Molecular Vision 2003; 9:601-605 <>
Received 17 July 2003 | Accepted 24 October 2003 | Published 11 November 2003

Evaluation of the canine RPE65 gene in affected dogs with generalized progressive retinal atrophy

Gabriele Dekomien, Jörg Thomas Epplen

Molecular Human Genetics, Ruhr-University, 44780 Bochum, Germany

Correspondence to: Gabriele Dekomien, Molecular Human Genetics, Ruhr-University, 44780 Bochum, Germany; Phone: (+49)234-32-25764; FAX: (+49)234-32-14196; email:


Purpose: The RPE65 gene was screened in 26 breeds of dogs in order to identify potential disease-causing mutations in dogs with generalized progressive retinal atrophy (gPRA).

Methods: Intronic sequences were obtained from canine genomic DNA by intron-overlapping polymerase chain reactions (PCRs). Mutation analysis was performed by PCR and demonstration of single strand conformation polymorphisms (SSCP). Genomic variations were verified by sequencing.

Results: A series of exonic and intronic single nucleotide polymorphisms (SNPs) were identified in the investigated breeds, but none of the dogs examined showed the typical RPE deletion for retinal dystrophy in Briards nor any other disease-causing mutation.

Conclusions: The informative SNPs provide evidence allowing indirect exclusion of mutations in the RPE65 gene as causing retinal degeneration in 25 of the 26 dog breeds investigated with presumed autosomal recessively transmitted gPRA.


Like retinitis pigmentosa (RP) in man, generalized progressive retinal atrophy (gPRA) represents a genetically heterogeneous disorder [1] in dogs that in most forms is inherited as an autosomal recessive (ar) trait. gPRA shows homologous disease symptoms like RP and is characterized by degeneration of the peripheral retina leading to night blindness and loss of visual fields. The age of onset and the progression rate varies in different dog breeds, but typically the disease progresses to complete blindness [1,2]. Until now, causal mutations for ar gPRA have been identified only in a few dog breeds [3-7]. A number of photoreceptor genes have been excluded as the primary genetic cause of the trait in the many of 26 dog breeds [7-14] investigated here.

The RPE65 gene is expressed in the RPE, a monolayer apposed to the outer surface of the retinal photoreceptor cells. Despite studies in RPE65-deficient transgenic mice [15], the precise function of the protein remains unknown, but it is required for the regeneration of 11-cis retinal in the visual cycle [16]. Mutations in the RPE65 gene are associated with several human inherited retinal degenerations: Lebers congenital amaurosis (LCA), childhood-onset severe retinal dystrophy (CSRD), or autosomal recessive retinitis pigmentosa (arRP) [17-20]. In Swedish briards, a homozygous 4 bp deletion after codon 153 of the RPE65 gene leads to congenital stationary blindness (csnb). Gene therapy with a recombinant adeno-associated virus (AAV) carrying wild type RPE65 restored the visual function in dogs [21,22].

Here we investigated the RPE65 gene for mutations leading to ar gPRA in several breeds of dogs, including the briard-specific mutation [23,24].


Blood of 821 dogs from 26 different breeds including 128 gPRA-affected animals Table 1 was received from the owners in cooperation with the breed clubs (Verband für das Deutsche Hundewesen [VDH]; Nederlandse Vereiniging van Saarlooswolfhonden; Schweizer Kynologischen Gesellschaft [SKG]). The blood of most of the dogs was sent from different regions of Germany. In addition, several Saarloos/Wolfhounds (Sa), Schapendoes (SD), Sloughis (Sl) and Tibetan Terriers (TT) originated from the Netherlands (Sa, SD), Switzerland (Sl, TT), Sweden (Sl) and USA (Sl). Dog breeds with known mutation localisation were included as controls. Breeders have assumed ar inheritance in the aforementioned breeds by observing the cases of gPRA in the pedigrees. Experienced veterinarians confirmed the gPRA status of affected and unaffected dogs by ophthalmoscopy. This was documented in certificates of the eye examinations. Genomic DNA was extracted from peripheral blood according to standard protocols [25].

Exon/intron boundaries were analysed by comparison with the mRNA sequence of the canine RPE65 gene ([24]; EMBL accession number AF084537) with the genomic sequences of chromosome 1 (EMBL accession number AL139413). Sequences of intron 1, 4-9, and 11-13 of the RPE65 gene were amplified (Table 2) by overlapping PCRs including neighboring exons in a thermocycler (Biometra, Goettingen, Germany). PCRs were performed under standard PCR conditions [11-13] with Taq Polymerase (Genecraft, Münster, Germany) and varying concentrations of MgCl2 (Table 3). For SSCP analysis, 0.06 μl of [α32P] dCTP (10 mCi/ml) was included in the PCR.

Amplified fragments were extracted from 1% agarose gels using the Easy Pure extraction kit (Biozym, Hess. Oldendorf, Germany) and sequenced with intron-overlapping primers (Table 3). Sequencing reactions were carried out by the dideoxy-chain termination method using the Big Dye Terminator kit (BDT; Perkin-Elmer, Norwalk, CT) according to the manufacturer's instructions in order to identify the intronic sequences. All sequencing reactions were run on an automated DNA sequencer (Applied Biosystems 373 XL, Foster City, USA) and analysed using the ABI PrismTM 373XL Collection and Sequencing Analysis 3.0 software.

Primer pairs for mutation screening were designed after DNA sequence analysis of the intron-overlapping RPE65 fragments (Table 3). PCR products were digested dependent on the lengths of the fragments with different restriction enzymes (Table 3) to optimize the mutation screening by SSCP analysis [26]. SSCP samples were treated according to [7-9]. PCR products were separated through two sets of 6% polyacrylamide (acrylamide / bisacrylamide: 19/1) gels, one set containing 10% glycerol, another containing 5% glycerol and 1 M urea. Autoradiographs were examined for band alterations, which were confirmed after purification and cycle sequencing as described above.

Results & Discussion

Intronic sequences of canine RPE65 gene have not yet been published. The alignment of mRNA sequences of the dog with genomic sequences of chromosome 1 of the human retinal RPE65 gene shows 14 exons with a size between 65 and 149 bp and introns mostly under 2 kb in the human gene. The canine intron/exon organisation corresponds to that of the human gene including high homologies in the exon-intron borders (Table 2). But after sequencing of the overlapping PCR products, introns 6, 8, 9, 11, 12, and 13 turned out to be longer than the human counterparts. This is in contrast according to our previous experience with other gPRA candidate genes, e.g. RCV1 and PDC ([12,13]; Table 2).

Introns 2, 3, and 10 could not be identified, probably because of excessive size (or special sequence conformation). Nevertheless we detected many informative SNPs of the RPE65 gene that allowed indirect gene analysis in the breeds. Alignment of the canine sequences with the BLAST search program pointed to short repeat areas in introns 6 (position 4-66 EMBL accession number AJ506756) and 12 (position 580-704 EMBL accession number AJ251207) corresponding to a canine tRNA-derived short interspersed nucleotide element (SINE; [27]). In addition, two fragmented long interspersed nucleotide elements (LINE; position 139-224 and 312-359 EMBL accession number AJ251207; [28]) were localized in intron 11.

For PCR-SSCP analysis, primers were designed flanking the exons including whenever possible the conserved splice sites (Table 3). Thirteen of 14 exons of the canine RPE65 gene were investigated for disease-causing mutations in all gPRA affected and some unaffected dogs. The 4 bp deletion that leads to CSNB in Briards was not found in the gPRA affected dogs. Many novel SNPs were observed. The published SNPs (exons 5 (T459C) and 6; [23]) were identified in 20 and 8 different dog breeds, respectively (Table 4). Additionally, in the ORF single nucleotide substitutions in exons 9 (C900T) and 10 (A1026T), lack of amino acid exchanges were verified in 4 breeds (Table 4). Further SNPs were identified within different introns: deletions within introns 1, 8, 11, and the 3'UTR, insertions in introns 6 and 8 as well as nucleotide substitutions within introns 6, 7, 8, 11, and 13 (Table 4). After sequencing of intron 11, breed-specific haplotypes (CAA or ATG) could be defined. The SNPs were present in heterozygous state in affected dogs in 25 of the 26 investigated breeds and are therefore informative markers for the exclusion of the RPE65 gene in these breeds with assumed ar transmitted gPRA. The Rottweiler investigated shows SNPs exclusively in homozygous state in introns 7 and 8. This fact does not allow to exclude the RPE65 gene as a cause for gPRA in this breed.

The founder effect in connection with inbreeding (i.e., effective genetic separation) leads to breed-specific gPRA mutations. Therefore, identical mutations are not likely to have happened independently in different breeds. Heteroallelism is an unlikely cause of ar gPRA within a single breed, as one would expect such disease to result from a single founder mutation. Therefore, we excluded the RPE65 gene as a cause for gPRA in Afghan Hounds, American Cocker Spaniels, Australian Cattle dogs, Berger des Pyrénées, Bologneses, Curly Coated Retrievers, English Cocker Spaniels, English Springers, Entlebucher Mountain Dogs, Giant Schnauzers, Golden Retrievers, Irish Setters, Labrador Retrievers, Miniature poodles, Newfoundlanddogs, Polish Lowland Sheepdogs, Saarloos, Salukis, Scottish Collies, Scottish Terriers, Schapendoes, Sloughis, Yorkshire Terrier, Tibetan Terriers and Wire-haired Dachshunds.


We thank the owners of the dogs for blood samples, the veterinarians of the Dortmunder Ophthalmologenkreis (DOK) for the ophthalmologic investigations of the dogs and for the support of different breeding clubs. These studies were supported in part by the Gesellschaft für kynologische Forschung, Bonn (Germany).


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