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1.  IQCB1 and PDE6B Mutations Cause Similar Early Onset Retinal Degenerations in Two Closely Related Terrier Dog Breeds 
Purpose.
To identify the causative mutations in two early-onset canine retinal degenerations, crd1 and crd2, segregating in the American Staffordshire terrier and the Pit Bull Terrier breeds, respectively.
Methods.
Retinal morphology of crd1- and crd2-affected dogs was evaluated by light microscopy. DNA was extracted from affected and related unaffected controls. Association analysis was undertaken using the Illumina Canine SNP array and PLINK (crd1 study), or the Affymetrix Version 2 Canine array, the “MAGIC” genotype algorithm, and Fisher's Exact test for association (crd2 study). Positional candidate genes were evaluated for each disease.
Results.
Structural photoreceptor abnormalities were observed in crd1-affected dogs as young as 11-weeks old. Rod and cone inner segment (IS) and outer segments (OS) were abnormal in size, shape, and number. In crd2-affected dogs, rod and cone IS and OS were abnormal as early as 3 weeks of age, progressing with age to severe loss of the OS, and thinning of the outer nuclear layer (ONL) by 12 weeks of age. Genome-wide association study (GWAS) identified association at the telomeric end of CFA3 in crd1-affected dogs and on CFA33 in crd2-affected dogs. Candidate gene evaluation identified a three bases deletion in exon 21 of PDE6B in crd1-affected dogs, and a cytosine insertion in exon 10 of IQCB1 in crd2-affected dogs.
Conclusions.
Identification of the mutations responsible for these two early-onset retinal degenerations provides new large animal models for comparative disease studies and evaluation of potential therapeutic approaches for the homologous human diseases.
We describe two genome-wide association studies in two closely related dog breeds affected with retinal degeneration, the pathology of the diseases and the discovery of a novel deletion mutation in PDE6B and an insertion mutation in IQCB1 as the causality for these diseases.
doi:10.1167/iovs.13-12915
PMCID: PMC3809947  PMID: 24045995
retina; mutation; GWAS
2.  Genetic and phenotypic variations of inherited retinal diseases in dogs: the power of within- and across-breed studies 
Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20 years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision.
doi:10.1007/s00335-011-9361-3
PMCID: PMC3942498  PMID: 22065099
3.  EXONIC SINE INSERTION IN STK38L CAUSES CANINE EARLY RETINAL DEGENERATION (erd) 
Genomics  2010;96(6):362-368.
Fine mapping followed by candidate gene analysis of erd - a canine hereditary retinal degeneration characterized by aberrant photoreceptor development - established that the disease cosegregates with a SINE insertion in exon 4 of the canine STK38L/NDR2 gene. The mutation removes exon 4 from STK38L transcripts and is predicted to remove much of the N-terminus from the translated protein, including binding sites for S100B and Mob Proteins, part of the protein kinase domain, and a Thr-75 residue critical for autophosphorylation. Although known to have roles in neuronal cell function, the STK38L pathway has not previously been implicated in normal or abnormal photoreceptor development. Loss of STK38L function in erd provides novel potential insights into the role of the STK38L pathway in neuronal and photoreceptor cell function, and suggests that genes in this pathway need to be considered as candidate genes for hereditary retinal degenerations.
doi:10.1016/j.ygeno.2010.09.003
PMCID: PMC2996878  PMID: 20887780
Retinal degeneration; Leber Congenital Amaurosis; STK38L; animal model
5.  On the origin of a domesticated species: Identifying the parent population of Russian silver foxes (Vulpes vulpes) 
The foxes at Novosibirsk, Russia, are the only population of domesticated foxes in the world. These domesticated foxes originated from farm-bred silver foxes (Vulpes vulpes), whose genetic source is unknown. In this study we examined the origin of the domesticated strain of foxes and two other farm-bred fox populations (aggressive and unselected) maintained in Novosibirsk. To identify the phylogenetic origin of these populations we sequenced two regions of mtDNA, cytochrome b and D-loop, from 24 Novosibirsk foxes (8 foxes from each population) and compared them with corresponding sequences of native red foxes from Europe, Asia, Alaska and Western Canada, Eastern Canada, and the Western Mountains of the USA. We identified seven cytochrome b - D-loop haplotypes in Novosibirsk populations, four of which were previously observed in Eastern North America. The three remaining haplotypes differed by one or two base change from the most common haplotype in Eastern Canada. ΦST analysis showed significant differentiation between Novosibirsk populations and red fox populations from all geographic regions except Eastern Canada. No haplotypes of Eurasian origin were identified in the Novosibirsk populations. These results are consistent with historical records indicating that the original breeding stock of farm-bred foxes originated from Prince Edward Island, Canada. Mitochondrial DNA data together with historical records indicate two stages in the selection of domesticated foxes: the first includes captive breeding for ~50 years with unconscious selection for behaviour; the second corresponds to over 50 further years of intensive selection for tame behaviour.
doi:10.1111/j.1095-8312.2011.01629.x
PMCID: PMC3101803  PMID: 21625363
domestication; mitochondrial DNA; phylogeography; red fox; tameness
6.  Structural Organization and Expression Pattern of the Canine RPGRIP1 Isoforms in Retinal Tissue 
This study characterized the complete structure of six cRPGRIP1 splicing variants expressed in adult canine retina, identified a novel 5′ and 3′ splicing pattern of the gene, and investigated the expression of the cRPGRIP1 isoforms in four additional canine tissues.
Purpose.
To examine the structure and expression of RPGRIP1 in dog retina.
Methods.
Determination of the structural analysis and expression pattern of canine RPGRIP1 (cRPGRIP1) was based on cDNA amplification. Absolute quantification of the expression level of cRPGRIP1 splice variants was determined by qRT-PCR. Regulatory structures were examined by computational analysis of comparative genomics.
Results.
cRPGRIP1 encompasses 25 exons that harbor a 3627-bp open reading frame (ORF) encoding a 1209-amino-acid (aa)–predicted protein. In addition to the main transcript, five full-length and several partial cRPGRIP1 isoforms were identified revealing four alternative 3′-terminal exons—24, 19a, 19c, and 19d—three of which could potentially produce C-terminally truncated proteins that lack the RPGR-interacting domain. A complex organization of the 5′-UTR for the cRPGRIP1 splice products have been described, with a common promoter driving multiple isoforms, including four full-length transcripts using the 3′-terminal exon 24. In addition, a potential alternative internal promoter was revealed to initiate at least two cRPGRIP1 splice variants sharing the same 3′-terminal exon 19c. Transcription initiation sites were highly supported by conserved arrangements of cis-elements predicted in a bioinformatic analysis of orthologous RPGRIP1 promoter regions.
Conclusions.
The use of alternative transcription start and termination sites results in substantial heterogeneity of cRPGRIP1 transcripts, many of which are likely to have tissue-specific expression. The identified exon–intron structure of cRPGRIP1 isoforms provides a basis for evaluating the gene defects underlying inherited retinal disorders in dogs.
doi:10.1167/iovs.10-6094
PMCID: PMC3109012  PMID: 21282582
7.  COL9A2 and COL9A3 mutations in canine autosomal recessive Oculo-skeletal Dysplasia 
Oculo-skeletal dysplasia segregates in two canine breeds, the Labrador retriever and samoyed, in which the causative loci have been termed drd1 and drd2, respectively. Affected dogs exhibit short-limbed dwarfism together with severe ocular defects, and this phenotype is inherited as an autosomal recessive trait in both breeds. The clinical and pathological appearance resembles human hereditary arthro-ophthalmopathies such as Stickler syndrome, or Marshall Syndrome, although these human disorders are usually dominant. Linkage studies in drd1-informative pedigrees mapped the locus to canine chromosome 24, and led to the identification of an insertional mutation in exon 1 of the gene COL9A3 that cosegregates with the disease. The drd2 locus was similarly mapped to canine chromosome 15 and shown to cosegregate with a 1,267 bp deletion mutation in the 5′ end of COL9A2. Both mutations affect the COL3 domain of the respective gene. Northern analysis showed reduced RNA expression in affected retina compared to normal. These models offer potential for studies such as protein-protein interactions between different members of the collagen gene family; regulation and expression of these genes in retina and cartilage, and even opportunities for gene therapy.
doi:10.1007/s00335-010-9276-4
PMCID: PMC2954766  PMID: 20686772
8.  Gene therapy rescues cone function in congenital achromatopsia 
Human Molecular Genetics  2010;19(13):2581-2593.
The successful restoration of visual function with recombinant adeno-associated virus (rAAV)-mediated gene replacement therapy in animals and humans with an inherited disease of the retinal pigment epithelium has ushered in a new era of retinal therapeutics. For many retinal disorders, however, targeting of therapeutic vectors to mutant rods and/or cones will be required. In this study, the primary cone photoreceptor disorder achromatopsia served as the ideal translational model to develop gene therapy directed to cone photoreceptors. We demonstrate that rAAV-mediated gene replacement therapy with different forms of the human red cone opsin promoter led to the restoration of cone function and day vision in two canine models of CNGB3 achromatopsia, a neuronal channelopathy that is the most common form of achromatopsia in man. The robustness and stability of the observed treatment effect was mutation independent, but promoter and age dependent. Subretinal administration of rAAV5–hCNGB3 with a long version of the red cone opsin promoter in younger animals led to a stable therapeutic effect for at least 33 months. Our results hold promise for future clinical trials of cone-directed gene therapy in achromatopsia and other cone-specific disorders.
doi:10.1093/hmg/ddq136
PMCID: PMC2883338  PMID: 20378608
9.  Mapping loci for fox domestication: deconstruction/reconstruction of a behavioral phenotype 
Behavior genetics  2010;41(4):593-606.
During the second part of the 20th century, Belyaev selected tame and aggressive foxes (Vulpes vulpes), in an effort known as the “farm-fox experiment”, to recapitulate the process of animal domestication. Using these tame and aggressive foxes as founders of segregant backcross and intercross populations we have employed interval mapping to identify a locus for tame behavior on fox chromosome VVU12. This locus is orthologous to, and therefore validates, a genomic region recently implicated in canine domestication. The tame versus aggressive behavioral phenotype was characterized as the first principal component (PC) of a PC matrix made up of many distinct behavioral traits (e.g. wags tail; comes to the front of the cage; allows head to be touched; holds observer’s hand with its mouth; etc.). Mean values of this PC for F1, backcross and intercross populations defined a linear gradient of heritable behavior ranging from tame to aggressive. The second PC did not follow such a gradient, but also mapped to VVU12, and distinguished between active and passive behaviors. These data suggest that 1) there are at least two VVU12 loci associated with behavior; 2) expression of these loci is dependent on interactions with other parts of the genome (the genome context) and therefore varies from one crossbred population to another depending on the individual parents that participated in the cross.
doi:10.1007/s10519-010-9418-1
PMCID: PMC3076541  PMID: 21153916
behavior genetics; domestication; social behavior; Vulpes vulpes; Canis familiaris
10.  Transcriptional Profile Analysis of RPGRORF15 Frameshift Mutation Identifies Novel Genes Associated with Retinal Degeneration 
In this study, several genes were identified that had differential expression in mutant retinas that are directly or indirectly active in nonclassic apoptotic processes or are related to mitochondrial functions. The findings indicate that these organelles may play a relevant role in disease progression.
Purpose.
To identify genes and molecular mechanisms associated with photoreceptor degeneration in a canine model of XLRP caused by an RPGR exon ORF15 microdeletion.
Methods.
Expression profiles of mutant and normal retinas were compared by using canine retinal custom cDNA microarrays. qRT-PCR, Western blot analysis, and immunohistochemistry (IHC) were applied to selected genes, to confirm and expand the microarray results.
Results.
At 7 and 16 weeks, respectively, 56 and 18 transcripts were downregulated in the mutant retinas, but none were differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct pathways. Downregulated genes included the known retina-relevant genes PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, the DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. qRT-PCR of 18 genes confirmed the microarray results and showed DE of additional genes not on the array. Only GFAP was DE at 3 weeks of age. Western blot and IHC analyses also confirmed the high reliability of the transcriptomic data.
Conclusions.
Several DE genes were identified in mutant retinas. At 7 weeks, a combination of nonclassic anti- and proapoptosis genes appear to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks, the expression of mitochondria-related genes indicates that they may play a relevant role in the disease process.
doi:10.1167/iovs.10-5443
PMCID: PMC3061521  PMID: 20574030
11.  CREB1/ATF1 Activation in Photoreceptor Degeneration and Protection 
The CREB1/ATF1 pathway is activated in canine and human rods and cones undergoing degeneration. The pathway is also activated by exposure to the neuroprotective agent CNTF. These data suggest that CREB1/ATF1 contributes to an innate protective response and is of potential therapeutic value in the treatment of RP and AMD.
Purpose.
The cAMP response element binding protein 1 (CREB1) and activating transcription factor 1 (ATF1) are closely related members of the bZIP superfamily of transcription factors. Both are activated in response to a wide array of stimuli, including cellular stress. This study was conducted to assess the CREB1/ATF1 pathway in photoreceptor disease and protection.
Methods.
The expression levels of p-CREB1, CREB1, and ATF1 were examined by immunoblot and immunohistochemistry in normal canine retina and retinas of several canine models of retinal degeneration (rcd1, rcd2, erd, prcd, XLPRA1, XLPRA2, T4R RHO). Humans retinas affected with age-related macular degeneration (AMD) were also examined. p-CREB1/ATF1 immunolabeling was assessed in normal and rcd1 dogs treated with ciliary neurotrophic factor (CNTF), to examine the effect of a neuroprotective stimulus on activation of CREB1/ATF1.
Results.
Native CREB1 and ATF1 as well as phosphorylated CREB1/ATF1 was examined in normal canine retina by immunoblot. The p-CREB1 antibody identified phosphorylated CREB1 and ATF1 and labeled the inner retina only in normal dogs. In degenerate canine and human retinas, strong immunolabeling appeared in rod and cone photoreceptors, indicating increased expression of native CREB1 and ATF1, as well as increased phosphorylation of these proteins. Retinal protection by CNTF in rcd1 dogs was accompanied by a significant increase in the number of p-CREB1/ATF1-labeled photoreceptor nuclei.
Conclusions.
Positive association of CREB1/ATF1 phosphorylation with photoreceptor protection suggests that it may contribute to an innate protective response. These data identify a signaling mechanism in rods and cones of potential importance for therapies of RP and AMD.
doi:10.1167/iovs.09-3741
PMCID: PMC3172238  PMID: 19643965
12.  Genome-wide association identifies a deletion in the 3’ untranslated region of Striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy 
Human genetics  2010;128(3):315-324.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins.
We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine-mapping and direct DNA sequencing identified an eight base pair deletion in the 3’ untranslated region (UTR) of the striatin (STRN) gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3’ UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, plakophilin- 2, plakoglobin and desmoplakin, all involved in the pathogenesis of ARVC in human beings.
We suggest that striatin may serve as a novel candidate gene for human ARVC.
doi:10.1007/s00439-010-0855-y
PMCID: PMC2962869  PMID: 20596727
13.  Using the NAFX to Measure the Effectiveness over Time of Gene Therapy in Canine LCA 
Purpose
To use ocular motility recordings to determine the changes over time of infantile nystagmus syndrome (INS) in RPE65-deficient canines with Leber Congenital Amaurosis (LCA) and assess the time course of the recalibration of the ocular motor system (OMS).
Methods
Nine dogs were treated bilaterally with AAV-RPE65. A second cohort of four dogs was treated with AAV2.RPE65, an optimized vector. Their fixation eye movements were recorded before treatment and at 4-week intervals for 3 months, by using high-speed (500 Hz) digital videography. The dogs were suspended in a sling and encouraged to fixate on distant (57 inches) targets at gaze angles varying between ±15° horizontally and ±10° vertically. The records for each eye were examined for qualitative changes in waveform and for quantitative changes in centralisation with the expanded nystagmus acuity function (NAFX) and compared with ERG results for restoration of receptor function.
Results
First group: Before treatment, five of the dogs had clinically apparent INS with jerk, pendular, or both waveforms and with peak-to-peak amplitudes as great as 15°. One dog had intermittent nystagmus. At the 1- and 2-month examinations, no change in nystagmus waveform or NAFX was observed in any of the initial dogs, while at 10 weeks, one dog treated bilaterally with the standard dosage showed reduced nystagmus in only one eye. The other eye did not respond to treatment, as confirmed by ERG. This result was unexpected since it was previously documented that unilateral treatment leads to bilateral reduction of INS. The other dog treated with the standard dosage showed no reduction of its small-amplitude, high-frequency pendular nystagmus despite positive ERG responses. Second group: Only one dog of the four had clinically detectable INS, similar in characteristics to that seen in the affected dogs of the first group. Unlike any previous dog studied, this one showed a damping of the nystagmus within the first 4 weeks after treatment.
Conclusions
In all but one of the cases in which OMS recalibration occurred, as measured by the clinical appearance of nystagmus and by quantitative measurement using the NAFX, the improvement was apparent no sooner than 10 weeks after treatment. Longer term, dose-related studies are needed to determine the minimum necessary degree of restored receptor functionality, the duration after rescue for recalibration of the OMS, and the conditions under which recalibration information can successfully affect the contralateral eye.
doi:10.1167/iovs.09-3387
PMCID: PMC2849152  PMID: 19458334
14.  Steroids Do Not Prevent Photoreceptor Degeneration in the Light-Exposed T4R Rhodopsin Mutant Dog Retina Irrespective of AP-1 Inhibition 
PURPOSE
AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model.
METHODS
The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed (∼15-17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to 3 hours after exposure. Twenty-four hours before exposure to light, some dogs were treated with systemic dexamethasone or intravitreal/subconjunctival triamcinolone. AP-1 DNA-binding activity was determined by electrophoresis mobility shift assay (EMSA) and phosphorylation of c-Fos and activation of ERK1/2 were determined by immunoblot analyses. The eyes were collected 1 hour and 2 weeks after exposure to light, for histopathology and immunocytochemistry.
RESULTS
Inhibition of AP-1 activation, and phosphorylation of ERK1/2 and c-Fos were found after dexamethasone treatment in light-exposed T4R RHO mutant dog retinas. In contrast, increased AP-1 activity and phosphorylation of c-Fos and ERK1/2 were found in triamcinolone-treated mutant retinas. Similar extensive rod degeneration was found after exposure to light with or without treatment, and areas with surviving photoreceptor nuclei consisted primarily of cones. Only with systemic dexamethasone did the RPE cell layer remain.
CONCLUSIONS
Intraocular or systemic steroids fail to prevent light-induced photoreceptor degeneration in the T4R RHO dog retina. Finding that systemic dexamethasone prevents AP-1 activation, yet does not prevent retinal light damage, further supports the hypothesis that AP-1 is not the critical player in the cell-death signal that occurs in rods.
doi:10.1167/iovs.08-3111
PMCID: PMC2742955  PMID: 19234347
15.  Chromosomal Mapping of Canine-Derived BAC Clones to the Red Fox and American Mink Genomes 
Journal of Heredity  2009;100(Suppl 1):S42-S53.
High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene–containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.
doi:10.1093/jhered/esp037
PMCID: PMC3139363  PMID: 19546120
Canis lupus familiaris; comparative genomics; FISH; Mustela vison; Vulpes vulpes
16.  Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer 
We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 × 1010 vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.
doi:10.1038/sj.mt.6300389
PMCID: PMC2842085  PMID: 18209734
17.  An Expressed Fgf4 Retrogene Is Associated with Breed-Defining Chondrodysplasia in Domestic Dogs 
Science (New York, N.Y.)  2009;325(5943):995-998.
Retrotransposition of processed mRNAs is a frequent source of novel sequence acquired during the evolution of genomes. The vast majority of retroposed gene copies are inactive pseudogenes that rapidly acquire mutations that disrupt the reading frame, while precious few are conserved to become new genes. Utilizing a multi-breed association analysis in the domestic dog, we demonstrate that a recently acquired fgf4 retrogene causes chondrodysplasia, a short-legged phenotype that defines several common dog breeds including the dachshund, corgi and basset hound. The discovery that a single evolutionary event underlies a breed-defining phenotype for 19 diverse dog breeds demonstrates the importance of unique mutational events in constraining and directing phenotypic diversity in the domestic dog.
doi:10.1126/science.1173275
PMCID: PMC2748762  PMID: 19608863
18.  Canine RD3 mutation establishes rod cone dysplasia type 2 (rcd2) as ortholog of human and murine rd3 
Rod cone dysplasia type 2 (rcd2) is an autosomal recessive disorder that segregates in collie dogs. Linkage disequilibrium and meiotic linkage mapping were combined to take advantage of population structure within this breed, and to fine map rcd2 to a 230 kb candidate region that included the gene C1orf36 responsible for human and murine rd3, and within which all affected dogs were homozygous for one haplotype. In one of three identified canine retinal RD3 splice variants, an insertion was found that cosegregates with rcd2, and is predicted to alter the last 61 codons of the normal open reading frame and further extend the ORF. Thus combined meiotic linkage and LD mapping within a single canine breed can yield critical reduction of the disease interval when appropriate advantage is taken of within breed population structure. This should permit a similar approach to tackle other hereditary traits that segregate in single closed populations.
doi:10.1007/s00335-008-9163-4
PMCID: PMC2652121  PMID: 19130129
dog genetics; progressive retinal atrophy; retinitis pigmentosa; linkage disequilibrium mapping
20.  An ADAM9 mutation in canine cone-rod dystrophy 3 establishes homology with human cone-rod dystrophy 9 
Molecular Vision  2010;16:1549-1569.
Purpose
To identify the causative mutation in a canine cone-rod dystrophy (crd3) that segregates as an adult onset disorder in the Glen of Imaal Terrier breed of dog.
Methods
Glen of Imaal Terriers were ascertained for crd3 phenotype by clinical ophthalmoscopic examination, and in selected cases by electroretinography. Blood samples from affected cases and non-affected controls were collected and used, after DNA extraction, to undertake a genome-wide association study using Affymetrix Version 2 Canine single nucleotide polymorphism chips and 250K Sty Assay protocol. Positional candidate gene analysis was undertaken for genes identified within the peak-association signal region. Retinal morphology of selected crd3-affected dogs was evaluated by light and electron microscopy.
Results
A peak association signal exceeding genome-wide significance was identified on canine chromosome 16. Evaluation of genes in this region suggested A Disintegrin And Metalloprotease domain, family member 9 (ADAM9), identified concurrently elsewhere as the cause of human cone-rod dystrophy 9 (CORD9), as a strong positional candidate for canine crd3. Sequence analysis identified a large genomic deletion (over 20 kb) that removed exons 15 and 16 from the ADAM9 transcript, introduced a premature stop, and would remove critical domains from the encoded protein. Light and electron microscopy established that, as in ADAM9 knockout mice, the primary lesion in crd3 appears to be a failure of the apical microvilli of the retinal pigment epithelium to appropriately invest photoreceptor outer segments. By electroretinography, retinal function appears normal in very young crd3-affected dogs, but by 15 months of age, cone dysfunction is present. Subsequently, both rod and cone function degenerate.
Conclusions
Identification of this ADAM9 deletion in crd3-affected dogs establishes this canine disease as orthologous to CORD9 in humans, and offers opportunities for further characterization of the disease process, and potential for genetic therapeutic intervention.
PMCID: PMC2925905  PMID: 20806078
21.  Age-dependent disease expression determines remodeling of the retinal mosaic in carriers of RPGR exon ORF15 mutations 
Purpose
To characterize the retinal histopathology in carriers of X-linked progressive retinal atrophy (XLPRA1 & XLPRA2), two canine models of X-linked retinitis pigmentosa caused, respectively, by a stop and a frameshift mutation in RPGRORF15.
Methods
Retinas of XLPRA2 and XLPRA1 carriers of different ages were processed for morphologic evaluation, TUNEL assay, and immunohistochemistry. Cell-specific markers were used to examine retinal remodeling events.
Results
A mosaic pattern composed of patches of diseased and normal retina was first detected in XLPRA2 carriers at 4.9 weeks of age. A peak of photoreceptor cell death led to focal rod loss; however, in these patches an increased density of cones was found to persist over time. Patches of disease gradually disappeared such that by 39 weeks of age the overall retinal morphology, albeit thinner, had improved lamination. In older XLPRA2 carriers (≥8.8 years), extended regions of severe degeneration occurred in the peripheral/mid-peripheral retina.
In XLPRA1 carriers, opsin mislocalization and rare events of rod death were detected by TUNEL assay at 20 weeks of age, however patchy degeneration was only seen by 1.4 years, and was still apparent at 7.8 years.
Conclusion
The time of onset and the progression of the disease differed between the two models. In the early onset form (XLPRA2) the morphologic appearance of the retinal mosaic changed as a function of age, suggesting that structural plasticity persists in the early postnatal canine retina as mutant photoreceptors die. In the late onset form (XLPRA1), patches of disease persisted until later ages.
doi:10.1167/iovs.08-3364
PMCID: PMC2718058  PMID: 19255154
22.  Intravitreal injection of ciliary neurotrophic factor (CNTF) causes peripheral remodeling and does not prevent photoreceptor loss in canine RPGR mutant retina 
Experimental eye research  2007;84(4):753-771.
Ciliary neurotrophic factor (CNTF) rescues photoreceptors in several animal models of retinal degeneration and is currently being evaluated as a potential treatment for retinitis pigmentosa in humans. This study was conducted to test whether CNTF prevents photoreceptor cell loss in XLPRA2, an early onset canine model of X-linked retinitis pigmentosa caused by a frameshift mutation in RPGR exon ORF15.
Four different treatment regimens of CNTF were tested in XLPRA2 dogs. Under anesthesia, the animals received at different ages an intravitreal injection of 12 μg of CNTF in the left eye. The right eye served as a control and was injected with a similar volume of phosphate buffered saline (PBS). Ocular examinations were performed regularly during the treatment periods. At termination, the dogs were euthanatized, eyes collected and the retinas were processed for embedding in optimal cutting temperature (OCT) medium. The outer nuclear layer (ONL) thickness was evaluated on H&E sections and values in both CNTF- and PBS-treated eyes were compared. Morphologic alterations in the peripheral retina were characterized by immunohistochemistry using cell-specific markers. Cell proliferation in the retinas was examined on semi-thin plastic sections, and by BrdU pulse-labeling and Ki67 immunohistochemistry on cryosections.
All CNTF-treated eyes showed early clinical signs of corneal epitheliopathy, subcapsular cataracts and uveitis. No statistically significant difference in ONL thickness was seen between the CNTF- and PBS-injected eyes. Prominent retinal remodeling that consisted in an abnormal increase in the number of rods, and in misplacement of some rods, cones, bipolar and Müller cells, was observed in the peripheral retina of CNTF-treated eyes. This was only seen when CNTF was in injected before the age at which the canine retina reaches full maturation.
In XLPRA2 dogs, intravitreal injections of CNTF failed to prevent photoreceptors from undergoing cell death in the central and mid-peripheral retina. CNTF also caused ocular side-effects and morphologic alterations in the periphery that were consistent with cell dedifferentiation and proliferation. Our findings suggest that some inherited forms of retinal degeneration may not respond to CNTF’s neuroprotective effects.
doi:10.1016/j.exer.2006.12.019
PMCID: PMC2709826  PMID: 17320077
neuroprotection; ciliary neurotrophic factor; retina; photoreceptors; RPGR; X-linked retinitis pigmentosa; remodeling; dog
23.  Operating in the Dark: A Night Vision System for Surgery in Retinas Susceptible to Light Damage 
Archives of ophthalmology  2008;126(5):714-717.
A standard Zeiss OPMI 6 operating microscope was modified with a bandpass infrared filter in the light path, and infrared image intensifiers for each of the two eye pieces. The aim of the study was to evaluate this system for subretinal injections in normal control and rhodopsin mutant dogs. These dogs are a model for human autosomal dominant retinitis pigmentosa (adRP), and their retinas degenerate faster when exposed to modest light levels as used in routine clinical examinations. We showed that the mutant retinas developed severe generalized degeneration when exposed to the standard operating microscope light, but not the infrared light. The modified operating microscope provided excellent view of the ocular fundus under infrared illumination, and allowed us to perform subretinal injections in the rhodopsin mutant dog retinas without any subsequent light-induced retinal degeneration.
doi:10.1001/archopht.126.5.714
PMCID: PMC2665164  PMID: 18474785
24.  Clinical Light Exposure, Photoreceptor Degeneration, and AP-1 Activation: A Cell Death or Cell Survival Signal in the Rhodopsin Mutant Retina? 
Purpose
The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas.
Methods
Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), super-shift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling.
Results
Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells.
Conclusions
The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa.
doi:10.1167/iovs.07-0428
PMCID: PMC2377016  PMID: 17962438
25.  Bestrophin Gene Mutations Cause Canine Multifocal Retinopathy: A Novel Animal Model for Best Disease 
Purpose
Canine multifocal retinopathy (cmr) is an autosomal recessive disorder of multiple dog breeds. The disease shares a number of clinical and pathologic similarities with Best macular dystrophy (BMD), and cmr is proposed as a new large animal model for Best disease.
Methods
cmr was characterized by ophthalmoscopy and histopathology and compared with BMD-affected patients. BEST1 (alias VMD2), the bestrophin gene causally associated with BMD, was evaluated in the dog. Canine ortholog cDNA sequence was cloned and verified using RPE/choroid 5′- and 3′-RACE. Expression of the canine gene transcripts and protein was analyzed by Northern and Western blotting and immunocytochemistry. All exons and the flanking splice junctions were screened by direct sequencing.
Results
The clinical phenotype and pathology of cmr closely resemble lesions of BMD. Canine VMD2 spans 13.7 kb of genomic DNA on CFA18 and shows a high level of conservation among eukaryotes. The transcript is predominantly expressed in RPE/choroid and encodes bestrophin, a 580-amino acid protein of 66 kDa. Immunocytochemistry of normal canine retina demonstrated specific localization of protein to the RPE basolateral plasma membranes. Two disease-specific sequence alterations were identified in the canine VMD2 gene: a C73 T stop mutation in cmr1 and a G482A missense mutation in cmr2.
Conclusions
The authors propose these two spontaneous mutations in the canine VMD2 gene, which cause cmr, as the first naturally occurring animal model of BMD. Further development of the cmr models will permit elucidation of the complex molecular mechanism of these retinopathies and the development of potential therapies.
doi:10.1167/iovs.06-1374
PMCID: PMC1931491  PMID: 17460247

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