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

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

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

Lack of exclusion of <i>SLC45A2</i> with white Doberman pinschers as assessed by genotyping microsatellites by agarose gel electrophoresis.

<p>2.0% agarose gel comparisons of (A) <i>SLC45A2</i> microsatellite marker 1 (192 kb upstream of <i>SLC45A2</i>), white Doberman pinschers (WDPs, left hand portions of panel A) and standard color Doberman pinschers (SDPs, right hand portion of panel A) and (B) <i>SLC45A2</i> microsatellite marker 2 (46 kb downstream of <i>SLC45A2</i>), for WDPs and SDPs. Note the high variability in the markers in the SDPs compared with the almost complete lack of variation seen for WDPs (the extra band seen in lane 1 is a heteroduplex band caused by a one-step tetranucleotide repeat mutation [see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092127#pone.0092127.s006" target="_blank">Table S2</a>]). Lanes for the two gels in each panel: L, 100 bp DNA ladder (New England Biolabs, Inc.), WDP lanes 1–14, WDP samples 1–14; SDP lanes: L, 100 bp DNA ladder, lanes 1–12, SDP samples 1–12. Samples in these gels correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092127#pone.0092127.s007" target="_blank">Table S3</a>, which contains the high resolution genotyping data.</p

Primers used for exonic sequencing of <i>SLC45A2</i>.

1<p>Exon 1 was too large to sequence as one product so two products were used to cover the region (Exon1–1 and 1–2). The reverse primer for sequencing Exon 7 differs between WDPs and SDPs due to the deletion.</p>2<p>Amplicon size as predicted by the UCSC Genome Browser canine reference genome, CanFam2.0.</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

A Partial Gene Deletion of <i>SLC45A2</i> Causes Oculocutaneous Albinism in Doberman Pinscher Dogs

<div><p>The first white Doberman pinscher (WDP) dog was registered by the American Kennel Club in 1976. The novelty of the white coat color resulted in extensive line breeding of this dog and her offspring. The WDP phenotype closely resembles human oculocutaneous albinism (OCA) and clinicians noticed a seemingly high prevalence of pigmented masses on these dogs. This study had three specific aims: (1) produce a detailed description of the ocular phenotype of WDPs, (2) objectively determine if an increased prevalence of ocular and cutaneous melanocytic tumors was present in WDPs, and (3) determine if a genetic mutation in any of the genes known to cause human OCA is causal for the WDP phenotype. WDPs have a consistent ocular phenotype of photophobia, hypopigmented adnexal structures, blue irides with a tan periphery and hypopigmented retinal pigment epithelium and choroid. WDPs have a higher prevalence of cutaneous melanocytic neoplasms compared with control standard color Doberman pinschers (SDPs); cutaneous tumors were noted in 12/20 WDP (<5 years of age: 4/12; >5 years of age: 8/8) and 1/20 SDPs (<i>p</i><0.00001). Using exclusion analysis, four OCA causative genes were investigated for their association with WDP phenotype; <i>TYR, OCA2, TYRP1</i> and <i>SLC45A2. SLC45A2</i> was found to be linked to the phenotype and gene sequencing revealed a 4,081 base pair deletion resulting in loss of the terminus of exon seven of <i>SLC45A2</i> (chr4∶77,062,968–77,067,051). This mutation is highly likely to be the cause of the WDP phenotype and is supported by a lack of detectable <i>SLC45A2</i> transcript levels by reverse transcriptase PCR. The WDP provides a valuable model for studying OCA4 visual disturbances and melanocytic neoplasms in a large animal model.</p></div

Markers used in exclusion analysis.

1<p>Gene location and amplicon based on the UCSC Genome Browser canine reference genome, CanFam2.0. Primer sequences available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092127#pone.0092127.s005" target="_blank">Table S1</a>.</p>2<p>Amplicon size as predicted by the UCSC Genome Browser canine reference genome, CanFam2.0.</p>3<p>Location of the marker in respect to the gene. D, downstream of the coding regions, U, upstream of the coding regions and MD, downstream of the middle of the gene, in an intronic region.</p>4<p>Distance from the furthest end of the gene to the beginning of the marker.</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