IDENTIFICATION OF THE CAUSATIVE MUTATION FOR INHERITED CONNECTIVE TISSUE DISORDERS IN EQUINES AND METHODS FOR TESTING FOR SAME

Abstract

Provided is a description of a mutation which is positively correlated with Warmblood Fragile Foal Syndrome Type 1 (WFFST1). The mutation is a G to A change at a specific location in the equine lysyl hydroxylase 1 (LH1) gene. Compositions and methods for use in diagnosing WFFST1 are provided.

Description

[0001] This application claims priority to U.S. patent application Ser. No. 61/486,464, filed on May 16, 2011, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to inherited disease observed in horses. More particularly, the invention relates to detecting a point mutation in a gene associated with Fragile Foal Syndrome Type 1.

DESCRIPTION OF RELATED ART

[0003] Inherited connective tissue disorders occur in a variety of species. They are known by many names and are associated with many different underlying genetic defects. Nonetheless, they all present with skin hyperextensibility. Warmblood Fragile Foal Syndrome Type 1 (WFFST1) occurs in the Warmblood horse population which are a group of mid-sized horse types primarily originating in Europe, developed with the aim of competing in Olympic equestrian sports (often called Sport Horses). The presently available pedigree data shows that Warmblood Fragile Foal Syndrome Type 1 segregates in the Hanoverian, Selle Francais, KWPN (Dutch), Oldenburg, and Westphalian lines. These registries, as well as the Holsteiner, Wurtemberger, Rhinelander, Gelderlander, Zweibrucker and Bavarian Warmblood are central to all Warmblood/Sport Horse registries, placing all horses among the broader group of registries at risk for carrying this disorder. A partial list of many of the registries includes: Hanoverian; Selle Francais; KWPN; Oldenburg; Rhinelander; Holsteiner; Westphalian; Gelderlander; American Warmblood; Anglo-Norman; Austrian Warmblood; Bavarian Warmblood; Belgian Warmblood (including Belgian Half-blood); Canadian Sport Horse; Danish Warmblood; Brazilian Sport Horse; Friesian Sport Horse; German Warmblood ZfDP; Czech Warmblood; Irish Sport Horse; Karossier/Ostfriesen/Alt-Oldenburger; Hungarian Warmblood; Romanian Sport HorseSwedish Warmblood; Swiss Warmblood; Wurttemberger; and Zweibrucker. There is an ongoing an unmet need to identify the mutation that causes WFFST1 and to develop compositions and methods for determining which horses carry it and which horses do not. The present invention meets these and other needs.

SUMMARY OF THE INVENTION

[0004] The present invention is based on the discovery of the mutation associated with WFFST1. The method comprises testing a biological sample obtained or derived from a horse to identify the presence of a G to A mutation in the equine lysyl hydroxylase 1 (LH1) gene. The location of the change will be apparent to those skilled in the art from the amino acid and nucleotide sequences described herein. In one embodiment, the mutation is at a nucleotide position 2086 of the cDNA produced using mRNA transcribed from an LH1 gene which contains the mutation. This cDNA is presented in FIG. 4 as SEQ ID NO:4. Determining a G to A mutation in only one allele establishes that the horse is heterozygous for the WFFST1 mutation and can thus be identified as a carrier. Determining the G to A mutation in both alleles establishes that the horse is homozygous for the WFFST1 mutation and can thus be identified as affected or predisposed to developing WFFST1. Determining an absence of the G to A mutation in both alleles establishes that the horse is homozygous for the absence of the allele and is indicative that the horse is genetically normal with respect to WFFST1.

[0005] The mutation can be determined from DNA or RNA, or from a cDNA amplified from RNA, or from protein obtained from a horse. In one embodiment, the presence of the mutation is identified by determining the presence of a polynucleotide comprising the sequence of SEQ ID NO:1, which is a PCR product obtained by amplification of a segment of the LH1 gene from a horse, the genome of which comprised the mutation. In another embodiment, determining the presence of the mutation comprises determining a change from G to A at position 4 in the sequence of SEQ ID NO:6, which is the wild type sequence of exon 19 of the equine LH1 gene. The sequence of exon 19 comprising the mutation is presented as SEQ ID NO:7.

[0006] In various embodiments, determining the presence of the WFFST1 mutation (irrespective of heterozygosity or homozygosity for it) establishes that the horse is not genetically normal with respect to WFFST1. Determining the absence of the mutation (irrespective of heterozygosity or homozygosity for its absence) establishes that the horse is not affected with WFFST1.

[0007] Also provided is a method for selecting horses for breeding. This aspect of the invention comprises obtaining a biological sample from a horse, testing the biological sample to detect the WFFST1 mutation and, based on analysis of the allelic status for the WFFST1 mutation, breeding horses that are genetically normal or heterozygous for it with horses that are genetically normal with respect to WFFST1.

[0008] Isolated nucleic acids and proteins which comprise the mutation are also provided by the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1A provides a photographic representation of excessive skin elasticity that is typical of horses affected with WFFST1.

[0010] FIG. 1B provides a photographic representation of a skin lesion that is typical of horses affected with WFFST1.

[0011] FIG. 2 provides the sequence of wild type equine LH1 cDNA (SEQ ID NO:2). The wild type bases at the positions of the mutation (base 2086) is bold and enlarged. Non-informative non-coding region single nucleotide polymorphisms (SNPs) are located at bases 2320 and 2580 (when the A of the ATG translational start codon beginning at position 55 is designated as nucleotide 1). Either G or A may occur at these positions in wild-type alleles. Thus there are two additional nucleotides depicted in this sequence in FIG. 2 than would exist in the cDNA, which is for convenience of illustrating this variability. In the depicted sequence the A of the ATG codon is at position 55. The translational stop codon is position 2182-2184 when designating the A at position 55 as nucleotide number 1.

[0012] FIG. 3 provides the wild type equine LH1 polypeptide sequence (727 amino acids; SEQ ID NO:3). The position of the amino acid in the normal peptide corresponding to the position of the change in mutation is bold and enlarged.

[0013] FIG. 4 provides the sequence of a mutant equine LH1 cDNA (SEQ ID NO:4). The position of the mutation is 2032 (when the A at position 55 is designated nucleotide 1), and non-informative, non-coding region SNPs are bold and enlarged. These are respectively at 2320 and 2580. The A of the ATG codon at 55 is bold and enlarged. The translational stop codon is position 2182-2184, and is bold and enlarged. When the first nucleotide of the sequence is counted as nucleotide number 1, the mutation is at position 2086 and the other features of the sequence described above can take their numerical positions in relation to this numbering convention.

[0014] FIG. 5 provides the amino acid sequence of the mutant equine LH1 peptide (SEQ ID NO:5). Arginine replaces the evolutionarily conserved Glycine at position 678 (R is bold and enlarged).

[0015] FIG. 6 provides the sequence of the wild type equine LH1 exon 19 (SEQ ID NO:6). The wild type base at the position of the mutation (base 4) is bold and enlarged and non-coding region SNPs are also shown in bold and enlarged. These are respectively at bases 292 and 552, and either G or an A may occur at these positions in wild-type alleles, while an A occurs at each of these positions in the WFF1 mutant allele. The translational stop codon is bold and enlarged.

[0016] FIG. 7 provides the sequence of the mutated equine LH1 exon 19 (SEQ ID NO:7). The mutation (base 4) is shown in bold and enlarged. The non-informative non-coding region SNPs are also bold and enlarged and occur at bases 292 and 552. The translational stop codon is shown in bold and is enlarged.

[0017] FIG. 8 provides the sequences of exons 1-18 of the equine LH1 gene (SEQ ID NOs 8-25, respectively).

[0018] FIG. 9A provides the nucleotide sequence of a representative amplicon obtained by PCR amplification of an equine LH1 gene that does not contain the mutation (SEQ ID NO:26).

[0019] FIG. 9B provides the nucleotide sequence of a representative amplicon obtained by PCR amplification of an equine LH1 gene that does contain the mutation. The mutation is at position 147 and is enlarged and shown in bold (SEQ ID NO:1).

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based on the discovery of a mutation which is positively correlated with WFFST1. The mutation is a G to A change in the equine LH1 gene. The mutation is referred to herein as the "WFFST1" mutation. The invention comprises a method for analysis of WFFST1 status in a horse. The method comprises obtaining a biological sample from a horse and testing nucleic acids and/or proteins from the biological sample to determine the presence or absence of the WFFST1 mutation. Determining a G to A mutation in only one allele establishes that the horse is heterozygous for the WFFST1 mutation. The horse can thus be identified as a carrier of WFFST1. Determining the G to A mutation in both alleles establishes that the horse is homozygous for the WFFST1 mutation. The horse can thus be identified as affected or predisposed to developing WFFST1. Determining an absence of the G to A mutation in both alleles establishes that the horse is homozygous for the absence. The horse can accordingly be identified as genetically normal with respect to WFFST1. An LH1 allele that does not comprise the WFFST1 mutation and is thus genetically normal with respect to WFFST1 is also referred to herein as "wild type." In one embodiment of the invention, determining the presence of a wild type WFFST1 allele establishes that the horse is not affected or predisposed to developing WFFST1. In another embodiment, determining the presence of a WFFST1 mutation establishes that the horse is not genetically normal with respect to WFFST1.

[0021] The location of the G to A change in any particular sample of equine nucleic acids can be ascertained by the skilled artisan via reference to the nucleotide sequences presented herein. The invention encompasses detecting the presence or absence of the WFFST1 mutation in any polynucleotide from any suitable biological sample obtained or derived from a horse. Detecting the mutations by testing the polypeptide encoded by the LH1 gene is also included. Further, for each DNA sequence described herein its RNA equivalent (replacing each T with a U) is included in the invention. All polynucleotides which encode the equine LH1 polypeptides presented herein are also included with the scope of the invention.

[0022] The WFFST1 mutation is shown in one illustrative embodiment as an A at position 147 of SEQ ID NO:1 (FIG. 9B). SEQ ID NO:1 is the sequence of an amplicon obtained by PCR amplification of a sample comprising nucleic acids which contained the mutation. The A at position 147 of SEQ ID NO:1 represents a change from a wild type G to the mutated sequence which comprises an A at position 147. The sequence of an amplicon obtained by PCR amplification of a sample which did not contain the mutation in presented in SEQ ID NO:26 (FIG. 9B).

[0023] Other polynucleotide sequences presented herein show the mutation in extended equine genetic sequences. For example, the WFFST1 mutation is also shown as an A at position 2086 in the sequence of SEQ ID NO:4. This WFFST1 mutation can be designated to be at position 2032 of SEQ ID NO:4 if nucleotide numbering in that sequence begins with the A at position 55, which is the A of the ATG translation initiation codon. SEQ ID NO:4 is the sequence of a cDNA produced from mRNA transcribed from an LH1 gene which contains the mutation. Non-informative, non-coding region SNPs are present at positions 2320 and 2580 (numbering of nucleotides as if the A of the ATG translational start codon is designated as nucleotide position 1). The non-informative SNPs can be A or G at these positions. The pertinent translational stop codon is position 2182-2184 (again where numbering using A at position 55 of the cDNA is designated as nucleotide position 1).

[0024] In another embodiment, determining the presence of the mutation comprises determining a change from G to A at position 4 in the sequence of SEQ ID NO:6. In connection with this, the wild type sequence of exon 19 of the equine LH1 gene as shown in SEQ ID NO:4 comprises G at position 4. The sequence of exon 19 comprising the WFFST1 mutation is provided in SEQ ID NO:7. Thus, the invention includes determining the presence of the mutation by determining the sequence of SEQ ID NO:7 from a sample obtained from a horse, or determining the absence of the mutation by determining the sequence of SEQ ID NO:6 from a sample obtained from a horse.

[0025] Those skilled in the art will recognize from the foregoing description that position 2086 in SEQ ID NO:2, position 2086 in SEQ ID NO:4, position 147 in SEQ ID NO:1, position 147 in SEQ ID NO:26, position 4 in SEQ ID NO:6, and position 4 in SEQ ID NO:7 are equivalent expressions of the location of the site where the G to A change that constitutes the WFFST1 mutation takes place at the genomic DNA level.

[0026] Those skilled in the art will also recognize that probes and primers can be developed for detecting LH1 sequences that contain the mutation or for LH1 sequences that do not contain the mutation based on the nucleotide sequences described herein. For example, any fragment of SEQ ID NO:2 or SEQ ID NO:4, or their complements, that is at least 12 nucleotides long and does not encompass the mutation site can be used as a primer for any conventional amplification or sequencing reaction to perform the method of the invention. Any fragment that is at least 12 nucleotides long and which does encompass the mutation site can be used for detecting the presence or absence of the mutation using any conventional technique that involves hybridization of a diagnostic probe that can discriminate between an A or a G at the mutation site. In specific embodiments, PCR can be performed on DNA obtained from a horse with a first primer that is identical or complementary to nucleotides 1-24 of SEQ ID NO:1 and a second primer which is identical or complementary to the 25 nucleotides at the 3' end of SEQ ID NO:1. Those skilled in the art will recognize that the sense orientation of the first and second primers will be opposite according to conventional PCR methods.

[0027] The WFFST1 mutation results in a change in the amino acid sequence encoded by the LH1 gene which comprises it. In particular, an equine LH1 polypeptide sequence encoded by an equine LH1 gene which contains the mutation is presented in SEQ ID NO:5. This polypeptide differs from the wild type sequence (a polypeptide sequence encoded by an equine LH1 gene that does not contain the mutation, depicted in SEQ ID NO:3) at amino acid position 678. In the mutant sequence the amino acid at position 678 is Arginine, while in the wild type sequence this amino acid position is occupied by Glycine. Thus, in various embodiments, the invention provides for testing LH1 protein from a biological sample obtained or derived from a horse to determine the amino acid at position 678. In one embodiment, detecting LH1 protein comprising Arginine at position 678 establishes that the horse that produced it is not genetically normal with respect to the WFFST1 mutation.

[0028] The invention can be carried out using any suitable biological sample obtained from a horse. In various embodiments, the biological sample contains nucleic acids encoding the LH1 protein or portions thereof, or a biological sample that contains the LHI protein, or combinations of such polynucleotides and protein. Suitable sources of biological sample include but are not limited to blood, hair, mucosal scrapings, semen, tissue biopsy, or saliva. In one embodiment, the biological sample is blood. In one embodiment, the biological sample is obtained from the horse and used directly in analyzing the polynucleotides and/or protein. In another embodiment, the biological sample is obtained from the horse and subjected to a processing step before the analysis. In some examples, the processing step can be carried out to isolate or purify the polynucleotides and/or proteins to be analyzed, or to amplify the polynucleotides. Thus, the invention in certain embodiments comprises extracting a biological sample from a horse so that the sample is separated from its naturally occurring environment, and mixing it with a non-naturally occurring reagent, such as an extraction buffer. The sample can be mixed with organic solvents, such as formaldehyde, chloroform, alcohols, and the like. The sample may also be subjected to successive heating and cooling steps, or freezing temperatures, such as -20 Celsius.

[0029] While any suitable technique can be used to detect the presence or absence of the WFFST1 mutation, presently preferred techniques include polymerase-chain based reactions, such as PCR or real-time PCR (RT-PCR), or reverse-transcriptase based PCR, or restriction digestions. These processes involve well known techniques and generally include providing a composition comprising isolated nucleic acids for testing and mixing the nucleic acids with non-naturally occurring reagents, such as recombinant or isolated bacterial, viral or bacteriophage polymerases, buffers comprising pre-fixed concentrations of free deoxyribonucleotides, including those that are unlabeled and/or those that are detectably labeled, and/or recombinant bacterial restriction endonucleases, and combinations of the foregoing. The compositions can comprise one or more synthetic oligonucleotides that have complete or partial complementarity to the isolated equine polynucleotide so that they can function as probes, such as primers. In another embodiment, an isolated polynucleotide which comprises or might comprise the WFFST1 mutation can be annealed to a synthetic oligonucleotide that is fixed to a substrate, such as glass or a resin. For example, an isolated polynucleotide which comprises or is suspected of comprising the WFFST1 mutation can be annealed to a synthetic oligonucleotide that is present on an array which comprises a plurality of distinct oligonucleotides. Such arrays can distinguish polynucleotides that differ from one another by a single nucleotide and will produce a signal that is detectable by an individual and/or a machine to signify the presence or the absence of the mutation.

[0030] In another embodiment, the presence or the absence of the WFFST1 mutation in a polynucleotide can be determined using a restriction endonuclease digestion. For example, when present the WFFST1 mutation introduces a unique AlwN1 site. Using a polynucleotide consisting of SEQ ID NO:1 hybridized to its complement as a non-limiting example, digesting this amplicon with AlwN1 yields fragments of ˜150 and 200 by on digestion, as compared to the uncut 350 by fragment obtained when the wild-type allele is present. Furthermore, the WFFST1 mutation ablates an AciI site, yielding fragments of ˜269, 10 and 72 bp, while digestion of the wild-type fragment produces ˜149, 120, 10 and 72 by bands in this illustrative example. Thus, digestion with AciI cleaves the wild-type product in three places while the mutant product is only cleaved in two places. Those skilled in the art will recognize that there are a multitude of restriction endonuclease assays that can be developed if desired from these observations.

[0031] As with polynucleotides, detecting a protein which comprises the WFFST1 mutation can be performed using any known technique. Such techniques include but are not limited to immunodetection assays, which entail forming a complex between a protein that comprises or might comprise the amino acid change encoded by the WFFST1 mutation with a monoclonal antibody that is produced by a hybridoma or by recombinant methods. The complex can include a detectably labeled ligand, such as a detectably labeled monoclonal antibody that is specific for the protein. The invention includes generation and use of monoclonal antibodies that can discriminate between the wild type and mutant forms of the LS1 protein.

[0032] Also provided in the present invention are kits for detecting the presence or absence of the WFFST1 mutation. The kits comprise reagents for nucleic acid based detection of the presence or absence of the mutation(s), or antibodies for detecting the presence or absence of the mutation. In one embodiment, the kits comprise reagents for extraction/preparation of nucleic acid samples and pair(s) of specific primers for use in identification of the mutation. In another embodiment, the kits provide antibodies and compositions used for probing samples with the antibodies to determine whether or not the WFFST1 mutation is present in a horse from which a biological sample is obtained and tested according to the invention. Any of the aforementioned kit components can be provided in one or more sealed, sterile containers, such as vials.

[0033] It is contemplated that any horse at any developmental stage (i.e., fetal, neonatal, postnatal, juvenile, adult, etc.) may be tested according to the method of the invention. Further, any horse, regardless of breed, could be homozygous or heterozygous for the WFFST1 mutation, or homozygous for the absence of the mutation, and thus is suitable for testing. In various embodiments, the horse may be a Warmblood, Thoroughbred, Standardbred, Tennessee Walking Horse, Mustang, Quarter Horse, Arabian, Draft breed, Miniature Horse, or a pony.

[0034] By using the tools and method described herein, horses which are genetically normal for the disease, carriers of the WFFST1 disease and horses which are affected by (or predisposed to) WFFST1 can be identified. Upon identification, affected or predisposed, or carrier horses can be eliminated from the breeding stock. Alternatively, horses which are affected or predisposed with WFFST, or carriers of the WFFST mutation, can be mated with genetically normal horses to avoid producing affected foals.

[0035] In one embodiment, the method further comprises fixing the result of determining the presence or absence of the WFFST1 mutation in a tangible medium, such as a paper report, or a portable medium on which electronic files can be stored, such as a hard drive, a computer disk, a thumb drive, and the like. In another embodiment, the invention includes communicating the result of determining the presence or absence of the WFFST1 mutation 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 horse owner, an equine registry, a veterinarian, or a provider of genetic test results.

[0036] The WFFST1phenotype is clinically detectable and present at birth (premature birth has been reported). Affected foals are euthanized within days to weeks of life due to the poor prognosis. Horses that are clinically affected can be recognized by skin traits that are characteristic of WFFST1. FIGS. 1A and 1B illustrate typical manifestations of WFFST1, where the skin lacks tensile strength (FIG. 1A) and extreme skin fragility characterized by tearing, ulceration, etc. from contact with normal surroundings; FIG. 1B). Lesions can occur anywhere on the body, but are most noted on pressure points and in addition to skin wounds, lesions are found on the gums and other oral cavity mucous membranes and the perineum. Skin is hyperextensible (stretchy) over essentially the whole body, and especially the head, neck, thorax and limbs. Limb joints are lax and hyperextensible. Fetlocks are the most dramatically affected and affected foals cannot stand normally. Ears may be soft, overly flexible, bent or floppy presumably due to failure of collagen in the supporting cartilage. At post mortem exam hematomas and seromas are noted under the skin, particularly over pressure points. Supporting structures around joints (capsule, tendons, ligaments) are lax. Lesions of articular cartilage are reported in some joints.

[0037] The invention will be further understood by the following Example, which is intended to be illustrative and not restrictive in any way.

EXAMPLE 1

[0038] This Example demonstrates a 100% correlation between homozygosity (AA) for the causative mutation and the disease phenotype. The disease phenotype is recognizable on clinical examination and is characterized by the presence at birth of soft skin lacking adequate tensile strength to withstand even normal environmental contact. The skin is hyperextensible (lacking normal recoil when traction is applied/released) over the whole body, especially the head, neck, thorax and limbs. Over the first hours to days of life, focal seromas, hematomas, and lacerations/ulcerations result from incidental environmental contact. Such lesions are predominantly found over pressure points such as fetlocks (metacarpal-phalangeal joints), carpi (knees), and tarsi (hocks), but may occur anywhere on the body (including oral cavity and perineum). Limb joints, particularly the fetlocks) are lax and hyperextensible, and affected foals cannot typically stand normally. The ears are typically bent or floppy. Also demonstrated is a 100% correlation between heterozygosity (AG) and the normal phenotype, as well as a 100% correlation between homozygosity for wild type (GG) and the normal phenotype. The normal phenotype lacks the clinical criteria specified above for the disease phenotype.

[0039] Results from determining genotype and phenotype are presented in Table 1. To obtain the results presented in Table 1, the WFFST1 genotype was established by PCR amplification of a 350 by fragment of the LH1 gene containing the WFFST1 mutation site from genomic DNA, followed by automated direct sequencing of the resulting PCR products. The sequences of amplified fragments (amplicons) representing the wild-type and WFFST1 alleles are given in SEQ ID NO: 26 and SEQ ID NO: 1, respectively. Genomic DNA was extracted from whole blood/serum or mane/tail hair root samples using respectively, the QIAamp DNA Blood Mini Kit or the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's recommended protocols. Amplifications were performed in 50 microliters total volume containing 3 microliters genomic DNA (˜40 ng), 1 X Platinum Taq Hi Fi buffer, 200 micromolar dNTPs, 0.2 micromolar each primer, and 1 unit Platinum Taq Hi Fi (Invitrogen). Primer sequences for PCR were 5'-atagctgtcactccacaaggcaca-3' (sense primer; SEQ ID NO:27) and 5'-cagtggttgttggcaacgaggaaaa-3' (antisense primer; SEQ ID NO:28). DNA amplification was performed in an ABI 2720 thermal cycler using the following program: Initial denaturation: 5 min at 95°, followed by 40 cycles of: 30 sec at 95°; 30 sec at 55°; 30 sec at 72°; followed by a final extension segment of 5 min at 72°. Unincorporated primers and nucleotides were eliminated from PCR reactions using the DNA Clean & Concentrator-5 Kit (Zymo Research) according to the manufacturer's recommended protocol. DNA sequencing was performed by cycle sequencing using a primer having the sequence 5'-atagctgtcactccacaaggcaca-3', using dye terminator chemistry (Big Dye v3.1, Applied Biosystems, Foster City Calif.), followed by dye terminator removal using Edge Performa DTR gelfiltration plates (Edge Biosystems, Gaithersburg Md.) and automated data collection on an Applied Biosystems 3730x1 DNA Analyzer.

[0040] The results presented in Table 1 demonstrate a 100% association between the presence of a G to A mutation in the LH1 gene at position 2086 in the sequence of SEQ ID NO:4. More specifically, there is a 100% association between homozygosity (A/A) for the causative mutation and the disease phenotype; a 100% association between heterozygosity (A/G) and the normal phenotype, as well as a 100% association between homozygosity for wild type (G/G) and the normal phenotype.

TABLE-US-00001 TABLE 1 Homozygous for Heterozygous for Homozygous for the mutant allele the mutant allele the normal allele Genotype (A/A) (A/G) (G/G) Number Tested 2 8 149 Clinical 2 Affected 8 Normal 149 Normal Phenotype

[0041] The data summarized in Table 1 show that two foals homozygous for the mutant (A) allele were clinically affected. The phenotype of affected foals was determined by clinical examination over the initial days of life. Eight Warmblood horses were determined to be heterozygous for the mutant allele (A/G) and all eight were clinically normal. Three of the eight heterozygotes were obligate carrier parents of the affected foals. The five additional heterozygotes were identified by screening DNA samples from 104 clinically normal Warmbloods from the local population. One hundred and forty-nine horses were determined to be homozygous for the normal allele (G) and all were clinically normal. This group of horses included 50 horses of various breeds (Thoroughbred, Standardbred, Tennessee Walking Horse, Mustang, Quarter Horse, Arabian, Drafts and ponies) in addition to 99 clinically normal Warmbloods.

[0042] The results in Table 1 show clearly that there is a 100% association (2/2 horses) between homozygosity for the causative mutation and the affected phenotype. The frequency of the mutant allele (A) appears to be approximately 3.77% in the test population. The frequency of the mutant allele (A) in the "at-risk" population (Warmbloods) appears to be approximately 5.50%, while the carrier frequency among the group of clinically normal adult Warmbloods studied is 7.48%. Mutations in lysyl hydroxylase 1 (LH1) are known to cause the human disorder Ehlers-Danlos Syndrome VI (EDSVI). This is an autosomal recessive connective tissue fragility syndrome characterized by premature birth, infant floppiness/joint laxity, development of kyphosis and/or scoliosis, skin fragility, abnormal bruisability and scarring, ocular abnormalities, recurrent joint dislocation, cardiac valvular failure, catastrophic arterial and/or bowel rupture. The present invention is believed to represent the first discovery of a spontaneously occurring EDSVI analog in a non-human animal species, and mutations causative of deficient/abnormal LH1 are believed to have not previously been described in non-human animals.

[0043] 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.

Sequence CWU 1

1

281350DNAhorse 1atagctgtca ctccacaagg cacaagggct gcttggctgg ggtggctcag atgggagaat 60ggggggtact aggggaaggg cccagcttcc tcttggggaa actgacgctt cctgttggga 120aactgacact tcctgtctcc cagggcaggg gctgtcggtt cctgcgttac aactgctcca 180tccgagcccc acggaagggc tggaccctca tgcaccccgg acgactcacg cactaccacg 240agggactccc caccaccaag ggcacccgct acatcgcggt ctccttcgtc gatccctaat 300cggccaagcc tggccacttc ggaccttttc ctcgttgcca acaaccactg 35022992DNAhorsemisc_feature(2374)..(2374)n is a, c, g, or t 2agtttccagc ctgccagcgc ctgctggctg cccggacgtc cccagacctc ggccatgcgg 60cctctgctgc tcctggcccc gctgggctgg ctgctcctgg ccgaagcgaa gggcgacgcc 120aagccggagg acaacctctt agtcctcacg gtggccacga aggagtccga ggggttccga 180cgcttcaagc gctcagccca gttcttcaac tacaagatcc aggcgctggg cctgggggag 240gactgggacg gggacaagga gacgtcagcg ggcggcgggc tgaaggttcg gctgctgaag 300aaagctctgg agaagcatgc agacaaagag aacctggtca ttctcttcac agacagctat 360gatgtggtat ttgcctcggg gccccgagag ctcctgaaga agttccggca ggccaggagc 420caggtggtct tctcggccga ggagctcatc taccccgacc gcaggctgga ggccaagtac 480ccggtggtgt ccgatggcaa gaggttcctg ggctctggag gcttcatcgg ttatgccccc 540aacctcagca aactggtggc tgagtgggag ggccaggaca gcgacagtga ccagctgttt 600tataccaaga tcttcttgga cccagagaag agggagcgga tcaacatcac cctggaccac 660cgctgccgta tcttccagaa cctggttgga gccttagatg aggtcgtgct caagtttgaa 720atgggccatg tgagggcgcg gaacctggcc tacgacaccc tccccgtcct gattcatggc 780aacgggccca ccaagctgca gctgaactac ctgggcaact atatccctcg cttctggacc 840ttcgagacgg gctgcacggt gtgtgacgag ggcctgcgca gcctcaaggg cattggggat 900gaagctctgc ctgtggtctt ggtcggcgtg ttcatcgagc agcccacgcc gttcctgtcc 960ctgttcttcc agcggcttct gcgcctgcat tacccccgga aacagctgcg gctttttatt 1020cataaccatg agcagcacca caaggctcag gtggagcagt tcctggcaga gcatggcggc 1080gagtacaagt ctgtgaaact ggtgggcccc gaggtgcggg tggcaaacgc cgatgccagg 1140aacatgggcg cggacctgtg ccggcaggac cgtggctgca cctactactt cagtgtggat 1200gccgacgtgg ccctgaccga gcccaagacc ctgcgactgc tgattgagca gaacaagaat 1260gtcatcgccc cgttgatgac ccgccacggg aggctgtggt cgaacttctg gggggcaatg 1320agtgcagatg gctactacgc ccgctccgag gactacgtgg acattgtgca ggggcggcgt 1380gttggcgtct ggaacgtgcc ctacatctcg aacatttacc tgatcaaggg cagtgccctg 1440cgggctgagc tgcagcagac agatctgttc caccacagca agctggatgc cgacatggcc 1500ttctgtgcca atatccggca gcaggatgtg ttcatgttcc tgaccaaccg gcacaccttc 1560ggccacctgc tctccctgga cagctaccag accacccacc tccacaacga cctctgggag 1620gtgtttagca accccgagga ctggaaggag aagtacatcc atgagaacta caccagggcc 1680ctggcgggga agctggtgga gatgccttgc ccggatgtct actggttccc catcttcacg 1740gagacggcct gtgacgagct ggtggaggag atggagcact acggccagtg gtctctggga 1800gacaataagg acaaccgcat ccagggtggc tacgaaaatg tgccgaccat cgacatccac 1860atgaaccaga tcagctttga gcgggagtgg cacaagttcc tggtggagta catcgccccc 1920atgacagaga agctgtatcc aggctactac accagggccc agttcgacct ggcctttgtt 1980gtccgctaca agcctgacga gcagccctcg ctgatgcccc accacgatgc ctccaccttc 2040actgtcaaca tcgccctgaa ccgggtcggg gtggattacg agggcggggg ctgtcggttc 2100ctgcgttaca actgctccat ccgagcccca cggaagggct ggaccctcat gcaccccgga 2160cgactcacgc actaccacga gggactcccc accaccaagg gcacccgcta catcgcggtc 2220tccttcgtcg atccctaatc ggccaagcct ggccacttcg gaccttttcc tcgttgccaa 2280caaccactgc ccagcagcct ctggggcctt ggggtcccag ggaacctggt ccagcctccg 2340ggctcttgac ctcccattgc tttcggagcc gccntcggag agactgggcc gcaggccaga 2400ggcagagcac acctccttgg ctggggcttt cctggtgttc tgctccccac cccgggagat 2460ggggtccacg ctcactgcct tgtaacagct catcctctcc cacctgttct cctgaaaagc 2520ccggtccctc ttcctctgcc tcttccatgg gcccagacct gagcagaacc gggcttaccc 2580agctgcccag agagactcta ggggccagaa gccatgcccc agagctccca ggcngggctg 2640ccacccggga acttctgctt caagcttcag ggtagacaca gagacctgga tgagactcaa 2700gtcccctccc tgatcctggg cctgctgaag ccccttcctc catggctcct gtcatgagag 2760caaaacattg tcgcctggag acggtgactc ggaaagcctc ctgggagaca ggaaaggcat 2820cgatgccaca gctccatcct ctacttgacc cttgctggcg ggaggggagt gatatgtcca 2880cacactgcac tgcgtcatcc tgttccggat gcctccggag agagggacgg acagtcagaa 2940acaagggagt ttctattaaa ggccatccaa accaaaaaaa aaaaaaaaaa aa 29923727PRThorse 3Met Arg Pro Leu Leu Leu Leu Ala Pro Leu Gly Trp Leu Leu Leu Ala 1 5 10 15 Glu Ala Lys Gly Asp Ala Lys Pro Glu Asp Asn Leu Leu Val Leu Thr 20 25 30 Val Ala Thr Lys Glu Ser Glu Gly Phe Arg Arg Phe Lys Arg Ser Ala 35 40 45 Gln Phe Phe Asn Tyr Lys Ile Gln Ala Leu Gly Leu Gly Glu Asp Trp 50 55 60 Asp Gly Asp Lys Glu Thr Ser Ala Gly Gly Gly Leu Lys Val Arg Leu 65 70 75 80 Leu Lys Lys Ala Leu Glu Lys His Ala Asp Lys Glu Asn Leu Val Ile 85 90 95 Leu Phe Thr Asp Ser Tyr Asp Val Val Phe Ala Ser Gly Pro Arg Glu 100 105 110 Leu Leu Lys Lys Phe Arg Gln Ala Arg Ser Gln Val Val Phe Ser Ala 115 120 125 Glu Glu Leu Ile Tyr Pro Asp Arg Arg Leu Glu Ala Lys Tyr Pro Val 130 135 140 Val Ser Asp Gly Lys Arg Phe Leu Gly Ser Gly Gly Phe Ile Gly Tyr 145 150 155 160 Ala Pro Asn Leu Ser Lys Leu Val Ala Glu Trp Glu Gly Gln Asp Ser 165 170 175 Asp Ser Asp Gln Leu Phe Tyr Thr Lys Ile Phe Leu Asp Pro Glu Lys 180 185 190 Arg Glu Arg Ile Asn Ile Thr Leu Asp His Arg Cys Arg Ile Phe Gln 195 200 205 Asn Leu Val Gly Ala Leu Asp Glu Val Val Leu Lys Phe Glu Met Gly 210 215 220 His Val Arg Ala Arg Asn Leu Ala Tyr Asp Thr Leu Pro Val Leu Ile 225 230 235 240 His Gly Asn Gly Pro Thr Lys Leu Gln Leu Asn Tyr Leu Gly Asn Tyr 245 250 255 Ile Pro Arg Phe Trp Thr Phe Glu Thr Gly Cys Thr Val Cys Asp Glu 260 265 270 Gly Leu Arg Ser Leu Lys Gly Ile Gly Asp Glu Ala Leu Pro Val Val 275 280 285 Leu Val Gly Val Phe Ile Glu Gln Pro Thr Pro Phe Leu Ser Leu Phe 290 295 300 Phe Gln Arg Leu Leu Arg Leu His Tyr Pro Arg Lys Gln Leu Arg Leu 305 310 315 320 Phe Ile His Asn His Glu Gln His His Lys Ala Gln Val Glu Gln Phe 325 330 335 Leu Ala Glu His Gly Gly Glu Tyr Lys Ser Val Lys Leu Val Gly Pro 340 345 350 Glu Val Arg Val Ala Asn Ala Asp Ala Arg Asn Met Gly Ala Asp Leu 355 360 365 Cys Arg Gln Asp Arg Gly Cys Thr Tyr Tyr Phe Ser Val Asp Ala Asp 370 375 380 Val Ala Leu Thr Glu Pro Lys Thr Leu Arg Leu Leu Ile Glu Gln Asn 385 390 395 400 Lys Asn Val Ile Ala Pro Leu Met Thr Arg His Gly Arg Leu Trp Ser 405 410 415 Asn Phe Trp Gly Ala Met Ser Ala Asp Gly Tyr Tyr Ala Arg Ser Glu 420 425 430 Asp Tyr Val Asp Ile Val Gln Gly Arg Arg Val Gly Val Trp Asn Val 435 440 445 Pro Tyr Ile Ser Asn Ile Tyr Leu Ile Lys Gly Ser Ala Leu Arg Ala 450 455 460 Glu Leu Gln Gln Thr Asp Leu Phe His His Ser Lys Leu Asp Ala Asp 465 470 475 480 Met Ala Phe Cys Ala Asn Ile Arg Gln Gln Asp Val Phe Met Phe Leu 485 490 495 Thr Asn Arg His Thr Phe Gly His Leu Leu Ser Leu Asp Ser Tyr Gln 500 505 510 Thr Thr His Leu His Asn Asp Leu Trp Glu Val Phe Ser Asn Pro Glu 515 520 525 Asp Trp Lys Glu Lys Tyr Ile His Glu Asn Tyr Thr Arg Ala Leu Ala 530 535 540 Gly Lys Leu Val Glu Met Pro Cys Pro Asp Val Tyr Trp Phe Pro Ile 545 550 555 560 Phe Thr Glu Thr Ala Cys Asp Glu Leu Val Glu Glu Met Glu His Tyr 565 570 575 Gly Gln Trp Ser Leu Gly Asp Asn Lys Asp Asn Arg Ile Gln Gly Gly 580 585 590 Tyr Glu Asn Val Pro Thr Ile Asp Ile His Met Asn Gln Ile Ser Phe 595 600 605 Glu Arg Glu Trp His Lys Phe Leu Val Glu Tyr Ile Ala Pro Met Thr 610 615 620 Glu Lys Leu Tyr Pro Gly Tyr Tyr Thr Arg Ala Gln Phe Asp Leu Ala 625 630 635 640 Phe Val Val Arg Tyr Lys Pro Asp Glu Gln Pro Ser Leu Met Pro His 645 650 655 His Asp Ala Ser Thr Phe Thr Val Asn Ile Ala Leu Asn Arg Val Gly 660 665 670 Val Asp Tyr Glu Gly Gly Gly Cys Arg Phe Leu Arg Tyr Asn Cys Ser 675 680 685 Ile Arg Ala Pro Arg Lys Gly Trp Thr Leu Met His Pro Gly Arg Leu 690 695 700 Thr His Tyr His Glu Gly Leu Pro Thr Thr Lys Gly Thr Arg Tyr Ile 705 710 715 720 Ala Val Ser Phe Val Asp Pro 725 42992DNAhorse 4agtttccagc ctgccagcgc ctgctggctg cccggacgtc cccagacctc ggccatgcgg 60cctctgctgc tcctggcccc gctgggctgg ctgctcctgg ccgaagcgaa gggcgacgcc 120aagccggagg acaacctctt agtcctcacg gtggccacga aggagtccga ggggttccga 180cgcttcaagc gctcagccca gttcttcaac tacaagatcc aggcgctggg cctgggggag 240gactgggacg gggacaagga gacgtcagcg ggcggcgggc tgaaggttcg gctgctgaag 300aaagctctgg agaagcatgc agacaaagag aacctggtca ttctcttcac agacagctat 360gatgtggtat ttgcctcggg gccccgagag ctcctgaaga agttccggca ggccaggagc 420caggtggtct tctcggccga ggagctcatc taccccgacc gcaggctgga ggccaagtac 480ccggtggtgt ccgatggcaa gaggttcctg ggctctggag gcttcatcgg ttatgccccc 540aacctcagca aactggtggc tgagtgggag ggccaggaca gcgacagtga ccagctgttt 600tataccaaga tcttcttgga cccagagaag agggagcgga tcaacatcac cctggaccac 660cgctgccgta tcttccagaa cctggttgga gccttagatg aggtcgtgct caagtttgaa 720atgggccatg tgagggcgcg gaacctggcc tacgacaccc tccccgtcct gattcatggc 780aacgggccca ccaagctgca gctgaactac ctgggcaact atatccctcg cttctggacc 840ttcgagacgg gctgcacggt gtgtgacgag ggcctgcgca gcctcaaggg cattggggat 900gaagctctgc ctgtggtctt ggtcggcgtg ttcatcgagc agcccacgcc gttcctgtcc 960ctgttcttcc agcggcttct gcgcctgcat tacccccgga aacagctgcg gctttttatt 1020cataaccatg agcagcacca caaggctcag gtggagcagt tcctggcaga gcatggcggc 1080gagtacaagt ctgtgaaact ggtgggcccc gaggtgcggg tggcaaacgc cgatgccagg 1140aacatgggcg cggacctgtg ccggcaggac cgtggctgca cctactactt cagtgtggat 1200gccgacgtgg ccctgaccga gcccaagacc ctgcgactgc tgattgagca gaacaagaat 1260gtcatcgccc cgttgatgac ccgccacggg aggctgtggt cgaacttctg gggggcaatg 1320agtgcagatg gctactacgc ccgctccgag gactacgtgg acattgtgca ggggcggcgt 1380gttggcgtct ggaacgtgcc ctacatctcg aacatttacc tgatcaaggg cagtgccctg 1440cgggctgagc tgcagcagac agatctgttc caccacagca agctggatgc cgacatggcc 1500ttctgtgcca atatccggca gcaggatgtg ttcatgttcc tgaccaaccg gcacaccttc 1560ggccacctgc tctccctgga cagctaccag accacccacc tccacaacga cctctgggag 1620gtgtttagca accccgagga ctggaaggag aagtacatcc atgagaacta caccagggcc 1680ctggcgggga agctggtgga gatgccttgc ccggatgtct actggttccc catcttcacg 1740gagacggcct gtgacgagct ggtggaggag atggagcact acggccagtg gtctctggga 1800gacaataagg acaaccgcat ccagggtggc tacgaaaatg tgccgaccat cgacatccac 1860atgaaccaga tcagctttga gcgggagtgg cacaagttcc tggtggagta catcgccccc 1920atgacagaga agctgtatcc aggctactac accagggccc agttcgacct ggcctttgtt 1980gtccgctaca agcctgacga gcagccctcg ctgatgcccc accacgatgc ctccaccttc 2040actgtcaaca tcgccctgaa ccgggtcggg gtggattacg agggcagggg ctgtcggttc 2100ctgcgttaca actgctccat ccgagcccca cggaagggct ggaccctcat gcaccccgga 2160cgactcacgc actaccacga gggactcccc accaccaagg gcacccgcta catcgcggtc 2220tccttcgtcg atccctaatc ggccaagcct ggccacttcg gaccttttcc tcgttgccaa 2280caaccactgc ccagcagcct ctggggcctt ggggtcccag ggaacctggt ccagcctccg 2340ggctcttgac ctcccattgc tttcggagcc gccatcggag agactgggcc gcaggccaga 2400ggcagagcac acctccttgg ctggggcttt cctggtgttc tgctccccac cccgggagat 2460ggggtccacg ctcactgcct tgtaacagct catcctctcc cacctgttct cctgaaaagc 2520ccggtccctc ttcctctgcc tcttccatgg gcccagacct gagcagaacc gggcttaccc 2580agctgcccag agagactcta ggggccagaa gccatgcccc agagctccca ggcagggctg 2640ccacccggga acttctgctt caagcttcag ggtagacaca gagacctgga tgagactcaa 2700gtcccctccc tgatcctggg cctgctgaag ccccttcctc catggctcct gtcatgagag 2760caaaacattg tcgcctggag acggtgactc ggaaagcctc ctgggagaca ggaaaggcat 2820cgatgccaca gctccatcct ctacttgacc cttgctggcg ggaggggagt gatatgtcca 2880cacactgcac tgcgtcatcc tgttccggat gcctccggag agagggacgg acagtcagaa 2940acaagggagt ttctattaaa ggccatccaa accaaaaaaa aaaaaaaaaa aa 29925727PRThorse 5Met Arg Pro Leu Leu Leu Leu Ala Pro Leu Gly Trp Leu Leu Leu Ala 1 5 10 15 Glu Ala Lys Gly Asp Ala Lys Pro Glu Asp Asn Leu Leu Val Leu Thr 20 25 30 Val Ala Thr Lys Glu Ser Glu Gly Phe Arg Arg Phe Lys Arg Ser Ala 35 40 45 Gln Phe Phe Asn Tyr Lys Ile Gln Ala Leu Gly Leu Gly Glu Asp Trp 50 55 60 Asp Gly Asp Lys Glu Thr Ser Ala Gly Gly Gly Leu Lys Val Arg Leu 65 70 75 80 Leu Lys Lys Ala Leu Glu Lys His Ala Asp Lys Glu Asn Leu Val Ile 85 90 95 Leu Phe Thr Asp Ser Tyr Asp Val Val Phe Ala Ser Gly Pro Arg Glu 100 105 110 Leu Leu Lys Lys Phe Arg Gln Ala Arg Ser Gln Val Val Phe Ser Ala 115 120 125 Glu Glu Leu Ile Tyr Pro Asp Arg Arg Leu Glu Ala Lys Tyr Pro Val 130 135 140 Val Ser Asp Gly Lys Arg Phe Leu Gly Ser Gly Gly Phe Ile Gly Tyr 145 150 155 160 Ala Pro Asn Leu Ser Lys Leu Val Ala Glu Trp Glu Gly Gln Asp Ser 165 170 175 Asp Ser Asp Gln Leu Phe Tyr Thr Lys Ile Phe Leu Asp Pro Glu Lys 180 185 190 Arg Glu Arg Ile Asn Ile Thr Leu Asp His Arg Cys Arg Ile Phe Gln 195 200 205 Asn Leu Val Gly Ala Leu Asp Glu Val Val Leu Lys Phe Glu Met Gly 210 215 220 His Val Arg Ala Arg Asn Leu Ala Tyr Asp Thr Leu Pro Val Leu Ile 225 230 235 240 His Gly Asn Gly Pro Thr Lys Leu Gln Leu Asn Tyr Leu Gly Asn Tyr 245 250 255 Ile Pro Arg Phe Trp Thr Phe Glu Thr Gly Cys Thr Val Cys Asp Glu 260 265 270 Gly Leu Arg Ser Leu Lys Gly Ile Gly Asp Glu Ala Leu Pro Val Val 275 280 285 Leu Val Gly Val Phe Ile Glu Gln Pro Thr Pro Phe Leu Ser Leu Phe 290 295 300 Phe Gln Arg Leu Leu Arg Leu His Tyr Pro Arg Lys Gln Leu Arg Leu 305 310 315 320 Phe Ile His Asn His Glu Gln His His Lys Ala Gln Val Glu Gln Phe 325 330 335 Leu Ala Glu His Gly Gly Glu Tyr Lys Ser Val Lys Leu Val Gly Pro 340 345 350 Glu Val Arg Val Ala Asn Ala Asp Ala Arg Asn Met Gly Ala Asp Leu 355 360 365 Cys Arg Gln Asp Arg Gly Cys Thr Tyr Tyr Phe Ser Val Asp Ala Asp 370 375 380 Val Ala Leu Thr Glu Pro Lys Thr Leu Arg Leu Leu Ile Glu Gln Asn 385 390 395 400 Lys Asn Val Ile Ala Pro Leu Met Thr Arg His Gly Arg Leu Trp Ser 405 410 415 Asn Phe Trp Gly Ala Met Ser Ala Asp Gly Tyr Tyr Ala Arg Ser Glu 420 425 430 Asp Tyr Val Asp Ile Val Gln Gly Arg Arg Val Gly Val Trp Asn Val 435 440 445 Pro Tyr Ile Ser Asn Ile Tyr Leu Ile Lys Gly Ser Ala Leu Arg Ala 450 455 460 Glu Leu Gln Gln Thr Asp Leu Phe His His Ser Lys Leu Asp Ala Asp 465 470 475 480 Met Ala Phe Cys Ala Asn Ile Arg Gln Gln Asp Val Phe Met Phe Leu 485 490 495 Thr Asn Arg His Thr Phe Gly His Leu Leu Ser Leu Asp Ser Tyr Gln 500 505 510 Thr Thr His Leu His Asn Asp Leu Trp Glu Val Phe Ser Asn Pro Glu 515 520 525 Asp Trp Lys Glu Lys Tyr Ile His Glu Asn Tyr Thr Arg Ala Leu Ala 530 535 540 Gly Lys Leu Val Glu Met Pro Cys Pro Asp Val Tyr Trp Phe Pro Ile 545 550 555 560 Phe Thr Glu Thr Ala Cys Asp Glu Leu Val Glu Glu Met Glu His Tyr 565 570 575 Gly Gln Trp Ser Leu Gly Asp Asn Lys Asp Asn Arg Ile Gln Gly Gly 580 585 590

Tyr Glu Asn Val Pro Thr Ile Asp Ile His Met Asn Gln Ile Ser Phe 595 600 605 Glu Arg Glu Trp His Lys Phe Leu Val Glu Tyr Ile Ala Pro Met Thr 610 615 620 Glu Lys Leu Tyr Pro Gly Tyr Tyr Thr Arg Ala Gln Phe Asp Leu Ala 625 630 635 640 Phe Val Val Arg Tyr Lys Pro Asp Glu Gln Pro Ser Leu Met Pro His 645 650 655 His Asp Ala Ser Thr Phe Thr Val Asn Ile Ala Leu Asn Arg Val Gly 660 665 670 Val Asp Tyr Glu Gly Arg Gly Cys Arg Phe Leu Arg Tyr Asn Cys Ser 675 680 685 Ile Arg Ala Pro Arg Lys Gly Trp Thr Leu Met His Pro Gly Arg Leu 690 695 700 Thr His Tyr His Glu Gly Leu Pro Thr Thr Lys Gly Thr Arg Tyr Ile 705 710 715 720 Ala Val Ser Phe Val Asp Pro 725 6891DNAhorsemisc_feature(292)..(292)n is a, c, g, or t 6ggcgggggct gtcggttcct gcgttacaac tgctccatcc gagccccacg gaagggctgg 60accctcatgc accccggacg actcacgcac taccacgagg gactccccac caccaagggc 120acccgctaca tcgcggtctc cttcgtcgat ccctaatcgg ccaagcctgg ccacttcgga 180ccttttcctc gttgccaaca accactgccc agcagcctct ggggccttgg ggtcccaggg 240aacctggtcc agcctccggg ctcttgacct cccattgctt tcggagccgc cntcggagag 300actgggccgc aggccagagg cagagcacac ctccttggct ggggctttcc tggtgttctg 360ctccccaccc cgggagatgg ggtccacgct cactgccttg taacagctca tcctctccca 420cctgttctcc tgaaaagccc ggtccctctt cctctgcctc ttccatgggc ccagacctga 480gcagaaccgg gcttacccag ctgcccagag agactctagg ggccagaagc catgccccag 540agctcccagg cngggctgcc acccgggaac ttctgcttca agcttcaggg tagacacaga 600gacctggatg agactcaagt cccctccctg atcctgggcc tgctgaagcc ccttcctcca 660tggctcctgt catgagagca aaacattgtc gcctggagac ggtgactcgg aaagcctcct 720gggagacagg aaaggcatcg atgccacagc tccatcctct acttgaccct tgctggcggg 780aggggagtga tatgtccaca cactgcactg cgtcatcctg ttccggatgc ctccggagag 840agggacggac agtcagaaac aagggagttt ctattaaagg ccatccaaac c 8917891DNAhorse 7ggcaggggct gtcggttcct gcgttacaac tgctccatcc gagccccacg gaagggctgg 60accctcatgc accccggacg actcacgcac taccacgagg gactccccac caccaagggc 120acccgctaca tcgcggtctc cttcgtcgat ccctaatcgg ccaagcctgg ccacttcgga 180ccttttcctc gttgccaaca accactgccc agcagcctct ggggccttgg ggtcccaggg 240aacctggtcc agcctccggg ctcttgacct cccattgctt tcggagccgc catcggagag 300actgggccgc aggccagagg cagagcacac ctccttggct ggggctttcc tggtgttctg 360ctccccaccc cgggagatgg ggtccacgct cactgccttg taacagctca tcctctccca 420cctgttctcc tgaaaagccc ggtccctctt cctctgcctc ttccatgggc ccagacctga 480gcagaaccgg gcttacccag ctgcccagag agactctagg ggccagaagc catgccccag 540agctcccagg cagggctgcc acccgggaac ttctgcttca agcttcaggg tagacacaga 600gacctggatg agactcaagt cccctccctg atcctgggcc tgctgaagcc ccttcctcca 660tggctcctgt catgagagca aaacattgtc gcctggagac ggtgactcgg aaagcctcct 720gggagacagg aaaggcatcg atgccacagc tccatcctct acttgaccct tgctggcggg 780aggggagtga tatgtccaca cactgcactg cgtcatcctg ttccggatgc ctccggagag 840agggacggac agtcagaaac aagggagttt ctattaaagg ccatccaaac c 8918130DNAhorse 8agtttccagc ctgccagcgc ctgctggctg cccggacgtc cccagacctc ggccatgcgg 60cctctgctgc tcctggcccc gctgggctgg ctgctcctgg ccgaagcgaa gggcgacgcc 120aagccggagg 130991DNAhorse 9acaacctctt agtcctcacg gtggccacga aggagtccga ggggttccga cgcttcaagc 60gctcagccca gttcttcaac tacaagatcc a 9110135DNAhorse 10ggcgctgggc ctgggggagg actgggacgg ggacaaggag acgtcagcgg gcggcgggct 60gaaggttcgg ctgctgaaga aagctctgga gaagcatgca gacaaagaga acctggtcat 120tctcttcaca gacag 13511164DNAhorse 11ctatgatgtg gtatttgcct cggggccccg agagctcctg aagaagttcc ggcaggccag 60gagccaggtg gtcttctcgg ccgaggagct catctacccc gaccgcaggc tggaggccaa 120gtacccggtg gtgtccgatg gcaagaggtt cctgggctct ggag 16412113DNAhorse 12gcttcatcgg ttatgccccc aacctcagca aactggtggc tgagtgggag ggccaggaca 60gcgacagtga ccagctgttt tataccaaga tcttcttgga cccagagaag agg 1131364DNAhorse 13gagcggatca acatcaccct ggaccaccgc tgccgtatct tccagaacct ggttggagcc 60ttag 641498DNAhorse 14atgaggtcgt gctcaagttt gaaatgggcc atgtgagggc gcggaacctg gcctacgaca 60ccctccccgt cctgattcat ggcaacgggc ccaccaag 9815102DNAhorse 15ctgcagctga actacctggg caactatatc cctcgcttct ggaccttcga gacgggctgc 60acggtgtgtg acgagggcct gcgcagcctc aagggcattg gg 10216132DNAhorse 16gatgaagctc tgcctgtggt cttggtcggc gtgttcatcg agcagcccac gccgttcctg 60tccctgttct tccagcggct tctgcgcctg cattaccccc ggaaacagct gcggcttttt 120attcataacc at 13217122DNAhorse 17gagcagcacc acaaggctca ggtggagcag ttcctggcag agcatggcgg cgagtacaag 60tctgtgaaac tggtgggccc cgaggtgcgg gtggcaaacg ccgatgccag gaacatgggc 120gc 12218105DNAhorse 18ggacctgtgc cggcaggacc gtggctgcac ctactacttc agtgtggatg ccgacgtggc 60cctgaccgag cccaagaccc tgcgactgct gattgagcag aacaa 10519126DNAhorse 19gaatgtcatc gccccgttga tgacccgcca cgggaggctg tggtcgaact tctggggggc 60aatgagtgca gatggctact acgcccgctc cgaggactac gtggacattg tgcaggggcg 120gcgtgt 12620142DNAhorse 20tggcgtctgg aacgtgccct acatctcgaa catttacctg atcaagggca gtgccctgcg 60ggctgagctg cagcagacag atctgttcca ccacagcaag ctggatgccg acatggcctt 120ctgtgccaat atccggcagc ag 14221117DNAhorse 21gatgtgttca tgttcctgac caaccggcac accttcggcc acctgctctc cctggacagc 60taccagacca cccacctcca caacgacctc tgggaggtgt ttagcaaccc cgaggac 1172263DNAhorse 22tggaaggaga agtacatcca tgagaactac accagggccc tggcggggaa gctggtggag 60atg 6323105DNAhorse 23ccttgcccgg atgtctactg gttccccatc ttcacggaga cggcctgtga cgagctggtg 60gaggagatgg agcactacgg ccagtggtct ctgggagaca ataag 10524147DNAhorse 24gacaaccgca tccagggtgg ctacgaaaat gtgccgacca tcgacatcca catgaaccag 60atcagctttg agcgggagtg gcacaagttc ctggtggagt acatcgcccc catgacagag 120aagctgtatc caggctacta caccagg 14725126DNAhorse 25gcccagttcg acctggcctt tgttgtccgc tacaagcctg acgagcagcc ctcgctgatg 60ccccaccacg atgcctccac cttcactgtc aacatcgccc tgaaccgggt cggggtggat 120tacgag 12626350DNAhorse 26atagctgtca ctccacaagg cacaagggct gcttggctgg ggtggctcag atgggagaat 60ggggggtact aggggaaggg cccagcttcc tcttggggaa actgacgctt cctgttggga 120aactgacact tcctgtctcc cagggcgggg gctgtcggtt cctgcgttac aactgctcca 180tccgagcccc acggaagggc tggaccctca tgcaccccgg acgactcacg cactaccacg 240agggactccc caccaccaag ggcacccgct acatcgcggt ctccttcgtc gatccctaat 300cggccaagcc tggccacttc ggaccttttc ctcgttgcca acaaccactg 3502724DNAhorse 27atagctgtca ctccacaagg caca 242825DNAhorse 28cagtggttgt tggcaacgag gaaaa 25

Claims

1. A method for determining whether a horse is heterozygous, homozygous or has an absence of a Warmblood Fragile Foal Syndrome Type 1 (WFFST1) mutation, wherein the WFFST1 mutation comprises a change from G to A at position 147 in the sequence of SEQ ID NO:26, the method comprising: obtaining a biological sample from the horse and determining from the biological sample the presence or absence of the WFFST1 mutation, and identifying the horse as WFFST1 mutation homozygous by determining homozygosity for the WFFST1 mutation; or identifying the horse as WFFST1 mutation heterozygous by determining heterozygosity for the WFFST1 mutation; or identifying the horse as genetically normal with respect to WFFST1 by determining a homozygous absence of the WFFST1 mutation.

2. The method of claim 1, wherein the determining the presence or absence of the WFFST1 mutation comprises isolating DNA or RNA from the biological sample and testing the isolated DNA and/or RNA for the presence or absence of the WFFST1 mutation.

3. The method of claim 1, wherein the determining the presence or absence of the WFFST1 mutation comprises processing the biological sample to isolate DNA which comprises or might comprise the WFFST1 mutation, subsequently amplifying a segment of the isolated DNA which comprises or might comprise the WFFST1 mutation to obtain a DNA amplification product, and testing the DNA amplification product to determine the presence or absence of the WFFST1 mutation.

4. The method of claim 3, wherein the determining the presence or absence of the WFFST1 mutation comprises separating RNA from the biological sample, creating a cDNA from the separated RNA, and testing the cDNA to determine the presence or absence of the WFFST1 mutation.

5. The method of claim 1, wherein the determining the presence or absence of the WFFST1 mutation comprises isolating protein from the biological sample and testing the isolated protein for the presence of a protein comprising the sequence of SEQ ID NO:5, wherein the presence of a protein comprising the sequence of SEQ ID NO:5 establishes the presence of the WFFST1 mutation.

6. A method for determining whether a genome of a horse comprises a Warmblood Fragile Foal Syndrome Type 1 (WFFST1) mutation, wherein the WFFST1 mutation comprises a change from G to A at position 4 in the sequence of SEQ ID NO:6, the method comprising: obtaining a biological sample from the horse and testing the biological sample to determine the presence or absence of the WFFST1 mutation.

7. The method of claim 6, wherein the determining the presence of the mutation comprises determining the presence of SEQ ID NO:7.

8. The method of claim 6, wherein the determining the absence of the mutation comprises determining the presence of SEQ ID NO:6.

9. The method of claim 6, comprising determining the absence of the WFFST1 mutation and identifying the horse from which the biological sample was obtained as not affected with WFFST1.

10. The method of claim 6, comprising determining the presence of the WFFST1 mutation and identifying the horse from which the biological sample was obtained as not genetically normal with respect to WFFST1.

11. The method of claim 6, comprising determining homozygosity for the WFFST1 mutation and identifying the horse from which the biological sample was obtained as affected with or predisposed to WFFST1.

12. The method of claim 6, comprising determining heterozygosity for the WFFST1 mutation and identifying the horse from which the biological sample was obtained as a carrier of the WFFST1 mutation.

13. The method of claim 6, wherein the determining the presence of the WFFST1 mutation comprises detecting from the biological sample a polypeptide comprising the sequence of SEQ ID NO:5.

14. A composition comprising an isolated nucleic acid, wherein the isolated nucleic acid comprises the sequence of SEQ ID NO:1.

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