Journal of Heredity Advance Access published online on July 23, 2007
Journal of Heredity, doi:10.1093/jhered/esm060
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Independent Origin and Restricted Distribution of RPGR Deletions Causing XLPRA
From the Section of Ophthalmology, Department of Clinical Studies-Philadelphia, University of Pennsylvania, Philadelphia, PA (Zangerl and Aguirre); and the J.A. Baker Institute, Cornell University, Ithaca, NY (Johnson and Acland)
Address correspondence to B. Zangerl, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104, or e-mail: bzangerl{at}vet.upenn.edu.
Canine X-linked progressive retinal atrophy (XLPRA) is an inherited blinding disorder caused by mutations in the ORF15 of the RPGR gene and homolog to human retinitis pigmentosa 3 (RP3). The disease is observed in 2 variations, XLPRA1 in Siberian husky and samoyed and XLPRA2 derived from mongrel dogs. A third, neutral, deletion has been described in red wolves. Haplotype analysis of the 633-kbp RP3 interval in 6 different canidae confirmed the same decent for the XLPRA1 mutation in both affected breeds but suggests a recent and independent origin for both forms of XLPRA. The RP3 interval was excluded from causative associations with blindness in the red wolf and akita, a breed closely related to Nordic sled dogs. Overall, these data suggest a limited distribution of the affected haplotypes and indicate that mutations in the ORF15 are likely to be limited to the described dog breeds.
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) contribute the most frequent subtype of X-linked RP in man (Buraczynska et al. 1997), with a majority of disease causing changes in the ORF15 region of the gene (Souied et al. 1997; Vervoort et al. 2000; Rozet et al. 2002). Recently, a canine model was postulated describing 3 distinct microdeletions in the canine RPGR ORF15, 2 of which are associated with retinal degenerations X-linked progressive retinal atrophy 1 (XLPRA1) and XLPRA2 (Zhang et al. 2002) and proposed as canine homologs of human retinitis pigmentosa 3 (RP3). A 5-bp deletion causing XLPRA1 is shared between at least 2 different dog breeds; Siberian huskies and samoyeds. XLPRA2 is caused by a 2-bp deletion originally derived from a mongrel dog. An additional 3-bp deletion was observed in red wolf, not causing a change in the protein sequence other than the removal of one amino acid. All 3 deletions are within 82 bp from each other, located in a 723-bp highly repetitive sequence that is still missing from the current canine genome draft (Lindblad-Toh et al. 2005). To date, the above described models are the only canine forms of progressive retinal atrophy (PRA) linked to the RP3 region, despite the obvious hot spot in the ORF15. The unique structure of dog breeds and therefore high potential to share disorders between breeds due to a founder effect (e.g., Aguirre et al. 1998; Goldstein et al. 2006) raised the question about the origin and distribution of the observed RPGR deletions among dog breeds.
Given the evolutionary relationship between the Nordic sled dogs (Parker et al. 2004), it was assumed that the deletion causing XLPRA1 was shared by descent between the Siberian husky and the samoyed. However, PRA was previously observed in the closely related akita (O'Toole and Roberts 1984), and a small pedigree available to us implied potential X-linked inheritance. Similarly, anecdotal reports of preferential male blind red wolves (G.D. Aguirre, personal communication with the Fossil Rim and North Western Carolina Wild Life Center and the Ross Park Zoo) suggested involvement of the observed deletion or another yet to be identified variation in the RP3 interval with a retinal phenotype in this species. Utilizing the previously established physical map of the canine RP3 linkage disequilibrium (LD) interval (Zhang et al. 2002), we identified 6 novel markers for this area defining 19 distinct haplotypes within 6 canine species screened. The XLPRA1 and XLPRA2 mutations were completely linked to an individual haplotype each, highly supporting recent, single, and independent mutation events for each of the diseases. The neutral 3-bp deletion seems private to the red wolf, however, as no correlation between the deletion and the blind phenotype was found. Based on haplotype analysis, blindness in the akita and the red wolf was excluded from association with the RP3 interval based on the current data set.
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Bacterial Artificial Chromosome Subcloning and Sequencing
Bacterial artificial chromosome (BAC) clones 188B12, 255O20, and 119B21 from the RPCI81 library (Li et al. 1999) were grown in 200-ml lysogeny broth medium plus 20 µg/ml chloramphenicol at 37 °C overnight. BAC DNA was isolated using Qiagen Maxi Plasmid Purification Kit (Qiagen, Valencia, CA), and 1 µg of DNA was digested with Sau3AI in a 200-µl volume at 37 °C overnight. Digested BAC DNA was concentrated by ethanol, precipitated and loaded onto a 1% agarose gel. DNA fragments between 700 and 1000 bp were isolated from the agarose gel using a DNA Purification Kit (Qiagen) and cloned into pUC19 vector at the BamHI restriction site with T4 ligase at 4 °C overnight followed by 1 h incubation at 25 °C. Ligated BAC DNA was transformed into Inv5
F' cells following the TA Cloning® Kit (Invitrogen, Carlsbad, CA) protocol. Positive clones were directly amplified using M13F (5'-GTAAAACGACGGCCAG-3') and M13R (5'-CAGGAAACAGCTATGAC-3') primers. PCR products of different sizes were selected, purified with the QIAquick PCR Purification Kit (Qiagen) and sequenced with PCR primers in both directions on an ABI3700 capillary sequencer.
Single Nucleotide Polymorphism Identification and Typing
Sequences were filtered for repetitive elements (www.repeatmasker.org) and vector contamination, and primers were designed for 21 individual BAC fragments. Fifteen primer pairs were optimized to work from genomic DNA, and corresponding PCR products were amplified in 2 known carriers for each of the 3 RPGR deletions. Additionally, the same individuals were screened for 13 BAC end sequences previously used for the physical mapping of the RP3 interval (Zhang et al. 2002). Two polymorphisms were known from previous investigation of the RPGR gene (Zeiss et al. 1998). Primer pairs listed in Table 1 were used to amplify PCR products from 12 akitas (6 females and 6 males), 12 Siberian huskies (9 females and 3 males), 23 samoyeds (9 females and 14 males), 10 miniature schnauzer (8 females and 3 males), 11 mixed breed dogs (8 females and 4 males), and 11 red wolves (Canis rufus = CRU; 9 females and 2 males) originally collected for the mapping of XLPRA (Zhang et al. 2001) and selected samples from other canidae (Vulpus marcotis = VMA, Canis latrans = CLA, Canis lupus = CLU, Lycaon pictus = LPI). Polymorphisms were typed either by restriction enzyme test with the specified enzyme or the ABI Prism® SNaPshotTM Multiplex Kit (Applied Biosystems, Foster City, CA) using the indicated fluorescent labeled primer (Table 1). In addition, the primer extension method was modified to report the presence or absence of each of the original mutations through the next following nucleotide that allows differentiation (Table 1, ORF15). The position of the original deletions on CFAX was calculated based on neighboring sequence as it is not available in the current genome draft (May 2005 release). Alleles were evaluated manually and haplotypes established based on phase established from male individuals.
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A physical map of the RP3 LD interval has been established previously (Zhang et al. 2002) identifying a series of RPCI81 BAC clones (Li et al. 1999) covering this area. Three BAC clones, 118B12, 255O20, and 119B21, located at each end and the middle of the established interval (Figure 1) were subcloned and screened for polymorphisms in 2 carriers for each of the 3 RPGR deletions. In addition, all BAC ends used in the previous study were examined in the same individuals (e.g., BAC 126H3, Figure 1), allowing us to cover a total of 11 kbp of the 633-kbp interval in this screen. Overall, 7 changes were recorded between dogs and red wolves, but not further investigated. Four polymorphisms were identified in a heterozygous state in at least one of the carriers for either XLPRA1 or XLPRA2 (TCT1-3, SRX1) and no heterozygosity was observed in the 2 red wolves tested. None of these 4 markers or 2 previously reported single nucleotide polymorphisms in the RPGR gene (RPGR1 and 2; Zeiss et al. 1998; Zangerl B, Johnson JL, personal communication to Zeiss C) are included in the current dog single nucleotide polymorphism (SNP) map (Lindblad-Toh et al. 2005).
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A total of 144 chromosomes from 4 different dog breeds, mongrel dogs, and 5 other canidae species (Table 2) were typed for the allelic status at each of the polymorphisms in addition to the 3 RPGR ORF15 deletions to confirm disease status and amplification of the SRY gene to confirm sex. Within the RP3 LD interval, 19 individual haplotypes were observed in all 6 species; 12 of these were present in the dog only, which displayed a total of 15 haplotypes (Table 2). All 16 chromosomes carrying the XLPRA1 mutation (2 affected male, 1 affected female, and 6 carrier Siberian huskies; 4 affected male and 2 carrier samoyeds) presented haplotype A (Table 2), whereas the 8 chromosomes carrying the XLPRA2 mutation were linked to haplotype B (3 affected males and 5 carrier). Neither of these 2 haplotypes was presented in any of the other 120 chromosomes typed, including akitas and red wolves clinically diagnosed with retinal atrophy. The 3-bp deletion originally observed in the red wolf was found in heterozygote state in 6 red wolf females who carried at least 2 different haplotypes (D and E). Whereas the deletion itself was only observed in red wolves, the corresponding haplotypes were shared between species. Neither the females carrying the deletion in heterozygous state nor 2 male red wolves carrying haplotype E showed any clinical signs of retinal disease, excluding the RP3 interval from association with this phenotype. This finding was further supported by exclusion of the cosegregation of a retinal degeneration phenotype with 3 previously reported microsatellite marker (CUX40002 reported in Zangerl et al. 2002; FH2548 reported in Breen et al. 2001; and FH2916 reported in Guyon et al. 2003) in a subset of red wolves (data not shown).
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XLPRA1 and XLPRA2 in the dog have been proposed as unique model to enhance our knowledge of the molecular function and biological relevance of disease causing mutations in the RPGR gene (Zhang et al. 2002). The ORF15 of this gene provides a hot spot for mutations that accounts for the majority of human RP patients linked to this interval (Vervoort et al. 2000). It was of particular interest to investigate whether these mutations occur on a defined genetic background that would allow screening for populations at risk. Each of the 2 mutations and the deletion originally observed in the red wolf were linked to individual haplotypes (Table 2, A, B, D, and E) and no LD was observed with any of the markers within these haplotypes. Thus, we could not identify a particular genetic background in the dog or red wolf that would predict higher risk of accumulating mutations in the RPGR ORF15. The neutral deletion identified in the red wolf was observed on at least 2 different haplotypes (Table 2, D and E), both of which are shared among different species. The high frequency with which this mutation was observed in the red wolves suggests this to be an old mutation. The restriction to the species, therefore, might reflect a bias in the samples tested. However, the mutation was not found in homozygous state in females or in any male individuals, although another 11 males were screened for the mutation. The 2 male red wolves included in the haplotype study were genotyped with haplotype E but did not display a retinal phenotype. Additionally, a subset of red wolves was typed for 3 microsatellite markers in the RP3 interval, including 3 males diagnosed with retinal degeneration and no cosegregation was found. Thus, we conclude that the retinal phenotype observed in the red wolf is not causatively associated with the RP3 interval.
XLPRA1 was linked to a single haplotype (Table 2, A), which was not observed in other individuals. Thus, the disorders described in the Siberian husky and samoyed are truly identical by descent and clearly distinguishable from the XLPRA2 haplotype at loci TCT3 and SRX1 (Table 2, B). It is worthwhile to point out that one male Siberian husky with a clinical diagnosis of PRA was not found to carry the RPGR mutation. This individual carries haplotype I; the same haplotype was present in homozygous state in a female of the same pedigree, which was not affected beyond the typical age of onset for XLPRA1. We conclude that this pedigree segregates a form of PRA not caused by mutations in the RPGR gene. The same was true for 2 male akitas diagnosed with PRA. Although this breed was previously shown to be genetically close to Nordic sled dogs (Parker et al. 2004), these animals did share neither the XLPRA1 mutation nor the corresponding RP3 haplotype. One of the dogs typed for haplotype N, which it shared with a phenotypical normal, heterozygote Siberian husky female (I, N). The second animal was part of a small pedigree that included several animals homozygous for haplotype C and was the only one displaying disease. In addition, this haplotype was the most common one in the individuals examined. Thus, we do not have evidence that the disease segregating in the akita is caused by mutations in the RP3 interval.
XLPRA2 was derived from a mongrel dog. Because the corresponding haplotype (B) was only observed with the disease, we are unable to link the disease to a particular breed at this point. In fact, it might have been a de novo mutation in the particular mongrel line. Interestingly, the disease relevant haplotypes consisting of the 3 polymorphisms closest to either XLPRA deletion (SRX1, RPGR1, and RPGR2) were not identified in any of the non-dog populations, suggesting that the mutation carrying chromosomes are unique to the dog.
In conclusion, we were able to develop polymorphic markers specifically designed to investigate linkage to previously described mutations in the RPGR ORF15 that are not included in the current dog SNP map (Lindblad-Toh et al. 2005). These markers established that the RP3 interval is not associated with retinal disease in the akita or red wolf. Both XLPRA1 and XLPRA2 arose independently as single mutation events. Although these canine RPGR models are extremely important to our understanding of the retinal function and development of therapy, they seem rather recent acquisitions in the diseased dog populations and are likely not observed in any breeds other than reported.
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National Eye Institute/National Institutes of Health (EY06855 and EY13132), the Foundation Fighting Blindness, and the Van Sloun Fund for Canine Genetic Research.
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The authors are appreciative to Dr Q. Zhang for insight in his RPGR work and the Fossil Rim and North Western Carolina Wild Life Center as well as the Ross Park Zoo for collaboration to retrieve red wolf samples. Additionally, we would like to thank Dr R. Wayne and J. Leonard, Department of Organismic Biology, University of California, for providing DNA samples from diverse other canine species. We gratefully acknowledge Dr V.N. Meyers-Wallen from Cornell University for support with sex determination using the Sry gene. The authors also highly value Kathie Weeks for significant contributions in facilitating communication with Wild Life Center, Zoo, and dog breeders.
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This paper was delivered at the 3rd International Conference on the Advances in Canine and Feline Genomics, School of Veterinary Medicine, University of California, Davis, CA, August 3–5, 2006.
Corresponding Editor: Steven Hannah
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