A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB

Retinal examination shows no abnormalities over time.

<p>Fundus photographs of the same dog show normal retinal integrity and vasculature. The tapetal retina (green-yellow color above the optic disc) shows a different color in the photographs due to the different light intensities used. The retina remains normal and unchanged during a 4 year observation period. The fluorescein angiogram (top right) shows normal vascular perfusion during the late arteriolar phase; the venules are just beginning to fill with contrast.</p

Outer retinal immunolabeling in normal, carrier and affected dogs.

<p>Cone arrestin, rod opsin and synaptophysin labeling are similar in the three genotypes. Punctate labeling of OPL synaptic terminals with SNAP-25 and CtBP2 was the same although the affected and carrier retinas showed more dispersed labeling interpreted as being a fixation artifact. Calbindin antibody clearly labels the horizontal cells in the scleral border of the INL. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer, IPL: inner plexiform layer.</p

CSNB colony pedigree.

<p>Three beagle dogs, F5858, M4 and M6233, were used as founders for the CSNB colony that was then expanded by outcrossing, backcrossing and intercrossing. Breeding affected to obligate heterozygous animals resulted in non-affected and affected progeny, while affected to affected matings always produced affected progeny. The disease is inherited as an autosomal recessive trait.</p

Normal retinal structure in CSNB affected dogs.

<p><b>A</b>) Sections from normal, carrier and affected retinas stained with H&E show no structural abnormalities. The images are representative of four normal and two of each carrier and affected dogs. <b>B</b>) Graphical representations of outer (ONL) and inner (INL) nuclear layer thickness in the superior retina expressed as rows of nuclei. Number of nuclei was counted at positions 1000μm apart starting at 1000μm from the ora serrata in 4 normal, 2 carrier and 2 affected retinas. Only three representative positions are shown. <b>C</b>) Rod spherule ultrastructure of affected retina. The pre- and post-synaptic regions are normal. The bars indicate 200nm. RPE: retinal pigment epithelium; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer.</p

Immunofluorescence labeling of selected key proteins and modulators of mGluR6 cascade.

<p>mGluR6 showed punctate labeling at the tips of bipolar cells in the normal and affected retina. GNB3 only labeled the cone photoreceptors in normal and affected dogs; bipolar labeling was negligible as a result of the longer fixation time [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137072#pone.0137072.ref071" target="_blank">71</a>]. GPR179 and TRPM1 showed comparable labeling at the tips of bipolar cells in the normal and affected retinas. RGS7 labeling was comparable in bipolar cells of the normal and affected retinas. Normal retina labeled RGS11 but not the affected one. Goα labeling was increased in affected retina, and bipolar cell dendritic arbors extended into the OPL. PKCα labeling was decreased around the rod bipolar somata, and the axon terminals formed a thicker and intensely labeled layer in IPL. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer, IPL: inner plexiform layer.</p

Scotopic and photopic ERGs show a defect in signal transmission from rods and cones to ON-bipolar cells.

<p>Electroretinograms (ERGs) recorded from CSNB dogs. <b>A</b>) Dark-adapted (scotopic) ERGs elicited by different stimulus intensities. Affected dog shows normal a-wave but loss of the b-wave. <b>B</b>) Light-adapted (photopic) ERGs elicited by different stimulus intensities. Affected dog shows a reduction of the b-wave at higher stimulus intensities of 1.0–1.5 log cd-s/m². <b>C</b>) Photopic long-flash ERG using 200 ms stimuli of 400 cd/m². Affected dog shows reduced ON-response (b-wave), but normal OFF-response (d-wave). <b>D</b>) Standard ERGs recommended by ISCEV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137072#pone.0137072.ref070" target="_blank">70</a>] recorded from the same affected dog at 2 and 7years-of-age showed no progressive ERG changes in the 5 year interval.</p

Haplotype ana lyses of candidate genes in CSNB pedigree.

<p>CSNB informative pedigree and haplotypes for the candidate genes studied. This pedigree is showing a subset of the beagle colony used for molecular genetic study. Phenotypic status was assigned by ERG and vision testing. Haplotypes for those genes with informative SNPs and microsatellites are shown in 5 representative animals out of the 10 samples examined (note that for affected animal 6F2 only <i>TRPM1</i> is illustrated as it shows a different haplotype from the other affecteds in the figure). Only 4 markers for each gene are shown to illustrate exclusion of candidate gene from disease. Markers in bold are intragenic, and flanking markers at either end of the haplotype block are in italics. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137072#pone.0137072.s002" target="_blank">S1 Table</a> for the details of all the markers studied.</p