AU2016204268B2 - Compositions and methods for diagnosing and treating kidney disorders in a canine - Google Patents

Abstract

The present invention provides methods for: diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine, wherein the kidney disorder is detectable by utilizing at least one relevant biomarker isolated and measured from a biological test sample taken from such canine. The invention additionally relates to compositions, reagents and kits for carrying out the specified methods.

Description

The present invention provides methods for: diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine, wherein the kidney disorder is detectable by utilizing at least one relevant biomarker isolated and measured from a biological test sample taken from such canine. The invention additionally relates to compositions, reagents and kits for carrying out the specified methods.

2016204268 22 Jun 2016

COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING KIDNEY DISORDERS IN A CANINE

FIELD OF THE INVENTION [0001] This application is a divisional application of Australian Patent Application No. 2014202164, the contents of which are incorporated herein by reference.

[0002] The present invention relates to compositions and methods for: diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine, wherein the kidney disorder is detectable by utilizing at least one relevant biomarker isolated and measured from a biological test sample taken from such canine. A relevant biomarker for practice of the compositions and methods of the present invention comprises an RNA transcript or its translation product taken from such biological test sample of such canine. A biological test sample for the practice of the method of the invention comprises a tissue sample of a kidney of such canine or a specimen of a biological fluid taken from such canine.

[0003] The present invention also relates to the identification of combinations of novel biomarkers for use in diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of, a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine.

BACKGROUND OF THE INVENTION [0004] Glomerulonephritis or glomerular nephritis (“GN”) is a renal disease which is characterized by inflammation of the glomeruli or capillary loops of the kidney. It is a pathologic process associated with a number of diverse underlying diseases. The condition occurs in acute, sub-acute and chronic forms and also secondary to an infection. The former conditions, where a concurrent illness cannot be found, are generally referred to as idiopathic glomerulonephritis. The latter conditions are generally referred to as secondary GN. Whatever the underlying cause, immune complexes form and result in a series of events leading to glomerular injury and loss of renal function, proteinuria and ultimately, in some cases, renal failure.

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8693-OO-WO-HL [0005] Nephritis is an inflammation of the kidney, which may be a focal or diffuse proliferative or destructive disease involving the glomerulus, renal tubule or the kidney interstitial (or connective) tissue. The most common form of nephritis is glomerulonephritis. Nephritis may progress through a number of stages ending in end-stage kidney disease or endstage renal failure.

[0006] Renal failure results from the inability of the kidney to maintain its normal functions. As a result, metabolic waste products and metabolites accumulate in the blood. These waste products and metabolites may adversely affect most bodily systems. Disturbances in the maintenance of fluid and electrolyte balances are characteristics of renal failure.

[0007] Acute renal failure may occur suddenly due to trauma, infection, inflammation or exposure to nephrotoxic substances. This condition may result in dehydration, hypotension and circulatory collapse. Acute renal failure is frequently segregated into three categories: (1) prerenal failure, which is associated with decreased renal blood flow; (2) intra-renal failure, which is associated with ischemia and toxins; and (3) post-renal failure, which results from obstruction of urine flow.

[0008] Chronic renal failure involves a progressive loss of kidney function that may eventually progress to end-stage renal disease or failure. At inception, chronic renal failure begins as a diminishing kidney function, without appreciable accumulation of metabolic waste products in the blood. As the glomerular filtration rate slows due to inflammation, waste products begin to accumulate. The disease progresses to uremia due to low kidney function, and high levels of protein end products start to accumulate and impair bodily functions. Common causes of chronic renal failure include: inflammation, infection, urinary tract obstruction and certain systemic diseases and toxicities, including hypercalcemia, lupus erythematosus, diabetes mellitus and hypertension.

[0009] End-stage renal disease is marked by irreversible chronic renal failure. Serum creatinine and blood urea nitrogen levels continue to rise and the resulting uremia impairs all bodily systems. The kidney can suffer permanent and almost complete loss of function on the order of 10% or less of normal kidney function. One cause of end-stage kidney disease is glomerulonephritis. Other causes include those mentioned for chronic renal failure.

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8693-OO-WO-HL [0010] Glomerulonephritis may arise as a result of a biological insult to the immune system. Foreign substances may adhere to the basement membrane and cause an immune response resulting in the production of antibodies. These antibodies may combine with the foreign substances to cause immune complexes that become deposited on the walls of the tiny glomerular capillaries, resulting in damage to the nephron. Alternatively, in some individuals the immune system can create autoantibodies which are immunoglobulins that may attack kidney cells resulting in a so-called autoimmune response. If proteins in the body are altered, an autoantibody response may ensue because the autoantibodies recognize the altered proteins as non-self. These autoantibody-protein complexes may likewise be deposited on the basement membrane of the glomerulus causing a disruption of the functioning of the nephron.

[0011] Glomerulonephritis is a common cause of proteinuria in dogs and may be either the idiopathic or secondary form of the condition. In the latter situation, the condition may develop secondary to neoplasia, inflammatory diseases, endocrine malfunctions, infections or familial nephropathies. As in humans, GN in dogs is mediated immunologically, involving immunoglobulins and complement factors in the body of the animal. Injury occurs within the glomeruli of the kidney resulting in morphological changes to the glomeruli. Eventually the injury is irreversible and leads to malfunction of the nephrons.

[0012] It is generally accepted in the scientific community that the regulation of gene expression plays a key role in the development of some diseases or conditions that affect an animal’s health and well being. Similarly, a differential expression of genes is one factor in the development of such diseases and conditions and the evaluation of gene expression patterns has become widely recognized as important to understanding the development and control of such diseases and conditions at the molecular level. To advance the understanding of genes and their relationship to disease, a number of methods have been developed for studying differential gene expression, e.g., DNA microarrays, expressed tag sequencing (EST), serial analysis of gene expression (SAGE), subtractive hybridization, subtractive cloning and differential display (DD) for mRNA, RNA-arbitrarily primed PCR (RAP-PCR), real-time PCR (RT-PCR), representational difference analysis (RDA), two-dimensional gel electrophoresis, mass spectrometry, and protein microarray based antibody-binding for proteins.

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8693-OO-WO-HL [00131 Due to the complexity of the biological pathways implicated in renal disease and the inherent molecular interactions and intercellular signaling processes, it is highly desirable to understand at a genetic level the interactions that are taking place. Detection of dysregulated genes in the early stages of loss of renal function in canines is helpful in understanding the biology of renal disease, especially glomerulonephritis on a genome-wide basis. The fact that gene dysregulation may be detected at an early stage of disease development in animals subjected to repeated ischemic injury is helpful in designing methods for diagnosing of, and devising and monitoring a treatment plan for, an abnormal loss of kidney function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine.

[0014] A more detailed understanding of the biological pathways involved through gene expression profiling will aid in the development of diagnostic procedures, reagents and test kits as well as salutary pharmaceutical, nutraceutical and nutritional (dietary) interventions in the disease pathways. These approaches may enable early detection and potentially prevention or treatment of the underlying kidney disorder, particularly glomerulonephritis, as well as in monitoring the prognosis of early stage renal failure and glomerulonephritis, especially in canines. Dysregulated genes involved in the pathology of such disorders may serve as important biomarkers for diagnosis and potentially prevention or treatment of the disorder and to optimize selection of appropriate pharmaceutical, nutraceutical and nutritional (dietary) interventions.

[0015] The level of gene expression and/or the determination of the level of functioning of an expressed gene product in a canine may be used to select an appropriate agent for therapeutic or prophylactic use. This data may be employed by the skilled worker in selecting appropriate drugs as agents for the prevention or treatment of renal diseases in canines through gene expression profiling. Gene expression data and analysis may also be used to select nutritional compositions, dietary supplements, and nutraceuticals having a salutary effect on kidney performance by utilizing biomarkers indicative of a healthy state of kidney functioning.

[0016] Only very limited work has been done to date in screening the canine genome for gene expression profiles in connection with the diagnosis of diseases in dogs. Certain work employing animal models has utilized cDNA array technologies to screen for gene expression in renal tissues associated with renal disease.

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8693-OO-WO-HL [0017] Studies in healthy populations of canines versus populations having a disease such as a kidney disease and loss of kidney function as described in this specification have not been extensively conducted. Little data is available with respect to the expression profile of the canine genome, especially with respect to the development of renal diseases in canines over time. Gene expression data contained in this specification identifies the genes associated with renal function in dogs. Such gene expression data enables development of compositions and methods for diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of, a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine, wherein the kidney disorder is detectable by utilizing at least one relevant biomarker isolated and measured from a biological test sample taken from such canine.

[0018] Gene expression data contained in the specification and examples enables a variety of desirable inventions based on the gene expression profiles described in the specification and examples of this patent application. The data permits identification and quantification of gene expression products as biomarkers of disease prevention, identification and treatment of the underlying renal disease. Gene expression data elicited as a result of the practice of the methods of the invention also permits monitoring the progression of such renal disease. These inventions further include genetic testing to identify susceptible subpopulations of animals likely to be afflicted with such renal disease, to identify optimal diets for the prevention or treatment of such renal diseases, to identify pharmaceutical, nutraceutical and nutritional (dietary) interventions based on the findings set forth on this specification in order to treat the underlying renal diseases. The inventions also include biomarkers for early disease detection, targeted therapeutics, diagnostic reagents and kits for the analysis of tissue and blood samples from canines susceptible to or having such a renal disease.

SUMMARY OF THE INVENTION [0019] The present invention relates to compositions and methods for: diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine, wherein the kidney disorder is detectable by utilizing at least one relevant biomarker isolated and measured from a biological test sample taken from such canine.

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8693-OO-WO-HL [0020] A relevant biomarker for practice of the compositions and methods of the present invention comprises an RNA transcript or its translation product taken from such biological test sample of such canine. A biological test sample for the practice of the method of the invention comprises a tissue sample of a kidney of such canine or a specimen of a biological fluid taken from such canine.

[0021] In particular, this invention relates to methods for: diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of, a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine, through use of gene expression analysis.

[0022] The present invention also relates to the identification of combinations of novel biomarkers for use in determining diagnosing of, devising and monitoring a treatment plan for, and monitoring the status of, a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis, in a canine.

[0023] The invention additionally relates to compositions, reagents and kits for carrying out the specified methods.

[0024] The invention is based, in part, on the discovery that particular gene expression profiles in canines correlate with a change in such animal from a normal to an abnormal biological process in the kidney that may lead to a decline in renal function over time. A correlation of a particular gene expression profile with the risk of experiencing a decline in renal function can be predicted, detected and diagnosed in a canine without rendering a conventional clinical diagnosis based on art-recognized clinical signs and symptoms of renal disease. An altered gene expression profile in a canine is, therefore, predictive of a decline in renal function, as might otherwise be diagnosed at a later time by art-recognized measurements of renal function. Such art-recognized measurements of renal function typically may include, for example, one of the following measurements: glomerular filtration rate, creatinine clearance rate, urinary protein levels, serum creatinine levels, urinary creatinine levels, blood urea nitrogen (BUN) levels, radioisotope metabolic labeling, soft tissue imaging, including sonography, magnetic resonance imaging and/or computed tomography. Non-intrusive assays such as serum creatinine and BUN

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8693-OO-WO-HL levels typically show poor correlation with kidney histopathology and generally would not be predictive of future changes in the kidney.

[0025] The methods of evaluating a canine to diagnose a kidney disorder involve evaluating the gene expression level or activity of one or more of the following genes or the expression (translation) product of such genes in a canine, selected from the group consisting of: secreted frizzled-related protein 2 (sFRP2); matrilin-2 (Matn2); lumican (LUM); collagen alpha 1 (III) chain, variant 12 (COL3A1); decorin (DCN); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA).

[0026] The methods of evaluating a canine to diagnose a kidney disorder involve evaluating the gene expression level or activity of one or more of the following genes or the expression product of such gene in a canine selected from the group consisting of: secreted frizzled-related protein 2 (sFRP2) or matrilin-2 (Matn2); and optionally, a second group consisting of: lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA).

[0027] The biomarkers useful in the practice of the present invention are: lumican (LUM); collagen alpha 1 (III) chain, variant 12 (COL3A1); decorin (DCN); secreted frizzled-related protein 2 (sFRP2); matrilin-2 (Matn2); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA), as more fully described below and in the sequence listings appended to this specification.

[0028] Canine lumican. Affymetrix probe CfaAFFx.lO198.1.Sl_s_at corresponds to NCBI Reference Sequence: XP_539716.1, accession XP_539716, predicted to be similar to Lumican precursor (keratin sulfate proteoglycan lumican [KSPG lumican]) [Canis familiaris]. Canine lumican is a 338 amino acid leucine-rich repeat protein coded by XM_539716.2 at GeneID:482599 [SEQ. ID. NO: 1], [0029] The terms “Lumican” and “LUM” and “Lum” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 1. Preferably, lumican is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 1. Lumican also includes

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8693-OO-WO-HL protein variants of the sequence as shown in SEQ. ID. NO: 1, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term lumican also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to XM_539716.2 and appended SEQ. ID. NO: 12. Lumican also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 12 and to fragments of SEQ. ID. NO: 12.

[0030] Canine collagen alpha 1 (III), variant 12 (COL3A1). Affymetrix probe CfaAffx.22804.1Sl_s_at corresponds to NCBI Reference Sequence: XP_863148.1, accession XP_863148, predicted to be similar to Collagen alpha (III) chain precursor isoform 12. Canine COL3A1 is a 1446 amino acid protein coded by XM_863148.1 at GenelD: 478835 [SEQ. ID. NO: 2], [0031] The terms “Collagen alpha (III) chain precursor isoform 12,” “Col3al” and “COL3A1” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 2. Preferably, COL3A1 is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 2. COL3A1 also includes protein variants of the sequence as shown in SEQ. ID. NO: 2, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term COL3A1 also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to XM_863148.1 and appended SEQ. ID. NO: 13. COL3A1 also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 13 and to fragments of SEQ. ID. NO: 13.

[0032] Canine decorin (DCN). Affymetrix probe Cfa.6065.1.Al_s_at corresponds to NCBI Reference Sequence: NP_001003228.1, accession NP_001003228, decorin [Canis lupus familiaris]. Canine decorin is a 360 amino acid leucine-rich repeat protein coded by NM 001003228.1 at GenelD: 403904 [SEQ.ID. NO: 3].

[0033] The terms “Decorin,” “Den” and “DCN” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 3. Preferably, decorin is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 3. Decorin also includes protein variants of the sequence as shown in SEQ. ID. NO: 3, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term decorin also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to NM_001003228.1 and

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8693-OO-WO-HL appended SEQ. ID. NO: 14. Decorin also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 14 and to fragments of SEQ. ID. NO: 14.

[0034] Canine secreted frizzled-related protein 2 (sFRP2). Affymetrix probe Cfa.l200.1.Sl_s_at corresponds to NCBI Reference Sequence: NP_001002987.1, accession NP_001002987 XP_532695, secreted frizzled-related protein 2 (sFRP2) [Canis lupus familiaris]. Canine secreted frizzled-related protein 2 (sFRP2) is a 294 amino acid protein coded by NM_001002987.1 at GenelD: 475471 [SEQ.ID. NO: 4], [0035] The terms “secreted frizzle-related protein 2,” “SFRP2,” and “sFRP2” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 4. Preferably, sFRP2 is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 4. sFRP2 also includes protein variants of the sequence as shown in SEQ. ID. NO: 4, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term sFRP2 also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to NM_001002987.1 and appended SEQ. ID. NO: 15. sFRP2 also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 15 and to fragments of SEQ. ID. NO: 15.

[0036] Canine matrilin-2 (Matn2). Affymetrix probe Cfa.9487.1Al_at corresponds to NCBI Reference Sequence: XP_5485552.2, accession NP_5485552, similar to matrilin 2 isoform a precursor [Canis lupus familiaris]. Canine matrilin-2 is a 978 amino acid protein coded by XM_5485552.2 at GenelD: 491431 [SEQ.ID. NO: 5], [0037] The terms “matrilin 2 isoform a precursor,” “matrilin 2” and “Matn2” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 5. Preferably, Matn2 is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 5. Matn2 also includes protein variants of the sequence as shown in SEQ. ID. NO: 5, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term Matn2 also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to XM_5485552.2 and appended SEQ. ID. NO: 16. Matn2 also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 16 and to fragments of SEQ. ID. NO: 16.

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8693-00-WO-HL [0038] Canine retinol binding protein 4 (rbp4). Affymetrix probe Cfa.l5489.1.Sl_at corresponds to NCBI Reference Sequence: XP_534969.2 accession NP 534969, similar to retinol binding protein 4, plasma precursor (rbp4) [Canis lupus familiaris]. Canine retinol binding protein 4 (rbp4) is a 267 amino acid protein coded by XM_534969.2 at GenelD: 477775 [SEQ.ID.NO: 6].

[0039J The terms “retinol binding protein 4,” “plasma precursor,” “retinol” and “Rbp4” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 6. Preferably, Rbp4 is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 6. Rbp4 also includes protein variants of the sequence as shown in SEQ. ID. NO: 6, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term Rbp4 also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to XM_534969.2 and appended SEQ. ID. NO: 17. Rbp4 also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 17 and to fragments of SEQ. ID. NO: 17.

[0040] Canine matrix metalloproteinase 9 (MMP-9). Affymetrix probe Cfa.3470.1Sl_at corresponds to NCBI Reference Sequence: NP_001003219.1 accession NP_001003219, matrix metalloproteinase 9 [Canis lupus familiaris]. Canine matrix metalloproteinase 9 (MMP9) is a 704 amino acid protein coded by NM_001003219.1 at GenelD: 403885 [SEQ.ID. NO: 7], [0041] The terms “matrix metalloproteinase 9” and “MMP9” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 7. Preferably, MMP9 is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 7. MMP9 also includes protein variants of the sequence as shown in SEQ. ID. NO: 7, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term MMP9 also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to NM_001003219.1 and appended SEQ. ID. NO: 18. MMP9 also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 18 and to fragments of SEQ. ID. NO: 18 [0042] Canine clusterin (CLU). Affymetrix probe Cfal254.Sl_s_at corresponds to NCBI Reference Sequence: NM_001003370.1 accession NM_001003370 canine clusterin. Canine clusterin is a 445 amino acid protein coded by NM_001003370.1 at GenelD: 442971 [SEQ.ID. NO: 8],

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8693-OO-WO-HL [0043] The terms “clusterin’’ and “Clu” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 8. Preferably, clusterin is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 8. Clusterin also includes protein variants of the sequence as shown in SEQ. ID. NO: 8, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term clusterin also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to NM_001003370.1 and appended SEQ. ID. NO: 19 Clusterin also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 19 and to fragments of SEQ. ID. NO: 19 [0044] Canine transferrin (TF). Affymetrix probe Cfa2217.1.Al_at corresponds to NCBI Reference Sequence: XP_534268.2 accession XP_534268 similar to canine serotransferrin precursor (Transferrin) isoform 1. Canine transferrin is a 705 amino acid protein coded by XM_534268.2 at GenelD: 477072 [SEQ.ID. NO: 9].

[0045] The terms “canine serotransferrin precursor (Transferrin) isoform 1,” “transferrin” and “TF” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 9. Preferably, transferrin is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 9. Transferrin also includes protein variants of the sequence as shown in SEQ. ID. NO: 9, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term transferrin also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to XM_534268.2 and appended SEQ. ID. NO: 20 Transferrin also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 20 and to fragments of SEQ. ID. NO: 20.

[0046] Canine apolipoproetin C-l (ApoCl). Affymetrix probe Cfal254.Sl_s_at corresponds to NCBI Reference Sequence: XP 533643.2 accession XP_533643 similar to canine apolipoproetin C-I precursor (ApoCl). Canine apolipoproetin C-I (ApoCl) is an 88 amino acid protein coded by NM_533643.2 at GenelD: 113459 [SEQ.ID. NO: 10], [0047] The terms “apolipoproetin C-l” and “ApoCl” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 10. Preferably, transferrin is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 10. ApoCl also includes protein variants of the sequence as shown in SEQ. ID. NO: 10, such as allelic variants

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8693-OO-WO-HL and other mutations such as substitutions, additions and/or deletions. The term ApoCl also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to NM_533643.2 and appended SEQ. ID. NO: 21 ApoCl also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 21 and to fragments of SEQ. ID. NO: 21.

[0048] Inhibin beta A (INHBA). Affymetrix probe Cfa596.1Al_at corresponds to NCBI Reference Sequence: XP_540364 accession XP_540364 similar to inhibin beta A chain precursor (INHBA) (Activin beta-A-chain) (erythroid differentiation protein; EDF). Canine inhibin beta A chain precursor (INHBA) is a 424 amino acid protein coded by XM_540364.2 at GenelD: 483245 [SEQ.ID. NO: 11], [0049] The terms “Inhibin beta A chain precursor” and “INHBA” denote a protein having substantially the amino acid sequence as shown in SEQ. ID. NO: 11. Preferably, inhibin is a protein substantially consisting of the amino acid sequence as shown in SEQ. ID. NO: 11. Inhibin also includes protein variants of the sequence as shown in SEQ. ID. NO: 11, such as allelic variants and other mutations such as substitutions, additions and/or deletions. The term inhibin also refers to the nucleic acid sequence encoding the protein. The sequence corresponds to XM_540364.2 and appended SEQ. ID. NO: 22 Transferrin also refers to nucleic acid sequences that hybridize to SEQ. ID. NO: 22 and to fragments of SEQ. ID. NO: 22.

[0050] In a preferred embodiment of the present invention, a method for diagnosing a kidney disorder in a canine comprises the steps of: (a) measuring the level of expression of at least one biomarker in a biological sample from the canine, wherein the at least one biomarker is selected from secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA); and (b) identifying the canine as having a kidney disorder wherein differences in expression of the one or more biomarkers in the sample relative to a control value for expression in a sample from a normal animal indicates the existence of a kidney disorder.

[0051] In a another preferred embodiment of the present invention, the canine has normal kidney function as defined by art-recognized clinical measurements, e.g., glomerular flirtation rate, creatinine clearance, urinary protein levels, blood creatinine levels, urinary creatinine levels

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8693-OO-WO-HL and/or blood urea nitrogen levels and the methods of the invention may be used to detect and diagnose in such canine a change from a normal state to an abnormal state leading to a kidney disorder characterized by reduced renal function, renal failure, reduced glomerular filtration rate and glomerulonephritis.

[0052] In another preferred embodiment, an elevated level of activity or expression of one or more genes in a canine selected from the group consisting of: lumican (LUM); collagen alpha 1 (III) chain, variant 12 (COL3A1); decorin (DCN); secreted frizzle-related protein 2 (sFRP2); and matrilin-2 (Matn2) correlates with a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis. The level of activity or expression of one or more genes may be determined by measuring the expression product of such genes which may be a polynucleotide or a polypeptide or protein, typically an RNA transcript or its translation product.

[0053] In another preferred embodiment, differential expression of one or more genes in a canine selected from at least one RNA transcript or its translation product selected from the group consisting of: secreted frizzled-related protein 2 (sFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA) correlates with an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis.

[0054] In another preferred embodiment, differential expression of one or more genes in a canine selected from a first group of at least one RNA transcript or its translation product selected from the group consisting of: secreted frizzled-related protein 2 (sFRP2) orrmatrilin-2 (Matn2); and optionally, a second group of at least one RNA transcript or its translation product selected from the group consisting of: lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA) correlates with an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis. It is to be understood that the present invention contemplates combination of biomarkers comprising genes or their expression products that are both (i) selected from a first group of at least one

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RNA transcript or its translation product selected from the group consisting of: secreted frizzledrelated protein 2 (sFRP2) or matrilin-2 (Matn2); and optionally, a second group of at least one RNA transcript or its translation product selected from the group consisting of: lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA); as well as (ii) selected from a first group of at least one RNA transcript or its translation product selected from the group consisting of; secreted frizzled-related protein 2 (sFRP2) or matrilin-2 (Matn2); and a second group of at least one RNA transcript or its translation product selected from the group consisting of: lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA). Embodiments of the invention contemplate constructing panels of biomarkers from various combinations of the two groupings of genes and their expression products.

[0055] In another preferred embodiment, the invention encompasses measurement and correlation of differential expression in a canine of one or more genes selected from at least one RNA transcript or its translation product selected from the group consisting of: secreted frizzledrelated protein 2 (sFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA), with accelerated or early loss of renal function in canines as evidenced by an abnormal process leading to a kidney disorder characterized by reduced renal function, reduced glomerular filtration rate, glomerulonephritis or renal failure.

[0056] In one aspect the invention includes contacting a tissue sample or bodily fluid specimen with an agent that detects in a canine one or more genes or the expression product of such one or more genes selected from at least one RNA transcript or its translation product selected from the group consisting of: secreted frizzled-related protein 2 (sFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA). The agent can be an antibody or a nucleic acid probe used in conjunction with

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8693-OO-WO-HL conventional assay means such as immobilization on a solid phase, mierotiter wells, tubes, dipsticks or other conventional means.

[0057] Another embodiment of the method of the invention encompasses use of conventional assay means to determine gene expression in a canine either alone or in conjunction with gene expression array displays employing polypeptides and/or polynucleotides, such conventional assay means comprising one or more of ELISA, RIA, immunoblot assays, in situ hybridization, Northern blot analysis, Western blot analysis and Luminex X-Map® analysis.

[0058] Another embodiment of the invention encompasses diagnosing a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis over time by determining gene expression profiles for one or more genes or their expression products selected from at least one RNA transcript or its translation product selected from the group consisting of: secreted frizzled-related protein 2 (sFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA) in conjunction with one or more conventional diagnostic measurements selected from the group consisting of: determining levels of albumin, serum cystatin C, blood creatinine, urinary creatinine, creatinine clearance, urinary protein, blood urea nitrogen and glomerular filtration rate, to choose a course of treatment for such canine.

[0059] In another embodiment, the invention features a kit for diagnosing and/or monitoring a kidney disorder in a canine over time, the kit comprising one or more agents capable of detecting expression profiles for one or more genes or their expression products selected from at least one RNA transcript or its translation product selected from the group consisting of: secreted frizzledrelated protein 2 (sFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (111) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA), and instructions for using said one or more agents to evaluate the risk of such canine developing a kidney disorder characterized by an abnormal loss of renal function, renal failure, reduced glomerular filtration rate or glomerulonephritis over time.

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8693-OO-WO-HL [0060] In another aspect, the invention includes contacting a tissue sample or bodily fluid specimen with an agent that detects in one or more genes or their expression products selected from at least one RNA transcript or its translation product selected from the group consisting of: secreted frizzled-related protein 2 (sFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA). The agent can be an antibody or a nucleic acid probe used in conjunction with conventional assay means such as immobilization on a solid phase, microtiter wells, tubes, dipsticks or other conventional means.

[0061] Particularly preferred embodiments of the present invention follow below. In each of the following embodiments, the present invention contemplates a test sample comprising either solid kidney tissue samples, or samples of biological fluids from a canine. Differential expression of the recited genes contemplates a significant difference in absolute terms. In preferred embodiments the differential expression may be greater than about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or 3.0 fold or greater. Especially preferred differential expression values are greater than about 2 fold or one standard deviation around the mean. Also, the embodiments of the invention may employ a variety of art-recognized detection means, including an array of one or more hybridization probes, panels of one or more antibodies and combinations of these technologies. When immunoassays are desired, the skilled worker can select among many art-recognized techniques, including immunoassays selected from the group consisting of a competitive binding assay, a non-competitive binding assay, a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), a sandwich assay, a precipitin reaction, a gel diffusion immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, chemiluminescence immunoassay, immunoPCR immunoassay, a protein A or protein G immunoassay and an Immunoelectrophoresis assay. When conventional diagnostic measurements are specified, these may be selected from the group consisting of: determining levels of albumin, serum cystatin C, blood creatinine, urinary creatinine, creatinine clearance, urinary protein, blood urea nitrogen and glomerular filtration rate

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8693-OO-WO-HL [0062] In addition, the methods and compositions of the invention contemplate utilizing at least one RNA transcript or its translation product which may be an altered form of the gene or its translation product.

[0063] A still further embodiment of the invention comprises a method for diagnosing a kidney disorder in a canine, comprising the steps of: (a) measuring the level of expression of at least one biomarker in a biological sample from the canine, wherein the at least one biomarker is selected from at least one gene or the translation product of such gene selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA); and (b) identifying the canine as having a kidney disorder wherein difference in expression of the one or more biomarkers in the sample relative to a control value for expression in a sample from a normal animal indicates the existence of a kidney disorder.

[0064] Another embodiment comprises a kit for the diagnosis, prognosis or monitoring a kidney disorder in a canine, comprising at least one or more agent capable of detecting at least one or more RNA transcript or its translation product selected from a first group of at least one gene or the translation product of such gene selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM), decorin (DCN), collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA); and instructions for using such at least one or more agent to measure the expression of the one or more biomarkers and to diagnose a kidney disease in such canine.

[0065] In a further aspect, the invention relates to compositions comprising one or more nucleic acid probes that specifically hybridize to a nucleic acid, or fragment thereof, encoding a biomarker of the present invention selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA).

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8693-OO-WO-HL [0066] In an additional aspect, the invention relates to compositions comprising antibodies that specifically bind to a polypeptide encoded by a gene expressing a biomarker of the present invention selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DON); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA).

[0067] It is further contemplated herein that the methods of the present invention may be used in combination with traditional diagnostic techniques that are able to detect the physical and morphological characteristics of kidney disorders. Thus, for example, the characterization of differential expression in genes for kidney in cells obtained from a tissue samples or bodily fluid specimens of a canine may be combined with conventional diagnostic (e.g., radiological) techniques in order to corroborate a diagnosis of a kidney disorder in a canine, including, for example, glomerulonephritis.

[0068] The invention also relates to kits to diagnose an abnormal kidney disorder in a canine comprising components that can be used to detect expression of the biomarkers of the present invention, including, but not limited to, the compositions and microarrays described herein.

[0069] A further aspect of the invention is a method for diagnosis and/or prognosis of kidney disorder in a canine, wherein the method comprises the steps of: obtaining at least one tissue sample or bodily fluid specimen from the animal; determining the amount of one or more biomarkers selected from Table 3 in said at least one sample or specimen obtained from the animal, wherein said biomarker is a polypeptide, protein, RNA, DNA, polynucleotide or metabolite thereof.

[0070] Yet another embodiment of the invention is a kit for diagnosis and/or prognosis of a kidney disorder in a canine, particularly for carrying out the method for diagnosis and/or prognosis of glomerulonephritis in a canine wherein the method comprises the steps of: obtaining at least one tissue sample or bodily fluid specimen from the animal; determining the amount of one or more biomarkers selected from Table 3 in said at least one sample or specimen obtained from the animal, wherein said biomarker is a polypeptide, protein, RNA, DNA,

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8693-OO-WO-HL polynucleotide or metabolite thereof, and optionally, further comprising a detectable agent linked to said biomarker.

[0071] A still further embodiment of the invention is a reagent for diagnosis and/or prognosis of glomerulonephritis in a canine particularly for carrying out the method for diagnosis and/or prognosis of glomerulonephritis in a canine, wherein the method comprises the steps of: obtaining at least one tissue sample or bodily fluid specimen from the animal; determining the amount of one or more biomarkers selected from Table 3 in said at least one sample or specimen obtained from a canine, wherein said biomarker is a polypeptide, protein, RNA, DNA, polynucleotide or metabolite thereof, and optionally further comprising a detectable agent linked to said biomarker.

[0072] A further aspect of the invention is a method for diagnosis and/or prognosis of kidney disorder in a canine, wherein the method comprises the steps of: obtaining at least one tissue sample or bodily fluid specimen from the animal; determining the amount of one or more biomarkers selected from Tables 3 and 4 in said at least one sample or specimen obtained from the animal, wherein said biomarker is a polypeptide, protein, RNA, DNA, polynucleotide or metabolite thereof.

[0073] Another embodiment of the invention is the use of one or more polypeptides, proteins, RNAs, DNAs, polynucleotides or metabolites thereof selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA) as a biomarker for diagnosis and/or prognosis of a kidney disorder, particularly for forming a kit for diagnosis or prognosis of a kidney disorder in a canine.

[0074] Another embodiment of the invention is the use of one or more polypeptides, proteins, RNAs, DNAs, polynucleotides or metabolites thereof selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin

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8693-OO-WO-HL beta A (INHBA) as a biomarker for diagnosis and/or prognosis of a kidney disorder, particularly for forming a kit for diagnosis or prognosis of a kidney disorder in a canine.

[0075] Yet another embodiment is such kit, wherein the reagents and equipment comprise DNA microarray analysis materials including oligonucleotide microarray, c-DNA microarray, and focused gene chip, or a combination thereof.

[0076] Another embodiment of the invention is a method of assessing the progression of a course of nutritional management for a canine suffering from a kidney disorder, the method comprising (a) measuring a first level of one or more biomarker polypeptides, proteins, RNAs, DNAs, polynucleotides or metabolites thereof selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA) in a tissue sample or a specimen of bodily fluid from said canine at a first point in time during the course of treatment, (b) measuring a second level of said biomarker in said sample or specimen from said canine at a second point in time during the course of treatment, and (c) comparing the measurements of the biomarker at said first point and said second point; wherein expression of the biomarker has at least about a 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, or 3.0 fold differential or greater difference in gene expression compared with expression in a cell of a control animal.

[0077] Still another embodiment of the invention is a method for identifying a plurality of genes that are differentially expressed between tissue samples or specimens of a bodily fluid for use in an informative array, comprising: providing a first set of heterogeneous nucleic acid probes derived from a first tissue sample; providing a second set of heterogeneous nucleic acid probes derived from a second tissue sample; hybridizing a nucleic acid array comprising a plurality of sequences derived from genes of a biological process with the first set of probes and determining a first level of expression for sequences of the array; hybridizing the array with said second set of probes and determining a second level of expression for sequences of the array; identifying a plurality of genes that are differentially expressed in said biological process by comparing the first level of expression with said second level of expression for hybridized

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8693-OO-WO-HL sequences; and establishing a ranking of the identified genes by a step selected from the group of steps consisting of: determining an absolute value of the difference between the first level of expression and the second level of expression, and ranking genes having a higher difference over genes having a lower difference; and determining a standard deviation of the difference between the first level of expression and the second level of expression, and ranking genes having a higher standard deviation over genes having a lower standard deviation, wherein the genes are selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2); matrilin-2 (Matn2); lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA).

[0078] Another embodiment of the invention is a method for converting a nucleic acid array into an informative array, comprising: providing a first set of heterogeneous nucleic acid probes derived from a first tissue sample; providing a different, second set of heterogeneous nucleic acid probes derived from a second tissue sample; hybridizing a nucleic acid array comprising a plurality of sequences with the first set of probes and determining a first level of expression for sequences of the array; hybridizing the array with said second set of probes and determining a second level of expression for sequences of the array; identifying a plurality of genes that are differentially expressed in said biological process based on a difference between the first level of expression and the second level of expression for identified genes, by a step selected from the group of steps consisting of: determining an absolute value for the difference between the first level of expression and the second level of expression, and ranking genes having a higher difference over genes having a lower difference; and determining a standard deviation of the difference between the first level of expression and the second level of expression, and ranking genes having a higher standard deviation over genes having a lower standard deviation; and selecting genes from the plurality of identified differentially expressed genes for inclusion on the informative array, wherein said genes are selected from the group consisting of: secreted frizzlerelated protein-2 (SFRP2) and matrilin-2 (Matn2); and, optionally, one or more genes selected from the group consisting of: lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA).

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8693-OO-WO-HL [0079] Another embodiment of the invention is a method of determining the efficacy of a treatment for a kidney disorder, comprising the steps of: (a) providing a biological sample from a canine affected by a kidney disorder, who has been subjected to said treatment, (b) determining the level in said sample of one or more biomarkers for glomerulonephritis, to create an expression profile for said canine, and (c) comparing said expression profile with: i) a comparable expression profile obtained from said test canine before initiation of said treatment, and/or ii) a comparable expression profile obtained from said test canine at an earlier stage of said treatment, and/or iii) a comparable expression profile characteristic of a subject who is unaffected by a kidney disorder, wherein the one or more biomarkers for a kidney disorder, comprise expression products of one or more gene selected from the group consisting of: secreted frizzle-related protein-2 (SFRP2) and matrilin-2 (Matn2); and, optionally, one or more genes selected from the group consisting of: lumican (LUM); decorin (DCN); collagen alpha 1 (III) chain, variant 12 (COL3A1); retinol binding protein 4 (rbp4); matrix metalloproteinase 9 (MMP9); clusterin (CLU); transferrin (TF); Apo-C-1 (ApoCl); and inhibin beta A (INHBA).

[0080] Other and further objects, features, and advantages of the present invention will be readily apparent to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION [0081] As used herein, an abnormal canine is a canine that has a risk of developing, predisposition for, or an abnormal loss of kidney function, renal failure, reduced glomerular filtration rate or glomerulonephritis, wherein the abnormality is detectable by utilizing at least one relevant biomarker isolated and measured from a biological specimen taken from such canine.

[0082] Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as

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8693-OO-WO-HL individual numbers within that range, for example, 1, 2, 3, 4, and 5. This applies regardless of the breadth of the range.

[0083] The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label.

[0084] The present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics.

[0085] Those skilled in the art will recognize that the products and methods embodied in the present invention may be applied to a variety of systems, including commercially available gene expression monitoring systems involving nucleic acid probe arrays, membrane blots, microwells, beads and sample tubes, constructed with various materials using various methods known in the art. Accordingly, the present invention is not limited to any particular environment, and the following description of specific embodiments of the present invention are for illustrative purposes only.

[0086] The gene expression monitoring system, in a preferred embodiment, may comprise a nucleic acid probe array (including an oligonucleotide array, a cDNA array, a spotted array, and the like), membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, beads or fibers (or any solid support comprising bound nucleic acids). The gene expression monitoring system may also comprise nucleic acid probes in solution.

[0087] The present invention also contemplates sample preparation involving amplification. A genomic sample may be amplified by a variety of mechanisms, some of which may employ PCR. The sample may be amplified on the array.

[0088] Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer

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8693-OO-WO-HL et al. Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and W090/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA).

[0089] The gene expression monitoring system according to the present invention may be used to facilitate a comparative analysis of expression in different cells or tissues, different subpopulations of the same cells or tissues, different physiological states of the same cells or tissue, different developmental stages of the same cells or tissue, or different cell populations of the same tissue. In a preferred embodiment, the proportional amplification methods of the present invention can provide reproducible results (i.e., within statistically significant margins of error or degrees of confidence) sufficient to facilitate the measurement of quantitative as well as qualitative differences in the tested samples.

[0090] The term “antibody” means any immunoglobulin that binds to a specific antigen, including IgG, IgM, IgA, IgD, and IgE antibodies. The term includes polyclonal, monoclonal, monovalent, humanized, heteroconjugate, antibody compositions with polyepitopic specificity, chimeric, bispecific antibodies, diabodies, single-chain antibodies and antibody fragments such as Fab, Fab’, F(ab’)2, and Fv, or other antigen-binding fragments.

[0091] The term “array” means an ordered arrangement of at least two probes on a substrate. At least one of the probes is a control or standard and at least one of the probes is a diagnostic probe. The arrangement of from about two to about 40,000 probes on a substrate assures that the size and signal intensity of each labeled complex formed between a probe and a sample polynucleotide or polypeptide is individually distinguishable. The collection of molecules deposited on the array may be prepared either synthetically or biosynthetically. The array may take a variety of forms including libraries of soluble molecules, libraries of compounds tethered to resin beads, silica chips or other solid supports. The nucleic acid array may include libraries of nucleic acids which can be prepared by spotting nucleic acids in essentially any length (for

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8693-OO-WO-HL example, from 1 to about 1,000 nucleotides in length) onto a substrate. A nucleic acid probe array preferably comprises nucleic acids bound to a substrate in known locations. In other embodiments, the system may include a solid support or substrate, such as a membrane, filter, microscope slide, microwell, sample tube, bead, bead array, or the like. The solid support may be made of various materials, including paper, cellulose, nylon, polystyrene, polycarbonate, plastics, glass, ceramic, stainless steel, or the like. The solid support may preferably have a rigid or semi-rigid surface, and may preferably be spherical (e.g., bead) or substantially planar (e.g., flat surface) with appropriate wells, raised regions, etched trenches, or the like. The solid support may also include a gel or matrix in which nucleic acids may be embedded.

[0092] The term “biomarker” refers to genes and gene products encoded by the gene of the invention, i.e., where the gene has been determined to have been differentially regulated as a result of a kidney disorder. In addition, the term can be generally used to refer to any portion of such gene or protein that can identify or correlate with the full-length gene or protein, for example, in an assay or other method of the invention.

[0093] Biomarker expression can also be identified by detection of biomarker translation (i.e., detection of biomarker protein in a sample). Methods suitable for the detection of biomarker protein include any suitable method for detecting and/or measuring proteins from a cell or cell extract. Such methods include, but are not limited to, immunoblot (e.g., Western blot), enzymelinked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry and immunofluorescence. Particularly preferred methods for detection of proteins include any single-cell assay, including immunohistochemistry and immunofluorescence assays. Such methods are well known in the art. Furthermore, antibodies against certain of the biomarkers described herein are known in the art and are described in the public literature, and methods for their preparation are well known to the skilled worker.

[0094] The term “differential expression” or “differentially expressed” means increased or unregulated gene expression or means decreased or downregulated gene expression as detected by the absence, presence, or at least two-fold, or at least a 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 or 1.0-fold change in the amount of transcribed messenger RNA or translated protein in a sample.

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8693-OO-WO-HL [0095] The term “fold” when used as a measure of differential gene expression means an amount of gene expression in a canine that is a multiple or a fraction of gene expression compared to the amount of gene expression against a control value. For example, a gene that is expressed 2 times as much in the animal as compared to the control value has a 2-fold differential gene expression and a gene that is expressed one-third as much in the animal as compared to the control value also has a 2-fold differential gene expression.

[0096] The term “fragment” means (1) an oligonucleotide or polynucleotide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polynucleotide sequence or (2) a peptide or polypeptide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polypeptide sequence. Such fragments can comprise any number of nucleotides or amino acids deemed suitable for a particular use. Generally, oligonucleotide or polynucleotide fragments contain at least about 10, 50, 100, or 1000 nucleotides and polypeptide fragments contain at least about 4, 10, 20, or 50 consecutive amino acids from the complete sequence. The term encompasses polynucleotides and polypeptides variants of the fragments. A polynucleotide, for example, can be broken up, or fragmented into, a plurality of segments.

[0097] Various methods of fragmenting nucleic acid are well known in the art. These methods may be, for example, either chemical or physical in nature. Chemical fragmentation may include partial degradation with a DNase; partial depurination with acid; the use of restriction enzymes; intron-encoded endonucleases; DNA-based cleavage methods, such as triplex and hybrid formation methods, that rely on the specific hybridization of a nucleic acid segment to localize a cleavage agent to a specific location in the nucleic acid molecule; or other enzymes or compounds which cleave DNA at known or unknown locations. Physical fragmentation methods may involve subjecting the DNA to a high shear rate. High shear rates may be produced, for example, by moving DNA through a chamber or channel with pits or spikes, or forcing the DNA sample through a restricted size flow passage, e.g., an aperture having a cross sectional dimension in the micron or submicron scale. Other physical methods include sonication and nebulization. Combinations of physical and chemical fragmentation methods may likewise be employed such as fragmentation by heat and ion-mediated hydrolysis. These methods can be optimized to digest a nucleic acid into fragments of a selected size range. Useful size ranges may

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8693-OO-WO-HL be from 100, 200, 400, 700 or 1000 to 500, 800, 1500, 2000, 4000 or 10,000 base pairs. However, larger size ranges such as 4000, 10,000 or 20,000 to 10,000, 20,000 or 500,000 base pairs may also be useful.

[0098] The term “gene” or “genes” means a complete or partial segment of DNA involved in producing a polypeptide, including regions preceding and following the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). The term encompasses any DNA sequence that hybridizes to the complement of gene coding sequences.

[0099] The term “homolog” means (1) a polynucleotide, including polynucleotides from the same or different animal species, having greater than 30%, 50%, 70%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to a polynucleotide and having the same or substantially the same properties and performing the same or substantially the same function as the complete polynucleotide, or having the capability of specifically hybridizing to a polynucleotide under stringent conditions or (2) a polypeptide, including polypeptides from the same or different animal species, having greater than 30%, 50%, 70%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity to a polypeptide identified by the expression of polynucleotides and having the same or substantially the same properties and performing the same or substantially the same function as the complete polypeptide, or having the capability of specifically binding to a polypeptide identified by the expression of polynucleotides. Sequence similarity of two polypeptide sequences or of two polynucleotide sequences is determined using methods known to skilled artisans, e.g., the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403410 (1990)). To obtain gapped alignments for comparison purposes, Gapped Blast can be utilized as described in Altschul et al. (Nucl. Acids Res. 25: 3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

[00100] The term “hybridization refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term

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8693-OO-WO-HL hybridization may also refer to triple-stranded hybridization. The resulting (usually) doublestranded polynucleotide is a hybrid. The proportion of the population of polynucleotides that forms stable hybrids is referred to herein as the degree of hybridization.

[00101] Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, polynucleotides derived from one sample are hybridized to the probes in a nucleic acid array. Signals detected after the formation of hybridization complexes correlate to the polynucleotide levels in the sample. In the differential hybridization format, polynucleotides derived from two samples are labeled with different labeling moieties. A mixture of these differently labeled polynucleotides is added to a nucleic acid array. The nucleic acid array is then examined under conditions in which the emissions from the two different labels are individually detectable. In one embodiment, the fluorophores Cy3 and Cy5 (Amersham Pharmacia Biotech, Piscataway, N.J.) are used as the labeling moieties for the differential hybridization format.

[00102] Signals gathered from nucleic acid arrays can be analyzed using commercially available software, such as those provided by Affymetrix or Agilent Technologies. Controls, such as for scan sensitivity, probe labeling and cDNA or cRNA quantization, are preferably included in the hybridization experiments. Hybridization signals can be scaled or normalized before being subject to further analysis. For instance, hybridization signals for each individual probe can be normalized to take into account variations in hybridization intensities when more than one array is used under similar test conditions. Hybridization signals can also be normalized using the intensities derived from internal normalization controls contained on each array. In addition, genes with relatively consistent expression levels across the samples can be used to normalize the expression levels of other genes. In one embodiment, probes for certain maintenance genes are included in a nucleic acid array of the present invention. These genes are chosen because they show stable levels of expression across a diverse set of tissues. Hybridization signals can be normalized and/or scaled based on the expression levels of these maintenance genes.

[00103] The term “hybridization complex” means a complex that is formed between sample polynucleotides when the purines of one polynucleotide hydrogen bond with the pyrimidines of the complementary polynucleotide, e.g., 5’-A-G-T-C-3’ base pairs with 3’-T-C-A-G-5’. The

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8693-OO-WO-HL degree of complementarily and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.

[00104] The term “hybridization probes includes nucleic acids (such as oligonucleotides) capable of binding in a base-specific manner to a complementary strand of nucleic acid.

[00105] The term “kidney disease” or “kidney disorder” or analogously “renal disease” or “renal disorder” is intended to cover an acute or chronic abnormal loss of kidney function, such as renal failure, reduced glomerular filtration rate and glomerulonephritis. Glomerulonephritis may take the form of membranous glomerulonephritis which involves thickening of the glomerular basement membrane. Alternatively, glomerulonephritis may take the form of proliferative or mesangioproliferative glomerulonephritis, which is characterized by proliferation of cells in the mesangial matrix. In addition, glomerulonephritis may take the form of membranoproliferative glomerulonephritis which involves a combination of the foregoing changes. Glomerulosclerosis is a severe form of glomerulonephritis. Kidney disease or kidney disorders also include nephritis, nephropathy, hyperfdtration, mild microalbuminuria, clinical albuminuria, advanced clinical nephropathy, chronic renal insufficiency, injuries to renal papilla, tubular necrosis and diabetic nephropathy, all as differentially diagnosed by veterinarians of ordinary skill in the art. The term is not intended to encompass polycystic kidney disease of genetic origin.

[00106] The term “polynucleotide” or “oligonucleotide” means a polymer of nucleotides. The term encompasses DNA and RNA (including cDNA and mRNA) molecules, either single or double stranded and, if single stranded, its complementary sequence in either linear or circular form. The term also encompasses fragments, variants, homologs, and alleles, as appropriate for the sequences that have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. The sequences may be fully complementary (no mismatches) when aligned or may have up to about a 30% sequence mismatch. Preferably, for polynucleotides, the chain contains from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. Preferably, for oligonucleotides, the chain contains from about 2 to 100 nucleotides, more preferably from about 6 to 30 nucleotides. The exact size of a polynucleotide or oligonucleotide will depend on various

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8693-OO-WO-HL factors and on the particular application and use of the polynucleotide or oligonucleotide. The term includes nucleotide polymers that are synthesized and that are isolated and purified from natural sources. The term “polynucleotide” is inclusive of “oligonucleotide.” [00107] The term “polypeptide,” “peptide,” or “protein” means a polymer of amino acids. The term encompasses naturally occurring and non-naturally occurring (synthetic) polymers and polymers in which artificial chemical mimetics are substituted for one or more amino acids. The term also encompasses fragments, variants, and homologs that have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. The term encompass polymers of any length, preferably polymers containing from about 2 to 1000 amino acids, more preferably from about 5 to 500 amino acids. The term includes amino acid polymers that are synthesized and that are isolated and purified from natural sources.

[00108] The term “probe” means (1) an oligonucleotide or polynucleotide, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, that is capable of annealing with or specifically hybridizing to a polynucleotide with sequences complementary to the probe or (2) a peptide or polypeptide capable of specifically binding a particular protein or protein fragment to the substantial exclusion of other proteins or protein fragments. An oligonucleotide or polynucleotide probe may be either single or double stranded. The exact length of the probe will depend upon many factors, including temperature, source, and use. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe typically contains about 10 to 100, 15 to 50, or 15 to 25 nucleotides. In certain diagnostic applications, a polynucleotide probe contains about 100-1000, 300-600, nucleotides, preferably about 300 nucleotides. The probes herein are selected to be “substantially” complementary to different strands of a particular target sequence. This means that the probes must be sufficiently complementary to specifically hybridize or anneal with their respective target sequences under a set of predetermined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a noncomplementary nucleotide fragment may be attached to the 5’ or 3’ end of the probe, with the remainder of the probe sequence being complementary to the target sequence. Alternatively, noncomplementary bases or longer sequences can be interspersed into

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8693-OO-WO-HL the probe provided that the probe sequence has sufficient complementarity with the sequence of the target polynucleotide to specifically anneal to the target polynucleotide. A peptide or polypeptide probe may be any molecule to which the protein or peptide specifically binds, including DNA (for DNA binding proteins), antibodies, cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organellar membranes.

[00109] The terms “sample” and “specimen” mean any animal tissue or fluid containing, e.g., polynucleotides, polypeptides, antibodies, metabolites, and the like, including cells and other tissue containing DNA and RNA. Examples include: blood, cartilage, connective, epithelial, lymphoid, muscle, nervous, sputum, and the like. A sample may be solid or liquid and may be DNA, RNA, cDNA, bodily fluids such as blood or urine, cells, cell preparations or soluble fractions or media aliquots thereof, chromosomes, organelles, and the like.

[00110] The term “single package” means that the components of a kit are physically associated in or with one or more containers and considered a unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, bottles, shrink wrap packages, stapled or otherwise affixed components, or combinations thereof. A single package may be containers of individual food compositions physically associated such that they are considered a unit for manufacture, distribution, sale, or use.

[00111] The term “specifically bind” means a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.

[00112] The term “specifically hybridize” means an association between two single stranded polynucleotides of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”). For example, the term may refer to hybridization of a polynucleotide probe with a substantially complementary sequence contained within a single stranded DNA or RNA

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8693-OO-WO-HL molecule according to an aspect of the invention, to the substantial exclusion of hybridization of the polynucleotide probe with single stranded polynucleotides of non-complementary sequence.

[00113] The term “stringent conditions” means (1) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C, (2) hybridization in 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt’s solution, sonicated salmon sperm DNA (50 pg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C; with washes at 42°C in 0.2x SSC and 0.1% SDS or washes with 0.015 M NaCl, 0.0015 M sodium citrate, 0.1% NaaSCL at 50°C or similar procedures employing similar low ionic strength and high temperature washing agents and similar denaturing agents.

[00114] The term “substance” means an element, compound, molecule, or a mixture thereof or any other material that could potentially be useful for diagnosing, prognosing, or modulating the onset or severity of a kidney disorder in an animal, including any drug, chemical entity, or biologic entity.

[00115] The term “useful variations” means (1) for a polynucleotide, the complements of the polynucleotide; the homologs of the polynucleotide and its complements; the variants of the polynucleotide, its complements, and its homologs; and the fragments of the polynucleotide, its complements, its homologs, and its variants and (2) for a polypeptide, the homologs of the polypeptide; the variants of the polypeptide and its homologs; and the fragments of the polynucleotide, its homologs, and its variants.

[00116] The term “variant” means (1) a polynucleotide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more nucleotides from or to a polynucleotide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence and (2) a polypeptide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more amino acids from or to a polypeptide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence. The term therefore includes single nucleotide polymorphisms (SNPs) and

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8693-OO-WO-HL allelic variants and includes conservative and non-conservative amino acid substitutions in polypeptides. The term also encompasses chemical derivatization of a polynucleotide or polypeptide and substitution of nucleotides or amino acids with nucleotides or amino acids that do not occur naturally, as appropriate.

[00117] The term “virtual package” means that the components of a kit are associated by directions on one or more physical or virtual kit components instructing the user how to obtain the other components, e.g., in a bag containing one component and directions instructing the user to go to a website, contact a recorded message, view a visual message, or contact a caregiver or instructor to obtain instructions on how to use the kit.

[00118] The invention is not limited to the particular methodology, protocols, and reagents described herein because they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise, e.g., reference to “a variant” includes a plurality of variants. Further, defined terms include variations of the terms used in the proper grammatical context, e.g., the term “specifically binds” includes “specific binding” and other forms of the term. Similarly, the words “comprise,” “comprises,” and “comprising” are to be interpreted inclusively rather than exclusively.

[00119] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein.

[00120] All patents, patent applications, publications, and other references cited or referred to herein are incorporated herein by reference to the extent allowed by law. The discussion of those references is intended merely to summarize the assertions made therein. No admission is made that any such patents, patent applications, publications or references, or any portion thereof, is

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8693-OO-WO-HL relevant prior art for the present invention and the right to challenge the accuracy and pertinence of such patents, patent applications, publications, and other references is specifically reserved.

[00121] In one embodiment, the present invention encompasses one or more genes or gene segments (“genes” as defined herein) that are differentially expressed in abnormal animals compared to a control value obtained from one or more normal animals. The invention is based upon the discovery of polynucleotides that are differentially expressed in abnormal animals compared to normal animals. The genes were identified by comparing the expression of genes in tissue samples taken from animals diagnosed as abnormal with genes in tissue samples from animals diagnosed as normal using Affymetrix GeneChip® technology.

[00122] The polynucleotides and genes are identified by measuring differences in gene expression from tissue samples taken from canines diagnosed as abnormal and having a kidney disorder against a control value, wherein the control value is obtained from a sample from one or more normal animals. Changes in gene expression can be determined by any method known to skilled artisans. Generally, changes in gene expression are determined by measuring transcription (determining the amount of mRNA produced by a gene) or measuring translation (determining the amount of protein produced by a gene). The amount of RNA or protein produced by a gene can be determined using any method known to skilled artisans for quantifying polynucleotides and proteins.

[00123] Generally, mRNA expression is determined using polymerase chain reaction (PCR) (including, without limitation, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR)), short or long oligonucleotide arrays, cDNA arrays, EST sequencing, Northern blotting, SAGE, MPSS, MS, bead arrays and other hybridization methods. The RNA measured is typically in the form of mRNA or reverse transcribed mRNA.

[00124] Protein or polypeptide expression is determined using various colormetric and spectroscopic assays and methods such as quantitative Western blots, ELISA, 2D-gels, gas or liquid chromatography, mass-spec, the lowry assay, the biuret assay, fluorescence assays, turbidimetric methods, the bicinchoninic assay, protein chip technology, infrared absorbance, ninhydrin, the Bradford assay, and ultraviolet absorbance.

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8693-OO-WO-HL [00125] In a preferred method, changes in gene expression are determined using Affymetrix Canine-1 and Canine-2 gene chips available from Affymetrix, Inc. and the instructions for using such chips to determine gene expression. Gene chips allow a large-scale study of biological processes and the measurement of activity within a cell at a certain point in time. Microarray analysis permits one to account for differences in phenotypes on a large-scale genetic basis. Actual measurement of gene expression products is a more accurate indicator of gene function than determining sequences per se. Microarray analysis is based upon quantifying the concentration of a gene’s mRNA transcript in a cell at a given time. DNA is immobilized on a medium and labeled target mRNA is hybridized with probes on the array. Binding of the labeled mRNA to the probes is measured by laser analysis. The measurement is a count of photons emitted. The entire chip is scanned and digitally imaged. The image is processed to locate probes and to assign intensity measurements to each probe. In this manner up- and downregulated genes may be determined. The analysis enables the skilled person to find groups of genes with similar expression profiles and to determine tissues with similar expression profiles. In this manner, genes that explain the observed differences in tissue samples can be identified.

[00126] Affymetrix Gene Chips typically employ probes of 25bp and probe sets of 11 to 20 probes corresponding to a particular gene or EST. The chip is constructed with a perfect match and mismatch probe of 25bp each, the former being perfectly complementary to a specific region of a gene and the latter having the 13^th bp substituted to make a mismatch. A probe summarization algorithm is used to determine background correction, normalization and probe summarization, which is the conversion of probe values to probeset expression values. RMA is one of the algorithms that may be used for this purpose. The algorithm performs the last two steps of analysis, normalization and summarization of probe-level intensity measurements. The perfect match values are, therefore, background corrected, normalized and summarized into a set of expression measurements.

[00127] The raw data is analyzed using GeneSpring version 7.0 (GS) software (Agilent Corporation) and validated using the R-Bioconductor (RB) freeware. Both software packages are used to compute probe intensities from the CEL files generated by the Affymetrix Instrument. The Present/Absent/Marginal calls per probe and P-values are computed using the RBioconductor and GeneSpring software separately.

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8693-OO-WO-HL [00128] Generally, differential gene expression is determined by measuring the expression of at least one gene. Preferably, the expression of two or more differentially expressed genes is measured to provide a gene expression pattern or gene expression profile. More preferably, the expression of a plurality of differentially expressed genes is measured to provide additional information for a more significant gene expression pattern or profile.

[00129] The present invention provides a plurality of markers that together or alone are or can be used as markers of renal disease. In especially useful embodiments of the invention, a plurality of these markers can be selected and their mRNA expression may be measured simultaneously to provide expression profiles for use in various aspects of the inventions described in this application. In a preferred embodiment of the present methods and compositions, at least 2, 3, 4, 5, 6, 7, 8, 9,10, or 11 markers are selected among lumican (LUM); collagen alpha 1 (III) chain, variant 12 (COL3A1); decorin (DCN); secreted frizzled-related protein 2 (sFRP2); matrilin-2 (Matn2); retinol binding protein 4 (rbp4); MMP-9; clusterin (CLU); transferrin (TF), Apo-C-1 (ApoCl); and inhibin beta A (INHBA) and can be used for determination of gene expression profiles employed in the practice of the methods of the invention. Each marker can be particularly linked to certain aspects of kidney disease.

[00130] In another embodiment of the invention, the level of gene expression can be alternatively determined by detecting the protein corresponding to the gene expression product. Secreted proteins of the denominated genes are preferred.

[00131] In another aspect, the invention provides a device suitable for detecting the expression of a plurality of genes differentially expressed in abnormal animals compared to a control values. The device comprises a substrate having a plurality of the oligonucleotide or polynucleotide probes of the present invention affixed to the substrate at known locations. The device is essentially an immobilized version of the oligonucleotide or polynucleotide probes described herein. The device is useful for rapid and specific detection of genes and polynucleotides and their expression patterns and profiles. Typically, such probes are linked to a substrate or similar solid support and a sample containing one or more polynucleotides (e.g., a gene, a PCR product, a ligase chain reaction (LCR) product, a DNA sequence that has been synthesized using amplification techniques, or a mixture thereof) is exposed to the probes such that the sample

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8693-OO-WO-HL polynucleotide(s) can hybridize to the probes. Either the probes, the sample polynucleotide(s), or both, are labeled, typically with a fluorophore or other tag such as streptavidin, and detected using methods known to skilled artisans. If the sample polynucleotide(s) is labeled, hybridization may be detected by detecting bound fluorescence. If the probes are labeled, hybridization is typically detected by label quenching. If both the probe and the sample polynucleotide(s) are labeled, hybridization is typically detected by monitoring a color shift resulting from proximity of the two bound labels. A variety of labeling strategies and labels are known to skilled artisans, particularly for fluorescent labels. Preferably, the probes are immobilized on substrates suitable for forming an array (known by several names including DNA microarray, gene chip, biochip, DNA chip, and gene array) comparable to those known in the art.

[00132] The polypeptide probes may be made according to conventional methods, e.g., using the nucleotide sequence data provided for polynucleotides of the present invention and methods known in the art. Such methods include, but are not limited to, isolating polypeptide directly from cells, isolating or synthesizing DNA or RNA encoding the polypeptides and using the DNA or RNA to produce recombinant products, synthesizing the polypeptides chemically from individual amino acids, and producing polypeptide fragments by chemical cleavage of existing polypeptides.

[00133] In another aspect, the invention provides a device suitable for detecting the expression of a plurality of genes differentially expressed in abnormal animals compared to a control value. The device comprises a substrate having a plurality of the peptide or polypeptide probes of the present invention affixed to the substrate at known locations. The device is essentially an immobilized version of the peptide or polypeptide probes described herein. The device is useful for the rapid and specific detection of proteins and their expression patterns. Typically, such probes are linked to a substrate and a sample containing one or more proteins is exposed to the probes such that the sample proteins can hybridize to the probes. In certain embodiments, the probes, the sample proteins, or both, are labeled and detected, typically with a fluorophore or other agent known to skilled artisans. Generally, the same methods and instrumentation used for reading polynucleotide microarrays is applicable to protein arrays. Preferably, the probes are immobilized on a substrate suitable for forming an array.

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8693-OO-WO-HL [00134] Methods for determining the amount or concentration of protein in a sample are known to skilled artisans. Such methods include radioimmunoassays, competitive-binding assays, Western blot analysis, and ELISA assays. For methods that use antibodies, polyclonal and monoclonal antibodies are suitable. Such antibodies may be immunologically specific for a protein, protein epitope, or protein fragment.

[00135] Some embodiments of the invention utilize antibodies for the detection and quantification of proteins produced by expression of the polynucleotides of the present invention. Although proteins may be detected by immunoprecipitation, affinity separation, Western blot analysis, protein arrays, and the like, a preferred method utilizes ELISA technology wherein the antibody is immobilized on a solid support and a target protein or peptide is exposed to the immobilized antibody. Either the probe, or the target, or both, can be labeled using known methods.

[00136] In some embodiments, expression patterns or profiles of a plurality of genes differentially expressed in abnormal animals are observed utilizing an array of probes for detecting polynucleotides or polypeptides. In one embodiment, arrays of oligonucleotide or polynucleotide probes may be utilized, whereas another embodiment may utilize arrays of antibodies or other proteins that specifically bind to the differentially expressed gene products of the present invention. Such arrays may be commercially available or they may be custom made using methods known to skilled artisans, e.g., in-situ synthesis on a solid support or attachment of pre-synthesized probes to a solid support via micro-printing techniques. In various embodiments, arrays of polynucleotides or polypeptides probes are custom made to specifically detect transcripts or proteins produced by the differentially expressed genes of the present invention.

[00137] In one embodiment, arrays of polynucleotide or polypeptide probes are custom made to specifically detect transcripts or proteins produced by two or more polynucleotides or genes identified in Table 2. These probes are designed to detect genes associated with lipid and glucose metabolism pathways in animals. In another embodiment, arrays of polynucleotide or polypeptide probes are custom made to specifically detect transcripts or proteins produced by

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8693-OO-WO-HL two or more polynucleotides or genes identified in Tables 3-5. These probes are designed to detect genes that are particularly relevant to abnormal canines compared to normal canines.

[00138] These probes are exposed to a sample to form hybridization complexes that are detected and compared with those of a standard. The differences between the hybridization complexes from the sample and standard indicate differential expression of polynucleotides and therefore genes differentially expressed in abnormal canines compared to a control value in the sample. In a preferred embodiment, probes are made to specifically detect polynucleotides or fragments thereof produced by one or more of the genes or gene fragments identified by the present invention. Methods for detecting hybridization complexes are known to skilled artisans.

[00139] In another aspect, the invention provides a method for detecting the differential expression of genes differentially expressed in abnormal canines compared to normal canines in a sample. The method comprises (a) reacting a combination comprising a plurality of polypeptide probes with proteins in the sample under conditions that allow specific binding between the probes and the proteins to occur, wherein the proteins bound by the probes are differentially expressed in an abnormal canine compared to a normal canine; (b) optionally, reacting a combination comprising a plurality of polypeptide probes with proteins in a standard under conditions that allow specific binding between the probes and the proteins to occur, wherein the proteins bound by the probes are differentially expressed in an abnormal canine compared to a normal canine; (c) detecting specific binding in the sample and, optionally, the standard from step (b); and (d) comparing the specific binding in the sample with that of a standard, wherein differences between the specific binding in the standard and the sample indicate differential expression of genes differentially expressed in abnormal canines compared to normal canines in the sample.

[00140] These probes are exposed to a sample to form specific binding that is detected and compared with those of a standard. The differences between the specific binding from the sample and standard indicate differential expression of proteins and therefore genes differentially expressed in abnormal canines compared to normal canines, particularly abnormal-associated genes, in the sample. In a preferred embodiment, probes are made to specifically detect proteins

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8693-OO-WO-HL or fragments thereof produced by one or more of the genes or gene fragments identified by the present invention.

[00141] In one embodiment, the method further comprises exposing the canine or sample to a test substance before reacting the polypeptides with the proteins. Then, the comparison is indicative of whether the test substance altered the expression of genes differentially expressed in abnormal canines compared to normal canines, particularly abnormal-associated genes, in the sample.

[00142] Example 1: Classification of canines with chronic kidney disease according to the guidelines of the International Renal Interest Society.

[00143] In the Examples that follow, canines exhibiting clinical signs of chronic kidney disease were tested versus animals not exhibiting signs or symptoms of chronic kidney disease. Pathological diagnoses of chronic kidney disease were made based on the criteria set forth in Tables 1 and 2 below and in accordance with the guidelines of the International Renal Interest Society (IRIS).

[00144] Staging of chronic kidney disease (CKD) is undertaken following the diagnosis of CKD in order to facilitate appropriate treatment and monitoring of the subject animal. Staging is based initially on fasting plasma creatinine, assessed on at least two occasions in the stable animal. Canines demonstrating normal renal function and no clinical signs or symptoms of CKD were grouped as non-disease canines. Stage 1 in canines corresponds to prior classifications of early renal disease with no biochemical evidence of CKD to renal insufficiency, where no azotemia is detected, but where glomerular filtration rate (GFR) may be reduced and there may be a poor concentrating ability of the kidneys. Stage 2 corresponds to the prior classification of early renal failure. In Stage 2, mild azotemia is noted. Stage 3 corresponds to prior classification of uremic renal failure, where moderate azotemia is detected. Systemic signs of uremic renal failure may be present such as bone pain, uremic gastritis, anemia and metabolic acidosis. Stage 4 corresponds to end-stage renal failure, which is characterized by severe azotemia and increasing systemic clinical signs of uremic crisis.

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8693-OO-WO-HL [00145] Table 1 identifies the five categories of canines studied, respectively. A total of 42 dogs diagnosed as not having CKD were studied. A total of 14 Stage 1 canines exhibited minimal glomerulonephritis (GN). The number of dogs studied exhibiting advanced stages of CKD were: Stage 2 mild GN = 24; Stage 3 moderate GN =8 and Stage 4 marked GN = 13. Plasma creatinine levels for each of the groups of dogs are shown in Table 2 as mean and median plasma creatinine levels for each group of dogs.

Table 1: Staging for Canines

IRIS CKD Staging	Plasma Creatinine range mg/dl
Non-Disease	<1.4 with no firm evidence of disease
Stage 1	<1.4 (<125 pmol/l) with evidence of disease. Non-azotemic. Some other renal abnormality present (e.g. inadequate concentrating ability without identifiable non-renal cause; abnormal renal palpation and/or renal imagining findings; proteinuria of renal origin; abnormal renal biopsy
Stage 2	1.4 to 2.0 (125-179 pmol/l) Mild renal azotemia. Clinical signs usual mild or absent.
Stage 3	2.1 to 5.0 (180-439 pmol/l) Moderate renal azotemia. Many clinical signs may be present.
Stage 4	>5.0 (>440pmol/l) Sever renal azotemia. Many extra-renal clinical signs present.

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Table 2 Creatinine Levels

Pathological Diagnosis	Mean Plasma Creatinine mg/dl	Median Plasma Creatinine mg/dl
Non-Disease (n = 42)	0.8	0.7
Minimal GN (n = 14)	0.7	0.7
Mild GN (n = 24)	0.9	0.7
Moderate GN (n = 8)	1.5	1.5
Marked GN (n = 13)	6.7	6.2

[00146] Example 2: Candidate selection criteria [00147] The raw data in the following examples was analyzed using GeneSpring version 7.0 (GS) software (Agilent Corporation) and validated using the R-Bioconductor (RB) freeware. Both software packages are used to compute probe intensities from the CEL files generated by the Affymetrix Instrument. The Present/Absent/Marginal calls per probe and P-values are computed using the R-Bioconductor and GeneSpring software separately.

[00148] The gene expression data is determined to be either “up” or “down” -regulated for any given analysis. The decision on whether a gene is “up” or “down” is based on the fold change, which is calculated as treatment intensity/control intensity for each individual probe. The fold change is considered down-regulated if its value is < 1/2 and is up-regulated if it is > 2.0. Also, a probe is considered significant for further scrutiny if it is called as present in only one of the conditions being compared (treatment or control) and is “absent” or “marginal” in the other and the fold change is significant according to the software used.

[00149] Example 3: RNA Isolation Procedures [00150] Materials and Methods. The following general procedures may be used to isolate RNA from tissue samples of dogs and cats for gene expression profiling utilizing gene chips as further described in the Examples of this specification. It will be apparent to a person of ordinary skill

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8693-OO-WO-HL in the art that these procedures or modifications thereof, as recognized within the art, can be applied to isolate RNA from tissue or body fluid samples for further gene expression analysis using a variety of analytical procedures available to a person of ordinary skill in the art, in particular microarray technologies.

[00151] Isolation of Ribonucleic Acid (RNA) from Tissue. Tissue samples may be collected, frozen in liquid nitrogen, thawed and then, ground in a mortal and pestle, homogenized and transferred to a 50ml conical flask. The homogenized tissue sample is then processed using a TRIzol® RNA extraction method according to the manufacturer’s instructions (Invitrogen) to produce good quality RNA which is then subjected to further genomic analysis.

[00152] Materials: ice, liquid nitrogen, frozen canine tissue, TRIzol® lysis reagent, chloroform minimum 99%, isopropyl alcohol, 70% ethanol (prepared with ethanol, absolute and deionized, RNase-free water), RNase Zap®, deionized water, RNA Storage Solution®, from Ambion.

[00153] Equipment: Ultra-Turrax T25 Power Homogenizer, Beckman Coulter Allegra 25R Centrifuge, Eppendorf Centrifuge, forceps, scalpel, hard cutting surface, i.e. cutting board, 1.5mL DNase and RNase free/sterile microcentrifuge tubes, 50mL DNase and RNase free/sterile disposable polypropylene tubes, Pl000, P200, P20, P10 and P2 Rainin Pipetman pipettes, filter pipette tips for Pl000, P200, P20, P10 and P2 pipettes, DNase and RNase free/sterile, and lint free wipes.

[00154] Preparations: Prepare 50mL polypropylene tubes with 4mL TRIzol® (one tube for each tissue selected for RNA isolation).

[00155] Tissue Homogenization: Fill a container capable of holding liquid nitrogen with 3-4 scoops of liquid nitrogen. Place a piece of frozen tissue immediately into the aforementioned container (the tissue should be about the size of a pea) and place the tissue into the appropriate labeled 50mL polypropylene tube (that already contains 4mL TRIzol®). Immediately begin homogenization using the Ultra-Turrax T25 Power Homogenizer. Homogenize on the highest setting (6) for 10-15 seconds. Cool the sample on ice for another 10-15 seconds and then repeat. Continue until the tissue is fully homogenized and the solution is cloudy. Upon complete

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8693-OO-WO-HL homogenization, cap the 50mL tube and return to the ice. Incubate the homogenized tissues at room temperature for 5 minutes before proceeding with the isolation procedure.

[00156] Example 4: RNA Preparation Procedures. RNA Isolation: The procedures given in the Invitrogen instructions provided with the TRIzol® reagent are generally followed. Separate the homogenized sample into four ImL aliquots in four 1.5mL microcentrifuge tubes. Add 200uL of chloroform to each ImL aliquot. Cap the tubes, vortex for 15 seconds and then shake up and down. The result should be a pink milky liquid. Incubate the tubes at room temperature for 2-3 minutes. Centrifuge the tubes for 15 minutes at 14,000 rpm and 4°C. Transfer the aqueous phase (top layer) to a sterile 1.5mL microcentrifuge tube. The typical volume of the aqueous phase which should be transferred to the new tube is about 500uL. Be sure not to transfer any of the intermediate or lower phase. Precipitate the RNA from solution by adding 500uL of Isopropyl Alcohol to each micro centrifuge tube containing the aqueous layer. Shake the tubes up and down for at least 20 seconds. Incubate the samples at room temperature for 10 minutes. Centrifuge the samples for 10 minutes, 14,000 rpm at 4°C. Remove the supernatant carefully by aspirating off the liquid being sure not to lose the pellet. Add ImL of 70% ethanol to wash the pellet. Dislodge the pellet by flicking the tube (or tapping the tube on the bench top) and shake to mix. Centrifuge for 5 minutes, 8,200 rpm at 4°C. Remove the supernatant carefully by aspirating off the liquid being sure not to lose the pellet. Use a lint free wipe to carefully soak up excess ethanol to make sure the pellet is dry. Resuspend each pellet into 30uL of RNA Storage Solution. Mix gently by pipetting until the RNA goes back into solution and then store at -80°C. It may be necessary to vortex the sample for a few seconds at a low speed to facilitate the resuspension of the RNA. If this is necessary, spin down the samples, using the microcentrifuge, prior to freezing.

[00157] RNA Cleaning: The procedures given in the RNeasy® Mini Handbook are followed.

[00158] RNA Isolation from Cells Cultured in OptiCell Chambers Using the RNeasy Mini Kit. Cells cultured from mammalian cell lines are used to isolate good quality RNA which is then used for future downstream genomic analysis. All work related to the culturing of the cells is to be done under strict aseptic conditions.

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8693-OO-WO-HL [00159] Reagents: 10X PBS, deionized H2O, absolute ethanol, RNA Storage Solution, βMercaptoethanol, RNase Zap®, Buffer RLT, and Buffer RW1 and Buffer RPE (provided in the RNeasy Mini Kit).

[00160] Equipment/Materials: RNeasy Mini Kit, QIAshredder spin columns, OptiCell knife, 20mL sterile syringe, OptiCell tips, Cell scraper, Pl000 Pipetman pipette, Rainin, P200 Pipetman pipette, Rainin, 100-100uL filtered pipette tips, l-200uL filtered pipette tips, sterile transfer pipettes, 55mL sterile solution basin, 1.5mL sterile microcentrifuge tubes, and Eppendorf Microcentrifuge.

[00161] Solutions: Buffer RLT (stock provided in RNeasy Mini Kit); -Add lOOuL of βMercaptoethanol per lOmL of Buffer RLT prior to beginning protocol. 70% Ethanol: Make 50mL of 70% ethanol by adding 35mL absolute ethanol to 15mL deionized, RNase-free water. IX PBS: RNase-free water. Lilter the solution using a ,22um filter.

[00162] Procedure: Removing Cells from the OptiCell Chamber (proceed one OptiCell at a time). Check the cells under a microscope to ensure that the cells are alive before isolating RNA. Remove and discard the cell culture medium. Using the OptiCell knife, cut away the top membrane exposing the cells on the lower membrane. Wash the membrane to which the cells are attached three times with IX PBS. Pipette 600uL of the Buffer RLT solution (containing βMercaptoethanol) onto the center of the membrane to which the cells are attached. Using the cell scraper, gently spread the Buffer RLT over the entire surface of the membrane, and then collect the liquid in one comer. Pipette off the entire volume of Buffer RLT and place into a QIAshredder spin column.

[00163] RNA Isolation: Centrifuge the QIAshredder spin columns at 14,000 rpm for 2 minutes. Discard the spin column but keep the collection tube and its contents. Add 600uL of 70% ethanol to the collection tube and mix well by pipetting (the total volume now = 1,2mL). Transfer 600uL of the cell lysate to an RNeasy mini column and centrifuge for 15 seconds at 14,000 rpm. Discard the flow through but keep the collection tube and the spin column. Transfer the remaining volume of cell lysate (~600uL) to the spin column and repeat the centrifugation. Discard the flow through but keep the collection tube and the spin column. Add 700uL Buffer RW1 to the spin column. Centrifuge for 15 seconds at 14,000 rpm to wash the column. Discard

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8693-OO-WO-HL the flow through and the collection tube. Transfer the spin column to a new 2mL collection tube and add 500uL Buffer RPE to the column. Centrifuge for 15 seconds at 14,000 rpm. Discard the flow through, keep the collection tube/column. Add another 500uL Buffer RPE to the column. Centrifuge for 2 minutes at 14,000 rpm. Transfer the spin column to a 1.5mL collection tube. Add 30uL of RNA Storage Solution directly to the silica gel membrane and centrifuge for 1 minute at 14,000 rpm to elute the RNA. Store the final RNA at -70°C.

[00164] RNA 6000 Nano Assay. Using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Assay, analyze RNA isolated from cultured mammalian cells, lymphocytes or tissues for quality.

[00165] Reagents: RNA 6000 Nano gel matrix, RNA 6000 Nano dye concentrate, RNA 6000 Nano Marker, (all of the above reagents are contained in the RNA 6000 Nano Assay kit, Agilent), RNA 6000 ladder, RNase Zap , and RNase-free water, from Ambion.

[00166] Equipment/Other Materials: Agilent Chip Priming Station, Agilent, RNA 6000 chip, Agilent, electrode cleaners, P2, P10, P200, and Pl000 Rainin Pipetman pipettes, sterile, DNase/RNase free filtered pipette tips, 1.5mL microcentrifuge tubes, sterile, vortex, IKA vortex mixer, microcentrifuge, and heating block.

[00167] Procedure: The procedure is given in the Reagent Kit Guide, RNA 6000 Nano Assay, Edition November 2003, by Agilent Technologies. The procedures are followed as given in the Guide, with the following modifications: Preparing the Gel, pg. 17- rather than separating the filtered gel into aliquots of 65uL each, keep the stock filtered gel in the original microcentrifuge tube and aliquot the 65uL as needed. Loading the RNA 6000 Nano Marker, pg. 22- add luL of RNase-free water (instead of RNA 6000 Nano Marker) to each sample well that will not contain sample. Not only will this conserve the amount of Marker used but also serves as a negative control to see that none of the reagents are contaminated, including the RNase-free water. Loading the Ladder and Samples, pg. 23- heat denature the samples and RNA 6000 Ladder for an additional 30 seconds (total of 2.5 minutes) at 71 °C. Starting the Chip Run, pg. 26- choose the “Eukaryote Total RNA Nano” option from the assay menu.

[00168] Example 5: Affymetrix GeneChip Expression Analysis. Gene expression is analyzed using Affymetrix Canine 1 and Canine 2 GeneChip ® Arrays which are commercially

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8693-OO-WO-HL available from Affymetrix, Inc., Santa Clara, CA 95051. Total RNA is reverse transcribed into cDNA. The cDNA is used to generate cRNA which is fragmented and used as probes for GeneChip hybridization. The gene chip is washed and the hybridization signal is measured with an Affymetrix laser scanner. The hybridization data is then validated and normalized for further analysis in accordance with instructions from the manufacturer.

[00169] Materials: Affymetrix provides most of the reagents and kit. Other reagents listed in the Affymetrix Manual but not supplied in the kit may be obtained separately (refer to GeneChip Expression Analysis Technical Manual (701021 Rev.4) for details), RNase Zap® and deionized water.

[00170] Equipment: Eppendorf microcentrifuge, 1.5mL DNase and RNase free/sterile microcentrifuge tubes, 50mL DNase and RNase free/sterile disposable polypropylene tubes, Pl000, P200, P20, P10 and P2 Rainin Pipetman pipettes, Filter pipette tips for Pl000, P200, P20, P10 and P2 pipettes, DNase and RNase free/sterile, and Peltier Thermal Cycler PTC-200.

[00171] Procedure: follow all procedures exactly as described in GeneChip Expression Analysis Technical Manual (Affymetrix Copyright 1999-2003). Use 5 microgram of total RNA for the first strand cDNA synthesis. Use either Peltier Thermal Cycler PTC-200 or heat block for temperature control on reactions and probe denaturing. The quality control is performed using RNA NanoDrop chips with BioAnalyer 2100. Use 100 Format (Midi Array) for the canine genechip.

[00172] Example 6: Gene Expression in Dogs with Chronic Kidney Disease [00173] Studies were conducted in accordance with the previous Examples 1-5 using dogs having various stages of chronic kidney disease to determine the underlying gene expression differences between dogs with normal renal function and dogs having minimal, mild, moderate and marked glomerulonephritis corresponding to Stages 1 through 4 as presented on Table 1. Procedures as described in the Examples of this specification were used to prepare tissue and bodily fluid samples from 42 dogs having normal renal function, 14 dogs having minimal glomerulonephritis, 24 dogs having mild glomerulonephritis, 8 dogs having moderate

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8693-OO-WO-HL glomerulonephritis and 13 dogs having marked glomerulonephritis, as determined by plasma creatinine levels presented in Table 2 and by clinical observation.

[00174] Based on gene expression data comparing the dogs with normal renal function versus dogs having glomerulonephritis, as defined in the preceding examples, the five genes listed on Table 3 were identified as meeting the selection criteria of Example 2 as potential biomarkers of chronic kidney disorders in dogs. The genes include lumican (LUM); collagen alpha 1 (III) chain, variant 12 (COL3A1); Decorin (DCN); secreted frizzled-related protein 2 (SFRP2); and Matrilin-2 (Matn2). Analogous human synonyms and mRNA and protein accession numbers are listed on Table 3 for each gene. Each of the proteins is a secreted protein.

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Table 3: List of Potential Biomarkers

Gene	Symbol in Canine/Human	Human Synonyms	Canine Synonym	Gene Description	mRNA	Protein
Lumican	LUM	LDC; SLRR2D			XM_539716.2	XP_539716.1
Collagen alpha 1 (III) chain, variant 12	COL3A1	EDS4A; FLJ34534			XM_858055.1	XP_863148
Decorin	DCN	CSCD; DSPG2; PG40; PGII; PGS2; SLRR1B			NM_001003228.1	NP_001003228.1
SFRP2	SFRP2	FRP-2; SARP1; SDF-5		secreted frizzledrelated protein 2	NM_001002987.1	NP_001002987.1
Matrilin-2	Matn2	Crtm2; MGC102477; matrilin-2		matrilin 2	XM_548552.2	XP_548552.2

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Table 4: List of Potential Biomarkers

Gene	Symbol In Canine/Human	Human Synonyms	Canine Synonym	Gene Description	mRNA	Protein
Retinol Binding Protein 4	Rbp4	Rbp-4		retinol binding protein 4, plasma	XM_534969.2	XP_534969.2
MMP-9	MMP9	CLG4B; GELB; MMP-9		matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)	NM_001003219.1	NP_001003219.1
Clusterin	CLU	AAG4; APOJ; CLI; KUB1; MGC24903; SGP-2; SGP2; SP40: TRPM-2; TRPM2	GP80		NM_001003370.1	NP_001003370.1

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Table 5:

List of Potential Biomarkers

Gene	Symbol in Canine/Human	Human Synonyms	Canine Synonym	Gene Description	mRNA	Protein
T ransferrin	TF	DKFZp781D0156; PRO1557; PRO2086			XM_534268	XP_534268
Apo-C-1	APOC1			apolipoprotein C-l	XM_533643	XP_533643
Inhibin beta A	INHBA	EDF; FRP			XM_540364	XP_540364

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Claims (16)

1. CLAIMS:

   1. A method for diagnosing a kidney disorder in a canine, comprising the steps of: measuring the level of expression of a biomarker in a biological sample from the canine, wherein the biomarker is lumican; and identifying the canine as having a kidney disorder wherein differences in expression of the biomarker in the sample relative to a control value for expression in a sample from a normal animal indicates the existence of a kidney disorder, wherein the kidney disorder is glomerulonephritis.
2. 2. The method of claim 1, wherein the test sample is a sample of solid kidney tissue or a bodily fluid.
3. 3. The method of claim 1, wherein the differential expression of step (b) is determined with an array of one or more hybridization probes.
4. 4. The method of claim 1, wherein the differential expression of step (b) is determined with a panel of one or more antibodies that specifically bind to the at least one RNA transcript or its translation product.
5. 5. The method of claim 1, wherein the differential expression of step (b) is determined by immunoassay to detect the at least one RNA transcript or its translation product.
6. 6. The method of claim 5, wherein the immunoassay is selected from the group consisting of a competitive binding assay, a non-competitive binding assay, a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), a sandwich assay, a precipitin reaction, a gel diffusion immunodiffusion assay, an agglutination assay, a fluorescent immunoassay, chemiluminescence immunoassay, immunoPCR immunoassay, a protein A or protein G immunoassay, Northern blot analysis, Western blot analysis,

   Luminex(TM)xMAP(TM) detection and an Immunoelectrophoresis assay.

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7. 7. The method of any preceding claim wherein the canine has normal renal function, wherein normal renal function is determined by one or more of: glomerular filtration rate, urine protein level, blood creatinine level, urine creatinine level, creatinine clearance and blood urea nitrogen.
8. 8. The method of claim 1, further comprising use of one or more conventional diagnostic measurement selected from the group consisting of: determining levels of albumin, serum cystatin C, blood creatinine, urinary creatinine, creatinine clearance, urinary protein, blood urea nitrogen and glomerular filtration rate.
9. 9. The method of any preceding claim wherein the kidney disorder is indicated by a significant difference in expression of a biomarker relative to control expression values wherein a significant difference in the case of increased expression is an increase of at least twofold and in the case of decreased expression is a decrease of at least 50% and wherein the biomarkers is lumican.
10. 10. A method of treating, ameliorating, or delaying the progression of glomerulonephritis in a canine, comprising diagnosing a kidney disorder by the method of any preceding claim.
11. 11. The method of claim 1, wherein the method comprises using a kit comprising at least one or more agent capable of detecting lumican and instructions for using such at least one or more agent to measure the expression of the biomarker and to diagnose a kidney disease in such canine.
12. 12. The method of claim 12, wherein the at least one or more agent is an antibody.
13. 13. The method of claim 11, wherein the at least one or more agent is attached to a detectable label.

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14. 14. The method of claim 1, wherein the method comprises using an antibody recognizing lumican.
15. 15. The method of claim 15, wherein the antibody is comprised within a kit.
16. 16. A nucleic acid probe, when used for diagnosing a kidney disorder in a canine, capable of hybridizing to a canine gene wherein the canine gene is lumican.

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    8693-sequence-listi ng.txt SEQUENCE LISTING <110> AL-MURRANI, SAMEER WALEED MALLADI, SUKHASWAMI GAO, XIANGMING <120> COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING KIDNEY DISORDERS IN AN ANIMAL <130> 27794-8693 <140>

    <141>

    <160> 22 <170> Patentln version 3.5 <210> 1 <211> 338 <212> PRT <213> Canis familiaris <400> 1

    Met 1 Asn Leu Gly val 5 Phe Thr Leu Leu Leu Ala Leu lie Gly Gly Al a 10 15 Ser Gly Gin Tyr Tyr Asp Tyr Asp Phe Ser Leu Pro lie Tyr Gly Leu 20 25 30 Ser Ser Pro Asn Cys Ala Pro Glu Cys Asn Cys Pro Gl u Ser Tyr Pro 35 40 45 Ser Ala Met Tyr Cys Asp Glu Leu Lys Leu Lys Ser Val Pro Met Val 50 55 60 Pro Pro Gly lie Lys Tyr Leu Tyr Leu Arg Asn Asn Gin lie Asp Hi s 65 70 75 80 lie Asp Glu Lys Al a Phe Glu Asn Val Thr Asp Leu Gin T rp Leu lie 85 90 95 Leu Asp Hi s Asn Leu Leu Glu Asn Ser Lys lie Lys Gly Arg lie Phe 100 105 110 Ser Lys Leu Lys Gin Leu Lys Lys Leu Hi s lie Asn Hi s Asn Asn Leu 115 120 125 Thr Glu Ser Val Gly Pro Leu Pro Lys Ser Leu val ASp Leu Gin Leu 130 135 140 Thr Hi s Asn Lys lie Gin Lys Leu Gly Ser Phe Asp Gly Leu Val Asn 145 150 155 160

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    8693-sequence-listi ng.txt

    Leu Thr Phe Val His 165 Leu Gin His Asn Gin 170 Leu Lys Glu Asp Thr 175 Val Ser Ala Al a Phe Lys Gly Leu Lys Ser Leu Glu Tyr Leu Asp Leu Ser 180 185 190 Tyr Asn Gin Met Al a Lys Leu Pro Ser Gly Leu Pro Al a Ser Leu Leu 195 200 205 Thr Leu Tyr Leu Asp Asn Asn Lys lie Ser Asn lie Pro Asp Glu Tyr 210 215 220 Phe Lys Arg Phe Asn Gly Leu Gin Tyr Leu Arg Leu Ser Hi s Asn Glu 225 230 235 240 Leu Ala Asp Ser Gly Val Pro Gly Asn Ser Phe Asn val Ser Ser Leu 245 250 255 Leu Glu Leu Asp Leu Ser Tyr Asn Lys Leu Lys Asn lie Pro Thr val 260 265 270 Asn Glu Asn Leu Glu Asn Tyr Tyr Leu Glu val Asn Glu Leu Glu Lys 275 280 285 Phe Glu Val Lys Ser Phe Cys Lys Il e Leu Gly Pro Leu Ser Tyr Ser 290 295 300 Lys lie Lys Hi s Leu Arg Leu Asp Gly Asn Arg Leu Thr Hi s Thr Ser 305 310 315 320 Leu Pro Pro Asp Met Tyr Glu Cys Leu Arg Val Al a Asn Glu lie Thr 325 330 335

    val Asn <210> 2 <211> 1466 <212> PRT <213> Cam's familiaris <400> 2

    Met Thr Ser Phe val Gin Lys Gly Thr Trp Leu Leu Leu Ala Leu Leu 1 5 10 15

    Gin Pro Ala Val lie Ser Ala Gin Gin Gin Ala lie Asp Gly Gly Cys 20 25 30

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    Ser Hi s Leu Gly Gl n Ser 8693-sequenceTyr Ala Asp Arg Asp 40 1i sti ng. txt Lys Pro Glu Val T rp 45 35 Pro Cys Gin lie Cys val Cys Asp Ser Gly Ser val Leu Cys Asp Asp 50 55 60 lie lie Cys Asp Glu Gin Glu Leu Asp Cys Pro Asn Pro Glu lie Pro 65 70 75 80 Phe Gly Glu Cys Cys Al a Val Cys Pro Gin Pro Pro Thr Ser Pro Pro 85 90 95 Arg Pro Pro Asn Gly His Gly Pro Gin Gly Pro Lys Gly Asp Pro Gly 100 105 110 Pro Pro Gly lie Pro Gly Arg Asn Gly Asp Pro Gly lie Pro Gly Gin 115 120 125 Pro Gly Ser Pro Gly Ser Pro Gly Pro Pro Gly lie Cys Glu Ser Cys 130 135 140 Pro Thr Gly Pro Gin Pro Asn Tyr Ser Pro Gin Phe Glu Ser Tyr Asp 145 150 155 160 Val Lys Al a Gly Val Ala Gly Gly Gly lie Gly Gly Tyr Pro Gly Pro 165 170 175 Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly Hi s Pro 180 185 190 Gly Ser Pro Gly Ser Pro Gly Tyr Gin Gly Pro Pro Gly Glu Pro Gly 195 200 205 Gin Ala Gly Pro Al a Gly Pro Pro Gly Pro Pro Gly Al a Met Gly Pro 210 215 220 Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro 225 230 235 240 Gl y Gl u Arg Gly Leu Pro Gly Pro Pro Gly Met Lys Gly Pro Al a Gly 245 250 255 Met Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg 260 265 270 Asn Gly Glu Lys Gly Asp Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn 275 280 285

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    8693-sequence-listi ng.txt

    Gly Leu Pro 290 Gly Glu Asn Gly 295 Ala Pro Gly Pro Met 300 Gly Pro Arg Gly Al a Pro Gly Gl u Arg Gly Arg Pro Gly Leu Pro Gly Al a Ala Gly Ala 305 310 315 320 Arg Gly Asn Asp Gly Al a Arg Gly Ser Asp Gly Gin Pro Gly Pro Pro 325 330 335 Gly Pro Pro Gly Thr Al a Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly 340 345 350 Glu Val Gly Pro Al a Gly Ser Pro Gly Ser Asn Gly Ser Pro Gly Gin 355 360 365 Arg Gly Gl u Pro Gly Pro Gin Gly Hi s Ala Gly Ala Pro Gly Pro Pro 370 375 380 Gly Pro Pro Gly Ser Asn Gly Ser Pro Gly Gly Lys Gly Glu Met Gly 385 390 395 400 Pro Ala Gly lie Pro Gly Ala Pro Gly Leu lie Gly Al a Arg Gly Pro 405 410 415 Pro Gly Pro Pro Gly Thr Asn Gly Ala Pro Gly Gin Arg Gly Gly Al a 420 425 430 Gly Gl u Pro Gly Lys Asn Gly Ala Lys Gly Glu Pro Gly Pro Arg Gly 435 440 445 Glu Arg Gly Glu Al a Gly Ser Pro Gly lie Pro Gly Pro Lys Gly Glu 450 455 460 Asp Gly Lys Asp Gly Ser Pro Gly Glu Pro Gly Al a Asn Gly Leu Pro 465 470 475 480 Gly Ala Al a Gly Glu Arg Gly Al a Pro Gly Phe Arg Gly Pro Ala Gly 485 490 495 Al a Asn Gly Leu Pro Gly Glu Lys Gly Pro Al a Gly Gl u Arg Gly Gly 500 505 510 Pro Gly Pro Ala Gly Pro Arg Gly Al a Pro Gly Gl U Pro Gly Arg Asp 515 520 525 Gly val Pro Gly Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly 530 535 540

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    8693-sequence-listi ng.txt

    Gly 545 Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro Gly Ser Gin Gly Glu 560 550 555 Ser Gly Arg Pro Gly Pro Pro Gly Pro Ser Gly Pro Arg Gly Gin Pro 565 570 575 Gly Val Met Gly Phe Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro Gly 580 585 590 Lys Asn Gly Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro Gin Gly Pro 595 600 605 Al a Gly Lys Asn Gly Glu Thr Gly Pro Gin Gly Pro Pro Gly Pro Thr 610 615 620 Gly Pro Al a Gly Asp Lys Gly Asp Al a Gly Pro Pro Gly Pro Gin Gly 625 630 635 640 Leu Gin Gly Leu Pro Gly Thr Ser Gly Pro Pro Gly Glu Asn Gly Lys 645 650 655 Pro Gly Glu Pro Gly Pro Lys Gly Glu Ser Gly Ser Pro Gly Val Pro 660 665 670 Gly Gly Lys Gly Asp Ser Gly Al a Pro Gly Glu Arg Gly Pro Pro Gly 675 680 685 Ala Ala Gly Pro Met Gly Pro Arg Gly Gly Al a Gly Pro Pro Gly Pro 690 695 700 Glu Gly Gly Lys Gly Al a Al a Gly Pro Pro Gly Pro Pro Gly Ser Al a 705 710 715 720 Gly Thr Pro Gly Leu Gin Gly Met Pro Gly Glu Arg Gly Gly Pro Gly 725 730 735 Gly Pro Gly Pro Lys Gly Asp Lys Gly Glu Pro Gly Ser Ala Gly Al a 740 745 750 Asp Gly Al a Pro Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro lie 755 760 765 Gly Pro Pro Gly Pro Ala Gly Gin Pro Gly Asp Lys Gly Gl u Gly Gly 770 775 780

    Ala Pro Gly Leu Pro Gly lie Ala Gly Pro Arg Gly Gly Pro Gly Glu Page 5

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    8693-sequence-listi ng.txt

    785 790 795 800

    Arg Gly Glu Hi s Gly 805 Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro 810 815 Gly Gin Asn Gly Glu Pro Gly Al a Lys Gly Glu Arg Gly Ala Pro Gly 820 825 830 Glu Lys Gly Glu Gly Gly Pro Pro Gly Val Al a Gly Pro Pro Gly Gly 835 840 845 Al a Gly Pro Ala Gly Pro Pro Gly Pro Gin Gly val Lys Gly Glu Arg 850 855 860 Gly Ser Pro Gly Gly Pro Gly Ala Ala Gly Phe Pro Gly Gly Arg Gly 865 870 875 880 Leu Pro Gly Pro Pro Gly Asn Asn Gly Asn Pro Gly Pro Pro Gly Ser 885 890 895 Ser Gly Al a Pro Gly Lys Asp Gly Pro Pro Gly Pro Pro Gly Asn Asn 900 905 910 Gly Ala Pro Gly Ser Pro Gly val Ser Gly Pro Lys Gly Asp Al a Gly 915 920 925 Gin Pro Gly Glu Lys Gly Ser Pro Gly Pro Gin Gly Pro Pro Gly Ala 930 935 940 Pro Gly Pro Leu Gly lie Al a Gly lie Thr Gly Al a Arg Gly Leu Al a 945 950 955 960 Gly Pro Pro Gly lie Pro Gly Pro Arg Gly Ser Pro Gly Pro Gin Gly 965 970 975 Val Lys Gly Glu Asn Gly Lys Pro Gly Pro Ser Gly Hi s Asn Gly Glu

    980 985 990

    Arg Gly Pro Pro Gly Pro Gin Gly Leu Pro Gly Leu Ala Gly Thr Ala 995 1000 1005

    Gly Glu Pro Gly Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro 1010 1015 1020

    Gly Arg Asp Gly Ala Pro Gly Gly Lys Gly Asp Arg Gly Glu Asn 1025 1030 1035

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    Gly Ser Pro Gly Ala Pro Gly 1045 8693-sequence-li sti ng Pro 1050 . txt Gly Pro Pro Al a Pro Gly Hl S 1040 Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr 1055 1060 1065 Gly Pro Al a Gl y Pro Ser Gly Al a Pro Gl y Pro Al a Gly Ser Arg 1070 1075 1080 Gly Pro Pro Gly Pro Gin Gly Pro Arg Gly Asp Lys Gly Glu Thr 1085 1090 1095 Gly Gl u Arg Gly Leu Asn Gly lie Lys Gly Hi s Arg Gly Phe Pro 1100 1105 1110 Gly Asn Pro Gly Ala Pro Gly Ser Pro Gly Pro Al a Gly Hi s Gin 1115 1120 1125 Gly Ala lie Gl y Ser Pro Gly Pro Al a Gl y Pro Arg Gly Pro Val 1130 1135 1140 Gly Pro Ser Gly Pro Pro Gly Lys Asp Gly Thr Ser Gly Hi s Pro 1145 1150 1155 Gly Pro lie Gly Pro Pro Gly Pro Arg Gly Asn Arg Gly Gl u Arg 1160 1165 1170 Gly Ser Glu Gly Ser Pro Gly Hi s Pro Gly Gin Pro Gly Pro Pro 1175 1180 1185 Gly Pro Pro Gl y Ala Pro Gly Pro Cys Cys Gl y Gl y Gly Al a Al a 1190 1195 1200 Al a Leu Al a Al a Ala Gly Gly Glu Lys Al a Gly Gly Phe Al a Pro 1205 1210 1215 Tyr Tyr Gl y Asp Gl u Pro Met Asp Phe Lys lie Asn Thr Gl u Glu 1220 1225 1230 lie Met Thr Ser Leu Lys Ser Val Ser Gly Gin lie Glu Ser Leu 1235 1240 1245 Val Ser Pro Asp Gl y Ser Arg Lys Hi s Pro Al a Arg Asn Cys Arg 1250 1255 1260 Asp Leu Lys Phe cys Hi s Pro Glu Leu Lys Ser Gly Glu Tyr Trp

    1265 1270 1275

    Page 7

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    Val Asp 1280 Pro Asn Gin Gly Cys 1285 8693-sequence-li sti ng lie 1290 . txt Lys Leu Asp Al a Lys val Phe Cys Asn Met Glu Thr Gly Gl u Thr Cys Leu Asn Al a Ser Pro Gly 1295 1300 1305 Ser Val Pro Arg Lys Asn T rp T rp Thr Asp Ser Gly Al a Glu Lys 1310 1315 1320 Lys Hi s Val T rp Phe Gly Glu Ser Met Asp Gl y Gl y Phe Gin Phe 1325 1330 1335 Gly Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val Leu Asp val Gin 1340 1345 1350 Leu Ala Phe Leu Arg Leu Leu Ser Ser Arg Al a Ser Gin Asn lie 1355 1360 1365 rhr Tyr Hi s Cys Lys Asn Ser lie Al a Tyr Met Asp Hi s Al a Ser 1370 1375 1380 Gly Asn val Lys Lys Ala Leu Arg Leu Met Gly Ser Asn Glu Gly 1385 1390 1395 Glu Phe Lys Al a Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu 1400 1405 1410 Glu Asp Gl y Cys Thr Lys Hi s Thr Gl y Glu T rp Gly Lys Thr Val 1415 1420 1425 Phe Glu Tyr Arg Thr Arg Lys Al a Val Arg Leu Pro lie lie Asp 1430 1435 1440 lie Ala Pro Tyr Asp Val Gly Gl y Pro Asp Gin Glu Phe Gly val 1445 1450 1455 Asp Val Gly Pro Val Cys Phe Leu

    1460 1465 <210> 3 <211> 360 <212> PRT <213> Canis familiaris <400> 3

    Met Lys Ala Thr lie lie Phe Leu Leu Leu Ala Gin val Ser Trp Ala 1 5 10 15

    Page 8

    2016204268 22 Jun 2016

    Gly Pro Phe Gl n Gl n 20 Arg Gly 8693-sequence-li sti ng. txt Gl u 30 Asp Glu Leu Phe Asp Phe 25 Met Leu Ά1 a Ser Gly lie Gly Pro Glu Asp Arg Al a Pro Asp Met Pro ASP Leu 35 40 45 Glu Leu Leu Gly Pro Val Cys Pro Phe Arg Cys Gin Cys Hi s Leu Arg 50 55 60 Val Val Gin Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu 65 70 75 80 Pro Pro Asp Thr Thr Leu Leu Asp Leu Gin Asn Asn Lys lie Thr Glu 85 90 95 lie Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu Hi s Thr Leu lie 100 105 110 Leu Val Asn Asn Lys lie Ser Lys lie Ser Pro Gly Al a Phe Thr Pro 115 120 125 Leu Leu Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn His Leu Lys Glu 130 135 140 Leu Pro Gl u Lys Met Pro Lys Thr Leu Gin Glu Leu Arg Ala Hi s Glu 145 150 155 160 Asn Glu lie Thr Lys Val Arg Lys Al a Val Phe Asn Gly Leu Asn Gin 165 170 175 Met lie val Val Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly lie ISO 185 190 Glu Asn Gly Ala Phe Gin Gly Met Lys Lys Leu Ser Tyr lie Arg lie 195 200 205 Al a Asp Thr Asn lie Thr Thr lie Pro Gin Gly Leu Pro Pro Ser Leu 210 215 220 Thr Glu Leu His Leu Glu Gly Asn Lys lie Thr Lys Val Asp Al a Ser 225 230 235 240 Ser Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn 245 250 255 Ser lie Ser Ala Val Asp Asn Gly Thr Leu Al a Asn Thr Pro Hi s Leu 260 265 270

    Page 9

    2016204268 22 Jun 2016

    8693-sequence-li sti ng.txt

    Arg Glu Leu 275 Hi s Leu Asp Asn Asn 280 Leu Ala 290 Gl u Hi s Lys Tyr lie 295 Gin lie 305 Ser Al a Val Gly Ser 310 Asn Asp Lys Lys Al a Ser Tyr 325 Ser Gly Val Tyr T rp Glu lie 340 Gin Pro Ser Thr Al a lie Gl n 355 Leu Gly Asn Tyr Lys 360 <21O> 4 <211> 294 <212> PRT <213> Canis fami1i ari s <400> 4 Met Pro 1 Arg Gly Pro 5 Gly Ser Leu Cys Cys Leu Gly 20 Ser Ala Arg Gly Ser Tyr Lys 35 Arg Ser Asn Cys Lys 40 Cys Hi s 50 Gly lie Gl u Tyr Gin 55 Asn Hi s 65 Glu Thr Met Lys Glu 70 Val Leu Leu val Met Lys Gin 85 Cys Hi s Pro Leu Phe Al a Pro 100 Val Cys Leu Asp Cys Hi s Ser 115 Leu Cys Val Gin Val 120

    Lys Leu Il e Arg Val 285 Pro Gly Gly Val val Tyr Leu 300 Hi s Asn Asn Asn Phe Cys Pro 315 Pro Gly Tyr Asn Thr 320 Ser Leu 330 Phe Ser Asn Pro val 335 Gin Phe 345 Arg Cys Val Tyr Val 350 Arg Ser

    Leu Leu 10 Leu Val Leu Ala Ser 15 Hi s Leu 25 Phe Phe Gly Gin Pro 30 Asp Phe Pro lie Pro Al a Asn 45 Leu Gin Leu Met Arg Leu Pro 60 Asn Leu Leu Gly Glu Gin Al a 75 Gly Al a Trp He Pro 80 Asp Thr 90 Lys Lys Phe Leu Cys 95 Ser Asp 105 Leu Asp Glu Thr lie 110 Gin Pro Lys Asp Arg Cys Al a 125 Pro Val Met

    Page 10

    2016204268 22 Jun 2016

    Ser Ala 130 Phe Gly Phe Pro T rp 135 8693-sequencePro Asp Met Leu 1i sti ng. txt Asp Arg Phe Gl U 140 Cys Pro Gin Asp Asn Asp Leu Cys lie Pro Leu Al a Ser Ser Asp Hi s Leu 145 150 155 160 Leu Pro Al a Thr Glu Glu Ala Pro Lys Val Cys Glu Al a Cys Lys Asn 165 170 175 Lys Asn Glu Asp Asp Asn Asp lie Met Glu Thr Leu Cys Lys Asn Asp 180 185 190 Phe Ala Leu Lys lie Lys Val Lys Glu lie Thr Tyr lie Asn Arg Asp 195 200 205 Thr Lys lie lie Leu Glu Thr Lys Ser Lys Thr lie Tyr Lys Leu Asn 210 215 220 Gly Val Ser Glu Arg Asp Leu Lys Lys Ser val Leu T rp Leu Lys Asp 225 230 235 240 Ser Leu Gin Cys Thr Cys Glu Glu Met Asn Asp lie Asn Ala Pro Tyr 245 250 255 Leu Val Met Gly Gl n Lys Leu Gly Gly Glu Leu Val lie Thr Ser Val 260 265 270 Lys Arg T rp Gin Lys Gly Gin Arg Glu Phe Lys Arg lie Ser Arg Ser 275 280 285 lie Arg Lys Leu Gin Cys

    290 <210> 5 <211> 978 <212> PRT

    <213> Cam's fami1i ari s <400> 5 Met Glu Al a Glu Val Pro T rp Lys Val Glu Hi s Val Val Thr Val Al a 1 5 10 15 Ser Lys Gin Arg Gin Arg His Phe Gin Gly Thr Ser Cys Hi s Leu Glu 20 25 30 Leu Gin Thr Met Glu Lys Met Leu Val Gly Cys Phe Leu Leu Val Leu 35 40 45 Gly Gin lie Leu Leu val Leu Pro Al a Glu Al a Lys Glu Arg Thr Pro

    Page 11

    2016204268 22 Jun 2016

    8693-sequence-listi ng.txt 50 55 60

    Trp Arg 65 Ser lie Ser Arg 70 Gly Arg His Ala Trp Thr 75 Hi s Pro Gin Thr 80 Al a Leu Leu Glu Ser Ser Cys Glu Asn Lys Gin Al a Asp Leu Val Phe 85 90 95 lie lie Asp Ser Ser Arg Ser Val Asn Thr His Asp Tyr Ala Lys Val 100 105 110 Lys Glu Phe lie Leu Asp lie Leu Gin Phe Leu Asp lie Ser Pro Asp 115 120 125 Leu Thr Arg val Gly Leu Leu Gin Tyr Gly Ser Thr He Lys Asn Glu 130 135 140 Phe Ser Leu Lys Thr Phe Lys Lys Lys Ser Glu Val Glu Arg Al a Val 145 150 155 160 Lys Arg Met Arg Hi s Leu Ser Thr Gly Thr Met Thr Gly Leu Al a lie 165 170 175 Gin Tyr Al a Leu Asn He Al a Phe Ser Glu Al a Gl U Gly Ala Arg Pro 180 185 190 Leu Arg Glu Asn Val Leu Arg val lie Met lie val Thr Asp Gly Arg 195 200 205 Pro Gin Asp Ser Val Al a Glu Val Al a Al a Lys Al a Arg Asp Thr Gly 210 215 220 lie Leu lie Phe Al a lie Gly Val Gly Gin val Asp Leu Asn Thr Leu 225 230 235 240 Lys Ala lie Gly Ser Glu Pro Hi s Glu Asp Hi s val Phe Leu Val Al a 245 250 255 Asn Phe Ser Gin Met Glu Ser Leu Thr Ser Val Phe Gin Lys Lys Leu 260 265 270 Cys Met val Hi s Met Cys Ser Val Leu Glu Hi s Asn Cys Ala Hi s Phe 275 280 285 Cys lie Asn Thr Pro Gly Ser Tyr Val Cys Arg Cys Lys Gin Gly Tyr 290 295 300

    Page 12

    2016204268 22 Jun 2016

    lie Leu Asn 305 Ser Asp 8693-sequenceGln Thr Thr Cys Arg lie 1i sti ng. txt Leu Cys Ala 320 Gin Asp 310 315 Ala Lys Ala His Gly Cys Glu Gin Leu Cys val Asn Val Leu Gly Ser 325 330 335 Phe Val Cys Gin Cys Tyr Ser Gly Phe Thr Leu Al a Glu Asp Gly Lys 340 345 350 Thr Cys Ala Ala Val Asp Tyr Cys Ala Ser Glu Asn His Gly Cys Glu 355 360 365 His Glu Cys Val Asn Ala Asp Ser Ser Tyr Phe Cys Arg Cys Arg Lys 370 375 380 Gly Phe Thr Leu Asn Pro Asp Lys Lys Thr Cys Al a Lys lie Asp Tyr 385 390 395 400 Cys Ala Ser Pro Asn Gin Gly Cys Gin His Glu Cys val Asn Thr Asp 405 410 415 Asp Ser Tyr Ser Cys Arg Cys Leu Lys Gly Phe Thr Leu Asn Pro Asp 420 425 430 Lys Lys Thr Cys Arg Arg lie Asn Tyr Cys Ala Leu Asn Lys Pro Gly 435 440 445 Cys Gl u Hi s Glu Cys lie Asn lie Glu Asp Gly Tyr Tyr Cys Arg Cys 450 455 460 Arg Gin Gly Tyr Thr Leu Asp Pro Asn Gly Lys Thr Cys Ser Arg Val 465 470 475 480 Asp His Cys Ala Glu Gin Asp His Gly Cys Glu Gin Leu Cys Leu Asn 485 490 495 Thr Glu Asp Ser Tyr Val Cys Gin Cys Ser Glu Gly Phe Leu lie Asn 500 505 510 Asp Asp Leu Lys Thr Cys Ser Arg Ala Asp Tyr Cys Leu Leu Ser Asn 515 520 525 His Gly Cys Glu Tyr Ser Cys val Asn Thr Asp Arg Ser Phe Val Cys 530 535 540 Arg Cys Pro Glu Gly Tyr Val Leu Arg Ser Asp Gly Lys Thr Cys Al a 545 550 555 560

    Page 13

    8693-sequence-listi ng.txt

    2016204268 22 Jun 2016

    Lys Leu Asp Ser Cys 565 Al a Val Gly Asp His 570 Gly Cys Glu Hi s Ser 575 Cys Val Ser Ser Gly Asp Ser Phe Val Cys Gin Cys Phe Gl u Gly Tyr lie 580 585 590 Leu Arg Glu Asp Gly Lys Thr Cys Arg Arg Lys Gly Val Cys Gin Ser 595 600 605 val Asn Hi s Gly Cys Glu Hi s lie Cys Val Asn Ser Asp Glu Ser Tyr 610 615 620 lie Cys Lys Cys Arg Glu Gly Phe Arg Leu Ala Glu Asp Gly Lys Arg 625 630 635 640 Cys Arg Arg Lys Asp Val Cys Lys Ser Thr Tyr Hi s Gly Cys Glu His 645 650 655 lie Cys Val Asn Hi s Gly Asn Ser Tyr lie Cys Lys Cys Ser Glu Gly 660 665 670 Phe Val Leu Ala Glu Asp Gly Lys Arg Cys Lys Arg Cys Thr Glu Gly 675 680 685 Pro Leu Asp Leu Val Phe Val lie Asp Gly Ser Lys Ser Leu Gly Glu 690 695 700 Glu Asn Phe Gl u lie val Lys Gin Phe val Al a Gly lie lie Asp Ser 705 710 715 720 Leu Ala Val Ser Pro Lys Al a Al a Arg val Gly Leu Leu Gin Tyr Ser 725 730 735 Thr Gin val Arg Thr Glu Phe Thr Leu Gly Asp Phe Ser Ser Ala Arg 740 745 750 Asp Met Lys Lys Al a Val Ala His Met Lys Tyr Met Gly Lys Gly Ser 755 760 765 Met Thr Gly Leu Al a Leu Lys His Met Phe Glu Arg Ser phe Asn Pro 770 775 780 Val Glu Gly Ala Arg Pro Val Ser Pro Gly Val Ser Arg val Al a lie 785 790 795 800 Val Phe Thr Asp Gly Arg Ala Gin Asp Asp Val Ser Ala T rp Ala Arg

    805 810 815

    Page 14

    2016204268 22 Jun 2016

    86 93-s eque nce- 1 i st ing. txt Arg Ala Arg Ala Gly Gly lie Thr Met Tyr Al a Val Gly Val Gly Lys 820 825 830 Ala lie Gl u Glu Gl u Leu Gin Glu lie Ala Ser Glu Pro Thr Asp Lys 835 840 845 Hi s Leu Phe Tyr Al a Glu Asp Phe Ser Thr Met Gly Glu lie Ser Glu 850 855 860 Lys Leu Lys Lys Gly lie Cys Glu Ala Leu Glu Asp Ser Asp Gly Ser 865 870 875 880 Gin Asp Ser Pro Al a Gly Glu Leu Pro Lys Arg Val Hi s Gin Pro Thr 885 890 895 Glu Ser Gl u Pro Val Thr lie Asn lie Arg Asp Leu Leu Ser Cys Ser 900 905 910 Asn Phe Al a Val Gin Hi s Arg Tyr Leu Phe Glu Gl U Asp Ser Val Ser 915 920 925 Arg Ser Thr Gin Lys Leu Phe Hi s Ser Thr Lys Ser Ser Gly Ser Pro 930 935 940 Leu Glu Glu Lys Hi s Asp Gin Cys Lys Cys Glu Asn Leu lie Met Phe 945 950 955 960 Gin Asn Leu Ala Asn Glu Glu Val Arg Lys Leu Thr Gl n Arg Tyr lie

    965 970 975

    Leu Phe <210> 6 <211> 267 <212> PRT

    <213> Canis farm i 1 i ari s <400> 6 Met Gin Val Leu Al a Al a Gly Arg Arg Leu Pro Ser Val Phe Hi s Pro 1 5 10 15 Gly Arg Tyr Glu Ser Glu Thr Pro Ser Arg Pro Gly Tyr Lys Ala Gly 20 25 30 Gly Pro Al a Ala Pro Al a Asp Arg Arg Leu Pro Trp Leu Hi s Al a Arg 35 40 45

    Page 15

    8693-sequence-listi ng.txt

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    Pro Ala 50 Pro Arg Pro Gly Leu Arg Ala Ala Pro 55 Val 60 Gly Gly Leu Pro Gly Thr Met Gl u T rp Val T rp Ala Leu Val Leu Leu Al a Ala Leu Gly 65 70 75 80 Ser Ala Arg Ala Glu Ser Asp Cys Arg Val Ser Asn Phe Gin val Lys 85 90 95 Lys Asn Phe Asp Lys Al a Arg Phe Ala Gly Thr Trp Tyr Ala Met Al a 100 105 110 Lys Lys Asp Pro Glu Gly Leu Phe Leu Gin Asp Asn lie Val Ala Glu 115 120 125 Phe Ser Val Asp Gl u Asn Gly Arg Met Ser Al a Thr Al a Lys Gly Arg 130 135 140 Val Arg Leu Leu Asn Asn Trp Asp Val Cys Al a Asp Met val Gly Thr 145 150 155 160 Phe Thr Asp Thr Glu Asp Pro Al a Lys Phe Lys Met Lys Tyr T rp Gly 165 170 175 Val Ala Ser Phe Leu Gin Lys Gly Asn Asp Asp His T rp lie Il e Asp 180 185 190 Thr Asp Tyr Asp Thr Tyr Al a Val Gin Tyr Ser Cys Arg Leu Leu Asn 195 200 205 Leu Asp Gly Thr Cys Al a Asp Ser Tyr Ser Phe val Phe Ser Arg Asp 210 215 220 Pro Asn Gly Leu Pro Leu Glu Al a Gin Lys lie Val Arg Gin Arg Gin 225 230 235 240 Glu Glu Leu Cys Leu Al a Arg Gin Tyr Arg Leu lie val Hi s Asn Gly 245 250 255 Tyr Cys Asp Gly Arg Ser Glu Pro Asn Thr Leu 260 265

    <210> 7 <211> 704 <212> PRT <213> Canis familiaris <400> 7

    Page 16

    2016204268 22 Jun 2016

    Met 1 Ser Pro Arg Gl n 5 Pro Leu 8693-sequence-li sti ng. txt Leu Gly 15 Cys val Leu Val 10 Phe Leu val Cys Ser Al a Ala Pro Arg Pro Hi s Lys Pro Thr val Val val Phe Pro 20 25 30 Gly Asp Leu Arg Thr Asn Leu Thr Asp Lys Gin Leu Al a Glu Glu Tyr 35 40 45 Leu Phe Arg Tyr Gly Tyr Thr Gin Val Al a Glu Leu Ser Asp Asp Lys 50 55 60 Gl n Ser Leu Ser Arg Gly Leu Arg Leu Leu Gin Arg Arg Leu Al a Leu 65 70 75 80 Pro Glu Thr Gly Glu Leu Asp Lys Thr Thr Leu Glu Al a Met Arg Al a 85 90 95 Pro Arg Cys Gly val Pro Asp Leu Gly Lys Phe Gin Thr Phe Glu Gly 100 105 110 Asp Leu Lys T rp His His Asn Asp Il e Thr Tyr Trp lie Gin Asn Tyr 115 120 125 Ser Gl u Asp Leu Pro Arg Asp Val lie Asp Asp Ala Phe Ala Arg Ala 130 135 140 Phe Ala Val Trp Ser Al a Val Thr Pro Leu Thr Phe Thr Arg Val Tyr 145 150 155 160 Gly Pro Glu Ala Asp lie lie lie Gin Phe Gly Val Arg Glu Hi s Gly 165 170 175 Asp Gly Tyr Pro Phe Asp Gly Lys Asn Gly Leu Leu Al a Hi s Ala Phe 180 185 190 Pro Pro Gly Pro Gly lie Gin Gly Asp Ala Hi s Phe Asp Asp Glu Glu 195 200 205 Leu Trp Thr Leu Gly Lys Gly val Val Val Pro Thr Hi s Phe Gly Asn 210 215 220 Ala Asp Gly Ala Pro Cys Hi s Phe Pro Phe Thr Phe Glu Gly Arg Ser 225 230 235 240 Tyr Ser Al a Cys Thr Thr ASP Gly Arg Ser Asp Asp Thr Pro T rp Cys 245 250 255

    Page 17

    8693-sequence-listi ng.txt

    2016204268 22 Jun 2016

    Ser Thr Thr Ala Asp Tyr Asp Thr Asp Arg Arg Phe Gly Phe Cys Pro 260 265 270 Ser Gl u Lys Leu Tyr Thr Gin Asp Gly Asn Gly Asp Gly Lys Pro Cys 275 280 285 Val Phe Pro Phe Thr Phe Glu Gly Arg Ser Tyr Ser Thr Cys Thr Thr 290 295 300 Asp Gly Arg Ser Asp Gly Tyr Arg T rp Cys Ser Thr Thr Gly Asp Tyr 305 310 315 320 Asp Gin Asp Lys Leu Tyr Gly Phe Cys Pro Thr Arg Val Asp Ser Al a 325 330 335 Val Thr Gly Gly Asn Ser Al a Gly Glu Pro Cys Val Phe Pro Phe lie 340 345 350 Phe Leu Gly Lys Gin Tyr Ser Thr Cys Thr Arg Glu Gly Arg Gly Asp 355 360 365 Gly Hi s Leu Trp Cys Al a Thr Thr Ser Asn Phe Asp Arg Asp Lys Lys 370 375 380 T rp Gly Phe Cys Pro Asp Gin Gly Tyr Ser Leu Phe Leu Val Ala Al a 385 390 395 400 Hi s Gl u Phe Gly Hi s Ala Leu Gly Leu Asp Hi s Ser Ser Val Pro Glu 405 410 415 Al a Leu Met Tyr Pro Met Tyr Ser Phe Thr Glu Gly Pro Pro Leu Hi s 420 425 430 Glu Asp Asp Val Arg Gly lie Gin Hi s Leu Tyr Gly Pro Arg Pro Glu 435 440 445 Pro Glu Pro Gin Pro Pro Thr Al a Pro Pro Thr Al a Pro Pro Thr Val 450 455 460 Cys Ala Thr Gly Pro Pro Thr Thr Arg Pro Ser Glu Arg Pro Thr Ala 465 470 475 480 Gly Pro Thr Gly Pro Pro Al a Al a Gly Pro Thr Gly Pro Pro Thr Al a 485 490 495 Gly Pro Ser Glu Ala Pro Thr val Pro val Asp Pro Ala Glu Asp lie

    500 505 510

    Page 18

    2016204268 22 Jun 2016

    86 93-s eque nce- 1 i st ing. txt Cys Lys Val Asn lie Phe Asp Al a lie Al a Glu lie Arg Asn Tyr Leu 515 520 525 Hi s Phe Phe Lys Gl u Gly Lys Tyr Trp Arg Phe Ser Lys Gly Lys Gly 530 535 540 Arg Arg Val Gin Gly Pro Phe Leu He Thr Asp Thr Trp Pro Al a Leu 545 550 555 560 Pro Arg Lys Leu Asp Ser Ala Phe Glu Asp Gly Leu Thr Lys Lys Thr 565 570 575 Phe Phe Phe Ser Gly Arg Gin Val T rp Val Tyr Thr Gly Thr Ser Val 580 585 590 Val Gly Pro Arg Arg Leu Asp Lys Leu Gly Leu Gly Pro Glu Val Thr 595 600 605 Gin Val Thr Gly Al a Leu Pro Gin Gly Gly Gly Lys Val Leu Leu Phe 610 615 620 Ser Arg Gin Arg Phe τ rp Ser Phe Asp val Lys Thr Gin Thr Val Asp 625 630 635 640 Pro Arg Ser Ala Gly Ser Val Glu Gin Met Tyr Pro Gly Val Pro Leu 645 650 655 Asn Thr Hi s Asp lie Phe Gin Tyr Gin Glu Lys Ala Tyr Phe Cys Gin 660 665 670 Asp Arg Phe Tyr Trp Arg Val Asn Ser Arg Asn Gl U Val Asn Gin val 675 680 685 Asp Glu Val Gly Tyr val Thr Phe Asp lie Leu Gin Cys Pro Glu Asp

    690 695 700 <210> 8 <211> 445 <212> PRT

    <213> Cam's f ami 1i ari s <400> 8 Met Met Lys Thr Leu Leu Leu Leu Val Gl y Leu Leu Leu Thr T rp Asp 1 5 10 15 Asn Gly Arg Val Leu Gly Asp Gin Ala Val Ser Asp Thr Glu Leu Gin 20 25 30

    Page 19

    8693-sequence-listi ng.txt

    2016204268 22 Jun 2016

    Gl u Met Ser 35 Thr Glu Gly Ser Lys Tyr 40 lie Asn Lys Glu 45 lie Lys Asn Al a Leu Lys Gly Val Lys Gin lie Lys Thr Leu lie Gl u Gin Thr Asn 50 55 60 Glu Glu Arg Lys Ser Leu Leu Ser Asn Leu Glu Glu Al a Lys Lys Lys 65 70 75 80 Lys Glu Asp Ala Leu Asn Asp Thr Lys Asp Ser Glu Thr Lys Leu Lys 85 90 95 Al a Ser Gin Gly Val Cys Asn Asp Thr Met Met Al a Leu T rp Glu Glu 100 105 110 Cys Lys Pro Cys Leu Lys Gin Thr Cys Met Lys Phe Tyr Ala Arg val 115 120 125 Cys Arg Ser Gly Ser Gly Leu Val Gly Hi s Gin Leu Glu Glu Phe Leu 130 135 140 Asn Gin Ser Ser Pro Phe Tyr Phe T rp Met Asn Gly Asp Arg lie Asp 145 150 155 160 Ser Leu Leu Glu Asn Asp Arg Gin Gin Thr His Al a Leu Asp Val Met 165 170 175 Gl n Asp Ser Phe Asn Arg Al a Ser Ser lie Met Asp Gl u Leu Phe Gin 180 185 190 Asp Arg Phe Phe Thr Arg Glu Pro Gin Asp Thr Tyr Hi s Tyr Ser Pro 195 200 205 Phe Ser Leu Phe Gin Arg Arg Pro Phe Phe Asn Pro Lys Phe Arg lie 210 215 220 Al a Arg Asn lie lie Pro Phe Pro Arg Phe Gin Pro Leu Asn Phe His 225 230 235 240 Asp Met Phe Gin Pro Phe Phe Asp Met lie Hi s Gin Al a Gin Gin Ala 245 250 255 Met Asp Val Asn Leu Hi s Arg lie Pro Tyr Hi s Phe Pro lie Glu Phe 260 265 270 Pro Glu Glu Asp Asn Arg Thr val Cys Lys Glu lie Arg Hi s Asn Ser

    275 280 285

    Page 20

    2016204268 22 Jun 2016

    86 93-s eque nce- 1 i st ing. txt Thr Gly Cys Leu Lys Met Lys Asp Gin Cys Glu Lys Cys Gin Glu lie 290 295 300 Leu Ser val Asp Cys Ser Ser Asn Asn Pro Al a Gin val Gin Leu Arg 305 310 315 320 Gin Glu Leu Ser Asn Ser Leu Gin lie Al a Glu Lys Phe Thr Lys Leu 325 330 335 Tyr Asp Glu Leu Leu Gin Ser Tyr Gin Glu Lys Met Phe Asn Thr Ser 340 345 350 Ser Leu Leu Lys Gl n Leu Asn Glu Gin Phe Ser Trp Val Ser Gin Leu 355 360 365 Ala Asn Leu Thr Gl n Ser Glu Asp Pro Phe Tyr Leu Gl n Val Thr Thr 370 375 380 Val Gly Ser Gin Thr Ser Asp Ser Asn val Pro val Gly Phe Thr Lys 385 390 395 400 Val val Val Lys Leu Phe Asp Ser Asp Pro lie Thr Val Met lie Pro 405 410 415 Glu Ala Val Ser Arg Asn Asn Pro Lys Phe Met Glu Thr Val Al a Glu 420 425 430 Lys Ala Leu Gin Gl u Tyr Arg Gin Lys Hi s Arg Glu Gl u 435 440 445

    <210> 9 <211> 705 <212> PRT

    <213> Canis <400> 9 Met Arg Leu 1 familiari s Ala Val 5 Arg Al a Leu Leu Ala Cys 10 Ala Val Leu Gly 15 Leu Cys Leu Ala val Ser Pro Glu Lys Thr Val Arg T rp Cys Thr Val Ser 20 25 30 Asn His Glu Ala Ser Lys Cys Ser Ser Phe Met Glu Asn Met Lys Thr 35 40 45 Val Leu Glu Asn Gly Pro Phe Val Ser Cys Val Lys Arg Thr Ser Tyr 50 55 60

    Page 21

    8693-sequence-listi ng.txt

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    Leu Glu 65 Cys lie Lys Ala lie Trp Ala 70 Asn Glu 75 Al a Asp Ala Val Thr 80 Leu Asp Al a Gly Leu Val Phe Glu Al a Gly Leu Asn Pro Tyr Asn Leu 85 90 95 Lys Pro Val Val Al a Glu Phe Tyr Gly Lys Asp Lys Gin Thr Arg Tyr 100 105 110 Tyr Ala val Ala Val Val Lys Lys Ser Ser Thr Phe Asn Leu Asn Gin 115 120 125 Leu Gin Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly 130 135 140 T rp Asn lie Pro Met Gly Leu Leu Tyr T rp Lys Leu Pro Gl u Pro Arg 145 150 155 160 Glu Ser Leu Gin Lys Al a Al a Ser Ser Phe Phe Thr Al a Ser Cys Val 165 170 175 Pro Cys val Asp Arg Thr Ala Phe Pro Lys Leu Cys Gin Leu Cys Val 180 185 190 Gly Lys Gly Thr Asp Lys Cys Al a Cys Ser Asn His Glu Pro Tyr Phe 195 200 205 Gly Tyr Ser Gly Al a Phe Lys Cys Leu Met Glu Asp Al a Gly Glu val 210 215 220 Al a Phe Val Lys Hi s Ser Thr val Leu Glu Asn Leu Pro Asn Lys Al a 225 230 235 240 Asp Leu Asp Gin Tyr Glu Leu Leu Cys Pro Asp Asn Lys Arg Lys Pro 245 250 255 Val Gly Glu Tyr Lys Gin Cys Tyr Leu Al a Lys Val Pro Ser His Al a 260 265 270 val val Al a Arg Ser Val Gly Gly Lys Glu Asp Leu lie Trp Glu Leu 275 280 285 Leu Asn Gin Ala Gin Glu Hi s Tyr Gly Lys Asp Lys Ser Lys Val Phe 290 295 300 Gin Leu Phe Ser Ser Thr Leu Gly Lys Asp Leu Leu Phe Lys Asp Ser 305 310 315 320 Page 22

    8693-sequence-listi ng.txt

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    Ala Glu Gly Phe Phe Arg lie Pro Pro Lys Met Asp Thr Trp Leu Tyr 325 330 335 Leu Gly Tyr Glu Tyr Val Thr Ala Leu Arg Asn Leu Arg Glu Asp Val 340 345 350 Arg Pro Asp Thr Pro Arg Asp Glu Cys Lys Lys val Lys Trp Cys Al a 355 360 365 val Gly Hi s Hi s Glu lie Ala Lys Cys Asp Glu T rp Ser Val Asn Ser 370 375 380 Glu Gly Lys lie Glu Cys Glu Ser Al a Glu Ser Thr Glu Asp Cys lie 385 390 395 400 Ala Lys lie Ala Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly 405 410 415 Tyr lie Tyr lie Al a Gly Gin Cys Gly Leu Val Pro Val Leu Al a Glu 420 425 430 Asn Tyr Lys Thr Gin Gly Ser Thr Cys Ser Asn Thr Ala Glu Glu Gly 435 440 445 Tyr Leu Al a Val Al a Val Val Lys Arg Leu Asp Lys Thr lie Ser Trp 450 455 460 Asn Asn Leu Gin Gly Arg Lys Ser Cys Hi s Thr Ala val Asp Arg Thr 465 470 475 480 Al a Gly Trp Asn lie Pro Met Gly Leu Leu Tyr Asn Arg He Asn Hi s 485 490 495 Cys Glu Phe Asp Lys Phe Phe Ser Gin Gly Cys Ala Pro Gly Ser Met 500 505 510 Arg Asn Ser Ser Leu Cys Al a Leu cys lie Gly Ser Al a Asn Val Pro 515 520 525 Gly Lys Glu cys val Pro Asn Asn His Glu Arg Tyr Tyr Gly Tyr Thr 530 535 540 Gly Ala Phe Arg Cys Leu Val Glu Lys Gly Asp Val Al a Phe Val Lys 545 550 555 560 Asp Gin Thr val Leu Gin Asn Thr Gly Gly Lys Asn Thr Glu Asp T rp

    Page 23

    2016204268 22 Jun 2016

    8693-sequence-listi ng.txt 565 570 575

    Ala Lys Asp Leu 580 Lys Glu Glu Asp Phe Glu 585 Leu Leu Cys Pro 590 Asp Gly Gin Arg Lys Ser Val Asp Lys Al a Pro Glu Cys Phe Leu Ala Lys Al a 595 600 605 Pro Asn Hi s Ala Val Val Ser Arg Lys Asp Lys Ala Ser Cys val Ser 610 615 620 Lys Met Leu Leu Asp Gin Gin Leu Leu Phe Gly Arg Asn Gly Asn Asp 625 630 635 640 Cys Ser Gly Lys Phe Cys Leu Phe Hi s Ser Ala Thr Lys Asp Leu Leu 645 650 655 Phe Arg Asp Asp Thr Gin Cys Leu Al a Lys Leu Pro Glu Asp Thr Thr 660 665 670 Tyr Lys Ser Tyr Leu Gly Al a Glu Tyr lie Thr Ala Val Ala Asn Leu 675 680 685 Arg Gin Cys Ser Thr Ser Lys Leu Leu Glu Al a Cys Thr Phe Hi s Thr

    690 695 700

    Ser

    705 <210> 10 <211> 88 <212> PRT

    <213> Cam's farm i1i ari s <400> 10 Met Arg Leu lie Leu Ser Leu Pro val Leu val Val val Leu Ser Met 1 5 10 15 Val Leu Glu Gly Pro Al a Pro Al a Gin Al a Ala Gly Glu lie Ser Ser 20 25 30 Thr Phe Gl u Arg lie Pro Asp Lys Leu Lys Glu Phe Gly Asn Thr Leu 35 40 45 Glu Asp Lys Ala Arg Al a Al a lie Glu Ser He Lys Lys Ser Asp lie 50 55 60 Pro Ala Lys Thr Arg Asn T rp Phe Ser Glu Ala Phe Lys Lys Val Lys

    65 70 75 80

    Page 24

    8693-sequence-listi ng.txt

    2016204268 22 Jun 2016

    Glu His Leu Lys Thr Ala Phe Ser 85 <210> 11 <211> 424 <212> PRT <213> Cam's familiaris <400> 11

    Met Pro 1 Leu Leu T rp 5 Leu Arg Gly Phe Leu val 10 Ala Ser Cys T rp 15 lie lie Val Arg Ser Ser Pro Thr Pro Gly Pro Glu Gly Pro Gly Al a Ala 20 25 30 Pro Ala Cys Pro Al a Cys Al a Leu Thr Al a Leu Pro Arg Asp Al a Pro 35 40 45 Asn Ser Gin Pro Glu Met val Glu Ala Val Lys Lys Hi s lie Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Glu Val Thr Gin Pro Val Pro Lys 65 70 75 80 Al a Ala Leu Leu Asn Ala lie Arg Lys Leu Hi s Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Phe Val Glu lie Glu Asp Asp lie Gly Arg Arg Al a Glu 100 105 110 Met Asn Glu Leu Met Glu Gin Thr Ser Glu lie lie Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu lie Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val val Glu Arg Al a Glu Val T rp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr lie Arg Leu Leu 165 170 175 Gin Lys Hi s Pro Gin Gly Ser Leu Asp Ala Gly Glu Glu Ala Glu Asp 180 185 190 Met Gly Phe Pro Glu Glu Arg Asn Glu Val Leu lie Ser Glu Lys Val

    195 200 205

    Page 25

    8693-sequence-listi ng.txt

    2016204268 22 Jun 2016

    val Asp Ala Arg 210 Lys Ser Thr 215 Trp His lie Phe Pro Val 220 Ser Ser Ser lie Gin Arg Leu Leu Asp Gin Gly Arg Ser Ser Leu Asp Val Arg lie 225 230 235 240 Ά1 a Cys Glu Gin Cys Hi s Glu Thr Gly Al a Ser Leu Val Leu Leu Gly 245 250 255 Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Asp Gly 260 265 270 Gly Asp Al a Gly Al a Gly Gly Asp Glu Asp Lys Glu Gin Ser His Arg 275 280 285 Pro Phe Leu Met Leu Gin Al a Arg Gin Ser Glu Asp Hi s Pro Hi s Arg 290 295 300 Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn lie Cys Cys 305 310 315 320 Lys Lys Gin Phe Phe Val Ser Phe Lys Asp lie Gly T rp Asn Asp T rp 325 330 335 lie lie Al a Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Gly Cys 340 345 350 Pro Ser Hi s lie Al a Gly Thr Ser Gly Ser Ser Leu Ser Phe Hi s Ser 355 360 365 Thr Val lie Asn Hi s Tyr Arg Leu Arg Gly Hi s Ser Pro Phe Thr Asn 370 375 380 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 385 390 395 400 Tyr Tyr Asp Asp Gly Gin Asn lie Il e Lys Lys Asp lie Gin Asn Met 405 410 415

    lie val Glu Glu Cys Gly Cys Ser 420 <210> 12 <211> 2028 <212> DNA <213> Canis familiaris <400> 12

    Page 26

    8693-sequence-listing. txt

    2016204268 22 Jun 2016

    agctagagta tttggactag ttaagcatga atctctctcc cattccatag ggaatgagct 60 gggcagtcct tcctccccac gtccacctgc acttcgttag agagcagaat gctcacatgc 120 caccccacaa gatccccaca gtgacataac tccatgcaga gactggcgtg actgggctgg 180 gtttcccccc cttcagctct tgtatcacta agaatctggc agccagttcc gtcctgacag 240 agttcacagc atatattggt ggattcttgt ccatagtgca tctgctttaa gaattaacga 300 aagcagtgtc aagacagaag gattcaaagc atttgccaaa aatgaatcta ggtgtgttta 360 ctctactctt ggcattaatt ggtggtgcca gcggccagta ctatgattac gatttctccc 420 taccgattta tgggctgtca tcaccaaact gtgcaccaga atgtaactgc cctgaaagct 480 acccatcggc catgtactgc gatgagctga aattgaaaag tgtaccaatg gtgcctcctg 540 gaatcaagta tctttacctt aggaataacc agategacca tattgatgaa aaggcctttg 600 aaaacgtaac tgacctgcag tggctcattc tggatcataa ccttctagaa aattccaaga 660 ttaaaggaag aattttctct aaactgaaac aactgaagaa gctgcatata aatcacaaca 720 acctgacaga gtctgtgggc ccgcttccca aatctctggt ggacctgcag cttacacaca 780 acaagatcca gaagctgggc tccttcgatg gactggtaaa cctgaccttt gtccaccttc 840 aacacaatca gctgaaagag gatactgtgt cagctgcttt taaaggtctt aagtccctcg 900 agtaccttga cttgagctac aatcagatgg ccaaactgcc ttctggtctc ccagcatctc 960 ttctgactct ctacttggac aacaataaga tcagcaacat ccctgatgag tatttcaagc 1020 gttttaatgg actgcagtat ctgcgtttat ctcataatga actggcggat agtggagtac 1080 ctggaaattc ttttaatgta tcatccctgc tggagctgga tctctcctac aataagctta 1140 aaaacatacc gactgtcaat gagaaccttg aaaactatta cctggaggtc aatgaactcg 1200 aaaagtttga ggtaaagagc ttctgtaaga tccttggacc attatcctac tccaagatta 1260 aacatttgcg tctggatggc aaccgtctta cccacaccag tctgccacct gatatgtacg 1320 aatgtctacg tgttgcaaat gaaatcactg ttaattaaca tctactaatt ccaattctat 1380 tgaggtgtac cctggagcaa cattttatgg tttatgtttt tgtgtgtgtc agttttcata 1440 gtattcaatt ttttgggtca ctttattagt ttcatgaatt tcagattctg agggaaatgt 1500 tttgtaaaca ttttactact ttttttttta agataagatg aaaggcaggc ctatttcatc 1560 acaagcacag acatagacac acagagacaa caaacttatg ctttatttgt aaatttagtg 1620 ttttctccat ctctactgtc aaatgatgtg caaagtcttc tcttggttga atggaagtca 1680 gccaaggatt ataactcctg aatcttaact taatatgcca aacagcatgg gctacacaca 1740 tatgaatatt taatatcttg aaccaaatga tctcaaattt caatgttcag atttgcttaa 1800 ctgaaaaata ggtggtaaat tttaagactg atgattttgc aaaatactag accaaattca 1860 tgaagccaca tacacttaga gcaatatttt tagtattaag Page aattatatag 27 ttacctagtg 1920

    8693-sequence-listi ng. txt

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    aaacttctct tttctagaat tatttttcac tctaagtcat gtgtatgttc ctttttaatc 1980 atttgcatgt tatgtttaac aagctcatag caaaataaaa catagcaa 2028 <210> 13 <211> 5511 <212> DNA <213> Cam's familiaris <400> 13 gaagccaaac tttttcctat ttaaggccga agcgaaggaa tctcagtggc tgagttttat 60 gacgggcccg gtgctgcagg gcagggaaca tctgatggtg ctactttgag ctgctgctct 120 cccctcctcg ctgcacaaag agtctcatgt ctcacattta gacatgacga gctttgtgca 180 aaaggggacc tggttgcttc tcgctctgct tcagcccgct gtcatctcgg cacagcagca 240 agctattgat ggaggatgct cccatcttgg tcagtcctat geggatagag atgtctggaa 300 gccagaaccg tgccaaatat gcgtctgtga ctcaggatct gttctctgcg atgacataat 360 atgtgacgaa caagaattag actgtcccaa cccagagatc ccatttggag aatgttgtgc 420 agtttgccca cagcctccaa catctcctcc tcgccctcct aatggtcatg gacctcaagg 480 ccctaaggga gatccaggcc ctcctggtat tcctgggaga aatggcgacc ccggtattcc 540 cggacagcca ggctcccctg gttctcctgg cccccctgga atctgtgaat cctgccctac 600 tggtcctcag cctaactatt ctccccagtt cgagtcctat gacgtcaagg ctggtgtcgc 660 aggaggcggc atcggaggct accccgggcc agctggtccc cctggtcctc ctggtccccc 720 tggtacatct ggtcatcctg gctccccggg ttccGcagga taccaaggec cccctggtga 780 gcctgggcaa gctggtcctg cgggccctcc aggaccccct ggtgctatgg gtccatctgg 840 tcctgctgga aaagatgggg aatcaggaag acctggacga ectggagagc gagggctgcc 900 tggacctcca ggtatgaaag gtccagctgg catgcctgga ttccccggta tgaaaggaca 960 tagaggcttt gatggacgaa atggagaaaa gggtgataca ggtgctcctg gattaaaggg 1020 tgaaaatggc ctcccaggtg aaaatggagc ccccggaccc atgggtcccc gaggtgctcc 1080 tggtgagcga ggacggccag gcctccccgg ggccgcgggt gctcgaggta acgatggtgc 1140 tcgtggaagt gacggacaac caggccctcc cggtccccct ggaactgcag gattecccgg 1200 ttcccctggt gctaagggtg aagttggacc cgcgggatct cctggttcaa acggcteccc 1260 tggacaaaga ggagaacctg gacctcaggg acatgctggt gctccaggcc ctcctggccc 1320 ccctgggagc aatggtagtc ctggcggtaa aggtgaaatg ggtcccgctg gcattcctgg 1380 agcccctgga ctgataggag cccggggccc tcctggacca cccggtacca atggtgctcc 1440 tggacagcga ggtggtgcag gtgaacccgg taaaaatggt gccaaaggag agccaggccc 1500 acgcggtgaa cgtggtgaag ctggttctcc aggtatccca Page ggacctaaag 28 gtgaagatgg 1560

    8693-sequence-listi ng. txt

    2016204268 22 Jun 2016

    caaagatggc tcgcctggag aaccaggtgc aaacggactt ccgggagctg ccggagaaag 1620 gggtgcacct ggattccgag gacctgctgg agcaaatggc cttccaggag aaaagggtcc 1680 cgctggggag cgtggtggtc caggccctgc agggcccaga ggagctcctg gagaacccgg 1740 ccgagatggt gtcccaggag gtccaggaat gaggggcatg cccggaagcc caggagggcc 1800 aggcagcgat gggaaaccag ggcctccegg aagteaagga gaaagtggtc gaccaggtcc 1860 tccaggcccg tctggtcccc gaggtcagcc tggcgtcatg ggtttccctg gtcctaaagg 1920 aaatgatggt gcacctggca agaatggaga gcgaggtggt cctggaggtc ccggccctca 1980 gggtcctgct ggaaagaatg gtgaaactgg acctcagggt cccccggggc ctactgggcc 2040 ggctggtgac aaaggagacg cgggaccccc tggtccccaa gggctacagg gtttgcccgg 2100 aaccagtgga cccccaggag aaaatggaaa acccggtgaa ccaggcccga agggtgaatc 2160 tggttcacct ggagttccag gaggcaaggg cgactcaggt gcaccgggeg agcgtggtcc 2220 tcccggggct gcaggcccca tgggcccccg aggtggagct ggacccccag gccccgaagg 2280 agggaagggc gctgctggcc cccctgggcc acccggctcg geaggtacec ctggtctgca 2340 ggggatgccc ggagagagag gaggccccgg cggccccggc cccaagggtg acaagggcga 2400 accaggcagt gcaggtgcag atggagcccc ggggaaggat ggtcccaggg gtcctaccgg 2460 tcccatcggc ccccctggcc cagccggtca gcctggagat aagggtgaag gtggtgcccc 2520 cggcctcccg ggcatagctg gtcctcgcgg tggccccggc gagagaggtg agcacggccc 2580 cccgggaccc gccggcttcc ccggagctcc cggacagaac ggcgagcccg gegetaaggg 2640 cgaaaggggc gcccccggcg agaagggtga aggaggcccc cccggagtgg cagggccccc 2700 cggaggagcc gggcctgcgg gtccccccgg tceccagggt gtcaaaggtg aacgtggcag 2760 tcccggcggt cccggtgccg ctggcttecc tggtggtcgt ggtcttcctg gccctcctgg 2820 caataatggt aacccaggcc ccccaggctc cagtggtgct ccaggcaagg atgggccccc 2880 cggtccaccc ggtaacaacg gcgctcctgg cagccctggg gtgtccggac ctaaaggtga 2940 tgctggccaa cccggtgaga agggatcgcc tggcccccag ggccctccgg gagccccagg 3000 ccctctcgga attgctggga tcaccggggc caggggtctc gcgggaccac caggcattcc 3060 aggtcctagg ggaagcccag gcccacaggg cgtcaagggt gaaaatggga agccaggacc 3120 cagtggtcac aacggagagc gcggtcctcc cgggccccag gggcttcccg gcctggccgg 3180 cacggctggt gaacctggaa gagatggaaa cccaggctcc gacgggctgc caggccgcga 3240 cggcgctccg ggcggcaagg gtgatcgtgg ggagaacggg tctcctggcg ccccgggcgc 3300 ccccggccac cccggccccc ccggccccgt gggcccggcg gggaagagcg gcgaccgagg 3360 cgagacgggc cccgctggtc cctccggcgc tcctggtcct gcaggctccc gaggcccccc 3420

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    tggtccccaa ggtccacgcg gtgacaaagg tgaaaccggt gaacgtggcc ttaacggcat 3480 caaaggccat cgaggattcc ccggtaaccc aggtgcccca ggctctccgg gtcctgcagg 3540 ccaceaggga geaatcggta gtccaggecc cgcaggcccc agaggaccag ttggacGcag 3600 tggaccccct ggtaaagacg gaacaagtgg acatccaggt cccattggac caccaggccc 3660 tcgaggtaac agaggagaaa gaggatctga gggctccccc ggccacccag gacagccagg 3720 tccccccggg cctcctggtg cccctggacc ctgctgtggt gggggcgctg ccgccctggc 3780 tgccgccgga ggggagaaag ctggcgggtt tgccccctat tacggagatg agccaatgga 3840 tttcaaaatc aacaccgagg agattatgac ttcgctcaaa tccgtcagtg gacaaataga 3900 aagcctcgtt agccccgacg ggtcccgcaa acaccccgct cggaactgcc gggacctcaa 3960 attctgccat ccagagctca agagcggaga gtactgggtt gaccccaacc aaggctgcaa 4020 gttggatgcg atcaaagtat tctgcaacat ggaaaccggg gagacgtgec tgaatgecag 4080 tcccgggagc gttccccgga agaactggtg gacagattct ggtgctgaga agaaacatgt 4140 ttggtttgga gaatccatgg atggtggctt ccagtttggc tacggcaatc ccgagctgcc 4200 tgaagacgtc ctcgatgtcc agctggcatt cctccgcctt ctctccagcc gggcctccca 4260 gaacatcacg tatcactgca agaatagcat cgcgtacatg gatcacgcca gtgggaatgt 4320 aaagaaagcc ctgaggctga tggggtcaaa tgaaggtgaa ttcaaggctg aaggaaacag 4380 caaattcaca tacacagtcc tggaggatgg ttgcactaaa cacactgggg aatggggcaa 4440 aacagtcttc gaataccgaa cacgcaaggc tgtgagacta cctattatag atattgcacc 4500 ctatgatgtt ggtggtcctg atcaagaatt tggtgtggac gttggccctg tttgcttttt 4560 ataaaccaaa ctctatctga aaccccagca aaaaatttca cactccatat gtgttcctct 4620 cgttctaacc ttgtcaacca gtacaagtga ccaactcaat tccagttatt tatttccaaa 4680 agttttggaa aaaagtataa tttggcaaaa aaaaagatac cttttttttt tttttttttg 4740 ctgttacacc aaatacagtt caaatgcttt ttgttctatt tttttaccaa tttcaatttc 4800 aaaatgtctc aatggtgcta taataaataa acttcaacac tcttacaata acactgtgtt 4860 atattctttg aatcctagcc catttgcaga gcaatgacta ggcttaccat taaaagataa 4920 cctttctttc tgaaatagtc aaacatgaaa ttagagaaga cctccctgtt tcaactacct 4980 caacetggtc agaaatacag atgaatteta gaagtcctgg aagatgaaaa aaatcataaa 5040 ttagcaataa tttcctaaaa tattgataaa aaagtatagt acgagaattt aaagaaaaaa 5100 tttttttaaa tctacgattt taatcttgga tatcaactgc ttttaaaggt gcttttctcc 5160 tttcttgtca ttgctgaaat ggtcaagatt acccacattt gggaaggctt taaagacaca 5220 tgttatggtg ctaatgtact ttcactttaa aaactccagg tcagaattgt tgccttgcat 5280 tcaaaacata aatgcacacc atctttcaaa aaaatttatt Page gecgtgtcac 30 ttcaggggac 5340

    8693-sequence-listi ng. txt

    2016204268 22 Jun 2016

    tctctctccc ttgatcctgt aaaagtcaac aacaacaaca acaacaacaa aaaattatgg 5400 ggctgctttt gtcacagtaa cacagagaat gtgctgaaat ttaactttgt aagcttgtat 5460 gtggttgttg gtcttttttt ttttttcctc cagacaccca taataaaata t 5511 <210> 14 <211> 1470 <212> DNA <213> Cam's familiaris <400> 14 taaaaccagg aggttctcaa cctattaaca ttcacaaaac agcacctacc ccctcctcct 60 ttccacacct gcaaactctt ttgcttgggc tgaatattta gtttaattac atctcacctt 120 taagggctcc tgtggcaaat ccccggatta aaaggttcct tggctgtgaa aatacataac 180 ctaaaccatg aaggcaacta tcatcttcct cctgcttgca caagtttcct gggctgggcc 240 gttccaacag agaggcttat ttgactttat gctagaagat gaggcttccg ggataggccc 300 ggaggaccgt gcacctgaca tgcctgacct cgagcttctg ggacctgtgt gtcccttccg 360 ctgtcagtgc catctccgag tggtccagtg ttccgacctg ggtctggaca aagtaccaaa 420 agatcttccc cctgacacta cgctgctcga cttgcaaaac aacaaaatca ccgaaatcaa 480 agatggagac ttcaagaacc tcaagaacct gcataccttg attcttgtaa acaacaaaat 540 tagcaaaatc agccctggag catttacacc tttgttgaaa ttggaacgac tttatctgtc 600 caagaatcat ctgaaggaat tgccagaaaa aatgcccaaa actcttcagg agctgcgtgc 660 ccatgagaat gagatcacca aagttcgaaa agctgtgttc aatggactga accagatgat 720 cgtcgtagag ctgggcacca atcccctgaa gagttcaggg attgaaaatg gagccttcca 780 gggaatgaag aagctctcct atatccgcat tgctgatacc aatataacta ccatccctca 840 aggtcttcct ccttccctta ctgaattaca tcttgaaggc aacaaaatca ccaaggttga 900 tgcatctagc ctgaaaggac tgaataattt ggctaagttg ggactgagtt ttaacagcat 960 ctccgctgtt gacaatggca ctctagccaa cactcctcat ctgagggagc ttcacttgga 1020 caacaataag ctcatcagag tacccggtgg gctggcggag cataagtaca tccaggttgt 1080 ctaccttcat aacaacaata tatctgcagt cggatctaat gacttctgcc cacctggata 1140 caacaccaaa aaggcttctt attcaggtgt gagccttttc agcaacccag tgcagtactg 1200 ggagatccag ccatccacct tccggtgtgt ctacgtgcgc tctgccatcc agcttggaaa 1260 ttataaataa ttcccaagaa agccctcgtt tttataacct ggcaaaatcc cattaatatc 1320 attgctcaaa aaaaaaaaaa aaaaaaacta aatacgggaa acttagctgc aatgtgaatg 1380 ttttccccct aatttataat actaaaccaa attgcattta aaatgccaaa aaaaaaaaat 1440 tttggccgcc aaaaaaaaaa aaaaaaaaaa 1470

    Page 31

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    <210> 15 <211> 1760 <212> DNA <213> Canis familiaris <400> 15 attcggcacg aggatgccgc ggggccccgg ctcgctgctg ctgctcgtcc tagegtegea 60 ctgctgcttg ggctcggcgc gcgggctctt cttcggccag cccgacttct cctacaagcg 120 cagcaactgc aagcccatcc cggccaacct gcagctgtgc cacggcatcg agtaccagaa 180 catgcggctg cccaacctgc tgggccacga gaccatgaag gaggtgctgg ageaggeggg 240 cgcctggatc ccgctggtca tgaagcagtg ccacccggac accaagaagt tcctctgctc 300 gctcttcgcc cccgtgtgcc tcgacgacct ggacgagacc atccagccgt gccaetetct 360 ctgcgtgcag gtgaaggacc gctgcgcccc ggtcatgtcg gccttcggct tcccctggcc 420 ggacatgctc gagtgcgacc gattccccca ggacaacgac ctctgcatac ccctcgctag 480 cagcgaccat ctcctgccgg ccaccgagga agctccaaag gtatgegaag cctgcaaaaa 540 taaaaatgag gatgacaacg acataatgga aactctttgt aaaaatgatt ttgcactgaa 600 aataaaagtg aaggagataa cctacatcaa cagagatacc aaaatcatcc tggagaccaa 660 gagcaagacc atttacaagc tgaacggtgt gtctgaaagg gacctgaaga aatccgtgct 720 gtggctgaaa gacagcctgc agtgcacctg cgaggagatg aatgacatca acgcgcctta 780 tctggtcatg ggacagaaac taggtgggga gctggtgatc acctcagtga agcggtggca 840 aaaggggcag agagagttca agcgcatctc ccgaagcatc cgcaagttgc agtgctagtt 900 tgcccccccc tccccaggcc gcctccagag ccaggctgac cacttccgct ctgggtcccc 960 agctctcatc cccccaagca caggcccttg cagetctggc cccagcctgg agcagcttcc 1020 cttgcctttt gcacgtttgc accctggcat ctcctgaatt ataaggeett aggaagette 1080 aggagtggat actgttttaa acatacggga ggaacccacc cagatcttgt agaaatgttc 1140 aaactaataa aatcatggaa tatttttacg gaagttttta aatagctcaa ctttagtttt 1200 gaataggtac agctgtgact tgggtctgac tttccttctt tctgtttggt ttgggccaac 1260 tgattttcaa tttcgtggta aggttgccgt aaegtgcaaa tggcttcatt tttcgatgtg 1320 gcccaaaatg tggtgggtca caatccttcg ttgagataaa gctggctgtt attegaegtt 1380 tctcagctcc agcctgagac ttcagagcct aagtettata ataattcacc tgtcatttta 1440 tgcccccgtt gggaacttac aacagtagca gcatgtactg atttccgggt agagtaette 1500 catttatcaa aagcacatta accaccatag catgattctt egaataaagg gcaaaacaga 1560 ttttataatt gacctgagta ctttaagctt ttgtttaaaa cattttttac ttaattttgc 1620 aaattaaacc attgtagctt acctgtaata tacatagtag Page ttgaccttaa 32 aaagttgtaa 1680

    8693-sequence-listi ng. txt

    2016204268 22 Jun 2016

    aaatattgct ttaaccaaca ctgtaaatat ttcagataag cattatattc ttgtatataa 1740 1760 actttgcttc ctgttttaaa <210> 16 <211> 2937 <212> DNA <213> Cam's familiaris <400> 16 atggaggctg aagtgccatg gaaagtggag catgtcgtca ctgttgctag taaacagcgc 60 cagaggcact tccaagggac cagttgccac ttggagctcc agactatgga aaagatgctg 120 gtgggctgct ttctgttggt cctcggacag atcctccttg tcctccctgc tgaggccaaa 180 gagcggactc cetggaggtc catctecaga gggagacatg cttggaecea tccccagacg 240 gccctcctgg agagctcctg tgagaataaa caggcagacc tggtcttcat catcgacagc 300 tcccgcagtg tcaacaccca tgactatgca aaagttaagg agttcatcct ggacatcttg 360 cagttcttgg acatcagtcc tgacctcacc cgagtgggcc tgctgcagta tggcagcacc 420 ateaagaacg agttctccct caagaccttc aagaagaagt ctgaagtgga gcgtgccgtc 480 aagaggatga ggcacctgtc cacaggcacc atgacggggc tggccatcca gtacgccctg 540 aacattgcat tctcagaagc agagggggca cggcccctga gggagaacgt gctgcgggtc 600 ataatgattg tgaccgacgg gaggccacag gactccgtgg ccgaggtggc tgccaaggca 660 cgtgacacag gcatcctgat ctttgccatc ggtgtgggcc aggtggacet caacacactg 720 aaggecattg ggagtgagcc ccatgaggac catgtcttcc tggtggccaa cttcagecag 780 atggagtccc tgacctcggt gttccagaag aagttgtgca tggtccacat gtgcagcgtc 840 ctggagcata actgtgccca cttctgcatc aatacccccg gctcatacgt ctgcaggtgc 900 aaacaagggt acatcctcaa ctcagatcag aegaettgea gaatccagga tctgtgtgcg 960 gccaaggccc atgggtgtga gcagctctgt gtcaatgtgc tcggctcctt tgtctgccag 1020 tgttacagcg gcttcacact ggctgaggac gggaagacgt gtgcggctgt ggactactgt 1080 gcctcagaga accacggatg tgaacatgag tgtgtgaatg ctgacagctc ctacttttgc 1140 cggtgccgta aaggatttac tcttaaccca gataaaaaaa catgtgcaaa gatagactac 1200 tgtgcctcgc ctaatcaagg atgtcagcac gagtgtgtta acacagatga ctcctattcc 1260 tgtcgctgcc taaaaggttt taccttgaat ccagataaga aaacctgcag aaggatcaac 1320 tactgtgcat tgaacaaacc gggctgtgag catgaatgca tcaacataga ggatggctac 1380 tactgccgct gccgccaggg ctacaccctg gaccccaatg gcaagacttg cagcagggtg 1440 gatcactgcg cggagcagga ccatggctgt gagcagctgt gcctgaacac tgaggattet 1500 tacgtctgcc agtgctcaga aggcttcctc atcaacgacg Page acctcaagac 33 ctgctceagg 1560

    2016204268 22 Jun 2016

    gcggattatt gcttgctgag 8693-sequence- caaccatggt tgcgaatact listing.txt cctgcgtcaa cactgaccga 1620 tcctttgttt gtcggtgtcc tgagggatac gtgctccgca gtgacgggaa gacctgtgcc 1680 aaattggact cctgtgctgt gggagaccat ggctgtgaac attcatgtgt aagcagtgga 1740 gaetcttttg tgtgccagtg ctttgaaggg tatatactcc gtgaagatgg gaaaacctgc 1800 agaaggaaag gtgtctgcca atcagtaaae catggctgtg agcatatttg tgtgaaeagt 1860 gatgagtcgt acatctgcaa gtgccgggaa ggattcaggc ttgctgaaga tgggaagcgc 1920 tgcagaagga aggatgtctg caaatcgacc taccacggct gtgaacacat ttgtgttaat 1980 catggeaact cctacatctg caaatgctca gagggatttg ttctagctga ggatggaaaa 2040 cggtgcaaga gatgcactga aggcccgctt gacctggtct ttgtgatcga tgggtccaag 2100 agcctcgggg aagagaattt cgagattgtg aagcagttcg tcgcgggcat tatagattcc 2160 ttggcggttt cccccaaagc cgcccgagtg gggctgctgc agtactccac gcaggtccgc 2220 acggagttca ccctgggaga cttcagctcg gceagagaca tgaaaaaagc cgtggcccac 2280 atgaagtaca tgggcaaggg ctctatgacg gggctggccc tgaaacacat gtttgagaga 2340 agttttaacc cagtggaagg ggccagaccc gtgtcccccg gggtgtccag agtggccatc 2400 gtgttcacgg acggccgggc ccaggacgac gtctccgcgt gggcccgcag agcccgggcc 2460 ggcggeataa ctatgtatgc tgtcggggta ggaaaagcta ttgaggaaga gctacaagag 2520 attgcctctg agcccacaga caagcatctc ttctatgccg aagacttcag cacaatggga 2580 gagataagtg aaaaactaaa gaagggtata tgtgaagctc tggaagactc tgatggaagt 2640 caggactccc cagcagggga actgccaaag agggtccacc agccaacaga atctgagcca 2700 gteaccataa atatccgaga cctactttcc tgttctaatt ttgcagtgca acaceggtat 2760 ctgtttgaag aagacagtgt ttcacggtcc acacaaaaac tttttcattc aacaaaatct 2820 tcaggaagtc ctttggaaga aaaacacgat cagtgcaaat gtgaaaatct tataatgttc 2880 cagaaccttg cgaatgaaga agtgagaaag ttaacacaac gctatatcct tttctaa 2937

    <210> 17 <211> 1054 <212> DNA <213> Canis familiaris

    <400> 17 ttatgcaagt gctggccgcc ggccggcgcc tcccctcggt ctttcacccc ggccgttacg 60 aaagcgagac cccctcccgc cccggctata aagcgggcgg cccggcggcc ccggcggatc 120 gccgccttcc ctggctccac gcgcgccccg caccgcggcc aggcttgcgc gcagctccgg 180 tgggcggact cccgggcaeg atggagtggg tgtgggcgct cgtgctgctg gcggegctgg 240 gcagegcccg ggcggagagc gactgccgag tgageaactt ceaagtcaag aagaacttcg 300

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    8693-sequence-listi ng. txt acaaggctcg cttcgccggg acctggtacg ccatggccaa gaaggacccc gagggcctct 360 ttctgcagga caacatcgtc gctgagttct cggtggatga gaatggccgg atgagcgcca 420 cggccaaggg ccgagtccgg cttttgaata actgggacgt gtgtgcagac atggttggca 480 ccttcacaga caccgaggac cctgctaaat tcaagatgaa gtattggggc gtagcgtcct 540 tcctecagaa aggaaatgat gaccactgga tcategacac ggactatgac acctatgccg 600 tgcagtattc ctgccgcctc ctgaacctcg atggcacctg tgctgacagc tactccttcg 660 tgttttcccg tgaccccaat ggccttcccc tggaggcaca aaagatcgtg aggcagaggc 720 aggaggagct gtgcctggcc aggcagtaca ggctgatcgt tcacaatggg tattgtgacg 780 gcagatcaga accaaacact ttgtagcaac gtggagtttc atttgaaaag ttcccattaa 840 ttcatgcagc cttcaattgt ctcgtatctt aggggtttag tttgccctgc tctgcacccc 900 tgccctccgc aaacatggaa tctcagcaca cctaaaaata tcggtgggga tcagtgaatc 960 tgeactcaga tgactgtett tcctggagtt ttctaaggaa ttgtttcaag cttaggattc 1020 cagactttga tttattaaaa tgtagtcacc tgtt 1054 <210> 18 <211> 2138 <212> DNA <213> Cam's familiaris <400> 18 gagacacacc actaccctca ccatgagccc caggcagccc ctggtcctgg tgttcctggt 60 gctgggctgc tgctctgcag ctcccagacc acacaagccc accgttgtgg tctttceagg 120 agacctgaga actaatctca ctgacaagca gctggcagag gaatatctgt ttcgctatgg 180 ctacactcaa gtggccgagc tgagcgacga caagcagtcc ctgagtcgcg ggctgcggct 240 tctccagagg cgcctggctc tgcctgagac tggagagctg gacaaaacca ccctggaggc 300 catgcgggcc ccgcgctgcg gcgtcccgga cctgggcaaa ttccagacct ttgagggcga 360 cctcaagtgg caccacaacg acatcactta ctggatacaa aactactcgg aagacttgcc 420 ccgcgacgtg atcgacgacg cctttgcccg agccttcgcg gtctggagcg cggtgacacc 480 gctcaccttc actcgcgtgt acggccccga agecgacatc atcattcagt ttggtgttag 540 ggagcacgga gatgggtatc ccttcgatgg gaagaacggg cttctggctc acgcctttcc 600 tcccggcccg ggcattcagg gagacgccca cttcgacgac gaggagttat ggactctggg 660 caagggcgtc gtggttccga cccacttcgg aaacgcagat ggcgccccct gccacttccc 720 cttcaccttc gagggccgct cctactcggc ctgcaccacg gacggccgct ccgatgacac 780 gccctggtgc agcaccacgg ccgactatga caccgaccgt cggttcggct tctgccccag 840 cgagaaactc tacacccagg acggcaatgg ggacggcaag ccctgcgtgt ttccgttcac 900

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    cttcgagggc cgctcctact 8693-sequence- ccacgtgcac caccgacggc 1isting.txt cgctcggacg gctaccgctg 960 gtgctccacc accggcgact acgaccagga caaactctac ggcttctgcc caacccgagt 1020 cgattccgcg gtgaccgggg gcaactccgc cggggagccg tgtgtcttcc ccttcatctt 1080 cctgggcaag cagtactega cgtgcaccag ggagggccgc ggagatgggc acctctggtg 1140 cgccaccact tcgaactttg acagagacaa gaagtggggc ttctgcccgg accaaggata 1200 cagcctgttc cttgtggccg cccatgagtt cggccacgcg ctgggtttag atcattcatc 1260 ggtgccagaa gcgctcatgt accccatgta cagcttcacc gagggccccc ccctgcatga 1320 agacgacgtg aggggcatcc agcatctgta cggtcctcgc cctgaacctg agccacagcc 1380 tccaaccgcc ccgcccaccg ccccgcccac cgtctgcgct actggtcctc ccaccacccg 1440 cccctcagag cgccccactg ctggccccac aggcccccct gcagctggcc ccacgggtcc 1500 ccccactgct ggcccctctg aggcccctac agtgcctgtg gatccggcag aggatatatg 1560 caaagtgaac atcttcgacg ccatcgcgga gatcaggaac tacttgcatt tcttcaagga 1620 agggaagtac tggcgattct ccaagggcaa gggacgccgg gtgcagggcc ccttccttat 1680 caccgacacg tggcctgcgc tgccccgcaa gctggactcc gcctttgagg acgggctcac 1740 caagaagact ttcttcttct ctgggcgcca agtgtgggtg tacacaggca cgtcggtggt 1800 aggcccgagg cgtctggaca agctgggcct gggcccggag gttacccaag tcaccggcgc 1860 cctcccgcaa ggcgggggta aggtgctgct gttcagcagg cagcgcttct ggagtttcga 1920 cgtgaagacg cagaccgtgg atcccaggag cgccggctcg gtggaacaga tgtaccccgg 1980 ggtgcccttg aacacgcatg acatcttcca gtaccaagag aaagcctact tctgccagga 2040 ccgcttctac tggcgtgtga attctcggaa tgaggtgaac caggtggacg aagtgggcta 2100 cgtgaccttt gacattttgc agtgccctga ggattaga 2138

    <210> 19 <211> 1636 <212> DNA <213> Cam's familiaris

    <400> 19 cgccggcgcc gctgaccgag gcctgctggg attccagaat tggagaagga ggcatgatga 60 agactctcct gctgctggtg gggctgctgc tgacctggga caatggacgg gttctgggag 120 accaggctgt ctcagacacc gagctccagg aaatgtccac cgaaggtagt aagtacatta 180 ataaggaaat aaaaaatgct ctcaaggggg tgaaacagat aaagacccta atagaacaga 240 caaacgaaga gcgcaaatca ctgctcagca acctggagga agccaagaag aagaaagagg 300 atgcactaaa tgataccaag gattctgaaa cgaagctgaa ggcgtcccag ggggtgtgca 360 atgacaccat gatggccctc tgggaggagt gcaagccctg cctgaaacag acctgcatga 420

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    agttctacgc acgtgtctgc 8693-sequence- agaagtggct cggggctagt listing.txt tggccaccag ctegaggagt 480 tcctgaacca gagttctccc ttetaettet ggatgaatgg egategtate gactccctgc 540 tggagaacga ccggcagcag acccacgccc tggaegteat gcaggacagc ttcaaccggg 600 catecagcat catggatgag cttttecagg acagattctt cacccgggag ccccaggata 660 cttaccacta ctcacccttc agcttattcc agaggaggee tttcttcaat cccaaatttc 720 gcatcgcccg gaacataata cctttcccta gattccagcc ettaaaette catgacatgt 780 ttcagccctt ctttgacatg atacaccagg ctcaacaggc catggatgtt aacctgcata 840 gaattcctta ccacttccca attgaattcc cagaagaaga taaccgcacc gtgtgcaagg 900 agatccgtca caactccaca ggttgcctga aaatgaagga ccagtgtgaa aagtgccagg 960 agatcttgtc agtggattgt tcttccaaca accctgcgca ggtccagctg cgacaggaac 1020 ttagtaattc cctccagatt geggagaagt tcaccaagct etaegaegag ctgctgcagt 1080 cctaccagga gaagatgttc aacacgtcct ccctgctgaa gcagctgaac gagcagttta 1140 gctgggtgtc ceaactggct aacctcactc aaagtgaaga cccattctat ctccaggtca 1200 cgacggtggg ttctcagact tctgactcca atgttcccgt gggcttcact aaggtggttg 1260 tgaagetett tgattctgac cccatcacgg tgatgatccc agaagcagtc tccaggaaca 1320 atectaaatt tatggagact gtggcagaga aggetettea ggagtatege caaaagcacc 1380 gagaggaatg agatgtgaac attgcttctc caaatatggg agggctgagt tcctttgacc 1440 ccaagatgag tgctaggccc ctagagagag ctctgcatgt caccaagtga ccaggccttg 1500 cctcgaggcg ctcctgtctc ctaacccagc tttccttcct ctggactccg cactgtaacg 1560 cctacgtttg ctgatcatgg gaagaactcc tgtgtgccac taaetcaata aaaccgccaa 1620 ggaatctgaa aaaaat 1636

    <210> 20 <211> 2321 <212> DNA <213> Cam's familiaris

    <400> 20 caggtctgtg ctccaggetc ctagccacag gcccaggaag atgaggeteg ccgtccgcgc 60 tctgctggcc tgcgcggtcc tggggctgtg tctggctgtc teccctgaga aaactgtgag 120 atggtgcact gtctcaaatc atgaggccag taagtgttcc agtttcatgg aaaatatgaa 180 aaccgtcctt gaaaatggtc cttttgtcag ctgtgtgaag agaacctcct accttgagtg 240 catcaaggct atttgggcaa atgaagcaga tgctgtgaca ettgatgeag gtttggtgtt 300 tgaggcaggc ctgaacceet acaacctaaa gcctgtagtg gcagagttct atgggaaaga 360 taagcaaacc egetattatg ctgtggctgt ggtgaagaag ageagtaegt tcaatctgaa 420

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    8693-sequence-listi ng. txt

    tcagctccaa ggcaagaaat cctgccacac aggccttggc aggtctgctg ggtggaacat 480 ccccatgggc ttactttatt ggaagttgcc agagccacgt gaatctcttc agaaggcagc 540 gtccagtttc ttcacggcca gctgtgttcc ctgtgtggat cggacagctt tccccaaact 600 gtgtcaactg tgtgtgggga aagggacgga caagtgtgcc tgctccaacc atgagceata 660 cttcggctac tcgggtgcct tcaagtgtct gatggaggac gctggggaag tggcctttgt 720 caagcattca acagtgttgg agaacctgcc aaacaaagct gacctggacc agtatgagct 780 gctctgccca gacaacaaac gaaagccagt gggggaatat aagcagtgct acctggccaa 840 ggtcccttcc catgctgttg tggcccgaag tgtggggggc aaggaagact tgatctggga 900 gcttctaaac caggcccagg aacattatgg caaagacaaa tctaaagtct tccagctctt 960 cagctcaact ttggggaagg acctgctgtt taaagactct gccgaagggt tttttaggat 1020 tcctcctaaa atggacacct ggctgtacct gggatatgag tatgtcactg ctcttcggaa 1080 cctaagggaa gatgtgcgcc cagatacecc aagggatgaa tgcaagaagg tgaaatggtg 1140 tgcagtaggt caccatgaga ttgccaagtg tgatgagtgg agtgtaaaca gcgaagggaa 1200 aatagagtgt gaatcagcag agtctactga agactgtatt gccaagattg cgaaaggaga 1260 ggctgatgcc atgagcttgg atggaggcta tatctacata gcgggccagt gcggtctggt 1320 gcctgtcctg gcagagaact acaaaactca gggctctaca tgtagcaaca cagcagagga 1380 agggtatctt gctgtggccg tagttaagag attagataaa accatctctt ggaataatct 1440 gcaaggcagg aagtcctgcc acactgcagt agatagaact gcaggctgga acattcccat 1500 gggcctgctc tacaacagga tcaaccactg tgaatttgat aaatttttct ctoaaggctg 1560 tgcccccgga tctatgcgga attccagtct ctgtgctctg tgtattggct cagcaaatgt 1620 tccaggaaag gagtgtgttc ccaataacca tgagagatac tatggctaca caggggcttt 1680 caggtgtctg gttgagaagg gagatgtggc ctttgtgaaa gaccagactg tcttgcagaa 1740 cactggggga aagaacactg aagattgggc taaggatctg aaggaagagg actttgagct 1800 gctgtgccct gatggccaaa ggaagtctgt ggacaaggct cctgagtgct tcctagccaa 1860 agccccaaat catgctgtgg tctcaaggaa agacaaggca tcttgtgtta gcaaaatgtt 1920 acttgaccag cagcttttgt ttggaagaaa tggaaatgac tgctcgggca agttttgctt 1980 gttccactca gcaaccaagg accttctgtt cagggacgac acacaatgtt tggccaaact 2040 tccagaggac acaacatata aatcatactt aggagcagag tatatcacgg ctgttgctaa 2100 cctgagacaa tgctccacct cgaaactcct ggaggcctgc actttccata catcttaaaa 2160 tccaagaggc ggagacacac cagatggtga tgggagctca cgtgacccat aagttccctc 2220 ggggcctcac tggcctgggt ggtttgtctg gtttcacagt tcagcggtgg aacctctgta 2280

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    8693-sequence-listing. txt gaacattaaa taaacattat tattggtgtt atcttttaaa a 2321 <210> 21 <211> 576 <212> DNA <213> Cam's familiaris <400> 21

    2016204268 22 Jun 2016 ctttccgaga cegctccagg ttggtgctcg atacctgatg cecctgatgc cccgggaggg 60 ctgcccctcc aagaacacta gaaaaggggt ggagggaact ccggggaggg aggaggggtg 120 tcactgatcg ctcccacccc tgcttctgct tccagagagc ccctccggcc ccgccatgag 180 gctcattctg tcgctcccgg ttttggtggt ggttctgtcg atggttttgg aaggtccagc 240 ccccgcccag gcggccggag aaatctccag cacttttgag cgcatcccgg ataagctgaa 300 ggagtttggt aacaccctgg aagacaaggc ccgggcagcc attgagagca tcaagaagag 360 cgacattcct gcaaagaccc gaaactggtt ttctgaggct ttcaagaaag tgaaggagca 420 tctcaaaact gccttctcct gaacaccagg agagccgccc ctctactctg gcctgtgtgc 480 cccaggaggg ggctctgaaa tttcccatcc cctggctcct tgccaaggac ttcatgatgt 540 tcatgtctac ccccaacctc caataaaaat cctata 576 <210> 22 <211> 1275 <212> DNA <213> Canis familiaris <400> 22 atgcccttgc tctggctgag aggattcttg gtggcgagtt gctggattat agtgcggagc 60 tccccgaccc cggggcccga ggggcccggc gcggcccccg cctgcccggc ctgcgcgctc 120 accgccctgc ccagggatgc ccccaactcc cagcccgaga tggtggaggc cgtcaagaag 180 cacatcctca acatgctgca cttgaagaag agacccgaag tcacccagcc ggtgcccaag 240 gcggcgcttc tgaacgcgat ccgcaagctg cacgtaggca aagtcgggga gaacgggttc 300 gtggagatag aggatgacat cggcaggagg gcagaaatga atgaactcat ggagcagacc 360 tcggagatca tcacgttcgc ggaatcaggc acagccagga aaacgctgca ctttgagatt 420 tccaaagaag gcagtgaeet gtcggtggtg gagcgtgcag aagtctggct cttcctcaaa 480 gtccGcaagg ccaacaggac caggaccaaa gtcaccatcc ggctcttgca gaagcacccc 540 cagggcagct tggatgcggg ggaggaggcc gaggacatgg gcttcccgga ggagaggaac 600 gaggtgttga tttctgaaaa ggtggtggac gcacggaaga gcacctggca catcttccct 660 gtctccagca gcatccagcg cttgctggac cagggcagga gctccctgga cgttcggatt 720 gcctgcgagc agtgccacga gacgggcgcc agectggtgc tcctgggcaa gaagaagaag 780 aaggaggagg agggggaagg gaagaagaag gacggaggag acgcaggggc cgggggggac 840

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    8693-sequence-listi ng. txt gaggacaagg agcagtccca cagacctttc ctcatgctgc aggcccgcca gtctgaagac 900 caccctcatc ggcggcggcg gcggggcctg gagtgtgacg gcaaggtcaa catctgctgt 960 aagaaacagt tctttgtgag cttcaaggac attggctgga acgactggat cattgccccc 1020 tccggctatc acgccaacta ctgcgagggt gggtgcccga gccacatagc aggcacgtcg 1080 gggtcctcgc tctcctttca ctcgaccgtc atcaaccact accgcctgcg gggtcacagc 1140 cccttcacca acctcaagtc gtgctgtgtg cccaccaagc tgagaccaat gtccatgctg 1200 tactacgatg atgggcaaaa catcatcaaa aaggacattc agaacatgat cgtggaggag 1260 tgcgggtgct catag 1275

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