Journal of Heredity 2003:94(1)
© 2003 The American Genetic Association 94:27-30
Canine Models of Ocular Disease: Outcross Breedings Define a Dominant Disorder Present in the English Mastiff and Bull Mastiff Dog Breeds
From the James A. Baker Institute for Animal Health, Cornell University, Ithaca, NY 14853.
Address correspondence to Gregory M. Acland at the address above, or e-mail: gma2{at}cornell.edu.
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Abstract |
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Progressive retinal atrophies (PRA) are a heterogeneous group of inherited eye diseases common to both dogs and man. Over 100 individual canine breeds display some sort of retinal degeneration, making the dog an extremely valuable resource both for finding the genetic determinants of inherited blindness and for developing naturally occurring animal models that mimic human disease. Progressive retinal atrophies within the English mastiff displayed an ambiguous mode of inheritance. By conducting outcross matings between affected English mastiffs and normal animals from other breeds, the mode of inheritance was confirmed as dominant. This directed candidate gene analysis and led to identification of two synonymous mutations and one nonsynonymous mutation within the canine rhodopsin gene. The nonsynonymous mutation (T4R) is the cause of PRA in the English mastiff, and a test was developed to investigate its presence in 17 additional breeds. Testing of PRA-affected animals from 16 breeds revealed that none carry the T4R mutation, indicating a different cause of PRA. Analysis of two affected bull mastiffs revealed one heterozygote (+/T4R) and one homozygous normal individual (+/+). These findings suggest that the genetic origin of PRA is often breed specific and underline the value of outcross mating to circumvent problems that act to mask the mode of inheritance.
Animal models of human disease facilitate experiments aimed at the elucidation of disease mechanisms and the evaluation of strategies for treatment. The canine population represents a rich source of animal models for a diverse range of human diseases, such as cancers, defects of the central nervous system, cardiac abnormalities, blood disorders, and diseases of the eye and ear (reviewed by Brooks and Sargan 2001). An important criteria for any animal model is the faithful reproduction of the human disease phenotype under investigation. This is well illustrated by the recent identification of a canine model for human dominant retinitis pigmentosa (RP). The phenotypic features displayed in a subset of human RP patients with rhodopsin (RHO) mutations include a nonuniform structural degeneration of the retina accompanied by an abnormal recovery of photoreceptor function after exposure to bright light (termed Class B patients) (Cideciyan et al. 1998). The canine RHO mutant animals display exactly the same set of phenotypic characteristics, making the dog the perfect model for investigation of a disease with a large social impact in human populations (Kijas et al. 2002).
In addition to the fact that a large number of canine diseases have a human homolog, the advantages offered by canine populations for the study of biomedical traits is receiving increasing attention (Ostrander et al. 2000; Ostrander and Kruglyak 2000). The majority of described canine diseases display a recessive mode of inheritance (70%) (Patterson 1980). Considering retinal disorders, all of the canine progressive retinal atrophies (PRAs) characterized are recessive with the exception of two X-linked forms (Acland et al. 1994; Zhang et al. 2002). The genetic cause of retinal disease in approximately 80 breeds remains to be determined. This potentially represents a large and untapped pool of extremely valuable large animal models of human ocular disease.
A number of factors contribute to the prevailing view that inherited eye disease in the dog is almost invariably autosomal recessive. First, it is easier to successfully select against and remove adverse dominant diseases within a purebred population and second, the genes contributing to the majority of dominant retinal disease in human (rhodopsin, RDS/peripherin) have been excluded as causal to PRA within a growing number of canine breeds (Gould et al. 1995; Ray et al. 1996, 1999). The highly inbred nature of many breeds means that a recessive disorder at high frequency can be difficult to distinguish from one with a dominant mode of inheritance. In addition, a number of factors may mask the mode of inheritance for a disease that exists within a pet population. These may include differences in clinical diagnostic criteria, a variable age of disease onset, differences in the severity of disease, and the unavailability of key pedigree members.
This report aims to illustrate the usefulness of using a targeted outcross breeding strategy to circumvent the problems associated with investigating a disease within a population. We used this strategy to investigate a form of PRA observed in the natural population of English mastiffs, which is caused by a mutation in the rhodopsin gene (Kijas et al. 2002). Specifically, outcross breedings were conducted to distinguish between a disease showing a simple, dominant mode of inheritance from either a recessive disease or disorder with a multigenic basis. The study also aimed to investigate the frequency of the English mastiff RHO mutation in other canine populations containing PRA.
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Materials and Methods |
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Animals
Examination of privately owned purebred English mastiffs for the presence of retinal disease was initiated at the request of the English Mastiff Club of America. Indirect ophthalmoscopy was conducted on approximately 500 English mastiffs over a 3-year period to search for increased tapetal reflectivity and/or retinal thinning diagnostic of retinal atrophy. Blood samples were obtained from 29 affected purebred mastiffs and a further 23 related but unaffected animals.
Candidate Gene Analysis and Mutation Detection
Amplification of the five exons of the canine rhodopsin gene was performed using previously published primers (Gould et al. 1995). Sequencing was performed with dye terminator chemistry on an ABI377 Prism DNA sequencer (Applied Biosystems, Foster City, CA). Detection of the C to G transversion at nucleotide 11 followed PCR using primers OPIAF (5'–GCA GCA CTC TTG GGA CTG AG) and OPIAR (5'–TGT AGT TGA GAG GTG TAC GC). Digestion of the 275 bp product using BsmFI results in fragments of 202, 47, and 26 bp (wildtype), or 249 and 26 bp (nt11C to G mutant).
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Results |
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Ambiguous Mode of Inheritance Resolved by Testcross Matings
Investigation of PRA within the English mastiff population revealed a disease with an ambiguous mode of inheritance. Examination of pedigree data showed that in the majority of cases, affected individuals had an affected parent indicating dominance (22 of 29 cases). However, Figure 1A shows a six-generation subset of animals segregating for the disorder and contains an example suggestive of a recessive disorder (animal V-7 does not have an affected parent). To define the mode of inheritance, controlled outcross matings were performed between an affected purebred English mastiff dam and a beagle-derived laboratory strain sire (Figure 1B). The sire was specifically selected as previous studies have shown this animal to be free of all known alleles which cause recessive canine PRA (Acland et al. 1998). The appearance of affected offspring from the mating indicates the presence of a dominant allele being transmitted from the English mastiff dam. Affected progeny were also observed from a second outcross breeding using a clinically normal Irish wolfhound (Figure 1C). The observation that the affected English mastiff produced disease against two independent genetic backgrounds expected not to carry recessive PRA alleles confirms the presence of a dominant form of PRA.
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Directed Analysis of Candidate Genes
The outcross breeding results served to direct candidate gene analysis as mutations in three genes (RHO, RP1, and RDS/peripherin) together account for approximately one half of all human cases of autosomal dominant retinitis pigmentosa (RetNet: www.sph.uth.tmc.edu/Retnet). Amplification and sequence analysis of each exon of the RDS/peripherin gene revealed no sequence variants when compared between a normal and affected purebred English mastiff. Analysis of the second candidate, RHO, identified two synonymous and a single nonsynonymous substitution (Figure 2). The two synonymous substitutions are both A to G transitions within exon 2 and occur at nucleotide positions 492 and 504. These were not investigated further, as neither alters the predicted amino acid sequence (residue 164 and 168) (Figure 2), nor associates specifically with affected English mastiffs. They may, however, prove useful to test for association with PRA in other canine populations, as polymorphism was detected in the Labrador retriever, poodle, and beagle. The identified nonsynonymous substitution occurs at position 11 and is a C to G transversion, which replaces threonine at residue four with arginine (T4R). Association testing within the English mastiff population demonstrated the T4R rhodopsin mutation is the cause of disease; this, and a detailed examination of the resulting disease characteristics, are published in detail elsewhere (Kijas et al. 2002). Mutation analysis within the pedigree in Figure 1A revealed that each affected animal carried the T4R mutation in the heterozygous state (+/T4R). In addition, the phenotypically normal animal III-7 also had genotype +/T4R, meaning this animal likely displayed either late onset or a reduced rate of disease progression. This exemplifies the potential difficulties involved with establishing the mode of inheritance within a natural purebred population and underscores the value in conducting an outcross mating.
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Screening for T4R Rhodopsin Across 17 PRA Breeds
In order to determine if T4R rhodopsin was causal to PRA in breeds other than the English mastiff, 47 PRA affected dogs from a total of 17 other breeds were tested. Progressive retinal atrophies affected individuals from the following 16 breeds were tested and found to be homozygous normal: Akita (1), Airedale (1), American cocker spaniel (4), American eskimo dog (3), American Staffordshire terrier (3), Australian cattle dog (3), basenji (5), Bernese mountain dog (1), English springer spaniel (4), Entelbucher sennehund (1), Glen of Imaal terrier (4), Italian greyhound (2), papillon (4), poodle (3), Tibetan terrier (5), and soft-coated wheaten terrier (1). Testing of two PRA affected animals within a 17th breed, the bull mastiff, revealed one heterozygote (T4R/+) and one homozygous normal animal.
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Discussion |
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Breeding strategies within purebred canine populations usually aim to maintain physical uniformity for breed-specific traits. As a result, inbreeding is common and pressure exists to prevent interbreed matings. The outcome is that the vast majority of canine diseases are likely to be breed-specific, making the use of interbreed matings informative for a number of reasons. First, such matings can quickly establish the mode of inheritance of a disease. The use in this report of unaffected dams from breeds other than English mastiff was essential to defining the disease as dominant. A similar strategy has been used to investigate other diseases, a prominent example being hip dysplasia common to German shepherds. Todhunter and colleagues constructed a three-generation family using dysplastic Labrador retrievers mated to unaffected greyhounds that confirmed the complex basis of the condition (Todhunter et al. 1999). During development of a canine model for an inherited renal cancer syndrome, a single-affected German shepherd-derived sire was outcrossed to five normal English setter dams, resulting in localization of the mutation to a small region of CFA5 (Jonasdottir et al. 2000). The foundation of any resource pedigree using more than one breed also serves to increase the background genetic heterozygosity at the molecular markers necessary to identify linkage with the trait of interest. As a large number of canine diseases await molecular characterization, both the unique properties of purebred populations and the ability to conduct controlled matings will continue to be important.
A collection of PRA-affected animals from 17 breeds was tested for the presence of the rhodopsin T4R mutation originally identified within the English mastiff. The finding that this mutation does not account for PRA in 16 of the breeds is consistent with the history of canine breed development. Most breeds were founded by a small number of individuals and subsequent population expansion proceeded under the "pedigree barrier." The stipulation that for registration an animal must have two registered purebred parents has ensured an effective barrier exit to prevent gene flow between breeds. This pedigree barrier has resulted in confinement of T4R rhodopsin to English mastiff and disease homogeneity within the breed. The cause of PRA within the 16 breeds tested is therefore presumably due either to mutations in genes other than RHO or conceivably independent mutations elsewhere in the molecule. The former appears true within the Tibetan terrier, where testing using a synonymous RHO exon 3 mutation did not show association with PRA (Gould et al. 1995), and for the basenji and soft-coated wheaten terrier, where exon scanning failed to identify pathogenic sequence variants (Ray et al. 1999).
Interestingly, the T4R mutation was found in the heterozygous state within one of two PRA-affected bull mastiffs tested. Bull mastiffs first arose in the United Kingdom in the 1850s after crossing the English mastiff with fighting bulldogs (Fiennes and Fiennes 1969). The T4R mutation likely arose after the foundation of the English mastiff and is present within the bull mastiff due to gene flow, as opposed to an independent mutational event. As a second PRA-affected bull mastiff lacked the T4R mutation, disease heterogeneity is present, and a second disease causing mutation/gene exists within this breed.
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Acknowledgments |
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This work was supported by grants EY06855 and EY13132 from the National Institutes of Health, the Morris Animal Foundation/the Seeing Eye, Inc., the Foundation Fighting Blindness, the Van Sloun Fund for Canine Genetic Research, and the New York State Office of Science, Technology, and Research. We thank Barbara Dukelow for technical assistance. This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002.
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Footnotes |
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Corresponding Editor: Urs Giger
Received July 15, 2002
Accepted October 9, 2002
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