Expression of GFP beyond the final extent of the retinal detachment.

<p>Dog 1 OD – this eye received an injection of AAV2(quadY-F). A. Color fundus image following retinal reattachment after the subretinal injection. The final extent of the detachment can be seen as a color change in the tapetal fundus as indicated by arrows. B. The same eye imaged to show GFP expression. C. An overlay of the fundus images in A and B. D. An enlarged view of the region in C that is within the white box. The white arrows correspond to the arrows in A. The black arrow heads point out the extent of the GFP expression which extends slightly beyond the final boundary of the retinal detachment.</p

Results of spread of injection bleb and extent of final GFP expression.

*<p>- indicates that bleb contacted edge of ONH following injection.</p><p>Key: NFL = Nerve fiber layer; OD = right eye; OS = left eye; ONH = optic nerve head.</p

A. Low power retinal section from eye 8-OD through the optic nerve (ON).

<p>Green shows GFP expression and blue is DAPI staining. Note that there is GFP expression in axons within the optic nerve and also within the part of the inner retina corresponding to the nerve fiber layer. The section is taken through the dorsal vasculature radiating from the optic nerve head and the blood vessels are the reason that the nerve fiber layer is not a continuous layer in this section. This region is away from the subretinal injection bleb which is why only the nerve fiber layer is expressing GFP and the outer retina is not. Inl = inner nuclear layer; onl = outer nuclear layer. Size bar 100 µm. B. Section through the optic nerve from eye 2-OD. This eye received an injection of AAV2(quadY-F). Green is GFP expression, blue DAPI staining. GFP expression is seen in the axons of the optic nerve. Size bar 250 µm.</p

Typical appearance of white Doberman pinschers.

<p>A group of three white Doberman pinschers demonstrate the typical cream coat-coloration. Note that all three dogs are squinting in the bright sunlight; photophobia was observed in all WDPs examined for this study.</p

Primers for amplification of cDNA in a WDP and a tri-colored dog.

1<p>Amplicons were desgined across exons to ensure no contamination of genomice DNA.</p>2<p>Amplicon size as predicted by the UCSC Genome Browser canine reference genome, CanFam2.0.</p

Ocular phenotype of white Doberman pinschers.

<p>Images taken from WDP (top row) and black standard-color Doberman pinscher (bottom row). An image of WDP head (A) demonstrates lightly-pigmented nose, lips, and eyelid margins compared with the same darkly pigmented structures in SDP (E). A close-up image of WDP eye (B) shows: non-pigmented leading edge of the nictitating membrane (NM), tan-colored iris base transitioning to blue at pupillary margin, and oval-shaped dyscoric pupil aperture. The black arrowheads (in B) demarcate a region of significant iridal stromal thinning that was noted on examination to transilluminate (not shown in image) with retroillumination by light reflected from the tapetum lucidum. SDP eye (F) shows: darkly pigmented margin of the nictitating membrane (NM) and brown iris with a round pupil aperture. WDP gonioscopy image (C), which allows visualization of structures lying within the iridocorneal angle (in images C & G, this region lies between the words “LIMBUS” and “IRIS”) shows fibers of the pectinate ligament (demarcated by black arrowheads) are of a similar tan-color to the iris base, whereas fibers of the pectinate ligament (demarcated by white arrowheads) are dark brown in SDP (G). WDP fundus image (D) shows yellow-colored tapetum lucidum (labeled “TAPETUM”) and significant hypopigmentation of the retinal pigment epithelium and choroid allowing visualization of the choroidal vasculature. SDP fundus image (H) shows green-colored tapetum lucidum (labeled “TAPETUM”) and heavy pigmentation of the non-tapetal fundus. For orientation purposes, images taken at higher magnification (B–D and F–H) have the superior (S) and inferior (I) globe positions labeled.</p

Cutaneous and ocular lesions of white Doberman pinschers.

<p>A close-up image of WDP eye (A) shows multiple pigmented nevus-like lesions on the eyelids and iris. A close-up image of another WDP eye (B) shows a large, lobulated, pigmented eyelid mass. Image of the dorsum of a WDP (C) shows numerous pigmented nevus-like lesions. Ventrum of the same WDP (D) shows a large, pedunculated, pigmented cutaneous mass. Image taken through a 4× objective microscope lens of an hematoxylin and eosin stained histopathology section of cutaneous tissue from WDP (E); note the infiltration of poorly-pigmented cells in small sheets pushed between collagen fibers and aggregated clusters within and abutting the epithelium. Immunohistochemistry with primary antibody recognizing Melan-A (melanocyte marker) from the same cutaneous biopsy sample (F); note strong labeling of the subepithelial cellular infiltrates, indicating that these poorly pigmented cells are melanocytes. Image taken through a 40× objective microscope lens of an hematoxylin and eosin stained histopathology section of a dermal mass from WDP (G) demonstrates cellular morphology of the atypical melanocytes. Although some intracellular pigment is present in (G), it is relatively sparse compared to an image taken through a 40× objective microscope lens of an hematoxylin and eosin stained histopathology section from a representative canine dermal melanoma (H) submitted to the Diagnostic Center for Population and Animal Health at Michigan State University. The amount of pigment present in (H) is considered typical for canine dermal melanoma/melanocytoma; the relative absence of pigmentation noted comparing image (G) to image (H) demonstrates why multiple examining pathologists characterized the lesions submitted from WDP as “amelanotic”. Size bars 100 um.</p

Deletion identified that includes part of exon 7 of <i>SLC45A2</i> in white Doberman pinschers.

<p>A 4,081 base pair deletion was identified in white Doberman pinschers (WDPs). The deletion occurs between chr4∶77,062,970 and chr4∶77,067,051 (UCSC Genome Browser, CanFam2.0). (A) <i>SLC45A2</i> canine exons (light blue boxes) and WDP sequenced exons (black boxes). Note that a partial deletion of exon 7 (the last exon) is seen in WDPs. The yellow box in (A) identifies the downstream break point of the WDP deletion; the WDP deletion ends before the human gene <i>RXFP3</i>. The yellow box (A) corresponds to the yellow box in (B) for WDPs. The deletion is marked with a purple box in (A) and corresponds to the beginning of the normal sequence seen in (B) for SDPs.</p

Single nucleotide variant locations in genomic WDP DNA.

1<p>Gene location is based on the UCSC Genome Browser canine reference genome, CanFam2.0.</p>2<p>Reference allele from UCSC Genome Browser canine reference genome, CanFam2.0.</p>3<p>Variant allele from sequenced gDNA of WDP.</p>4<p>Protein change due to the SNV change in WDP.</p>5<p>SNP number from Broad Instituse SNP collection (<a href="http://www.broadinstitute.org/mammals/dog/snp2" target="_blank">http://www.broadinstitute.org/mammals/dog/snp2</a>).</p

cDNA PCR-amplicons for <i>SLC45A2, ACTB</i> and <i>TYR</i> shows extremely low or absent <i>SLC45A2</i> transcript in white Doberman pinscher skin sample.

<p>cDNA was made from mRNA extracted from skin samples for lanes 1, WDP; 2, white skin from a tri-colored dog; and 3, pigmented skin from a tri-colored dog. Each lane 4 is a water blank used as a negative control. (A) <i>SLC45A2</i> product from exon 2–4, i.e. upstream of the deletion start site, (B) <i>SLC45A2</i> product from exon 6–7, i.e. bridging the deletion start site, (C) <i>ACTB</i> used for mRNA integrity control and (D) <i>TYR</i> used for mRNA integrity control as well as to show proof of concept of this assay. The two <i>SLC45A2</i> products are undetectable in the WDP sample, while the <i>ACTB</i> and <i>TYR</i> products are present with bands at similar intensity to controls. The white skin from a tri-colored dog showed undetectable <i>TYR</i> product but a similar band intensity from the pigmented skin for all other products. The L lane shows a 100 bp ladder (New England Biolabs, Ipswich, MA, USA).</p