cGMP Phosphodiesterase-a Mutation Causes Progressive Retinal Atrophy in the Cardigan Welsh Corgi Dog Simon M. Petersen–Jones,1 David D. Entz, and David R. Sargan PURPOSE. To screen the a-subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6A) as a potential candidate gene for progressive retinal atrophy (PRA) in the Cardigan Welsh corgi dog. METHODS. Single-strand conformation polymorphism (SSCP) analysis was used to screen short introns of the canine PDE6A gene for informative polymorphisms in members of an extended pedigree of PRA-affected Cardigan Welsh corgis. After initial demonstration of linkage of a poly- morphism in the PDE6A gene with the disease locus, the complete coding region of the PDE6A gene of a PRA-affected Cardigan Welsh corgi was cloned in overlapping fragments and sequenced. SSCP-based and direct DNA sequencing tests were developed to detect the presence of a PDE6A gene mutation that segregated with disease status in the extended pedigree of PRA-affected Cardigan Welsh corgis. Genomic DNA sequencing was developed as a diagnostic test to establish the genotype of Cardigan Welsh corgis in the pet population. RESULTS. A polymorphism within intron 18 of the canine PDE6A gene was invariably present in the homozygous state in PRA-affected Cardigan Welsh corgis. The entire PDE6A gene was cloned from one PRA-affected dog and the gene structure and intron sizes established and compared with those of an unaffected animal. Intron sizes were identical in affected and normal dogs. Sequencing of exons and splice junctions in the affected animal revealed a 1-bp deletion in codon 616. Analysis of PRA-affected and obligate carrier Cardigan Welsh corgis showed that this mutation cosegregated with disease status. CONCLUSIONS. A single base deletion at codon 616 in the PDE6A gene cosegregated with PRA status with zero discordance in Cardigan Welsh corgis with PRA. A lod score of 4.816 with a recombi- nation fraction (u) of zero strongly suggests that this mutation is responsible for PRA in the breed. The mutation is predicted to lead to a frame shift resulting in a string of 28 altered codons followed by a premature stop codon. The authors suggest that this type of PRA be given the name rod–cone dysplasia 3 (rcd3). (Invest Ophthalmol Vis Sci. 1999;40:1637–1644) ene mutations causing autosomal recessive retinitis report is that which causes rod–cone dysplasia type 1 (rcd1)in pigmentosa (ARRP) in humans have been reported in the Irish setter breed. Rcd1 is caused by an amber mutation in opsin,1 the a-2 and b-3 subunits of cyclic guanosine the b-subunit of the cGMP phosphodiesterase gene G 11–13 monophosphate (cGMP) phosphodiesterase, the a- subunit of (PDE6B). Mutations in the homologous gene have been cGMP-gated channel,4 RPE65,5 adenosine triphosphate (ATP)- identified in the retinal degeneration (rd) mouse,14,15 and a binding cassette transferase protein,6,7 tubby-like protein 1 subset of autosomal recessive retinitis pigmentosa pa- (TULP1),8 and cellular retinaldehyde-binding protein.9 The tients.3,16–18 Rcd1 is characterized by the absence of cGMP- analogous group of conditions in the dog are the progressive phosphodiesterase activity, leading to a 10-fold increase in retinal atrophies (PRAs) and are known to occur in several cGMP levels.19 This results in arrested development of photo- breeds of dog (see Ref. 10 for a review). Despite investigating receptors followed by a progressive rod-led photoreceptor genes known to cause similar retinal dystrophies in other degeneration.20 PRA in the collie is characterized by similar species, the only causal gene mutation identified before this biochemical and histopathologic changes.21–23 However, breeding studies have shown the two forms of PRA to be nonallelic,24 leading to the designation of the form in the collie From The Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge, United as rod–cone dysplasia type 2 (rcd2). Both rcd1 and rcd2 are Kingdom. early-onset forms of PRA. Supported by Wellcome Trust Veterinary Research Career Devel- PRA in the Cardigan Welsh corgi has a similar early onset opment Fellowship 041623/z (SMP–J). leading to blindness in the young adult dog and, similar to most Submitted for publication October 27, 1998; revised February 2, forms of canine PRA, is inherited in an autosomal recessive 1999; accepted March 2, 1999. Proprietary interest category: N. manner. PRA in this breed was first recorded in the veterinary 1Present address: Department of Small Animal Clinical Sciences, literature in 197225 and has recently undergone a resurgence, College of Veterinary Medicine, Michigan State University, East Lan- with cases occurring in The Netherlands, New Zealand, and sing, Michigan. the United States. The affected lines in all three countries can Reprint requests: Simon M. Petersen–Jones, Department of Small Animal Clinical Sciences, Michigan State University, D-208 Veterinary be traced back to stock imported from the United Kingdom. Medical Center, East Lansing, MI 48824-1314. Detailed electrophysiological, histopathologic, and biochemi- Investigative Ophthalmology & Visual Science, July 1999, Vol. 40, No. 8 Copyright © Association for Research in Vision and Ophthalmology 1637 Downloaded from jov.arvojournals.org on 09/28/2021 1638 Petersen–Jones et al. IOVS, July 1999, Vol. 40, No. 8 cal analysis of PRA-affected Cardigan Welsh corgis has not yet in the canine PDE6A gene. Sequencing to obtain the en- been performed. tire sequence of the coding region and intron–exon bound- We report here that PRA in the Cardigan Welsh corgi is aries was performed. Initially, PCR products were cloned caused by a 1-bp deletion in codon 616 of PDE6A. We predict using the TA cloning kit (Invitrogen, NV Leek, Groningen, that the histopathogenesis of this form of PRA is similar to that The Netherlands) and at least two independently derived resulting from the amber mutation in PDE6B in rcd1, and if this clones from each region were sequenced by double- proves to be the case, we propose that the condition in the stranded sequencing using a Thermo-Sequenase core se- Cardigan Welsh corgi be designated rod–cone dysplasia type 3 quencing kit (Vistra DNA Systems; Amersham) with a se- (rcd3). quencing primer labeled with Texas red (Amersham) and PRA in the Cardigan Welsh corgi is only the second form run on a Vistra 725 DNA sequencer (Vistra DNA Systems; of canine PRA for which the causal gene mutation has been Amersham). identified and represents the only naturally occurring animal model of ARRP due to a PDE6A mutation. Linkage Analysis Two-point lod scores were calculated for segregation of PRA and an exon 15 1-bp deletion in two-generation families METHODS (phase unknown) represented in the pedigree.29 MLINK Dogs from the LINKAGE program package (Human Genome Map- ping Project, Medical Research Council, UK) was used for Genomic DNA was extracted from blood samples obtained these calculations.30 from dogs within pedigrees of Cardigan Welsh corgis in which The allele frequencies within each group of blood samples PRA was segregating and also from unaffected breeding lines. were used to perform a simple binomial analysis of the prob- The diagnosis of PRA was made by veterinary ophthalmologists ability of homozygosity for a given allele in affected animals, on the basis of clinical history and ophthalmoscopic signs. A given independent assortment of trait and allele. Contingent pedigree showing the relationship between affected animals is probabilities for allele frequencies in the carrier dogs were not shown in Figure 4. Treatment of animals conformed to the included. Therefore, the real probability of independence (no ARVO Statement on the Use of Animals in Ophthalmic and linkage) is lower than the figure quoted in all cases. Vision Research. Sequencing of a PCR Product Spanning the Site of Single-Strand Conformation Polymorphism the Codon 616 1-bp Deletion Analysis of Presumptive Intron 18 of the Canine PDE6A Gene PCR amplification of a genomic fragment containing part of intron 14 and the mutation site in exon 15 was performed Using information about the intron sizes and position of the using sense primer 59-TCATTCCATCGCCGACTC-39 (primer human alpha PDE gene (kindly supplied by Steven Pittler positioned –81––64 from intron–exon boundary) and anti- before publication) a primer pair (sense 59-GTGATCTCT- sense primer 59-CCTCATCTCGCAGCAACGTT-39 (corre- CAGCCATCACC-39 and antisense 59-GATTCTGCTGCAG- sponds to first nucleotide of intron 15 plus nucleotides CACTGTG-39) was designed from the canine cDNA se- 2019–2001). Initially, a radioactively labeled PCR was car- 26–28 quence to amplify the presumptive intron 18 of the ried out using 50 ng genomic DNA in the presence of 2 mCi canine gene by polymerase chain reaction (PCR). The 32P-dATP and SSCP analysis performed as before. Subse- PCR product was found to be a suitable length for single- quently, an unlabeled PCR reaction was used, and 2 mlofthe strand conformation polymorphism (SSCP). PCR-SSCP was reaction product was sequenced directly by a Taq-based 32 performed by the inclusion of 2 mCi [a- P]dATP (Amer- method (Thermo-Sequenase, Amersham) as before, using a sham Life Science, Amersham, UK) in a 50-ml PCR reac- Texas red–labeled oligonucleotide 59-GGTGTCTTTCCAA- tion. After thermal cycling, 2 ml of the resultant product GATGGAG-39 (corresponds to nucleotide numbers 1984– was placed into 10 ml SSCP loading buffer (95% deion- 1965) as primer. ized formamide; 10 mM NaOH; 0.05% bromphenol blue; 0.05% xylene cyanol). The samples were denatured by heating to 95°C for 2 minutes and placing on ice for 15 RESULTS minutes and electrophoresis performed on a 0.53 MDE- hydrolink polyacrylamide gel (Hoefer, Newcastle-under- Screening of Intron 18 of PDE6A for Lyme, UK), with a running buffer of 0.63 TBE, at 0.12 Polymorphisms by SSCP W/cm for 14 hours at room temperature.