. PROCEDURE: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFß1 to induce myofibroblast formation. The cultures were treated with either 5 µm or 10 µm SAHA for 72 h in the presence of TGFß1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying α-smooth muscle actin (αSMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays. RESULTS: Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGFß1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in αSMA mRNA and 86-88% (P < 0.001) decrease in αSMA+ immunocytochemical staining. SAHA treatment also resulted in 4.5- to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 µm dose; 0.5-fold decrease with 10 µm dose). No change in fibroblast viability was observed with a 5 µm SAHA dose, whereas a 10 µm dose resulted in a moderate 17% decrease in cell viability. CONCLUSIONS: Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGFß-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.

Decorin-PEI nanoconstruct attenuates equine corneal fibroblast differentiation.

OBJECTIVE: To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. PROCEDURE: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduced into ECFs using PEI nanoparticles to confirm gene delivery, then DCN was introduced to evaluate for antifibrotic effects. GFP gene delivery was confirmed with real-time qPCR and ELISA. Changes in fibrosis after DCN therapy were quantified by measuring α-smooth muscle actin (αSMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined by evaluating cell morphology, cellular viability, and TUNEL assay. RESULTS: Polyethylenimine-green fluorescent protein-treated cultures showed 2.2 × 10(4) GFP plasmid copies/µg of cellular DNA and 2.1 pg of GFP/100 µL of lysate. PEI-DCN delivery significantly attenuated TGFß-induced transdifferentiation of fibroblasts to myofibroblasts (2-fold decrease of αSMA mRNA; P = 0.05) and significant inhibition of αSMA (49 ± 14.2%; P < 0.001) in immunocytochemical staining and immunoblotting were found. Furthermore, PEI-DNA nanoparticle delivery did not alter cellular phenotype at 24 h and cellular viability was maintained. CONCLUSIONS: Twenty-two kilo dalton Polyethylenimine nanoparticles are safe and effective for equine corneal gene therapy in vitro. PEI-mediated DCN gene delivery is effective at inhibiting TGFß-mediated fibrosis in this model.