BACKGROUND OF THE INVENTION

Field of the Invention

[0001] l ^" hc invention relates generally to methods for diagnosing and predicting chronic valvular disease and particularly to methods for diagnosing and predicting chronic valv ular disease by measuring gene expression markers associated with chronic valvular disease.

Description of Related Art

[0002] Cardiac disease is one of the most common disorders in dogs Approximately 1 1 % of dogs suffer cardiac disease, 95% of which have adult onset. One third of dogs ages 10 or over has Chronic Valvular Disease (CVD). CVD is characterized by a progressive degeneration and deformation of the atrioventricular valves, most commonly the mitral valves, resulting in early mitral valve insufficiency, This in turn leads to the appearance of a systolic heart murmur due to mitral regurgitation, wherein inadequate closure of the mitral valve causes blood to flow back to the left atrium. The affected dogs finally develop left atrioventricular volume overload, pulmonary edema, atrial dilatation and supraventricular arrhythmias.

[0003] Although surgical or medical treatment of affected valves is possible, nutritional intervention is preferred by pet owners. Early detection and treatment are imperative however detection can be difficult due to the lack of symptoms. Biomarkers are useful for detecting conditions when an animal is displaying minimal symptoms or asymptomatic. Gene expression markers arc useful biomarkers. Currently there are no known biomarkers useful as diagnostic agents to measure chronic valvular disease in animals.

[0004] Therefore there remains a need for diagnosing and predicting chronic valvular disease in animals to provide the most appropriate and effective level of treatment. Such treatment will improve the animal's quality of life. The present invention satisfies this need.

SUMMARY OF THE I VENTION

[0005] It is, therefore, an object of the present invention to provide methods for diagnosing and predicting chronic valvular disease in animals.

[0006] This and other objects arc achieved using methods for diagnosing and predicting chronic valvular disease in an animal that involve obtaining a sample from (he animal; analyzing the sample for the presence of one or more gene expression markers associated with chronic valvular disease; comparing the amount of gene expression markers identified in the sample to a corresponding amount of the same gene expression markers present in a sample from one or more comparable control animals that do not suffer from chronic valvular disease; and using said comparison to diagnose chronic valvular disease in the animal if the gene expression markers found in the animal's sample is differentially expressed in the control animal's sample.

[0007] Other and further objects, features, and advantages of the present invention will be readily apparent to those skilled in the art.

DETAILED DESCRIPTION OH THE INVENTION

Definitions

[0003] The term "animal" means any animal susceptible to or suffering from chronic valvular disease, including human, avian, bovine, canine, equine, feline, hicrinc, lupine, murine, ovine, or porcine animals.

[0009] The term "differential expression" or "differentially expressed" means increased or upregulated gene expression or means decreased or downregulatcd gene expression as detected by the absence, presence, or change in the amount of transcribed messenger RNA or translated protein in a sample, or means an increase or decrease in the amount of protein present in a sample.

[0010] The term "comparable control animal" means an animal of the same species and type or an individual animal evaluated at two different times.

[0011] The term "companion animals" means domesticated animals such as dogs, cats, birds, rabbits, guinea pigs, ferrets, hamsters, mice, gcrbils, pleasure horses, cows, goats, sheep, donkeys, pigs, and more exotic species kept by humans for company, amusement, psychological support, extrovert display, and all of the other functions that humans need to share with animals of other species.

[0012] The term "diagnosing" means determining if an animal is suffering from or predicting if the animal is susceptible to developing chronic valvular disease.

[0013] As used herein, ranges arc used herein in shorthand, so as to avoid having to list and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range.

[0014] As used herein, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references "a", "an", and "the" are generally inclusive of the plurals of the respective terms. For example, reference to "a method" includes a plurality of such ""methods." Similarly, the words " comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. Likewise the terms "include", "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context.

[0015] The methods and compositions and other advances disclosed here are not limited to particular methodology, protocols, and reagents described herein because, as the skilled artisan will appreciate, they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to, and does not. limit the scope of that which is disclosed or claimed

[0016] Unless defined otherw ise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by one of ordinary skill in the art in the ficld(s) of the invention, or in the field(s) where the term is used.

[0017] All patents, patent applications, publications, technical and/or scholarly articles, and other references cited or referred to herein are in their entirety 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, arc relevant, material, or prior art. The right to challenge the accuracy and pertinence of any assertion of such patents, patent applications, publications, and other references as relevant, material, or prior art is specifically reserved.

The Invention

[0018] In one aspect, the invention provides methods for diagnosing chronic valvular disease in an animal. The methods comprise obtaining a biological sample from the animal; analyzing the sample for the presence of one or more gene expression markers associated with chronic valvular disease; comparing the amount of gene expression markers identified in the sample to a corresponding amount of the same gene expression markers present in a sample from one or more comparable control animals that do not suffer from chronic valvular disease; and using said comparison to diagnose chronic valvular disease in the animal if the gene expression markers found in the animal's sample are differentially expressed in the control animal's sample.

[0019] In one aspect, the invention provides methods for diagnosing chronic valvular disease in an animal. The methods comprise obtaining a biological sample from the animal; analyzing the sample for the presence of one or more gene expression markers associated with chronic valvular disease; comparing the amount of gene expression markers identified in the sample to a corresponding amount of the same gene expression markers present in a sample from one or more comparable control animals that do not suffer from chronic valvular disease; and using said comparison to diagnose chronic valvular disease in the animal if the gene expression markers found in the animal's sample arc greater than the amount present in the control animal's sample.

[0020] In one aspect, the invention provides methods for diagnosing chronic valvular disease in an animal. The methods comprise obtaining a biological sample from the animal; analyzing the sample for the presence of one or more gene expression markers associated with chronic valvular disease; comparing the amount of gene expression markers identified in the sample to a corresponding amount of the same gene expression markers present in a sample from one or more comparable control animals that do not suffer from chronic valvular disease; and using said comparison to diagnose chronic valvular disease in the animal if the gene expression markers found in the animal's sample are less than the amount present in the control animal's sample.

[0021] The inventions is based upon the discovery that the gene expression markers of the invention are present in the biological sample of an animal and that the amount of the gene expression markers in the sample serves as a biochemical indicator for diagnosing chronic valvular disease by indicating or predicting the threshold for chronic valvular disease. The invention allows veterinary and other clinicians to perform tests for these ~biomarkers" in a sample and determine whether the animal is susceptible to or suffering from chronic valvular disease and whether there is a need for further diagnostics or treatment. Having established the need for further diagnostics or treatments, the cost and risk of such further diagnostics or treatments arc justified.

[0022] In various embodiments, one or more comparable control animals that arc not the animal being evaluated for chronic valvular disease and that have been determined not to suffer from chronic valvular disease are evaluated for at least one of the gene expression markers and the results of such evaluations are used as a baseline value for comparison with the results from an animal being evaluated for one or more of the gene expression markers. In preferred embodiments, the baseline value for the gene expression markers is determined by evaluating numerous comparable control animals.

[0023] In another embodiment, the amount of at least one of the gene expression markers are determined for an animal at various times throughout the animal's life and the results used to determine if the animal is susceptible to or suffering from chronic valvular disease, e.g.. if the amount of at least one of the gene expression markers increases or decreases as the animal ages, the animal can be diagnosed as susceptible to or suffering from chronic valvular disease. In preferred embodiments, the animal is evaluated periodically and the results for the gene expression markers are recorded. Then, if a subsequent evaluation shows that the amount of one or more gene expression markers has increased or decreased since the last evaluations), the animal is diagnosed as susceptible to or suffering from chronic valvular disease.

[0024] Any sample that is of biological origin may be useful in the present invention. Examples include, but are not limited to, blood (scrum plasma), cerebral spinal fluid (CSF), urine, stool, breath, saliva, or biops of any tissue In one embodiment the sample is a tissue sample. In another embodiment, the sample is cardiac tissue. In one embodiment the tissue is mitral valve tissue. In another embodiment, the tissue is left ventricle tissue. In one embodiment, the sample is a serum sample. While the term "scrum" is used herein, those skilled in the art will recognize that plasma or whole blood or a sub-fraction of whole blood may also be used.

[0025] In various embodiments of the invention, changes in gene expression may be measured in one or both of two ways: ( 1 ) measuring transcription through detection of mRNA produced by a particular gene; and (2) measuring translation through detection of protein produced by a particular transcript

[0026] Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR (including, without limitation, RT-PCR and qPCR), R asc protection. Northern blotting, microarrav. macroarray. and other hybridization methods. The genes that are assayed or interrogated according to the invention are typically in the form of mRNA or reverse transcribed mRNA. The genes may be cloned and/or amplified. The cloning itself does not appear to bias the representation of genes within a population. However, it may be preferable to use polyA+ RNA as a source, as it can be used with fewer processing steps.

[0027] Decreased or increased expression can be measured at the protein level using any of the methods wrll known in the art for protein quantitation, such as, for example, western Wotting, ELISA. mass spectrometry, etc.

[0028] While the use of one of the gene expression markers is sufficient for diagnosing chronic valvular disease, the use of one or more, two or more, three or more, or four or more of such gene expression markers is encompassed within the invention and may be preferred in many circumstances. The gene expression markers can be evaluated and used for a diagnosis in any combination.

[0029] In one embodiment, the diagnosis is based upon determining the amount of one or more gene expression markers in the animal's sample is selected from NOS3, COL6A5 (COL29A I ). Serpincl (PAI- I ), SELP, SLC27A6. ED I CANFA, CD74, , MYC, MT2 CANFA, IL8 CANFA, IL6, ACSL 1. ADIPOQ, and AGT.

[0030] In one embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's sample are greater compared to the amount present in the control animal's sample, wherein the gene expression markers are NOS3, COL6A5 (COL29A1 ), Serpinel (PAl-1 ). SELP, SLC27A6. EDN I CANFA, CD74, , MYC. MT2_CANFA, IL8 CANFA, and 1L6.

[0031] In one embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal s sample arc less than compared to the amount present in the control animal's sample, wherein the gene expression markers are ACSLI . ADIPOQ, and AGT.

[0032] In one embodiment, the diagnosis is based upon determining the amount of one or more gene expression markers in the animal's mitral valve sample is selected from NOS3, COL6A5 (COL29AI ), Serpinel (PAI-I ), SELP, ACSLI , ADIPOQ, 03FAR I , FABP4. SLC27A6. EDNI CANFA. CD74, GSTPI . MGSTI . MYC, MT2 CANFA, IL8 CANFA, IL6, PLIN I , CLDN 1 , and AGT.

[0033] In one embodiment, the diagnosis is based upon determining the amount of one or more gene expression markers in the animal's Left Ventricle sample is selected from N0S2, NOS3, MMPI 5. MMP8, MMP9 .TIMP I , NPPA, COL14AI , HOPX, Serpine l (PAI-1 ) .TGFB3. LIPE. MLYCD, FADS I , ACSLI , LOX. TGFBR2. WNT9B, WNT5A, WISP2, FZD8, OSMR, OSM, ELOVL7, ACOTI , MT2_CANFA, MT 1 CANFA, STAT3, EDNRB, TAGLN2 , ADIPOQ, RETN. PLA2G5. PLA2G4A, FFAR2, AGT, NFKBIA, TLR4, FOS, JUNB, AQP9, SOAT1 , SMAD6. EDNRB, N X2-5, GATA4 CANFA, PTGS2 (COX-2). IL8_CANFA. IL6, and MYH I CANFA.

[0034] In one embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's mitral valve sample arc greater compared to the amount present in the control animal's mitral valve sample, wherein the gene expression markers arc NOS3, COL6A5 (COL29A1 ). Serpincl (PAI-1 ). SELP. SLC27A6, EDN I CANFA, CD74, , MYC, MT2 CANFA, IL8 CANFA, IL6. and CLDN I In a preferred embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's mitral valve sample are greater compared to the amount present in the control animal's mitral valve sample, wherein the gene expression markers are NOS3, COL6A5 (COL29A1 ), Scrpincl (PAI- 1 ). and SELP.

[0035] In one embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's left ventricle sample are greater compared to the amount present in the control animal's left ventricle sample, wherein the gene expression markers are NOS3, P15, MMP8, M P9 ,ΤΙΜΡΙ , NPPA. HOPX. Serpinel (PAI-1 ) , LOX, TGFBR2. WNT9B. OSMR. OSM. ELOVL7, MT2_CANFA, MT I CANFA, STAT3. EDNRB, TAGLN2 , RETN, FFAR2, NFKBIA. TLR4. FOS. JUNB, AQP9, SOAT1 , SMAD6, EDNRB, PTGS2 (COX-2). IL8 CANFA, and IL6. In a preferred embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's left ventricle sample are greater compared to the amount present in the control animal's left ventricle sample, wherein the gene expression markers arc NOS3, MMPI 5. MMP8, MP9, TFMPI , NPPA, HOPX, Serpinel (PAI-1 ). LOX. TGFBR2. WNT9B, OSMR. OSM, ELOVL7, MT2 CANFA. MT1 CANFA. STAT3. EDNRB. and TAGLN2.

[0036] In one embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's mitral valve sample arc less than compared to the amount present in the control animal's mitral valve sample, wherein the gene expression markers are ACSL1. ADIPOQ, 03FAR I , FABP4. GSTP I, MGSTI , PLIN 1 , and AGT. In a preferred embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's mitral valve sample are less than compared to the amount present in the control animal's mitral valve sample, wherein the gene expression markers are.

[0037] In one embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's left ventricle sample are less than compared to the amount present in the control animal's left ventricle sample, wherein the gene expression markers are NOS2, C0L I4A I . TGFB3, LIPE, MLYCD, FADS I , ACSL1 , WNT5A, WISP2. FZD8. ACOTl . ADIPOQ, PLA2G5. PLA2G4A, AGT, N X2-5, GATA4 CANFA, and MYH l CANFA. In a preferred embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's left ventricle sample arc less than compared to the amount present in the control animal's sample, wherein the gene expression markers are NOS2, COL14A1 , TGFB3, LIPE. MLYCD. FADS1 , ACSLI . WNT5A, WISP2, FZD8, and A OTJ . In a more preferred embodiment, the diagnosis is based upon determining if the gene expression markers found in the animal's left ventricle sample are less than compared to the amount present in the control animal's sample, wherein the gene expression markers are NOS2. COL I4AI , T0FB3, LIPE, MLYCD. FADS I . and ACSL I .

[0038] In various embodiments, the animal is a human or companion animal. Preferably, the companion animal is a canine such as a dog or a feline such as a cat.

EXAMPLES

[0039] The invention can be further illustrated by the following examples, although it will be understood that these examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifically indicated.

Example 1

[0040] Biomarkers were identified through a differential gene expression profiling study comparing diseased and normal cardiac tissues.

[0041] Study Design. Mitral valve necropsies were collected from 3 nondiseased dogs and 3 diseased dogs, while left ventricle necropsies were collected from 4 nondiseased dogs and 2 diseased dogs. Echocardiograms were utilized to classify disease status. NA sample was extracted from each of the necropsy samples, and was sequenced using RNA-seq technology to quantitate the global gene expression levels. Differentially expressed genes (DEG) were also determined

[0042] Results. The study identified eight hundred and twelve (812) differentially expressed genes in left ventricle tissues (Table 5), and two hundred and sixty-three (263) differentially expressed genes in mitral valve tissues (Table 6). Genes were further selected and ranked based on their biological relevance in cardiomyopathy and Chronic Valvular Disease (CVD). A list of top 50 left ventricle genes (Table I ), and a list of top 20 mitral valve genes (Tabic 3) were generated.

[0043] The top 50 left ventricle gene expression markers differentially expressed in left ventricle tissue are identified in Table 1.

Table I

[0044] The top 15 left ventricle gene expression markers differentially expressed (Table 2) were selected from the Table I for real time quantitative reverse transcription PCR assay (qPCR). The results showed 12 genes with p values of less than 0.05 (p 0.05), while 14 with p values of less than 0.1 (pO.I O). In addition, 14 genes showed consistency in the direction of gene expressional changes with RNA-seq.

Table 2

[0045] The top 20 mitral valve gene expression markers differentially expressed in mitral valve tissue are identified in Tabic 3.

Table 3

[0046] The top 5 left mitral valve gene expression markers differentially expressed and shown in Table 4 were selected from the Tabic 2 for qPCR assay. The results showed 2 genes with p values of less than 0.05. In addtion. all five genes showed consistency in the direction of gene expressional changes with RNA-seq.

Table 4

[0047] The 812 differentially expressed genes in the left ventricle tissue arc identified in Table

Table 5

[0048] The 263 differentially expressed genes in mitral valve tissue are identified in Table 6

Table 6

[0049] The gene expression differentially expressed in both left ventricle and mitral valve tissue arc identified in Table 7.

Table 7

[0050] In the specification, there have been disclosed typical preferred embodiments of the invention. Although specific terms arc employed, they are used in a generic and descriptive sense only and not for purposes of limitation. The scope of the invention is set forth in the claims. Obviously many modifications and variations of the invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.