WO2018218049A1 - Methods of identifying equine immune-mediated myositis - Google Patents

Abstract

This invention provides reaction mixtures, kits and methods for the diagnosis and treatment of equine immune-mediated myositis.

Description

METHODS OF IDENTIFYING EQUINE

IMMUNE-MEDIATED MYOSITIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

62/510,877, filed on May 25, 2017, which is hereby incorporated herein by reference in its entirety.

BACKGROUND

[0002] Immune mediated myositidies (IMMs) are an important cause of morbidity and, in some cases, mortality in several species including humans (1), dogs (2), and horses (3, 4). Common clinical features include malaise, muscle atrophy and weakness with a histopathologic hallmark of inflammatory infiltrates, particularly lymphocytes, surrounding blood vessels and within myocytes (5, 6). There are several different IMM subtypes including inclusion body myositis in humans (7), polymyositis and dermatomyositis in dogs and humans (5) and canine masticatory myositis (8). Causes of autoimmune diseases such as IMM are not well understood, but an environmental stimulus combined with genetic predilection appears to be important initiating factors (9, 10). The precise environmental trigger for equine EVIM is not clear, but 39% of horses with IMM are reported to have a recent history of infection, particularly with Streptococci, or vaccination with influenza, herpes virus- 1 or Streptococcus equi subspecies equi three to four weeks prior to onset (3, 4)

SUMMARY

[0003] In one aspect, provided are reaction mixtures. In some embodiments, the reaction mixtures comprise (i) a biological sample from an equine comprising a nucleic acid template, and (ii) one or more polynucleotides or oligonucleotide pairs for differentiating a single nucleotide polymorphism (SNP) indicative of equine immune-mediated myositis (PMM), wherein the SNP is at position 1126 of SEQ ID NO:2, wherein the one or more polynucleotides or oligonucleotide pairs differentiate AA, AG and GG genotypes at position 1126 of SEQ ID NO:2; or (ii) one or more polynucleotides or oligonucleotide pairs for differentiating a single nucleotide polymorphism at position 52,993,878 of equine chromosome 11 (NC 009154.2; EquCab 2.0), wherein the one or more polynucleotides or oligonucleotide pairs differentiate AA, AG and GG genotypes at position 52,993,878 of equine chromosome 11. In some embodiments, the one or more oligonucleotides comprises forward primer: CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4), GGATCATCTGAGGGGGAAAT (SEQ ID NO:5),

TGGAACCTTGTGGGAACATT (SEQ ID NO: 6) and ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7). In some embodiments, the nucleic acid template comprises genomic DNA. In some embodiments, the nucleic acid template comprises mRNA. In some embodiments, the reaction mixture further comprises a polymerase and dNTPs. In some embodiments, the reaction mixture further comprises a reverse-transcriptase. In some embodiments, the reaction mixture further comprises a positive and/or negative control nucleic acid template.

[0004] In a further aspect, provided are solid supports attached to one or more oligonucleotides that specifically differentiate a single nucleotide polymorphism (SNP) indicative of equine immune-mediated myositis (EVIM). In some embodiments, the SNP is at position 1126 of SEQ ID NO:2, wherein the one or more oligonucleotides differentiate AA, AG and GG genotypes at position 1126 of SEQ ID NO:2. In some embodiments, the (SNP) is at position 52,993,878 of equine chromosome 11 (NC_009154.2; EquCab 2.0), wherein the one or more oligonucleotides differentiate AA, AG and GG genotypes at position 52,993,878 of equine chromosome 11. In some embodiments, the solid support is attached to an oligonucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:8. In certain embodiments, the solid support is a microarray, e.g., a genotyping array. In some embodiments, the solid support is attached to 10,000 or fewer, e.g., 5000 or fewer, 1000 or fewer, oligonucleotides.

[0005] In a further aspect, provided are methods for identifying an equine suffering from, having a predisposition to develop, or at risk of suffering from immune-mediated myositis (IMM). In some embodiments, the methods comprise: a) obtaining a biological sample comprising a nucleic acid template from the equine, determining the genotype of myosin heavy chain 1 (MYH1) at position 1126 of SEQ ID NO:2; and b) selecting an equine with a GG or AG genotype at position 1126 of SEQ ID NO:2, wherein a GG or AG genotype at position 1126 of SEQ ID NO:2 identifies an equine suffering from or at risk of suffering from immune-mediated myositis (EVIM) relative to an AA genotype. In some embodiments, the methods comprise: a) obtaining a biological sample comprising a nucleic acid template from the equine, determining the genotype of myosin heavy chain 1 (MYHl) at position 52,993,878 of equine chromosome 11 (NC_009154.2; EquCab 2.0); and b) selecting an equine with a GG or AG genotype at position 52,993,878 of equine

chromosome 11, wherein a GG or AG genotype at position 52,993,878 of equine chromosome 11 identifies an equine suffering from or at risk of suffering from immune- mediated myositis (IMM) relative to an AA genotype. In some embodiments, the methods comprise: a) obtaining a biological sample comprising a nucleic acid template from the equine, determining the genotype of myosin heavy chain 1 (MYHl) at position 52,993,878 of equine chromosome 11 (NC 009154.2; EquCab 2.0); and b) selecting an equine with an AA genotype at position 52,993,878 of equine chromosome 11, wherein an AA genotype at position 52,993,878 of equine chromosome 11 identifies an equine having reduced risk of suffering from immune-mediated myositis (EVIM) relative to an AG or GG genotype. [0006] In another aspect, provided are methods of treating an equine for immune- mediated myositis (IMM). In some embodiments, the methods comprise the steps of:

a) identifying an equine exhibiting signs of skeletal muscle damage; b) determining or detecting in a biological sample comprising a nucleic acid template from the equine a GG or AG genotype at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11; and c) administering a therapeutically effective amount of an

immunosuppressant agent to the equine. In some embodiments, the IMM genotype is detected by an amplification reaction using polynucleotides that distinguish between alleles. In some embodiments, the amplification reaction is selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), T7 polymerase mediated amplification, T3 polymerase mediated amplification and SP6 polymerase mediated amplification. In some embodiments, amplifying the nucleic acid sequence comprises using reverse transcription and amplification of the mRNA molecule. In some embodiments, the A to G substitution is detected by: a) specifically amplifying a nucleic acid sequence comprising position 1126 of SEQ ID NO:2 or position 52,993,878 of equine chromosome 11, thereby amplifying nucleic acids comprising the single nucleotide polymorphism (S P) indicative of EVIM; and b) detecting the amplified nucleic acids, thereby detecting the SNP indicative of EVIM. In some embodiments, the nucleic acid sequence is specifically amplified using forward primer: CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4), GGATCATCTGAGGGGGAAAT (SEQ ID NO:5), TGGAACCTTGTGGGAACATT (SEQ ID NO:6) and

ATGTGGAACCTTGTGGGAAC (SEQ ID NO: 7). In some embodiments, the FMM genotype is detected by hybridization using polynucleotides which distinguish between alleles. In some embodiments, the IMM genotype is detected by sequencing. In some embodiments, the equine is a domesticated equine. In some embodiments, the equine is a Quarter Horse, an equine of Quarter Horse lineage (e.g., a Quarter Pony, a Quarab), an equine breed selected from Paint Horse, Appaloosa, Akhal-Teke, Arabian, Belgian, Clydesdale, Franches-Montagnes, Friesian dwarf, German Warmblood, Hanoverian, Icelandic, Lusitano, Shetland pony, Standardbreds, Swedish Warmbood, Yukatian, or a mixture thereof. In some embodiments, the equine is of Arabian descent. In some embodiments, identifying or detecting an AG or GG genotype confirms the IMM genotype and excludes or indicates the requirement for further testing to determine the presence of malignant hyperthermia, glycogen branching enzyme deficiency and a dominant gain of function mutation in glycogen synthase 1 as causes of skeletal muscle damage

(rhabdomyolysis). In some embodiments, the equine is additionally negative for one or more of Polysaccharide Storage Myopathy (PSSM1), Hyperkalemic Periodic Paralysis Disease (HYPP), Glycogen Branching Enzyme Deficiency (GBED), Malignant

Hyperthermia (MH), Hereditary Equine Regional Dermal Asthenia (HERD A), and Overo Lethal White Syndrome (OLWS).

[0007] In another aspect, provided are kits. In some embodiments, the kits comprising one or more polynucleotides or oligonucleotide pairs for differentiating a single nucleotide polymorphism (SNP) indicative of equine immune-mediated myositis (FMM), wherein the SNP is at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1, wherein the one or more polynucleotides or oligonucleotide pairs differentiate AA, AG and GG genotypes at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1. In some embodiments, the kits comprises forward primer: CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4), GGATCATCTGAGGGGGAAAT (SEQ ID NO: 5), TGGAACCTTGTGGGAACATT (SEQ ID NO:6) and ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7). Further provided are kits comprising the solid supports, as described above and herein. Further provided are kits comprising one or more antibodies, or antibody fragments, capable of distinguishing mutated MYH1 protein from the wild-type MYH1 protein, wherein the mutated MYH1 protein has a glycine residue at the position corresponding to position 320 of SEQ ID NO: l and the wild-type MYH1 protein has a glutamic acid residue at the position corresponding to position 320 of SEQ ID NO: 1.

DEFINITIONS

[0008] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Green and Sambrook et al.

MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel, ed., Current Protocols in Molecular Biology, 1990-2017, John Wiley Interscience), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well-known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

[0009] The terms "Quarter Horse" and "American Quarter Horse" refer to a breed of horse. Breed characteristics include small, short, refined head with a straight profile, and a strong, well-muscled body, featuring a broad chest and powerful, rounded hindquarters. They usually stand between 14 and 16 hands (56 and 64 inches, 142 and 163 cm) high, although some Halter-type and English hunter-type horses may grow as tall as 17 hands (68 inches, 173 cm). There are two main body types: the stock type and the hunter or racing type. The stock horse type is shorter, more compact, stocky and well-muscled, yet agile. The racing and hunter type Quarter Horses are somewhat taller and smoother muscled than the stock type. Horses shown in-hand in Halter competition are larger yet, with a very heavily muscled appearance, while retaining small heads with wide jowls and refined muzzles. Quarter Horses come in nearly all colors. The primary breed registry for

American Quarter Horses is with the American Quarter Horse Associate (aqha.com).

[0010] The terms "immune-mediated myositis" or "IMM" to interchangeably refer to a cause of rhabdomyolysis, stiffness, and muscle atrophy predominantly affecting Quarter Horses and equines of Quarter Horse bloodlines. Clinical signs included rapid atrophy, particularly of the epaxial and gluteal muscles, depression, stiffness and fever. Equines with IMM exhibit elevated creatine kinase (CK) activity and aspartate transaminase (AST) activity, as detected on a serum biochemistry panel. Deficiencies of dysferlin, dystrophin, and a-sarcoglycan are not associated with IMM. Sarcolemmal MHC I and II expression in a proportion of myofibers of EVIM horses in conjunction with lymphocytic infiltration supports an immune-mediated etiology for IMM. See, e.g., Lewis, et al, J Vet Intern Med (2007) 21 : 495-503 ; Durward-Akhurst, et al, J Vet Intern Med (2016) 30: 1313-1321 ; and Hunyadi, et al, J Vet Intern Med (2017) 31 : 170-175.

[0011] "Skeletal muscle myosin heavy chain 1" or "MYHl" interchangeably refer to nucleic acids and polypeptide polymorphic variants (including single nucleotide polymorphisms involving displacement, insertion, or deletion of a single nucleotide that may or may not lead to a change in an encoded polypeptide sequence), alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) or 99%) or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, or over the full-length, to an amino acid sequence encoded by a MYHl nucleic acid (see, e.g., GenBank Accession Nos.

NM_001081759.1→ NP_001075228.1 (Equus caballus)), and conservatively modified variants thereof, (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a MYHl protein, and conservatively modified variants thereof, (4) have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, or over the full-length, to a MYHl nucleic acid. MYHl nucleic acids include polynucleotides comprising the SNPs described herein.

[0012] Positions within a MYHl coding nucleic acid sequence can be counted, for example, with reference to the coding nucleic acid sequence represented by SEQ ID NO:2 (NCBI RefSeq No: XM 005597036.3). Positions within a MYHl amino acid sequence can be counted, for example, with reference to the equine MYH1 protein sequence represented by SEQ ID NO: 1 (NCBI RefSeq No: XP 005597093.1). A MYH1 polynucleotide or polypeptide sequence is typically from a domesticated equine. MYH1 nucleic acids and proteins include both naturally occurring and recombinant molecules. The equine MYH1 genomic nucleic acid sequence is published as ENSEMBL accession number

ENSECAG00000022909 (which correlates to Chromosome 1 1 : 52,973,970-52,998,897 reverse strand; EquCab2:CM000387.2; transcript, ENSECAT00000026554.1). As used herein, a "MYH1 gene" will have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 500, 1000, 2000, 3000, 6000, 10,000, 20,000, 24,000 or more nucleotides, or over the full-length, to ENSECAG00000022909 or the complement thereof.

[0013] The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0014] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0015] Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0016] A "single nucleotide polymorphism" or "SNP" refers to polynucleotide that differs from another polynucleotide by a single nucleotide exchange. For example, without limitation, exchanging one A for one C, G or T in the entire sequence of polynucleotide constitutes a SNP. Of course, it is possible to have more than one SNP in a particular polynucleotide. For example, at one locus in a polynucleotide, a C may be exchanged for a T, at another locus a G may be exchanged for an A and so on. When referring to SNPs, the polynucleotide is most often DNA and the SNP is one that usually results in a change in the genotype that is associated with a corresponding change in phenotype of the organism in which the SNP occurs.

[0017] A "variant" is a difference in the nucleotide sequence among related polynucleotides. The difference may be the deletion of one or more nucleotides from the sequence of one polynucleotide compared to the sequence of a related polynucleotide, the addition of one or more nucleotides or the substitution of one nucleotide for another. The terms "mutation," "polymorphism" and "variant" are used interchangeably herein to describe such variants. As used herein, the term "variant" in the singular is to be construed to include multiple variances; i.e., two or more nucleotide additions, deletions and/or substitutions in the same polynucleotide. A "point mutation" refers to a single substitution of one nucleotide for another.

[0018] A nucleic acid "that distinguishes" as used herein refers to a

polynucleotide(s) that (1) specifically hybridizes under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a MYHl protein, and conservatively modified variants thereof, or (2) has a nucleic acid sequence that has greater than about 80%, 85%, 90%, 95%, preferably greater than about 96%, 97%, 98%, 99%), or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a MYHl nucleic acid (e.g., a sequence as set forth in SEQ ID NO:2, or complements or a subsequences thereof. A nucleic acid that distinguishes a first MYHl polymorphism from a second MYHl polymorphism at the same position in the MYHl sequence will allow for polynucleotide extension and amplification after annealing to a MYHl polynucleotide comprising the first polymorphism, but will not allow for will not allow for polynucleotide extension or amplification after annealing to a MYHl polynucleotide comprising the second polymorphism. In other embodiments, a nucleic acid that distinguishes a first MYH1 polymorphism from a second MYH1 polymorphism at the same position in the MYH1 sequence will hybridize to a MYH1 polynucleotide comprising the first polymorphism but will not hybridize to a MYH1 polynucleotide comprising the second polymorphism. [0019] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tij ssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point I for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5 x SSC, and 1% SDS, incubating at 42° C, or, 5xSSC, 1% SDS, incubating at 65° C, with wash in

0.2xSSC, and 0.1% SDS at 65° C.

[0020] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in 1 X SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

[0021] The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).

[0022] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated MYHl nucleic acid is separated from open reading frames that flank the MYHl gene and encode proteins other than MYHl . The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0023] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0024] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, . alpha. -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0025] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB

Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0026] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0027] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles.

[0028] The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine I, Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

[0029] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., share at least about 80% identity, for example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region to a reference sequence, e.g., SEQ ID NOs: l-8), when compared and aligned for maximum

correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50-100 amino acids or nucleotides in length, or over the full-length of a reference sequence.

[0030] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins to MYHl nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.

[0031] An indication that two nucleic acid sequences or polypeptides are

substantially identical is that the polypeptide encoded by the first nucleic acid is

immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically

substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0032] "Array" as used herein refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, U.S. Patent Nos. 5, 143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al, Science, 251 :767-777 (1991). These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase synthesis methods. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Patent No. 5,384,261. Arrays may comprise a planar surface or may be nucleic acids or peptides on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate as described in, e.g., U.S. Patent No. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device, as described in, e.g., U.S. Patent Nos. 5,856, 174 and 5,922,591.

[0033] As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of

immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad

immunoglobulin variable region genes. Light chains are typically classified as either kappa or lambda. Heavy chains are typically classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. [0034] A typical full-length (intact) immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.

[0035] Antibodies exist as intact immunoglobulins or as a number of well- characterized fragments that can be produced, inter alia, by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2, a dimer of Fab which itself is a light chain joined to V_H- C_HI by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')₂ dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region {see,

Fundamental Immunology, W E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes whole antibodies, antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. In certain embodiments antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), for example, single chain Fv antibodies (scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. In certain embodiments the single chain Fv antibody is a covalently linked V_H- V_L heterodimer that may be expressed from a nucleic acid including V_H and V_L encoding sequences either joined directly or joined by a peptide-encoding linker {see, e.g., Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883). While the V_H and V_L are connected to each as a single polypeptide chain, the V_H and V_L domains associate non- covalently. The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv's (scFv), however, alternative expression strategies have also been successful. For example Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused, for example, to g3 capsid protein and the

complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons. The important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p {see, e.g., U.S. Patent No: 5,733,743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778).

Accordingly, in certain embodiments, anti-multimeric MYHl antibodies include, but are not limited to all that have been displayed on phage or yeast (e.g., scFv, Fv, Fab and disulfide linked Fv (see, e.g., Reiter et al. (1995) Protein Eng. 8: 1323-1331)), and in addition to monospecific antibodies, also include bispecific, trispecific, quadraspecific, and generally polyspecific antibodies (e.g., bs scFv).

[0036] An "antibody fragment" refers to a portion of an intact antibody. In certain embodiments the antibody fragment comprises or consists of the antigen binding and/or the variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab ₎₂ and Fv fragments, diabodies, linear antibodies (see, e.g., U.S. Patent 5,641,870; Zapata et al. (19950 Protein Eng. 8(10): 1057-1062), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0037] The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., multimeric MYHl protein and/or endogenous MYHl or fragment(s) thereof). It has been shown that the antigen-binding function of an antibody can be performed by various fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, CL and CHI domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked, e.g., by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and CHI domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb including V_H and V_L domains; (vi) a dAb fragment (Ward et al. (1989) Nature 341, 544-546), which consists of a V_H domain; (vii) a dAb which consists of a V_H or a V_L domain; (viii) an isolated complementarity determining region (CDR) or (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker; and the like. In certain embodiments although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules known as single chain Fv (scFv) {see, e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. £7X4 85: 5879- 5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by methods such as recombinant DNA techniques, or enzymatic or chemical cleavage of intact immunoglobulins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 illustrates that within the cohort of genome-wide association horses affected with IMM, 23/36 had available pedigree information. All affected horses (red) could be traced back to a common sire within 8 generations. Pedigree analysis supported either an autosomal dominant or autosomal recessive mode of inheritance. Circles=females, squares=males. [0039] Figure 2 illustrates a Manhattan plot and (insert) QQ-plot demonstrating a genome-wide significant association with the IMM phenotype on chrl 1. Minimal genomic inflation was present.

[0040] Figure 3 illustrates genotypes for the MYHl E321G variant across the four cohorts examined; GWA cohort (35 IMM-affected and 53 at risk), Follow-up cohort (36 IMM-affected and 22 at risk), Random QH cohort (n=28) and other breeds (n=175).

[0041] Figures 4A-D illustrate pedigrees from 4 subsets of the larger pedigree

(Figure 1) demonstrating a founder effect of 4 stallions for the FMM phenotype.

Circles=females, squares=males. Open circles=phenotype unknown. Colored

circles=FMM-affected with red=G/G genotype, blue=G/A genotype and gray=A/A phenotype. Two of these founder stallions were traced back to the stallion in Family A within 1-3 generations. For Family D, the dam line traced back to the stallion in Family A within 4 generations (Figure 1).

[0042] Figures 5 A-B illustrate schematic representation of MYHl with wild type

(E317E, wt) and MYHl mutation (E317G, mut). Panel A. Wild type (wt) variant is shown as light green (red arrow) and E317G mutation (mut) is shown on the adjacent panel as teal (red arrow). In both panel helices are represented as blue and additionally loop, SWITCH regions and actin binding sites are labelled. Panel B. Detailed alignments of the query protein (upper sequence line) and structure hits showing the protein sequence (PDB

SEQRES record) and the resolved coordinates (PDB ATOM record). The input variant is indicated by green arrows (below and above the alignment) and highlighted amino acids represent residues that appear in dbSNP as well as common associated somatic mutation in cancer (COSMIC) variants.

[0043] Figures 6A-D illustrate a side chain and stability analysis of MYHl E317G mutation. Panel A. Contact surface areas between atoms and solvent accessible surfaces. Table includes contact surface area (A2) and minimal atomic distance (A) between two atoms of the two residues for the 15 amino acid residues that were determined to be impacted by the mutation. Panel B. A pictorial presentation of contact/residues affected by the FH7 E317G mutation and its association to SWITCH1, helix I and helix J regions of MYHl. The residues affected are represented with an asterisk (*). Helix K is partially represented. The input variant is indicated by green arrows (below and above the alignment) and highlighted amino acids represent residues that appear in dbSNP as well as common associated somatic mutation in cancer (COSMIC) variants. Panel C. G23D model displaying a close up of combined MYHl mutation (teal only) and wild type (green). Blue represent the helices and shows the proximity of t e MYHl mutation to the SWITCH 1 region and to the G677 and P710 residues found between Loop2 and SH2 helix domain of MYHl. Panel D. Stability analysis of the E317G variant using I-Mutant-2 at physiological pH, directly accessed from G23D, which predicts decreased stability of the mutant protein. Maximal RI score is 10.

[0044] Figures 7A-K. A. Marked CD4+ lymphocytic infiltrates (red) within myofibers of horse 1, an E321G MYH1 homozygote. IHC x 20. B. Serial section to c demonstrating inflammatory cell infiltrates (arrows) in horse 1. HE x 20. C. Serial section to b demonstrating that fibers infiltrated by lymphocytes (vertical arrows) are 2X fibers (red) and that some fibers with cellular infiltrates (horizontal arrows) do not have any remaining myosin staining. IF x 20. D. Serial section to e from horse 2 showing inflammatory cells (arrows). HE 10. E. Serial section to D demonstrating a complete absence of 2X fibers. Dark regions with no staining (arrows) correspond to regions of inflammation in D. IF x 10. F. Normal fiber type distribution and fiber sizes in another homozygous wild-type horse for comparison. IF x 20. G. Section of muscle from horse 3 that had a 4-month history of IMM showing a small amount of inflammatory cell infiltrates. HE x 10. H. Another section of horse 3 demonstrating atrophic type 2X fibers (brown). IF x 10. I. Preponderance of type 2A (yellow) or 2AX fibers (intermediate) over type 2X fibers (brown) in another region of the sample from horse 3. IF x 10. J. Normal fiber type distribution of muscle from a wild-type horse (type 1 blue, type 2A yellow, type 2X brown) showing type 2X fibers that are larger than type 2A fibers. IF x 10. K. Normal fiber type distribution and fiber sizes in the semimembranosus muscle that lacks inflammation from horse 4 that was homozygous for the E321G variant. IF x 20. Bars in all figures represents 100 μιη.

[0045] Figures 8 A-B illustrate a protein alignment of MYH1 gene and Streptococcus equi (AHI46575.1) showing similarities in regions of the S. equi alignment and the MYH1 gene. Sequences were aligned using CLUSTALX (version 2).

DETAILED DESCRIPTION

1. Introduction

[0046] Quarter Horses (QH) develop recurrent, rapid-onset muscle atrophy as a result of immune-mediated myositis (IMM) of unknown origin. The histopathologic hallmark of IMM is lymphocytic infiltration of myofibers. Immune-mediated myositis (FMM) results in a sudden onset of acute severe wasting of topline muscles in Quarter Horses and the disease has a very strong familial relationship. IMM is found particularly in cutting and reining horses as well as pleasure horses but has also been identified in other performance subtypes. Following exposure to respiratory infection, vaccination or tying up, affected horses rapidly lose 30% of their muscle mass. When muscle biopsies of topline muscles are taken during the first few weeks of atrophy, specific immune cells are observed destroying a subset of muscle fibers. After weeks of atrophy, muscle samples do not have inflammation and merely show a nonspecific decrease in muscle fiber sizes. Horses will regain muscle mass in 2-4 months, however, repeated episodes of atrophy can be so severe that some horses are eventually euthanized. Until now, the cause of FMM was unknown and diagnosis only effectively made through muscle biopsy of acutely affected horses.

[0047] Genetic associations of IMM have been found with various major histocompatibility complex loci in humans and dogs (9, 11). Because FMM affects predominantly one breed, Quarter Horse (QH), and since certain stallions appear to be overrepresented in the genetic lineage of QHs with IMM, we investigated whether there is an underlying genetic variant that causes susceptibility to IMM (3, 12). The first objective of this study was to identify variants underlying risk for developing equine IMM by performing a genome wide association (GWA) study of QHs and related breeds with and without IMM that were housed in the same environment and therefore exposed to the same risk factors that may result in the EVIM phenotype. The second objective was to evaluate the region of association from the GWA using whole genome sequencing to identify a putative functional variant associated with the equine IMM phenotype. The third objective evolved following identification of a putative functional variant and was designed to determine if the protein encoded by the candidate gene altered protein structure and was targeted by inflammation.

[0048] The present compositions and methods are based, in part, on the discovery of a functional variant associated with equine EVIM. A genome wide association study (GWA) was performed on 36 IMM QHs and 54 age/breed matched unaffected QHs from the same environment using the Equine S P50 and S P70 genotyping arrays. A mixed model analysis identified nine S Ps within a -2.87 Mb region on ECA11 that were significantly (Punadjusted < 1.4 x 10^"6) associated with the IMM phenotype. Associated haplotypes within this region encompassed 36 annotated genes, including four myosin genes {MYH2, MYH3, MYH8 and MYH13). Whole genome sequencing of four IMM and four unaffected QHs identified a single segregating nonsynonymous E321G mutation mMYHl encoding myosin heavy chain 2X. Genotyping of additional 35 IMM and 22 unaffected QHs confirmed an association (P=2.9 x 10^"5) and the mutation was not found in 175 horses from 21 non-QH breeds. Type 2X myofibers of IMM horses contained lymphocytic infiltrates and were present in lesser numbers in the presence of inflammation. Protein modeling and contact/stability analysis identified 15 residues affected by the mutation with significantly decreased stability. We conclude that a mutation m MYHl is highly associated with the IMM phenotype in the Quarter Horse. This is the first report of a mutation mMYHl and the first link between a skeletal muscle myosin mutation and autoimmune disease.

[0049] The IMM susceptibility mutation was found in a highly conserved region of the MYHl gene, one of the most common proteins in skeletal muscle. The mutation causes a substitution of amino acids which appears to change the structure of myosin. Exposure of this mutated protein to the immune system, in the face of specific environmental stimuli, can trigger autoimmune destruction of the muscle cells. A similar phenomena occurs in humans with rheumatic fever and inflammation in the heart following Strep infections. The IMM susceptibility mutation appears to be codominant.

2. Reaction Mixtures [0050] Provided are reaction mixtures for identifying a mutation in the equine

MYH1 gene or protein correlated with immune-mediated myositis (IMM). In some embodiments, the reaction mixtures comprise (i) a biological sample from an equine comprising a nucleic acid template, and (ii) one or more polynucleotides or oligonucleotide pairs for differentiating a single nucleotide polymorphism (S P) indicative of equine immune-mediated myositis (IMM), wherein the SNP is at position 1126 of SEQ ID NO:2, wherein the one or more oligonucleotide pairs differentiate AA, AG and GG genotypes at position 1126 of SEQ ID NO:2; or (ii) one or more polynucleotides or oligonucleotide pairs for differentiating a single nucleotide polymorphism at position 52,993,878 of equine chromosome 11 (NC 009154.2; EquCab 2.0), wherein the one or more oligonucleotide pairs differentiate AA, AG and GG genotypes at position 52,993,878 of equine chromosome 11. In some embodiments, the one or more oligonucleotides comprises forward primer: CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4),

GGATC ATCTGAGGGGGAAAT (SEQ ID NO : 5), TGGAACCTTGTGGGAAC ATT (SEQ ID NO:6) and ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7).

[0051] The nucleic acid template in the biological sample can comprise genomic

DNA and/or mRNA. In some embodiments, the reaction mixtures further can comprise appropriate buffers, salts, polymerases, reverse-transcriptases, dNTPs, nuclease inhibitors, and other reagents to facilitate amplification and/or detection reactions {e.g., primers, labels) for amplifying the MYH1 gene from genomic DNA or the MYH1 coding sequence from mRNA.

[0052] Because the mutation in equine MYH1 discovered to be correlated with immune-mediated myositis results in a coding substitution of the amino acid residue at position 320 of SEQ ID NO: 1 from glutamic acid (E) to glycine (G), it is possible to distinguish the mutated MYH1 protein from the wild-type MYH1 protein by immunoassay. In such embodiments, the reaction mixtures comprise a biological sample comprising equine MYH1 protein, one or more antibodies, or fragments thereof, capable of

distinguishing mutated MYH1 protein from the wild-type MYH1 protein, wherein the mutated MYH1 protein has a glycine residue at the position corresponding to position 320 of SEQ ID NO: 1 and the wild-type MYH1 protein has a glutamic acid residue at the position corresponding to position 320 of SEQ ID NO: 1. As appropriate, such reaction mixtures can further comprise buffers, protease inhibitors and secondary antibodies, or fragments thereof. As appropriate, the primary or secondary antibodies can be labeled.

3. Solid Supports

[0053] Further provided are solid supports attached to one or more polynucleotides or oligonucleotides that specifically differentiate a single nucleotide polymorphism (S P) indicative of equine immune-mediated myositis (IMM).

[0054] In some embodiments, the SNP is at position 1126 of SEQ ID NO:2, wherein the one or more oligonucleotides differentiate AA, AG and GG genotypes at position 1126 of SEQ ID NO:2. In some embodiments, the (SNP) is at position 52,993,878 of equine chromosome 11 (NC 009154.2; EquCab 2.0), wherein the one or more oligonucleotides differentiate AA, AG and GG genotypes at position 52,993,878 of equine chromosome 11. In some embodiments, the solid support is attached to an oligonucleotide having at least about 80% sequence identity, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 8. [0055] In certain embodiments, the solid support is a microarray, e.g., a genotyping array. Microarrays suitable for genotyping are commercially available, e.g., from Illumina (illumina.com), ThermoFisher (thermofisher.com), ACGT (acgtinc.com). In some embodiments, the one or more polynucleotides or oligonucleotides that specifically differentiate a single nucleotide polymorphism (SNP) indicative of equine immune- mediated myositis (FMM) can be further or additionally attached to an equine genotyping array. In some embodiments, the solid support or genotyping array is attached to 10,000 or fewer, e.g., 5000, 4000, 3000, 2000, 1000 or fewer, oligonucleotides. Construction and use of microarrays is known in the art and described, e.g., in Bowtell and Sambrook, "DNA Microarrays: A Molecular Cloning Manual," Cold Spring Harbor Laboratory Press; 1st edition (September 15, 2002). In some embodiments, the solid support is a microbead.

[0056] In some embodiments, the solid support (e.g., microarray or microbead) is attached to one or more antibodies, or fragments thereof, capable of distinguishing mutated MYHl protein from the wild-type MYHl protein, wherein the mutated MYHl protein has a glycine residue at the position corresponding to position 320 of SEQ ID NO: l and the wild- type MYHl protein has a glutamic acid residue at the position corresponding to position 320 of SEQ ID NO: 1. 4. Methods of Diagnosis a. Obtaining a Biological Sample

[0057] The present diagnostic methods are useful for identifying whether an equine is suffering symptoms of muscle damage due to immune-mediated myositis (IMM), or has increased susceptibility to suffer muscle damage due to immune-mediated myositis based on its MYHl genotype, particularly at SNP at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1 (NC_009154.2; EquCab 2.0). Equines are diploid organisms possessing pairs of homologous chromosomes. Thus, at a typical genetic locus, an equine has three possible genotypes that can result from the combining of two different alleles (e.g. A and B). The equine may be homozygous for one or another allele, or heterozygous, possessing one of each of the two possible alleles (e.g. AA, BB or AB). The present methods are based, in part, on the discovery that a homozygous "GG" or heterozygous "AG" genotype at the SNP at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1 (NC_009154.2; EquCab 2.0) within the MYHl gene is statistically correlated with a predisposition to develop immune mediated myositis in comparison to a homozygous "AA" genotype at the same position. The methods can involve obtaining a biological sample from an equine suspected of suffering IMM.

[0058] The biological sample suitable for testing by the methods described herein comprises a template nucleic acid, e.g., genomic DNA or mRNA. The biological sample can include body fluids including whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, semen, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas, and the like; and biological fluids such as cell extracts, cell culture supernatants; fixed tissue specimens; and fixed cell specimens. Biological samples can also be from solid tissue, including hair bulb, skin, muscle, biopsy or autopsy samples or frozen sections taken for histologic purposes. These samples are well known in the art. A biological sample is obtained from any equine to be tested for MYHl SNP(s) as described herein, including, e.g., a Quarter Horse, an equine of Quarter Horse lineage (e.g., a Quarter Pony, a Quarab), an equine breed selected from Paint Horse, Appaloosa, Akhal-Teke, Arabian, Belgian, Clydesdale, Franches- Montagnes, Friesian dwarf, German Warmblood, Hanoverian, Icelandic, Lusitano, Shetland pony, Standardbreds, Swedish Warmbood, Yukatian, or a mixture thereof. In some embodiments, the equine is a neonate, a colt or a foal. A biological sample can be suspended or dissolved in liquid materials such as buffers, extractants, solvents and the like. [0059] The biological sample may be obtained from an equine exhibiting symptoms of skeletal muscle weakness or damage. In some embodiments, the equine is asymptomatic, but is suspected of being predisposed to developing immune-mediated myositis, e.g., due to breed, parentage or lineage. In some embodiments, the biological sample is from an equine who has a parent, grandparent or sibling that is or has suffered from immune-mediated myositis. In certain embodiments, a biological sample is also obtained from an equine is not suffering from or suspected of developing immune-mediated myositis as a negative control. In certain embodiments, a biological sample is also obtained from an equine known to be suffering from immune-mediated myositis as a positive control. b. Detecting the Genotype

[0060] The MYH1 S Ps can be detected using any methods known in art, including without limitation amplification, sequencing and hybridization techniques. Detection techniques for evaluating nucleic acids for the presence of a single base change involve procedures well known in the field of molecular genetics. Methods for amplifying nucleic acids find use in carrying out the present methods. Ample guidance for performing the methods is provided in the art. Exemplary references include manuals such as PCR

Technology: PRINCIPLES AND APPLICATIONS FOR DNA AMPLIFICATION (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR PROTOCOLS: A GUIDE TO

METHODS AND APPLICATIONS (eds. Innis, et al., Academic Press, San Diego, Calif, 1990); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, 1990-2017, including supplemental updates; Green and Sambrook, Molecular Cloning, A Laboratory Manual (4th Ed, 2012).

[0061] The nucleic acid template is isolated from the biological sample and a region of the MYHl gene is amplified using an oligonucleotide pair to form nucleic acid amplification products of MYHl gene polymorphism sequences. Amplification can be by any of a number of methods known to those skilled in the art including PCR, and the methods are intended to encompass any suitable techniques of DNA amplification. A number of DNA amplification techniques are suitable for use with the present methods. Conveniently such amplification techniques include methods such as polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), rolling circle amplification, T7 polymerase mediated

amplification, T3 polymerase mediated amplification and SP6 polymerase mediated amplification. The precise method of DNA amplification is not intended to be limiting, and other methods not listed here will be apparent to those skilled in the art and their use is within the scope of the invention.

[0062] In some embodiments, the polymerase chain reaction (PCR) process is used

(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR involves the use of a

thermostable DNA polymerase, known sequences as primers, and heating cycles, which separate the replicating deoxyribonucleic acid (DNA), strands and exponentially amplify a gene of interest. Any type of PCR, including quantitative PCR, RT-PCR, hot start PCR, LA-PCR, multiplex PCR, touchdown PCR, finds use. In some embodiments, real-time PCR is used. [0063] The amplification products are then analyzed in order to detect the presence or absence of at least one polymorphism in the MYH1 gene that is associated with immune- mediated myositis, as discussed herein. By practicing the methods of the present methods and analyzing the amplification products it is possible to determine the genotype of individual equines with respect to the MYH1 IMM-correlated polymorphism. [0064] In some embodiments, analysis may be made by restriction fragment length polymorphism (RFLP) analysis of a PCR amplicon produced by amplification of genomic DNA with the oligonucleotide pair. In order to simplify detection of the amplification products and the restriction fragments, those of skill will appreciate that the amplified DNA will further comprise labeled moieties to permit detection of relatively small amounts of product. A variety of moieties are well known to those skilled in the art and include such labeling tags as fluorescent, bioluminescent, chemiluminescent, and radioactive or colorigenic moieties.

[0065] A variety of methods of detecting the presence and restriction digestion properties of MYH1 gene amplification products are also suitable for use with the present methods. These can include methods such as gel electrophoresis, mass spectroscopy or the like. The present methods are also adapted to the use of single stranded DNA detection techniques such as fluorescence resonance energy transfer (FRET). For FRET analysis, hybridization anchor and detection probes may be used to hybridize to the amplification products. The probes sequences are selected such that in the presence of the SNP, for example, the resulting hybridization complex is more stable than if there is a G or C residue at a particular nucleotide position. By adjusting the hybridization conditions, it is therefore possible to distinguish between animals with the SNP and those without. A variety of parameters well known to those skilled in the art can be used to affect the ability of a hybridization complex to form. These include changes in temperature, ionic concentration, or the inclusion of chemical constituents like formamide that decrease complex stability. It is further possible to distinguish animals heterozygous for the SNP versus those that are homozygous for the same. The method of FRET analysis is well known to the art, and the conditions under which the presence or absence of the SNP would be detected by FRET are readily determinable.

[0066] Suitable sequence methods of detection also include e.g., dideoxy

sequencing-based methods and Maxam and Gilbert sequence (see, e.g., Green and

Sambrook, supra). Suitable FtPLC-based analyses include, e.g., denaturing FIPLC (dHPLC) as described in e.g., Premstaller and Oefner, LC-GC Europe 1-9 (July 2002); Bennet et al., BMC Genetics 2: 17 (2001); Schrimi et al., Biotechniques 28(4):740 (2000); and Nairz et al., PNAS USA 99(16): 10575-10580 (2002); and ion-pair reversed phase HPLC-electrospray ionization mass spectrometry (ICEMS) as described in e.g., Oberacher et al.; Hum. Mutat. 21(1):86 (2003). Other methods for characterizing single base changes in MYH1 alleles include, e.g., single base extensions (see, e.g., Kobayashi et al, Mol. Cell. Probes, 9: 175- 182, 1995); single-strand conformation polymorphism analysis, as described, e.g, in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989), allele specific oligonucleotide

hybridization (ASO) (e.g., Stoneking et al., Am. J. Hum. Genet. 48:70-382, 1991; Saiki et al., Nature 324, 163-166, 1986; EP 235,726; and WO 89/11548); and sequence-specific amplification or primer extension methods as described in, for example, WO 93/22456; U.S. Pat. Nos. 5, 137,806; 5,595,890; 5,639,611; and U.S. Pat. No. 4,851,331; 5'- nuclease assays, as described in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280.

[0067] Methods for detecting single base changes well known in the art often entail one of several general protocols: hybridization using sequence-specific oligonucleotides, primer extension, sequence-specific ligation, sequencing, or electrophoretic separation techniques, e.g., singled-stranded conformational polymorphism (SSCP) and heteroduplex analysis. Exemplary assays include 5' nuclease assays, template-directed dye-terminator incorporation, molecular beacon allele-specific oligonucleotide assays, single-base extension assays, and SNP scoring by real-time pyrophosphate sequences. Analysis of amplified sequences can be performed using various technologies such as microchips, fluorescence polarization assays, and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. In addition to these frequently used methodologies for analysis of nucleic acid samples to detect single base changes, any method known in the art can be used to detect the presence of the MYHl S Ps described herein.

[0068] For example FRET analysis can be used as a method of detection.

Conveniently, hybridization probes comprising an anchor and detection probe, the design of which art is well known to those skilled in the art of FRET analysis, are labeled with a detectable moiety, and then under suitable conditions are hybridized a MYHl amplification product containing the site of interest in order to form a hybridization complex. A variety of parameters well known to those skilled in the art can be used to affect the ability of a hybridization complex to form. These include changes in temperature, ionic concentration, or the inclusion of chemical constituents like formamide that decrease complex stability. The presence or absence of the MYHl S P is then determined by the stability of the hybridization complex. The parameters affecting hybridization and FRET analysis are well known to those skilled in the art. The amplification products and hybridization probes described herein are suitable for use with FRET analysis. [0069] In one embodiment, the polymorphism or allele or SNP at position 1126 of

SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11 (NC 009154.2; EquCab 2.0) within the MYHl gene is detecting using an oligonucleotide pair comprising forward primer: CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4), GGATC ATCTGAGGGGGAAAT (SEQ ID NO : 5), TGGAACCTTGTGGGAAC ATT (SEQ ID NO:6) and ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7). c. Identifying or Selecting the Equine Based on Genotype

[0070] The methods identify individual equines based on the knowledge of the equine' s MYHl genotype. A homozygous "GG" or heterozygous "AG" genotype at the SNP at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11 (NC_009154.2; EquCab 2.0) within the MYHl gene is statistically correlated with a predisposition to develop immune mediated myositis in comparison to a homozygous "AA" genotype at the same position.

[0071] With the knowledge of the equine' s MYHl genotype, one can then identify and sort equines into groups of like phenotype(s), or otherwise use the knowledge of the genotype in order to predict which equines will have the desired phenotypes, for example, decreased susceptibility to develop FMM. Knowledge of the equine' s genotype at the SNP at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1 (NC_009154.2; EquCab 2.0) within the MYHl gene allows a breeder to encourage breeding between equines with a desired MYHl genotype (e.g., a homozygous "AA" genotype), and to discourage breeding between equines with an undesirable MYHl genotype (e.g., a homozygous "GG" or heterozygous "AG" genotype). Knowledge of the equine' s MYHl genotype can also guide a veterinarian to provide efficacious medical treatment to the equine, e.g., by administering an immunosuppressant.

[0072] Selecting or sorting can be taken to mean placing equines in physical groupings such as pens, so that equines of like genotype are kept separate from equines of a different genotype. This would be a useful practice in the case of breeding programs where it would be desirable to produce equines of particular genotypes. For example, it may be desirable to breed equines that are homozygous "AA" at the S P at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1 (NC_009154.2; EquCab 2.0) within the MYHl gene, such that breeding among these equines would only produce equines with a desired MYHl genotype. On the other hand, it may also be desirable to decrease production of equines with an undesired MYHl genotype. Separating out equines with the desired MYHl genotype(s) would prevent animals with an undesired MYHl genotype from breeding with equines possessing a desired MYHl genotype, facilitating the reproduction of equines with an increased susceptibility to develop IMM, which is associated with a homozygous "GG" or heterozygous "AG" genotype at the SNP at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1 (NC_009154.2; EquCab 2.0) within the MYHl gene. Furthermore, ensuring that at least one equine in a breeding pair possesses desired MYHl allele allows for the frequency of the desired MYHl allele to be increased in the next, and subsequent generations. [0073] Sorting may also be of a "virtual" nature, such that an equine' s genotype is recorded either in a notebook or computer database. In this case, equines could then be selected based on their known genotype without the need for physical separation. This would allow one to select for equines of desired phenotype where physical separation is not required. For example, the AQHA and many horse breed registries perform parentage verification using a set of alleles each time a horse is registered. The AQHA also requires that stallions used for breeding registered foals have genotype results on file for several diseases including Polysaccharide Storage Myopathy (PSSM1), Hyperkalemic Periodic Paralysis Disease (HYPP), Glycogen Branching Enzyme Deficiency (GBED), Malignant Hyperthermia (MH) and Hereditary Equine Regional Dermal Asthenia (HERD A). The Paint horses registry has similar requirements with the addition of Overo Lethal White Syndrome (OLWS).

5. Methods of Treatment [0074] Further provided are methods of treating an equine determined to be suffering from immune-mediated myositis. As stated above, knowledge of the equine' s MYHl genotype at the SNP at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11 (NC_009154.2; EquCab 2.0) within the MYHl gene can guide a veterinarian to provide efficacious medical treatment to the equine, e.g., by administering an immunosuppressant. In particular, the clinical symptoms of IMM, e.g., rapid atrophy, particularly of the epaxial and gluteal muscles, depression, stiffness and fever, could also be attributed to another muscle disorder that occurs in an equine in the absence of genetic and/or serum tests confirming a diagnosis of IMM. Equines are predisposed to a number of causes of muscle damage (rhabdomyolysis) including malignant hyperthermia, glycogen branching enzyme deficiency and a dominant gain of function mutation in glycogen synthase 1. Knowing that a horse was homozygous "GG" or heterozygous "AG" genotype at the SNP at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine

chromosome 11 (NC_009154.2; EquCab 2.0) within the MYHl gene would guide a veterinarian to exclude or indicate the requirement for further testing to determine the presence of other disorders as the underlying cause of the muscle damage, and would indicate immuosuppressive drugs (e.g., corticosteroids such as prednisolone,

dexamethasone, combinations thereof) as the appropriate medical treatment needed for these equines, whereas immunosuppressive drugs would not be appropriate treatment for the other aforementioned non-immune-mediated muscle damage disorders in this breed of horse.

[0075] Accordingly, further provided are methods of treating an equine for immune- mediated myositis (IMM). In some embodiments, the methods comprise the steps of: a) identifying an equine exhibiting clinical signs of skeletal muscle damage; b) determining or detecting in a biological sample comprising a nucleic acid template from the equine a GG or AG genotype at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11, as described above and herein; and c) administering a therapeutically effective amount of an immunosuppressant agent to the equine. In some embodiments, the immuosuppressive drug is selected from a corticosteroid (e.g., prednisolone),

dexamethasone, and combinations.

6. Kits

[0076] Further provided are kits. In some embodiments, the kits comprise one or more polynucleotides or oligonucleotide pairs for differentiating a single nucleotide polymorphism (SNP) indicative of equine immune-mediated myositis (IMM), wherein the SNP is at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1, wherein the one or more polynucleotides or oligonucleotide pairs differentiate AA, AG and GG genotypes at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1. In some embodiments, the kits comprises forward primer:

CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4),

GGATCATCTGAGGGGGAAAT (SEQ ID NO: 5), TGGAACCTTGTGGGAACATT (SEQ ID NO:6) and ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7). In some embodiments, the kits comprise a solid support (e.g., a microarray, microbeads) that are attached to one or more polynucleotides or oligonucleotide pairs for differentiating a single nucleotide polymorphism (SNP) indicative of equine immune-mediated myositis (FMM), wherein the SNP is at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1, wherein the one or more polynucleotides or oligonucleotide pairs differentiate AA, AG and GG genotypes at position 1 126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 1 1. In some embodiments, the kits comprise one or more antibodies, or antibody fragments, capable of distinguishing mutated MYH1 protein from the wild-type MYH1 protein, wherein the mutated MYH1 protein has a glycine residue at the position corresponding to position 320 of SEQ ID NO: 1 and the wild-type MYH1 protein has a glutamic acid residue at the position corresponding to position 320 of SEQ ID NO: 1. In addition, the kit can comprise appropriate buffers, salts and other reagents to facilitate amplification and/or detection reactions (e.g., primers, labels, secondary antibodies). EXAMPLES

[0077] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

A mutation in MYH1 is associated with susceptibility to immune-mediated myositis in

Quarter Horses

This example is published as Finno, et al. Skeletal Muscle (2018) 8:7, which is hereby incorporated herein by reference in its entirety for all purposes.

MATERIALS AND METHODS [0078] IMM Case and Control Selection. GWA cohort: IMM QHs (n=36; 21 males and 15 females) were selected based on a history of rapid onset of muscle atrophy

(particularly of the epaxial or gluteal muscles) and the presence of lymphocytes invading myofibers or cuffing blood vessels in a muscle biopsy as previously described (3). Horses with type 1 polysaccharide storage myopathy based on amylase-resistant polysaccharide in myofibers or the presence of the H309A GYS1 mutation (13) were excluded. The median age at the time of biopsy was 2 years (range 0.1-19 years) for IMM affected QHs. Due to the importance of environmental triggers, unaffected QHs (n=54; median age 2 years, range 1-27; 21 males and 33 females) were selected from two herds that had active IMM cases. Unaffected horses had no history of muscle atrophy or stiffness consistent with IMM. Horses were selected such that they were not related at least within one generation. Of the horses used for the GWA, 1/36 affected and 41/54 unaffected horses were used in a previous genetic study of neuroaxonal dystrophy (14).

[0079] Whole-genome sequencing: From the GWA cohort, four of the most severely affected FMM QH (2 females and 2 males) and four unaffected (2 females and 2 males) were selected for whole-genome sequencing.

[0080] Follow-up cohort: An additional 22 unaffected QHs (7 male, 15 female, >2 years) and 35 IMM QHs (24 male, 11 female; median age at biopsy 2.5 years [range 0.5-18 years]), phenotyped in muscle samples by mild lymphocytic infiltrates in myocytes or vascular cuffing, were genotyped for the nonsynonymous MYH1 E321G variant. The unaffected QH in this follow-up cohort were housed on the same property as IMM-affected cases and therefore at risk for developing IMM. [0081] Random QH cohort: A cohort of 28 healthy Quarter Horse (n=22) and the related breed, Paint horses,(6), that were embryo transfer recipient horses of unknown bloodlines were genotyped for the putative variant to assess the prevalence of X eMYHl E321G variant in distantly related or unrelated cohorts. There was no history of IMM in this herd.

[0082] Across breed cohort: Additional genotyping of the MYH1 E321G variant was performed in a total of 69 horses across 6 breeds (Table 1). Additionally, publically mapped whole-genome sequences from the Sequence Read Archive (SRA;

ncbi.nlm.nih.gov/sra) was available for 106 horses across 17 breeds.

Table 1

Table 1

Table 1

[0083] Pedigree analysis. From the original GWA cohort, pedigrees were available for 23/36 QH. Pedigrees were analyzed using pedigraph (15). Following genotyping of all horses for the MYH1 E321G variant, additional pedigrees were created of individual families.

[0084] Genome-wide association (GWA). DNA isolations were prepared from whole blood or muscle samples according to the protocol provided through the

ARCHIVEPURE™ DNA Blood Kit (5 Prime, Hilden, Germany). 35 FMM QHs and 13 unaffected QHs were genotyped across 74,500 SNP markers with the Equine SNP 70K BeadChip (Illumina, San Diego, CA) and 1 horse with IMM and 41 control horses were genotyped across 54,602 S Ps with the Equine S P 50K BeadChip (Illumina). Datasets were merged and only SNPs that passed quality control settings (minor allele frequency >1%, genotyping across individuals >90% and Hardy-Weinberg p>0.001) were selected. A genome-wide efficient mixed model association was performed using GEMMA software using the standardized relatedness matrix option (-gk 2) (16). Population stratification was estimated by assessing the genomic control inflation factor (λ). A Bonferroni correction for 39,589 tests (the number of useable SNPs) from the GEMMA analysis, based on a

Pgenome-wide of 0.05, was determined as 1.26 x 10^"6. [0085] Haplotype analysis. For ECA11, which demonstrated the only genome-wide significant associations on the GWA, haplotypes were reconstructed on the individual chromosome using Haploview (17). SNPs were filtered based on genotyping (>90%) and minor allele frequency (>1%). Association testing of both the single markers and haplotypes was performed using 1000 permutations. The adjusted haplotype-wide significance threshold was Ppemmted = 0.05.

[0086] Whole genome sequencing. Using Illumina' s TruSeq DNA PCR-free library preparation kit (Illumina, San Diego, CA, USA) and following manufacturer's instructions, libraries were prepared with median insert size of 300-400 bp from the four IMM QHs and four unaffected QHs. The eight libraries were barcoded and pooled across eight lanes of a 125PE flow cell on an Illumina HiSeq2500, generating an average of 10.2x coverage per horse. Following quality trimming, reads were mapped to the EquCab2.0 reference genome using the Burrows-Wheeler Aligner (BWA) version 0.7.5a (18) using default settings. After sorting the mapped reads by the coordinates of the sequence, PCR duplicates were labeled with Picard tools (http://sourceforge.net/projects/picard/). The Genome Analysis Tool Kit (GATK version v.2.7.4) was used to perform local realignment (19). Variant calls were made across all eight samples simultaneously using standard hard filtering parameters or variant quality score recalibration with Haplotype Caller according to GATK Best Practices Recommendations (20, 21). SnpEFF (22) and SnpSift (23) were used to predict the functional effects of detected variants across the genome and within ECA11 candidate region and filter by segregation using Fisher's Exact Test. Functional homozygous variants segregating in IMM with "moderate" or "high" effects, as defined by SnpEFF (22), were further evaluated using all publically available mapped whole-genome sequences in the NCBI Sequence Read Archive (ncbi.nlm.nih.gov/sra). Visual inspection of the raw reads using the Integrated Genomics Viewer (24) within the ECA11 region of association was additionally performed. Whole genome sequences were deposited in the NCBI SRA (SRP).

[0087] Genotyping. Primer pairs were designed using Primer3Plus software (25) (F and R) to amplify and subsequently genotype the nonsynonymous MYH1 E321G variant in an additional cohort of FMM-affected and unaffected QHs and a cohort of unaffected Arabian horses. Amplification of products was performed using end-point PCR and visualized with the QIAxcel Advance System (QIAGEN, Valencia, CA, USA) and the QIAxcel DNA Screening Kit (QIAGEN, Valencia, CA, USA). The 20μΙ. PCR reactions were comprised of 2U of Hot-start TAQ and 2.0μΙ. of lOx Buffer (Applied Biosystems, Foster City, CA, USA), 0.25 mM dNTPs (Thermo Fisher, Waltham, MA, USA), 0.5 μΜ of both forward and reverse primers (Invitrogen Life Technologies, Carlsbad, CA, USA) and 20 ng genomic DNA. Standard PCR conditions were performed as follows: 95°C for 10 minutes, 35 cycles of 95°C denaturation for 30 seconds, 60°C annealing for 1 minute, 72°C extension for 1 minute and a final extension at 72°C for 10 minutes. PCR products were purified using ExoS AP-IT® PCR Product Cleanup Kit (Affymetrix, San Diego, CA, USA). Sanger sequencing was performed using ABI 2500 automated sequencers. Resulting sequences were aligned to EquCab2.0 (ncbi.nlm.nih.gov/genome/145) and analyzed with SEQUENCER® software (Gene Codes Corporation, Ann Arbor, MI, USA).

[0088] Protein modeling and side chain analysis. Conformational changes caused by the identified E321G FH7 variant were modelled using online G23D tool (26) and Homo sapiens myosin gene chain A, (PDB ID: 4pa0). The amino acid in the mutated position was modeled using SCcomp (27). Contact surface areas and solvent accessible surface areas were calculated using G23D, which applies Voronoi tessellation to allocate contact surfaces between neighboring atoms (28, 29). Stability analysis of the E317G variant was performed using I-Mutant-2 (30), directly accessed from G23D.

[0089] Inflammation and muscle fiber type composition. Control horses

homozygous wild type for the MYH1 E321G variant was selected based on a history of muscle atrophy and absence of inflammation in muscle biopsies. Frozen muscle samples were selected for fiber typing from 10 IMM and 5 control horses. Serial sections were stained with hematoxylin and eosin (HE) to identify inflammatory infiltrates and

immunofluorescent labelling for fiber type. Frozen samples from semimembranosus (4 E321G FH7 homozygotes, 4 wild type) and middle gluteal (3 E321G FH7

homozygotes, 1 wild type) muscles were utilized. Type 1, 2 A, 2 AX and 2X muscle fiber types were identified by multiple fluorescent labelling according to Tulloch et al 2011. Briefly, sections were incubated with a goat polyclonal anti-collagen V IgG antibody (1350- 01 Southern Biotech) 1 : 100 for 1 hour at room temperature. Next, three separate mouse monoclonal antibodies to detect type 1, slow myosin IgG 1 : 100 (MAB1628 Millipore), type 2a IgG 1 :6 (A4.74 DSHB) and both type 2a and 2x IgG 1 : 10 (NCL-MHCf Leica

Biosystems) were conjugated to fluorescent IgGl Fab fragments using Zenon ® Mouse IgG labeling kits (Life Technologies) Alexa Fluor® 488 (A4.74), Alexa Fluor® 594 (NCL- MHCF) and Pacific Blue™ (MAB1628). The three Zenon® labelled antibodies were mixed together, added to the tissue sections and incubated at 4°C overnight. A secondary antibody for Collagen V, FITC-rabbit anti-goat IgG (61-1611, Invitrogen) 1 : 500 was applied to the cryosections and incubated for 1 hour at room temperature. Sections were subsequently mounted using VECTASHIELD mounting medium (HI 000, Vector Labs) and examined using a fluorescence microscope (Olympus) with filters designed for each of the different emitting wavelengths. Images were captured and pseudo-colored composites generated.

[0090] The total number of type 1, 2 A, 2 AX and 2X muscle fibers was determined for the entire muscle section (range 447 to 3,244 muscle fibers/sample) and fiber type compositions were determined by dividing total number of fibers of each type by the total number of muscle fibers counted. The fiber type composition of IMM samples with inflammation and wild type samples were compared by genotype and disease status using a two way ANOVA.

RESULTS

[0091] Pedigree Analysis. From the original GWA cohort, pedigrees were available for 23/36 QH. All affected horses could be traced back to a common sire within 8 generations (Figure 1). Pedigree analysis supported either an autosomal dominant or autosomal recessive mode of inheritance.

[0092] Genome-wide association study. Following quality control of the 73,706

SNPs, 39,589 SNPs remained (1,601 excluded for minor allele frequency <1%, 32,439 excluded for genotyping <90% and 77 excluded for failing hardy Weinberg equilibrium [p<0.001]). Genomic inflation (λ) was estimated at 1.98, indicative of population stratification. Due to the elevated genomic inflation, a mixed model analysis was performed utilizing GEMMA with the same filters (16). Using the GEMMA relationship matrix, genomic inflation was controlled for in the population (λ = 1.02). Nine SNPs on chromosome 11 reached genome-wide significance (Table 2 and Fig. 2).

Table 2

Genome-wide significant SNPs for IMM in the QH

[0093] Haplotype analysis. Haplotype analysis of 1109 SNPs on ECA11 that passed quality control (838 removed for genotyping <90% and 64 removed for minor allele frequency <1%) using Haploview (17) identified four highly significant haplotype blocks (Ppermuted O.001) spanning -3.1 Mb from chrl 1 :52379156-55487290. This extended region overlapped the majority of the -2.87 Mb region flanked by the 9 genome-wide significant SNPs from the GWA and was therefore prioritized for further evaluation.

[0094] Evaluation of Candidate Region. The 3.1 Mb region identified through

GWA and subsequent haplotype analysis encompassed 33 Ensembl annotated genes. Of these, only 18 genes were validated in the custom equine transcriptome, which includes RNA-seq data from skeletal muscle (31). Of these 18 genes, four myosin genes {MYHl, MYH2, MYH3 and MYHl 3) and myocardin (MYOCD) are located within the associated region. MYHl, MYH2 are expressed in adult equine skeletal muscle while MYH3 is expressed at the embryonic stage and FH73 is expressed primarily in ocular skeletal muscle (32). [0095] Whole genome sequencing. Whole genome sequencing was performed on four FMM and four unaffected QHs at ~10x coverage. Visual inspection of the raw reads using the Integrated Genomics Viewer (Robinson et al., 2011) within the GWA region did not identify any structural variants across the eight horses. Using Haplotype Caller according to GATK Best Practices Recommendations (20, 21), an average of 5, 107, 127 single nucleotide variants [SNVs] and 655,690 insertions/deletions were identified across all eight horses. Variants were filtered based on an extended region of interest from the GWA and haplotype analysis (chrl 1 :49-56 Mb) and 840 variants within this filtered region were significantly associated with the EVIM phenotype (Fisher's Exact Test, Pumdjusted <0.0001). Of these variants, 0 were classified as "high effect" and 9 as "moderate effect" by SNPEff (22) (Table 3). Of the 9 "moderate effect" variants, two (chrl 1 : 50909195 and chrl 1 :

53989868) were identified as the FMM QHs were homozygous at the reference allele and the unaffected QHs were homozygous at the alternate allele. As EquCab2.0 is a

Thoroughbred with no history of IMM, these two variants were excluded from further analysis. The 7 segregating "moderate effect" SNVs were then prioritized for further evaluation (Table 3). Based on the phenotype of FMM, the MYH1 and MYH3 missense mutations were the most likely candidate functional variants. These 7 variants were then examined in all publically available mapped whole-genome sequences (n=106 across 17 breeds) from the Sequence Read Archive (SRA; ncbi.nlm.nih.gov/sra). The MYH1 and MYH3 missense variants (E321G and Rl 130H, respectively) were the only variants not found in other breeds (Table 3).

Table 3

Segregrating Variants for IMM

* Mansour, et al., BMC Genomics. 2017;18(1): 103. The bold entries are the prioritized region based on significance of haplotype association testing. T entries are genotyped in additional cohort.

[0096] In order to further prioritize these two variants, the custom equine

transcriptome recently published by our laboratory was utilized (31). These RNA-seq datasets included skeletal muscle and embryo (both inner cellular mass and

trophoectoderm). Within all available sets, the H3 variant was non-coding (Table 3). Therefore, the MYH1 variant was prioritized for further evaluation in a larger group of phenotyped horses.

[0097] Genotyping: GWA, Follow-up and Random QH cohorts. DNA from 46/48 horses used in the GWA study was available for genotyping XheMYHl E321G variant and a significant association was identified (P=1.06 x 10^"13). Within the follow-up cohort of mildly affected IMM horses, a significant allelic association was validated (P=2.95 x 10^"5) with the MYH1 E321G variant (Table 4). In the random cohort of QH without a history of IMM, 3 heterozygotes were identified. Genotyping of 175 horses across 21 non-QH breeds did not identify any other breeds with the MYH1 E321G variant (Fig. 3).

[0098] Pedigree Evaluation based on MYH1 E321G Genotype. Available pedigrees were re-evaluated on the 23 FMM-affected horses based on XheMYHl E321G genotype and could be distinctly summarized as linking to four founder stallions (Fig 4). Two of these founder stallions were traced back to the stallion in Family A within 1-3 generations. For Family D, the dam line traced back to the stallion in Family A within 4 generations.

[0099] Protein Modeling, contact area and stability analysis . The missense mutation identified mMYHl was located in a highly conserved region of the myosin globular head in subfragment-1 between the helix loop-helix region of HJ and HK (Fig. 5 A, B). The helix loops lay between SWITCHl and SWITCH2 motifs that have been identified as RAS GTP proteins, and play a role in the binding of ATP (26). The MYH1 E321G mutation substitutes a negatively charged glutamic acid (E) for a non-polar glycine (G) that lacks side chains necessary for hydrogen bond formation. Contact area and solute accessibility analyses showed that 15 residues were directly affected by X e MYHl E321G mutation (Fig. 6A) with the largest reduction of contact between the SWITCHl and helix 1 domains oiMYHl (Fig. 6B, C). Furthermore, this single mutation of E317G is responsible for significantly decreasing the stability of the protein at physiological conditions with a RI score of 8/10 (Fig. 6D).

[0100] Inflammation and muscle fiber type composition: CD4+ and CD8+ lymphocytes and macrophages were identified within and surrounding myofibers in all horses homozygous and heterozygous for the E321G MYH1 variant (Fig. 7A). Inflammatory cells were present in type 2X fibers which contain myosin heavy chain 2X that is encoded by MYH1 (Fig. 7B, 7C). Significantly fewer type 2X fibers (mean 27%; range 0-50%) were found in E321G MYH1 homozygous and heterozygous samples with inflammation compared to controls (mean 60; range 43-87%; P < 0.0001) (Fig. 7D, 7E) Small numbers of inflammatory cells, atrophic type 2X fibers, and a predominance of type 2A and 2AX fibers over 2X fibers were identified in the semimembranosus muscle of an IMM homozygote with a 4-month long history of FMM (Fig. 7F-H). In contrast, in the complete absence of inflammation, the proportion of type 1, 2A, 2AX, and 2X fibers of control horses did not differ significantly from FMM muscle samples (Fig. 7I-K). The mean percentage of type 2X fibers in FMM E321G horse muscle without inflammatory infiltrates (4 semimembranosus, 2 gluteal) was 45%, ranging from 30 to 56%, as compared to control samples (3 semimembranosus, 2 gluteal), where the mean percentage of type 2X fibers was 60%, ranging from 43-87%.

DISCUSSION [0101] This is the first report of a mutation in MYH1 gene associated with susceptibility to a specific myopathy. A GWA study initially identified a -3.1 Mb region on ECA11 associated with IMM that encompassed myosin genes expressed in adult equine muscle, namely MYH1 and MYH2. Both myosin genes were of interest because they encode type 2A and type 2X MyHC, which respectively comprise ~ 30-40%) and 40-80%) of muscle fibers present in muscles typically affected with FMM (33). One nonsynonymous variant was identified in MYH1 that resulted in substitution of a charged glutamic acid for a non-polar amino acid, glycine at 321 in the myosin heavy chain globular head. Credence for the significance of the variant was provided by the fact that glutamic acid is highly conserved, present in this position in seven other species (Sus, Bos, Homo, Oryctol, Rat, Canis) and in the corresponding position of MyHC 2 A, perinatal, extraocular, embryonic and cardiac/slow MyHC (34) (Fig. 8). A strong selection pressure against mutations in skeletal muscle myosin heavy chains appears to exist based on the remarkable orthology of myosin heavy chain genes across species (34; 35) Thus, the E321G MYH1 variant identified in MYH1 in IMM horses is novel and significantly associated with a

susceptibility to develop IMM.

[0102] The MYH1 E321G variant appears to be variably penetrant, conferring susceptibility to disease potentially depending upon whether other factors needed to trigger an immune mediated disease are present. A significant proportion of both IMM and in contact control horses were heterozygous for the MYH1 E321G variant. Heterozygosity, however, was much less in the unrelated group of QH and absent in the Arabian breed of horse as well as in the publically available mapped NCBI SRA database of 17 breeds. The high degree of heterozygosity in the original cohort of horses can be explained by the fact that the in contact group of horses were closely related to the affected horses. The unadjusted genomic inflation factor for the initial GWA was 1.98. In order to control for environmental exposure to factors that trigger EVIM, we selected horses in the same environment, which was a breeding farm in one case and a private farm with preference for certain bloodlines in another. Lack of expression of IMM in some heterozygotes could be the result of differential expression of the affected allele, subthreshold environmental triggering of auto-immunity or a lack of initial priming of the immune system from a first exposure to mutant MyHC2X. Together, these results suggest that rather than consistently causing disease, homozygosity and, in some cases heterozygosity, for the MYH1 variant predisposed horses to a myopathy under certain environmental triggers. Eleven of 71 horses diagnosed with IMM and genotyped for the MYH1 E321G were homozygous reference. Of these, 3 were from the initial GWA cohort, where horses were phenotyped more stringently (i.e. moderate to severe lymphocytic infiltrate on muscle biopsy) and eight were from the follow-up cohort where EVIM-affected cases had a milder degree of myofiber lymphocytic infiltrates and included those with only perivascular lymphocytes. These 11 horses were likely phenocopies in that they presented with muscle atrophy or high serum CK activity, had mild inflammatory infiltrates in muscle biopsies but did not possess the MYH1 E321G variant. Phenocopies were to be expected because differentiation of immune-mediated versus inflammatory myopathies of infectious origin is difficult based solely on muscle histopathology (2; 12). [0103] Mutations in MYH2 have been reported in a small number of human patients. Recessive truncating deletions in MYH2 result in early-onset weakness confined to extraocular, semitendinosus, gracilis, vastus lateralis and medial gastrocnemius muscles whereas dominant point mutations in MYH2 result in a later onset of mild progressive weakness, (36-38). Both recessive and dominant MYH2 mutations result in mild myopathic changes such as variability of fiber size, internalized nuclei, and increased interstitial connective and adipose tissue; however, only recessive deletions produce a total absence of type 2A myofibers (36; 38). Unlike patients with recessive MYH2 mutations, in horses with IMM, the reduction in type 2X myofibers was dependent upon inflammation and in the absence of inflammation IMM horses have a normal proportion of type 2X fibers.

Lymphocytic destruction of MyHC 2X fibers appeared to be a prerequisite for acute inflammation in IMM horses. Thus, the clinical signs and muscle histopathology of FMM are distinct from those previously reported for MYH2 mutations. [0104] In contrast to the low frequency of mutations in MYH1 and MYH2, more than 500 disease-causing point mutations have been described in MYH7, with the majority producing hypertrophic or dilated cardiomyopathy (39). A minority of MYH7 mutations are reported to cause skeletal myopathies such as myosin storage myopathies or Laing distal myopathy (39-41). One family with a p.K1729del in MYH7 had similar inflammatory changes to those seen in equine FMM although the clinical presentation was that of distal limb weakness not the rapid proximal muscle atrophy seen in equine IMM (41). Similar to equine IMM, increased skeletal muscle MHC Class I expression and perivascular and endomysial lymphocytic infiltrates (CD3+, CD4+, and CD8+) were found in this family with Laing distal myopathy, along with rimmed vacuoles, which are not a feature of equine IMM (6; 41). Laing distal myopathy has highly variable muscle pathology, however, and inflammation is not a consistent feature of skeletal muscle in most patients (42).

[0105] The best characterized link between myosin, inflammation and muscle disease would be immune-mediated myocarditis (43). Fragments of cardiac myosin have been shown to activate Toll like receptors (TLR2), which strongly drive reactivity to self and subsequently determine the type of adaptive immune response (i.e., Thl, Th2) that occurs (44). Synergy between the activated innate immune response and the adaptive response of pathogenic T cell epitopes appear to be important in the generation of chronic myocardial inflammation (44). Similar to human myocarditis, the adaptive immune response could be triggered in IMM horses by shared epitopes between bacteria such as the M protein of group A Streptococcus sp. and myosin (Fig. 8) (45). The innate immune response could be triggered in IMM by release of the mutant form of MyH2X from myofibers following muscle damage (trauma, vaccination). The loss of hydrogen bonds with the MYHl mutation could possibly lead to conformational changes in myosin that activate TLRs and autoimmunity. Of note, a nonsynonymous mutations in MYH7 (S545A) in DBA/2 mice appears to predisposes these mice to immune-mediated myocarditis (46; 47). When DBA/2 mice with the MYH7 S545A variant and BALB/c mice not possessing the variant are auto-inoculated with cardiac myosin, chronic myocarditis only occurs when serum from the DBA/2 strain is injected into DBA/2 not BALB/c mice (46). The authors concluded that susceptibility to autoimmune myocarditis was dependent not only on the activation of self-reactive lymphocytes but also on genetically determined target organ sensitivity. Because both the DBA/2 MYH7 mutation and the equine MYH1 variant are located in the globular head of myosin, it is possible that a mutation in this highly conserved region somehow confers target host susceptibility to myositis.

[0106] Perhaps the most intriguing link between myosin and autoimmunity comes from canine masticatory muscle myositis (CMM). CMM presents with painful swelling followed by rapid atrophy of masticatory muscles (8). Similar to equine IMM, biopsies of CMM masseter muscle are characterized by MHC I upregulation, B cells and a

predominance of CD4+ over CD8+ T lymphocytic infiltrates in masticatory muscles (2; 6; 8; 46). Both within the masseter muscle and in the circulation, autoantibodies for masticatory muscle myosin (2M) or myosin binding protein-C are evident with CMM, suggesting that myosin isoforms unique to masseter muscles have antigenic potential and can serve as target antigens for inflammatory cells (48). Genetic analysis of CMM dogs has yet to be performed to assess the potential for putative mutations in masticatory muscle myosin encoding genes to enhance susceptible to CMM.

[0107] In conclusion, an E321G MYH1 mutation is highly associated with susceptibility to EVIM in horses. In the absence of inflammation, type 2X muscle fiber type composition is within normal limits in EVIM horses, however, with particular environmental stimuli, the MYH1 mutation results in invasion and destruction of type 2X myofibers by lymphocytes and rapid onset of gross muscle atrophy.

DISCUSSION

1. Dalakas MC. 1991. Polymyositis, dermatomyositis and inclusion-body myositis. N Engl J Med 325: 1487-98 2. Evans J, Levesque D, Shelton GD. 2004. Canine inflammatory myopathies: a clinicopathologic review of 200 cases. J Vet Intern Med 18:679-91

3. Lewis SS, Valberg SJ, Nielsen IL. 2007. Suspected immune-mediated myositis in horses. J Vet Intern Med 21 :495-503

4. Hunyadi L, Sundman EA, Kass PH, Williams DC, Aleman M. 2017. Clinical Implications and Hospital Outcome of Immune-Mediated Myositis in Horses. J Vet Intern Med 31 : 170-5 5. Shelton GD. 2007. From dog to man: the broad spectrum of inflammatory myopathies. Neuromuscul Disord 17:663-70

6. Durward-Akhurst SA, Finno CJ, Barnes N, Shivers J, Guo LT, et al. 2016. Major Histocompatibility Complex I and II Expression and Lymphocytic Subtypes in Muscle of Horses with Immune-Mediated Myositis. J Vet Intern Med 30: 1313-21

7. Schmidt J, Dalakas MC. 2013. Inclusion body myositis: from immunopathology and degenerative mechanisms to treatment perspectives. Expert Rev Clin Immunol 9: 1125-33

8. Shelton GD, Cardinet GH, 3rd, Bandman E. 1987. Canine masticatory muscle disorders: a study of 29 cases. Muscle Nerve 10:753-66 9. Rothwell S, Cooper RG, Lamb JA, Chinoy H. 2013. Entering a new phase of immunogenetics in the idiopathic inflammatory myopathies. Curr Opin Rheumatol 25:735- 41

10. Davidson A, Diamond B. 2001. Autoimmune diseases. N Engl J Med 345:340-50

11. Massey J, Rothwell S, Rusbridge C, Tauro A, Addicott D, et al. 2013. Association of an MHC class II haplotype with increased risk of polymyositis in Hungarian Vizsla dogs.

PLoS One 8:e56490

12. Durward-Akhurst SA, Valberg SJ. 2017. Immune-Mediated Muscle Diseases of the Horse. Vet Pathol:300985816688755

13. McCue ME, Valberg SJ, Miller MB, Wade C, DiMauro S, et al. 2008. Glycogen synthase (GYSl) mutation causes a novel skeletal muscle glycogenosis. Genomics 91 :458-

66

14. Finno CJ, Aleman M, Higgins RJ, Madigan JE, Bannasch DL. 2014. Risk of false positive genetic associations in complex traits with underlying population structure: a case study. Vet J 202:543-9 15. Garbe JR, Da Y. 2008. Pedigraph: A software tool for the graphing and analysis of large complex pedigrees. User manual version 2.4. Minneapolis, MN: Department of Animal Science, University of Minnesota

16. Zhou X, Stephens M. 2012. Genome-wide efficient mixed-model analysis for association studies. Nat Genet 44:821-4 17. Barrett JC, Fry B, Mailer J, Daly MJ. 2005. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21 :263-5

18. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25: 1754-60 19. McKenna A, Hanna M, B anks E, Sivachenko A, Cibul ski s K, et al . 2010. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20: 1297-303

20. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, et al. 2011. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43 :491-8

21. Van der Auwera GA, Carneiro MO, Haiti C, Poplin R, Del Angel G, et al . 2013. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics 43 : 11 0 1-33

22. Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, et al. 2012. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain wl 118; iso-2; iso-3. Fly (Austin) 6:80-92

23. Cingolani P, Patel VM, Coon M, Nguyen T, Land SJ, et al. 2012. Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift. Front Genet 3 :35 24. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, et al. 2011. Integrative genomics viewer. Nat Biotechnol 29:24-6

25. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, et al. 2012. Primer3— new capabilities and interfaces. In Nucleic Acids Res, p. el 15

26. Solomon O, Kunik V, Simon A, Kol N, Barel O, et al. 2016. G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures. BMC Genomics

17:681

27. Eyal S, Yagen B, Sobol E, Altschuler Y, Shmuel M, Bialer M. 2004. The activity of antiepileptic drugs as histone deacetylase inhibitors. Epilepsia 45:737-44

28. McConkey BJ, Sobolev V, Edelman M. 2003. Discrimination of native protein structures using atom-atom contact scoring. P Natl Acad Sci USA 100:3215-20 29. McConkey BJ, Sobolev V, Edelman M. 2002. Quantification of protein surfaces, volumes and atom-atom contacts using a constrained Voronoi procedure. Bioinformatics 18: 1365-73

30. Capriotti E, Fariselli P, Casadio R. 2005. 1-Mutant2.0: predicting stability changes upon mutation from the protein sequence or structure. Nucleic Acids Research 33 :W306-

W10

31. Mansour TA, Scott EY, Finno CJ, Bellone RR, Mienaltowski MJ, et al. 2017. Tissue resolved, gene structure refined equine transcriptome. BMC Genomics 18: 103

32. Oldfors A. 2007. Hereditary myosin myopathies. Neuromuscul Disord 17:355-67 33. Valberg SJ. 2014. Muscle anatomy, physiology, and adaptations to exercise and training. In The Athletic Horse: Principles and practice of equine sports medicine, ed. dR Hodgson, McGowan,C, McKeever,K.: 174-201. London: Elsevier. Number of 174-201 pp.

34. Maccatrozzo L, Caliaro F, Toniolo L, Patruno M, Reggiani C, Mascarello F. 2007. The sarcomeric myosin heavy chain gene family in the dog: analysis of isoform diversity and comparison with other mammalian species. Genomics 89:224-36

35. Weiss A, Schiaffino S, Leinwand LA. 1999. Comparative sequence analysis of the complete human sarcomeric myosin heavy chain family: implications for functional diversity. J Mol Biol 290:61-75

36. Martinsson T, Oldfors A, Darin N, Berg K, Tajsharghi H, et al. 2000. Autosomal dominant myopathy: missense mutation (Glu-706— > Lys) in the myosin heavy chain Ila gene. Proc Natl Acad Sci U S A 97: 14614-9

37. Tajsharghi H, Darin N, Rekabdar E, Kyllerman M, Wahlstrom J, et al. 2005.

Mutations and sequence variation in the human myosin heavy chain Ila gene (MYH2). Eur J Hum Genet 13 :617-22 38. Tajsharghi H, Hilton-Jones D, Raheem O, Saukkonen AM, Oldfors A, Udd B. 2010. Human disease caused by loss of fast Ila myosin heavy chain due to recessive MYH2 mutations. Brain 133 : 1451-9

39. Colegrave M, Peckham M. 2014. Structural implications of beta-cardiac myosin heavy chain mutations in human disease. Anat Rec (Hoboken) 297: 1670-80 40. Meredith C, Herrmann R, Parry C, Liyanage K, Dye DE, et al. 2004. Mutations in the slow skeletal muscle fiber myosin heavy chain gene (MYH7) cause laing early-onset distal myopathy (MPD1). Am J Hum Genet 75:703-8

41. Roda RH, Schindler AB, Blackstone C, Mammen AL, Corse AM, Lloyd TE. 2014. Laing distal myopathy pathologically resembling inclusion body myositis. Ann Clin Transl

Neurol 1 : 1053-8

42. Lamont PJ, Udd B, Mastaglia FL, de Visser M, Hedera P, et al. 2006. Laing early onset distal myopathy: slow myosin defect with variable abnormalities on muscle biopsy. J Neurol Neurosurg Psychiatry 77:208-15 43. Root-Bernstein R, Fairweather D. 2015. Unresolved issues in theories of

autoimmune disease using myocarditis as a framework. J Theor Biol 375: 101-23

44. Zhang P, Cox CJ, Alvarez KM, Cunningham MW. 2009. Cutting edge: cardiac myosin activates innate immune responses through TLRs. J Immunol 183 :27-31

45. Pahlman LI, Morgelin M, Eckert J, Johansson L, Russell W, et al. 2006.

Streptococcal M protein: a multipotent and powerful inducer of inflammation. J Immunol 177: 1221-8

46. Liao L, Sindhwani R, Rojkind M, Factor S, Leinwand L, Diamond B. 1995.

Antibody-mediated autoimmune myocarditis depends on genetically determined target organ sensitivity. J Exp Med 181 : 1123-31 47. Quinn-Laquer BK, Kennedy JE, Wei SJ, Beisel KW. 1992. Characterization of the allelic differences in the mouse cardiac alpha-myosin heavy chain coding sequence.

Genomics 13 : 176-88

48. Wu X, Li ZF, Brooks R, Komives EA, Torpey JW, et al. 2007. Autoantibodies in canine masticatory muscle myositis recognize a novel myosin binding protein-C family member. J Immunol 179:4939-44

[0108] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. INFORMAL SEQUENCE LISTING

Sequence ID No: 1 - Equine myosin heavy chain-1 (MYH1) amino acid sequence - NCBI Reference Sequence: XP 005597093.1 mssdqemaif geaapylrks ekerieaqnk pfdaktsvfv adpkesfvka tvqsreggkv takteagatv tvkedqcfpm nppkydkied mammthlhep avlynlkery aawmiytysg Ifcvtvnpyk wlpvynaevv tayrgkkrqe apphifsisd nayqfmltdr enqsilitge sgagktvntk rviqyfatia vtgekkkeep tsgkmqgtle dqiisanpll eafgnaktvr ndnssrfgkf irihfgttgk lasadietyl leksrvtfql kaersyhify qimsnkkpdl iemllittnp ydyafvsqg [e/g] itvpsiddqeelmatdsaieilgftsderv siykltgavm hygnlkfkqk qreeqaepdg tevadkaayl qglnsadllk alcyprvkvg nefvtkgqtv eqvynavgal akavydkmfl wmvarinqql dtkqprqyfi gvldiagfei fdfnsleqlc inftneklqq ffnhhmfvle qeeykkegie wefidfgmdl aacieliekp mgifsileee cmfpkatdts fknklyeqhl gksnnfqkpk pvkgkpeahf slihyagtvd ynitgwldkn kdplnet vg lyqkssvktl allfsgpasa daeaggkkgg kkkgssfqtv salfrenlnk lmtnlrsthp hfvrciipne tktpgamehe lvlhqlrcng vlegiricrk gfpsrilyad fkqrykvlna saipegqfid skkasekllg sididhtqyk fghtkvffka gllglleemr ddklaqiitr tqarcrgfla rveyqrmver resifciqyn vrafmnvkhw pwmklyfkik pllksaetek emanmkeefe ktkeslakae akrkeleekm valmqekndl qlqvqaeads ladaeercdq liktkiqlea kikeaterae deeeinaelt akkrkledec selkkdiddl eltlakveke khatenkvkn Iteemaglde tiakltkekk alqeahqqtl ddlqaeedkv ntltkaktkl eqqvddlegs leqekklrmd lerakrkleg dlklaqestm diendkqqld eklkkkefem snlqskiede qalamqlqkk ikelqariee leeeieaera srakaekqrs dlsreleeis erleeaggat saqiemnkkr eaefqkmrrd leeatlqhea taaalrkkha dsvaelgeqi dnlqrvkqkl ekeksemkme iddlasnmet vskakgnlek mcrtledqls elkskeeeqq rlvndltgqr arlqteagey srqldekdsl vsqlsrgkqa ftqqieelkr qleeeikaks alahalqsar hdcdllreqy eeeqeakael qramskanse vaqwrtkyet daiqrteele eakkklaqrl qdaeehveav nakcaslekt kqrlqneved lmidvertna acaaldkkqr nfdkilsewk hkyeethael easqkesrsl stelfkvkna yeesldqlet lkrenknlqq eisdlteqia eggkrihele kvkkqieqek seiqaaleea easleheegk ilriqlelnq vkseidrkia ekdeeidqlk rnhvrwetm qtmldaeirs rndairikkk megdlnemei qlnhanrmaa ealrnyrntq gilkdtqlhl ddalrgqedl keqlamverr anllqaeiee lratleqter srkiaeqell daservqllh tqntslintk kkletdisql qgemedivqe ahnaeekakk aitdaammae elkkeqdtsa hlermkknle qtvkdlqhrl deaeqlalkg gkkqiqklea rvrdlegeve seqkrnveav kglrkherrv keltyqteed rknilrlqdl vdklqskvka ykrqaeeaee qsnvnlskfr kiqheleeae eradiaesqv nklrvksrev htkiisee

Sequence ID No: 2 - Equine myosin heavy chain-1 (MYH1) nucleic acid sequence - NCBI Reference Sequence:

XM 005597036.3 ttaggaaaga ggaggaggct atgtgttttc catatttgat cgtataaaag tacccttgga atgagtatga cctgtctttc ctcataaagc ttcaagttct gacccacttc aaggccgcat ctctaaggca gggtctttga ctgggcctcc atcaataacc cgcagccatg agttcagacc aggaaatggc tatatttggg gaggctgctc cttacctccg aaagtctgaa aaggagcgaa ttgaagccca gaataagcct ttcgatgcca agacatcagt ttttgtggct gaccctaagg agtcctttgt gaaagcaaca gtgcagagca gggaaggagg gaaggtgaca gccaagactg aagctggagc tacagtaact gtgaaagaag accaatgctt ccccatgaac cctcccaaat atgacaagat cgaggacatg gccatgatga ctcacctgca tgagcctgct gtgctgtaca acctcaaaga gcgctacgca gcctggatga tctacaccta ctcaggtctt ttctgtgtca ccgtcaaccc ctacaagtgg ttgccagtgt acaacgccga ggtggtgacg gcctaccgag gcaaaaagcg ccaggaggcc ccgccccaca tcttctccat ctctgacaac gcctatcagt tcatgctgac tgatcgggag aatcagtcta tcttaatcac tggagaatct ggtgccggga agactgtgaa taccaagcgt gtcatccagt actttgcaac aattgcagtt actggggaga agaagaagga ggaacctact tccggcaaaa tgcaggggac tctggaagat cagatcatca gtgccaaccc cctactggag gcctttggca acgccaagac cgtgaggaat gacaactcct ctcgctttgg taaattcatc aggatccact tcggtaccac agggaaactg gcttctgctg atattgaaac atatcttctg gagaagtcta gagttacttt ccagctaaag gcggaaagaa gctaccacat tttttatcag atcatgtcta acaagaagcc agatctaatt gaaatgctcc tgatcaccac caacccgtatgactatgcctttgtcagtcaagggg [a/g] gatcacagtcccca gcattgatga ccaagaagag ttgatggcca cagatagtgc cattgagatc ttgggcttca cttctgatga aagagtgtcc atctataagc tcacaggggc agtaatgcat tacgggaacc tgaaattcaa gcagaagcag cgtgaggagc aagctgagcc agatggcact gaagttgctg acaaggctgc ctatcttcag ggtctgaact ctgctgacct gctcaaagct ctctgctacc ccagggtcaa ggtcggcaat gagttcgtca ccaaaggcca gactgtagaa caggtgtaca atgcggtggg tgctctggcc aaagccgtct acgataagat gttcctctgg atggtcgccc gcatcaacca gcagctggac accaagcagc ccaggcagta cttcatcggg gtcttggaca tcgctggctt tgagatcttt gatttcaaca gcctggagca gctgtgcatc aacttcacca acgagaaact gcaacagttt ttcaaccacc acatgttcgt gctggagcag gaggagtaca agaaggaagg catcgagtgg gagttcatcg acttcggcat ggacctggct gcctgcattg agctcatcga gaagccgatg ggcatcttct ccatcctgga agaggagtgc atgttcccca aggccacaga cacctccttc aagaacaagc tgtatgaaca gcatcttgga aagtccaaca atttccagaa gcctaaacct gtcaaaggca agcctgaggc ccacttctcc ctgattcact acgccggcac tgtggactac aacattactg gctggcttga caagaacaag gaccccctga atgagaccgt ggtcgggctg taccagaagt cttcagtgaa gactctggct ttgctcttct ctgggccagc aagtgctgat gcggaggctg gtggaaagaa aggaggcaag aagaagggtt cttctttcca gaccgtgtct gcgctcttca gggagaattt gaataagctg atgaccaacc tgaggagcac tcaccctcac tttgtacggt gcatcatccc caatgaaacc aaaactcctg gtgccatgga gcatgaactt gtcctgcacc agctgaggtg caatggtgtg ctggaaggca tccgcatctg cagaaaggga ttcccaagca ggatccttta tgcagacttc aaacagagat acaaggtatt aaatgcaagt gctatccctg aaggacaatt catcgatagc aagaaggcgt ctgagaagct ccttgggtcc attgacattg accacaccca gtacaaattt ggtcacacca aggtcttctt caaagctggt ctcctggggc tcctagagga gatgcgagat gacaagctgg cccagataat tacccgaacc caggccaggt gcagagggtt cttggcaaga gtggagtacc agaggatggt ggagagaaga gagtccatct tctgcatcca gtacaatgtc cgtgccttca tgaacgtgaa gcactggccc tggatgaagc tgtatttcaa gatcaagccc ctcctcaaga gtgcagagac agagaaagag atggccaaca tgaaggaaga attcgagaag accaaagaaa gccttgcaaa ggctgaggcc aaaagaaaag agctggaaga aaaaatggta gctctgatgc aagagaaaaa cgacctgcaa cttcaggttc aagctgaagc agacagtttg gctgatgcag aggaaagatg tgaccagctg attaaaacca aaatccagct ggaggccaaa atcaaggagg cgactgagag agctgaggat gaggaagaga tcaacgctga gctgacggcc aagaagagga aactggagga cgaatgctca gagctcaaga aggacattga tgaccttgag ctgacactgg ccaaggttga aaaggagaaa catgccacag aaaataaggt gaaaaacctc acagaggaga tggcaggcct ggacgaaacc atcgctaagc tgaccaagga gaagaaggcc ctccaagagg cccaccagca gaccctggat gacctgcagg cagaagagga caaggtcaac actctgacca aagctaaaac caagctagag cagcaagtgg atgatcttga aggatctcta gagcaagaaa agaaacttcg aatggatcta gaaagagcaa agaggaaact ggagggtgac ctaaaattgg cccaagaatc cacaatggac atagaaaatg acaaacagca acttgatgaa aaactgaaaa agaaagagtt tgaaatgagc aatctgcaaa gcaagattga agatgagcag gcccttgcga tgcagctgca gaagaagatc aaggagttac aggcccgcat cgaggagctg gaggaggaaa tcgaggcaga gcgcgcctcc cgggccaaag cagagaagca gcgctccgac ctctcccggg aactggagga gatcagcgag aggctcgaag aagccggtgg ggcgacttca gcccagattg agatgaacaa gaagcgggag gctgagttcc agaaaatgcg cagggacctg gaggaggcca ccctgcagca tgaagccacg gcggccgctc tgcggaagaa gcacgcggac agtgtggcag agctcgggga gcagatagac aacctgcaga gagtcaagca gaaactggag aaggagaaga gcgaaatgaa gatggagatt gacgacctgg cgagcaacat ggagactgtc tccaaagcca agggcaacct tgaaaagatg tgccgcaccc tggaagatca actgagtgaa cttaagagca aagaggaaga gcagcagagg ctggtcaatg acctgacggg ccagagagcg cgcctgcaga cagaagcagg tgaatattca cgccagctag atgaaaagga ctcattagtt tctcagctct caaggggcaa acaagcattc acacaacaga ttgaggaact gaaaaggcag cttgaagagg agataaaggc caagagtgcc ctggcccatg ccctgcagtc agcccgccat gattgtgacc tgctgcggga acagtacgag gaggagcagg aagccaaggc cgagctgcag agggcaatgt ccaaggccaa cagtgaggtt gcccagtgga ggaccaagta cgagacggac gccatccagc gcacggagga gctggaggag gccaagaaga agctggctca gcggctgcag gatgctgagg aacacgtaga agctgtgaat gccaaatgtg cttcccttga gaagaccaag cagcggctcc agaatgaagt ggaggacctc atgattgatg ttgagagaac caatgctgcc tgtgcagccc tggacaaaaa gcaaaggaac ttcgataaga tcctgtcaga atggaagcac aagtatgaag aaactcatgc tgaacttgaa gcttcccaaa aggagtccag gtcactcagc acagagctgt tcaaggttaa gaatgcttat gaggaatcct tagaccaact tgaaaccttg aagcgggaaa ataagaattt gcaacaggag atttctgatc tcactgagca gattgcagaa ggagggaagc gtatccatga actggaaaaa gtaaagaagc aaattgagca agaaaagtct gaaattcagg ctgctttaga agaagcagag gcatctcttg aacacgaaga gggaaagatc ctgcgcatcc aactggagtt gaaccaagtc aagtctgaaa ttgataggaa aattgctgaa aaggatgagg aaattgacca gctgaagaga aaccatgtca gagttgtgga gacgatgcag accatgctgg atgctgagat caggagccgg aatgatgcca tcaggatcaa gaagaagatg gagggagacc tcaatgaaat ggaaatccag ctgaaccacg ccaaccgcat ggctgcagag gccctgagga actacaggaa cactcaaggc atcctcaagg acacccagct gcacctggat gacgctctcc ggggccagga ggacctgaag gagcagctgg ccatggtgga gcgcagagcc aacctgctgc aggccgagat cgaggagctg cgggccactc tggagcagac cgagaggagc aggaaaatcg cagaacagga gctcctggat gccagtgagc gcgtccagct cctgcacacc cagaacacca gcctgatcaa caccaagaag aagctggaga cagacatttc ccagctgcag ggagagatgg aagatatcgt ccaggaagct cacaatgcag aagagaaggc caagaaggcc atcactgatg cggccatgat ggctgaggag ctgaagaagg agcaggacac cagcgcccac ctggagcgga tgaagaagaa tctggagcag acggtgaagg acctgcagca ccgtctggat gaggccgagc agctggccct gaagggtggg aagaagcaga tccagaaact ggaggccagg gtacgtgacc ttgaaggaga agttgaaagt gaacagaagc gcaatgttga ggctgtcaag ggtctgcgca aacatgagag aagagtaaag gaactcactt accagactga ggaagaccgc aagaatatac tcaggctcca ggatctggtg gataaactgc aatcaaaggt gaaagcttac aagagacaag ctgaggaagc ggaggaacag tccaatgtca atctctccaa attccgcaag atccagcacg agctggagga ggccgaggaa cgggctgaca tcgccgagtc ccaggtcaac aagctgcggg tgaagagccg ggaggttcac acaaaaatca ttagtgaaga gtaa

forward primer: CCCAAGATCTCAATGGCACT

reverse primer: ACCTTGTGGGAACATTCAGC

reverse primer: GGATCATCTGAGGGGGAAAT SEQ ID NO: 6 - reverse primer: TGGAACCTTGTGGGAACATT

SEQ ID NO: 7 - reverse primer: ATGTGGAACCTTGTGGGAAC

SEQ ID NO: 8 - illustrative oligonucleotide for genotyping array; SNP identified with brackets

TGAAACCCTTGGCCGCCGAAGTGGAGTGCGTGAACTGAACCACTCAGCCGTGGGCCCGGCCCACTAA AGGAGTTTTTTAAAAAGCTGCATGTGTAACTTACATCTGTGGCCATCAACTCTTCTTGGTCATCAAT GCTGGGGACTGTGATC [T/C] CCCCTTGACTGACAAAGGCATAGTCATACGGGTTGGTGGTGATCAG GAGCATTTCTGGGTACAGAGAATTCAGGGTATGTGTTTTACATTAGAAGATGTTAATAAAACAGTTA ACTTCTAGCTTATAAATTTAAACTCTTTCTTACCAAT

Claims

CLAIMS What is claimed is:

1. A reaction mixture comprising (i) a biological sample from an equine comprising a nucleic acid template, and (ii) one or more oligonucleotide pairs for differentiating a single nucleotide polymorphism (SNP) indicative of equine immune- mediated myositis (IMM), wherein the SNP is at position 1126 of SEQ ID NO:2, wherein the one or more oligonucleotide pairs differentiate AA, AG and GG genotypes at position 1126 of SEQ ID NO:2.

2. A reaction mixture comprising (i) a biological sample from an equine comprising a nucleic acid template, and (ii) one or more oligonucleotide pairs for differentiating a single nucleotide polymorphism at position 52,993,878 of equine chromosome 11 (NC 009154.2; EquCab 2.0), wherein the one or more oligonucleotide pairs differentiate AA, AG and GG genotypes at position 52,993,878 of equine

chromosome 11.

3. The reaction mixture of any one of claims 1 to 2, wherein the one or more oligonucleotides comprises forward primer: CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of:

ACCTTGTGGGAACATTCAGC (SEQ ID NO:4), GGATCATCTGAGGGGGAAAT (SEQ ID NO:5), TGGAACCTTGTGGGAACATT (SEQ ID NO:6) and

ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7).

4. The reaction mixture of any one of claims 1 to 3, wherein the nucleic acid template comprises genomic DNA.

5. The reaction mixture of any one of claims 1 to 4, wherein the reaction mixture further comprises a polymerase and dNTPs.

6. A solid support attached to one or more oligonucleotides that specifically differentiate a single nucleotide polymorphism (SNP) indicative of equine immune-mediated myositis (IMM), wherein the SNP is at position 1126 of SEQ ID NO:2, wherein the one or more oligonucleotides differentiate AA, AG and GG genotypes at position 1126 of SEQ ID NO:2.

7. A solid support attached to one or more oligonucleotides that specifically differentiate a single nucleotide polymorphism (S P) at position 52,993,878 of equine chromosome 11 (NC_009154.2; EquCab 2.0), wherein the one or more

oligonucleotides differentiate AA, AG and GG genotypes at position 52,993,878 of equine chromosome 11.

8. The solid support of any one of claims 6 to 7, wherein the solid support is attached to an oligonucleotide having at least about 80% sequence identity to SEQ ID NO:8.

9. The solid support of any one of claims 6 to 8, wherein the solid support is a microarray.

10. The solid support of any one of claims 6 to 9, wherein the solid support is attached to 10,000 or fewer oligonucleotides.

11. A kit comprising the solid support of any one of claims 6 to 10.

12. A method for identifying an equine suffering from or at risk of suffering from immune-mediated myositis (EVIM), the method comprising:

a) obtaining a biological sample comprising a nucleic acid template from the equine, determining the genotype of myosin heavy chain 1 (MYHl) at position 1126 of SEQ ID NO:2; and

b) selecting an equine with a GG or AG genotype at position 1126 of SEQ ID NO:2, wherein a GG or AG genotype at position 1126 of SEQ ID NO:2 identifies an equine suffering from or at risk of suffering from immune-mediated myositis (EVIM) relative to an AA genotype.

13. A method for identifying an equine suffering from or at risk of suffering from immune-mediated myositis (EVIM), the method comprising:

a) obtaining a biological sample comprising a nucleic acid template from the equine, determining the genotype of myosin heavy chain 1 (MYHl) at position 52,993,878 of equine chromosome 11 (NC_009154.2; EquCab 2.0); and

b) selecting an equine with a GG or AG genotype at position 52,993,878 of equine chromosome 11, wherein a GG or AG genotype at position 52,993,878 of equine chromosome 11 identifies an equine suffering from or at risk of suffering from immune- mediated myositis (IMM) relative to an AA genotype.

14. A method for identifying an equine with reduced risk of suffering from immune-mediated myositis (IMM), the method comprising:

a) obtaining a biological sample comprising a nucleic acid template from the equine, determining the genotype of myosin heavy chain 1 (MYHl) at position 52,993,878 of equine chromosome 11 (NC_009154.2; EquCab 2.0); and

b) selecting an equine with an AA genotype at position 52,993,878 of equine chromosome 11, wherein an AA genotype at position 52,993,878 of equine chromosome 11 identifies an equine having reduced risk of suffering from immune-mediated myositis (IMM) relative to an AG or GG genotype.

15. A method of treating an equine for immune-mediated myositis (EVIM), the method comprising:

a) identifying an equine exhibiting signs of skeletal muscle damage; b) determining in a biological sample comprising a nucleic acid template from the equine a GG or AG genotype at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11; and

c) administering a therapeutically effective amount of an immunosuppressant agent to the equine.

16. The method of any one of claims 12 to 15, wherein the EVIM genotype is detected by an amplification reaction using polynucleotides that distinguish between alleles.

17. The method of claim 16, wherein the amplification reaction is selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), T7 polymerase mediated amplification, T3 polymerase mediated amplification and SP6 polymerase mediated amplification.

18. The method of any one of claims 12 to 17, wherein amplifying the nucleic acid sequence comprises using reverse transcription and amplification of the mRNA molecule.

19. The method of any one of claims 12 to 18, wherein the A to G substitution is detected by:

a) specifically amplifying a nucleic acid sequence comprising position 1 126 of SEQ ID NO:2 or position 52,993,878 of equine chromosome 1 1, thereby amplifying nucleic acids comprising the single nucleotide polymorphism (S P) indicative of IMM; and b) detecting the amplified nucleic acids, thereby detecting the SNP indicative of IMM.

20. The method of any one of claims 12 to 19, wherein the nucleic acid sequence is specifically amplified using forward primer: CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4), GGATCATCTGAGGGGGAAAT (SEQ ID NO:5), TGGAACCTTGTGGGAACATT (SEQ ID NO:6) and

ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7).

21. The method of any one of claims 12 to 13, wherein the FMM genotype is detected by hybridization using polynucleotides which distinguish between alleles.

22. The method of any one of claims 12 to 13, wherein the FMM genotype is detected by sequencing.

23. The method of any one of claims 12 to 13, wherein the equine is a domesticated equine.

24. The method of any one of claims 12 to 23, wherein the equine is a Quarter Horse, an equine of Quarter Horse lineage (e.g., a Quarter Pony, a Quarab), an equine breed selected from Paint Horse, Appaloosa, Akhal-Teke, Arabian, Belgian, Clydesdale, Franches-Montagnes, Friesian dwarf, German Warmblood, Hanoverian, Icelandic, Lusitano, Shetland pony, Standardbreds, Swedish Warmbood, Yukatian, or a mixture thereof.

25. The method of any one of claims 12 to 24, wherein the equine is of Arabian descent.

26. The method of any one of claims 12 to 25, further wherein the equine is negative for one or more of Polysaccharide Storage Myopathy (PSSMl), Hyperkalemic Periodic Paralysis Disease (HYPP), Glycogen Branching Enzyme Deficiency (GBED), Malignant Hyperthermia (MH), Hereditary Equine Regional Dermal Asthenia (HERD A), and Overo Lethal White Syndrome (OLWS).

27. A kit comprising one or more oligonucleotide pairs for differentiating a single nucleotide polymorphism (SNP) indicative of equine immune-mediated myositis (IMM), wherein the SNP is at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11, wherein the one or more oligonucleotide pairs differentiate AA, AG and GG genotypes at position 1126 of SEQ ID NO:2 or at position 52,993,878 of equine chromosome 11.

28. The kit of claim 27, comprising forward primer:

CCCAAGATCTCAATGGCACT (SEQ ID NO:3) and one or more reverse primers selected from the group consisting of: ACCTTGTGGGAACATTCAGC (SEQ ID NO:4),

GGATCATCTGAGGGGGAAAT (SEQ ID NO: 5), TGGAACCTTGTGGGAACATT (SEQ ID NO:6) and ATGTGGAACCTTGTGGGAAC (SEQ ID NO:7).