April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Molecular and immunohistochemical characterization of a canine model of complete congenital stationary night blindness (CSNB)
Author Affiliations & Notes
  • Gautami Das
    School of Vet Medicine, University of Pennsylvania, Philadelphia, PA
  • Keiko Miyadera
    School of Vet Medicine, University of Pennsylvania, Philadelphia, PA
  • Evelyn Santana
    School of Vet Medicine, University of Pennsylvania, Philadelphia, PA
  • Gustavo D Aguirre
    School of Vet Medicine, University of Pennsylvania, Philadelphia, PA
  • Mineo Kondo
    Ophthalmology, Mie University Graduate School of Medicine, Tsu, Japan
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3288. doi:
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      Gautami Das, Keiko Miyadera, Evelyn Santana, Gustavo D Aguirre, Mineo Kondo; Molecular and immunohistochemical characterization of a canine model of complete congenital stationary night blindness (CSNB). Invest. Ophthalmol. Vis. Sci. 2014;55(13):3288.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: CSNB is a clinically and genetically heterogeneous, non-progressive retinal disorder associated with impaired night vision that can be accompanied by nystagmus, myopia and strabismus. It is clinically subdivided into complete and incomplete forms. The majority of the diseases involve defects in signaling from photoreceptors to bipolar cells. The aim of this study is to perform immunohistochemical (IHC) and molecular characterization of an autosomal recessive naturally occurring complete (Schubert-Bornschein type) CSNB in the Beagle dog.

Methods: Disease association of CSNB candidate genes (CABP4, CACNA1F, CACNA2D4, SLC24A1, LRIT3, TRPM1, GPR179, GRM6, NYX, PDE6B, RHO and GNAT1) was done by haplotyping and qRTPCR expression analyses by ddCt method using GAPDH for normalization and an unpaired t-test (p<0.05 and fold change >±2.0). In addition, BHLHE23 (mouse KO resembles human CSNB phenotype) and GNB3 (required for the normal expression of all the important molecules in the mGLUR6 cascade across ON bipolar cells) were also included in both the analyses. Retinal structure and relevant proteins (RHO, hCAR, SNAP25, CtBP2, SYN, Go-alpha, PKC-alpha, GNB3, LRIT3, mGLUR6, TRPM1) were assessed by H&E staining and IHC in affected (n=2) and carrier (n=2), ages=13, 25 mon, and 4 control (n=4; age=8-24 mon) dogs.

Results: Haplotype analysis ruled out the known CSNB candidates in the disease pathogenesis. Results to date show no significant difference in the mRNA expression of the genes analyzed except for CABP4 where a 2.5 fold decreased expression was observed in affecteds compared to controls. There was no difference in structure between normal and mutant retinas. IHC showed a marked decrease in the intensity and localization of some proteins between normal and affected; hCAR expression was reduced in cone pedicles, CtBP2 labeling was less intense in OPL terminals, and TRPM1, mGLUR6 and LRIT3 in bipolar terminals were reduced. In addition, PKC-alpha expression was almost completely absent in both dendrites and synaptic boutons. In general, the carrier retinas showed intermediate labeling pattern although in some cases the results were comparable to the affecteds.

Conclusions: Our results exclude the causal association of known CSNB candidate genes with the disease in the canine model, and suggest the involvement of a yet unknown gene.

Keywords: 689 retina: distal (photoreceptors, horizontal cells, bipolar cells) • 440 candidate gene analysis • 554 immunohistochemistry  
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