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Genetics  |   August 2012
Exclusion of RPGRIP1 ins44 from Primary Causal Association with Early-Onset Cone–Rod Dystrophy in Dogs
Author Affiliations & Notes
  • Tatyana Kuznetsova
    From the Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; the
  • Simone Iwabe
    From the Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; the
  • Kathleen Boesze-Battaglia
    School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Sue Pearce-Kelling
    OptiGen LLC, Cornell Business and Technology Park, Ithaca, New York.
  • Yim Chang-Min
    School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Kendra McDaid
    From the Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; the
  • Keiko Miyadera
    From the Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; the
  • Andras Komaromy
    From the Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; the
  • Gustavo D. Aguirre
    From the Section of Ophthalmology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; the
  • Corresponding author: Gustavo D. Aguirre, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104; gda@vet.upenn.edu
Investigative Ophthalmology & Visual Science August 2012, Vol.53, 5486-5501. doi:10.1167/iovs.12-10178
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      Tatyana Kuznetsova, Simone Iwabe, Kathleen Boesze-Battaglia, Sue Pearce-Kelling, Yim Chang-Min, Kendra McDaid, Keiko Miyadera, Andras Komaromy, Gustavo D. Aguirre; Exclusion of RPGRIP1 ins44 from Primary Causal Association with Early-Onset Cone–Rod Dystrophy in Dogs. Invest. Ophthalmol. Vis. Sci. 2012;53(9):5486-5501. doi: 10.1167/iovs.12-10178.

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

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Abstract

Purpose.: Canine cone–rod dystrophy 1 (cord1) has been previously mapped to CFA15, and a homozygous 44-bp insertion in exon 2 (Ins44) of canine RPGRIP1 (cRPGRIP1Ins/Ins ) has been associated with the disease. However, from the recent identification of a significant discordance in genotype–phenotype association, we have reexamined the role of cRPGRIP1 in cord1.

Methods.: Retinal structure and function was assessed by clinical retinal examination, noninvasive imaging, electroretinography, and histopathology/immunohistochemistry. cRPGRIP1 splicing was analyzed by RT-PCR. Retinal gene expression was determined by quantitative RT-PCR (qRT-PCR). Five markers spanning the entire cRPGRIP1 were identified and used for haplotyping.

Results.: Electroretinography demonstrated that cone responses were absent or present in cRPGRIP1Ins/Ins individuals. Moreover, performance in vision testing and optical coherence tomography (OCT) were comparable in cRPGRIP1Ins/Ins dogs, regardless of the cone ERG status. While histologic changes in retinal structure were minimal, immunohistochemistry demonstrated a lack of cone opsin labeling in cRPGRIP1Ins/Ins dogs. cDNA analysis revealed that Ins44 disrupts a putative exonic splicing enhancer that allows for skipping of exon 2, while retaining the functional RPGR-interacting domain (RID) of the protein. New cRPGRIP1 sequence changes were identified, including a 3-bp deletion affecting the 3′ acceptor splice site of alternative exon 19c. The extended haplotype spanning cRPGRIP1 was identical in cRPGRIP1Ins/Ins dogs with and without retinal degeneration. Gene expression analysis showed that expression levels were not associated with Ins44 genotype.

Conclusions.: The results indicated that cRPGRIP1 Ins44 is an unlikely primary cause of cord1, and that the causal gene and mutation are likely located elsewhere in the critical disease interval.

Introduction
Cone–rod dystrophies (CRDs) represent a group of progressive, inherited blinding diseases characterized by primary dysfunction and loss of cone photoreceptors preceding or accompanied by rod death. Humans with CRD exhibit reduced visual acuity followed by severe loss of central and color vision that often progresses to complete blindness. The onset of clinical disease ranges from early to late adulthood. Mutations in more than 20 genes have been associated with CRD in human patients (Retinal Network RetNet: http://www.sph.uth.tmc.edu/Retnet/disease.htm University of Texas Houston Health Science Center, Houston, TX). 
Canine models have been identified for a number of inherited retinal degenerations. 1,2 In terms of CRD, the standard wire-haired dachshund, miniature long-haired dachshund (MLHD), Glen of Imaal terrier, and pit bull terrier are the only dog breeds thus far affected, and the genes involved have been identified in all but pit bull terriers. 36 Of these, a severe early-onset CRD (cord1) was described in MLHDs 5 and the locus mapped to a region of canine chromosome 15 (CFA15) orthologous to human chromosome 14 (HSA14). This region includes RPGRIP1, which codes for the retinitis pigmentosa GTPase regulator (RPGR)–interacting protein, a ciliary protein important for maintenance and function of photoreceptor cilia. 7,8 Mutations in RPGRIP1 in humans are associated with Leber congenital amaurosis 9 and recessive cone–rod dystrophy. 10  
Sequence analysis of cRPGRIP1 in diseased dogs have identified a 44-nucleotide insertion in exon 2 (Ins44) that is proposed to generate a frameshift and premature stop codon in exon 3. 5 The strong phenotype–genotype correlation between the mutation and the disease in the original study colony suggests that the aberrant gene product promotes cord1 in MLHD. However, subsequent studies in a subset of the general MLHD pet population have shown that approximately 16% of apparently normal controls were, in fact, RPGRIP1 Ins44 homozygous mutants (RPGRIP1Ins/Ins ). 11 Furthermore, ophthalmic and ERG testing of RPGRIP1Ins/Ins MLHDs in the UK pet population have indicated that homozygosity is not invariably associated with early-onset cord1. 12 As well, the Ins44 insertion or its variant has also been found in dog breeds that are not predisposed to CRDs, for example, Beagle, English springer spaniel (ESS), Labrador retriever, and French bulldog. 11 Indeed, 42% of >1100 ESSs tested are RPGRIP1Ins/Ins (provided in the public domain by the English Springer Spaniel Field Trial Association, http://www.essfta.org/Health_Research/pra_news.htm), yet less than 0.6% of 18,129 dogs examined between 2000 and 2009 are affected with clinically evident inherited retinal degeneration. 13  
Together, these results suggest that there is not a simple cause and effect relationship between the cRPGRIP1 Ins44 sequence change and cord1. While it remains possible that the Ins44 represents a predisposing factor for disease, it seems more likely that Ins44 is a benign polymorphism, and that a second mutation in cRPGRIP1, or a different gene within the mapped cord1 critical region, is causal for disease. As such, it could interact with a recently identified second locus to determine the early-onset form of the disease. 14  
Previously we have characterized the structure, organization, and expression of the cRPGRIP1 reference sequence along with five alternatively spliced variants and showed a complex 5′- and 3′-splicing pattern. 15 With this information in hand, we set out to determine how cRPGRIP1 mutations and alteration in expression of cRPGRIP1 isoforms contribute to cord1 pathogenesis in dogs. 
Materials and Methods
Study Dogs
Several different populations of dogs were used for the studies. In all cases, the research was conducted in full compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and with Institutional Animal Care and Use Committee (IACUC) approval. 
cord1 Colony.
Experimental dogs were bred and maintained at the Retinal Disease Studies Facility (RDSF), Kennett Square, Pennsylvania. The colony was established by outcrossing two purebred MLHDs to unrelated mixed breed dogs (Fig. 1). Semen from one of the founders (C-UK1) was obtained from the Animal Health Trust (AHT), Lanwades Park, Newmarket, United Kingdom. C-UK1 is RPGRIP1Ins/Ins and affected with an early-onset generalized retinal degeneration. 5 The dog had 30-Hz cone flicker ERG responses that were very low in amplitude, but not absent when first recorded, and decayed exponentially at later ages (C. Mellersh, personal communication, July 11, 2011; see Fig. 4 “Casper” in Turney et al. 16 [2007]). C-UK1 was outcrossed to an unrelated normal, mixed breed female (N212), and intercross matings were subsequently performed. 
Figure 1. 
 
Pedigree of research colony informative for cRPGRIP1 Ins44. The dogs with clinical retinal degeneration are indicated by **. Dogs with absent 30-Hz ERG cone flicker responses are indicated by oblique black arrows; oblique gray dashed arrow indicates dog (R28) with markedly reduced but still present 30-Hz ERG cone flicker responses. All dogs with cone ERG abnormalities are cRPGRIP1Ins/Ins , but two dogs with this genotype (R17 and R42) have cone responses.
Figure 1. 
 
Pedigree of research colony informative for cRPGRIP1 Ins44. The dogs with clinical retinal degeneration are indicated by **. Dogs with absent 30-Hz ERG cone flicker responses are indicated by oblique black arrows; oblique gray dashed arrow indicates dog (R28) with markedly reduced but still present 30-Hz ERG cone flicker responses. All dogs with cone ERG abnormalities are cRPGRIP1Ins/Ins , but two dogs with this genotype (R17 and R42) have cone responses.
The second colony founder (R10) was a female RPGRIP1Ins/Ins MLHD affected with advanced end-stage retinal degeneration at 2.6 years of age (Figs. 1, 2A); the retinal vessels were attenuated or unapparent ophthalmoscopically, and the tapetal region of the fundus was hyperreflective. Clinically, R10 was severely impaired visually, with widely dilated pupils that were poorly responsive to light. Because of her advanced retinal degeneration and visual impairment, an ERG was not recorded. At the time of donation for research investigations, R10 had been bred to a male RPGRIP1 +/+ (wild type) MLHD (R9) that had a severe visual impairment from a retinal disease clinically different from cord1, and considered to be acquired and nonhereditary (asymmetric fundus abnormalities: end-stage retinal degeneration in right eye and midstage disease in the left eye; the left eye had a flat retinal detachment with multiple retinal holes and multifocal retinal scars indicative of diffuse, healed chorioretinitis of inflammatory origin). Five progeny (R11–R15) resulted from this mating; all were RPGRIP1+/Ins , clinically normal when first examined at 6 months of age, and remained normal subsequently (most recent examinations at 2.6–4.0 years of age). Subsequent matings among the progeny of these founders formed the basis of this study (Fig. 1). 
Figure 2. 
 
Fundus photographs of selected research colony dogs taken at different ages. Only dog R10 (A) has generalized retinal degeneration with marked vascular attenuation and tapetal hyperreflectivity. The retinas in the other dogs are clinically normal: R10, R17, and R29 are cRPGRIP1Ins/Ins ; R13, R14, and R30 are cRPGRIP1+/Ins . See Table 1 for details on clinical examination results and ages.
Figure 2. 
 
Fundus photographs of selected research colony dogs taken at different ages. Only dog R10 (A) has generalized retinal degeneration with marked vascular attenuation and tapetal hyperreflectivity. The retinas in the other dogs are clinically normal: R10, R17, and R29 are cRPGRIP1Ins/Ins ; R13, R14, and R30 are cRPGRIP1+/Ins . See Table 1 for details on clinical examination results and ages.
Table 1. 
 
Ins44/D3 Genotype and Clinical Retinal Phenotype for Dogs Examined
Table 1. 
 
Ins44/D3 Genotype and Clinical Retinal Phenotype for Dogs Examined
Dog Ins44 Genotype* D3 Genotype* Clinical Retinal Phenotype-Inherited Retinal Degeneration Clinical/ERG Examination Age (y) Pathology-IHC, Age (y) Breed
Research colony
 R1 12 12 Normal fundus; no ERG performed 4.4 MLHDx
 R2 12 12 Normal fundus; no ERG performed 4.4 4.4 MLHDx
 R4 12 12 Normal fundus; rod/cone ERG responses 0.4 MLHDx
 R5 22 22 Normal fundus; absent cone ERG 3.1/0.4 MLHDx
 R6 12 12 No eye exam; rod/cone ERG responses 0.4 MLHDx
 R7 12 12 No eye exam; rod/cone ERG responses 0.4 MLHDx
 R8 12 12 Normal fundus; rod/cone ERG responses 2.9/0.4 2.9 MLHDx
 R9 11 12 Acquired retinal disease—detachment, inflammation, degeneration/no ERG performed 2.8 MLHD
 R10 22 22 Generalized retinal degeneration; no ERG performed 2.6 MLHD
 R11 12 22 Normal fundus; no ERG performed 0.5 MLHD
 R12 12 22 Normal fundus; rod/cone ERG responses 1.8 MLHD
 R13 12 22 Normal fundus; rod/cone ERG responses 2.6 MLHD
 R14 12 12 Normal fundus; no ERG performed 4.0 MLHD
 R15 12 12 Normal fundus; no ERG performed 0.5 MLHD
 R16 12 12 Normal fundus; rod/cone ERG responses 0.6/0.4 MLHDx
 R17 22 22 Normal fundus; rod/cone ERG responses 1.9/1.8 MLHDx
 R18 22 22 Normal fundus; absent cone ERG 0.6/0.4 MLHDx
 R21 22 22 Normal fundus; absent cone ERG 0.6/0.4 0.7 MLHDx
 R22 12 12 Normal fundus; rod/cone ERG responses 0.6/0.4 0.7 MLHDx
 R25 11 Normal fundus; rod/cone ERG responses 0.4/0.5 MLHDx
 R26 12 12 Normal fundus; rod/cone ERG responses 1.5/1 MLHDx
 R27 12 12 Normal fundus (focal choroidal coloboma-OS); rod/cone ERG responses 1.4/1.4 1.6 MLHDx
 R28 22 22 Normal fundus; cone ERG responses markedly reduced 1.5/1.5 MLHDx
 R29 22 22 Normal fundus; absent cone ERG 1.5/1.3 1.5 MLHDx
 R30 12 Normal fundus; rod/cone ERG responses 1.1/1 MLHDx
 R35 12 12 Normal fundus; no ERG performed 1.4 MLHDx
 R36 12 12 No eye exam; rod/cone ERG responses 1/0.7 MLHDx
 R42 22 22 Normal fundus; rod/cone ERG responses 1/1 MLHDx
 R46 22 22 Normal fundus; absent cone ERG 1/1 MLHDx
Pet population‡
 Caroline 22 22 Normal fundus 3.5 MLHD
 Sandy 12 12 Normal fundus 4.4 MLHD
 Clair 22 22 PRA suspicious 1.3 MLHD
 Kaylee 12 12 Normal fundus 12 MLHD
 Molson 22 22 PRA 4.4 MLHD
 Corby 12 12 Normal fundus 8.8 MLHD
 Josie 22 22 Normal fundus 1 MLHD
 Maura 12 12 Normal fundus 6 MLHD
 Miper 12 12 Normal fundus 6 MLHD
 Bella 11 12 Normal fundus 1 MLHD
 08-7988 11 12 Normal fundus 7.5 MLHD
 08-7989 11 12 Normal fundus 2.1 MLHD
 08-7991 11 12 Normal fundus 5.7 MLHD
 Melody 11 11 Normal fundus 11.4 MLHD
 Chloe 11 12 Normal fundus 10.9 MLHD
 Kaylee 11 11 Normal fundus 1.2 MLHD
 Tori 11 22 Normal fundus 1 MLHD
 Spice 11 22 Normal fundus 1 MLHD
 Brandy 12 12 Normal fundus 7.1 MLHD
 Hamilton 11 22 Normal fundus 2 MLHD
 George 22 22 PRA 13.8 ESS
 Stoney 12 12 PRA 6.1 ESS
 Minnie 22 22 PRA 9.9 ESS
 Keah 22 22 PRA 15.5 ESS
 Stella 22 22 PRA 6.2 ESS
 Emma 22 22 PRA 2.8 ESS
 Holly 22 22 PRA 13 ESS
 Genesis 22 22 Normal fundus 7 ESS
 Mackenzie 22 22 Normal fundus 11.4 ESS
 Kayla 22 22 Normal fundus 11 ESS
 Tucker 22 22 Normal fundus 8.3 ESS
 Louie 22 22 Normal fundus 1.8 ECS
Purebred and Other Dogs.
In addition to the study colony, 20 MLHDs, 11 ESSs, and 1 English cocker spaniel (ECS) were included for genotype analysis of cRPGRIP1, and genotype–phenotype correlation (Table 1). The samples from the pet population were accompanied by documentation of ophthalmic examination and clinical diagnoses by board-certified veterinary ophthalmologists (diplomates, the American College of Veterinary Ophthalmologists [ACVO]) made through the Canine Eye Registration Foundation program. Thirty additional dogs, including 12 Siberian huskies, 8 Beagles, and 10 Papillons were also tested for Ins44. Of 10 Papillons analyzed, 5 were normal and 5 had retinal degeneration. The Siberian huskies and Beagles were from an X-linked retinal degeneration pedigree (XLPRA1; 5-nt RPGR° RF15 microdeletion) derived from an affected male dog outcrossed to unrelated normal mix bred or purebred females as previously described. 17  
Clinical Assessment and In Vivo Retinal Imaging
Routine ophthalmic examinations, including biomicroscopy, indirect ophthalmoscopy, and direct/indirect fundus photography, were conducted on a regular basis throughout the study. In 3 dogs that were retained for breeding, in vivo imaging was done under general anesthesia by using a combined confocal scanning laser ophthalmoscope/spectral-domain optical coherence tomography (cSLO/sdOCT) instrument (Spectralis HRA/OCT, Heidelberg, Germany). En face imaging was done with near-infrared or shortwave autofluorescent modes with the 55° lens, and overlapping images were acquired. sdOCT scans were done with the 30° lens in groups of parallel linear (“raster”) scans. In each retinal quadrant centered on the optic disc, a 30 × 20-degree area was imaged with 49 sequential B-scans, each separated by 120 μm. As well, single, high-resolution averaged (n = 100) sequential B-scans were taken in a 30° area (line) on the four cardinal points and centered in the optic disc. For sdOCT volume scans, each scanned image was the average of nine. 
Electroretinography
Standard Ganzfeld scotopic and photopic ERGs were recorded from anesthetized dogs as described, with a modified Ganzfeld dome fitted with the light-emitting diode (LED) light stimuli of a ColorDome stimulator (Diagnosys LLC, Lowell, MA). 18 The ERG was recorded with custom-made Burian-Allen bipolar contact lens electrodes (Hansen Labs, Coralville, IA) in monopolar mode, and platinum subdermal needle electrodes (Grass Safelead needle electrodes; Grass Technologies, West Warwick, RI) by using the Espion E 2 computer-based system (Diagnosys Systems, Inc., Littleton, MA). The dogs were premedicated with subcutaneous acepromazine maleate (0.5 mg/kg) and atropine sulfate (0.02 mg/kg), and anesthetized with intravenous propofol followed by isoflurane inhalation anesthesia. Rod- and mixed rod-cone–mediated responses were recorded after 20 minutes of dark adaptation with scotopic single white flash stimuli of increasing intensities (from 0.000577 to 10.26 cd.s/m2). Following 5 minutes of light adaptation to a background illumination of 34.26 cd/m2, 1-Hz single flash (from 0.00577 to 10.26 cd.s/m2) and 29.41-Hz flicker (from 0.00577 to 5.77 cd.s/m2) cone-mediated signals were recorded. Except for the brighter scotopic light stimuli (≥0.577 cd.s/m2), multiple responses were averaged. 
Objective Vision Testing
We used an objective means for quantifiable assessment of vision in dogs. The obstacle-avoidance course is 3.6-m long and has six panels, which are varied in position in a random order for every test run. Details of the apparatus and method of testing have been described; the testing modality permits quantitative detection of absent photopic vision in dogs with achromatopsia, and the recovery of cone function following gene therapy. 18,19 The luminance used for testing has now been expanded to lower light levels, and vision is tested at 0.003, 0.2, 1, 65, and 646 lux. 
Tissue Processing, Histology, and Immunohistochemistry
Eyes from selected dogs were collected immediately after enucleation and fixed in formaldehyde-containing Excalibur Alcoholic Z-Fix solution (Excalibur Pathology, Inc., Oklahoma City, OK) for 72 to 96 hours, and transferred to 70% ethanol. Tissue sections from the central (pupil–optic nerve) axis were cut in the dorsoventral plane and stained with hematoxylin-eosin (H&E). Unstained sections were deparaffinized by heat and xylene extraction before serial rehydration in decreasing ethanol concentrations. For immunohistochemistry, deparaffinized sections were incubated with primary antibodies in a solution 0.025% Triton X-100 and 1.5% normal goat serum in PBS overnight followed by incubation with appropriate fluorescent secondary antibodies (Alexa Fluor Dyes; Molecular Probes, Invitrogen, Carlsbad, CA). Primary polyclonal antibodies included antibodies against R/G cone opsin (anti-opsin red–green, 1:2000, catalog No. AB 5405; Chemicon [Millipore], Temecula, CA), human cone arrestin (1:10,000; LUMIf; provided by Cheryl Craft, Doheny Eye Institute, University of Southern California, Los Angeles, CA), and rod opsin (1:1000, catalog No. MAB 5316; Chemicon [Millipore]); 4′,6-diamidino-2-phenylindole (DAPI) stain was used to label cell nuclei. Slides were mounted with fluoromount G mounting media (catalog No. 01100-01; Southern Biotech, Birmingham, AL), coverslipped, and examined by epifluorescence microscopy with a Zeiss Axioplan microscope (Carl Zeiss Meditech, Oberkochen, Germany) by using transmitted light or epifluorescence. Images were digitally captured (Spot 4.0 camera; Diagnostic Instruments, Inc., Sterling Heights, MI), and imported into a graphics program (Photoshop; Adobe, Mountain View, CA) for display. 
Sample Collection, DNA/RNA Extraction, cDNA Synthesis
Retinas were collected under sterile and RNase-free conditions within 1 minute after euthanasia and enucleation, frozen by immersion in liquid nitrogen, and stored at −70°C until used. Total RNA was isolated from canine tissues by using TRIzol (Invitrogen) and a single chloroform extraction. First strand cDNA was synthesized in 20-μL reactions by using the High Capacity RNA-to-cDNA kit (Applied BioSystems, Foster City, CA) following the manufacturer's recommendations. Genomic DNA was isolated from blood samples by using the QIAamp DNA kit (Qiagen, Valencia, CA) following the manufacturer's directions. 
PCR Amplification and Sequencing
Primer sequences for all PCR experiments are represented in Supplementary Table S1. PCR reactions were performed on 50 ng genomic DNA or cDNA generated from 50 ng DNAase-treated retinal RNA, in a final volume of 20 μL containing 1× PCR buffer (Qiagen), 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μM forward and reverse primers, and 1 unit Platinum Taq DNA polymerase (Invitrogen). Cycling conditions were 5 minutes initial denaturation at 95°C followed by 35 cycles of 95°C for 20 seconds (denaturation), 55°C for 30 seconds (annealing), 68°C for 1 min/1000 bp (elongation). When necessary, PCRs were optimized by adding 6% Dimethyl sulfoxide (DMSO). PCR products were analyzed on a 6% polyacrylamide gel followed by ethidium bromide staining. For sequencing purpose, PCR products were analyzed on a 1.5% agarose gel, extracted with NucleoTrap Gel Extraction Kit (Clontech, Mountain View, CA) and directly sequenced. 
DNA Marker Selection
Sequence differences identified in cRPGRIP1 in individual dogs of different breeds were evaluated to select critical informative markers for haplotype assignment. 
3′RACE
The analysis was performed as previously described. 15  
Relative Quantification (ddCt) Assay
Real-time PCR was performed in a total volume of 25 μL by using 96-well microwell plates on the Applied Biosystems 7500 Real-Time PCR System. Amplification data were analyzed with the 7500 Software version 2.0.1 (Applied Biosystems). All PCRs were performed in microwells by using cDNA generated from 50 ng DNAase-treated retinal RNA from 5-month-old (2 females) and 7-month-old (1 male) normal dogs, 7-month-old RPGRIP1Ins/Ins (R18, male; R21, female) dogs, and 7-month-old RPGRIP1+/Ins (R16, male; R22, female) dogs. The SYBR green platform was used for gene expression analysis of nine RPGRIP1 splice variants by using a primer concentration of 0.15 μM. Primer sequences for amplicons 1-10EX, 19-19aEX, 21-22EX, and 19c-2 are listed in Supplementary Table S1. Primer sequences for amplicons 2-5a, 2-5b, 2-5c and for the reference sequence used have been previously described. 15 The TaqMan platform (Applied Biosystems) was used for the analysis of eight photoreceptor-expressed genes. SAG and GFAP primers and TaqMan Gene Expression Assay probes (Cf02628845_m1 and Cf02655695_m1, respectively) were used in the analysis. Primer and probe sequences for OPN1SW, OPN1LW, CNGB3, RHO, CNGA1, and CNGB1 have been previously validated and published (see Table S1 in Komaromy et al. 18 [2010]). The GAPDH primers and TaqMan Gene Expression Assay probe (Hs02786624_g1) were used to normalize the cDNA templates. 
Western Blot
Retinas (R18-cRPGRIP1Ins/Ins , 0.6 years; R16-cRPGRIP1+/Ins , 0.6 years; controls (n = 2), 7 and 16 weeks) were lysed in RIPA buffer (Abcam, Cambridge, MA) homogenized, and gently sonicated. Debris was pelleted at 13,500 rpm (15 minutes, 4°C) and the resulting cleared lysate was for Western blot analysis. Equal amounts of protein (35 μg per lane) were separated on a 4% to 12% Bis-Tris gel, transferred onto a nitrocellulose membrane, blocked with generic protein (milk), and probed with anti-RPGRIP1 antibody Ab38 against the conserved RPGR-interacting domain (RID) of RPGRIP1 7,20 at a 1:500 dilution. Secondary antibody used was horseradish peroxidase (HRP) anti-rabbit (31432; Thermo Scientific, Rockford, IL) at 1:1000 dilution. The membrane was imaged with a Kodak imaging station (Carestream Health, Inc., Rochester, NY). 
Results
Exclusion of RPGRIP1 Ins44 as the Primary Causative Mutation for cord1
To assess the role of cRPGRIP1Ins/Ins in cord1, we used dogs from our research colony, formed from two unrelated MLHDs that were cRPGRIP1Ins/Ins and had early-onset retinal degeneration (Fig. 1, C-UK1 and R10). The resulting pedigree of dogs studied is presented in Figure 1. The genotype of all dogs was confirmed by PCR. 
Clinical Findings.
Ophthalmic examinations were performed, and results of the most recent examination are presented in Table 1. All 16 cRPGRIP1+/Ins dogs were clinically normal at all examination time points. Notably, eight cRPGRIP1+/Ins dogs were examined at >1.5 years of age, and none showed any clinical evidence of retinal degenerative disease (Figs. 2B, 2C, 2F, and Table 1). Of the eight cRPGRIP1Ins/Ins dogs produced for this study, four were descendants of founder male C-UK1 (R5, R17, R18, R21), and four were intercross progeny descending from both founders (R28, R29, R42, R46) (Fig. 1). In contrast to previous results in the original AHT colony, 5 retinal degenerative changes were not observed in any of the dogs of any age, even those one year old or older (R42, R46, R28, R29, R17, and R5; Figs. 2D, 2E, and Table 1). 
Electroretinography.
ERGs were recorded from selected dogs to assess retinal functional abnormalities at different ages (Fig. 3, Table 1). Of the cRPGRIP1Ins/Ins dogs, five (R5, R18, R21, R29, R46) had nonrecordable cone-mediated 30-Hz flicker responses and one had markedly reduced amplitude (R28). However, two dogs (R17, R42) had cone-mediated 30-Hz flicker responses that were normal in waveform, but with amplitudes that were slightly lower than some cRPGRIP1+/Ins or wild-type controls (Fig. 3). In addition, rod-mediated, and mixed rod–cone responses were similar among cRPGRIP1 +/Ins and cRPGRIP1Ins/Ins dogs, although the amplitude was slightly lower than what was observed in the limited number of wild-type control dogs. However, even within the cRPGRIP1 +/Ins group, there was variability of response amplitudes among dogs (e.g., compare the 30-Hz cone-mediated flicker responses of R8 and R16 [relatively higher amplitude] to those of dogs R6 and R22 [relatively lower amplitude] in Fig. 3). 
Figure 3. 
 
ERG data for MLHD dogs with different Ins44 genotypes. ERGs were recorded at 0.4 years of age (R4–R8; top panel), and at 0.4 years (R16–R22) or 0.3 years (R25; lower panel). All cRPGRIP1Ins/Ins dogs are also homozygous for the D3 deletion. Among the cRPGRIP1Ins/Ins dogs, R5, R18, and R21 lack recordable cone ERG responses, but these are present in R17.
Figure 3. 
 
ERG data for MLHD dogs with different Ins44 genotypes. ERGs were recorded at 0.4 years of age (R4–R8; top panel), and at 0.4 years (R16–R22) or 0.3 years (R25; lower panel). All cRPGRIP1Ins/Ins dogs are also homozygous for the D3 deletion. Among the cRPGRIP1Ins/Ins dogs, R5, R18, and R21 lack recordable cone ERG responses, but these are present in R17.
Finally, to determine if there was any age-related decrement in ERG function, recordings were repeated in four cRPGRIP1Ins/Ins dogs: R5, R17, R42, and R46. Such analysis revealed that responses remained virtually unchanged over time in dogs with demonstrable cone-mediated ERG responses (R17 between 0.7 and 1.8 years; R42 between 0.3 and 1 year). In dogs with nonrecordable cone-mediated ERG responses, rod-mediated responses remained consistent over time (R5 at 0.4 and 3.1 years; R46 at 0.3 and 1 year) (Figs. 3, 4, and Supplementary Fig. S1). 
Figure 4. 
 
ERG and objective vision testing in cRPGRIP1Ins/Ins dogs. (A) Both dogs have rod-mediated responses that show minimal to no changes at the two time points examined. Cone 30-Hz flicker responses are absent in one of the dogs. (B) Objective vision testing evaluated with an obstacle avoidance course. The graph shows the transit time in seconds for dogs R42 (white bar) and R46 (black bar) as a function of ambient light intensity; results are shown for the 6-, 8-, and 10-month time periods. Both dogs show comparable and normal visual performance under scotopic and photopic illumination, and no change with time. Values in gray are the mean ± 1 SD for transit times in normal dogs. 19
Figure 4. 
 
ERG and objective vision testing in cRPGRIP1Ins/Ins dogs. (A) Both dogs have rod-mediated responses that show minimal to no changes at the two time points examined. Cone 30-Hz flicker responses are absent in one of the dogs. (B) Objective vision testing evaluated with an obstacle avoidance course. The graph shows the transit time in seconds for dogs R42 (white bar) and R46 (black bar) as a function of ambient light intensity; results are shown for the 6-, 8-, and 10-month time periods. Both dogs show comparable and normal visual performance under scotopic and photopic illumination, and no change with time. Values in gray are the mean ± 1 SD for transit times in normal dogs. 19
Objective Vision Testing.
To objectively assess vision in cRPGRIP1Ins/Ins dogs, dogs with recordable (R42) or nonrecordable (R46) 30-Hz cone-mediated responses were selected for training in an obstacle avoidance course. Testing was done every 4 to 8 weeks between 4 and 10 months of age, and ERGs were performed at 4-month and 1-year time points (Fig. 4). Both dogs performed equally well in the test, and there was no decrease in performance over time. Note that dogs with achromatopsia and total absence of cone function due to CNGB3 mutations have significant increases in obstacle course transit times at the 65 and 646 lux intensities, which are reversed to normal following corrective gene therapy. 18,19  
Retinal Histopathology.
To determine if cRPGRIP1Ins/Ins was associated with retinal abnormalities, eyes from six study dogs, four cRPGRIP1+/Ins and two cRPGRIP1Ins/Ins , were examined; both Ins44 homozygotes had absent cone ERG function. The four cRPGRIP1+/Ins dogs (age range, 0.7–4.4 years) had normal retinal structure as illustrated for the 0.7-year-old dog (Figs. 5A, 6A). Retinas from the two cRPGRIP1Ins/Ins dogs (aged 0.7 and 1.5 years) were similar, although specific regional abnormalities were observed (Figs. 5B, 6B). To further evaluate such abnormalities, morphologic assessment was made at six defined locations in the superior (S) and inferior (I) meridians: Superior area 1 (S1) and inferior area 1 (I1) were within 1000 μm of the optic disc edge; superior area 2 (S2) and inferior area 2 (I2) were at the midpoint (±500 μm) between the optic disc and ora serrata; and superior area 3 (S3) and inferior area 3 (I3) were within 1000 μm of the ora serrata. Both cRPGRIP1Ins/Ins dogs had normal to near-normal preservation of retinal structure near the optic disc (area 1; compare Figs. 5A1 to 5B1); however, the inner and outer segments were shortened and compressed, with occasional macrophages present in the subretinal space in areas 2 and 3 (Figs. 6B1, 6B2; 6C, respectively). These abnormalities were not progressive, as there was no further loss of photoreceptor cells during the 0.7- to 1.5-year time period (Fig. 7). 
Figure 5. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A4) or cRPGRIP1Ins/Ins (R21, B1B4) dogs. Images are taken from region near and inferior to the optic disc (area 1), and stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2), hCAR (A3, B3), or R/G opsin (A4, B4) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the central regions, retinal integrity is normal in the cRPGRIP1+/Ins or cRPGRIP1Ins/Ins dogs (A1, A2), and opsin immunolabeling is comparable although focal delocalization of opsin is present (B2, arrow). In the control, hCAR (A3, oblique arrows) and R/G opsin (A4) antibodies label the cones. In contrast, the cRPGRIP1Ins/Ins dog shows that hCAR labels only the outer perinuclear region of cone somata (B3, short arrows) and synaptic terminals, but labeling of cone IS and OS is not present, and only one cone OS labels with R/G opsin antibody (B4, arrow). Calibration marker in A1 = 20 μm and applies to all figures except those highlighted by boxed region in A3, A4, B3, B4, which is shown to the right of the main panels (calibration marker = 5 μm). GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium.
Figure 5. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A4) or cRPGRIP1Ins/Ins (R21, B1B4) dogs. Images are taken from region near and inferior to the optic disc (area 1), and stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2), hCAR (A3, B3), or R/G opsin (A4, B4) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the central regions, retinal integrity is normal in the cRPGRIP1+/Ins or cRPGRIP1Ins/Ins dogs (A1, A2), and opsin immunolabeling is comparable although focal delocalization of opsin is present (B2, arrow). In the control, hCAR (A3, oblique arrows) and R/G opsin (A4) antibodies label the cones. In contrast, the cRPGRIP1Ins/Ins dog shows that hCAR labels only the outer perinuclear region of cone somata (B3, short arrows) and synaptic terminals, but labeling of cone IS and OS is not present, and only one cone OS labels with R/G opsin antibody (B4, arrow). Calibration marker in A1 = 20 μm and applies to all figures except those highlighted by boxed region in A3, A4, B3, B4, which is shown to the right of the main panels (calibration marker = 5 μm). GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium.
Figure 6. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A2) or cRPGRIP1Ins/Ins (R21, B1B2, C) dogs. Images are taken from area 2, midpoint between the optic disc and ora serrata (A1A2, B1B2), or from the periphery (area 3, C). Sections stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2, C) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the cRPGRIP1Ins/Ins dog, there is progressive shortening and loss of rod outer segments, and opsin delocalization into the ONL (B2, C, oblique arrows and *, respectively). A subretinal macrophage is present in areas of rod outer segment shortening and loss (B1, arrowhead). Calibration marker in A1 = 20 μm and applies to all figures. NFL, nerve fiber layer; PR, photoreceptor layer.
Figure 6. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A2) or cRPGRIP1Ins/Ins (R21, B1B2, C) dogs. Images are taken from area 2, midpoint between the optic disc and ora serrata (A1A2, B1B2), or from the periphery (area 3, C). Sections stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2, C) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the cRPGRIP1Ins/Ins dog, there is progressive shortening and loss of rod outer segments, and opsin delocalization into the ONL (B2, C, oblique arrows and *, respectively). A subretinal macrophage is present in areas of rod outer segment shortening and loss (B1, arrowhead). Calibration marker in A1 = 20 μm and applies to all figures. NFL, nerve fiber layer; PR, photoreceptor layer.
Figure 7. 
 
Outer nuclear layer counts from cRPGRIP1Ins/Ins (R21, R29) and cRPGRIP1+/Ins (R22, R8) dogs. Y-axis values represent the mean of three ONL nuclear layer thickness counts within a single microscopic field per location; for each dog, four defined retinal locations (two areas in the superior and two areas in the inferior meridian) are shown. There are no apparent differences between the two genotypes, or between dogs of different ages.
Figure 7. 
 
Outer nuclear layer counts from cRPGRIP1Ins/Ins (R21, R29) and cRPGRIP1+/Ins (R22, R8) dogs. Y-axis values represent the mean of three ONL nuclear layer thickness counts within a single microscopic field per location; for each dog, four defined retinal locations (two areas in the superior and two areas in the inferior meridian) are shown. There are no apparent differences between the two genotypes, or between dogs of different ages.
Figure 8. 
 
In vivo images of retinal structure of cRPGRIP1+/Ins (A) and cRPGRIP1Ins/Ins (B, C) dogs. Each row of images compares the near-infrared en face view (left panel) with a spectral domain OCT image from the same retinal location. The vertical green arrow in each en face image indicates the specific location illustrated by sdOCT and presented in the right-adjacent panel. Retinal layer organization is similar in all dogs INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 8. 
 
In vivo images of retinal structure of cRPGRIP1+/Ins (A) and cRPGRIP1Ins/Ins (B, C) dogs. Each row of images compares the near-infrared en face view (left panel) with a spectral domain OCT image from the same retinal location. The vertical green arrow in each en face image indicates the specific location illustrated by sdOCT and presented in the right-adjacent panel. Retinal layer organization is similar in all dogs INL, inner nuclear layer; ONL, outer nuclear layer.
To evaluate the localization of photoreceptor proteins, single immunolabeling and DAPI nuclear staining were carried out following paraffin removal and dehydration of the sections. The fixative used permits excellent immunolabeling of rod opsin, while preserving labeling of cones with hCAR and R/G opsin antibodies. 21 For other antibodies, it is marginally or not effective. The immunolabeling results of cRPGRIP1+/Ins used as controls were comparable to those obtained previously in retinas of cRPGRIP1+/+ dogs (data not shown). 
Retinal area 1 showed similar rod opsin immunolabeling in cRPGRIP1+/Ins and cRPGRIP1Ins/Ins dogs, with opsin labeling predominantly restricted to the outer segment layer (Figs. 5A2, 5B2). While an occasional rod cell showed opsin mislocalization in area 1 of cRPGRIP1Ins/Ins dogs, areas 2 and 3 showed more extensive opsin mislocalization, with opsin also localized to the outer nuclear layer (ONL) and synaptic terminals (areas 2 and 3; Figs. 6B2, 6C). In the cRPGRIP1+/Ins dog, antibodies directed against cones labeled the outer/inner segments, perinuclear region, and synaptic terminals (hCAR), or outer segments (R/G cone opsin) (Figs. 5A3, 5A4). However, in the cRPGRIP1Ins/Ins dog, the cone outer and inner segments did not label with hCAR, and labeling was concentrated in the apical perinuclear region. In addition, R/G cone opsin–positive outer segments were rarely evident even in area 1, the region that appeared normal in H&E sections and had minimal rod opsin mislocalization (Figs. 5B3, 5B4). 
In Vivo Retinal Imaging.
Noninvasive in vivo retinal imaging using high-resolution sdOCT was undertaken to assess retinal integrity in dogs retained for breeding. For this we used one cRPGRIP1+/Ins (R27) and two cRPGRIP1Ins/Ins dogs, one with normal (R17) and one with absent (R5) cone ERG responses. Qualitatively, both the 30 × 20-degree area parallel linear scans of each quadrant centered on the optic disc (data not shown) and the single high-resolution averaged scans (Fig. 8) were normal and were similar to scans of the heterozygous control and other nonaffected dogs (data not shown). However, direct measurements of ONL thickness indicated that the heterozygous control had a thicker ONL in the S and I quadrants near the optic nerve head (S = 62 μm per I = 53 μm) than did the two cRPGRIP1Ins/Ins dogs (R17: S = 50 μm per I = 43 μm; R5: S = 49 μm per I = 41 μm). These differences could be associated with the Ins44 genotype irrespective of the phenotype, or could result from aging, as the control dog was the youngest of the ones imaged. The histopathology and in vivo retinal imaging studies in cRPGRIP1Ins/Ins dogs demonstrated that photoreceptors are not progressively lost over time, at least within the time frame (between 1.5 and 3.5 years) of the current study. 
Ins44 Mutation Produces Skipping of Exon 2, but Does Not Prevent Expression of RPGRIP1 RID Domain.
The studies described above do not support a causative role for the Ins44 in cord1. However, owing to the vital role of RPGRIP1 in photoreceptor function, we decided to determine if Ins44 mutation is indeed associated with the generation of a stop codon, as originally predicted. 5 Molecular analysis of cRPGRIP1 retinal transcripts indicated that Ins44 was associated with exon 2 skipping, which eliminated the mutation from the transcript (Fig. 9A). Skipping of exon 2 in the 344-bp RT-PCR product was verified by direct sequencing of the product (Fig. 9B). In silico translation of the aberrant transcript, that is, the reference sequence without exon 2, predicts that it encodes a protein product nearly identical to the reference full-length protein, but with a truncated 5′-region and the putative start codon (first Met) now encoded by exon 5. 
Figure 9. 
 
Analysis of splicing pattern and protein expression of cRPGRIP1 in cRPGRIP1Ins/Ins dogs. (A) Analysis of splicing variants of cRPGRIP1 by RT-PCR in cRPGRIP1Ins/Ins and wild-type retinas. Primers 1EX, F and 5EX, R were used for the experiment (see Supplementary Table S1). In cRPGRIP1Ins/Ins, exon 2 is “skipped” during splicing and is not included in the final processed mRNA. The truncated PCR product is indicated by white arrow. Heteroduplex formed during PCR from mixtures of DNA templates is indicated by asterisk. (B) Sequence of the truncated cDNA containing exons 1 and 3 spliced together. (C) Western blot analysis performed with Ab38 antibody against RPGRIP1 RID domain did not show a significant difference between cRPGRIP1Ins/Ins, cRPGRIP1+/Ins, and wild-type dogs. NTC, no template control.
Figure 9. 
 
Analysis of splicing pattern and protein expression of cRPGRIP1 in cRPGRIP1Ins/Ins dogs. (A) Analysis of splicing variants of cRPGRIP1 by RT-PCR in cRPGRIP1Ins/Ins and wild-type retinas. Primers 1EX, F and 5EX, R were used for the experiment (see Supplementary Table S1). In cRPGRIP1Ins/Ins, exon 2 is “skipped” during splicing and is not included in the final processed mRNA. The truncated PCR product is indicated by white arrow. Heteroduplex formed during PCR from mixtures of DNA templates is indicated by asterisk. (B) Sequence of the truncated cDNA containing exons 1 and 3 spliced together. (C) Western blot analysis performed with Ab38 antibody against RPGRIP1 RID domain did not show a significant difference between cRPGRIP1Ins/Ins, cRPGRIP1+/Ins, and wild-type dogs. NTC, no template control.
Because mutation-associated exon skipping is predominantly associated with disruption of an exonic splicing enhancer (ESE), we attempted to determine if Ins44 interrupts an ESE motif. To do so, we analyzed the sequence of exon 2 with the ESE prediction software RESCUE-ESE. 22 Among the ESEs predicted in this region, one (hexamer GATGAG) is disrupted by the Ins44 (GATG-Ins44-AG), and therefore could provide a putative molecular mechanism leading to exon 2 skipping in cRPGRIP1
Western blot analysis was then undertaken on retinal tissue extracts from 7-month-old cRPGRIP1+/Ins and cRPGRIP1Ins/Ins dogs, and 7- and 16-week-old wild-type samples, using antibody Ab38 specific for the conserved RID domain of RPGRIP1 (Fig. 9C). Ab38 has been previously shown to identify approximately 33-, 45-, and 160-kDa protein isoforms in canine retina, but the 160-kDa isoform is clearly observed only upon longer exposure of the blot. 20 In our hands, two predominant RPGRIP1 immunoreactive isoforms with apparent masses of 37 kDa and 45 kDa were detected in retinal extracts of all samples tested, but the 160-kDa band was not visible. Thus, despite exon 2 skipping in cRPGRIP1Ins/Ins dogs, the resulting protein pattern detected by Ab38 is the same as in cRPGRIP1+/+ and cRPGRIP1+/Ins retinal samples, and the RPGR-interacting domain, critical for RPGRIP1 function, is preserved. 
Lack of Correlation between Ins44 and Retinal Gene Expression.
Although our results indicate that production of the aberrant C-terminally truncated protein is highly unlikely, Ins44 could still affect the expression of cRPGRIP1 isoforms, and directly or indirectly contribute to the disease. To address this possibility, expression studies were carried out on total retinal RNA from cRPGRIP1Ins/Ins (R18, R21), cRPGRIP1+/Ins (R16, R22), and three nonaffected wild-type dogs to examine the relative expression level of nine cRPGRIP1 splice variants and other photoreceptor-specific genes. The samples analyzed came from littermate dogs (R16, R18, R21, R22) that had normal clinical retinal examination findings, but absent 30-Hz cone ERG signals (R18 and R21) (Figs. 1, 3). In addition, dogs R21 and R22 were used for the morphologic studies that showed cone and midperipheral/peripheral rod abnormalities in R22 (see Figs. 5, 6). 
We first investigated the expression profile of nine cRPGRIP1 splice variants; owing to the substantial heterogeneity, gene expression data for each dog are presented individually (Fig. 10A). As expected, Ins44 homozygous dogs showed negligible 1/2-3EX transcript levels, while transcript levels in Ins44 heterozygotes were reduced by approximately 0.4- to 0.8-fold from control values. The lowest levels of 2-5a, 2-5b, 2-5c, and 21-22EX transcripts were observed in both cRPGRIP1Ins/Ins retinas and one cRPGRIP1+/Ins retina. Isoform 19c-2 was reduced in the two male dogs, irrespective of their Ins44 genotype. The other cRPGRIP1 isoforms showed expression levels comparable to normal control in female retinas and decreased expression level in male regardless of Ins44 genotype. The results suggest that Ins44 does not change the transcription of cRPGRIP1 but affects only splicing of exon 2, and excludes any evidence of nonsense-medicated decay pathway. 2325  
Figure 10. 
 
Gene expression analysis in Ins44-homozygous and heterozygous dogs compared to normal wild-type control. (A) Relative quantification of nine cRPGRIP1 splice variants expressed in the retina: 5- and 7-month-old controls (n = 3), 7-month-old Ins44-homozygous (R18, male; R21, female) and heterozygous (R16, male; R22, female) dogs. (B) Relative quantification of cone-expressed (OPN1SW, OPN1LW, CNGB3), rod-expressed (RHO, CNGA1, CNGB1), photoreceptor/pineal gland–specific (SAG), and astrocyte/Müller cell–specific (GFAP) gene markers to characterize photoreceptor gene expression in the corresponding dogs. Note: Because of heterogeneity in gene expression levels among the four siblings, the gene expression data are presented individually.
Figure 10. 
 
Gene expression analysis in Ins44-homozygous and heterozygous dogs compared to normal wild-type control. (A) Relative quantification of nine cRPGRIP1 splice variants expressed in the retina: 5- and 7-month-old controls (n = 3), 7-month-old Ins44-homozygous (R18, male; R21, female) and heterozygous (R16, male; R22, female) dogs. (B) Relative quantification of cone-expressed (OPN1SW, OPN1LW, CNGB3), rod-expressed (RHO, CNGA1, CNGB1), photoreceptor/pineal gland–specific (SAG), and astrocyte/Müller cell–specific (GFAP) gene markers to characterize photoreceptor gene expression in the corresponding dogs. Note: Because of heterogeneity in gene expression levels among the four siblings, the gene expression data are presented individually.
We next examined the expression of three cone-specific (OPN1SW, OPN1LW, CNGB3), four rod-specific (SAG, RHO, CNGA1, CNGB1), and one inner retinal (GFAP) gene, the expression of which are known to be altered in a variety of inherited retinal degenerations. With the exception of GFAP, which showed increased expression in 3 of the 4 retinas examined (two homozygous [R18, R21] and one heterozygous [R22]), the expression patterns for these genes generally paralleled those of cRPGRIP1 isoforms in that the two retinas with the lowest levels of cRPGRIP1 expression (one homozygous and one heterozygous male) also had lower expression of rod- or cone-specific genes (compare Fig. 9A with Fig. 9B). Analysis of additional heterozygous dogs did not show sex-related differences (data not shown). The results showed that Ins44 affects expression of only a restricted set of cRPGRIP1 isoforms, which normally have exon 2 in their structure. Moreover, the relative abundance of the remaining RPGRIP1 transcripts, as well as transcripts from eight photoreceptor-specific genes analyzed, was not associated with Ins44 genotype. 
Exclusion of the RPGRIP1 Gene from Direct Causal Association with cord1
Identification of a Novel Mutation Affecting Splicing of 3′-Terminal Alternative Exon 19C.
We next assessed if alternative mutations in the cRPGRIP1 gene could instead be responsible for the disease, either independently of or synergistically with Ins44. To this end, we identified a homozygous three-nucleotide deletion (D3) in cRPGRIP1Ins/Ins dogs that eliminates the 3′ acceptor splice site and the first nucleotide of alternative 3′-terminal exon 19c, which is common for the group of transcripts driven by an internal promoter and which potentially encodes proteins without RID.15 To address the possibility that the D3 sequence change reduces the efficiency of polyadenylation26,27 and cRPGRIP1 splicing, we performed comparative RT-PCR analysis with cDNA generated from a polyT primer from wild-type and D3-homozygous dogs. From our previous 3′RACE data, the 3′UTR sequence following exon 19c was believed to be incomplete since a stretch of oligo-A (A15) is present in the DNA sequence, suggesting possible mispriming of the polyT primer.15 Therefore, to estimate the putative size of the 3′UTR, we next used a forward primer designed to bind to exon 19 and four reverse primers (Rev1, Rev2, Rev3, Rev4) binding to different positions in the 3′UTR flanking exon 19c (Fig. 11A, Supplementary Table S1). Such primer design allows for detection of spliced RNA that has not yet been cleaved for polyadenylation. Each primer pair yielded a diverse population of products from wild-type dog cDNA (Fig. 11B). The F-Rev1 pair of primers gave eight products (1s–8s), F-Rev2 produced six products (1s–6s), while F-Rev3 and F-Rev4 gave rise to only one product each (6s and 9s, respectively) (Figs. 11B, 11C; Supplementary Table S2). These results indicated that different transcripts may derive from alternate use of polyadenylation signals in the 3′UTR flanking exon 19c. A 3′RACE experiment with a forward primer located in exon 19 showed four bands corresponding to the four cRPGRIP1 transcripts with different alternative 3′-terminal exons (19a, 19c, 19d, or 24) as previously described15 (Fig. 11D). 
Figure 11. 
 
Analysis of cRPGRIP1 splicing pattern in D3-homozygous dogs. (A) Localization of primers (F, Rev1-4) used in RT-PCR. (B) RT-PCR performed in wild-type dog produced multiple transcripts. Several new exons were identified (for exon boundaries see Supplementary Table S2). In D3-homozygous retina, the number of transcripts detected is greatly reduced in comparison to normal control. PCR products detected are listed only by number because of space limitation. (C) Schematic representation of transcripts detected by RT-PCR in wild-type and D3-homozygous dogs. Asterisks represent position of a predicted stop codon. Note: Supplementary Table S2 indicates how transcript numbers in Figure 11C correspond to PCR product numbers in Figure 11B. (D) 3′RACE data show absence of the main transcript having alternative exon 19c. Asterisk shows PCR products heteroduplex.
Figure 11. 
 
Analysis of cRPGRIP1 splicing pattern in D3-homozygous dogs. (A) Localization of primers (F, Rev1-4) used in RT-PCR. (B) RT-PCR performed in wild-type dog produced multiple transcripts. Several new exons were identified (for exon boundaries see Supplementary Table S2). In D3-homozygous retina, the number of transcripts detected is greatly reduced in comparison to normal control. PCR products detected are listed only by number because of space limitation. (C) Schematic representation of transcripts detected by RT-PCR in wild-type and D3-homozygous dogs. Asterisks represent position of a predicted stop codon. Note: Supplementary Table S2 indicates how transcript numbers in Figure 11C correspond to PCR product numbers in Figure 11B. (D) 3′RACE data show absence of the main transcript having alternative exon 19c. Asterisk shows PCR products heteroduplex.
The same RT-PCR analysis was carried out in a cRPGRIP1 D3-homozygous dog. In addition to the correct splicing patterns, we observed several different splicing abnormalities, including exon 19c skipping and cryptic splicing that greatly reduced transcript diversity. A comparable 3′RACE analysis on 2 retinas of dogs homozygous for D3 and Ins44 showed no band corresponding to the main transcript-containing exon 19c (Fig. 11D), suggesting abnormal polyadenylation. 
We next investigated potential correlations between the Ins44/D3 genotype and the disease phenotype. We used a set of 59 dogs, all of which had been previously genotyped for Ins44, and which included 47 MLHD or MLHD-crossbred dogs from the study pedigree and the pet population, 11 ESSs, and 1 ECS. All dogs were genotyped for the D3 variant (Table 1). All cRPGRIP1Ins/Ins dogs studied also were homozygous for D3. Several, but not all, of these dogs had retinal cone functional abnormalities (study pedigree) or clinically evident retinal degeneration (colony founders and dogs from the same or other breeds in the pet population). Several cRPGRIP1+/Ins or cRPGRIP1+/+ dogs were homozygous for D3; none of these had evidence of retinal dysfunction or disease. A further 30 dogs, comprising 12 Siberian Husky–derived crossbreds, 8 Beagles, and 10 Papillons were genotyped only for Ins44, and all were cRPGRIP1+/+ . As clinically normal dogs can be homozygous for both D3 and Ins44, we concluded that neither is sufficient to cause cord1
Haplotype Analysis of cRPGRIP1 for Disease Association.
To further explore the association between cRPGRIP1 and retinal disease, we extended the mutation analysis of cRPGRIP1, and the results provided data to develop a gene-specific haplotype for association studies. We searched for mutations in the coding region, promoter (P1, P2, 1000 bp each), and 3′UTR (600 bp) of this gene in two cRPGRIP1Ins/Ins dogs (R5, R10) and several clinically normal wild-type dogs (MLHD, Siberian Husky, and Beagle breeds). In addition to Ins44 and D3, eight other sequence changes were detected in the analysis. We also performed a genotyping pilot study of cRPGRIP1 sequence changes in additional dogs of different breeds. Table 2 represents the summary of the results. 
Table 2. 
 
Sequence Changes Identified in Canine RPGRIP1 Gene
Table 2. 
 
Sequence Changes Identified in Canine RPGRIP1 Gene
Location of Sequence Change Nucleotide Change Amino Acid Change Breed
Promoter (P1) −197C > T Siberian Husky, Beagle
Intron 1 c.19+19T > A MLHD/MLHDx, ESS, ECS
Exon 2 c.142_143ins44 MLHD/MLHDx, ESS, ECS
Intron 2 c.152+186_227del42 Siberian Husky
Intron2 c.152+227_228ins205 Siberian Husky
Exon 14 c.1740C > T p.P580P Siberian Husky
Exon 17 c.2548G > A p.E850K Siberian Husky, Beagle
Intron/exon 19c* c.548-2_c.548del3 MLHD/MLHDx, ESS, ECS
Exon 24 c.3494T > C p.V1165A MLHD, Siberian Husky, Beagle
Exon 24 c.3531(GGGAGCCGA)2-7 p.1178(GAE)2-7 MLHD, Siberian Husky, Beagle, Papillon
Five of these sequence changes were found in purebred or MLHD-derived dogs (MLHDx), and were chosen for haplotype analysis. The haplotype panel for analysis included: c.19+19T > A; c.142_143ins44 (Ins44); c.548-2_c.548del3 (D3); c.3494T > C, and c.3531(GGGAGCCGA)2-7 (Table 2). These markers covered a total of 63.7 kb of cRPGRIP1 genomic region and were used for comparative haplotyping of 8 MLHD or MLHDx crossbreds (0.6–4.4 years), 10 ESSs (2.8–15.5 years), and 1 ECSs (1.8 years), all cRPGRIP1Ins/Ins
From previously published data of strong association of Ins44 with cord15 , homozygosity for Ins44 was an inclusion selection criterion in the group analyzed. The selected retinal phenotypes ranged from clinically normal, absent ERG 30-Hz cone function, or advanced retinal degeneration. Results of genotyping and phenotype characteristics are presented in Table 3. Without exception, all dogs had a single 5-locus homozygous haplotype (2-2-2-1-3; see Table 3). Results indicated that there is no association between extended haplotype of cRPGRIP1 and retinal degeneration. 
Table 3. 
 
cRPGRIP1 Haplotype and Phenotype of cRPGRIP1 Ins/Ins Dogs
Table 3. 
 
cRPGRIP1 Haplotype and Phenotype of cRPGRIP1 Ins/Ins Dogs
Sample ID c.19+19T > A Ins44 D3 p.V1165A p.1178(GAE)2-7 Phenotype* Age (y) Breed
R5 22 22 22 11 33 NVL/absent cone ERG 1.8 MLHDx
R10 22 22 22 11 33 Retinal degeneration (late stage) 2.6 MLHD
R18 22 22 22 11 33 NVL, absent cone ERG 0.7 MLHDx
R21 22 22 22 11 33 NVL, absent cone ERG 0.7 MLHDx
Molson 22 22 22 11 33 PRA 4.4 MLHD
Josie 22 22 22 11 33 NVL 1 MLHD
Caroline 22 22 22 11 33 NVL 3.5 MLHD
Clair 22 22 22 11 33 “PRA suspicious” 1.3 MLHD
George 22 22 22 11 33 PRA 13.8 ESS
Minnie 22 22 22 11 33 PRA 9.9 ESS
Keah 22 22 22 11 33 PRA 15.5 ESS
Stella 22 22 22 11 33 PRA 6.2 ESS
Emma 22 22 22 11 33 PRA 2.8 ESS
Holly 22 22 22 11 33 PRA 13 ESS
Genesis 22 22 22 11 33 NVL 7 ESS
Mackenzie 22 22 22 11 33 NVL 11.4 ESS
Kayla 22 22 22 11 33 NVL 11 ESS
Tucker 22 22 22 11 33 NVL 8.3 ESS
Louie 22 22 22 11 33 NVL 1.8 ECS
Discussion
The original studies that established a causal association between cRPGRIP1Ins/Ins and cord1 have been carried out in a closed research colony in which affected dogs show ophthalmoscopic evidence of retinal disease at approximately 25 weeks of age, which progressed to advanced degeneration before 1 year of age. 16 Thirty-Hz cone ERG responses are markedly reduced at 6 weeks of age, and by 30 weeks of age, both rod and cone ERG responses are barely detectable. 16 Although there is considerable interanimal variation of cone responses, progression is the rule, not the exception. 16 Histologic abnormalities have also been observed in the photoreceptors of affected dogs by 6 weeks of age, with shortening of the inner and outer segments, and opsin delocalization. These changes are progressive, resulting in an approximately exponential decay in photoreceptor cell numbers in the outer nuclear layer with age. Similar abnormalities also have been reported in a colony of MLHDs in France that was derived from the AHT dogs. 28 All affected dogs have a homozygous 44-nucleotide insertion in the 3′ end of exon 2 that is predicted to alter the reading frame, thereby introducing a premature stop codon that could presumably lead to a truncated or absent protein that lacks the RID domain and is causal to the disease. 5 However, despite the highly consistent genotype–phenotype correlation in the original AHT research colony, significant discordance has been found in the general MLHD pet population, and the onset of disease is highly variable if at all present. 11,12,14  
To more definitively address the role of cRPGRIP1 in the disease, we established a research colony whose four founders included two affected dogs (cRPGRIP1Ins/Ins ) that had early-onset retinal degeneration, one of which came from the AHT research colony described above. 5,16 Of the Ins44 homozygous dogs produced, all six dogs examined after 1 year of age were ophthalmoscopically normal (age range: 1–3.5 years), a time at which advanced retinal degeneration would have been expected among the affected dogs in the original research colony. 5,16 Cone-mediated 30-Hz flicker ERG responses were variable in cRPGRIP1Ins/Ins dogs, being nonrecordable in five, normal in two, and markedly reduced in amplitude in one. Moreover, repeated ERGs in several of the dogs clearly indicated that these responses were stable and that rod-mediated responses were not lost over time. With the exception of the two colony founders that had retinal degeneration leading to early-onset blindness, the results indicated that the functional deficits are nonprogressive, at least in the time period of the study, and that ERG cone functional abnormalities, when present, do not predict photopic visual performance. The data were not consistent with the previously published phenotype of cord1 dogs 16 or RPGRIP1 knockout (RPGRIP1 tm1Tili and RPGRIP1 nmf247) mice 29,30 that have early and severe photoreceptor abnormalities. Together, these results indicated that Ins44 in RPGRIP1 is not the primary cause of the disease. 
In the dog, the Ins44 variant in exon 2 has been predicted to alter the reading frame of the transcript, thereby introducing a premature stop codon. 5 We established that, instead of altering the reading frame, the Ins44 mutation induces skipping of exon 2. Comparable expression of RPGRIP1 in cRPGRIP1Ins/Ins , cRPGRIP1+/Ins , and cRPGRIP1+/+ retinas was found by Western blot analysis using an antibody against the RID domain, a finding that argues against introduction of a premature stop codon. Moreover, preservation of the RID domain, which is critical for RPGRIP1–RPGR interaction, 7 means that the Ins44 variant does not prevent expression and translation of transcripts bearing a normal 3′ end that encodes RID, suggesting that the protein is likely functional. In our experiment, we only detected two highly abundant translated products of 37 kDa and 45 kDa. A higher molecular mass protein of approximately 160 kDa, previously observed only upon longer exposure of the blots, 20 was not found, probably because of proteolysis. Despite the fact that cRPGRIP1 gives rise to multiple isoforms, there are no data so far to support their efficient translation, since some of them have been detected at very low expression levels. 15 In addition, some cRPGRIP isoforms, such as 19c-1 and 19c-2, have no RID domain to be detected with Ab38, specific for the conserved RID. 
Web-based analysis using RESCUE-ESE to identify putative ESEs suggests that inactivation of an ESE is the most likely mechanism underlying exon skipping in the Ins44 variant in cRPGRIP1 exon 2. Moreover, bioinformatics analysis of aberrant cRPGRIP1 sequences also suggests that Ins44 will affect the N-terminus of some cRPGRIP1 isoforms. 15 Previous studies have shown that certain RPGRIP1 isoforms undergo limited proteolysis, leading to the relocation and nuclear accumulation of a small N-terminal domain encoded by first three exons of RPGRIP1. 31 In this scenario, alteration of the N-terminal sequence of cRPGRIP1 by Ins44 could affect nuclear import of the RPGRIP1 nuclear domain. Although the ultimate effect of such alterations in nuclear import is unclear, our data to date suggest that it does not cause disease or affect progression. 
To further determine if changes in cRPGRIP1 isoforms and/or photoreceptor-specific gene expression could contribute to disease progression, we used quantitative RT-PCR (qRT-PCR) to examine retinal mRNA expression levels in wild-type control and dogs homozygous or heterozygous for Ins44. The homozygous dogs used showed absent cone ERG responses, and morphologic examination in one cone function-deficient dog identified abnormalities consistent with loss of cone outer segments, and regional rod opsin mislocalization. While the expression results for exon 2 were consistent in dogs with Ins44 (negligible in cRPGRIP1Ins/Ins , and 0.4–0.8 reduction in cRPGRIP1+/Ins ), there was marked variability of expression for the other cRPGRIP1 isoforms, which was not dependent on the Ins44 genotype. Expression of other photoreceptor-specific genes was equally variable, and not associated with the Ins44 genotype. Initially, we suspected that the gene expression profiles could be sex dependent, but analysis of additional heterozygous dogs did not show any sex-related differences (data not shown). 
As our results did not support the previously proposed mechanism that cRPGRIP1 Ins44 is largely truncated and thus dysfunctional, we next sought to identify additional or alternative mutations in RPGRIP1 that could provide insight into its role in the clinical spectrum of retinal disease. To this end, we have reported nine previously uncharacterized sequence changes including three intronic mutations, a silent mutation in exon 14, missense mutations in exons 17 and 24, a microsatellite polymorphism in exon 24, and a deletion (D3) that removes the 3′ splice site and first nucleotide of alternative exon 19c. 15 Using RT-PCR and 3′RACE, we next demonstrated that the D3 mutation reduces the normal 3′ splicing pattern, activates a cryptic splice site, and promotes generation of erroneous splicing isoforms containing “pseudoexon” 19c-4. As both Ins44 and D3 alter the splicing of cRPGRIP1 and defects in alternative splicing contribute to disease, 3234 we next examined whether the D3 deletion can modify the Ins44 disease phenotype by assessing their distribution among 59 dogs that were either clinically normal or had retinal degeneration. In most cases, cRPGRIP1Ins/Ins dogs also are homozygous for D3, an indication that the two are in linkage disequilibrium. However, as clinically normal dogs can also be homozygous for both D3 and Ins44, we conclude that neither is sufficient to cause cord1; it is yet to be determined if the combined changes contribute to an increased risk for developing cord1
We have also extended the cRPGRIP1 disease-association analysis by using five polymorphic markers spanning almost the entire gene sequence. Notably, all 19 Ins44 homozygous dogs (MLHDs, MLHDx, ESSs, and ECSs) bore the same haplotype, regardless of their clinical phenotype. Moreover, the four ESSs without retinal degeneration ranged in age between 7 and 11.4 years, much older than the typical disease onset of either cord1 or the ESS breed-specific retinal disease (Table 3). For cRPGRIP1Ins/Ins MLHDs from the purebred pet population, one had overt retinal degeneration at 4.4 years, one was suspected to have degeneration (1.3 years), and two were normal (1 and 3.5 years). cord1 was ruled out in the dogs clinically normal at or above 1 year of age, as typical cord1 cases in the original research colony have shown recognizable retinal degeneration by this age. Given that retinal degeneration in ESSs and MLHDs has each been recognized as autosomal recessive, and the same cRPGRIP1 extended haplotype occurs in clinically normal and retinal degenerate animals, it is unlikely that this gene is causally associated with the disease by itself. 
Based on a previous study that has shown that not all cRPGRIP1Ins/Ins dogs develop clinically recognizable retinal degeneration, 11,12 a recent genome-wide association study in cRPGRIP1Ins/Ins dogs stratified by phenotype has mapped an independent locus which, in the homozygous state, correlates with an early-onset phenotype. 14 The newly identified locus is located approximately 35 Mb from RPGRIP1 on CFA15, and the genes in its 1.49-Mb interval are being analyzed to determine a role as a cord1 disease modifier (Keiko Miyadera et al., unpublished data). While the identification of the second locus does not necessarily lend further support to cRPGRIP1Ins/Ins being causal to cord1, it is essential to unequivocally establish the disease gene at the first locus. Mapping of the first locus to a 14.15-Mb interval by Mellersh et al. 5 benefited from the use of the research colony, which subsequently was shown to be fixed for the second locus. 14 The involvement of the first locus is definitive, based on the perfect association with cord1 in the AHT research colony. RPGRIP1 is located within the mapped cord1 region of 14.15 Mb on CFA15, 5 as well as within the narrowed 1.74-Mb critical disease interval, 11 and has been the most likely disease candidate given the predicted severity of the insertion. However, despite its important functional role in photoreceptors, and its interactions with a number of ciliary proteins in visual cells, 29,30,35 our study indicated that mutations in RPGRIP1 are not the primary or sole cause of cord1, and the real causative mutation representing the locus initially mapped by Mellersh et al. 5 is in physical proximity with RPGRIP1 in the narrowed 1.74-Mb critical disease interval. 11 In such a case, cRPGRIP1 Ins44 could represent a benign polymorphism that has been fixed in some populations, for example, AHT research colony 14 and many ESSs, or could be a cord1 disease modifier. For that reason, identification of the direct causal gene/mutation will provide new insights into animal and human retinopathies. 
Supplementary Materials
Acknowledgments
The authors thank Cathryn Mellersh, Animal Health Trust, United Kingdom, for providing semen for one of the colony founders (C-UK1) with cRPGRIP1 Ins44 and affected with early-onset cord1; Paulo Ferreira for anti-RPGRIP Ab38; Sem Genini and Jessica Rowlan for providing TaqMan probes for gene expression analysis; Gregory M. Acland, Jacob G. Appelbaum, and Leslie King for helpful discussions and comments; the RDSF staff for excellent animal care and technical support; Svetlana Savina for immunohistochemistry; and Mary Leonard of the Biomedical Art and Design facility at the University of Pennsylvania for illustrations. 
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Footnotes
 Supported by National Eye Institute, National Institutes of Health Grants EY-06855, -010420, -13132, -17549, -018705, P30 EY-001583, Foundation Fighting Blindness center grant and Career Development Award (KM), Van Sloun Fund for Canine Genetic Research, Hope for Vision, and The ONCE International Prize for R&D in Biomedicine and New Technologies for the Blind.
Footnotes
 Disclosure: T. Kuznetsova, None; S. Iwabe, None; K. Boesze-Battaglia, None; S. Pearce-Kelling, OptiGen LLC (E); Y. Chang-Min, None; K. McDaid, None; K. Miyadera, None; A. Komaromy, None; G.D. Aguirre, OptiGen LLC (C), P
Figure 1. 
 
Pedigree of research colony informative for cRPGRIP1 Ins44. The dogs with clinical retinal degeneration are indicated by **. Dogs with absent 30-Hz ERG cone flicker responses are indicated by oblique black arrows; oblique gray dashed arrow indicates dog (R28) with markedly reduced but still present 30-Hz ERG cone flicker responses. All dogs with cone ERG abnormalities are cRPGRIP1Ins/Ins , but two dogs with this genotype (R17 and R42) have cone responses.
Figure 1. 
 
Pedigree of research colony informative for cRPGRIP1 Ins44. The dogs with clinical retinal degeneration are indicated by **. Dogs with absent 30-Hz ERG cone flicker responses are indicated by oblique black arrows; oblique gray dashed arrow indicates dog (R28) with markedly reduced but still present 30-Hz ERG cone flicker responses. All dogs with cone ERG abnormalities are cRPGRIP1Ins/Ins , but two dogs with this genotype (R17 and R42) have cone responses.
Figure 2. 
 
Fundus photographs of selected research colony dogs taken at different ages. Only dog R10 (A) has generalized retinal degeneration with marked vascular attenuation and tapetal hyperreflectivity. The retinas in the other dogs are clinically normal: R10, R17, and R29 are cRPGRIP1Ins/Ins ; R13, R14, and R30 are cRPGRIP1+/Ins . See Table 1 for details on clinical examination results and ages.
Figure 2. 
 
Fundus photographs of selected research colony dogs taken at different ages. Only dog R10 (A) has generalized retinal degeneration with marked vascular attenuation and tapetal hyperreflectivity. The retinas in the other dogs are clinically normal: R10, R17, and R29 are cRPGRIP1Ins/Ins ; R13, R14, and R30 are cRPGRIP1+/Ins . See Table 1 for details on clinical examination results and ages.
Figure 3. 
 
ERG data for MLHD dogs with different Ins44 genotypes. ERGs were recorded at 0.4 years of age (R4–R8; top panel), and at 0.4 years (R16–R22) or 0.3 years (R25; lower panel). All cRPGRIP1Ins/Ins dogs are also homozygous for the D3 deletion. Among the cRPGRIP1Ins/Ins dogs, R5, R18, and R21 lack recordable cone ERG responses, but these are present in R17.
Figure 3. 
 
ERG data for MLHD dogs with different Ins44 genotypes. ERGs were recorded at 0.4 years of age (R4–R8; top panel), and at 0.4 years (R16–R22) or 0.3 years (R25; lower panel). All cRPGRIP1Ins/Ins dogs are also homozygous for the D3 deletion. Among the cRPGRIP1Ins/Ins dogs, R5, R18, and R21 lack recordable cone ERG responses, but these are present in R17.
Figure 4. 
 
ERG and objective vision testing in cRPGRIP1Ins/Ins dogs. (A) Both dogs have rod-mediated responses that show minimal to no changes at the two time points examined. Cone 30-Hz flicker responses are absent in one of the dogs. (B) Objective vision testing evaluated with an obstacle avoidance course. The graph shows the transit time in seconds for dogs R42 (white bar) and R46 (black bar) as a function of ambient light intensity; results are shown for the 6-, 8-, and 10-month time periods. Both dogs show comparable and normal visual performance under scotopic and photopic illumination, and no change with time. Values in gray are the mean ± 1 SD for transit times in normal dogs. 19
Figure 4. 
 
ERG and objective vision testing in cRPGRIP1Ins/Ins dogs. (A) Both dogs have rod-mediated responses that show minimal to no changes at the two time points examined. Cone 30-Hz flicker responses are absent in one of the dogs. (B) Objective vision testing evaluated with an obstacle avoidance course. The graph shows the transit time in seconds for dogs R42 (white bar) and R46 (black bar) as a function of ambient light intensity; results are shown for the 6-, 8-, and 10-month time periods. Both dogs show comparable and normal visual performance under scotopic and photopic illumination, and no change with time. Values in gray are the mean ± 1 SD for transit times in normal dogs. 19
Figure 5. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A4) or cRPGRIP1Ins/Ins (R21, B1B4) dogs. Images are taken from region near and inferior to the optic disc (area 1), and stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2), hCAR (A3, B3), or R/G opsin (A4, B4) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the central regions, retinal integrity is normal in the cRPGRIP1+/Ins or cRPGRIP1Ins/Ins dogs (A1, A2), and opsin immunolabeling is comparable although focal delocalization of opsin is present (B2, arrow). In the control, hCAR (A3, oblique arrows) and R/G opsin (A4) antibodies label the cones. In contrast, the cRPGRIP1Ins/Ins dog shows that hCAR labels only the outer perinuclear region of cone somata (B3, short arrows) and synaptic terminals, but labeling of cone IS and OS is not present, and only one cone OS labels with R/G opsin antibody (B4, arrow). Calibration marker in A1 = 20 μm and applies to all figures except those highlighted by boxed region in A3, A4, B3, B4, which is shown to the right of the main panels (calibration marker = 5 μm). GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium.
Figure 5. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A4) or cRPGRIP1Ins/Ins (R21, B1B4) dogs. Images are taken from region near and inferior to the optic disc (area 1), and stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2), hCAR (A3, B3), or R/G opsin (A4, B4) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the central regions, retinal integrity is normal in the cRPGRIP1+/Ins or cRPGRIP1Ins/Ins dogs (A1, A2), and opsin immunolabeling is comparable although focal delocalization of opsin is present (B2, arrow). In the control, hCAR (A3, oblique arrows) and R/G opsin (A4) antibodies label the cones. In contrast, the cRPGRIP1Ins/Ins dog shows that hCAR labels only the outer perinuclear region of cone somata (B3, short arrows) and synaptic terminals, but labeling of cone IS and OS is not present, and only one cone OS labels with R/G opsin antibody (B4, arrow). Calibration marker in A1 = 20 μm and applies to all figures except those highlighted by boxed region in A3, A4, B3, B4, which is shown to the right of the main panels (calibration marker = 5 μm). GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium.
Figure 6. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A2) or cRPGRIP1Ins/Ins (R21, B1B2, C) dogs. Images are taken from area 2, midpoint between the optic disc and ora serrata (A1A2, B1B2), or from the periphery (area 3, C). Sections stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2, C) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the cRPGRIP1Ins/Ins dog, there is progressive shortening and loss of rod outer segments, and opsin delocalization into the ONL (B2, C, oblique arrows and *, respectively). A subretinal macrophage is present in areas of rod outer segment shortening and loss (B1, arrowhead). Calibration marker in A1 = 20 μm and applies to all figures. NFL, nerve fiber layer; PR, photoreceptor layer.
Figure 6. 
 
Retinal photomicrographs of 0.7-year-old cRPGRIP1+/Ins (R22, A1A2) or cRPGRIP1Ins/Ins (R21, B1B2, C) dogs. Images are taken from area 2, midpoint between the optic disc and ora serrata (A1A2, B1B2), or from the periphery (area 3, C). Sections stained with H&E (A1, B1) or immunolabeled with anti–rod opsin (A2, B2, C) antibodies. DAPI nuclear stain was used with the immunolabeling, but only shown in half of the images. In the cRPGRIP1Ins/Ins dog, there is progressive shortening and loss of rod outer segments, and opsin delocalization into the ONL (B2, C, oblique arrows and *, respectively). A subretinal macrophage is present in areas of rod outer segment shortening and loss (B1, arrowhead). Calibration marker in A1 = 20 μm and applies to all figures. NFL, nerve fiber layer; PR, photoreceptor layer.
Figure 7. 
 
Outer nuclear layer counts from cRPGRIP1Ins/Ins (R21, R29) and cRPGRIP1+/Ins (R22, R8) dogs. Y-axis values represent the mean of three ONL nuclear layer thickness counts within a single microscopic field per location; for each dog, four defined retinal locations (two areas in the superior and two areas in the inferior meridian) are shown. There are no apparent differences between the two genotypes, or between dogs of different ages.
Figure 7. 
 
Outer nuclear layer counts from cRPGRIP1Ins/Ins (R21, R29) and cRPGRIP1+/Ins (R22, R8) dogs. Y-axis values represent the mean of three ONL nuclear layer thickness counts within a single microscopic field per location; for each dog, four defined retinal locations (two areas in the superior and two areas in the inferior meridian) are shown. There are no apparent differences between the two genotypes, or between dogs of different ages.
Figure 8. 
 
In vivo images of retinal structure of cRPGRIP1+/Ins (A) and cRPGRIP1Ins/Ins (B, C) dogs. Each row of images compares the near-infrared en face view (left panel) with a spectral domain OCT image from the same retinal location. The vertical green arrow in each en face image indicates the specific location illustrated by sdOCT and presented in the right-adjacent panel. Retinal layer organization is similar in all dogs INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 8. 
 
In vivo images of retinal structure of cRPGRIP1+/Ins (A) and cRPGRIP1Ins/Ins (B, C) dogs. Each row of images compares the near-infrared en face view (left panel) with a spectral domain OCT image from the same retinal location. The vertical green arrow in each en face image indicates the specific location illustrated by sdOCT and presented in the right-adjacent panel. Retinal layer organization is similar in all dogs INL, inner nuclear layer; ONL, outer nuclear layer.
Figure 9. 
 
Analysis of splicing pattern and protein expression of cRPGRIP1 in cRPGRIP1Ins/Ins dogs. (A) Analysis of splicing variants of cRPGRIP1 by RT-PCR in cRPGRIP1Ins/Ins and wild-type retinas. Primers 1EX, F and 5EX, R were used for the experiment (see Supplementary Table S1). In cRPGRIP1Ins/Ins , exon 2 is “skipped” during splicing and is not included in the final processed mRNA. The truncated PCR product is indicated by white arrow. Heteroduplex formed during PCR from mixtures of DNA templates is indicated by asterisk. (B) Sequence of the truncated cDNA containing exons 1 and 3 spliced together. (C) Western blot analysis performed with Ab38 antibody against RPGRIP1 RID domain did not show a significant difference between cRPGRIP1Ins/Ins , cRPGRIP1+/Ins , and wild-type dogs. NTC, no template control.
Figure 9. 
 
Analysis of splicing pattern and protein expression of cRPGRIP1 in cRPGRIP1Ins/Ins dogs. (A) Analysis of splicing variants of cRPGRIP1 by RT-PCR in cRPGRIP1Ins/Ins and wild-type retinas. Primers 1EX, F and 5EX, R were used for the experiment (see Supplementary Table S1). In cRPGRIP1Ins/Ins , exon 2 is “skipped” during splicing and is not included in the final processed mRNA. The truncated PCR product is indicated by white arrow. Heteroduplex formed during PCR from mixtures of DNA templates is indicated by asterisk. (B) Sequence of the truncated cDNA containing exons 1 and 3 spliced together. (C) Western blot analysis performed with Ab38 antibody against RPGRIP1 RID domain did not show a significant difference between cRPGRIP1Ins/Ins , cRPGRIP1+/Ins , and wild-type dogs. NTC, no template control.
Figure 10. 
 
Gene expression analysis in Ins44-homozygous and heterozygous dogs compared to normal wild-type control. (A) Relative quantification of nine cRPGRIP1 splice variants expressed in the retina: 5- and 7-month-old controls (n = 3), 7-month-old Ins44-homozygous (R18, male; R21, female) and heterozygous (R16, male; R22, female) dogs. (B) Relative quantification of cone-expressed (OPN1SW, OPN1LW, CNGB3), rod-expressed (RHO, CNGA1, CNGB1), photoreceptor/pineal gland–specific (SAG), and astrocyte/Müller cell–specific (GFAP) gene markers to characterize photoreceptor gene expression in the corresponding dogs. Note: Because of heterogeneity in gene expression levels among the four siblings, the gene expression data are presented individually.
Figure 10. 
 
Gene expression analysis in Ins44-homozygous and heterozygous dogs compared to normal wild-type control. (A) Relative quantification of nine cRPGRIP1 splice variants expressed in the retina: 5- and 7-month-old controls (n = 3), 7-month-old Ins44-homozygous (R18, male; R21, female) and heterozygous (R16, male; R22, female) dogs. (B) Relative quantification of cone-expressed (OPN1SW, OPN1LW, CNGB3), rod-expressed (RHO, CNGA1, CNGB1), photoreceptor/pineal gland–specific (SAG), and astrocyte/Müller cell–specific (GFAP) gene markers to characterize photoreceptor gene expression in the corresponding dogs. Note: Because of heterogeneity in gene expression levels among the four siblings, the gene expression data are presented individually.
Figure 11. 
 
Analysis of cRPGRIP1 splicing pattern in D3-homozygous dogs. (A) Localization of primers (F, Rev1-4) used in RT-PCR. (B) RT-PCR performed in wild-type dog produced multiple transcripts. Several new exons were identified (for exon boundaries see Supplementary Table S2). In D3-homozygous retina, the number of transcripts detected is greatly reduced in comparison to normal control. PCR products detected are listed only by number because of space limitation. (C) Schematic representation of transcripts detected by RT-PCR in wild-type and D3-homozygous dogs. Asterisks represent position of a predicted stop codon. Note: Supplementary Table S2 indicates how transcript numbers in Figure 11C correspond to PCR product numbers in Figure 11B. (D) 3′RACE data show absence of the main transcript having alternative exon 19c. Asterisk shows PCR products heteroduplex.
Figure 11. 
 
Analysis of cRPGRIP1 splicing pattern in D3-homozygous dogs. (A) Localization of primers (F, Rev1-4) used in RT-PCR. (B) RT-PCR performed in wild-type dog produced multiple transcripts. Several new exons were identified (for exon boundaries see Supplementary Table S2). In D3-homozygous retina, the number of transcripts detected is greatly reduced in comparison to normal control. PCR products detected are listed only by number because of space limitation. (C) Schematic representation of transcripts detected by RT-PCR in wild-type and D3-homozygous dogs. Asterisks represent position of a predicted stop codon. Note: Supplementary Table S2 indicates how transcript numbers in Figure 11C correspond to PCR product numbers in Figure 11B. (D) 3′RACE data show absence of the main transcript having alternative exon 19c. Asterisk shows PCR products heteroduplex.
Table 1. 
 
Ins44/D3 Genotype and Clinical Retinal Phenotype for Dogs Examined
Table 1. 
 
Ins44/D3 Genotype and Clinical Retinal Phenotype for Dogs Examined
Dog Ins44 Genotype* D3 Genotype* Clinical Retinal Phenotype-Inherited Retinal Degeneration Clinical/ERG Examination Age (y) Pathology-IHC, Age (y) Breed
Research colony
 R1 12 12 Normal fundus; no ERG performed 4.4 MLHDx
 R2 12 12 Normal fundus; no ERG performed 4.4 4.4 MLHDx
 R4 12 12 Normal fundus; rod/cone ERG responses 0.4 MLHDx
 R5 22 22 Normal fundus; absent cone ERG 3.1/0.4 MLHDx
 R6 12 12 No eye exam; rod/cone ERG responses 0.4 MLHDx
 R7 12 12 No eye exam; rod/cone ERG responses 0.4 MLHDx
 R8 12 12 Normal fundus; rod/cone ERG responses 2.9/0.4 2.9 MLHDx
 R9 11 12 Acquired retinal disease—detachment, inflammation, degeneration/no ERG performed 2.8 MLHD
 R10 22 22 Generalized retinal degeneration; no ERG performed 2.6 MLHD
 R11 12 22 Normal fundus; no ERG performed 0.5 MLHD
 R12 12 22 Normal fundus; rod/cone ERG responses 1.8 MLHD
 R13 12 22 Normal fundus; rod/cone ERG responses 2.6 MLHD
 R14 12 12 Normal fundus; no ERG performed 4.0 MLHD
 R15 12 12 Normal fundus; no ERG performed 0.5 MLHD
 R16 12 12 Normal fundus; rod/cone ERG responses 0.6/0.4 MLHDx
 R17 22 22 Normal fundus; rod/cone ERG responses 1.9/1.8 MLHDx
 R18 22 22 Normal fundus; absent cone ERG 0.6/0.4 MLHDx
 R21 22 22 Normal fundus; absent cone ERG 0.6/0.4 0.7 MLHDx
 R22 12 12 Normal fundus; rod/cone ERG responses 0.6/0.4 0.7 MLHDx
 R25 11 Normal fundus; rod/cone ERG responses 0.4/0.5 MLHDx
 R26 12 12 Normal fundus; rod/cone ERG responses 1.5/1 MLHDx
 R27 12 12 Normal fundus (focal choroidal coloboma-OS); rod/cone ERG responses 1.4/1.4 1.6 MLHDx
 R28 22 22 Normal fundus; cone ERG responses markedly reduced 1.5/1.5 MLHDx
 R29 22 22 Normal fundus; absent cone ERG 1.5/1.3 1.5 MLHDx
 R30 12 Normal fundus; rod/cone ERG responses 1.1/1 MLHDx
 R35 12 12 Normal fundus; no ERG performed 1.4 MLHDx
 R36 12 12 No eye exam; rod/cone ERG responses 1/0.7 MLHDx
 R42 22 22 Normal fundus; rod/cone ERG responses 1/1 MLHDx
 R46 22 22 Normal fundus; absent cone ERG 1/1 MLHDx
Pet population‡
 Caroline 22 22 Normal fundus 3.5 MLHD
 Sandy 12 12 Normal fundus 4.4 MLHD
 Clair 22 22 PRA suspicious 1.3 MLHD
 Kaylee 12 12 Normal fundus 12 MLHD
 Molson 22 22 PRA 4.4 MLHD
 Corby 12 12 Normal fundus 8.8 MLHD
 Josie 22 22 Normal fundus 1 MLHD
 Maura 12 12 Normal fundus 6 MLHD
 Miper 12 12 Normal fundus 6 MLHD
 Bella 11 12 Normal fundus 1 MLHD
 08-7988 11 12 Normal fundus 7.5 MLHD
 08-7989 11 12 Normal fundus 2.1 MLHD
 08-7991 11 12 Normal fundus 5.7 MLHD
 Melody 11 11 Normal fundus 11.4 MLHD
 Chloe 11 12 Normal fundus 10.9 MLHD
 Kaylee 11 11 Normal fundus 1.2 MLHD
 Tori 11 22 Normal fundus 1 MLHD
 Spice 11 22 Normal fundus 1 MLHD
 Brandy 12 12 Normal fundus 7.1 MLHD
 Hamilton 11 22 Normal fundus 2 MLHD
 George 22 22 PRA 13.8 ESS
 Stoney 12 12 PRA 6.1 ESS
 Minnie 22 22 PRA 9.9 ESS
 Keah 22 22 PRA 15.5 ESS
 Stella 22 22 PRA 6.2 ESS
 Emma 22 22 PRA 2.8 ESS
 Holly 22 22 PRA 13 ESS
 Genesis 22 22 Normal fundus 7 ESS
 Mackenzie 22 22 Normal fundus 11.4 ESS
 Kayla 22 22 Normal fundus 11 ESS
 Tucker 22 22 Normal fundus 8.3 ESS
 Louie 22 22 Normal fundus 1.8 ECS
Table 2. 
 
Sequence Changes Identified in Canine RPGRIP1 Gene
Table 2. 
 
Sequence Changes Identified in Canine RPGRIP1 Gene
Location of Sequence Change Nucleotide Change Amino Acid Change Breed
Promoter (P1) −197C > T Siberian Husky, Beagle
Intron 1 c.19+19T > A MLHD/MLHDx, ESS, ECS
Exon 2 c.142_143ins44 MLHD/MLHDx, ESS, ECS
Intron 2 c.152+186_227del42 Siberian Husky
Intron2 c.152+227_228ins205 Siberian Husky
Exon 14 c.1740C > T p.P580P Siberian Husky
Exon 17 c.2548G > A p.E850K Siberian Husky, Beagle
Intron/exon 19c* c.548-2_c.548del3 MLHD/MLHDx, ESS, ECS
Exon 24 c.3494T > C p.V1165A MLHD, Siberian Husky, Beagle
Exon 24 c.3531(GGGAGCCGA)2-7 p.1178(GAE)2-7 MLHD, Siberian Husky, Beagle, Papillon
Table 3. 
 
cRPGRIP1 Haplotype and Phenotype of cRPGRIP1 Ins/Ins Dogs
Table 3. 
 
cRPGRIP1 Haplotype and Phenotype of cRPGRIP1 Ins/Ins Dogs
Sample ID c.19+19T > A Ins44 D3 p.V1165A p.1178(GAE)2-7 Phenotype* Age (y) Breed
R5 22 22 22 11 33 NVL/absent cone ERG 1.8 MLHDx
R10 22 22 22 11 33 Retinal degeneration (late stage) 2.6 MLHD
R18 22 22 22 11 33 NVL, absent cone ERG 0.7 MLHDx
R21 22 22 22 11 33 NVL, absent cone ERG 0.7 MLHDx
Molson 22 22 22 11 33 PRA 4.4 MLHD
Josie 22 22 22 11 33 NVL 1 MLHD
Caroline 22 22 22 11 33 NVL 3.5 MLHD
Clair 22 22 22 11 33 “PRA suspicious” 1.3 MLHD
George 22 22 22 11 33 PRA 13.8 ESS
Minnie 22 22 22 11 33 PRA 9.9 ESS
Keah 22 22 22 11 33 PRA 15.5 ESS
Stella 22 22 22 11 33 PRA 6.2 ESS
Emma 22 22 22 11 33 PRA 2.8 ESS
Holly 22 22 22 11 33 PRA 13 ESS
Genesis 22 22 22 11 33 NVL 7 ESS
Mackenzie 22 22 22 11 33 NVL 11.4 ESS
Kayla 22 22 22 11 33 NVL 11 ESS
Tucker 22 22 22 11 33 NVL 8.3 ESS
Louie 22 22 22 11 33 NVL 1.8 ECS
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