WO2001038578A1 - Inherited retinal diseases at the canine rp3 locus: linkage, marker- and mutation-based tests - Google Patents

Abstract

The present invention relates to methods for idenfifying or selecting dogs which are genetically normal, are carriers of, or are affected with X-linked progressive retinal atrophy (XLPRA) by testing a biological sample with genetic markers which co-segregate with a XLPRA gene locus. The present invention also relates to methods for identifying or selecting dogs which are genetically normal, are carriers of, or are affected with LPRA by testing a biological sample for a gene encoding canine retinitis pigmentosa GTPase regulator (RPGR) having a nucleotide mutation in one or both alleles indicative of a carrier of or a dog affected with XLPRA. Other aspects of the present invention relate to isolated nucleic acid molecules encoding the ORF 15 of the canine RPGR in normal, XLPRA1-affected, and XLPRA2-affected dogs.

Description

INHERITED RETINAL DISEASES AT THE CANINE RP3 LOCUS: LINKAGE, MARKER- AND MUTATION-BASED TESTS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/167,365, filed November 24, 1999. This work was supported by NEI/NIH grant EY 06855. The U.S. Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods of identifying inherited retinal diseases in canines, termed X-linked progressive retinal atrophy (XLPRA), using genetic, mutation- and marker-based tests. Nucleic acid molecules encoding the gene and mutations thereof which are responsible for XLPRA in canines are also disclosed.

BACKGROUND OF THE INVENTION

In 1996, mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) were identified as the cause of the RP3 form of X-linked retinitis pigmentosa (XLRP) in man and the RP3 gene was found to be located on human Xp21.1 , in approximately a 520 kb interval between OTC and DXS1110 (Figure 1; Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentosa (RP3)," Nature Genet., 13:35-42 (1996)). However, RPGR mutations account for only 20-30% of cases whose disease maps to the RP3 interval (Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentosa (RP3),^*' Nature Genet., 13:35-42 (1996); Roepman et al., "Positional Cloning of the Gene for X-Linked Retinitis Pigmentosa 3: Homology With the Guanine Nucleotide Exchange Factor RCC1," Hum. Mol. Genet., 5:1035-1041 (1996); Fujita et al., "Analysis of the RPGR Gene in 11 Pedigrees With the Retinitis Pigmentosa Type 3 Genotype: Paucity of Mutations in the Coding Region but Splice Defects in Two Families," Am. J. Hum. Genet.. 61 :571-580 (1997)). RPGR is expressed at low levels, in particular in the retinal pigment epithelium (RPE) and retina (Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentosa (RP3)," Nature Genet., 13:35-42 (1996); Yan et al., "Biochemical Characterization and Subcellular Localization of the Mouse Retinitis Pigmentosa GTPase Regulator (mRpgr)," J. Biol. Chem.. 273:19656-19663 (1998)), thus making it difficult to assess levels of the transcript in neuroretinal target tissues. Despite ubiquitous expression of RPGR, the disease caused by mutations in this gene is limited to the retina. RPGR has a complex splicing pattern in both human and mouse (Yan et al., "Biochemical Characterization and Subcellular Localization of the Mouse Retinitis Pigmentosa GTPase Regulator (mRpgr),'- J. Biol. Chem., 273:19656-19663 (1998); Holinski- Feder et al., "The RPGR Gene in Retinitis Pigmentosa Type 3," Invest. Ophthalmol. Vis. Sci., 39:S292 (1998); Kirschner et al., "RPGR Transcription Studies in Mouse and Human Tissues Reveal a Retina-Specific Isoform That is Disrupted in a Patient With X-Linked Retinitis Pigmentosa," Hum. Mol. Genet.. 8: 1571-1578 (1999); Vervoort et al., "Mutational Hot Spot within a New RPGR Exon in X-Linked Retinitis Pigmentosa," Nature Genet. 25:462-466 (2000)). Of the splice variants described, it is not known which, if any, are specific or most biologically relevant to the retina, although one retina-specific isoform has been described which, when deleted, results in the RP3 phenotype (Kirschner et al., "RPGR Transcription Studies in Mouse and Human Tissues Reveal a Retina-Specific Isoform That is Disrupted in a Patient With X-Linked Retinitis Pigmentosa," Hum. Mol. Genet., 8:1571-1578 (1999)). Neither is it known what is the proportional expression of the different splice variants in the retina, or whether these proportions are altered in RP3. The mechanism by which mutations in RPGR cause retinal degeneration is unclear. RPGR initially was thought to play a role as a guanine nucleotide exchange factor for a small G-protein in the RPE or retina (Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentosa (RP3)," Nature Genet.. 13:35-42 (1996); Roepman et al., "Positional Cloning of the Gene for X-Linked Retinitis Pigmentosa 3: Homology With the Guanine Nucleotide Exchange Factor RCC 1 ," Hum. Mol. Genet.. 5:1035-1041 (1996)). Because mutations in the RCC- 1 (regulator of chromosome condensation) domain prevent binding to phosphodiesterase-δ, which may play a role in membrane insertion or solubilization of prenylated proteins (Linari et al., "The Retinitis Pigmentosa GTPase Regulator, RPGR, Interacts With the Delta Subunit of Rod Cyclic GMP Phosphodiesterase," Proc. Natl. Acad. Sci. USA. 96:1315-1320 (1999)), a role for RPGR as a facilitator of intracellular protein trafficking has been proposed. Progressive retinal atrophy (PRA) represents a heterogenous group of phenotypically similar retinal disorders. Each such disorder shows the same general ophthalmoscopic abnormalities and visual deficits. These are characterized initially by rod dysfunction followed by loss of day vision; in the late stages of disease, the dogs are blind, have end-stage retinal degenerative changes, and secondary cataracts. PRA can be subdivided into developmental and degenerative diseases (Acland et al., "Non-Allelism of Three Genes (rcdl, rcd2 and erd) for Early-Onset Hereditary Retinal Degeneration," Exp. Eve Res. 49: 983-998 (1989); Aguirre et al., "Variation in Retinal Degeneration Phenotype Inherited at the prcd Locus," Exp. Eve Res. 46: 663-687 (1988)). The developmental class represents a large aggregate of genetically distinct disorders which are expressed cytologically in the postnatal period, when visual cells are beginning to differentiate. These include several gene loci among which are early retinal degeneration (erd; Acland and Aguirre, "Retinal Degenerations in the Dog: IV. Early Retinal Degeneration (erd) in Norwegian Elkhounds," Exp. Eve Res. 44: 491-521 (1987)), and rod cone dysplasia 2 (rcdl; Acland et al., "Non-Allelism of Three Genes (rcdl, rcd2 and erd) for Early-Onset Hereditary Retinal Degeneration," Exp. Eve Res. 49: 983-998 (1989)). In contrast, the degenerative class of diseases represents defects in which photoreceptor cells degenerate after having differentiated normally - this class includes mutations at the progressive rod-cone degeneration (prcd) and X-linked PRA (XLPRA) gene loci (Acland et al., "XLPRA: A Canine Retinal Degeneration Inherited As an X-Linked Trait," Am. J. Med. Genet. 52: 27-33 (1994); Aguirre et al., "Variation In Retinal Degeneration Phenotype Inherited At the prcd Locus," Exp. Eye Res. 46: 663-687 (1988)).

XLPRA is the only known naturally occurring animal model for XLRP, a blinding disorder in humans (Acland et al., "XLPRA: a Canine Retinal

Degeneration Inherited as an X-Linked Trait," Am. J. Med. Genet.. 52: 27-33 (1994)). Retinal development appears to occur normally, and affected male dogs first develop abnormal rod- and cone-mediated electroretinograph (ERG) responses between 6 and 12 months of age. The ERG responses then deteriorate rapidly. Ophthalmoscopic evidence of generalized retinal degeneration (hyperreflectivity due to retinal thinning, vascular attenuation, optic disc pallor) is present by 18 months of age. Histologically, hemizygous males develop lesions as early as 12 months of age. There is then a rapid progression of the disease with photoreceptors showing outer segment disorientation followed by shortening and loss. These changes are initially present in rods; with progression, diffuse loss of rods predominates, followed by cone degeneration and finally complete retinal atrophy by 2 years or later (Zeiss et al., "Retinal Pathology of Canine X-Linked Progressive Retinal Atrophy, the Locus Homolog of RP3," Inv. Qphthalm. Vis. Sci. 40:3292-3304 (1999)). Carrier females have reduced ERG amplitudes and develop ophthalmoscopic evidence of patchy retinal thinning presumed to be due to random X chromosome inactivation. Histologically, the lesions range from scattered patches of severe rod loss with partial to complete cone preservation, to diffuse and random loss of cells and their nuclei in the outer nuclear layer.

Two canine models of XLRP have now been identified: XLPRAi in the Siberian Huskies and Samoyeds, and XLPRA₂ in the Miniature Schnauzers. The first model, XLPRA] , is derived from a Siberian Husky male by outcrossing to non- affected females (Acland et al., "XLPRA: a Canine Retinal Degeneration Inherited as an X-Linked Trait," Am. J. Med. Genet.. 52: 27-33 (1994)). This disease is present in the general dog population from which the original dogs used to develop the research colony originated. The second model, XLPRA2_. ^lS derived from a Miniature Schnauzer pedigree, in which an inherited retinal disorder is segregating, by outcrossing and backcrossing to non-affected dogs of other breeds. This disease is present in the general dog population from which the original dogs used to develop the research colony originated.

Based on previous work (Parshall et al., "Photoreceptor Dysplasia: An Inherited Progressive Retinal Atrophy of Miniature Schnauzer Dogs," Prog. Vet. Comp. Ophth., 1 :187 (1991)), the disease in the Miniature Schnauzer was initially suspected to be inherited as an autosomal recessive retinal disorder and, based on this assumption, had been named photoreceptor dysplasia (pd). Molecular studies of this disease did not come up with any definitive gene/mutation responsible for the disease, although a sequence change inherited as an autosomal recessive trait that resulted in a non-conservative amino acid substitution in the phosducin gene was identified (Zhang et al., "Characterization of Canine Photoreceptor Phosducin cDNA and Identification of a Sequence Variant in Dogs With Photoreceptor Dysplasia," Gene, 215:231-239 (1998)). Genetic studies indicate that pd in the Miniature Schnauzer breed is not likely to be autosomal recessive, but rather an X-linked recessive disorder which is now referred to as XLPRA₂. This conclusion is based on the fact that some of the descendants of the dogs used in the original studies were used to develop the outbred pedigree used to characterize XLPRA₂. However, since at least a second form of PRA also exists in the breed, it is not definitive that the disease called pd is XLPRA2 or another inherited PRA. Unlike XLPRA _\ in the Siberian Husky, XLPRA₂ is a developmental abnormality which shows extensive structural and functional abnormalities of the rod and cone photoreceptors by 4-6 weeks after birth; thereafter, the abnormally developed retina slowly degenerates. Diagnosis of breeds affected with the XLPRA group of diseases is complicated by the need for sophisticated testing methods such as ERG, and by the late onset of the disease in the case of XLPRA _\. Regarding the latter, the age by which the disease can be diagnosed by current methods may be an age which is later than the onset of a dog's reproductive life. This late age of diagnosis results in the dissemination of the undesirable trait within the population and an increase in the disease frequency. The only effective control measure now available to dog breeders is to eliminate from the breeding pool all known affected and carrier dogs. Test mating, which is routinely used as a control measure in autosomal recessive disorders, is not practical in X-linked diseases. This is due to the fact that inheritance of genes located on (linked to) the X chromosome follows a pattern different from autosomal inheritance; which is central to the application of X-linked PRA discoveries. The canine sex chromosomes (like those of most mammals) are the X and Y chromosomes, which carry different sets of genes and are responsible for biological sex determination. The female has two X chromosomes, but no Y; designated as XX. The male has only one X chromosome and one Y; designated as XY. In females, the two alleles of an X-linked gene are present in three different combinations: (1) two normal alleles or XnXn with the normal phenotype; (2) one normal and one mutant allele or XnXm usually with the normal phenotype - the carrier state; or (3) two mutant alleles or XmXm with the affected phenotype. In contrast, for males there is only one X chromosome, thus the male has only two possible combinations: (1) one normal X allele and a Y chromosome or XnY with the normal phenotype; or (2) one mutant X allele and a Y chromosome or XmY with the affected phenotype. Males are never a carrier of an X-linked condition. As a result, there are special consequences of X-linked inheritance for breeding. Affected or carrier females with a normal mate can produce affected males, while affected males with a normal mate produce no affected offspring. Principals of X-linked inheritance must be applied to all mating combinations. Most X-linked genetic diseases are not expressed in the carrier female. It should be noted, however, that due to random inactivation of one X chromosome at an early developmental stage in the female, and due to the biology of the disease itself, some X-linked diseases are expressed in female carriers in varying degrees, which is the case for Miniature Schnauzer females carrying the X-linked PRA. Therefore, test matings are not effective as a control measure in XLPRA due to the difficulty of ascertaining the genetic status of affected females which can be either heterozygous or homozygous affected depending on the extent of random X- inactivation which occurs in females. Thus, there is a long-felt need in the canine breeding industry for a genetic test that permits identification of XLPRA and in various breeds of dogs before the detectable onset of clinical symptoms, as well as genotyping of dogs at risk for XLPRA.

The present invention is directed to overcoming these deficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to a method for identifying dogs which are genetically normal, are carriers of, or are affected with XLPRA. This method includes obtaining a biological sample from a dog and testing the biological sample for the presence of at least one genetic marker which co-segregates with an

XLPRA gene locus by linkage analysis, under conditions effective to determine the presence of a mutated XLPRA gene locus in one or both alleles. The present invention also relates to a method for selecting dogs for breeding. This includes obtaining a biological sample from a dog. The biological sample is then tested for the presence of at least one genetic marker which co- segregates with an XLPRA gene locus by linkage analysis, under conditions effective to determine the presence of a mutated XLPRA gene locus in one or both alleles. Dogs with the mutated XLPRA gene locus in one or both alleles are eliminated from a breeding stock. Alternatively, male dogs with the mutated XLPRA gene locus in one allele are bred with genetically normal female dogs. Another aspect of the present invention relates to a method for identifying dogs which are genetically normal, are carriers of, or are affected with XLPRA. This method involves obtaining a biological sample from a dog and testing the biological sample for a gene encoding canine RPGR having a nucleotide mutation in one or both alleles indicative of a carrier of or a dog affected with XLPRA. The present invention also relates to a method for selecting dogs for breeding. This includes obtaining a biological sample from a dog and testing the biological sample for a gene encoding canine RPGR having a nucleotide mutation in one or both alleles indicative of a carrier of or a dog affected with XLPRA. Dogs with the mutation are eliminated from a breeding stock or the dogs with the mutation are bred with genetically normal dogs.

Still another aspect of the present invention is directed to an isolated nucleic acid molecule encoding the intron 15 open reading frame ("ORF 15") of the canine RPGR in normal dogs, having a nucleotide sequence of SEQ. ID. No. 9.

Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the ORF 15 of the canine RPGR in XLPRA i -affected dogs, having a nucleotide sequence of SEQ. ID. No. 11.

Yet another aspect of the present invention pertains to an isolated nucleic acid molecule encoding the ORF 15 of the canine RPGR in XLPRA -affected dogs, having a nucleotide sequence of SEQ. ID. No. 13. The present invention has identified the gene mutations responsible for both XLPRA i and XLPRA₂ which are useful in a mutation-based test for both forms of PRA in dogs. Also disclosed is a marker-based test for XLPRA which can function alone as an equally powerful alternative to a mutation-based test for XLPRA, or as an independent and redundant confirmation of the mutation-based test in breeds for which the XLPRA mutation is known. Thus, in breeds for which the PRA mutation has not yet been identified, the marker-based test enables distinction of the XLPRA disease from all other forms of retinal degeneration and permits testing of dogs in all families segregating XLPRA. Furthermore, the marker-based test, in combination with the linkage-based test, also identifies XLPRA-affected dogs and families in which the mutation is novel (i.e. neither XLPRAi nor XLPRA ).

The present invention describes the development of XLPRA as a model for the RP3 form of XLRP and of a marker-based method which enables one (1) to establish whether PRA in any given dog breed, or family within a breed is a locus homolog of RP3 in man, (2) to identify which dogs in any such population are affected with, are carriers of, or are homozygous normal (wild-type) for XLPRA, and (3) to permit breeding advice to be given to dog breeders, owners, and breed organizations to allow breeding plans to be instituted that will eliminate the risk of producing dogs affected with XLPRA. Since it- has been proposed that the apparent deficiency in identifiable RPGR mutations in human RP3 patients might be due to the complex splicing pattern of this gene (Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentosa (RP3)," Nature Genet., 13:35-42 (1996); Fujita et al, "Analysis of the RPGR Gene in 11 Pedigrees With the Retinitis Pigmentosa Type 3 Genotype: Paucity of Mutations in the Coding Region but Splice Defects in Two Families," Am. J. Hum. Genet., 61 :571-580 (1997)), analysis of the splice variants became critical. Thus, the present invention also describes a mutation-based method which enables one (1) to identify the mutation present in XLPRAi and XLPRA₂, (2) to identify which dogs in any such population are affected with, are carriers of, or are homozygous normal (wild-type) for XLPRA] or XLPRA2, and (3) to permit breeding advice to be given to dog breeders, owners, and breed organizations to allow breeding plans to be instituted that will eliminate the risk of producing dogs affected with XLPRA] or XLPRA₂. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of the human RP3 interval, identifying the physical relationship among the genes CYBB (i.e. cytochrome b beta subunit), TCTE1L (i.e. the human homolog of mouse t complex gene), SRPX (i.e. sushi- repeat-containing protein, X chromosome), RPGR (i.e. retinitis pigmentosa GTPase regulator), and OTC (i.e. ornithine transcarbamylase). These genes reside in a chromosomal region encompassing approximately 500 kb. The exact size of the corresponding canine segment is not yet determined, but is clearly similar based on linkage and Radiation Hybrid mapping results. This figure is based on Figure 1 from Meindl et al., Hum. Mol. Genet.. 4(12):2339-2346 (1995) and Figure 1 of Nature Genet., 13(1): 35-42 (1996), which are hereby incorporated by reference.

Figure 2 is an ideogram of the canine and human X chromosomes. The canine X chromosome and locations of some of the genes used to type the XLPRA pedigree are indicated on the left. The human X chromosome and locations of the five X-linked RP loci are indicated on the right (adapted from RetNet, http://www.sph.uth.tmc.edu/Retnet/). There is no recombination between RPGR and XLPRA] or XLPRA₂; as RPGR occupies a similar location on the canine X chromosome to that on the human X chromosome, it can be concluded that XLPRA] and XLPRA₂ are the locus homologs of RP3. Chromosome band patterns were adapted from published reports for canines (Reimann et al., "An Extended Nomenclature of the Canine Karyotype," Cytogenet. Cell Genet., 73:140-144 (1996), which is hereby incorporated by reference) and humans (Harnden et al., ISCN. An International System for Human Cytogenetic Nomenclature (1985), which is hereby incorporated by reference).

Figure 3 shows a representative XLPRA] informative pedigree, demonstrating cosegregation of an RPGR Nla III RFLP with the disease phenotype. Marker allele a is in phase with the XLPRA] mutation. Circles = females, squares = males; black symbols = affected individuals, white symbols = nonaffected; dotted symbols = nonaffected obligate heterozygotes for XLPRA]. Although data are shown for only one marker locus in the XLPRA haplotype, identical results were obtained for each locus located within the zero recombination region that distinguish between the normal and affected haplotype.

Figure 4 shows a representative XLPRA2 informative pedigree, demonstrating cosegregation of an RPGR Nla III RFLP with the disease phenotype. Marker allele a is in phase with the XLPRA2 mutation. Circles = females, squares = males; black symbols = affected individuals, white symbols = nonaffected; dotted symbols = nonaffected obligate heterozygotes for XLPRA₂. Although data are shown for only one marker locus in the XLPRA haplotype, identical results obtained for each locus located within the zero recombination region are useful in distinguishing between the normal and affected haplotype. Figure 5 A illustrates the comparison of human, mouse (mRpgr- 1) and canine (2.4 kb variant) RPGR cDNAs. The numbers above each transcript indicate the exons spliced in the cDNA. Exon numbers are assigned based on the reported human (Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentosa (RP3)," Nature Genet.. 13:35-42 (1996); Roepman et al., "Positional Cloning of the Gene for X-Linked Retinitis Pigmentosa 3: Homology With the Guanine Nucleotide Exchange Factor RCC1," Hum. Mol. Genet., 5:1035-1041 (1996), which are hereby incorporated by reference); and mouse (Yan et al., "Biochemical Characterization and Subcellular Localization of the Mouse Retinitis Pigmentosa GTPase Regulator (mRpgr)," J. Biol. Chem.. 273: 19656-19663 (1998), which is hereby incorporated by reference) cDNA structures and their homology to the RPGR-coding sequence in dog. The portions of the coding sequence which encode putative functional domains of the protein are indicated as follows: the GTP phosphate-binding sites are indicated with two thick black vertical bars (exon 2), the RCC-1 domains are shaded with wavy horizontal lines (exons 3-10), and the positively charged regions are light gray (exons 14-16). The terminal isoprenylation site (exon 19) is indicated with oblique lines. UTRs are shaded in black. Figure 5B shows the alternative splice variants of canine RPGR. Arrows are used to depict the locations of primers used to amplify the canine RPGR- coding sequences. Forward primers generally are placed above the bar representing each transcript, reverse primers are placed below; arrowheads indicate the primer direction. Exons are numbered according to their homology to human exons and are shaded as in (Figure 5 A), with the addition of the following: the composite exon 14-14A-15 is indicated by cross-hatching, and exon 15A by a checkered pattern. Sites of alternative splicing in the 3' half of the gene are indicated as follows: 1.8 kb (Gen Bank Accession No. AF148799): exon 10 is spliced in-frame to genomic sequence (putative exon 10A) and a stop codon is present nine nucleotides downstream from the splice junction; 2.4 kb (GenBank Accession No. AF14898): exon 13 is spliced to exon 16, with omission of exons 14 and 15; 2.8 kb (GenBank Accession No. AF148800): exon 13 is spliced in- frame to a composite exon 14- 14A- 15- 15 A and no stop codon has been identified; 3.3 kb (GenBank Accession No. AF148801): exon 14-14A-15 is present between exons 13 and 16.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for identifying dogs which are genetically normal, are carriers of, or are affected with XLPRA. This method includes obtaining a biological sample from a dog and testing the biological sample for the presence of at least one genetic marker which co-segregates with a XLPRA gene locus by linkage analysis, under conditions effective to determine the presence of a mutated XLPRA gene locus in one or both alleles.

The recent developments made in the canine genome linkage map now offer a way of identifying linked markers to XLPRA. Linkage analysis is based on first finding the general chromosomal region in which the mutated gene is located, followed by identification of genetic markers to characterize a much smaller region of the chromosome containing the disease locus (the location of the mutated gene). The closer together the marker and the mutated gene are on the chromosome, the less likely a recombination event will occur between them during meioses; i.e., there is linkage between the marker and the mutated gene. The more closely linked the marker and mutated gene are, the more predictive and useful is the test for identifying carriers. Additionally, by using two or more marker loci, substantial additional information can be ascertained in a linkage analysis that can markedly increase the accuracy of the linkage test. Further, using multiple marker loci in a linkage analysis allows for the ability to screen various affected breeds of dogs to identify breed- specific haplotypes that characterize the XLPRA allele in the specific breed of dog.

The genetic markers here refer to a variable (polymorphic) nucleotide sequence that is present in canine genomic DNA on the X chromosome, in which polymorphisms are identifiable with specific oligonucleotides (e.g., distinguishable by nucleic acid amplification and observance of a difference in size or sequence of nucleotides due to the polymorphism). Markers can be identified by any one of several techniques known to those skilled in the art, including microsatellite or short tandem repeat (STR) amplification, analyses of restriction fragment length polymorphisms (RFLP), single nucleotide polymorphism (SNP), detection of deletion or insertion sites, and random amplified polymorphic DNA (RAPD) analysis (Cushwa et al., Animal Biotech., 7:11-31 (1996), which is hereby incorporated by reference). A genetic marker is indicative of a mutation in the XLPRA gene locus when the marker (1) is genetically linked and co-segregates with the XLPRA gene locus such that the linkage observed has a maximum logarithm of odds (LOD) score of at least 3.0, (2) comprises a region of canine X chromosome homologous to a gene or noncoding region mapping to the RP3 region of human X chromosome (the XLPRA- informative region; see Figure 2), (3) contains a polymorphism informative for the XLPRA phenotype, and (4) can be used in a linkage assay or other molecular diagnostic assays (DNA test) to identify normal alleles (wild type; (+)), and mutant (XLPRA) alleles (by the presence of the polymorphism), and hence can distinguish XLPRA-affected dogs (XLPRA/XLPRA), carriers of XLPRA (XLPRA/+), and normal dogs (+/+) in the case of female dogs, and XLPRA-affected dogs (XLPRA/Y) from normal dogs (+/Y) in the case of male dogs. In that regard, markers additional to those illustrative examples disclosed herein, that map either by linkage or by physical methods so close to the XLPRA gene locus (i.e. the zero recombination region) that any polymorphism in or with such derivative chromosomal regions, may be used in a molecular diagnostic assay for detection of XLPRA or the carrier status. Genetic markers of the present invention can be made using different methodologies known to those in the art. For example, using the maps illustrated in Figures 1 and 2, the region of canine X chromosome that is informative for XLPRA may be microdissected, and fragments cloned into vectors to isolate DNA segments which can be tested for linkage with the XLPRA gene locus once polymorphisms are found. Alternatively, using pairs of primers for markers in the XLPRA-informative region of canine X chromosome, isolated DNA segments can be obtained from the XLPRA-informative region of canine X chromosome by nucleic acid amplification (e.g., polymerase chain reaction) or by nucleotide sequencing of the relevant region of X chromosome. Using the linkage test of the present invention, the DNA segments may be assessed for their ability to co-segregate with the XLPRA gene locus (e.g., a LOD score may be calculated) and, thus, the usefulness of each DNA segment in a molecular diagnostic assay for detection of XLPRA or the carrier status can be assessed. To find the chromosomal location of the XLPRA gene locus, linkage of micro satellite markers in the XLPRA locus can be established by analyzing the segregation of polymorphic alleles of each marker to the XLPRA pedigree. Pedigrees informative for XLPRA, as well as pedigrees not informative for XLPRA, are developed to assist in the identification of XLPRA linked marker loci. Using primers for each linked microsatellite marker, genomic DNA is analyzed by nucleic acid amplification (Ostrander et al., Genomics 16:207-213 (1993); Ostrander et al., Mamm. Genome. 6:192-195 (1995); Francisco et al, Mamm. Genome. 7:359-362 (1996), which are hereby incorporated by reference). Using sets of primers specific for other microsatellite markers, and using similar techniques, it can be found that other microsatellite markers are associated with the XLPRA locus.

Polymorphisms in canine genes located on the RP3 region of the X chromosome can be detected by RFLP. Thus, primers from the gene are used to screen canine genomic DNA for polymorphisms. Briefly, segments of genomic DNA from female carriers of XLPRA are amplified using primers which bind to selected regions of the gene. The amplified products are sequenced and examined for sequence differences at the same position between the two alleles. These sequence difference can result in a change in restriction site which can be detected when the PCR product is digested with the specified restriction enzyme, and the digested products are electrophoresed in polyacrylamide gels (e.g., 6%) and stained with ethidium bromide for visual analysis. Alternatively, polymoφhisms associated with the canine genes can be identified by microsatellite analysis. Briefly, primer pairs which flank each microsatellite are used in a nucleic acid amplification reaction containing genomic DNA. One of the primers has been previously radiolabeled with P-ATP. Following nucleic acid amplification, the amplified product is analyzed by polyacrylamide gel electrophoresis with subsequent autoradiography. Polymorphic markers associated with the canine genes are used to establish linkage to the XLPRA gene locus by analyzing the markers in pedigrees informative of XLPRA. When the polymoφhism is a SNP caused by a change in a single nucleotide in one allele, the SNP also can be detected by electrophoresis. Primers are used in a nucleic acid amplification reaction containing canine genomic DNA. The presence of both alleles would give 2 bands: a lower band corresponding to a homoduplex for each allele (indistinguishable in a 6% polyacrylamide gel), and an upper band corresponding to the heteroduplex formed between the two alleles. With the knowledge demonstrated herein that DNA sequences (polymoφhic markers) on the canine X chromosome have been identified as being linked to the XLPRA locus, additional markers may be generated from the known sequences or indicated location on canine X chromosome, for use in the method of the present invention. This is because the strong homology of the canine X chromosome, in the XLPRA-informative region, to the RP3 region of the human X chromosome indicates that any gene, expressed sequence tag or other conserved sequence that is mapped to this homologous region in the human will also map to the XLPRA- informative region of the canine X chromosome. Thus, any gene, expressed sequence tag or conserved sequence that maps to the RP3 region of the human X chromosome may be analyzed for polymoφhisms and utilized to detect linkage to the XLPRA gene locus using the routine methods described herein. For example, using the sequence of any gene, expressed sequence tag or conserved sequence known to those skilled in the art to map to the RP3 region of human X chromosome, one skilled in the art can readily isolate fragments of canine genomic DNA from both XLPRA-affected and normal dogs, determine the sequence of both affected and normal fragments or digest both with restriction enzymes and find polymoφhic differences either at the sequence level or in the restriction enzyme digest pattern, respectively, of the two products. Once such a polymoφhic difference is found, further characterization of the polymoφhic sequence will yield a marker linked to the XLPRA gene locus.

When determining the XLPRA locus genotype of a dog, any tightly linked (LOD score > 3; zero recombination) marker or more than one such marker can be used. Segregation analysis will reveal which allele(s) for each such tightly linked marker cosegregate with the mutant allele at the XLPRA locus. Because males have only one X-chromosome, the genotype of affected males immediately reveals the alleles at each tightly linked marker that are in "coupling phase" with the XLPRA mutant allele. The "phase" refers to the physical relationship among individual alleles of multiple genes. Such alleles (one per locus) are said to be in "coupling phase" if they are on the same chromosome, in comparison to alleles that lie on opposite members of a chromosome pair and are said to be in "repulsion". Determination of which alleles at a set of loci tightly linked to XLPRA are in coupling phase with the XLPRA mutant allele identifies the XLPRA-informative haplotype(s). In any pedigree in which XLPRA segregates, it is thus possible by using one or more such tightly linked markers to accurately and reliably identify the chromosomal segment bearing the XLPRA mutant allele even if the specific XLPRA mutation has not been identified. Once this has been established, the XLPRA genotype of close relatives of the dog may be determined by evaluating their genotype using the informative markers. In different pedigrees, one skilled in the art will appreciate that different markers and combinations of markers may be more useful, depending on whether the affected dog has different alleles at the marker loci from nonaffected relatives. Further, new markers may be identified for use in pedigrees in which none of the prior markers show diagnostic utility. However, a strength of the diagnostic method according to the present invention is that the number of polymoφhic loci already identified essentially makes it improbable that the test will be non-diagnostic in any given pedigree.

For any one marker, the probability that a tested dog will receive from a parent the same XLPRA allele that was in phase with the marker allele in the parent, is a function of the linkage distance between the marker locus and the XLPRA locus. For example, TIMP-1 is approximately 13 centimorgans from the XLPRA gene locus. This means that approximately 87% of the offspring will receive from a parent both the XLPRA allele and the TIMP-1 locus allele that were in phase (on the same chromosome) in that parent. Conversely, approximately 13% of the offspring will receive the XLPRA allele and the TIMP-1 locus allele that were on the opposite chromosome arm of that parent. In contrast, marker RPGR is only 0 centimorgans from the XLPRA gene locus. Such close linkage means that the XLPRA allele and the RPGR locus allele will be transmitted in phase in over 99% of reproductive events (meioses). With only one marker, analyses are based on the probability of whether the 2 loci (marker and XLPRA) were passed from a parent to offspring maintaining this phase relationship or whether the phase relationship was altered, e.g., via a recombination event. Using one marker, one is not able to distinguish a genotype representing a phase-preserved chromosome from a genotype representing a recombination event. However, if a closely linked marker (e.g., RPGR, or TCTE1L, or SRPX, or OTC) is used for genotype analysis, and if one assumes that all pups receive the XLPRA allele in phase, the likelihood of error in such analysis is much less than 1%. Because such markers are extremely closely linked to the XLPRA gene locus, any one of these markers alone is sufficient to establish the XLPRA genotype for individuals in a pedigree. Thus, in one embodiment of the present invention, a single marker (a marker in the zero recombination region from the XLPRA gene locus) located in the XLPRA-informative region may be used to determine of the XLPRA locus genotype of a set of dogs that are closely related to a dog known to be affected with XLPRA. The identification of the zero recombination region allows one to search for sequence information for this region from other species, e.g., humans, and use this sequence information to identify new genes or conserved sequences from which disease-specific SNPs can be characterized and used to develop or modify a marker-based test that is specific for the mutant chromosome for each of the diseases. Knowing which gene-specific markers are in the zero recombination interval for the disease allows one to take the gene-specific BACs, cosmids or any other genomic DNA libraries, and sequence these genes (e.g. TCTE1L, SRPX, and OTC). This sequence allows one to compare the sequence between normal and affected animals to identify SNPs, insertions, and deletions which are specific to the mutant chromosome for those genes in the zero recombination region. It should be noted that there is a limitation in establishing a disease-specific haplotype for markers in the XLPRA interval when using microsatellites, as in the case for the microsatellite markers in the TCTE1L, SRPX, and OTC genes. The limitation is based on microsatellite "slippage" in different generations, thus increasing or decreasing the number of repeats (n) for each microsatellite.

Using the method of the present invention, either a single individual or multiple individuals in a pedigree in which one or more known and available near relatives are affected with XLPRA can be tested. In this situation, it is determined whether the individuals tested are normal (wildtype; +/+ or +/Y), carrier (XLPRA/+), or XLPRA-affected (XLPRA/XLPRA or XLPRA/Y) at the XLPRA gene locus. In order to make this determination, it is necessary to type both the known affected

0 dog(s) and the dogs to be tested at at least one (and usually several) of the genetic markers in the XLPRA-informative region of the canine X chromosome.

Genotyping is based on the analysis of genomic DNA which is extracted using standard methods known to those skilled in the art, such as using a lysing buffer (e.g., 10 mM Tris pH 8.3, 50 mM KC1, 1.5 mM MgCl₂, 0.01% gelatin, 0.45% NP40™, 0.045% Tween 20™, and 60 μg/ml proteinase K) to lyse cells containing the DNA. DNA is extracted from specimens which may include blood (e.g., fresh or frozen), tissue samples (e.g., spleen, buccal smears), and hair samples containing follicular cells. Once the genomic DNA is isolated and purified, nucleic acid amplification (e.g., polymerase chain reaction) is used to amplify the region of DNA corresponding to each genetic marker to be used in the analysis. While conditions may vary slightly depending on the primer sequences used, generally nucleic acid amplification is performed for 30-40 cycles in a volume of 25 μl containing reaction buffer (e.g., 50 mM KC1, 10 mM Tris-HCl, pH 8.3, 1.0 to 3.0 mM MgCl₂), 0.2 mM each dNTPs (dATP, dCTP, dGTP, and dTTP), 0.2 μM oligonucleotide primer, 10 ng template DNA, and 0.5 units of thermostable DNA polymerase.

Using more than one marker (e.g., a combination of linked markers) found in the XLPRA-informative region to determine the XLPRA locus genotype of a dog has several advantages over using a single linked marker. For example, a number of mutations in the RPGR gene may cause the XLPRA disease phenotype, and the nature of the mutation may vary amongst pedigrees and amongst affected breeds. Using multiple linked markers makes the method more informative than a single marker-based method for determination of the XLPRA locus genotype. Additionally, two or more marker loci from within the zero recombination region which are shown to be informative can markedly increase the accuracy of the test by reducing the risk of error. In a preferred embodiment, the dog is a Siberian Husky, Samoyed, Miniature Schnauzer, or any breed in which progressive retinal atrophy disease is X- linked and the disease locus maps to an RP3 region of an X chromosome.

In carrying out any of the methods of the present invention, the biological sample is any tissue containing genomic DNA. Suitable biological samples include blood, hair, mucosal scrapings, semen, tissue biopsy, or saliva. In a most preferred embodiment, the biological sample is blood.

The present invention also relates to a method for selecting dogs for breeding. This includes obtaining a biological sample from a dog. The biological sample is then tested for the presence of at least one genetic marker which co- segregates with an XLPRA gene locus by linkage analysis, under conditions effective to determine the presence of a mutated XLPRA gene locus in one or both alleles. While normal males or females can be bred to any dog, carrier or affected females with the mutated XLPRA gene locus in one or both alleles are eliminated from a breeding stock. Affected males can be bred to normal females with three different consequences: (1) no affected pups will be born, or (2) all sons will be normal and can be used for future breedings, or (3) all females will be carriers and should not be used for breeding.

Another aspect of the present invention is a method for identifying dogs which are genetically normal, are carriers of, or are affected with XLPRA. This method involves obtaining a biological sample from a dog and testing the biological sample for a gene encoding canine RPGR having a nucleotide mutation in one or both alleles indicative of a carrier of or a dog affected with XLPRA.

In carrying out this aspect of the present invention, the dog is any breed in which progressive retinal atrophy disease is X-linked and the disease locus maps to an RP3 region of an X chromosome. Such dogs include a Siberian Husky, Samoyed, or Miniature Schnauzer.

A gene encoding canine RPGR having a 5 -nucleotide deletion mutation disclosed in the present invention (see SEQ. ID. No. 1 1) in one allele is indicative of a female Siberian Husky or Samoyed carrier of XLPRAi. A gene encoding canine RPGR having a 5-nucleotide mutation disclosed in the present invention (see SEQ. ID. No. 1 1) in both alleles of a female (or the single allele of a male) is indicative of a Siberian Husky or a Samoyed affected with XLPRA]. A gene encoding canine RPGR having a 2-nucleotide deletion mutation disclosed in the present invention (see SEQ. ID. No. 13) in one allele is indicative of a female Miniature Schnauzer carrier of XLPRA₂. A gene encoding canine RPGR having a 2- nucleotide mutation disclosed in the present invention (see SEQ. ID. No. 13) in both alleles of a female (or the single allele of a male) is indicative of a Miniature Schnauzer affected with XLPRA .

Because XLPRA-indicative mutations are disease specific, formally they are not considered polymoφhisms and only segregate in families affected by XLPRA] (Siberian Husky and Samoyed) or XLPRA₂ (Miniature Schnauzer). Thus, the mutations are informative for diagnosis or exclusion of specific diseases, but in families not segregating the specific disease (i.e. they have a different mutation in this gene) they will not be positively informative.

Methods of screening a biological sample for mutated nucleic acids can be carried out using either DNA or mRNA isolated from the biological sample. During periods when the gene is expressed, mRNA may be abundant and more readily detected. However, these genes are temporally controlled and, at most stages of development, the preferred material for screening is DNA.

Oligonucleotide Ligation Assay ("OLA") (Landegren et el., "A Ligase- Mediated Gene Detection Technique," Science. 241 :1077-1080 (1988) ; Landegren et al., "DNA Diagnostics ~ Molecular Techniques and Automation," Science, 242:229- 237 (1988); U.S. Patent No. 4,988,617 to Landegren et al., which are hereby incoφorated by reference), as described below, is one method for testing the genetic material in the biological sample. The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is biotinylated, and the other is labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. OLA is capable of detecting deletion mutations. However, numerous methods for characterizing or detecting deletion mutations are known in the art and any of those methods are also suitable for the present invention.

Another method of characterizing a deletion mutation entails direct DNA sequencing of the genetic locus that flanks and includes the deletion. Such analysis can be accomplished using either the "dideoxy-mediated chain termination method," also known as the "Sanger Method" (Sanger et al., "DNA Sequencing with Chain-Terminating Inhibitors," Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977), which is hereby incorporated by reference) or the "chemical degradation method," also known as the "Maxam-Gilbert method" (Maxam et al., "A New Method for Sequencing DNA," Proc. Natl. Acad. Sci. USA. 74:560-564 (1977), which is hereby incoφorated by reference).

One example of a procedure for sequencing DNA molecules using arrays of oligonucleotides is disclosed in U.S. Patent No. 5,202,231 to Drmanac et al., which is hereby incoφorated by reference. This involves application of target DNA to a solid support to which a plurality of oligonucleotides are attached. Sequences are read by hybridization of segments of the target DNA to the oligonucleotides and assembly of overlapping segments of hybridized oligonucleotides. The array utilizes all possible oligonucleotides of a certain length between 1 1 and 20 nucleotides, but there is little information about how this array is constructed. See also Chetverin et al., "Sequencing of Pools of Nucleic Acids on Oligonucleotide Arrays," BioSystems 30:215-31 (1993); WO 92/16655 to Khrapko et al.; Kuznetsova et al., "DNA Sequencing by Hybridization with Oligonucleotides Immobilized in Gel. Chemical Ligation as a Method of Expanding the Prospects for the Method," Mol. Biol. 28(20): 290-99(1994); Livits et al., "Dissociation of Duplexes Formed by Hybridization of DNA with Gel-Immobilized Oligonucleotides," J. Biomolec. Struct. & Dynam. 11(4): 783-812 (1994), which are hereby incoφorated by reference.

WO 89/10977 to Southern, which is hereby incoφorated by reference, discloses the use of a support carrying an array of oligonucleotides capable of undergoing a hybridization reaction for use in analyzing a nucleic acid sample for known point mutations, genomic fingeφrinting, linkage analysis, and sequence determination. The matrix is formed by laying nucleotide bases in a selected pattern on the support. This reference indicates that a hydroxyl linker group can be applied to the support with the oligonucleotides being assembled by a pen plotter or by masking. Recently, single strand polymoφhism assay ("SSPA") analysis and the closely related heteroduplex analysis methods have come into use as effective methods for screening for single-base mutations (Orita et al., "Detection of Polymoφhisms of Human DNA by Gel Electrophoresis as Single-Strand Conformation Polymoφhisms," Proc. Natl. Acad. Sci. USA, 86:2766-2770 (1989), which is hereby incorporated by reference). In these methods, the mobility of PCR-amplified test DNA from clinical specimens is compared with the mobility of DNA amplified from normal sources by direct electrophoresis of samples in adjacent lanes of native polyacrylamide or other types of matrix gels. Single-base changes often alter the secondary structure of the molecule sufficiently to cause slight mobility differences between the normal and mutant PCR products after prolonged electrophoresis.

Ligase chain reaction is another method of screening for mutated nucleic acids (see Barany, "Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase," Proc. Natl. Acad. Sci. USA, 88:189-193 (1991); Barany, "The Ligase Chain Reaction (LCR) in a PCR World," PCR Methods and Applications, 1 :5-16 (1991); WO 90/17239 to Barany et al.; Barany et al., "Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNA Ligase-Encoding Gene," Gene, 109:1-11 (1991); and Barany, "Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase," Proc. Natl. Acad. Sci. USA, 88:189-193 (1991), which are hereby incoφorated by reference). In general, the LCR procedure is carried out with two pairs of oligonucleotide probes: one pair binds to one strand of the target sequence to be detected; the other pair binds to the other complementary strand of the target sequence to be detected. The reaction is carried out by, first, denaturing (e.g., separating) the strands of the target sequence, then reacting the separated strands with the two pairs of oligonucleotide probes in the presence of a heat stable ligase so that each pair of oligonucleotide probes hybridizes to target DNA and, if there is perfect complementarity at their junction, adjacent probes are ligated together. If such complementarity is lacking, no ligation occurs and the probes separate individually from the target sequence during denaturation. The ligated or unligated probes are then separated during the denaturation step. The process is cyclically repeated until the sequence has been amplified to the desired degree. Detection can then be carried out by electrophoresis or by capture hybridization on an array of DNA probes. Ligated and unligated probes can then be detected to identify the presence of a mutation.

The ligase detection reaction (LDR) process is another method for detecting a mutation. It is described generally in WO 90/17239 to Barany et al., Barany et al., "Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNA Ligase-encoding Gene," Gene, 109:1-11 (1991), and Barany, "Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase," Proc. Natl. Acad. Sci. USA, 88:189-193 (1991), the disclosures of which are hereby incoφorated by reference. The ligase detection reaction is similar to the LCR technique; however, in LDR, there is only one pair of oligonucleotide probes which are complementary to one strand of the target sequence. While LCR provides an opportunity for exponential amplification, LDR achieves linear amplification.

Mundy et al. (U.S. Pat. No. 4,656,127, which is hereby incoφorated by reference) discusses alternative methods for determining the identity of the nucleotide present at a particular polymoφhic site. Mundy's methods employ a specialized exonuclease-resistant nucleotide derivative. A primer complementary to the allelic sequence immediately 3 '-to the polymoφhic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymoφhic site on the target molecule contains a nucleotide that is complementary to the particular exonucleotide-resistant nucleotide derivative present, then that derivative will be incoφorated by a polymerase onto the end of the hybridized primer. Such incoφoration renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonucleotide-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymoφhic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. The Mundy method has the advantage that it does not require the determination of large amounts of extraneous sequence data. It has the disadvantages of destroying the amplified target sequences and unmodified primer and of being extremely sensitive to the rate of polymerase incoφoration of the specific exonuclease-resistant nucleotide being used.

Recently, several primer-guided nucleotide incoφoration procedures, i.e. microsequencing methods, for assaying polymoφhic sites (i.e., sites of mutations) in DNA have been described (Kornher et al., "Mutation Detection Using Nucleotide Analogs that Alter Electrophoretic Mobility," Nucl. Acids. Res., 17:7779-7784

(1989); Sokolov, "Primer Extension Technique for the Detection of Single Nucleotide in Genomic DNA," Nucl. Acids Res.. 18:3671 (1990); Syvanen et al., "A Primer- Guided Nucleotide Incoφoration Assay in the Genotyping of Apolipoprotein E," Genomics. 8:684-692 (1990); Kuppuswamy et al., "Single Nucleotide Primer Extension to Detect Genetic Diseases: Experimental Application to Hemophilia B (Factor IX) and Cystic Fibrosis Genes," Proc. Natl. Acad. Sci. USA. 88:1143-1147 (1991); Prezant et al., "Trapped-Oligonucleotide Nucleotide Incoφoration (TONI) Assay, a Simple Method for Screening Point Mutations," Hum. Mutat., 1 : 159- 164 (1992); Ugozzoli et al., "Detection of Specific Alleles by Using Allele-specific Primer Extension Followed by Capture on Solid Support," GATA, 9:107-112 (1992); Nyren et al., "Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay," Anal. Biochem., 208:171-175 (1993), which are hereby incoφorated by reference). These methods differ from Genetic Bit TM Analysis ("GBA TM " discussed extensively below) in that they all rely on the incoφoration of labeled deoxynucleotides to discriminate between bases at a polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incoφorated, polymoφhisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen et al., "Identification of Individuals by Analysis of Biallelic DNA Markers, Using PCR and Solid-Phase Minisequencing," Amer. J. Hum. Genet.. 52:46-59 (1993), which is hereby incoφorated by reference).

Cohen et al. (French Patent 2,650,840; PCT Application No. WO 91/02087, which are hereby incoφorated by reference) discuss a solution-based method for determining the identity of the nucleotide of a polymoφhic site. As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3'-to a polymoφhic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymoφhic site, will become incoφorated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis TM or GBA TM is described by Goelet et al. (PCT Application No. 92/15712, which is hereby incoφorated by reference). In a preferred embodiment, the method of Goelet et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymoφhic site. The labeled terminator that is incoφorated is thus determined by, and complementary to, the nucleotide present in the polymoφhic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Application No. WO 91/02087, which are hereby incoφorated by reference), the method of Goelet et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase. It is thus easier to perform, and more accurate than the method discussed by Cohen.

Other recently developed variations for detecting the presence of mutations include: differential restriction endonuclease digestion (DRED), allele- specific oligonucleotide probing (ASOP), and ligase-mediated gene detection (LMGD). Additional methods of analysis would also be useful in this context, such as fluorescence resonance energy transfer (FRET) as disclosed by Wolf et al.,

"Detection of Nucleic Acid Hybridization by Nonradiative Fluorescence Resonance Energy Transfer," Proc. Nat. Acad. Sci. USA, 85: 8790-94 (1988), which is hereby incoφorated by reference.

DRED analysis is accomplished in the following manner. If conditions occur including (1) a particular amplified cDNA segment contains a sequence variation that distinguishes an allele of a polymoφhism and (2) this sequence variation is recognized by a restriction endonuclease, then the cleavage by the enzyme of a particular polynucleotide segment can be used to determine the alloantigen phenotype. In accomplishing this determination, amplified cDNA derived from platelet or red blood cell mRNA is digested and the resulting fragments are analyzed by size. The presence or absence of nucleotide fragments, corresponding to the endonuclease-cleaved fragments, determines which phenotype is present.

In ASOP analysis according to conventional methods, oligonucleotide probes are synthesized that will hybridize, under appropriate annealing conditions, exclusively to a particular amplified cDNA segment that contains a nucleotide sequence that distinguishes one allele from other alleles of a red blood cell or platelet membrane glycoprotein. This specific probe is discernibly labeled so that when it hybridizes to the allele distinguishing cDNA segment, it can be detected, and the specific allele is thus identified. In the course of the third method of analysis, LMGD, as disclosed by

Landegren et al., "A Ligase-Mediated Gene Detection Technique," Science, 241 : 1077-80 (1988), which is hereby incoφorated by reference, a pair of oligonucleotide probes are synthesized that will hybridize adjacent to each other, i.e., to a cDNA segment under appropriate annealing conditions, at the specific nucleotide that distinguishes one allele from other alleles. Each of the pair of specific probes is labeled in a different manner, and when it hybridizes to the allele-distinguishing cDNA segment, both probes can be ligated together by the addition of a ligase. When the ligated probes are isolated from the cDNA segments, both types of labeling can be observed together, confirming the presence of the allele-specific nucleotide sequence. Where the above-described pair of differently labeled probes bind to a nucleotide sequence containing a distinguishing nucleotide of a different allele, the probe pair is not ligatable and, after the probes are isolated from the cDNA segments, both types of labeling are observed separately.

WO 94/11530 to Cantor, which is hereby incoφorated by reference, relates to the use of an oligonucleotide array to carry out a process of sequencing by hybridization. The oligonucleotides are duplexes having overhanging ends to which target nucleic acids bind and are then ligated to the non-overhanging portion of the duplex. The array is constructed by using streptavidin-coated filter paper which captures biotinylated oligonucleotides assembled before attachment.

WO 93/17126 to Chetverin, which is hereby incoφorated by reference, uses sectioned, binary oligonucleotide arrays to sort and survey nucleic acids. These arrays have a constant nucleotide sequence attached to an adjacent variable nucleotide sequence, both bound to a solid support by a covalent linking moiety. The constant nucleotide sequence has a priming region to permit amplification by PCR of hybridized strands. Sorting is then carried out by hybridization to the variable region. Sequencing, isolating, sorting, and manipulating fragmented nucleic acids on these binary arrays are also disclosed. In one embodiment with enhanced sensitivity, the immobilized oligonucleotide has a shorter complementary region hybridized to it, leaving part of the oligonucleotide uncovered. The array is then subjected to hybridization conditions so that a complementary nucleic acid anneals to the immobilized oligonucleotide. DNA ligase is then used to join the shorter complementary region and the complementary nucleic acid on the array. WO 92/10588 to Fodor et al., which is hereby incoφorated by reference, discloses a process for sequencing, fingeφrinting, and mapping nucleic acids by hybridization to an array of oligonucleotides. The array of oligonucleotides is prepared by a very large scale immobilized polymer synthesis which permits the synthesis of large, different oligonucleotides. In this procedure, the substrate surface is functionalized and provided with a linker group by which oligonucleotides are assembled on the substrate. The regions where oligonucleotides are attached have protective groups (on the substrate or individual nucleotide subunits) which are selectively activated. Generally, this involves imaging the array with light using a mask of varying configuration so that areas exposed are deprotected. Areas which have been deprotected undergo a chemical reaction with a protected nucleotide to extend the oligonucleotide sequence where imaged. A binary masking strategy can be used to build two or more arrays at a given time. Detection involves positional localization of the region where hybridization has taken place. See also U.S. Patent Nos. 5,324,633 and 5,424,186 to Fodor et al., U.S. Patent Nos. 5,143,854 and 5,405,783 to Pirrung et al., WO 90/15070 to Pirrung et al., Pease et al., "Light- generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis", Proc. Natl. Acad. Sci USA 91 : 5022-26 (1994), which are hereby incoφorated by reference. Beattie et al, "Advances in Genosensor Research," Clin. Chem. 41 (5): 700-09 (1995), which is hereby incoφorated by reference, discloses attachment of previously assembled oligonucleotide probes to a solid support.

Landegren et al., "Reading Bits of Genetic Information: Methods for Single-Nucleotide Polymoφhism Analysis," Genome Research, 8:769-776 (1998), which is hereby incoφorated by reference, discloses a review of methods for mutation analysis which are suitable for the present invention.

In another embodiment, testing the biological sample includes amplifying a region of the gene encoding canine RPGR to provide an amplified fragment before detecting any mutation present in the biological sample. Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means, either to facilitate sequencing or for direct detection of mutations. (See generally Kwoh et al., "Target Amplification Systems in Nucleic Acid-Based Diagnostic Approaches," Am. Biotechnol. Lab., 8:14-25 (1990) which is hereby incoφorated by reference.) Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction ("LCR") strand displacement amplification (see generally, Walker et al., "Strand Displacement Amplification — An Isothermal, In Vitro DNA Amplification Technique," Nucleic Acids Res.. 20:1691-1696 (1992); Walker et al., "Isothermal In- Vitro Amplification of DNA By a Restriction Enzyme-DNA Polymerase System," Proc. Natl. Acad. Sci. USA 89:392-396 (1992), which are hereby incoφorated by reference), transcription-based amplification (see Kwoh et al., "Transcription-Based Amplification System and Detection of Amplified Human Immunodeficiency Virus Type 1 With a Bead-Based Sandwich Hybridization Format," Proc. Natl. Acad. Sci. USA 86:1173-1177 (1989), which is hereby incoφorated by reference), self-sustained sequence replication (or "3SR") (see Guatelli et al., "Isothermal In- Vitro Amplification of Nucleic Acids By a Multienzyme Reaction Modeled After Retroviral Replication," Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990), which is hereby incoφorated by reference), the Qβ replicase system (see Lizardi et al., "Exponential Amplification of Recombinant RNA Hybridization Probes," Biotechnology, 6:1 197- 1202 (1988), which is hereby incoφorated by reference), nucleic acid sequence-based amplification (or "NASBA") (see Lewis, "Review of Progress in Developing Amplification Technologies Which May Compete With Roche Diagnostic Systems' Polymerase Chain Reaction (PCR)," Genetic Engineering News. 12(9):1, 8-9 (1992), which is hereby incoφorated by reference), the repair chain reaction (or "RCR") (see Lewis, "Review of Progress in Developing Amplification Technologies Which May Compete With Roche Diagnostic Systems' Polymerase Chain Reaction (PCR)," Genetic Engineering News, 12(9):1, 8-9 (1992), which is hereby incoφorated by reference), and boomerang DNA amplification (or "BDA") (see Lewis, "Review of Progress in Developing Amplification Technologies Which May Compete With Roche Diagnostic Systems' Polymerase Chain Reaction (PCRV'Genetic Engineering News, 12(9): 1, 8-9 (1992), which is hereby incoφorated by reference). Polymerase chain reaction is currently preferred. Genomic sequence-specific amplification technologies, such as the polymerase chain reaction (Mullis et al., "Specific Enzymatic Amplification of DNA in- Vitro the Polymerase Chain Reaction," Cold Spring Harbor Symp. Quant. Biol. 51 :263-274 (1986); European Patent Application No. 50,424 to Erlich et al.; European Patent Application No. 84,796 to Erlich et al.; European Patent Application 258,017 to Erlich et al.; European Patent Application No. 237,362 to Erlich et al.; European Patent Application No. 201,184 to Mullis; U.S. Patent No. 4,683,202 to Mullis et al.; U.S. Patent No. 4,582,788 to Erlich; Saiki et al., "Enzymatic Amplification of Beta Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia," Science 230:1350-1354 (1985); and U.S. Patent No. 4,683,194 to Saiki et al., which are hereby incoφorated by reference), may be employed to facilitate the recovery of the desired polynucleotides. In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication. The amplified sequence may be readily identified by a variety of techniques. This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body- fluid sample. In a preferred embodiment, testing the biological sample includes performing PCR using genomic DNA templates and polyacrylamide gel electrophoresis (PAGE). In particular, PCR is performed using primers spanning the location of the mutation. The sizes of the amplified DNA fragments from homozygous normal and affected dogs are different. Subsequently, the amplified DNA fragments are electrophoresed using PAGE.

In one embodiment of the invention, the testing of the genetic material in the biological sample is carried out by Taq cycle sequencing. The method for cycle sequencing, based on linear amplification of template DNA by polymerase chain reaction, was described by Murray, "Improved Double Stranded Sequencing Using the Linear Polymerase Chain Reaction," Nucleic Acids Research, 17:88-89 (1989), which is hereby incoφorated by reference. This technique essentially combines fhermocycling procedure using Taq polymerase with dideoxy sequencing. In principle, the sequencing reaction consists of primer annealing to the template DNA followed by repeated extension of the primer by Taq polymerase in the presence of dNTPs/ddNTPs, linearly amplifying the sequence reaction products. Currently, cycle sequencing is done almost exclusively by non-isotopic methods using an automated DNA sequencer. A popular format for the sequencing protocol developed by Probe et al., "A System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides," Science. 238:336-341 (1987), which is hereby incoφorated by reference, is based on the use of a set of four chain-terminating dideoxynucelotides, each coupled to a different fluorescent dye and distinguishable by fluorescence emission. The DNA fragments are resolved by gel electrophoresis in one sequencing lane and detected by a scanning fluorescence detection system with computer-based automatic sequence identification.

Another method that can be used to detect a mutation is polymerase chain reaction restriction fragment length polymoφhism (PCR-RFLP). Single nucleotide changes in the genes are common phenomenon. Such alterations, depending on their locations, can be innocuous or deleterious to the gene function. Single base changes can alter the recognition sequence of restriction enzymes resulting in creation of a new, or abolition of an existing, restriction site, giving rise to variation in DNA fragment length. The variants are called restriction fragment length polymoφhism (RFLP). These are inherited in a codominant fashion and are allelic variants, generating homozygous and heterozygous genotypes. Identification of RFLP in mammalian genome has been classically determined by Southern blot analysis. Use of polymerase chain reaction (PCR) to detect RFLP has dramatically accelerated the pace of initial identification and subsequent assaying of a large number of samples in an easy to use format. In short, two oligonucleotide primers are designed from the region of the genome flanking the suspected variation in the sequence between two alleles. These primer pairs are used to amplify the encompassing region of interest from genomic DNA by PCR using Taq polymerase and dNTPs in the presence of an optimal concentration of magnesium chloride. The PCR products are digested with the restriction enzyme with altered recognition sites between two alleles of the genome, and the digested DNA fragments are separated by electrophoresis in a solid matrix of choice (e.g., agarose or polyacrylamide) depending on the size of the fragments. (See, e.g., Ray et al., "Molecular Diagnostic Test for Ascertainment of Genotype at the Rod Cone Dysplasia (rcdl) Locus in Irish Setters," Current Eve Research. 14:243-247 (1995); Ray et al., "A Highly

Polymoφhic RFLP Marker in the Canine Transducin α-1 Subunit Gene," Anim. Genet.. 27:372-373 (1996); Ray et al., "PCR/RFLP Marker in the Canine Opsin Gene," Anim. Genet.. 27:293-294 (1996); Wang et al., "PCR/RFLP Marker in the Canine Transducin-γ Gene (GNGT1)," Anim. Genet., 28:319-320 (1997); Gu et al., "Detection of Single Nucleotide Polymoφhism," BioTechniques, 24:836-837 (1998) and Zeiss et al., "A Highly Polymoφhic RFLP Marker in the Canine Retinitis Pigmentosa GTPase Regulator (RPGR) Gene," Anim. Genet.. 29:409 (1998), which are hereby incoφorated by reference). In addition to the rapidity, the PCR-RFLP technique also offers the flexibility to create an allele specific restriction site when the nucleotide change does not naturally create a RFLP. This is routinely done by deliberately incoφorating a mismatch nucleotide in one of the primers such that a restriction site is created in one of the two alleles. Nickerson et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson et al., "Automated DNA Diagnostics Using an Elisa-Based Oligonucleotide Ligation Assay," Proc. Natl. Acad. Sci. USA, 87:8923-8927 (1990), which is hereby incoφorated by reference). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Another aspect of the present invention relates to a method for selecting dogs for breeding. This includes obtaining a biological sample from a dog and testing the biological sample for a gene encoding canine RPGR having a nucleotide mutation in one or both alleles indicative of a carrier of or a dog affected with XLPRA. Dogs with the mutation are eliminated from a breeding stock or the dogs with the mutation are bred with genetically normal dogs. This method allows affected or carrier dogs to be eliminated from the breeding stock or bred to genetically normal dogs which do not have a nucleotide mutation in a gene encoding RPGR. Yet another aspect of the present invention relates to an isolated nucleic acid molecule that encodes for the ORF 15 of the canine RPGR in normal dogs and comprises a nucleotide sequence corresponding to SEQ. ID. No. 9 as follows:

1 GTG AGT GAA GGC AAG GGA AAG GCA GGA GGT GGA GGT GAG GGA ATC CAG CGG 52 GAG GGT GAT TCA GGA GTG GAA CAG AGG CAA AGT GAG GAG GGG CAA GAG GAG

103 GAA GAC AAG AGA GGA GGA GAA ATG GAG GGC CTG GCT AAG GGA GAG AAA AAC

154 CTA GAA GAG GAG GAG GCC CAG GAA CAA AGG GAG AGG GAA CAA GGC CAT CGG

205 GAA GAA AGA AAC AAA GGA ACA GAA GGA GAG GAA GGG GAA GAG CAA AGA GAT

256 GAG AAG GAA GGG GAG GGG AGA GGA GGG GAA GAA GAC CGG GAA GAG GAA GAA 307 GAG GAG GAA GAA GGC AAG GAG GAA GGA GAA TGG AAA GAG GAA GGA CAG GGA

358 GAG GGG GAA GAA GAA GGA GAG GAG GAG GAA GCA GAG GAG AAA GAA GAA GAA 409 GCA GAG CAA GGA GGG GAG GAA GGA GAG GGG GAA GAG GAG GAA GGA GGA GAG 460 GGG GAA GGG AAG AAA AGA GAA GGG GAA GGG GAA GAG GAA GGA GCA GAG AAA 511 GAG AAG GAA GCA GAA GAA GTA GAG GAG GAA GGA GAG GGG GAA TTG GAA GAG 562 GAA GGG GAG GGG GAA TTA GAG GAA GGA GAG GGA AAA GGG GAA GAA AGA GAG 613 GGG GAA TTG GAG GGG GAA GAG GAG GAG GGG GAA GAG GAA GGG GAG GAG AAA 664 GGG GAG GCT GGA GGG GAG GAA GGA GAG GCA GAA TGG GAA GGA AAG GGG GAA

715 GTG GAG GAA GGA GAA GGG GAA GTG GAG GAG GAA GGA GAG GAG GAA GTG GAA

766 GAA GGA GAA GGG GAA GGA GAG GGG GAA GTG GAG GAA GGA GAA GCA GAA GAA

817 GAG GAT GCA GAA GAA GAA GAA AGG GAA GTG GAA GAA GAA GGA GAG GGG GAA 868 GGA AAC AAA AGA GAA TAT GAA GGG GAG GAG GAA GAG GGA GAA GAG GAG GAA

919 AGA GAA AAT GAA AGA GAG GAG GAA GAA GAA GGA GGA GAA GCA GGG GAA GGG

970 GAG GAG GAA GAA GAG GGG GAA GCA GGG CAG GAA GAA GGG GAG GGG GAA GAA

1021 GGG GAA GAG GAG GTA GGG GAG GAA GAG GGC GAA GGA GAG GAA GAA GAA GAA

1072 GGG GAG GAA GAA GAA GGA GAG GAG GAG GGG GAA GAG GAG GAA GGA GAG GGG 1123 GAG GAG GAA GGG AGA GGA AGG GAA GGT GAG AGG GAA GAG GAA GTA GGG GAG

1174 GAG GAG GAA ACA GAA GGG GAA GGG GAC AAG GAA GGA GAG GAG GAA GGA GAG

1225 GAA GAG GGA GAG GAA GCA GAG TGG GGA GTG AGG GAG AGG GAA GAG AAG GAC

1276 ACT GAA GAA GAG GGG AAA TAT GAG GAG ACA GGA GAT GAA GAG AGT GAA AAA

1327 CAG GGA AGA CGG GGT GAA GGA AGA GAG TCC AAC AGA GCA AGC AAA ATC AGA 1378 GGA TCT GTG AAA TAT AAC AAA GAT AAG GCA TAC CCC CAA AAG TTT ATT ACT

1429 AAC ACA GAG GGA AAG GGG AAA GAG CGT GAG GCA CAG AGG TTC AAA ATG CCA

1480 ATG CGG TCA AAA CAA CTT TTA AAA AAT GGG CCA CCG GGT TCT AAA AAA TTC

1531 TGG ACT AAT GTA TTA CCA CAT TAT TTA GAA TTG AAG TAA CAAACTTTAAAATTG

1585 ACTGGATTTGGTCCAGCCAAACAGAGTATCTTACACATAATAGGCACTAAATACATTTTATTAAAAT 1652 TGCTTTTAGGCCATGGTTACTTTATGTTTGGTATAGTAACAACTGATAAAGACAACATAAGGCACTG

1719 GTAAATTTAGCGCCTCTTAAACTAATATTTTTGGTATATTTCCCCCCAAAATTATGAATCTGTCACT

1786 AGAACATATCAAAGGGTCATAGCAGATAAATAACATTATATATTTCTCATTAAAGGCAGATTTAGGA

1853 GATTGTAGTATACTATTATAAGAGTGTTGCGTTTGTATATGCGAACTGGTCCCTTTAAAGACGGCAC

1920 AGATCCTTCAGGGAGATAAAAACATCTCATTTCTTATGACTTTATTTCAATCCCTGATGACAGTTTG 1987 CCTACCATAAGGAATAATTTTGTAAGATGGATGATATTCTAAAACTAAAATGAAGAATTCATATTTT

2054 TAAAAAATAGTGCTTTTTTTTTTTTTTTTTACCTTATATGTGTCCTTTTTGCAACAATCTACCAAGT

2121 GACAGCTGGATTCAAAGCTTAGATCTTAATGTGACATCTTTTAGTTACTGTCTGGGCAAATGACAGT

2188 ATATCTCATGAAATGGAGGTGAACCTAAACATATATTCTGGAAGTATGTAAAACTTGTAAGTAGTTA

2255 TTGGCCAGGCAGAGCACCTCACTGGGGTTCACTTTCCTCATGTGTAAGATGGAGTGGATTGAATGAT 2322 TGAACTAAGGTCTGTTCTAACTCTCAGATTCTTTTGTTCTAACAGGTGGTTTTTCGCCCATTGTGTT

2389 TGTCATTTGCATGAAAGTTTTTTAAAACTTTAATGTCACTTTAAAATCAATCTTTTCTATAAATTAT

2456 ATGTTCTTTACTCCTAAAAACTTAGGCTGAGCCTTCATGTCTCTTGTCTTCTGTTATTCTGTATTCT 2523 AAACTTTAACTTCTGAGCTTTACCTGGACTGTCAGAATCTTCATTCCCAATGTCTATTATGTACCCT 2590 CAGTGTGTTCTTCATTGACTATTCTGTAATTCTCAGTTCGTTGCTTGTTAGAATGTTCCAAGTAAAA 2657 TAAAGTGTAAATATATACTGTCAGTAATTGTTTGTGGAAAGAAAAACTAACATTAAGTATTGATTTT 2724 TCAAGAGGCTAGATTTCTATTAGCATAAATTCAGTCTTCCCTTAAAATTTTCAGTATGTATCATGCA 2791 GGTAGATCATTTGAT

The normal ORF 15 has a deduced amino acid sequence corresponding to SEQ. ID. No. 10 as follows:

Val Ser Glu Gly Lys Gly Lys Ala Gly Gly Gly Gly Glu Gly lie Gin Arg 17 Glu Gly Asp Ser Gly Val Glu Gin Arg Gin Ser Glu Glu Gly Gin Glu Glu 34 Glu Asp Lys Arg Gly Gly Glu Met Glu Gly Leu Ala Lys Gly Glu Lys Asn 51

Leu Glu Glu Glu Glu Ala Gin Glu Gin Arg Glu Arg Glu Gin Gly His Arg 68

Glu Glu Arg Asn Lys Gly Thr Glu Gly Glu Glu Gly Glu Glu Gin Arg Asp 85

Glu Lys Glu Gly Glu Gly Arg Gly Gly Glu Glu Asp Arg Glu Glu Glu Glu 102 Glu Glu Glu Glu Gly Lys Glu Glu Gly Glu Trp Lys Glu Glu Gly Gin Gly 119

Glu Gly Glu Glu Glu Gly Glu Glu Glu Glu Ala Glu Glu Lys Glu Glu Glu 136

Ala Glu Gin Gly Gly Glu Glu Gly Glu Gly Glu Glu Glu Glu Gly Gly Glu 153

Gly Glu Gly Lys Lys Arg Glu Gly Glu Gly Glu Glu Glu Gly Ala Glu Lys 170

Glu Lys Glu Ala Glu Glu Val Glu Glu Glu Gly Glu Gly Glu Leu Glu Glu 187 Glu Gly Glu Gly Glu Leu Glu Glu Gly Glu Gly Lys Gly Glu Glu Arg Glu 204

Gly Glu Leu Glu Gly Glu Glu Glu Glu Gly Glu Glu Glu Gly Glu Glu Lys 221

Gly Glu Ala Gly Gly Glu Glu Gly Glu Ala Glu Trp Glu Gly Lys Gly Glu 238

Val Glu Glu Gly Glu Gly Glu Val Glu Glu Glu Gly Glu Glu Glu Val Glu 255

Glu Gly Glu Gly Glu Gly Glu Gly Glu Val Glu Glu Gly Glu Ala Glu Glu 272 Glu Asp Ala Glu Glu Glu Glu Arg Glu Val Glu Glu Glu Gly Glu Gly Glu 289

Gly Asn Lys Arg Glu Tyr Glu Gly Glu Glu Glu Glu Gly Glu Glu Glu Glu 306

Arg Glu Asn Glu Arg Glu Glu Glu Glu Glu Gly Gly Glu Ala Gly Glu Gly 323

Glu Glu Glu Glu Glu Gly Glu Ala Gly Gin Glu Glu Gly Glu Gly Glu Glu 340

Gly Glu Glu Glu Val Gly Glu Glu Glu Gly Glu Gly Glu Glu Glu Glu Glu 357 Gly Glu Glu Glu Glu Gly Glu Glu Glu Gly Glu Glu Glu Glu Gly Glu Gly 374

Glu Glu Glu Gly Arg Gly Arg Glu Gly Glu Arg Glu Glu Glu Val Gly Glu 391

Glu Glu Glu Thr Glu Gly Glu Gly Asp Lys Glu Gly Glu Glu Glu Gly Glu 408

Glu Glu Gly Glu Glu Ala Glu Trp Gly Val Arg Glu Arg Glu Glu Lys Asp 425

Thr Glu Glu Glu Gly Lys Tyr Glu Glu Thr Gly Asp Glu Glu Ser Glu Lys 442 Gin Gly Arg Arg Gly Glu Gly Arg Glu Ser Asn Arg Ala Ser Lys lie Arg 459

Gly Ser Val Lys Tyr Asn Lys Asp Lys Ala Tyr Pro Gin Lys Phe lie Thr 476

Asn Thr Glu Gly Lys Gly Lys Glu Arg Glu Ala Gin Arg Phe Lys Met Pro 493

Met Arg Ser Lys Gin Leu Leu Lys Asn Gly Pro Pro Gly Ser Lys Lys Phe 510

Trp Thr Asn Val Leu Pro His Tyr Leu Glu Leu Lys Stop 522

Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes for the mutant ORF 15 of the canine RPGR in XLPRAj- affected dogs and comprises a nucleotide sequence of SEQ. ID. No. 1 1, which includes a GAGAA deletion of nucleotides 878 through 882, as follows:

1 GTG AGT GAA GGC AAG GGA AAG GCA GGA GGT GGA GGT GAG GGA ATC CAG CGG 52 GAG GGT GAT TCA GGA GTG GAA CAG AGG CAA AGT GAG GAG GGG CAA GAG GAG 103 GAA GAC AAG AGA GGA GGA GAA ATG GAG GGC CTG GCT AAG GGA GAG AAA AAC 154 CTA GAA GAG GAG GAG GCC CAG GAA CAA AGG GAG AGG GAA CAA GGC CAT CGG 205 GAA GAA AGA AAC AAA GGA ACA GAA GGA GAG GAA GGG GAA GAG CAA AGA GAT 256 GAG AAG GAA GGG GAG GGG AGA GGA GGG GAA GAA GAC CGG GAA GAG GAA GAA 307 GAG GAG GAA GAA GGC AAG GAG GAA GGA GAA TGG AAA GAG GAA GGA CAG GGA 358 GAG GGG GAA GAA GAA GGA GAG GAG GAG GAA GCA GAG GAG AAA GAA GAA GAA 409 GCA GAG CAA GGA GGG GAG GAA GGA GAG GGG GAA GAG GAG GAA GGA GGA GAG 460 GGG GAA GGG AAG AAA AGA GAA GGG GAA GGG GAA GAG GAA GGA GCA GAG AAA 511 GAG AAG GAA GCA GAA GAA GTA GAG GAG GAA GGA GAG GGG GAA TTG GAA GAG 562 GAA GGG GAG GGG GAA TTA GAG GAA GGA GAG GGA AAA GGG GAA GAA AGA GAG 613 GGG GAA TTG GAG GGG GAA GAG GAG GAG GGG GAA GAG GAA GGG GAG GAG AAA 664 GGG GAG GCT GGA GGG GAG GAA GGA GAG GCA GAA TGG GAA GGA AAG GGG GAA 715 GTG GAG GAA GGA GAA GGG GAA GTG GAG GAG GAA GGA GAG GAG GAA GTG GAA 766 GAA GGA GAA GGG GAA GGA GAG GGG GAA GTG GAG GAA GGA GAA GCA GAA GAA 817 GAG GAT GCA GAA GAA GAA GAA AGG GAA GTG GAA GAA GAA GGA GAG GGG GAA

868 GGA AAC AAA A|TA TGA AGGGGAGGAGGAAGAGGGAGAAGAGGAGGAAAGAGAAAATGAAAGAG

930 AGGAGGAAGAAGAAGGAGGAGAAGCAGGGGAAGGGGAGGAGGAAGAAGAGGGGGAAGCAGGGCAGGA

997 AGAAGGGGAGGGGGAAGAAGGGGAAGAGGAGGTAGGGGAGGAAGAGGGCGAAGGAGAGGAAGAAGAA

1064 GAAGGGGAGGAAGAAGAAGGAGAGGAGGAGGGGGAAGAGGAGGAAGGAGAGGGGGAGGAGGAAGGGA 1131 GAGGAAGGGAAGGTGAGAGGGAAGAGGAAGTAGGGGAGGAGGAGGAAACAGAAGGGGAAGGGGACAA 1198 GGAAGGAGAGGAGGAAGGAGAGGAAGAGGGAGAGGAAGCAGAGTGGGGAGTGAGGGAGAGGGAAGAG

1265 AAGGACACTGAAGAAGAGGGGAAATATGAGGAGACAGGAGATGAAGAGAGTGAAAAACAGGGAAGAC 1332 GGGGTGAAGGAAGAGAGTCCAACAGAGCAAGCAAAATCAGAGGATCTGTGAAATATAACAAAGATAA

1399 GGCATACCCCCAAAAGTTTATTACTAACACAGAGGGAAAGGGGAAAGAGCGTGAGGCACAGAGGTTC 1466 AAAATGCCAATGCGGTCAAAACAACTTTTAAAAAATGGGCCACCGGGTTCTAAAAAATTCTGGACTA

1533 ATGTATTACCACATTATTTAGAATTGAAGTAACAAACTTTAAAATTGACTGGATTTGGTCCAGCCAA 1600 ACAGAGTATCTTACACATAATAGGCACTAAATACATTTTATTAAAATTGCTTTTAGGCCATGGTTAC 1667 TTTATGTTTGGTATAGTAACAACTGATAAAGACAACATAAGGCACTGGTAAATTTAGCGCCTCTTAA 1734 ACTAATATTTTTGGTATATTTCCCCCCAAAATTATGAATCTGTCACTAGAACATATCAAAGGGTCAT 1801 AGCAGATAAATAACATTATATATTTCTCATTAAAGGCAGATTTAGGAGATTGTAGTATACTATTATA 1868 AGAGTGTTGCGTTTGTATATGCGAACTGGTCCCTTTAAAGACGGCACAGATCCTTCAGGGAGATAAA 1935 AACATCTCATTTCTTATGACTTTATTTCAATCCCTGATGACAGTTTGCCTACCATAAGGAATAATTT 2002 TGTAAGATGGATGATATTCTAAAACTAAAATGAAGAATTCATATTTTTAAAAAATAGTGCTTTTTTT 2069 TTTTTTTTTTACCTTATATGTGTCCTTTTTGCAACAATCTACCAAGTGACAGCTGGATTCAAAGCTT 2136 AGATCTTAATGTGACATCTTTTAGTTACTGTCTGGGCAAATGACAGTATATCTCATGAAATGGAGGT 2203 GAACCTAAACATATATTCTGGAAGTATGTAAAACTTGTAAGTAGTTATTGGCCAGGCAGAGCACCTC

2270 ACTGGGGTTCACTTTCCTCATGTGTAAGATGGAGTGGATTGAATGATTGAACTAAGGTCTGTTCTAA

2337 CTCTCAGATTCTTTTGTTCTAACAGGTGGTTTTTCGCCCATTGTGTTTGTCATTTGCATGAAAGTTT

2404 TTTAAAACTTTAATGTCACTTTAAAATCAATCTTTTCTATAAATTATATGTTCTTTACTCCTAAAAA 2471 CTTAGGCTGAGCCTTCATGTCTCTTGTCTTCTGTTATTCTGTATTCTAAACTTTAACTTCTGAGCTT

2538 TACCTGGACTGTCAGAATCTTCATTCCCAATGTCTATTATGTACCCTCAGTGTGTTCTTCATTGACT 2605 ATTCTGTAATTCTCAGTTCGTTGCTTGTTAGAATGTTCCAAGTAAAATAAAGTGTAAATATATACTG 2672 TCAGTAATTGTTTGTGGAAAGAAAAACTAACATTAAGTATTGATTTTTCAAGAGGCTAGATTTCTAT 2739 TAGCATAAATTCAGTCTTCCCTTAAAATTTTCAGTATGTATCATGCAGGTAGATCATTTGAT

The mutant ORF 15 in XLPRA-affected dogs has a deduced amino acid sequence corresponding to SEQ. ID. No. 12, where the GAGAA deletion of nucleotides 878 through 882 creates a premature stop, as follows: Val Ser Glu Gly Lys Gly Lys Ala Gly Gly Gly Gly Glu Gly lie Gin Arg 17

Glu Gly Asp Ser Gly Val Glu Gin Arg Gin Ser Glu Glu Gly Gin Glu Glu 34

Glu Asp Lys Arg Gly Gly Glu Met Glu Gly Leu Ala Lys Gly Glu Lys Asn 51 Leu Glu Glu Glu Glu Ala Gin Glu Gin Arg Glu Arg Glu Gin Gly His Arg 68

Glu Glu Arg Asn Lys Gly Thr Glu Gly Glu Glu Gly Glu Glu Gin Arg Asp 85

Glu Lys Glu Gly Glu Gly Arg Gly Gly Glu Glu Asp Arg Glu Glu Glu Glu 102

Glu Glu Glu Glu Gly Lys Glu Glu Gly Glu Trp Lys Glu Glu Gly Gin Gly 119

Glu Gly Glu Glu Glu Gly Glu Glu Glu Glu Ala Glu Glu Lys Glu Glu Glu 136 Ala Glu Gin Gly Gly Glu Glu Gly Glu Gly Glu Glu Glu Glu Gly Gly Glu 153

Gly Glu Gly Lys Lys Arg Glu Gly Glu Gly Glu Glu Glu Gly Ala Glu Lys 170

Glu Lys Glu Ala Glu Glu Val Glu Glu Glu Gly Glu Gly Glu Leu Glu Glu 187

Glu Gly Glu Gly Glu Leu Glu Glu Gly Glu Gly Lys Gly Glu Glu Arg Glu 204

Gly Glu Leu Glu Gly Glu Glu Glu Glu Gly Glu Glu Glu Gly Glu Glu Lys 221 Gly Glu Ala Gly Gly Glu Glu Gly Glu Ala Glu Trp Glu Gly Lys Gly Glu 238

Val Glu Glu Gly Glu Gly Glu Val Glu Glu Glu Gly Glu Glu Glu Val Glu 255

Glu Gly Glu Gly Glu Gly Glu Gly Glu Val Glu Glu Gly Glu Ala Glu Glu 272

Glu Asp Ala Glu Glu Glu Glu Arg Glu Val Glu Glu Glu Gly Glu Gly Glu 289

Gly Asn Lys lie Stop 293

Yet another aspect of the present invention relates to an isolated nucleic acid molecule that encodes for the mutant ORF 15 of the canine RPGR in XLPRA₂-affected dogs and comprises a nucleotide sequence of SEQ. ID. No. 13, which includes a GA deletion of nucleotides 932 through 933, as follows:

1 GTG AGT GAA GGC AAG GGA AAG GCA GGA GGT GGA GGT GAG GGA ATC CAG CGG

52 GAG GGT GAT TCA GGA GTG GAA CAG AGG CAA AGT GAG GAG GGG CAA GAG GAG

103 GAA GAC AAG AGA GGA GGA GAA ATG GAG GGC CTG GCT AAG GGA GAG AAA AAC

154 CTA GAA GAG GAG GAG GCC CAG GAA CAA AGG GAG AGG GAA CAA GGC CAT CGG 205 GAA GAA AGA AAC AAA GGA ACA GAA GGA GAG GAA GGG GAA GAG CAA AGA GAT

256 GAG AAG GAA GGG GAG GGG AGA GGA GGG GAA GAA GAC CGG GAA GAG GAA GAA

307 GAG GAG GAA GAA GGC AAG GAG GAA GGA GAA TGG AAA GAG GAA GGA CAG GGA 358 GAG GGG GAA GAA GAA GGA GAG GAG GAG GAA GCA GAG GAG AAA GAA GAA GAA

409 GCA GAG CAA GGA GGG GAG GAA GGA GAG GGG GAA GAG GAG GAA GGA GGA GAG 460 GGG GAA GGG AAG AAA AGA GAA GGG GAA GGG GAA GAG GAA GGA GCA GAG AAA

511 GAG AAG GAA GCA GAA GAA GTA GAG GAG GAA GGA GAG GGG GAA TTG GAA GAG 562 GAA GGG GAG GGG GAA TTA GAG GAA GGA GAG GGA AAA GGG GAA GAA AGA GAG 613 GGG GAA TTG GAG GGG GAA GAG GAG GAG GGG GAA GAG GAA GGG GAG GAG AAA 664 GGG GAG GCT GGA GGG GAG GAA GGA GAG GCA GAA TGG GAA GGA AAG GGG GAA 715 GTG GAG GAA GGA GAA GGG GAA GTG GAG GAG GAA GGA GAG GAG GAA GTG GAA 766 GAA GGA GAA GGG GAA GGA GAG GGG GAA GTG GAG GAA GGA GAA GCA GAA GAA 817 GAG GAT GCA GAA GAA GAA GAA AGG GAA GTG GAA GAA GAA GGA GAG GGG GAA 868 GGA AAC AAA AGA GAA TAT GAA GGG GAG GAG GAA GAG GGA GAA GAG GAG GAA

919 AGA GAA AAT GAA A|GA GGA GGA AGA AGA AGG AGG AGA AGC AGG GGA AGG GGA

970 GGA GGA AGA AGA GGG GGA AGC AGG GCA GGA AGA AGG GGA GGG GGA AGA AGG

1021 GGA AGA GGA GGT AGG GGA GGA AGA GGG CGA AGG AGA GGA AGA AGA AGA AGG 1072 GGA GGA AGA AGA AGG AGA GGA GGA GGG GGA AGA GGA GGA AGG AGA GGG GGA

1123 GGA GGA AGG GAG AGG AAG GGA AGG TGA GAGGGAAGAGGAAGTAGGGGAGGAGGAGGAA

1181 ACAGAAGGGGAAGGGGACAAGGAAGGAGAGGAGGAAGGAGAGGAAGAGGGAGAGGAAGCAGAGTGGG

1248 GAGTGAGGGAGAGGGAAGAGAAGGACACTGAAGAAGAGGGGAAATATGAGGAGACAGGAGATGAAGA

1315 GAGTGAAAAACAGGGAAGACGGGGTGAAGGAAGAGAGTCCAACAGAGCAAGCAAAATCAGAGGATCT 1382 GTGAAATATAACAAAGATAAGGCATACCCCCAAAAGTTTATTACTAACACAGAGGGAAAGGGGAAAG

1449 AGCGTGAGGCACAGAGGTTCAAAATGCCAATGCGGTCAAAACAACTTTTAAAAAATGGGCCACCGGG

1516 TTCTAAAAAATTCTGGACTAATGTATTACCACATTATTTAGAATTGAAGTAACAAACTTTAAAATTG

1583 ACTGGATTTGGTCCAGCCAAACAGAGTATCTTACACATAATAGGCACTAAATACATTTTATTAAAAT

1650 TGCTTTTAGGCCATGGTTACTTTATGTTTGGTATAGTAACAACTGATAAAGACAACATAAGGCACTG 1717 GTAAATTTAGCGCCTCTTAAACTAATATTTTTGGTATATTTCCCCCCAAAATTATGAATCTGTCACT

1784 AGAACATATCAAAGGGTCATAGCAGATAAATAACATTATATATTTCTCATTAAAGGCAGATTTAGGA

1851 GATTGTAGTATACTATTATAAGAGTGTTGCGTTTGTATATGCGAACTGGTCCCTTTAAAGACGGCAC

1918 AGATCCTTCAGGGAGATAAAAACATCTCATTTCTTATGACTTTATTTCAATCCCTGATGACAGTTTG

1985 CCTACCATAAGGAATAATTTTGTAAGATGGATGATATTCTAAAACTAAAATGAAGAATTCATATTTT 2052 TAAAAAATAGTGCTTTTTTTTTTTTTTTTTACCTTATATGTGTCCTTTTTGCAACAATCTACCAAGT 119 GACAGCTGGATTCAAAGCTTAGATCTTAATGTGACATCTTTTAGTTACTGTCTGGGCAAATGACAGT

2186 ATATCTCATGAAATGGAGGTGAACCTAAACATATATTCTGGAAGTATGTAAAACTTGTAAGTAGTTA

2253 TTGGCCAGGCAGAGCACCTCACTGGGGTTCACTTTCCTCATGTGTAAGATGGAGTGGATTGAATGAT 320 TGAACTAAGGTCTGTTCTAACTCTCAGATTCTTTTGTTCTAACAGGTGGTTTTTCGCCCATTGTGTT 2387 TGTCATTTGCATGAAAGTTTTTTAAAACTTTAATGTCACTTTAAAATCAATCTTTTCTATAAATTAT

2454 ATGTTCTTTACTCCTAAAAACTTAGGCTGAGCCTTCATGTCTCTTGTCTTCTGTTATTCTGTATTCT 2521 AAACTTTAACTTCTGAGCTTTACCTGGACTGTCAGAATCTTCATTCCCAATGTCTATTATGTACCCT 2588 CAGTGTGTTCTTCATTGACTATTCTGTAATTCTCAGTTCGTTGCTTGTTAGAATGTTCCAAGTAAAA 2655 TAAAGTGTAAATATATACTGTCAGTAATTGTTTGTGGAAAGAAAAACTAACATTAAGTATTGATTTT 2722 TCAAGAGGCTAGATTTCTATTAGCATAAATTCAGTCTTCCCTTAAAATTTTCAGTATGTATCATGCA 2789 GGTAGATCATTTGAT

The mutant ORF 15 in XLPRA₂-affected dogs has a deduced amino acid sequence corresponding to SEQ. ID. No. 14, where the GA deletion of nucleotides 932 through 933 causes change in amino acid sequence, as follows:

Val Ser Glu Gly Lys Gly Lys Ala Gly Gly Gly Gly Glu Gly lie Gin Arg 17

Glu Gly Asp Ser Gly Val Glu Gin Arg Gin Ser Glu Glu Gly Gin Glu Glu 34

Glu Asp Lys Arg Gly Gly Glu Met Glu Gly Leu Ala Lys Gly Glu Lys Asn 51 Leu Glu Glu Glu Glu Ala Gin Glu Gin Arg Glu Arg Glu Gin Gly His Arg 68

Glu Glu Arg Asn Lys Gly Thr Glu Gly Glu Glu Gly Glu Glu Gin Arg Asp 85

Glu Lys Glu Gly Glu Gly Arg Gly Gly Glu Glu Asp Arg Glu Glu Glu Glu 102

Glu Glu Glu Glu Gly Lys Glu Glu Gly Glu Trp Lys Glu Glu Gly Gin Gly 119 Glu Gly Glu Glu Glu Gly Glu Glu Glu Glu Ala Glu Glu Lys Glu Glu Glu 136

Ala Glu Gin Gly Gly Glu Glu Gly Glu Gly Glu Glu Glu Glu Gly Gly Glu 153

Gly Glu Gly Lys Lys Arg Glu Gly Glu Gly Glu Glu Glu Gly Ala Glu Lys 170

Glu Lys Glu Ala Glu Glu Val Glu Glu Glu Gly Glu Gly Glu Leu Glu Glu 187 Glu Gly Glu Gly Glu Leu Glu Glu Gly Glu Gly Lys Gly Glu Glu Arg Glu 204

Gly Glu Leu Glu Gly Glu Glu Glu Glu Gly Glu Glu Glu Gly Glu Glu Lys 221

Gly Glu Ala Gly Gly Glu Glu Gly Glu Ala Glu Trp Glu Gly Lys Gly Glu 238

Val Glu Glu Gly Glu Gly Glu Val Glu Glu Glu Gly Glu Glu Glu Val Glu 255

Glu Gly Glu Gly Glu Gly Glu Gly Glu Val Glu Glu Gly Glu Ala Glu Glu 272 Glu Asp Ala Glu Glu Glu Glu Arg Glu Val Glu Glu Glu Gly Glu Gly Glu 289

Gly Asn Lys Arg Glu Tyr Glu Gly Glu Glu Glu Glu Gly Glu Glu Glu Glu 306

Arg Glu Asn Glu Arg | Gly Gly Arg Arg Arg Arg Arg Ser Arg Gly Arg Gly 323

Gly Gly Arg Arg Gly Gly Ser Arg Ala Gly Arg Arg Gly Gly Gly Arg Arg 340

Gly Arg Gly Gly Arg Gly Gly Arg Gly Arg Arg Arg Gly Arg Arg Arg Arg 357 Gly Gly Arg Arg Arg Arg Gly Gly Gly Gly Arg Gly Gly Arg Arg Gly Gly 374

Gly Gly Arg Glu Arg Lys Gly Arg Stop 382

EXAMPLES

The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

Example 1 - Animals and DNA Samples

The XLPRAi colony was derived from a Siberian Husky male by outcrossing to non-affected females (Acland et al., "A Canine Retinal Degeneration Inherited as an X-Linked Trait," Amer. J. Med. Genet.. 52:27-33 (1994), which is hereby incoφorated by reference). This disease is present in the general dog population from which the original dogs used to develop the research colony originated. The XLPRA2 colony was derived from a Miniature Schnauzer pedigree, in which an inherited retinal disorder was segregating, by outcrossing and backcrossing to non-affected dogs of other breeds. This disease is present in the general dog population from which the original dogs used to develop the research colony originated. Animals were only included in the studies if clinically evident disease status could be confirmed unequivocally by clinical examination (ophthalmoscopy and electroretinogram) and/or histology (Acland et al., "A Canine Retinal Degeneration Inherited as an X-Linked Trait," Amer. J. Med. Genet., 52:27- 33 (1994), which is hereby incoφorated by reference). The genotype of dogs maintained through adulthood for breeding was established by clinical methods, combined with disease status of their progeny.

Genomic DNA was isolated using standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press (1989), which is hereby incoφorated by reference) from blood samples collected in citrate anticoagulant tubes.

Example 2 - Linkage Mapping of the Diseases to the RP3 Region of the Canine X-Chromosome

The XLPRA] pedigrees were typed with five widely spaced intragenic markers which were known to map to the canine X-chromosome. Initial linkage studies using the first three markers — androgen receptor (AR) (Shibuya et al., "Two Polymorphic Microsatellites in a Coding Segment of the Canine Androgen Receptor Gene," Anim. Genet., 24:345-348 (1993), which is hereby incoφorated by reference), dystrophin (DMD) (Valentine et al., "Canine X-Linked Muscular Dystrophy as an Animal Model of Duchenne Muscular Dystrophy: A Review," Am. J. Med. Genet., 42:352-356 (1992), which is hereby incoφorated by reference), and factor IX (Gu et al., "A Polymoφhic (TTTA)_n Tandem Repeat in an Intron of the Canine Factor IX Gene," Anim. Genet., 28:370-383 (1997), which is hereby incoφorated by reference) — indicated that the XLPRA] locus is located on the short arm of the canine X chromosome. To determine whether XLPRA] is a locus homolog of RP2 or RP3, two additional intragenic markers in the canine tissue inhibitor of metalloproteinase 1 (TIMP-1) (Zeiss et al., "TIMP-1 Expression is Increased in X-Linked Progressive Retinal Atrophy Despite Its Exclusion as a Candidate Gene," Gene, 225:67-76 (1998), which is hereby incoφorated by reference) and RPGR (Zeiss et al., "A Highly Polymoφhic RFLP Marker in the Canine Retinitis Pigmentosa GTP-ase Regulator (RPGR) Gene, Anim. Genet.. 29:409 (1998), which is hereby incoφorated by reference) genes were identified. Two-point linkage analysis was performed between the XLPRA] locus and each marker, and between each pair of markers using the

LINKAGE package of programs (Lathrop et al., "East Calculations of Lod Scores and Genetic Risks on Small Computers," Am. J. Hum. Genet.. 36:460-465 (1984); Terwilleger et al., Handbook of Human Genetic Linkage, Baltimore, Maryland: Johns Hopkins University Press (1994); Ott, Analysis of Human Genetic Linkage, Baltimore, Maryland: Johns Hopkins University Press (1991), which are hereby incoφorated by reference). The disease trait was coded as sex-linked recessive with full penetrance. Results of two-point linkage analysis (see Table 1) indicated that there were 5 recombinants between TIMP-1 and XLPRA] for the 36 dogs typed

(LOD 5.01, θ = 0.13), thus placing XLPRA] approximately 13 cM away from TIMP- 1. Of 48 dogs examined, there was no recombination between an intragenic polymoφhism within the canine RPGR gene and XLPRA (LOD 11.74, θ = 0). Thus, zero recombination between RPGR and XLPRA] indicated locus homology between XLPRA] and RP3, and placed XLPRA at the canine equivalent of human Xp21.

Table 1. Results of two-point linkage analysis between XLPRA) and five intragenic markers. Significant LOD scores (z) are indicated in bold face.

DMD RPGR TIMP-1 AR FIX

XLPRA θ 0.22 0.00 0.13 0.14 0.26

Z 0.93 11.74 5.01 4.54 1.9 n 14 48 36 36 38

RPRG θ 0.24 Z 0.74

TIMP-1 θ 0.24 0.13 z 0.74 5.01

AR θ 0.17 0.14 0.00 z 1.13 4.54 10.5

FIX θ 0.40 0.26 0.13 0.11 z 0.09 1.9 5.01 5.13

Once linkage of XLPRA j and RPGR was established, a 43 dog subset of the Miniature Schnauzer XLPRA2 pedigree whose disease was ascertained using moφhological criteria was typed. In these animals, tight linkage with zero recombination (LOD 12.9, 0 = 0) was observed between XLPRA2 and RPGR, thus establishing presumptive locus homology to RP3. Once linkage between XLPRA2 and RPGR was detected with zero combination, a linkage study with other markers used in Table 1 was not carried out as with XLPRA] as it was not necessary. Thus, X-linked inheritance was proven by genetic methods and tight linkage with zero recombinations was found with RPGR, confirming locus homology with RP3. Therefore, Figure 2 and the linkage results presented in Table 1 showed that because of the tight linkage between XLPRA] and the RPGR gene, XLPRA] is the locus homolog of RP3. The mapping of XLPRA2 also placed the disease as a locus homolog of RP3 as it was tightly linked to RPGR with zero recombinations.

Two-point linkage analysis between AR and the other markers used in the linkage mapping indicated that the order of these 5 genes on the X chromosome is preserved between human and dog. Of the genes included in the linkage analysis, the approximate physical locations on the canine X chromosome of two, AR (Deschenes et al., "Comparative Mapping of Canine and Human Proximal Xq and Genetic Analysis of Canine X-Linked Severe Combined Immunodeficiency," Genomics, 23:62-68 (1994), which is hereby incoφorated by reference) and factor IX (Dutra et al., "Gene Localization and Syntenic Mapping by FISH in the Dog," Cvtogenet. Cell Genet., 74:113-117 (1996), which is hereby incoφorated by reference), were known. In addition, two other genes (Choroideremia and Phosphoglycerolkinase) that are in the same linkage group with AR show the same gene order in dog, human and mouse (Deschenes et al., "Comparative Mapping of Canine and Human Proximal Xq and Genetic Analysis of Canine X-Linked Severe Combined Immunodeficiency," Genomics, 23:62-68 (1994), which is hereby incoφorated by reference). The dog X chromosome has been found to be more like the human than that of any other mapped species (bovine, sheep, goat, pig and rodent) and the gene order is conserved (Piumi et al., "Comparative Cytogenetic Mapping Reveals Chromosome Rearrangements Between the X Chromosomes of Two Closely Related Mammalian Species (Cattle and Goats)," Cvtogenet. Cell Genet., 81 :36-41 (1998); Galloway et al., "A Linkage Map of the Ovine X Chromosome," Genome Res., 6:667-677 (1996); de Gortari et al., "A Second-Generation Linkage Map of the Sheep Genome," Mamm. Genome. 9:204- 209 (1998); Flu et al., "Mapping Genes to Swine X Chromosome Provides Reference Loci for Comparative Mapping, Mamm. Genome, 8:608-610 (1997); Millwood et al., "A Gene-Based Genetic Linkage and Comparative Map of the Rat X Chromosome," Genomics, 40:253-261 (1997); Kuroiwa et al., "Comparative FISH Mapping of

Mouse and Rat Homologues of Twenty-Five Human X-Linked Genes," Cvtogenet. Cell Genet., 81 :208-212 (1998), which are hereby incoφorated by reference). Example 3 - Canine-Specific Polymorphic Markers Defining the XLPRA Zero Recombination Region

Linkage of microsatellite markers to the XLPRA was established by analyzing the segregation of polymoφhic alleles of the markers in the XLPRA pedigrees. Using sets of primers specific for each linked microsatellite marker, large pieces of genomic DNA and retinal cDNA that represent the canine homolog of the human RP3 interval and genes expressed from within this interval were identified, amplified and sequenced. As part of this effort, specific clones from a canine bacterial artificial chromosome (BAC) library that contain the canine homologs of genes mapping within this interval in humans (see Figure 1 ) were identified. Consequently, four polymoφhic markers that co-segregate with XLPRAi and XLPRA₂ were identified: TCTE1L (CA),₆ dinucleotide microsatellite, SRPX- (GAAA)₂₆ tetranucleotide microsatellite, RFLP marker from the RPGR gene, and OTC-(GAAA) _{] 2} tetranucleotide microsatellite.

The TCTE1L microsatellite marker [typical dinucleotide repeat number: (CA)]₆] was located in a BAC clone [BAC clone "391 N 14" from canine genomic BAC library <http://www.chori.org/bacpac/mcanine81.htm>], positive for TCTE1L., by performing PCR for 35 cycles in 1.5 mM MgCl₂ (94°C for 2 min, 94°C for 30 sec, 53°C for 30 sec, 72°C for 30 sec, with a final extension at 72°C for

15 min) using primers CUX20001-3 and CUX20001-4. The amplified PCR products were electrophoresed in a 6% polyacrylamide gel. Primer CUX20001-3 has a nucleotide sequence corresponding to SEQ. ID. No. 1 as follows:

5 ' GGGTCTGAGCATGGCTTTGA 3 '

Primer CUX20001-4 has a nucleotide sequence corresponding to SEQ. ID. No. 2 as follows:

5 ' TTGATGCCTCGGGCTTGGG 3 '

The SRPX microsatellite marker [typical tetranucleotide repeat number: (GAAA) ₆] was located in a BAC clone [BAC clone "255 O 20" from canine genomic BAC library <http://www.chori.org/bacpac/mcanine81.htm>] positive for SRPX, by performing PCR for 32 cycles (94°C for 15 sec, 62°C for 30 sec, and 72°C for lmin) using primers CMS1 and CMS2. The sizes of the expected bands ranged from 230-300 bp. The amplified PCR products were electrophoresed in a 6% polyacrylamide gel. Primer CMS1 has a nucleotide sequence corresponding to SEQ. ID. No. 3 as follows:

5 ' TGGCACGCGCGGATCCTTGG 3 '

Primer CMS2 has a nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:

5 ' TGTGCGCGCTGCCTGGGTAGG 3 '

The RFLP marker from the RPGR gene is within the intron 14 of the RPGR gene (Zeiss et al., "A Highly Polymoφhic RFLP marker in the canine retinitis pigmentosa GTPase regulator (RPGR) gene," Anim. Genet.. 29:409 (1998), which is hereby incorporated by reference). PCR was carried out for 34 cycles (94°C for

15 sec, 58°C for 30 sec, and 72°C for 2 min) using primers RPGR-27 and RPGR-28. The size of the amplified fragment of canine genomic DNA was 590 bp. Primer RPGR-27 has a nucleotide sequence corresponding to SEQ. ID. No. 5 as follows:

5 ' GACATAGGTAATGACTCAGGCCAG 3 '

Primer RPGR-28 has a nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:

5 ' AATTTGGACAGTATGTGTTCGGTC 3 '

PCR-amplified DNA was digested with Nla III (New England Biolab) at 37°C for 4 hours and the digests analyzed by electrophoresis on a 7% polyacrylamide gel.

Digestion of the 590 bp PCR product amplified from canine genomic DNA produced a 500 bp polymoφhic fragment (allele 1) and a 90 bp fragment. When the polymoφhic Nla III site was present, it cleaved the 500 bp DNA fragment into two fragments of 300 bp and 200 bp (allele 2).

The OTC microsatellite marker [typical tetranucleotide repeat number:

(GATA)] ] was located in a BAC clone [BAC clone "119 B 21 " from canine genomic BAC library <http://www.chori.org/bacpac/mcanine81.htm>] positive for OTC, by performing PCR for 35 cycles in 1.5 mM MgCl₂ (94°C for 2 min, 94°C for 15 sec, 62°C for 30 sec, 72°C for 1 min, with a final extension at 72°C for 15 min) using primers CUX40002-1 and CUX40002-2. The sizes of the expected bands ranged around 174 bp. The amplified PCR products were electrophoresed in a 6% polyacrylamide gel. Primer CUX40002-1 has a nucleotide sequence corresponding to SEQ. ID. No. 7 as follows:

5' GCATGGAGTTTCCTTGCTCCTC 3'

Primer CUX40002-2 has a nucleotide sequence corresponding to SEQ. ID. No. 8 as follows:

5' TATTCAAGGTGCTGAATGGGGA 3'

These markers corresponded to genes that flank or are located in the human RP3 interval, and established that this interval is conserved between dog and human in gene content. All mutations so far identified within the RP3 interval in humans reside in the RPGR gene, but it has not yet been formally excluded that some RP3 interval forms of XLRP might result from mutations in a different gene in this interval.

In extensive pedigrees, including those informative for XLPRA, and others in which XLPRA did not segregate, these markers were always found to cosegregate perfectly. That is, no recombinations were seen among these markers in over 80 observed meioses (LOD Score > 24.0). Linkage within this interval is so tight that it is not possible to establish a specific order with a significant confidence level, either by Radiation Hybrid mapping or by meiotic linkage mapping in the dog. Because of this extremely close cosegregation, the general term XLPRA marker was used to mean any one of or any combination of these four markers, where specific identification is not critical. Furthermore, recombination events were observed occasionally between this set of XLPRA markers and more distant flanking markers on the canine X (including DMD telomerically, TIMP 1 and AR centromerically, and several previously described microsatellite markers assigned to the canine X chromosome; see Table 2). In all such cases, when a recombination was observed between the telomeric marker and the XLPRA marker, there was never a recombination between the XLPRA marker and the centromeric marker, and vice versa (Table 2). These data firmly establish that the markers on the canine X chromosome are ordered DMD-[XLPRA markers]-TIMPl. The frequency of recombination events between these other markers and the XLPRA interval also make them inadequately useful for DNA testing in this class of disease.

Table 2. Recombinants found between X-linked canine markers and XLPRA in XLPRA, and XLPRA₂ informative pedigrees. Data for DMD, TIMP1, AR, and FIX are from Table 1. Markers FH2985, FH2548, FH2997, FH2916, and FH3027 are tetrasatellite microsatellite markers placed on the X- chromosome in the current genome map of the dog. Recombination events are those observed between each marker and XLPRA in informative pedigrees. Map order shown is assigned by recombination mapping and distances from XLPRA. Note that "combined theta" and "combined LOD scores" for markers in the zero recombination interval are highlighted in bold.

* : markers not typed in XLPRA₂ pedigree.

**: TCTE1L marker is not informative in XLPRA₂ pedigree.

Taken individually and together, the informativeness of all these markers is extremely high. Table 3 records the number of alleles for each of the following markers.

Table 3. Summary of Alleles identified at individual loci in the XLPRA interval

Because these markers form a cosegregating interval, they can be used in combination to create an XLPRA-interval haplotype (see Table 4). This is very straightforward for an X-chromosomal region compared to an autosomal region, because males are hemizygous (have only one X chromosome) and make the phase of alleles within the haplotype directly readable. Table 4 records haplotypes observed on affected and normal chromosomes from 17 different dogs.

Table 4. Examples of different haplotypes for genes in the zero recombination interval of XLPRAi ^and XLPRA₂, and comparison with haplotypes from normal animals or Siberian Huskies or Miniature Schnauzers which have non-XLPRA forms of retinal degeneration. Allele numbers refer to allele in Table 3.

As shown in Table 4, the number of distinguishable alleles, and thus the heterozygosity, for the haplotype is far greater than that of the individual loci, making this an extremely powerful way to establish a fϊngeφrint of the entire XLPRA interval. This method was used, both prior to and subsequent to finding the actual mutation in XLPRA j and XLPRA₂, to prove that the PRA disease phenotype cosegregates with XLPRA markers in an extended family of dogs derived from Siberian Huskies (Figure 3). The method was also used to prove that in a separate family of Siberian Huskies, PRA was not segregating with XLPRA markers (dogs 7 and 8, Table 4). For example, the haplotypes of male Siberian Husky dogs 1 and 7 (Table 4) were different, confirming that the retinal degeneration present in dog 7 is not XLPRAi. Further pedigree analyses using XLPRA markers in the family of dogs from which dogs 7 and 8 (Table 4) were derived confirmed lack of cosegregation of the PRA phenotype and XLPRA markers in this family. The method was also used to prove that in a family of Samoyed dogs, the PRA disease phenotype cosegregates with XLPRA markers. All affected dogs shared the same XLPRA haplotype (e.g. dog 2, Table 4) derived from a carrier female (dog 5, Table 4). None of the nonaffected males in this family received the affected haplotype. The method was also used to prove that in an extended family of Miniature Schnauzers and dogs derived from Miniature Schnauzers, the PRA disease phenotype cosegregates with XLPRA markers (Figure 4). The method was also used to prove that in two additional separate families of Miniature Schnauzers, PRA was not segregating with XLPRA markers. In these families, multiple XLPRA haplotypes were identified in both PRA affected males and PRA affected females (e.g. dogs 9 and 10, Table 4), confirming that the retinal degeneration in these dogs was not XLPRA₂. Therefore, these markers allow one to establish whether any particular breed or family within a breed is affected by XLPRA prior to or without identifying the actual mutation (or even the gene). If the disease in any such breed or family is transmitted as XLPRA, then one can use these markers and the haplotype formed thereby to identify dogs that are affected with, are carriers of, or normal for the disease. These specific markers are only specific examples of the type of markers and tests that can be developed, given the teaching of cosegregation of XLPRA with the XLPRA interval. As indicated earlier, large regions of genomic DNA (both from BACs and large amplified fragments) from within the XLPRA interval have been identified and partially sequenced. With this sequence information it would be straightforward and indeed desirable to develop additional markers from within this region for use in different populations. For example, if one wished to develop a marker-based test for a new breed in which the specific mutation was as yet unknown, or was extremely difficult to incoφorate into a direct test, one could sequence DNA from within this region from selected affected, normal, or carrier animals, and identify new polymoφhisms such as SNPs that would also serve to create haplotypes to fingeφrint the interval in the specific population.

Example 4 — Cloning of the Normal Canine RPGR cDNA and Characterization of Additional RPGR Splice Variants

Total RNA was prepared from retinal tissues of four normal and four XLPRA-affected male dogs by standard-guanidium thiocyanate extraction (Chomzynski et al., "Single-Step Method for RNA Isolation by Acid-Guanidinium Thiocyanate Phenol-Chloroform Extraction," Anal. Biochem.. 172: 156-159 (1987), which is hereby incoφorated by reference). Total RNA was also extracted from eight normal non-retinal tissues (liver, lung, kidney, heart, brain, testes, ovary and bone marrow). A 3 μg aliquot of total RNA was reverse transcribed using random hexamers or a reverse primer specific for the poly A tail. Primer sequences, primer locations, annealing temperatures and expected product sizes for selected RPGR PCRs are given in Table 5 and Figure 5B. A MgCl concentration of 1.5 mM was used for all reactions.

Table 5 Primers, locations and conditions used to amplify RPGR fragments.

Primer Sequence Location (nucleotide) T_m(°Q Product Sue (bp)

RPGR 17 (1) CGAA 1 TATCΛCCCCCACCGAA 1 AG 7-30 (SEQ ID No 18)

RPGR 1 4/3 (r) CAATATGAAGTGCCTAGATAGGCCTC 1599-1574 (1 8 kb) 64 1595 (SEQ ID No 19)

RPGR 17 (f) CGAATTA1 CACCCCCACCGAATAG 7-30 (SEQ ID No 20)

RPGR 16 (r) CCAACACTGAACAGTTAAGGCCAC 2374-2351 (2 4 kb) 64 2370 (SEQ ID No 21)

RPGR 17 (f) CGAATTA rCACCCCCACCGAATAG 7-30 (SEQ ID No 22)

RPGR 16 (r) CCAACACTGAACAGTTAAGGCCAC 3334-3311 (3 3 kb) 64 2820 (SEQ ID No 23)

RPGR 17 (f) CGAATTATCACCCCCACCGAATAG 7-30 (SEQ ID No 24)

RPGR 33 (r) TTTCCGATGGCCTTGTTCCCTCTC (SEQ 2824-2801 (2 8 kb) 64 3330 ID No 25)

RPGR 3 (f) GGCAGTAACAACTGGGGTCAG ITAG 258-282 (SEQ ID No 26)

RPGR 10 (r) CTCTCCCTCTCTCTTCGCCGCA (SEQ ID 1330-1309 (all) 64 1075 No 27)

RPGR 27 (f) GACATAGGTAATGCTCAGGCCAG (SEQ 1841 -1864 (2 8 kb) 60 986 ID No 28)

RPGR 33 (r) TTTCCGATGGCCTTGTTCCCTCTC (SEQ 2824-2801 ID No 29

RPGR 20 (0 ATGAGCCTGAATTCCAATGACAAG 1602-1625 (SEQ ID No 30)

RPGR 21 (r) CTGGAACAT . CTTCTTGCTTTCCA (SEQ 2703-2680 (3 3 kb) 60 1078 ID No 31) f, forward; r, reverse. Due to the low expression of RPGR transcripts, cloning and characterization of all splice variants was done using PCR-based methodology. The canine RPGR cDNA was obtained by a combination of RT-PCR , 5' RACE, and 3 'RACE (Gibco BRL, Gaithersburg, MD) from brain RNA. Canine-specific primers located at the 5'- and 3 '-untranslated region (UTR) of the sequence were designed and used to amplify a 2.4 kb fragment representing a full-length retinal RPGR cDNA by RT-PCR from canine retina. This, and subsequent PCR products, were cloned and sequenced from both the coding and noncoding strands. Additional splice variants were identified by PCR from testes (3.3 kb) and retina (1.8 kb, 2.8 kb). The exons (and introns) representing these splice variants are illustrated in Figure 5, and the primers used for characterizing these fragments and their position are noted. Analysis of the splice variants was critical since it has been proposed that the apparent deficiency in identifiable RPGR mutations in human RP3 patients might be due to the complex splicing pattern of this gene (Meindl et al., "A Gene (RPGR) With

Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentoase (RP3)," Nature Genet.. 13:35-42 (1996); Fujita et al., "Analysis of the RPGR Gene in 11 Pedigrees With the Retinitis Pigmentosa Type 3 Genotype: Paucity of Mutations in the Coding Region But Splice Defects in Two Families," Am. J. Hum. Genet.. 61 :571-580 (1997), which are hereby incoφorated by reference). Comparison of the deduced amino acid sequences of the four canine RPGR splice variants, and the published human and mouse RPGR sequences, revealed extensive preservation of amino acid homology over the RCC-1 repeat region. Homology in all three species dropped over the hydrophilic portion of the protein, encoded by exons 14-16, but was resumed over the terminal 120 amino acids. The open reading frame present in the canine 2.4 kb splice variant was characterized first among all the splice variants identified in dog. The characterized region of the 2.4 kb variant includes 2132 nucleotides of coding sequence, 80 nucleotides of 5'- UTR, and 280 nucleotides of 3'-UTR including the poly-A tail. The canine RPGR cDNA appeared to have a single initiation codon (nucleotide 81-83) which corresponds to that in the human cDNA. The preceding 80 nucleotides could be translated to generate an additional 26 amino acid sequence which are in-frame with the initial predicted protein sequence, thus suggesting that the dog, like the mouse, has two initiation codons (Yan et al., "Biochemical Characterization and Subcellular Localization of the Mouse Retinitis Pigmentosa GTPase Regulator (mRpgr)," J. Biol. Chem.. 273:19656-19663 (1998), which is hereby incoφorated by reference). The 2.4 kb splice variant shared 82% and 79% nucleotide sequence identity, respectively, with the human and mouse RPGR nucleotide sequences, and was thus highly homologous to the RPGR cDNA reported initially in human (Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentoase (RP3)," Nature Genet.. 13:35-42 (1996); Roepman et al., "Positional Cloning of the Gene For X-Linked Retinitis Pigmentosa 3 : Homology With the Guanine Nucleotide Exchange Factor RCC 1 ," Hum. Mol. Genet., 5:1035-1041 (1996), which are hereby incoφorated by reference) and mouse (Yan et al., "Biochemical Characterization and Subcellular Localization of the Mouse Retinitis Pigmentosa GTPase Regulator (mRpgr)," J. Biol. Chem.. 273:19656-19663 (1998), which is hereby incoφorated by reference). Highly conserved regions in all three species included the two GTP phosphate binding domains, the RCC-1 homology region and the terminal isoprenylation signal (Figure 5A).

In the dog, human, and mouse, all of the characterized canine RPGR splice variants shared the same 5' half of the coding region (exons 1-10). In fact, the 1.8 kb variant consisted almost exclusively of the first 10 exons, suggesting that the critical functional portion of the coding sequence resided within the RCC-1 repeat regions. Homology to the RCC-1 region prompted speculation that RPGR was a guanine nucleotide exchange factor (Meindl et al., "A Gene (RPGR) With Homology to the RCC1 Guanine Nucleotide Exchange Factor is Mutated in X-Linked Retinitis Pigmentoase (RP3)," Nature Genet., 13:35-42 (1996), which is hereby incorporated by reference). However, a BLAST search of the predicted amino acid sequence from the 2.4 kb canine RPGR cDNA against the Swiss protein database indicated that canine RPGR had approximately 30% amino acid identity to a protein with a putative role in ubiquitin conjugation (KIAA0032; GenBank Accession No. 2495699), and slightly lower (25-30%) identity with RCC-1 proteins of several species. The portions of the human RPGR protein encoded by exons 14 -16 are highly positively charged, but their function is unknown (Buraczynska et al., "Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families With X-linked Retinitis Pigmentosa," Am. J. Hum. Genet.. 61 : 1287-1292 (1997), which is hereby incoφorated by reference). The corresponding region in dog appears to be the major site of alternate splicing in the canine RPGR gene, suggesting that it may play a role in cellular localization or substrate specificity of the protein products of individual splice variants. Apart from the 1.8 kb variant, all other canine splice variants were characterized by variable in-frame inclusion of sequence encoded by exons 14, intron 14, exon 15 or intron 15. In the 2.8 kb variant, the coding sequence is terminated by a series of polylysine residues. Despite much effort, the true 3' UTR of this variant was initially unobtainable; now, it has been found to be ORF 15. Reported sites of alternative splicing in the human RPGR gene include coding regions presumably derived from intron 15, and sequences downstream of the published 3'

UTR (Holinski-Feder et al., "The RPGR gene in Retinitis Pigmentosa Type 3," Invest. Qpthalmol. Vis. Sci.. 39: S292 (1998); Kirschner et al, "RPGR Transcription Studies in Mouse and Human Tissues Reveal a Retina-Specific Isoform that is Disrupted in a Patient with X-Linked Retinitis Pigmentosa," Hum. Mol. Genet.. 8: 1571-1578 (1999), which are hereby incoφorated by reference).

The inclusion of the composite exon 14-14A-15 to create the 3.3 kb variant in testes appeared to be developmentally regulated, and coincided with the onset of sexual maturity. The presence of similarly sized splice variants in the testes of both mouse (Yan et al., "Biochemical Characterization and Subcellular Localization of the Mouse Retinitis Pigmentosa GTPase Regulator (mRpgr)," J. Biol. Chem.. 273:19656-19663 (1998); Kirschner et al., "RPGR Transcription Studies in Mouse and Human Tissues Reveal a Retina-Specific Isoform that is Disrupted in a Patient with X-Linked Retinitis Pigmentosa," Hum. Mol. Genet.. 8: 1571-1578 (1999), which are hereby incoφorated by reference) and dog, as well as relatively abundant expression of RPGR in the dog testes, suggests a significant role for RPGR in this organ. The 3.3 kb transcript was detected at a lower level than the other 3 variants on Southern blots of retinal RT-PCR products, and could be amplified by quantitative PCR, thus indicating that this variant is expressed at very low levels in the retina. In summary, a graphic comparison of putative splicing patterns in all of the canine RPGR variants and the human and mouse (mRpgr- 1) RPGR cDNAs is given in the Figure 5, and includes all characterized canine splice variants. Alternative splice sites in the 2.4, 3.3, and 2.8 kb canine RPGR cDNA variants are clustered in the region encoding the hydrophilic (exons 14-16) portion of the protein. Whereas the 2.4 and 3.3 kb splice variants share common 5' and 3' termini, the 2.8 and 1.8 kb variants have unique 3' termini derived from introns 15 and 10, respectively. In contrast, the two additional murine RPGR splice variants described by Yan et al., "Biochemical Characterization and Subcellular Localization of the

Mouse Retinitis Pigmentosa GTPase Regulator (mRpgr)," J. Biol. Chem.. 273:19656- 19663 (1998), which is hereby incoφorated by reference, differ from the full length mouse cDNA due to deletions of exons 8-11, and 18 in mRpgr-2 and deletion of exons 18 and 19A in mRpgr-3.

Example 5 - Comparison of All RPGR Splice Variants in Normal and XLPRAj- and XLPRA₂- Affected Animals

The size and expression of all splice variants in normal and XLPRA- affected retinas were determined by RT-PCR. In addition, all transcripts were examined in RPE/choroid from an XLPRA-affected dog. Primers and conditions used are described in Table 5, and locations of primers indicated in Figure 5B. RPGR sequence from each variant was assessed by direct sequencing of PCR products. In order to minimize sequencing errors, all fragments were sequenced in both directions from at least two normal and two affected dogs. Primers were selected so that overlapping sequence from both coding and non-coding strands was obtained. Full- length sequence was assembled and compared for base differences.

The expression and sizes of the 1.8 kb, 2.4 kb and 2.8 kb variants were assessed in normal and XLPRA] -affected retinas, as well as in affected RPE/choroid, by RT-PCR. Bands of similar size were present in both genotypes for these three splice variants and there were no changes found in the coding sequences. Two nucleotide variations were identified by direct sequencing of overlapping PCR products; both were located in the composite exon 14-14A-15-15A. One was a previously described single nucleotide polymoφhism in exon 14A (nucleotide 1323 of the 2.8 kb variant) (Zeiss et al., "A Highly Polymoφhic RFLP Marker in the

Canine Retinitis Pigmentosa GTPase Regulator (RPGR) Gene," Anim. Genet.. 29:409 (1998), which is hereby incoφorated by reference) and the other was a single base change in exon 15A (nucleotide 2597 of the 2.8 kb variant). The first nucleotide change (A— G) resulted in a valine for methionine substitution in the predicted amino acid sequence. The polymoφhism was present at an allele frequency of approximately 50% in the normal canine population (Zeiss et al., "A Highly Polymoφhic RFLP Marker in the Canine Retinitis Pigmentosa GTPase Regulator (RPGR) Gene," Anim. Genet.. 29:409 (1998), which is hereby incoφorated by reference). The second nucleotide change was a benign polymoφhism, which was in phase with first polymoφhism, and did not result in a change in the amino acid sequence. In addition, the UTRs for all splice variants were also examined for sequence differences between affected and normal dogs and none were found. The principal retinal expressed 2.4 kb variant in XLPRA.2-affected retinas was sequenced and no nucleotide changes in the coding sequence were found.

Example 6 - Evaluation of Relative Expression Levels of Each Splice Variant in Normal and XLPRA-Affected Retina by Semi-Quantitative RT- PCR

Since no disease causing mutations were found within the splice variants identified, the hypothesis that an alteration in the proportional expression of the four splice variants could result in the XLPRA phenotype was tested. The expression levels of each variant in retinal tissues from three normal and three affected dogs of differing ages and disease severity were examined using a Perkin- Elmer 7700 PCR instrument. RPGR probes and primers were selected to span splice junctions which were specific for each variant. RPGR amplimers were normalized to 18S ribosomal RNA. Of the affected dogs, one was eight weeks old, an age where the retina is histologically normal; the other two were older (15 months and 18 months) and had photoreceptor degeneration. No significant decrease in any of the splice variants with respect to one another was present in retinal RNA of normal and affected dogs of any age; i.e. dogs with pre-degenerate or actively degenerating retinas. When the ΔCt values were transformed, the expression of all RPGR splice variants appeared to increase in the older affected dogs at the time that the retinas were actively degenerating. Similar increases in TIMP-1 expression were found in the XLPRA] model (Zeiss et al., "TIMP-1 Expression is Increased in X-Linked Progressive Retinal Atrophy Despite Its Exclusion As a Candidate Gene," Gene, 225:67-76, (1998), which is hereby incoφorated by reference). Example 7 -- Cloning of a Unique RPGR Exon (ORF 15) and Identification by Sequencing of the Disease Causing Mutations in XLPRAi ^ar|d XLPRA₂

After it was established that XLPRA] and XLPRA₂ were tightly linked to RPGR with zero recombinations, a search was conducted for a retina-specific or a retina expressed transcript. Since previous results did not find a stop codon in the 2.8 kb transcript, suggesting that the transcript cloned was only partially characterized and that additional coding sequence was present, the search focused on the 2.8 kb transcript. A portion of the previously identified intron 15 of the RPGR gene was found to contain coding sequence which was termed " ORF 15" and was found to be the site of the mutations in XLPRA j and XLPRA2. ORF 15 in normal animals had a nucleotide sequence corresponding to SEQ. ID. No. 9, and a deduced amino acid sequence corresponding to SEQ. ID. No. 10. The nucleotide and deduced amino acid sequences of ORF 15 in XLPRA] -affected animals were SEQ. ID. No. 11 and SEQ. ID. No. 12, respectively. The nucleotide and deduced amino acid sequences of ORF 15 in XLPRA₂-affected animals were SEQ. ID. No. 13 and SEQ. ID. No. 14, respectively.

Example 8 - PCR-Based Test for Identification of the Mutations in XLPRAi

A pair of canine specific primers (RGF14 and RGR13) were designed to flank the sequence region of mutation (with 5 bp deletion). RGF14, a forward primer, has a nucleotide sequence corresponding to SEQ. ID. No. 15 as follows:

5' AAGGGGAGGAGAAAGGGGAGGCT 3'

RGR13, a reverse primer, has a nucleotide sequence corresponding to SEQ. ID. No. 16 as follows:

5' ATGAAGGGGAGGAGGAAGAGGGA 3'

PCR was optimized with a new PCR system called FailSafe PCR kit with 2X preMix G (Epicentre Company, Madison, WI). 100 ng of template DNA was used for a 25 μl PCR reaction (PCR product size: 257 bp). The amplified PCR products were separated on a 8% acrylamide gel with DNA markers of 25 bp and φx 174. Affected dogs showed a PCR fragment that was smaller (by approximately 5 bp) than normal animals. Carrier dogs showed clearly the normal and mutant bands as well as the formation of heteroduplexes because the normal and mutant fragments annealed and their altered conformation resulted in a retardation of migration in the gel during electrophoresis.

Using this technique, a pedigree of Samoyed dogs with X-linked retinal degeneration was tested and the results were the same. Sequencing results showed that the mutation was identical to that found in Siberian Husky with

XLPRA], thus confirming the identification of XLPRA] mutation in the Samoyed breed as a cause of PRA and the application of the same test for diagnosis.

Example 9 - PCR-Based Test for Identification of the Mutations in XLPRA₂ A pair of canine specific primers (RGF14 and RGR12) were designed to flank the sequence region of mutation (with 2 bp deletion). RGF14, a forward primer, has a nucleotide sequence corresponding to SEQ. ID. No. 15. RGR12, a reverse primer, has a nucleotide sequence corresponding to SEQ. ID. No. 17 as follows:

5 ' TCCCCTACTTCCTCTTCCCTCTCA 3 '

PCR was optimized with a new PCR system called FailSafe PCR kit with 2X preMix D (Epicentre Company, Madison, WI). 100 ng of template DNA was used for a 25 μl PCR reaction (PCR product size: 257 bp). The amplified PCR products were digested with restriction enzyme Fok I and separated on a 8% acrylamide gel with

DNA markers of 25 bp and φx 174.

The affected dogs showed a PCR fragment that is smaller (by approximately 2 bp) than normal animals. Carrier dogs showed clearly the normal and mutant bands as well as the formation of heteroduplexes because the normal and mutant fragments annealed and their altered conformation resulted in a retardation of migration in the gel during electrophoresis. Although the invention has been described in detail for the puφoses of illustration, it is understood that such detail is solely for that puφose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

1. A method for identifying dogs which are genetically normal, are carriers of, or are affected with X-linked progressive retinal atrophy, said method comprising: obtaining a biological sample from a dog and testing the biological sample for the presence of at least one genetic marker which co-segregates with an X-linked progressive retinal atrophy gene locus by linkage analysis, under conditions effective to determine the presence of a mutated X-linked progressive retinal atrophy gene locus in one or both alleles.

2. The method according to claim 1, wherein the dog is a Siberian Husky, Samoyed, Miniature Schnauzer, or any breed in which progressive retinal atrophy disease is X-linked and the disease locus maps to an RP3 region of an X chromosome.

3. The method according to claim 1, wherein the biological sample is any tissue containing genomic DNA.

4. The method according to claim 1, wherein the biological sample is blood, hair, mucosal scrapings, semen, tissue biopsy, or saliva.

5. The method according to claim 1, wherein the at least one genetic marker is selected from the group consisting of TCTE1L microsatellite marker, SPRX microsatellite marker, OTC microsatellite marker, and a restriction fragment length polymoφhism marker from the retinitis pigmentosa GTPase regulator gene.

6. The method according to claim 5, wherein the at least one genetic marker is the TCTE1L microsatellite marker.

7. The method according to claim 6, wherein the polymoφhism is analyzed by using SEQ. ID. No. 1 and SEQ. ID. No. 2 as primers in a polymerase chain reaction.

8. The method according to claim 5, wherein the at least one genetic marker is the SPRX microsatellite marker.

9. The method according to claim 8, wherein the polymoφhism is analyzed by using SEQ. ID. No. 3 and SEQ. ID. No. 4 as primers in a polymerase chain reaction.

10. The method according to claim 5, wherein the at least one genetic marker is the OTC microsatellite marker.

11. The method according to claim 10, wherein the polymoφhism is analyzed by using SEQ. ID. No. 5 and SEQ. ID. No. 6 as primers in a polymerase chain reaction.

12. The method according to claim 5, wherein the at least one. genetic marker is the retinitis pigmentosa GTPase regulator gene.

13. The method according to claim 12, wherein the polymoφhism is analyzed by using SEQ. ID. No. 7 and SEQ. ID. No. 8 as primers in a polymerase chain reaction to obtain an amplified product, said method further comprising: digesting the amplified product with restriction enzyme Nla III.

14. The method according to claim 1, wherein the at least one genetic marker is a combination of genetic markers.

15. A method for selecting dogs for breeding comprising: obtaining a biological sample from a dog; testing the biological sample for the presence of at least one genetic marker which co-segregates with an X-linked progressive retinal atrophy gene locus by linkage analysis, under conditions effective to determine the presence of a mutated X-linked progressive retinal atrophy gene locus in one or both alleles; and eliminating dogs with the mutated X-linked progressive retinal atrophy gene locus in one or both alleles from a breeding stock or breeding male dogs with the mutated X-linked progressive retinal atrophy gene locus in one allele with genetically normal female dogs.

16. The method according to claim 15, wherein the dog is a Siberian Husky, Samoyed, Miniature Schnauzer, or any other breed in which progressive retinal atrophy disease is X-linked and the disease locus maps to an RP3 region of an X chromosome.

17. The method according to claim 15, wherein the biological sample is any tissue containing genomic DNA.

18. The method according to claim 15, wherein the biological sample is blood, hair, mucosal scrapings, semen, tissue biopsy, or saliva.

19. The method according to claim 15, wherein the at least one.. genetic marker is selected from the group consisting of TCTE1L microsatellite marker, SPRX microsatellite marker, OTC microsatellite marker, and a restriction fragment length polymoφhism marker from the retinitis pigmentosa GTPase regulator gene.

20. The method according to claim 19, wherein the at least one genetic marker is the TCTE1L microsatellite marker.

21. The method according to claim 20, wherein the polymoφhism is analyzed by using SEQ. ID. No. 1 and SEQ. ID. No. 2 as primers in a polymerase chain reaction.

22. The method according to claim 19, wherein the at least one genetic marker is the SPRX microsatellite marker.

23. The method according to claim 22, wherein the polymoφhism is analyzed by using SEQ. ID. No. 3 and SEQ. ID. No. 4 as primers in a polymerase chain reaction.

24. The method according to claim 19, wherein the at least one genetic marker is the OTC microsatellite marker.

25. The method according to claim 24, wherein the polymoφhism is analyzed by using SEQ. ID. No. 5 and SEQ. ID. No. 6 as primers in a polymerase chain reaction.

26. The method according to claim 19, wherein the at least one genetic marker is the RPGR gene.

27. The method according to claim 26, wherein the polymoφhism is analyzed by using SEQ. ID. No. 7 and SEQ. ID. No. 8 as primers in a polymerase chain reaction to obtain an amplified product, said method further comprising: digesting the amplified product with restriction enzyme Nla III.

28. The method according to claim 15, wherein the at least one genetic marker is a combination of genetic markers.

29. A method for identifying dogs which are genetically normal, are carriers of, or are affected with X-linked progressive retinal atrophy, said method comprising: obtaining a biological sample from a dog and testing the biological sample for a gene encoding canine retinitis pigmentosa GTPase regulator having a nucleotide mutation in one or both alleles indicative of a carrier of or a dog affected with X-linked progressive retinal atrophy.

30. The method according to claim 29, wherein the dog is a Siberian Husky, Samoyed, Miniature Schnauzer, or any breed in which progressive retinal atrophy disease is X-linked and the disease locus maps to an RP3 region of an X chromosome.

31. The method according to claim 30, wherein the dog is a Siberian Husky or a Samoyed and the nucleotide mutation is a 5-nucleotide deletion in ORF 15 of the retinitis pigmentosa GTPase regulator gene.

32. The method according to claim 31 , wherein the nucleotide mutation is a GAGAA deletion of nucleotides 878 through 882 in the nucleotide sequence of SEQ. ID. No. 9.

33. The method according to claim 30, wherein the dog is a Miniature Schnauzer and the nucleotide mutation is a 2-nucleotide deletion in ORF 15 of the retinitis pigmentosa GTPase regulator gene.

34. The method according to claim 33, wherein the nucleotide mutation is a GA deletion of nucleotides 932 through 933 in the nucleotide sequence of SEQ. ID. No. 9.

35. The method according to claim 29, wherein the biological sample is any tissue containing genomic DNA.

36. The method according to claim 29, wherein the biological sample is blood, hair, mucosal scrapings, semen, tissue biopsy, or saliva.

37. The method according to claim 29, wherein the nucleic acid is a deoxyribonucleic acid.

38. The method according to claim 29, wherein the nucleic acid is a messenger ribonucleic acid.

39. The method according to claim 29, wherein said testing is carried out by an oligonucleotide ligation assay.

40. The method according to claim 29, wherein said testing is carried out by direct sequence analysis.

41. The method according to claim 29, wherein said testing is carried out by single strand polymoφhism assay.

42. The method according to claim 29, wherein said testing is carried out by ligase chain reaction.

43. The method according to claim 29, wherein said testing is carried out by ligase detection reaction.

44. The method according to claim 29, wherein said testing is carried out by subjecting the biological sample to conditions effective to hybridize

DNA molecules with the mutation to a probe specific for the sequence of the mutation.

45. The method according to claim 29, wherein said testing is'; carried out by digesting the biological sample with a restriction endonuclease.

46. The method according to claim 29, wherein said testing comprises: amplifying a region of the gene encoding canine retinitis pigmentosa GTPase regulator to provide an amplified fragment before detecting any mutation present in the biological sample.

47. The method according to claim 29, wherein said testing is carried out by performing PCR using genomic DNA templates and polyacrylamide gel electrophoresis.

48. The method according to claim 29, wherein said testing is carried out by Taq cycle sequencing.

49. The method according to claim 29, wherein said testing is carried out by PCR-based restriction fragment length polymoφhism.

50. A method for selecting dogs for breeding comprising: obtaining a biological sample from a dog; testing the biological sample for a gene encoding canine retinitis pigmentosa GTPase regulator having a nucleotide mutation in one or both alleles indicative of a carrier of or a dog affected with X-linked progressive retinal atrophy; and eliminating dogs with the mutation from a breeding stock or breeding the dogs with the mutation with genetically normal dogs.

51. The method according to claim 50, wherein the dog is a Siberian Husky, Samoyed, Miniature Schnauzer, or any breed in which progressive retinal atrophy disease is X-linked and the disease locus maps to an RP3 region of an X chromosome.

52. The method according to claim 51 , wherein the dog is a Siberian Husky or a Samoyed and the nucleotide mutation is a 5-nucleotide deletion in ORF 15 of the retinitis pigmentosa GTPase regulator gene.

53. The method according to claim 52, wherein the nucleotide mutation is a GAGAA deletion of nucleotides 878 through 882 in the nucleotide sequence of SEQ. ID. No. 9.

54. The method according to claim 51 , wherein the dog is a Miniature Schnauzer and the nucleotide mutation is a 2-nucleotide deletion in ORF 15 of the retinitis pigmentosa GTPase regulator gene.

55. The method according to claim 54, wherein the nucleotide mutation is a GA deletion of nucleotides 932 through 933 in the nucleotide sequence of SEQ. ID. No. 9.

56. The method according to claim 50, wherein the biological sample is any tissue containing genomic DNA.

57. The method according to claim 50, wherein the biological sample is blood, hair, mucosal scrapings, semen, tissue biopsy, or saliva.

58. The method according to claim 50, wherein the nucleic acid is a deoxyribonucleic acid.

59. The method according to claim 50, wherein the nucleic acid is a messenger ribonucleic acid.

60. The method according to claim 50, wherein said testing is carried out by an oligonucleotide ligation assay.

61. The method according to claim 50, wherein said testing is carried out by direct sequence analysis.

62. The method according to claim 50, wherein said testing is carried out by single strand polymoφhism assay.

63. The method according to claim 50, wherein said testing is carried out by ligase chain reaction.

64. The method according to claim 50, wherein said testing is carried out by ligase detection reaction.

65. The method according to claim 50, wherein said testing is carried out by subjecting the biological sample to conditions effective to hybridize

DNA molecules with the mutation to a probe specific for the sequence of the mutation.

66. The method according to claim 50, wherein said testing is carried out by digesting the biological sample with a restriction endonuclease.

67. The method according to claim 50, wherein said testing comprises: amplifying a region of the gene encoding canine retinitis pigmentosa GTPase regulator to provide an amplified fragment before detecting any mutation present in the biological sample.

68. The method according to claim 50, wherein said testing is carried out by performing PCR using genomic DNA templates and polyacrylamide gel electrophoresis.

69. The method according to claim 50, wherein said testing is carried out by Taq cycle sequencing.

70. The method according to claim 50, wherein said testing is carried out by PCR-based restriction fragment length polymoφhism.

71. An isolated nucleic acid molecule encoding the ORF 15 of the canine RPGR in normal dogs, having a nucleotide sequence of SEQ. ID. No. 9.

72. The isolated nucleic acid molecule according to claim 71, having a deduced amino acid sequence of SEQ. ID. No. 10.

73. An isolated nucleic acid molecule encoding the ORF 15 of the canine RPGR in XLPRAi -affected dogs, having a nucleotide sequence of SEQ. ID. No. 11.

74. The isolated nucleic acid molecule according to claim 73, having a deduced amino acid sequence of SEQ. ID. No. 12.

75. An isolated nucleic acid molecule encoding the ORF 15 of the canine RPGR in XLPRA₂-affected dogs, having a nucleotide sequence of SEQ. ID. No. 13.

76. The isolated nucleic acid molecule according to claim 75, having a deduced amino acid sequence of SEQ. ID. No. 14.