# Patent

## Title
Method for identifying oculoskeletal dysplasia in dogs

## Abstract

Provided are methods for identifying dogs as likely to be genetically normal, carriers of, or affected with Oculo-skeletal dysplasia (OSD) by determining the presence or absence of a drd2 COL9A2 mutation and/or a drd1 COL9A3 mutation. Also provided is a method for selective breeding of dogs and kits useful for carrying out the methods of the invention.

## Background
This application claims priority to U.S. patent application Ser. No. 60/968,150, filed on Aug. 27, 2007, the disclosure of which is incorporated herein by reference.


This invention was supported by government funding under grant no. EY06855 from the National Institutes of Health. The Government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates generally to a canine disease termed Oculo-skeletal dysplasia. More particularly, the invention relates to compositions and methods for use in testing dogs for Oculo-skeletal dysplasia.
DESCRIPTION OF RELATED ART
Oculo-skeletal dysplasia (OSD) is an autosomal recessive disorder that has been observed in 2 dog breeds, Labrador retriever (Carrig et al. JAVMA (1977) 170:49-57) and Samoyed (Meyers et al., JAVMA (1983) 183:975-979), but mutations associated with the disease may be present in other breeds.
Dogs that are affected with OSD have skeletal abnormalities characterized by short-limbed dwarfism and ocular defects including vitreous dysplasia, retinal detachment and cataracts. Dogs affected with OSD can usually be recognized upon physical examination by an experienced veterinarian, particularly an experienced veterinary ophthalmologist, or an experienced veterinary orthopedist, and sometimes by experienced dog breeders. However, recognition of affected dogs is not sufficient to allow adequate selection pressure to be applied to significantly reduce the frequency of a mutation that may be associated with OSD in the population. Furthermore, while it is widely held that OSD carriers can usually be recognized upon physical examination by an experienced veterinary ophthalmologist via detecting retinal folds or retinal dysplasia, this is highly unreliable as both false positive and false negative diagnoses are common. Thus, ophthalmoscopic examination to detect carriers is too inaccurate to allow adequate selection pressure to be applied to significantly reduce frequency of mutation(s) in the population that may be associated with OSD. Thus, improved methods for determining the likelihood of a dog to be a carrier, affected with, or normal for OSD are needed.
SUMMARY OF THE INVENTION
The present invention is based on our discovery of mutations associated with oculo-skeletal dysplasia (OSD). The OSD mutations analyzed in the method of the invention are referred to as the drd1 COL9A3 mutation and the drd2 COL9A2 mutation. These mutations are also referred to herein as the drd1 mutation and the drd2 mutation, respectively. drd1 and drd2 refer to dwarfism-retinal-dysplasia 1 and 2, respectively. The method comprises obtaining a biological sample from a dog and determining from the sample the presence or absence of the drd1 mutation, the presence or absence of the drd2 mutation, or the presence or absence of both of these mutations. Dogs that do not have either of these mutations (homozygous for the wild type sequence) are considered normal for OSD. Dogs that are heterozygous for the drd1 mutation are considered drd1 mutation carriers. drd1 mutation carriers are considered to be carriers of a drd1 form of OSD. Dogs that are heterozygous for the drd2 mutation are considered drd2 mutation carriers. drd2 mutation carriers are considered to be carriers of a drd2 form of OSD. Dogs that are homozygous for either the drd1 mutation or the drd2 mutation are considered to be affected with OSD.
The present invention also provides a method for selective breeding of dogs, whereby dogs that are identified as carriers of either OSD mutation, or as affected with OSD can be removed from the breeding stock. Also provided are kits useful for carrying out the methods of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides the genomic sequence of the 5′ end of the canine COL9A2 gene.
FIG. 2 provides the nucleotide sequence of the region of the COL9A2 gene which encompasses the COL9A2 drd2 mutation.
FIG. 3 provides the canine COL9A2 cDNA sequence.
FIG. 4A provides the wild type canine COL9A3 drd1 cDNA coding sequence.
FIG. 4B provides the mutant canine COL9A3 drd1 cDNA coding sequence.
FIG. 4C provides the provides the genomic sequence of the 5′ end of COL9A3 gene in a normal CFA24 chromosome.
FIG. 4D provides the genomic sequence of the 5′ end of COL9A3 in a CFA24 chromosome that comprises the drd1 mutation.
FIG. 5A provides a pedigree of a colony of dogs descended from a purebred Samoyed affected with OSD.
FIGS. 5B and 5C provide photographic representation of electrophoretic separation of PCR amplification products from drd2 COL9A2 tests performed on biological samples obtained from the dogs depicted in the pedigree.
FIG. 6 provides a photographical representation of electrophoretic separation of PCR amplification products from drd1 COL9A3 tests.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for determining whether a dog is normal for OSD, or is a carrier of an OSD mutation, or is affected with OSD. The invention is based on our discovery of genetic mutations associated with OSD. In particular, we have discovered a mutation that is a 1,267 base pair deletion in the canine COL9A2 gene on canine chromosome 15 (CFA15) (the drd2 COL9A2 mutation) and a mutation that is a one nucleotide (a guanine; “G”) insertion in the canine COL9A3 gene on canine chromosome 24 (CFA24) (the drd1 COL9A3 mutation).
The term “drd1 COL9A3 mutation” is used interchangeably herein with “drd1 mutation.” Likewise, the term “drd2 COL9A2 mutation” is used interchangeably with “drd2 mutation.” The drd1 mutation and the drd2 mutation can each be referred to individually as an “OSD mutation” and collectively as “OSD mutations.”
As used herein, a dog is termed “normal” or “normal for OSD” if the dog does not have either OSD mutation (i.e., the dog is homozygous wild type for both OSD mutation sites). A dog is termed “affected” or “affected with OSD” if the dog is homozygous for either or both OSD mutations. A dog is a carrier of OSD if it is heterozygous for either the drd1 mutation or the drd2 mutation, or for both mutations. A “drd1 mutation carrier” as used herein means a dog that is heterozygous for the drd1 mutation. A drd1 mutation carrier is considered to be a carrier of drd1 form of OSD. A “drd2 mutation carrier” as used herein means a dog that is heterozygous for the drd2 mutation. A drd2 mutation carrier is considered to be a carrier of drd2 form of OSD. Determining that a dog is a drd1 mutation carrier does not indicate whether or not the dog is a drd2 mutation carrier, or whether or not the dog is affected via homozygosity for the drd2 mutation. Likewise, determining that a dog is a drd2 mutation carrier does not indicate whether or not the dog is a drd1 mutation carrier, or whether or not the dog is affected via homozygosity for the drd1 mutation.
It will be recognized by those skilled in the art that carriers of either OSD mutation that are not affected with OSD may nevertheless exhibit varying OSD traits, such as an ocular phenotype that ranges from localized retinal dysplasia characterized by focal or multifocal retinal folds to large plaques of dysplastic retinal tissue, but have a normal appendicular skeleton. The present invention addresses this and other difficulties in ascertaining the OSD status of dogs by providing a method for determining whether a dog is normal for OSD, or is a carrier of the drd1 mutation, or is a carrier of the drd2 mutation, or is affected with OSD. The method comprises the steps of obtaining a biological sample from a dog and determining from the biological sample the presence or absence of the drd1 mutation, the drd2 mutation, or both of these mutations. A determination that the dog is homozygous for either OSD mutation is indicative that the dog is affected with OSD. A determination that neither OSD mutation is present is indicative that the dog is normal for OSD. A determination that the dog is heterozygous for the drd1 mutation indicates that the dog is a drd1 mutation carrier, a carrier of drd1 form of OSD. A determination that the dog is heterozygous for the drd2 mutation indicates that the dog is a drd2 mutation carrier, and is therefore a carrier of drd2 form of OSD.
Any dog, regardless of breed, could be heterozygous or homozygous for either or both OSD mutations. To date, we have found the drd1 mutation in Labrador retrievers and the drd2 mutation in Samoyeds. Nevertheless, upon identification of heterozygosity for one OSD mutation, it is preferable to determine the status of the other OSD mutation. For example, upon a determination that a dog is a drd1 mutation carrier, it would be preferable to determine the drd2 status of the dog to ascertain whether or not the dog is also a drd2 mutation carrier, or whether the dog is homozygous for the drd2 mutation, and is therefore affected with OSD, in addition to being a drd1 mutation carrier.
In one embodiment, the method further comprises communicating the result of determining whether a dog is normal for OSD, a drd1 mutation carrier or drd2 mutation carrier, or is affected with OSD to an individual. The test result can be communicated to an individual by any method. Non-limiting examples of the individual to whom the test results may be communicated include the dog owner, a canine pedigree accreditization agency, a veterinarian, or a provider of genetic test results.
We have determined that the drd2 COL9A2 mutation is a 1,267 base pair deletion. The deletion and its genomic context is illustrated by the DNA sequences depicted in FIGS. 1-3.
FIG. 1 provides the genomic sequence of the 5′ end of the canine COL9A2 gene (SEQ ID NO:1). This sequence is a corrected version of the 5′ region of the canine COL9A2 genomic sequence, and extends from a location 1,310 nucleotides (nts) of SEQ ID NO:1 before the start codon (boxed), to the beginning of intron 2. In particular, we have determined that both the 2004 July CanFam1 and 2005 May CanFam2 assemblies (provided to the public through GenBank) have incorrect and incomplete sequences for this interval of CFA15. In particular, we have determined the 2005 assembly is missing exon 1 and surrounding sequence. We determined this using primers designed from sequence shared by both CanFam assemblies to amplify genomic DNA to retrieve the sequence presented as SEQ ID NO:1. In the corrected sequence presented in SEQ ID NO:1, nucleotide 1 corresponds to nucleotide (nt) 5,649,378 of canine chromosome 15 in the CanFam2 2005 assembly, and nt 5,641,234 of canine chromosome 15 in the CanFam1 2004 assembly. The start codon is at positions 1311-1313 (boxed), exon 1 ends at nt 1373 (and thus contains a 63 base coding sequence), intron 1 extends from nt 1374 through 2262 and exon 2 from nts 2263-2337. Capital letters signify coding regions.
FIG. 2 provides a DNA sequence (SEQ ID NO:2) that illustrates the sequence deleted in the drd2 COL9A2 mutation in the genomic CFA15 DNA context. The region of the canine genomic COL9A2 sequence presented in FIG. 2 encompasses the drd2 mutation (the deletion), and extends from the drd2 5′ region, through exon 1, and into the start of intron 1. The ATG of codon 1 is boxed; the coding sequence is shown in uppercase. The drd2 mutation is a deletion of the 1,267 nucleotides between nucleotide number 230 and nucleotide number 1,498 in the nucleotide sequence depicted in FIG. 2. Thus, determining a deletion of 1,267 nucleotides between nucleotide number 230 and nucleotide number 1,498 in the nucleotide sequence depicted in FIG. 2 (SEQ ID NO:2) identifies the presence of the drd2 mutation. The drd2 mutation removes the complete exon 1 of the transcript and its 5′ untranslated region (UTR), as well as part of intron 1.
The presence or absence of the drd2 mutation can be detected by any appropriate method, including but not limited to by analysis of canine genomic DNA, mRNA, cDNA, or protein.
In one embodiment, the presence or absence of the drd2 mutation can be determined by PCR analysis. Exemplary PCR primer binding sites useful for PCR based amplification across the deletion are underlined in FIG. 2 (primer pair 1) or double underlined (primer pair 2).
FIG. 3 provides the canine COL9A2 cDNA sequence (SEQ ID NO:3). The first methionine (ATG) and the normal stop (TGA) codons are boxed. Non-coding sequence is shown in lowercase, coding sequence in uppercase. A single nucleotide polymorphism (SNP) identified in exon 3 has 2 alleles (C or T) and is shown as a bold enlarged Y (indicating a pyrimidine). In CFA15 chromosomes that comprise the drd2 mutation, the C allele of this SNP is present, but this allele is also observed in normal dogs of various breeds.
Turning to the drd1 mutation, we have determined that the drd1 mutation is a one nucleotide (guanine) insertion in the canine COL9A3 gene on canine chromosome 24 (CFA24). This insertion is illustrated in FIG. 4B (SEQ ID NO:4), which depicts a cDNA sequence comprising the Canine drd1 COL9A3 mutation. FIG. 4A (SEQ ID NO:5) provides the wild type drd1 COL9A3 cDNA sequence. In FIGS. 4A and 4B, the first methionine (ATG) and the normal stop (TAA) codons are boxed. In a chromosome that contains the drd1 mutation, the extra guanine (G) is inserted into the string of 4 Gs shown at nucleotide positions 7-10 of the normal coding sequence (FIG. 4A), resulting in a string of 5 Gs in the mutant (FIG. 4B). In the mutant sequence shown in FIG. 4B, the inserted G is arbitrarily indicated (bolded and enlarged) as the 5th G in the string at nucleotide position 11 of SEQ ID NO:4, although the insertion could be any one of the 5 Gs. Thus, it is considered that determining a G in position 11 of SEQ ID NO:4 as depicted in FIG. 4B identifies the presence of the drd1 mutation. It is also considered that determining a G in position 11 of SEQ ID NO:4 as depicted in FIG. 4B identifies a nucleotide sequence consisting of GGGGG in positions 7-11 in the nucleotide sequence depicted in FIG. 4B. However, it will be recognized that the invention includes any other method of identifying the presence or absence of the drd1 mutation, including but not limited to by analysis of canine genomic DNA, mRNA, cDNA, or protein. In respect of genomic DNA, FIG. 4C provides the genomic sequence of the 5′ end of COL9A3 gene in a normal CFA24 chromosome (SEQ ID NO:6). FIG. 4D provides the genomic sequence of the 5′ end of COL9A3 in a CFA24 chromosome that comprises the drd1 mutation (SEQ ID NO:7), wherein the fifth G that signifies the presence of the GGGGG sequence that may be used to identify the drd1 mutation is shown in bold and enlarged. The atg start codon is boxed in FIGS. 4C and 4D.
It will be recognized that identifying the presence or absence of the drd1 mutation by analysis of genomic DNA sequence is well within the purview of one skilled in the art and is included in the present invention. It is accordingly considered that identification of the presence or absence of the drd1 mutation by analysis of genomic DNA sequence also identifies a G in position 11 of SEQ ID NO:4.
Representative primers useful for use in one embodiment of the invention for determining the presence or absence of the drd1 mutation from genomic DNA are underlined in FIGS. 4C and 4D (these primers are also shown as primer pair 3 in Table 2). In FIGS. 4C and 4D, SNPs are shown as bold where r=A or G, y=C or T. The first methionine in the COL9A3 gene is boxed.
The insertion of G in the drd1 mutation alters the drd 1 open reading frame by +1 nucleotide, which introduces a stop codon after 48 codons (TGA; shown in bold and enlarged in FIG. 4B). Thus, the G in position 11 of SEQ ID NO:4 is in the +1 open reading frame relative to the ATG start codon presented in nucleotide positions 1-3 of FIG. 4B. The invention therefore also includes identifying the drd1 COL9A3 mutation using any method by which a +1 translational frameshift introduced into the drd1 COL9A3 gene by the drd1 mutation can be detected.
In addition to the inserted G, we have also identified four SNPs that are shown as bolded and enlarged R or Y in the sequence depicted in FIG. 4A for the wild type drd1 COL9A3 cDNA sequence, where the R indicates a purine (alleles are A or G for the first, second and fourth SNPS shown as R) and Y indicates a pyrimidine (alleles are C or T for the third SNP shown as Y). In CFA24 chromosomes that have the drd1 mutation, we have determined the alleles present for the 4 SNPs are: G, G, T, A (in haplotype order). These are shown in bold and enlarged in the sequence depicted in FIG. 4B. However, this haplotype is also observed in normal dogs.
As noted above, the drd1 mutation creates a +1 translational frameshift and a premature stop codon (TGA) relative to the normal coding sequence. This results in a predicted protein that is altered relative to the normal protein. The predicted altered protein is shorter and has a predicted amino acid sequence that is different from that of the predicted normal drd1 protein. Therefore, the presence of the drd1 mutation could be determined by detecting this altered protein. Similarly, the drd2 mutation also results in a sequence encoding a predicted protein that is altered via a predicted different and shorter amino acid sequence relative to the predicted normal protein. Therefore, the presence of the drd2 mutation could also be determined by detecting this predicted altered protein. Detecting either predicted altered protein would be indicative that the dog is not normal. Such altered proteins could be detected using any conventional technique, such as by immunodetection methods, including but not limited to immunohistochemistry, Western blotting, ELISA, and fluorescent in situ hybridization (FISH).
The biological sample tested in the method of the invention can be any biological sample that contains nucleic acids or protein. For example, a sample of blood, hair, spleen, mucosal scrapings, semen, tissue biopsy, saliva or the like can be used. In one embodiment, the biological sample is blood. Suitable collection techniques for obtaining biological samples from dogs are known in the art.
Techniques for isolating and preparing nucleic acids in a form that is suitable for testing for OSD mutations are well known. Nucleic acids for use in testing for OSD mutations may be tested directly using any suitable method, or may be amplified before testing using a variety of techniques that are well known. For example, genomic DNA or mRNA may be amplified through use of PCR or RT-PCR, respectively (Saiki et al. Science 239:487-491 (1988)). Other suitable in vitro amplification methods include the ligase chain reaction (LCR) (Wu and Wallace Genomics 4:560-569 (1989)), strand displacement amplification (SDA) (Walker et al. PNAS USA 89:392-396 (1992)), and the self-sustained sequence replication (3SR) (Fahy et al. PCR Methods Appl. 1:25-33 (1992)).
Detecting the presence or absence of an OSD mutation in nucleic acids can be accomplished by a variety of methods. Such methods include but are not limited polymerase chain reaction (PCR), hybridization with allele-specific oligonucleotide probes (Wallace et al. Nucl Acids Res 6:3543-3557 (1978)), including immobilized oligonucleotides (Saiki et al. PNAS USA 86:6230-6234 (1989)) or oligonucleotide arrays (Maskos and Southern Nucl Acids Res 21:2269-2270 (1993)), allele-specific PCR (Newton et al. Nucl Acids Res 17:2503-25 16 (1989)), mismatch-repair detection (MRD) (Faham and Cox Genome Res 5:474-482 (1995)), denaturing-gradient gel electrophoresis (DGGE) (Fisher and Lerman et al. PNAS USA 80:1579-1583 (1983)), single-strand-conformation-polymorphism detection (Orita et al. Genomics 5:874-879 (1983)), chemical (Cotton et al. PNAS USA 85:4397-4401 (1988)) or enzymatic (Youil et al. PNAS USA 92:87-91 (1995)) cleavage of heteroduplex DNA, methods based on allele specific primer extension (Syvanen et al. Genomics 8:684-692 (1990)), genetic bit analysis (GBA) (Nikiforov et al. Nuci Acids Res 22:4167-4175 (1994)), the oligonucleotide-ligation assay (OLA) (Landegren et al. Science 241:1077 (1988)), the allele-specific ligation chain reaction (LCR) (Barrany PNAS USA 88:189-193 (1991)), dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), PyrosequencingTM, any of various DNA “chip” technologies, such as those offered by AFFYMETRIX (Santa Clara, Calif.), Polymorphism chipsgap-LCR (Abravaya et al. Nucl Acids Res 23:675-682 (1995)), and radioactive and/or fluorescent DNA sequencing using standard procedures well known in the art.
In one embodiment, amplification of genomic DNA for use in testing for an OSD mutation is performed by PCR. For this embodiment, PCR primers and a method of using the primers in amplification reactions are provided such that different amplification products are observed when DNA is amplified from affected, drd1 mutation carrier or drd2 mutation carrier, or normal dogs. It will be recognized by those skilled in the art that while particular sequences of PCR primers are provided herein, other PCR primer sequences can be designed to detect the presence or absence of an OSD mutation.
When PCR primers are used such that the amplification products are of distinct sizes, the amplification products can be analyzed by standard methods such as electrophoretic separation and detection using ethidium bromide and ultraviolet light, or any other suitable detection method. Alternatively, amplification products can be isolated and sequenced using any of a variety of techniques.
The method of the present invention can be carried out for any breed of dog. In general, dogs known to be affected with OSD include Labrador retrievers and Samoyeds. However, any other breed of dog, including mixed breeds, may be tested according to the method of the invention. Some non-limiting examples of dog breeds that could be tested in the method of the invention include Akita, American cocker spaniel, American eskimos, Australian cattle dog, Australian stumpy tailed cattle dog, basenji, Bernese mountain dog, border collie, Chesapeake bay retriever, Chinese crested, English cocker spaniel, English mastiff, English springer spaniel, Entlebucher mountain dog, Finnish lapphund, German shorthaired pointer, giant schnauzer, Havanese, lowchen, miniature poodle, miniature schnauzer, Nova scotia duck tolling retriever, Portuguese water dogs, silky terrier, spitz, standard poodle, standard wirehaired dachshund, Tibetan terriers and toy poodle.
Also provided in the present invention are kits for detecting the presence or absence of an OSD mutation in a biological sample from a dog or a nucleic acid sample extracted from the biological sample. The kits of the present invention comprise reagents for nucleic acid based detection of the presence of an OSD mutation. In one embodiment, the kits comprise reagents for extraction/preparation of nucleic acid samples and pair(s) of specific primers for identification of OSD mutations.
The presently provided OSD mutation tests will allow diagnosis of dogs of any age, such as a fetal dog (in utero), neonatal dogs, adult dogs, etc., and will eliminate the false positives and false negatives that have complicated previous identification of the OSD status of dogs. Accordingly, by using the tools and method described herein, dogs which are genetically OSD normal, drd1 mutation carriers or drd2 mutation carriers, or dogs affected with OSD, can be identified and selected for breeding. It is preferable to select dogs that are normal for OSD for mating. However, the method also permits removal of carrier or affected dogs from a breeding stock. Alternatively, dogs which are heterozygous for an OSD mutation can be mated with genetically normal dogs to ensure the absence of dogs affected with OSD in the litter.
The invention will be further understood by the following examples, which are intended to be illustrative and not restrictive in any way.
EXAMPLE 1
This Example illustrates discovery of the drd2 mutation and presents particular embodiments of the invention that are useful for detecting the presence or absence of the drd2 mutation.
The CanFam2 May 2005 dog whole genome shotgun assembly v2.0 (available at genome.ucsc.edu/cgi-bin/hgGateway) identifies 30 predicted exons of the canine homolog of the human COL9A2 gene, but fails to predict homologs of human exons 1 and 26. The earlier (July 2004) CanFam1 assembly does identify a predicted canine exon 1 homolog, but comparisons between the 2004 (CanFam1) and 2005 (CanFam2) assemblies identified sequence inconsistencies between them in the interval corresponding to the 5′ end, exon 1 and part of intron 1 of the predicted canine COL9A2 gene.
We designed primers based on the 2004 assembly to amplify overlapping cDNA fragments to cover the complete coding sequence of the canine COL9A2 by RT-PCR (Table 1).
Shown in Table 1 are primer sequences used to amplify the complete coding region of COL9A2 (primer pairs 1 to 10), as well as to amplify COL9A3 (primer pairs 11 to 21). Also shown are the sequences of primer pairs used to retrieve the correct 5′ end sequence of the Col9A2 gene. The primers were used to amplify cDNA from retina samples obtained from normal dogs, and dogs homozygous for the drd2 mutation which were therefore affected with OSD. These normal and affected dogs were members of a controlled breeding colony we established. Briefly, the colony was established from a purebred Samoyed affected with OSD (a dog that was homozygous for the drd2 mutation). This dog was bred to homozygous normal Irish-setter dogs and the heterozygous F1 progeny were then backcrossed to dogs homozygous for the drd2 mutation (affecteds) or intercrossed to drd2 mutation carriers to yield litters segregating the drd2 phenotype. Five related three-generation families from this colony (two intercrosses and three backcrosses), which included 63 progeny, were studied.












TABLE 1






Forward primer
Forward primer
Reverse primer
Reverse primer



Pair
name
sequence
name
sequence
















A. Col9A2 primers used to amplify the coding sequence.













 1
COL9A2_5UTR_F4
ccgccccgtccgagagcagc
COL9A2exon4R
gcttcccatcaggcccatctgg





(SEQ ID NO: 8)

(SEQ ID NO: 9)


 


 2
COL9A2exon1F
gagcctccgccgcccgcatg
COL9A2exon4_5R
gctccagttagaccatcaatcc




(SEQ ID NO: 10)

(SEQ ID NO: 11)


 


 3
COL9A2rg1F
ctggcgcagatcagaggt
COL9A2rg1R
cagttggtcggacacaagaa



(exon1_2)
(SEQ ID NO: 12)
(exon10_11)
(SEQ ID NO: 13)


 


 4
COL9A2exon2F
cctggatccgacggcatcgac
COL9A2exon7_8R
gtggtccaggaggtccagcaaa




(SEQ ID NO: 14)

g






(SEQ ID NO: 15)


 


 5
COL9A2exon4_5F
tgggattgatggtctaactgg
COL9A2exon22R
gggtccgatttctccttgag




(SEQ ID NO: 16)

(SEQ ID NO: 17)


 


 6
COL9A2exon21F
ctccctggattctctggtcc
COL9A2exon26R
gcccttctcgcctttctctc




(SEQ ID NO: 18)

(SEQ ID NO: 19)


 


 7
COL9A2exon24F
cttgccaggcatcaagggagac
COL9A2exon31R
ggtcacccttctctccacgtttt




(SEQ ID NO: 20)

(SEQ ID NO: 21)


 


 8
COL9A2exon24_25F
atcaagggagacaagggcttcc
COL9A2exon30R2
ctcacagcgacctctgccagt




(SEQ ID NO: 22)

(SEQ ID NO: 23)


 


 9
COL9A2exon29F
cgaccagcacatcgtgaccgt
COL9A2exon32R
tccttgccgttgattgcctg




(SEQ ID NO: 24)

(SEQ ID NO: 25)


 


10
COL9A2exon30_31F2
ccaagggaaaacgtggagaga
ColA2_3UTR_R2
gaaagctggcttcctggtctgag




ag

(SEQ ID NO: 27)




(SEQ ID NO: 26)


 








B. Col9A3 primers used to amplify the coding sequence.













11
COL9A3_5UTR_F
gcgcgcagagccgctgagag
COL9A3exon8R
attccaccggggacaccact





(SEQ ID NO: 28)

(SEQ ID NO: 29)


 


12


COL9A3rg1R
ggaggacccctgggtgac





(exon10)
(SEQ ID NO: 30)


 


13


COL9A3exon12R2
cgacctctccttgatctcctt






(SEQ ID NO: 31)


 


14
COL9A3rg1F
gcagaaagtgggacctcaag
COL9A3exon8R
attccaccggggacaccact



(exon12)
(SEQ ID NO: 32)

(SEQ ID NO: 29)


 


15


COL9A3rg1R
ggaggacccctgggtga





(exon10)
(SEQ ID NO: 30)c


 


16
COL9A3exon4F
aagccagggaagccaggaga
COL9A3exon8R
attccaccggggacaccact




(SEQ ID NO: 33)

(SEQ ID NO: 29)


 


17


COL9A3rg1R
ggaggacccctgggtgac





(exon10)
(SEQ ID NO: 30)


 


18
COL9A3exon8F
agaggcaggagagagtggtgt
COL9A3exon12R
cttgccgacctctccttgat




(SEQ ID NO: 34)

(SEQ ID NO: 35)


 


19


COL9A3exon25_26R
aacgccctggtctcccttag






(SEQ ID NO: 36)


 


20
COL9A3exon20_21F
gaagggagaactgctggagag
COL9A3exon29_30R
gatttgttcgctgagcatcc




(SEQ ID NO: 37)

(SEQ ID NO: 38)


 


21
COL9A3exon26F
ctggggacaaaggagagctg
COL9A3_3UTR_R2
cccgaggtacgatgttagagc




(SEQ ID NO: 39)

(SEQ ID NO: 40)


 








C. Col9A2 primers used to amplify genomic DNA.













22
Col9A2_5UTR_F2
tgtggtaggctttcctgaaga
A2i2R2
cccgacccaggttagagact





(SEQ ID NO: 41)

(SEQ ID NO: 42)


 


23
Col9A2_5UTR_F3
aaaagcctgacctcctagctg




(SEQ ID NO: 43)


 


24
Col9A2_5UTR_F7
ccggttctgtgctgctaagccag




(SEQ ID NO: 44)


 


25
A2_exon1_F
gagcctccgccgccccgcatg
A2int1R11
cgcctaacccgaaaggagcac




(SEQ ID NO: 45)

(SEQ ID NO: 46)


 


26
A2_exon1_F2
ctgctgctgctccaggggctc




(SEQ ID NO: 47)


 


27
A2_F11
gcccggttctgtgctgctaag




(SEQ ID NO: 48)


 


28
A2_exon1_F2
ctgctgctgctccaggggctc
A2int1R
gtgaatgggcaccattgtct




(SEQ ID NO: 47)

(SEQ ID NO: 49)


 


29
A2_F10
ccggttctgtgctgctaag
A2int1R10
cctaacccgaaaggagcac




(SEQ ID NO: 50)

(SEQ ID NO: 51)









Amplification of affected retina cDNA resulted in a lack of amplification product from PCR analysis using three primer pairs (primer pairs 1, 2 and 3 in table 1). This indicated that the genomic sequence at the 5′ end of the gene in affected dogs is mutated, which results in a lack of amplification products. After retrieving the correct 5′ end of the gene from a dog normal for OSD using primer pairs 22 to 29 in Table 1, a comparison of this region of COL9A2 to the drd2-affected (homozygous for the drd2 mutation) dog revealed a 1,267 bp deletion in the affected dog-(illustrated in FIGS. 1-3).
To identify dogs that were normal for OSD, that were drd2 mutation carriers, and that were affected with OSD via homozygosity for the drd2 mutation, as well as to analyze co-segregation of the COL9A2 mutation with the disease in the Samoyed derived colony dogs, genomic DNA of 70 Samoyed-colony-derived dogs were amplified using two primer pairs (primer pairs 1 and 2 in table 2). Analysis of the resulting PCR amplification products showed complete co-segregation with the disease: all affected dogs were homozygous for the drd2 mutation, all obligated carriers were heterozygous for the drd2 mutation, and all known normal dogs were homozygous for the wild type allele.
Thus, this Example demonstrates the discovery of the drd2 mutation and its association with OSD, and illustrates a method for detecting the presence or absence of this mutation in to ascertain the OSD status of dogs.
EXAMPLE 2
Based on our discovery of the drd2 mutation as set forth in Example 1, we designed another PCR based test. This test was performed on samples obtained from dogs obtained from the control colony described in Example 1. A pedigree for dogs analyzed in this Example is presented in FIG. 5A.
The primers used in this Example are presented in Table 2 (primer pairs 1 and 2). The two primer pairs were used in separate PCR reactions (Table 2). Representative PCR amplification products obtained using primer pair 1 are shown in FIG. 5B. Representative PCR amplification products obtained using primer pair 2 are shown in FIG. 5C.
To obtain the PCR results presented in FIGS. 5B and 5C, 50-100 ng of genomic DNA were mixed with 12.5 ul of GoTaq green master mix (Promega, Catalog number M7123), 1.25 ul of DMSO (5% final concentration) and 10 uM of forward and reverse primers in a final volume of 25 ul. The DNA was then denatured at 95° C. for 2 minutes and 35 cycles of 95° C. for 30 seconds, 58° C. (for primer pair number 1) or 60° C. (for primer pair number 2) for 30 seconds, 72° C. for 30 seconds were performed in a thermal cycler (MJ Research, Watertown, Mass., USA). An additional final extension of 5 minutes at 72° C. was performed to ensure full-length products.
Primer pair number 1 in Table 2 produces a 1,445 bp product from amplification of wild type (normal) chromosomes while a smaller molecular weight product (178 bp) is observed from amplification of affected chromosomes (FIG. 5B). Since in carrier dogs both alleles are present, this PCR reaction will usually produce only the smaller molecular weight product in carriers due to competition between the targets and the differences in size between the two amplicons. Therefore, a second PCR reaction can be performed using primer pair number 2 in Table 2. This primer pair yields a 504 bp product from amplification of wild type (normal) chromosomes and will not result in any product from affected chromosomes because the binding site for the reverse primer is located within the deletion (FIG. 5C). PCR products obtained using this strategy to amplify DNA obtained from normal dogs, dogs that are drd2 mutation carriers, and dogs that were affected with OSD via homozygosity for drd2. The PCR products were visualized on a 1.8% agarose gel and stained with Ethidium Bromide using standard protocols. A normal dog yielded either a faint 1,445 bp band in the reaction using primer pair number 1 or no visible band and a 504 bp band from PCR amplification using primer pair 2 (FIG. 5A dog 13). A carrier dog yielded a 178 bp band from PCR amplification using primer pair 1 and a 504 bp band using primer pair 2 (FIG. 5A dogs 1,3,4,5,6,9,10,11,12). Amplification from an OSD affected dog (homozygous for the drd2 mutation) yields only a 178 bp band from PCR amplification using primer pair 1 and no amplification when using primer pair 2 (FIG. 5A, dogs 2,7,8).









TABLE 2







Primer Pairs
Expected Size

















Forward


Reverse

Expected
Expected




primer
Forward primer
Reverse primer
primer
Expected
size in
size in


#
name
sequence
name
sequence
size in wt
carrier
affected





1
COL9A2—
gctgaccttgtggattttc
COL9A2intron1R
gtgaatgggcacca
1,445 bp
178 bp
178 bp




Part1 1F
tcc

ttgtct




SEQ ID NO: 52

SEQ ID NO: 53


 


2
COL9A2—
catctctccctcactccct
COL9A2_Part10R
tcacccctctcccag
  504 bp
504 bp
No band



Part10F
cct

tctcttag




SEQ ID NO: 54

SEQ ID NO: 55


 


3
COL9A3t
gctgccactgggctcctt
COL9A3test1R
agcaggagcaggg
  248 bp
248 bp
249 bp



est1F
tcttcg

ccagcgtg

and




SEQ ID NO: 56

SEQ ID NO: 57

249 bp


 


4
COL9A3t
gctgccactgggctcctt
COL9A3test1R3
cgctcacatgcgcc
  298 bp
298 bp
299 bp



est1F
tcttcg

ccggtc

and




SEQ ID NO: 56

SEQ ID NO: 58

299 bp


 


5
COL9A3—
ggcgcagccatggccg
COL9A3_AS_R
ggtcagggtggcg
   72 bp
 72 bp
No band



AS_WF
ggac

gccaggagc




SEQ ID NO: 59

SEQ ID NO: 60


 


6
COL9A3—
ggcgcagccatggccg
COL9A3_AS_R
ggtcagggtggcg
No band
 73 bp
 73 bp



AS_AF
ggcg

gccaggagc




SEQ ID NO: 61

SEQ ID NO: 60









Thus, this Example demonstrates an embodiment of the invention that is useful for determining OSD status by testing for the presence or absence of the COL9A2 drd2 mutation.
EXAMPLE 3
This Example demonstrates an embodiment of the invention using the PCR based method described in Example 2 to test unrelated Samoyeds (meaning the dogs had no parents or grandparents in common).
Fifty-five unrelated Samoyed dogs were screened for the presence or absence COL9A2 drd2 mutation using this test. One Samoyed was found to be a drd2 mutation carrier and is therefore considered to be a carrier of the drd2 form of OSD. Another 126 dogs, considered to be normal for OSD based on phenotypic analysis, from 26 additional breeds were screened for the deletion and all the dogs had the wild type (normal) genotype (Table 3).









TABLE 3






Number
drd2 mutation


Breed
of dogs
Carriers

















American Cocker Spaniel
5
0


American Eskimos
10
0


American Pitbull Terrier
5
0


American Staffordshire Terrier
5
0


Australian Cattle Dogs
5
0


Basenji
1
0


Border Collies
5
0


Boxer
1
0


Chesapeake Bay Retriever
5
0


Chinese Crested
5
0


English Cocker Spaniel
5
0


English Springer Spaniel
7
0


Entelbucher Mountain Dogs
7
0


Glen Of Immal Terrier
6
0


Golden Retriever
5
0


Golden Retriever/Labrador Retriever Crosses
5
0


Labrador Retriever
5
0


Nova Scotia Duck Tolling Retriever
4
0


Samoyed
55
1


Papillon
7
0


Poodle
7
0


Portuguese Water Dogs
5
0


Tibetan Terrier
8
0


American Bulldog
2
0


Pomeranian
1
0


Corgi
4
0


Dachshund
1
0


Total
181
1









Thus, this Example demonstrates another embodiment of the invention that is useful for determining OSD status in dogs by testing for the presence or absence of the drd2 mutation.
EXAMPLE 4
This Example describes discovery of the drd1 mutation and particular embodiments of methods for testing for the presence or absence of this mutation.
Our comparison of the 2005 CanFam2 dog sequence assembly to the human COL9A3 genomic sequence identified 29 predicted canine exons and failed to identify predicted exons homologous to human exons 16, 17 and 32. Based on this analysis, 12 primers (Table 1 primer pairs 11 to 21) were designed to amplify overlapping cDNAs obtained from one normal, and one affected dogs derived from Labrador retriever colony dogs to cover the complete coding region of the canine Col9A3. Briefly, the Labrador retriever colony dogs are descended from two unrelated purebred Labrador retrievers affected with OSD. These dogs were bred to homozygous normal unrelated Beagles, Beagle-crossbred dogs, Irish-Setter and Irish-setter-crossbreds dogs and poodle-crossbred dogs, and the heterozygous F1 progeny were then backcrossed to affected dogs or intercrossed to carriers of the drd1 mutation to yield litters segregating the OSD phenotype. Eight related three-generation families from this colony (five intercrosses and three backcrosses), which included 68 progeny, were analyzed using 12 primers were in 11 different combinations of primer pairs to produce redundant overlapping fragments.
Our comparison of retinal cDNA amplification products from normal dogs and dogs homozygous for the drd1 mutation revealed a one-base insertion (guanine) in the coding sequence (exon 1) that changes a string of 4 guanines (nucleotides 49,699,847-49,699,850 of canine chromosome 24 in the May 2005 CanFam2 assembly v2.0; <genome.ucsc.edu/cgi-bin/hgGateway>) to a string of 5 Gs. This mutation is indicated by the presence of the G at nucleotide position 11 in the nucleotide sequence depicted in FIG. 4B (SEQ ID NO:4). The string of five Gs is as depicted in FIG. 4B for nucleotides 7-11.
To identify drd1-affected (homozygous for the drd1 mutation), drd1 mutation carrier and normal dogs in the Labrador retriever derived colony, and to analyze co-segregation of the drd1 mutation with the disease, genomic DNA of 80 colony dogs was amplified using the primer pairs COL9A3test1F and COL9A3test1R (Table 2, primer pair 3). Sequencing of the 248 bp amplicon with the forward primer identified normal animals (4 Gs) or affected animals (5 G's, 249 bp amplicon) while an overlapping chromatogram was observed in carrier dogs at the insertion point. All outbred normal dogs were determined to be homozygous wild type. All affected dogs were determined to be homozygous for the drd1 mutation (5 Gs). All the obligated carriers were determined to be heterozygous for the drd1 mutation (overlapping chromatograms; not shown). 30 unaffected dogs from intercrosses were genotyped as follows: 5 were homozygous normal (4 Gs) and 25 were heterozygoug for the drd1 mutation. These results accordingly showed a complete co-segregation of the drd1 mutation with the disease in the colony.
In another test, we employed the primer pair COL9A3test1F and COL9A3test1R3 (Table 2, primer pair 4). Sequencing of the 298 bp product with the forward and the reverse primers identified normal sequence or affected sequence (299 bp) while an overlapping chromatogram was observed in drd1 mutation carrier dogs after the insertion point (sequencing data not shown).
Thus, this Example demonstrates the discovery of the drd1 mutation and its association with OSD, and illustrates a method for detecting the presence or absence of this mutation to determine the OSD status of dogs.
EXAMPLE 5
This Example describes another embodiment by which the presence or absence of the drd1 mutation can be determined. In particular, we designed an allele-specific PCR based test using primer pairs 5 and 6 presented in Table 2, namely: Forward (wild type primer): 5′-GGCGCAGCCATGGCCGGGAC-3′ (COL9A3_AS_WF; SEQ ID NO:59); Forward (mutant primer): 5′-GGCGCAGCCATGGCCGGGCG-3′ (COL9A3_AS_AF; SEQ ID NO:61); and Reverse primer: 5′-GGTCAGGGTGGCGGCCAGGAGC-3′ (COL9A3_AS_R; SEQ ID NO:60).
DNA samples were obtained from normal, drd1 mutation carrier and affected dogs from our control colony of Labrador retrievers. Each DNA sample was analyzed in separate PCR reactions, with primer pairs 5 (wild type forward and reverse primers) and 6 (mutant forward primer and wild type reverse primer). The reactions were performed in a total volume of 15 μl, containing 1×PCR buffer (Qiagen); 10-100 ng/μl total DNA; 0.3 μM forward (wild or mutant type) and reverse primers; 0.2 mM each of four dNTPs; 1.5 mM MgCl2; 17% DMSO and 0.375 units Taq-polymerase. The reaction mixture was overlaid with mineral oil. The enzyme Taq-pol (5 u/μl) is diluted 1:10 with water and 0.75 μl was added to the reaction in a “hot-start” manner: the enzyme was added after the denaturing step at 94C). The PCR cycles were: 1 cycle of 94C for 3 minutes; 32 cycles of 94C for 15 seconds, 58C for 30 seconds and 72C for 15 seconds. From each PCR amplification, 6-8 μl was analyzed by electrophoresis on a 6% PAGE in TBE buffer with  ̃1 mg/ml of ethidium bromide for staining. Representative photographs of electrophoretic separation of amplification products obtained from using this protocol are presented in FIG. 6, where M=mutant PCR (primer pair 6), W=Wild type PCR (primer pair 5). Samples 1, 2 and 6 are drd1 mutation carriers and are therefore considered carriers of the drd1 form of OSD, samples 3, 4 and 5 are affected with OSD, and samples 7, 8, 9, 10, and 11 are normal for OSD. The sizes of the amplification products are provided in Table 2.
Thus, this Example demonstrates another embodiment of the invention that is useful for determining OSD status in dogs by testing for the presence or absence of the drd1 mutation.
EXAMPLE 6
This Example provides an illustration of determining the presence or absence of the drd1 mutation in unrelated dogs to determine OSD status.
In particular, 59 phenotypically normal unrelated Labrador retrievers (no parents or grandparents in common), and 19 unrelated Labrador retrievers with retinal folds were tested for the presence or absence of the drd1 mutation. One dog with retinal folds was found to be homozygous for the mutation (affected). 78 dogs considered normal for OSD via phenotypic examination from 23 additional breeds were also screened and all 78 dogs were normal for the mutation (Table 4), meaning they were homozygous wild type for the drd1 gene.










TABLE 4








Affected




drd1
(homozygous



Number
mutation
for the drd1


Breed
of dogs
carriers
mutation)


















American cocker spaniel
2
0
0


American Eskimos
3
0
0


Australian cattle dogs
6
0
0


Basenji
2
0
0


Border Collies
10
0
0


Boxer
1
0
0


Chesapeake bay retriever
4
0
0


Chinese crested
4
0
0


Collie
5
0
0


English cocker spaniel
8
0
0


Entelbucher mountain dogs
1
0
0


Glen of Immal terrier
2
0
0


Golden Retriever
4
0
0


Irish Setter
1
0
0


Labrador retrievers
78
0
1


mix
6
0
0


Nova scotia duck tolling retriever
4
0
0


Poodle
3
0
0


Portuguese water dogs
4
0
0


American Bulldog
2
0
0


Pomeranian
1
0
0


Corgi
4
0
0


Dachshund
1
0
0


Total
156
0
1









Thus, this Example demonstrates yet another embodiment of the invention that is useful for determining OSD status by testing for the presence or absence of the drd1 mutation.
The invention has been described through specific embodiments. However, routine modifications to the compositions, methods and devices will be apparent to those skilled in the art and such modifications are intended to be covered within the scope of the invention.

## Claims

1. A method for determining whether a dog is heterozygous, homozygous or has an absence of a drd1 mutation identified as a G in position 11 as depicted in nucleotide sequence of SEQ ID NO:4 (mutation 1), and/or whether the dog is heterozygous, homozygous or has an absence of a drd2 mutation identified as a deletion of 1,267 nucleotides between nucleotide number 230 and nucleotide number 1,498 in nucleotide sequence of SEQ ID NO:2 (mutation 2), the method comprising: obtaining a biological sample from the dog determining the presence or absence of the drd1 or drd2 mutation and from the biological sample identifying the dog as:
drd1 mutation homozygous by determining homozygosity for mutation 1; or
drd1 mutation heterozygous by determining heterozygosity for mutation 1; or
drd1 mutation absent based by determining an absence of mutation 1; or
drd2 mutation homozygous by determining homozygosity for mutation 2; or
drd2 mutation heterozygous by determining heterozygosity for mutation 2; or
drd2 mutation absent based by determining an absence of mutation 2.

2. The method of claim 1, wherein identification of the G in position 11 is carried out by determining the presence of 5 consecutive Gs in SEQ ID NO:4 starting at position 7.

3. The method of claim 1, wherein the mutation 1 or the mutation 2 is detected by analysis of polymerase chain reaction amplification product.

4. The method of claim 1, wherein the dog is a breed of dog selected from the group of dog breeds consisting of Akita, American cocker spaniel, American eskimos, Australian cattle dog, Australian stumpy tailed cattle dog, basenji, Bernese mountain dog, border collie, Chesapeake bay retriever, Chinese crested, English cocker spaniel, Samoyed, English mastiff, English springer spaniel, Entlebucher mountain dog, Finnish lapphund, Labrador retriever, German shorthaired pointer, giant schnauzer, Havanese, lowchen, miniature poodle, miniature schnauzer, Nova scotia duck tolling retriever, Portuguese water dogs, silky terrier, spitz, standard poodle, standard wirehaired dachshund, Tibetan terriers and toy poodle.

5. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, hair, mucosal scrapings, semen, tissue biopsy and saliva.

6. The method of claim 5, wherein the biological sample is collected from a fetal dog, a neonatal dog, or an adult dog.

7. The method of claim 1, further comprising selecting a dog for breeding, wherein a dog selected for breeding exhibits an absence of mutation 1 and mutation 2, or is heterozygous for mutation 1 or mutation 2 and is selected for breeding with a dog that exhibits an absence of mutation 1 and mutation 2.

8. The method of claim 1, further comprising communicating to an individual a result obtained from determining the presence or absence of mutation 1 and/or mutation 2.

9. A method for determining whether a Laborador retriever is a carrier for drd1 form of Oculo-skeletal dysplasia (OSD) or is affected with OSD comprising the steps of obtaining a biological sample from the Laborador retriever and determining from the biological sample the presence of a G in position 11 of the nucleotide sequence depicted in SEQ ID NO:4, and
identifying the Laborador retriever as a carrier of drd1 form of OSD based on heterozygosity for the presence of the G in position 11 of the nucleotide sequence depicted in SEQ ID NO:4, or identifying the Laborador retriever as affected with OSO based on homozygosity for the presence of the G in position 11 of the nucleotide sequence of SEQ ID NO:4.

10. The method of claim 9 wherein upon determination of heterozygosity for the presence of the G in position 11 of the nucleotide sequence depicted in SEQ ID NO:4, a further step of determination of status of drd2 form of OSD is carried out.

11. The method of claim 10, wherein the status of drd2 form of OSD is determined by detecting the presence or absence of a deletion of 1,267 nucleotides between nucleotide number 230 and nucleotide number 1,498 of nucleotide sequence of SEQ ID NO:2.

12. The method of claim 9, wherein determining the presence of the G in position 11 of the nucleotide sequence as depicted in SEQ ID NO:4 comprises determining 5 consecutive Gs starting at position 7 of SEQ ID NO:4.

13. The method of claim 9, wherein determining the presence of the G in position 11 of the nucleotide sequence as depicted in SEQ ID NO:4 is carried out by polymerase chain reaction.

14. A method for determining whether a Samoyed is a carrier for drd2 form of Oculo-skeletal dysplasia (OSD) or is affected with OSD comprising the steps of obtaining a biological sample from the Samoyed and determining from the biological sample the presence of a deletion of 1,267 nucleotides between nucleotide number 230 and nucleotide number 1,498 of the nucleotide sequence as depicted in SEQ ID NO:2; and
identifying the Samoyed as a carrier of drd2 form of OSD based on heterozygosity for the presence of the deletion or identifying the Samoyed as affected with OSO based on homozygosity for the presence of the deletion.

15. The method of claim 14, wherein the presence or absence of the deletion is detected by analysis of polymerase chain reaction amplification product.

16. The method of claim 14 wherein upon identification of the deletion, a further step of determination of status of drd1 form of OSD is carried out.

17. The method of claim 16, wherein the status of drd1 form of OSD is determined by detecting the presence or absence of a G in position 11 of the nucleotide sequence depicted in SEQ ID NO:4.