WO2014071200A1

WO2014071200A1 - Beta blocker responder status assays and related materials and methods

Info

Publication number: WO2014071200A1
Application number: PCT/US2013/068087
Authority: WO
Inventors: Michael Bristow; Elaine EPPERSON; Anis KARIMPOUR-FARD; Brian LOWES; David Kao
Original assignee: The Regents Of The University Of Colorado, A Body Corporate
Priority date: 2012-11-02
Filing date: 2013-11-01
Publication date: 2014-05-08

Abstract

The present invention discloses that myocardial expression levels of certain mRNA sequences are able to distinguish patients who will respond favorably to β-one adrenergic blockade from those who will continue to experience cardiac hypertrophy and a continued decrease in left ventricle ejection fraction. Therefore, the present invention provides diagnostics, assays, panels, treatments, computer models, and related materials and methods.

Description

TITLE

BETA BLOCKER RESPONDER STATUS ASSAYS AND RELATED MATERIALS AND METHODS

Inventors: Michael Bristow, Elaine Epperson, Anis Karimpour-Fard, Brian Lowes, David Kao

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application No.

61/722,062, filed November 2, 2012, the entire disclosure of which is expressly incorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number HL048013 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates to the determination of β-blocker responder status in human patients. The invention additionally provides diagnostics, assays, panels, and treatments dependant on the patient's β-blocker responder status.

SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted via EFS- web and is hereby incorporated by reference in its entirety. The ASCII copy, created on October 31, 2013, is named 53-54477-CU3209H_SL.txt, and is 9,395 bytes in size.

BACKGROUND OF THE INVENTION

[0005] Heart failure is associated with poor quality of life, frequent hospitalizations, and a high mortality. Approximately 30% of patients with advanced heart failure will not respond to optimal medical therapy and the long term prognosis for these patients remains poor. Therapeutic options for these patients, including cardiac transplantation and mechanical cardiac support, are limited due to donor supply and cost. A better understanding of the mechanisms of myocardial dysfunction and the pathways by which dysfunction can be reversed would be an important contribution to clinical decision making, prognosis prediction and the development of new interventions.

[0006] The mechanisms responsible for progressive myocardial dysfunction and remodeling of the cardiomyopathic, intact, failing human heart are incompletely understood. The mechanism(s) behind β-blocker related improvements in myocardial function and reversal of remodeling also remain poorly understood.

SUMMARY OF THE INVENTION

[0007] The present invention discloses that myocardial expression levels of certain mRNA sequences are able to distinguish cardiac patients who will respond favorably to β-adrenergic blockade from those who will continue to experience left ventricular remodeling and no improvement in ejection fraction. The present invention provides a diagnostic that can distinguish between responders (who would not need expensive and invasive therapies) and non-responders (who would receive quicker follow up and appropriately more aggressive treatments).

[0008] The present invention provides methods to identify a human as having β-blocker responder status, comprising:

a) obtaining at least one myocardial tissue sample from a human who has heart failure due to a dilated cardiomyopathy;

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 1 1 , and SEQ ID NO: 12; and

c) identifying the human as having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 1 1 and SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 are higher compared to non-failing control mRNA expression levels.

[0009] The present invention also provides methods to identify a human as having β-blocker responder status, comprising:

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 1 1 , and SEQ ID NO: 13 ; and

c) identifying the human as having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 12 are lower compared to non- failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are higher compared to non-failing control mRNA expression levels. [00010] The present invention also provides methods to identify a human as having β-blocker responder status, comprising:

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13; and c) identifying the human has having β-blocker responder status if:

i) a generalized linear model algorithm including the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates the human as having β-blocker responder status; and

ii) a generalized linear model algorithm including the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates the human as having β-blocker responder status.

[00011] The present invention also provides methods to identify a human as having β-blocker responder status, further comprising obtaining mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 13, and identifying the human as having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 11 and SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are higher compared to non-failing control mRNA expression levels.

[00012] The present invention also provides methods wherein the sample is obtained from a human having a condition selected from the group consisting of: idiopathic dilated cardiomyopathy; left ventricular ejection fraction less than 40%; one or more biomarkers of heart failure; heart failure; elevated atrial natriuretic peptide; elevated beta-type natriuretic peptide; and elevated cholesteryl ester transfer protein.

[00013] The present invention also provides methods wherein the mRNA levels are obtained via use of a physical assay selected from the group consisting of: qPCR; competitive hybridization; and microarray.

[00014] The present invention also provides methods wherein a human identified as having β- blocker responder status has a higher likelihood of an outcome selected from the group consisting of: greater likelihood of shorter duration of heart failure prior to testing than non-responders; more myocyte reverse remodeling compared to non-responders; better prognosis compared to non- responders; more heart tissue repair compared to non-responders; improved left ventricle ejection fraction compared to non-responders; decreased end diastolic volume compared to non-responders; reductions in heart rate compared to non-responders; reductions in blood urea nitrogen compared to non-responders; reductions in atrial natriuretic peptide compared to non-responders; reductions in increases in body mass index compared to non-responders; and reductions in increases in systolic pressure compared to non-responders.

[00015] The present invention also provides methods further comprising administering to the human identified as having β-blocker responder status a therapeutic dose of β-blocker.

[00016] The present invention also provides methods further comprising administering to the human identified as having β-blocker responder status a therapeutic dose of metoprolol succinate, metoprolol succinate and doxazosin, and carvedilol.

[00017] The present invention also provides methods wherein the β-blocker is metoprolol succinate and is administered once daily at a dosage range selected from the group consisting of: 5 - 15 mg; 7.5 - 15 mg; 12.5 - -25 mg; 15 - 30 mg; 25 - 50 mg; 35 - 60 mg; 45 - 70 mg; 50 - 75 mg; 60 - 85 mg; 75 - 100 mg; 85 - 110 mg; 95 - 120 mg; 100 -125 mg; 110 - 135 mg; 120 - 145 mg; 125 - 150 mg; 135 - 160 mg; 145 - 170 mg; 150 - 175 mg; 160 - 185 mg; 170 - 195 mg; 175 - 200 mg; 185 - 210; and 195 - 220 mg.

[00018] The present invention also provides methods wherein the β-blocker is metoprolol succinate and doxazosin and is administered once daily at a metoprolol succinate dosage selected from the group consisting of: 5 - 15 mg; 7.5 - 15 mg; 12.5 - -25 mg; 15 - 30 mg; 25 - 50 mg; 35 - 60 mg; 45 - 70 mg; 50 - 75 mg; 60 - 85 mg; 75 - 100 mg; 85 - 110 mg; 95 - 120 mg; 100 -125 mg; 110

- 135 mg; 120 - 145 mg; 125 - 150 mg; 135 - 160 mg; 145 - 170 mg; 150 - 175 mg; 160 - 185 mg; 170 - 195 mg; 175 - 200 mg; 185 - 210; and 195 - 220 mg, and a doxazosin dosage selected from the group consisting of 0.25 - 1.25 mg; 0.5 - 1.5 mg; 1 - 2 mg; 1.25 - 2.25 mg; 1.5 -2.5 mg; 2- 3 mg; 2 - 4 mg; 3 - 5 mg; 4 - 6 mg; 4 - 8 mg; 5 -7 mg; 5 - 8 mg; 6 - 8mg; 6 - 10 mg; and 7 - 10 mg.

[00019] The present invention also provides methods wherein the β-blocker is carvedilol and is administered twice daily at a dosage selected from the group consisting of: 1 - 5 mg; 2 - 4 mg; 2.5

- 3.5 mg; 3 - 7 mg; 3.125 - 6.25 mg; 4 - 8 mg; 5 - 10 mg; 5 - 7 mg; 7.5 - 15 mg; 10 - 15 mg; 11.5 - 13.5 mg; 12.5 - 25 mg; 15 - 30 mg; 15 - 25 mg; 20 -30 mg; 25 - 50 mg; 30 - 50 mg; 40 - 50 mg; 30

- 60 mg; 40 - 60 mg; and 50 - 60 mg.

[00020] The present invention also provides methods further comprising recommending one or more lifestyle changes selected from the group consisting of: reducing sodium intake to 2,000 - 3,000 mg/day, or less; increase dietary fiber intake; increase potassium intake; moderate fluid intake; achieve and/or maintain a healthy body weight; begin and/or continue mild to moderate aerobic exercise; .smoking cessation; limit or cease alcohol intake. [00021] The present invention also provides methods to identify a human as having β-blocker non-responder status, comprising:

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; and

c) identifying the human as having β-blocker non-responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 1;1 and SEQ ID NO:

12 are substantially similar to non-failing control mRNA expression levels.

[00022] The present invention also provides methods to identify a human as having β-blocker non-responder status, comprising:

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 13; and

13 are substantially similar to non-failing control mRNA expression levels.

The present invention also provides methods to identify a human as having β-blocker non- responder status, comprising:

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13; and

c) identifying the human has having β-blocker non-responder status if:

i) a generalized linear model algorithm including the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates the human as having β-blocker non-responder status; and

ii) a generalized linear model algorithm including the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates the human as having β-blocker non- responder status.

[00023] The present invention also provides methods further comprising administering one or more therapeutic agents to the human identified as having β-blocker non-responder status that are not β-blockers.

[00024] The present invention also provides methods wherein the one or more therapeutic agents are selected from the group consisting of: angiotensin-converting enzyme (ACE) inhibitors; angiotensin II receptor blockers; diuretics; aldosterone antagonists; anticoagulants; cardiac glycosides; vasodilators; antiarrhythmics; human B-type natriuretic peptide; and inotropic agents.

[00025] The present invention also provides methods wherein a human identified as having non-responder status has a higher likelihood of an outcome selected from the group consisting of: greater likelihood of longer duration of heart failure prior to testing compared to responders; limited or no myocyte reverse remodeling compared to responders; poor prognosis compared to responders; more heart tissue damage compared to responders; longer duration of heart failure compared to responders; higher prevalence of atrial fibrillation compared to responders; wider QRS complex duration compared to responders; and lower renal function compared to responders.

[00026] The present invention also provides computer-assisted methods to generate a report of the responder/non-responder status of a human, comprising:

a) obtaining a myocardial tissue sample from a human;

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

c) inputting the mRNA expression levels and/or statistically-manipulated levels into a computer comprising a decision algorithm;

d) identifying the responder/non-responder status of the human by applying the decision algorithm to the mRNA expression data; and

e) generating a report of the responder/non-responder status of the human.

[00027] The present invention also provides computer-assisted methods to generate a report of the responder/non-responder status of a human, comprising:

a) obtaining a myocardial tissue sample from a human;

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 13;

c) inputting the mRNA expression levels and/or statistically-manipulated levels into a computer comprising a decision algorithm; d) identifying the responder/non-responder status of the human by applying the decision algorithm to the mRNA expression data; and

e) generating a report of the responder/non-responder status of the human.

[00028] Also provided are methods herein, wherein the decision algorithm comprises a decision selected from the group consisting of:

a) non-responder status if the sample mRNA expression levels are similar to non-failing control levels and/or non-responder control levels; or

b) responder status if the sample mRNA expression levels are different from non-failing control levels and/or non-responder control levels.

[00029] The present invention also provides methods wherein the method comprises the use of computer software.

[00030] The present invention also provides methods wherein the method is an automated method.

[00031] The present invention also provides kits comprising:

a) an isolated nucleic acid for each of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and

b) non-failing control RNA or non-responding control RNA.

[00032] Also provided are kits further comprising one or more additional containers comprising one or more of: wash reagent(s); and one or more detection reagents, capable of detecting presence of a bound nucleic acid.

[00033] The present invention also provides kits wherein the detection reagent is labeled with a reporter dye selected from the group consisting of: acridine; AMCA; BODIPY; cascade blue; Cy2; Cy3; Cy5; Cy7; dabcyl; edans; eosin; erythrosine; fluorescein; 6-Fam; tet; joe; hex; Oregon green; rhodamine; rhodol green; tamra; rox; and Texas red.

[00034] The present invention also provides kits wherein the detection reagent is labeled with a quencher dye.

[00035] The present invention also provides kits wherein the detection reagent is labeled with a label selected from the group consisting of: biotin; hapten; oligonucleotide; and luminescent tags.

[00036] The present invention also provides microarrays comprising two or more isolated nucleic acids comprising two or more sequences selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11 ; SEQ ID NO: 12; and SEQ ID NO: 13.

[00037] The present invention also provides microarrays comprising isolated nucleic acid sequences of SEQ ID NO : 10, SEQ ID NO : 11 , SEQ ID NO : 12, and SEQ ID NO : 13. [00038] The present invention also provides microarrays wherein the isolated nucleic acids are synthetic oligonucleotides and/or fragments of cDNA.

[00039] The present invention also provides microarrays , further comprising isolated nucleic acids comprising a variant of one or more sequences selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; and SEQ ID NO: 13, wherein the variant of the one or more sequences is a negative control.

[00040] The present invention also provides microarrays wherein the synthetic

oligonucleotides are 6 -60 nucleotides in length, 15 - 30 nucleotides in length, 20 - 25 nucleotides in length, or 7 -20 nucleotides in length.

[00041] Also provided herein are methods to identify a therapy for the amelioration of heart failure symptoms, comprising:

a) administering a test therapy to a human with heart failure due to dilated cardiomyopathy, wherein the patient is in need of therapy;

b) obtaining at least one myocardial tissue sample from the human;

c) conducting at least one assay of the sample so as to obtain one or more mRNA expression levels in the sample for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11 ; SEQ ID NO: 12; and SEQ ID NO: 13, and

d) identifying a therapy for the amelioration of heart failure symptoms based on the mRNA expression levels.

[00042] Also provided herein are methods to identify a therapy for the amelioration of heart failure symptoms wherein the therapy for the amelioration of heart failure is the administering to the patient a therapeutic dose of β-blocker if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 11 and SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 are higher compared to non-failing control mRNA expression levels.

[00043] Also provided herein are methods to identify a therapy for the amelioration of heart failure symptoms wherein the therapy for the amelioration of heart failure is the administering to the patient a therapeutic dose of β-blocker if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 12 are lower compared to non- failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are higher compared to non- failing control mRNA expression levels.

[00044] Also provided herein are methods to identify a therapy for the amelioration of heart failure symptoms wherein the therapy for the amelioration of heart failure is the administering to the patient a therapeutic dose of β-blocker if the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6 are lower compared to non-failing control mRNA expression levels and/or the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 are higher compared to non-failing control mRNA expression levels.

[00045] Also provided herein are methods to identify a therapy for the amelioration of heart failure symptoms wherein the therapy for the amelioration of heart failure comprises administering one or more therapeutic agents selected from the group consisting of: angiotensin-converting enzyme (ACE) inhibitors; angiotensin II receptor blockers; diuretics; aldosterone antagonists; digoxin; and anticoagulants, and/or recommending one or more lifestyle changes selected from the group consisting of: reducing sodium intake to 2,000 - 3,000 mg/day, or less; increase dietary fiber intake; increase potassium intake; moderate fluid intake; achieve and/or maintain a healthy body weight; begin and/or continue mild to moderate aerobic exercise; .smoking cessation; limit or cease alcohol intake, if mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; and SEQ ID NO: 13 are substantially similar to non-failing control mRNA expression levels.

[00046] Also provided herein are methods to treat idiopathic dilated cardiomyopathy, comprising administering at least one composition selected from the group consisting of: cholesteryl ester transfer protein agonist, or pharmaceutically-acceptable formulation thereof; cholesteryl ester transfer protein mimic, or pharmaceutically-acceptable formulation thereof; cholesteryl ester transfer protein enhancer, or pharmaceutically-acceptable formulation thereof; or cholesteryl ester transfer protein peptide, or pharmaceutically-acceptable formulation thereof.

[00047] Also provided herein are methods which further comprise repeating the method over time.

[00048] The present invention also provides methods which further comprise decreasing or increasing the dose of a therapeutic agent based on the human's identified β-blocker responder status.

[00049] The present invention also provides methods which further comprise communicating the data or β-blocker responder status to at least one human. [00050] The present invention also provides methods wherein the data and/or status is communicated in paper form or computer readable medium form.

[00051] Also provided herein are methods to identify a human as having β-blocker non- responder status, comprising:

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9; and

c) identifying the human as having β-blocker non-responder status if the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 are substantially similar to non- failing control mRNA expression levels.

[00052] Also provided herein are computer-assisted methods to generate a report of the responder/non-responder status of a human, comprising:

a) obtaining a myocardial tissue sample from a human;

b) conducting at least one assay of the sample so as to obtain mRNA expression levels in the sample for one or more mRNAs capable of hybridizing to a nucleic acid selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9; c) inputting the mRNA expression levels and/or statistically-manipulated levels into a computer comprising a decision algorithm;

d) identifying the responder/non-responder status of the human by applying the algorithm to the mRNA expression data; and

e) generating a report of the responder/non-responder status of the human.

[00053] The present invention also provides methods wherein the human is identified as having β-blocker responder status if the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6 are lower compared to non-failing control mRNA expression levels and/or the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 are higher compared to non-failing control mRNA expression levels. [00054] The present invention also provides kits comprising:

a) an isolated nucleic acid comprising one or more sequences selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9, and

b) non-failing control RNA or non-responding control RNA.

BRIEF DESCRIPTION OF THE FIGURES

[00055] FIG. 1 : mRNA expression data and correlations.

[00056] FIG. 2: mRNA expression data and correlations showing a nine biomarker signature.

[00057] FIGS. 3 - 11 are boxplot graphs representing data for expression of nine mRNAs from myocardial tissue from patients with idiopathic dilated cardiomyopathies, obtained at the indicated intervals during a multi-center, randomized trial, as described further herein. Each plot shows the p- value from the pairwise statistical test comparing baseline expression levels from patients with a favorable clinical response to beta-blockade to those without a favorable clinical response to beta- blockade. The groups are: NFC, in orange (non-failing control, n=4), patients without heart failure; NR, in blue (non-responders to beta-blockade, n=17); and R, in red (responders to beta-blockade, n=30)

[00058] Patient groups were defined using clinical parameters, at 0 months for NFC, and at 0, 3 and 12 months for NR and R. In data presentation, NR Omos = NR at 0 months, NR 3mos = NR at 3 months, etc. Statistics: non-parametric pairwise comparison (Mann- Whitney aka Wilcoxon tests.) All associated non-parametric ANOVA tests (i.e. Kruskal-Wallis comparing NFC, NR Omos, and R Omos OR NFC, NR 12mos, and R 12mos) returned p values below 0.05.

[00059] FIG. 3: Beta blocker response boxplot graph: BC040287 (SEQ ID NO: 1).

[00060] FIG. 4: Beta blocker response boxplot graph: BC040287.1 (SEQ ID NO: 2).

[00061] FIG. 5: Beta blocker response boxplot graph: AK025546 (SEQ ID NO: 3).

[00062] FIG. 6: Beta blocker response boxplot graph: AA605090 (SEQ ID NO: 4).

[00063] FIG. 7: Beta blocker response boxplot graph: AW970112 (SEQ ID NO: 5).

[00064] FIG. 8: Beta blocker response boxplot graph: AA420989 (alias C15orf61) (SEQ ID NO:

6) .

[00065] FIG. 9: Beta blocker response boxplot graph: NM 000078 (alias CETP) (SEQ ID NO:

7) .

[00066] FIG. 10: Beta blocker response boxplot graph: NM 014791 (alias MELK) (SEQ ID NO: 8).

[00067] FIG. 11: Beta blocker response boxplot graph: AF326731 (alias NUF2) (SEQ ID NO:

9).

[00068] FIG. 12: Beta blocker response boxplot graph: ANP and BNP (reference) [00069] FIG. 13: Beta blocker response boxplot graph: CETP (SEQ ID NO: 7).

[00070] FIG. 14: Schematic of the role of cholesteryl ester trafficking and cholesterol transport

[00071] FIGS. 15 - 18 are boxplot graphs representing data for expression of four mRNAs from myocardial tissue from patients with idiopathic dilated cardiomyopathies, obtained at the indicated intervals during a multi-center, randomized trial, as described further herein. The groups are: NFC, in orange (non-failing control, n=4), patients without heart failure; NR, in blue (non-responders to beta- blockade, n=17); and R, in red (responders to beta-blockade, n=30)

[00072] Patient groups were defined using clinical parameters, at 0 months for NFC, and at 0, 3 and 12 months for NR and R. In data presentation, NR Omos = NR at 0 months, NR 3mos = NR at 3 months, etc. Statistics: non-parametric pairwise comparison (Mann- Whitney aka Wilcoxon tests.) All associated non-parametric ANOVA tests (i.e. Kruskal-Wallis comparing NFC, NR Omos, and R Omos OR NFC, NR 12mos, and R 12mos) returned p values below 0.05.

[00073] FIG. 15: Beta blocker response boxplot graph: hCG 2045206 (SEQ ID NO: 10).

[00074] FIG. 16: Beta blocker response boxplot graph: 241584 at (SEQ ID NO: 11).

[00075] FIG. 17: Beta blocker response boxplot graph: C4orf49 (SEQ ID NO: 12).

[00076] FIG. 18: Beta blocker response boxplot graph: KLRC3 (SEQ ID NO: 13).

[00077] FIG. 19A: Receiver operating characteristic area under the curve: hCG 2045206 (SEQ ID NO: 10).

[00078] FIG. 19B: Receiver operating characteristic area under the curve: 241584 at (SEQ ID NO: 11)

[00079] FIG. 19C: Receiver operating characteristic area under the curve: C4orf49 (SEQ ID NO: 12).

[00080] FIG. 19D: Receiver operating characteristic area under the curve: KLRC3 (SEQ ID NO: 13).

[00081] FIG. 20A: Receiver operating characteristic area under the curve: hCG 2045206 (SEQ ID NO: 10); 241584 at (SEQ ID NO: 11); and C4orf49 (SEQ ID NO: 12).

[00082] FIG. 20B: Receiver operating characteristic area under the curve: hCG 2045206 (SEQ ID NO: 10); 241584 at (SEQ ID NO: 11); and KLRC3 (SEQ ID NO: 13).

[00083] FIG. 21: mRNA expression data and correlations showing a four biomarker signature. DETAILED DESCRIPTION OF THE INVENTION

[00084] A Beta-Blocker Effect on Remodeling and Gene Expression Trial ("BORG") was designed to compare changes in myocardial gene expression to alterations in myocardial phenotype in patients with heart failure from idiopathic dilated cardiomyopathy treated for 18 months with varying levels of adrenergic blockade with either metoprolol (βι-receptor blockade), metoprolol plus doxazosin (βι plus ai-receptor blockade) or carvedilol (βι plus ( i plus p₂-receptor blockade). The experiment was designed to measure gene expression by quantitative reverse transcription-polymerase chain reaction (RT-PCR) for a select number of nucleic acids. The primary objective of this research was to expand the analysis of measured genes within the BORG to include almost the entire transcriptome (mRNA expression profile) that is available by utilizing microarray technology.

[00085] The BORG trial was designed to utilize serial analysis of myocardial gene expression or repeated longitudinal measurements to help discriminate biologically active pathways from randomly elevated expressions. Inter-patient variability in gene expression is greater than intra-patient variability. Specifically, serial global gene expression measurements in the same patient over time are closer than those taken between patients with the same phenotype. Hence, the trial was designed to minimize random variations between individuals and focus on changes in gene expression that correlate with changes in remodeling.

[00086] The present invention provides means to identify a human's responder status depending on the mRNA transcript profile of the individual. The present responder status can be assayed according to known assays, such as qPCR, microarrays, and competitive hybridization.

[00087] The present invention therefore provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in the Sequence Listing.

[00088] As used herein "Arrays" or "Microarrays" refers to an array of distinct polynucleotides or oligonucleotides synthesized then immobilized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675- 1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

[00089] As used herein, "non-failing control" includes data and/or analysis of myocardial mRNA expression levels from a human with no heart failure and no dilated cardiomyopathy.

[00090] The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5', or 3' untranslated sequence, coding sequence, sequential oligonucleotides that cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit are oligonucleotides that are specific to a gene or genes of interest. Oligonucleotides present on the array may also be slight variants on the known sequence that act as negative controls.

[00091] In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5' or at the 3' end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair

(mismatched by one) serves as a control. The number of oligonucleotide pairs may range from one to two million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

[00092] In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between one to two million which lends itself to the efficient use of commercially available instrumentation.

[00093] The biological samples may be obtained from heart cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples. [00094] Using such arrays, the present invention provides methods to identify the utility and effectiveness of potential treatments, including drug candidates or other therapies, or a combination thereof.

[00095] Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted.

[00096] The test samples of the present invention include total RNA prepared from human tissue. The test sample preparation used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts from tissue are well known in the art and can readily be adapted in order to obtain a sample that is compatible with the system utilized.

[00097] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

[00098] Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container (including a chip) comprising a nucleic acid(s) disclosed in Figure 1 and/or 2 (one that is capable of identifying mRNAs associated with responder status) and (b) one or more containers comprising non-failing control RNA and/ or non-responder control RNA, and optionally (c) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

[00099] In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified biomarkers of the present invention can be identified using the sequence information disclosed herein and can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

[000100] Higher stringency conditions utilize buffers with lower ionic strength and/or a higher reaction temperature, and tend to require a more perfect match between probe/primer and a target sequence in order to form a stable duplex. If the stringency is too high, however, hybridization may not occur at all. In contrast, lower stringency conditions utilize buffers with higher ionic strength and/or a lower reaction temperature, and permit the formation of stable duplexes with more mismatched bases between a probe/primer and a target sequence. By way of example and not limitation, exemplary conditions for high stringency hybridization conditions using an allele-specific probe are as follows: prehybridization with a solution containing 5X standard saline phosphate EDTA (SSPE), 0.5% NaDodS0₄ (SDS) at 55°C, and incubating probe with target nucleic acid molecules in the same solution at the same temperature, followed by washing with a solution containing 2XSSPE, and 0.1% SDS at 55°C or room temperature.

[000101] Moderate stringency hybridization conditions may be used for allele-specific primer extension reactions with a solution containing, e.g., about 50 mM KC1 at about 46°C. Alternatively, the reaction may be carried out at an elevated temperature such as 60°C. In another embodiment, a moderately stringent hybridization condition suitable for oligonucleotide ligation assay (OLA) reactions wherein two probes are ligated if they are completely complementary to the target sequence may utilize a solution of about 100 mM KC1 at a temperature of 46°C.

[000102] Oligonucleotides may be prepared by methods well known in the art. Chemical synthetic methods include, but are limited to, the phosphotriester method described by Narang et al., 1979, Methods in Enzymology 68:90; the phosphodiester method described by Brown et al., 1979, Methods in Enzymology 68: 109, the diethylphosphoamidate method described by Beaucage et al., 1981, Tetrahedron Letters 22: 1859; and the solid support method described in U.S. Pat. No. 4,458,066.

[000103] In another embodiment of the invention, a nucleic acid detection reagent of the invention is labeled with a fluorogenic reporter dye that emits a detectable signal. While the preferred reporter dye is a fluorescent dye, any reporter dye that can be attached to a detection reagent such as an oligonucleotide probe or primer is suitable for use in the invention. Such dyes include, but are not limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Dabcyl, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and Texas Red.

[000104] In yet another embodiment of the invention, the detection reagent may be further labeled with a quencher dye such as Tamra, especially when the reagent is used as a self-quenching probe such as a TaqMan (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemless or linear beacon probe (Livak et al., 1995, PCR Method Appl. 4:357-362; Tyagi et al., 1996, Nature Biotechnology 14: 303-308; Nazarenko et al., 1997, Nucl. Acids Res. 25:2516-2521 ; U.S. Pat. Nos. 5,866,336 and 6,117,635).

[000105] The detection reagents of the invention may also contain other labels, including but not limited to, biotin for streptavidin binding, hapten for antibody binding, and oligonucleotide for binding to another complementary oligonucleotide such as pairs of zipcodes. Moreover, luminescent tags are also useful in the present invention.

[000106] Nucleic acid arrays are reviewed in the following references: Zammatteo et al., "New chips for molecular biology and diagnostics", Biotechnol Annu Rev. 2002; 8:85-101 ; Sosnowski et al., "Active microelectronic array system for DNA hybridization, genotyping and pharmacogenomic applications", Psychiatr Genet. 2002 December; 12(4): 181-92; Heller, "DNA microarray technology: devices, systems, and applications", Annu Rev Biomed Eng. 2002; 4: 129-53. Epub 2002 Mar. 22; Kolchinsky et al., "Analysis of SNPs and other genomic variations using gel-based chips", Hum Mutat. 2002 April; 19(4):343-60; and McGall et al., "High-density genechip oligonucleotide probe arrays", Adv Biochem Eng Biotechnol. 2002; 77:21-42.

[000107] Microfluidic devices, which may also be referred to as "lab-on-a-chip" systems, biomedical micro-electro-mechanical systems (bioMEMs), or multicomponent integrated systems, are exemplary kits/systems of the present invention for analyzing Nucleic acids. Such systems miniaturize and compartmentalize processes such as probe/target hybridization, nucleic acid amplification, and capillary electrophoresis reactions in a single functional device. Such microfluidic devices typically utilize detection reagents in at least one aspect of the system, and such detection reagents may be used to detect one or more Nucleic acids of the present invention. One example of a microfluidic system is disclosed in U.S. Pat. No. 5,589,136, which describes the integration of PCR amplification and capillary electrophoresis in chips. Exemplary microfluidic systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples may be controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts. Varying the voltage can be used as a means to control the liquid flow at intersections between the micro-machined channels and to change the liquid flow rate for pumping across different sections of the microchip. See, for example, U.S. Pat. No. 6,153,073, Dubrow et al., and U.S. Pat. No. 6,156,181, Parce et al.

[000108] Information on association/correlation between mRNA expression profiles, genotypes, and disease-related phenotypes can be exploited in several ways. For example, in the case of a highly statistically significant association between one or more mRNA expression levels with predisposition to a disease for which treatment is available, detection of such a genotype pattern in an individual may justify immediate administration of treatment, or at least the institution of regular monitoring of the individual.

[000109] The profile panels/assays/diagnostics of the invention may contribute to the identification of responder status in an individual in different ways. As used herein, the terms "diagnose,"

"diagnosis," and "diagnostics" include, but are not limited to any of the following: detection of drug overdose or underdose symptoms an individual may presently have, predisposition/susceptibility, determining a particular type or subclass of symptoms or propensity in an individual, confirming or reinforcing a previously made diagnosis symptoms or propensity, pharmacogenomic evaluation of an individual to determine which therapeutic strategy that individual is most likely to positively respond to or to predict whether a patient is likely to respond to a particular treatment such as a particular drug, predicting whether a patient is likely to experience toxic effects from a particular treatment or therapeutic compound, and evaluating the future prognosis of an individual having heart failure symptoms or propensity. Such diagnostic uses are based on the mRNA expression data, individually or in a unique combination(s).

[000110] Combined detection of a plurality of mRNA expression data (for example, mRNA which hybridize to 2, 3, 4, 5, 6, 7, 8, 9, or any other number in-between, or more, of the nucleic acids herein) typically increases the probability of an accurate diagnosis. To further increase the accuracy of diagnosis or predisposition screening, analysis of the nucleic acids of the present invention can be combined with that of other nucleic acids, polymorphisms or other risk factors of heart failure symptoms or propensity, such as disease symptoms, pathological characteristics, family history, diet, environmental factors or lifestyle factors.

[000111] It will, of course, be understood by practitioners skilled in the treatment or diagnosis of heart failure symptoms or propensity that the present invention generally does not intend to provide an absolute identification of individuals who are at risk (or less at risk) of developing heart failure symptoms or propensity, and/or pathologies related to such as heart failure symptoms or propensity, but rather to indicate a certain increased (or decreased) degree or likelihood of developing the pathology based on statistically significant association results. However, this information is extremely valuable as it can be used to, for example, initiate preventive treatments or to allow an individual carrying one or more significant nucleic acids to foresee warning signs such as minor clinical symptoms, or to increase the frequency of regularly scheduled physical exams to monitor for appearance of a condition in order to identify and begin treatment of the condition at an early stage in a personalized manner. Particularly with heart failure, a disease that is extremely debilitating or fatal if not treated in a timely way, the knowledge of a potential predisposition, even if this predisposition is not absolute, would likely contribute in a very significant manner to treatment efficacy.

[000112] The diagnostic techniques of the present invention may employ a variety of

methodologies to determine whether a test subject has an mRNA expression profile/pattern (aka trans criptome) associated with an increased or decreased risk of developing a detectable trait or whether the individual suffers from a detectable trait as a result of a particular pattern.

[000113] In another embodiment, the nucleic acid detection reagents of the present invention are used to determine whether an individual has one or more nucleic acid affecting the level (e.g., the concentration of mRNA or protein in a sample, etc.) or pattern (e.g., the kinetics of expression, rate of decomposition, stability profile, Km, Vmax, etc.) of gene expression (collectively, the "gene response" of a cell or bodily fluid). Such a determination can be accomplished by screening for mRNA or protein expression (e.g., by using nucleic acid arrays, RT-PCR, TaqMan assays, or mass spectrometry proteomics), identifying genes having altered expression in an individual, genotyping single nucleotide polymorphisms (SNPs) that could affect the expression of the genes having altered expression (e.g., SNPs that are in and/or around the gene(s) having altered expression, SNPs in regulatory/control regions, SNPs in and/or around other genes that are involved in pathways that could affect the expression of the gene(s) having altered expression, or all SNPs could be genotyped), and correlating genotypes with altered gene expression. In this manner, specific SNP alleles at particular SNP sites can be identified that affect gene expression.

[000114] A corresponding control tissue or blood sample can be obtained from normal (n on- failing, or not in heart failure) human individual or population of normal individuals, or from cultured normal cells. The control tissue may be processed along with the sample from the subject, so that the levels of mRNA in the subject's sample can be compared directly to the mRNA levels from cells of the control sample. A reference mRNA expression standard can also be used as a control, including those in databases.

[000115] The present invention provides methods for assessing the pharmacogenomics of a subject harboring a particular transcriptome to a particular therapeutic agent or pharmaceutical compound, or to a class of such compounds. Pharmacogenomics deals with the roles which clinically significant hereditary variations play in the response to drugs due to altered drug disposition and/or abnormal action in affected persons. See, e.g., Roses, Nature 405, 857-865 (2000); Gould Rothberg, Nature Biotechnology 19, 209-211 (2001); Eichelbaum, Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996); and Linder, Clin. Chem. 43(2):254-266 (1997). The clinical outcomes of these variations can result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the transcriptome of an individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. For example, mRNA expression in drug metabolizing enzymes can affect the availability and activity of these enzymes, which in turn can affect both the intensity and duration of drug action, as well as drug metabolism and clearance.

[000116] The discovery of unique transcriptomes in drug metabolizing enzymes, drug transporters, proteins for pharmaceutical agents, and other drug targets now explains why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages.

[000117] Pharmacogenomic uses of the present invention provide several significant advantages for patient care, particularly in treating heart failure symptoms or propensity. Pharmacogenomic characterization of an individual, based on an individual's transcriptome, can identify those individuals unlikely to respond to treatment with a particular medication and thereby allows physicians to avoid prescribing the ineffective medication to those individuals. On the other hand, the present transcriptome profiling of an individual may enable physicians to select the appropriate medication and dosage regimen that will be most effective based on an individual's transcriptome profile. This information increases a physician's confidence in prescribing medications and motivates patients to comply with their drug regimens. Furthermore, pharmacogenomics may identify patients predisposed to toxicity and adverse reactions to particular drugs or drug dosages. Adverse drug reactions lead to more than 100,000 avoidable deaths per year in the United States alone and therefore represent a significant cause of hospitalization and death, as well as a significant economic burden on the healthcare system (Pfost et. al., Trends in Biotechnology, August 2000.). Thus,

pharmacogenomics based on the transcriptome profiles disclosed herein has the potential to both save lives and reduce healthcare costs substantially.

[000118] Pharmacogenomics in general is discussed further in Rose et al., "Pharmacogenetic analysis of clinically relevant genetic polymorphisms", Methods Mol Med. 2003; 85:225-37.

Pharmacogenomics as it relates to cardiovascular disorders is discussed in Siest et al.,

"Pharmacogenomics of drugs affecting the cardiovascular system", Clin Chem Lab Med. 2003 April; 41(4):590-9, Mukherjee et al., "Pharmacogenomics in cardiovascular diseases", Prog Cardiovasc Dis. 2002 May-June; 44(6):479-98, and Mooser et al., "Cardiovascular pharmacogenetics in the SNP era", J Thromb Haemost. 2003 July; 1(7): 1398-402.

[000119] The present invention also can be used to identify novel therapeutic targets for cardiac drugs. The therapeutics may be composed of, for example, small molecules, proteins, protein fragments or peptides, antibodies, nucleic acids, or their derivatives or mimetics which modulate the functions or levels of the target genes or gene products.

[000120] A subject identified as having a therapeutically-targetable propensity ascribed to a mRNA profile(s) may be treated so as to correct the genetic defect (see Kren et al., Proc. Natl. Acad. Sci. USA 96:10349-10354 (1999)).

[000121] The invention further provides a method for identifying a compound or agent that can be used to overcome a negative responder status propensity. The nucleic acids disclosed herein are useful as targets for the identification and/or development of therapeutic agents. A method for identifying a therapeutic agent or compound typically includes assaying the ability of the agent or compound to modulate the activity and/or expression of a nucleic acid or the encoded product and thus identifying an agent or a compound that can be used to treat a disorder characterized by undesired activity or low expression of the nucleic acid or the encoded product. The assays can be performed in cell-based and cell-free systems. Cell-based assays can include cells naturally expressing the nucleic acid molecules of interest or recombinant cells genetically engineered to express certain nucleic acid molecules. [000122] Modulators of variant gene expression can be identified in a method wherein, for example, a cell is contacted with a candidate compound/agent and the expression of mR A determined. The level of expression of mRNA in the presence of the candidate compound is compared to the level of expression of mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of variant gene expression based on this comparison. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

[000123] The invention further provides methods of treatment using a compound identified through drug screening as a gene modulator to modulate variant nucleic acid expression. Modulation can include either up-regulation (i.e., activation or agonization) or down-regulation (i.e., suppression or antagonization) of nucleic acid expression.

[000124] Expression of mRNA transcripts and encoded proteins, either wild type or variant, may be altered in individuals with a particular regulatory/control element, such as a promoter or transcription factor binding domain, that regulates expression. In this situation, methods of treatment and compounds can be identified, as discussed herein, that regulate or overcome the variant regulatory/control element, thereby generating normal, or healthy, expression levels of either the wild type or variant protein.

[000125] The mRNA expression information of the present invention are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of a variant gene, or encoded product, in clinical trials or in a treatment regimen. Thus, the expression pattern can serve as an indicator for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance, as well as an indicator for toxicities. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

[000126] In another aspect of the present invention, there is provided a pharmaceutical pack comprising a therapeutic agent (e.g., a small molecule drug, antibody, peptide, antisense or RNAi nucleic acid molecule, etc.) and a set of instructions for administration of the therapeutic agent to humans diagnostically tested for one or more nucleic acids (eg. transcriptome profile) provided by the present invention. [000127] The transcriptomes of the present invention are also useful for improving many different aspects of the drug development process. For instance, an aspect of the present invention includes selecting individuals for clinical trials based on their trans criptome. For example, individuals with transcriptomes that indicate that they are likely to positively respond to a drug can be included in the trials, whereas those individuals whose transcriptomes indicate that they are less likely to or would not respond to the drug, or who are at risk for suffering toxic effects or other adverse reactions, can be excluded from the clinical trials. This not only can improve the safety of clinical trials, but also can enhance the chances that the trial will demonstrate statistically significant efficacy. Furthermore, the transcriptomes of the present invention may explain why certain previously developed drugs performed poorly in clinical trials and may help identify a subset of the population that would benefit from a drug that had previously performed poorly in clinical trials, thereby "rescuing" previously developed drugs, and enabling the drug to be made available to a particular patient population that can benefit from it.

[000128] Selecting Therapy Based on Responder Status

[000129] Many patients will respond favorably to β-adrenergic blockade by β-blockers such as metoprolol succinate, metoprolol succinate + doxazosin, and carvediol. These patients (responders) demonstrate increased ventricular chamber and myocyte reverse remodeling, increased heart tissue repair, improved left ventricle ejection fractions, decreased end diastolic volume, and improved prognosis when compared to patients who do not respond favorably to β-blockers (non-responders). Responders also see reductions in heart rate, blood urea nitrogen, atrial natriuretic peptide, body mass index, and reductions in increases in systolic pressure compared to non-responders.

[000130] Responders may be identified by comparing mRNA expression levels of particular genes with non-failing and non-responding controls. In certain embodiments, when a patient has expression levels for one or more mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NOs: 1 - 6 are lower compared to non-failing control mRNA expression levels and/or the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids of SEQ ID NOs: 7 - 9 are higher compared to non- failing control mRNA expression levels, the patient is identified as a responder. In another embodiment, when a patient has expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NOs: 11-12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression level for mRNAs which are capable of hybridizing to the nucleic acid of SEQ ID NO: 10 is higher compared to non- failing control mRNA expression levels, the patient is identified as a responder. In yet another embodiment, when a patient has expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NOs: 10 and 13 are higher compared to non- failing control mRNA expression levels and the sample mRNA expression level for mRNAs which are capable of hybridizing to the nucleic acid of SEQ ID NO: 12 is lower compared to non-failing control mRNA expression levels, the patient is identified as a responder.

[000131] mRNA levels are considered to be either "higher" or "lower" when mRNA levels relative to non-failing controls and/or non-responding failing controls are significantly different (K-W p<0.05 and Wilcoxon p<0.05, see Examples).

[000132] Patients identified as responders may be treated by administering a therapeutic dose of one or more β-blockers including, but not limited to, metoprolol succinate, metoprolol succinate and the a-blocker doxazosin, and carvediol. Standard dosing for all β-blockers may be followed. For example, metoprolol succinate may be administered at doses of approximately 12.5 mg QD to approximately 200 mg QD. Doxazosin may be administered at doses of approximately 1 mg QD to approximately 8 mg QD, along with a standard dose of metoprolol succinate. Carvediol may be administered at doses of approximately 3.125 mg BID to approximately 50 mg BID.

[000133] Non-responders demonstrate limited or no ventricular chamber reverse remodeling, more heart tissue damage, longer duration of heart failure, higher prevalence of atrial fibrillation, wider QRS complex duration, lower renal function, and poor prognosis when compared to responders.

[000134] As with responders, non-responders may be identified by comparing mRNA expression levels of particular genes with non-failing and non-responding failing controls. When a patient has expression levels for one or more mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NOs: 1 - 12 are substantially similar to non- failing control levels and/or non -responder control levels, the patient is identified as a non-responder.

[000135] mRNA levels are considered to be "substantially similar" when mRNA levels relative to non-failing controls and/or non-responding failing controls are not significantly different (K-W p>0.05 and Wilcoxon p>0.05, see Examples).

[000136] Patients identified as non-responders will not substantially benefit from treatment with β- blockers. These patients may be treated by one or more drugs classified as one of: angiotensis- converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, diuretics, aldosterone antagonists, anticoagulants, cardiac glycosides, vasodilators, antiarrhythmics, human B-type natriuretic peptide, and inotropic agents. Furthermore, since non-responders cannot fully benefit from treatment with β-blockers, certain lifestyle changes may be recommended in conjunction with pharmaceutical treatment. Such changes may include reducing sodium intake to 2,000 - 3,000 mg/day, or less, increasing dietary fiber intake, increasing potassium intake, moderating daily fluid intake, achieving a healthy bodyweight, maintaining a healthy body weight, starting and/or maintaining a mild to moderated aerobic exercise program, quitting smoking, and eliminating or limiting alcohol consumption. These modifications are known to benefit those suffering from heart failure, including dilated cardiomyopathy. Such lifestyle changes may also be recommended to responders, but may be even more important to non-responders. [000137] Computer-Related Embodiments

[000138] The nucleic acids identified as biomarkers in the present invention may be "provided" in a variety of mediums to facilitate identification of responder status in an individual. As used in this section, "provided" refers to a manufacture, other than an isolated nucleic acid molecule, that contains mRNA expression information of the present invention. Such a manufacture provides the mRNA information in a form that allows a skilled artisan to examine the manufacture using means not directly applicable to examining the nucleic acids or a subset thereof as they exist in nature or in purified form. The mRNA information that may be provided in such a form includes any of the mRNA information provided by the present invention such as, for example, mRNA expression data for the biomarkers herein; or any other information provided by the present invention in the Sequence Listing.

[000139] In one application of this embodiment, the nucleic acids of the present invention can be recorded on a computer readable medium. As used herein, "computer readable medium" refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable media can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.

[000140] As used herein, "recorded" refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the mRNA information of the present invention.

[000141] A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide/amino acid sequence information of the present invention on computer readable medium. For example, the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in the form of an ASCII file, or stored in a database application, such as OB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the mRNA information of the present invention. [000142] By providing the mRNA information of the present invention in computer readable form, a skilled artisan can routinely access the information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. Examples of publicly available computer software include BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203- 207 (1993)) search algorithms.

[000143] The present invention further provides systems, particularly computer-based systems, which contain the mRNA information described herein. Such systems may be designed to store and/or analyze information on, for example, a large number of nucleic acid expression information, or information on nucleic acid expression from a large number of individuals. The nucleic acid expression information of the present invention represents a valuable information source. The nucleic acid expression information of the present invention stored/analyzed in a computer-based system may be used for such computer-intensive applications as determining or analyzing nucleic acid expression in a population, mapping disease genes, genotype-phenotype association studies, grouping nucleic acid expression into categories, correlating nucleic acid expression with response to particular drugs, or for various other bioinformatic, pharmacogenomic, drug development, or human

identification/forensic applications.

[000144] As used herein, "a computer-based system" refers to the hardware means, software means, and data storage means used to analyze the nucleic acid expression information of the present invention. The minimum hardware means of the computer-based systems of the present invention typically comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. Such a system can be changed into a system of the present invention by utilizing the nucleic acid expression information provided on the CD-R, or a subset thereof, without any experimentation.

[000145] As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein nucleic acid expression patterns of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, "data storage means" refers to memory which can store nucleic acid expression information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleic acid expression information of the present invention.

[000146] As used herein, "search means" refers to one or more programs or algorithms that are implemented on the computer-based system to identify or analyze mRNA expression profile in a target based on the expression information stored within the data storage means. Search means can be used to determine responder status. [000147] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. An exemplary format for an output means is a display that depicts the presence or absence of specified nucleic acid expression profile(s). Such presentation can provide a rapid, binary scoring system for many nucleic acids simultaneously.

[000148] One exemplary embodiment of a computer-based system comprising nucleic acid expression information of the present invention includes a processor connected to a bus. Also connected to the bus are a main memory (preferably implemented as random access memory, RAM) and a variety of secondary storage devices, such as a hard drive and a removable medium storage device. The removable medium storage device may represent, for example, a floppy disk drive, a CD- ROM drive, a magnetic tape drive, etc. A removable storage medium (such as a floppy disk, a compact disk, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device. The computer system includes appropriate software for reading the control logic and/or the data from the removable storage medium once inserted in the removable medium storage device.

[000149] The nucleic acid expression information of the present invention may be stored in a well- known manner in the main memory, any of the secondary storage devices, and/ or a removable storage medium. Software for accessing and processing the nucleic acid expression information (such as scoring tools, search tools, comparing tools, etc.) preferably resides in main memory during execution.

[000150] Also provided is a research system in which embodiments may be implemented. The research system includes a study data analysis system. The study data analysis system may be used, for example, to store, recall, access, implement, or otherwise use datasets or other information obtained from study data.

[000151] The study data analysis system may be used, for example, to identify agent(s) associated with one or more treatment targets which are associated with a specific subpopulation(s) of individuals for whom the incidence of one or more adverse events is acceptable at a defined level. The study data analysis system may identify such agent(s) by, for example, storing, analyzing and/ or providing datasets or other information obtained from study data as to the safety and optionally, the effectiveness, of the agent(s).

[000152] An agent, as used herein, can be, for example, a medical or non-medical intervention, including, for example, administration of prescription or non-prescription medications, small molecule drugs or biologies, nutraceuticals, or dietary supplements. An agent may also be, for example, alcohol or an illicit substance. A treatment target, as used herein, can be, for example, a medical condition, treatment goal or disorder meriting clinical, nutraceutical or alternative medical intervention. Treatment targets may also be voluntary procedures, for example, cosmetic procedures. Treatment, as used herein, can refer to treating and/or prevention. A treatment target is search of an agent is a treatment target of interest (e.g., a medical condition) for which the incidence and/or severity of an adverse event(s) under a standard of care is high and/or unacceptable.

[000153] Ongoing, prospective and completed clinical trials for various agents may be found in databases such as government or private clinical trial websites, which lists specific details for clinical trials, including primary and secondary outcomes, enrollment size, inclusion and exclusion criteria, and other parameters. In addition, clinical trial results are generally available in journal publications that are known to, and accessible by, persons of ordinary skill in the art.

[000154] Examples

[000155] Example 1. Materials, Methods

[000156] A. General description of BORG study.

[000157] A multi-center, randomized trial recruited 51 patients with idiopathic dilated cardiomyopathies and a LVEF < 40 %. After their baseline evaluations subjects were randomized (1 : 1 : 1) to metoprolol (βΐ blockade), metoprolol plus doxazosin (βΐ + al blockade), or carvedilol (βΐ plus β2 plus al blockade). Cardiac function was measured at baseline, 3 months, and 12 months, by radionucleotide ventriculography. At baseline, 3 months and 12 months patients had endomyocardial biopsies performed, with biopsies taken from the right ventricular septum. Total RNA was extracted, and gene expression was quantified using the Affymetrix HGU133 plus 2 chip.

[000158] B. β-blocker Therapy

[000159] After randomization, patients were immediately started on study medication starting dosage twice daily or once daily as indicated (Table 1). At each visit, if the patient tolerated the previous week's dosing, the dose of study drug was increased until the patient reached the target dose.

Table 1 : Dosing Schedule of β-blocker Therapy

[000160] C. mm A Extraction

[000161] Total RNA was extracted from 1 to 4 RV endomyocardial biopsies obtained during cardiac catheterization, flash-frozen in liquid nitrogen and stored at -80°C. A glass-fiber filter (GFF) based purification method was utilized (mirVana™ miRNA Isolation Kit, Ambion, Inc), following the protocol designed for Total RNA Isolation. Tissue disruption was carried out in 10mm diameter polypropylene tubes using a high-speed, 8mm diameter rotor/stator homogenizer (Ultra-Turrax™, IKA). The one modification to the mirVana protocol was that the Lysis/Binding Buffer used for tissue homogenization was replaced with a different phenol :guanidine isothiocyanate reagent (TRIzol™, Invitrogen) that foamed less during homogenization. The use of the TRIzol Reagent was primarily necessitated by the concomitant proteomic analyses on these samples whose methods were developed specifically utilizing the phenol: chloroform "sub-natant" fraction of the TRIzol tissue homogenate. This step was carried out according to the TRIzol Reagent protocol (but with the inclusion of the mirVana Kit Homogenate Additive) to the point of producing the first aqueous supernatant.

[000162] The TRIzol aqueous supernatant was then diluted per the mirVana protocol with ethanol and applied to the GFF spin-column to bind the RNA from solution. Washes were carried out according to the mirVana protocol using a vacuum manifold, finally drying the columns by centrifugation. Elution of the RNA from the GFF was accomplished by applying 100 microliters of nuclease-free water heated to 95°C. [NOTE: A positive-displacement pipettor was used to apply a more consistent volume (sample-to-sample) of the heated water.] Centrifugation for 2 minutes eluted the RNA into a clean microfuge tube.

[000163] The resultant RNA solution was then treated to remove contaminating genomic DNA by employing a DNase digestion step (TURBO DNA-free™ kit, Ambion, Inc.). After completing the digestion the DNase was removed by an enzyme-binding Inactivation Reagent included in the kit. [NOTE: As with other enzyme-removal resins we have used in the past, it was found that this reagent also bound any proteins still remaining in the RNA solution, thereby improving the apparent RNA purity as monitored by spectrophotometric assay at A260/A280/A230.] The entire inactivated reaction was then filtered though a 0.45 μιη PVDF spin-filter (Ultrafree-MC® Durapore®, Millipore) and the remaining RNA was then stored at -80°C in a solution of ammonium acetate and ethanol.

[000164] D. Affymetrix GeneChip Expression Analysis

[000165] The stored total RNA was thawed, pelleted by centrifugation and resuspended in nuclease-free water to begin the processing for analysis. The Ovation® RNA Amplification System (NuGEN Technologies, Inc.; San Carlos, CA) was employed to amplify the RNA samples to quantities sufficient for analysis by Affymetrix GeneChip Microarray (Santa Clara, CA). After verifying RNA quantity and quality (Agilent 2100 Bioanalyzer, Agilent RNA 6000 Nano-LabChip) the samples (5-100 ng) were subjected to primer-initiated reverse transcription and nick/fragmentation-primed second strand synthesis resulting in a double-stranded cDNA molecule. All of the molecules in this cDNA pool, representing the transcriptome, were equipped with a uniquely sequenced priming site composed of RNA, incorporated during the first strand synthesis by using a chimeric oligo-dT(DNA)/RNA primer to initiate the RT reaction. The specificity of this unique-sequenced RNA -based priming site was utilized in the subsequent single primer isothermal amplification (ribo-SPIA) cDNA amplification step. This DNA polymerase-based amplification was initiated by the RNA portion of the chimeric primer whose complementary site was exposed on the sense-strand of the double-stranded cDNA by RNase H digestion. The resulting multiple copies of each cDNA molecule accumulated to a total amplification of 10,000-fold. The amplification reaction was then terminated and cDNAs were collected and purified with the Zymo Research DNA Clean and Concentrator-25 (Zymo Research). Purified cDNA was analyzed for quantity and quality (Agilent 2100 Bioanalyzer/Agilent RNA 6000 Nano LabChip).

[000166] Before application of the cDNA samples to the Affymetrics HG-U133 Plus 2.0 Human Expression Microarray, the molecules were fragmented and biotin-end-labelled. 3.75 μg of each cDNA was used on each microarray. The cDNAs were subjected to a chemical/enzymatic fragmentation process (FL-Ovation® cDNA Biotin Module V2, NuGEN Technologies) that produced single-stranded cDNA fragments in the 50-100 base size range. Subsequently, this fragmented cDNA pool was labeled by enzymatically attaching a 3' biotin-labeled nucleotide.

[000167] These labeled, fragmented cDNAs were applied to the microarray after they were diluted into the Hybridization Cocktail composed of Hybridization Buffer, DMSO and control oligonucleotides as well as acetylated-BSA and herring sperm DNA to control non-specific hybridization signal. Hybridization was performed by incubating 200 μΐ, of this Hybridization Cocktail with the Affymetrix U133 Plus 2.0 GeneChip for 16-20 hours at 45°C. After hybridization, the hybridization solutions were removed and the GeneChip was washed and stained with

Streptavidin-phycoerythrin, and read at a resolution of 6 microns with an HP Gene Array Scanner.

[000168] E. Statistical Analysis

[000169] Three patient groups were defined: responder (R, n=30), non-responder (NR, n=l 7), and non-failing controls (NFC, n=4). Data were normalized using the RMA method from Affymetrix but separately controlling for batch effect, and low variance probesets were removed. The remaining 16,383 probesets were analyzed initially by ANOVA comparing all 6 groups followed by Tukey pairwise tests to compare R to NR at baseline. A number of (35) significant differences at baseline between R and NR were found to be predictive for beta-blocker response. Upon further analysis, all candidate mRNAs were analyzed (and including NFC), using non-parametric ANOVA (Kruskal- Wallis) followed by non-parametric t-test (Wilcoxon) to compare all the baseline groups and then the same comparing NFC to the 12 month data. Nine of the 35 mRNAs that distinguish NR from R and baseline also separated R from NFC at baseline according to t-test. All nine of these returned to NFC levels by 12 months (Figure 1).

[000170] Example 2. mRNA expression in patient groups.

[000171] Patients with nonischemic dilated cardiomyopathy and LVEF < 40% underwent endomyocardial biopsy and radionuclide ventriculography at 0, 3, and 12 months of β-blocker therapy with either carvedilol or metoprolol CR/XL (or metoprolol + doxazosin). Response was defined as an increase in EF of >8% at 12 months or if not available, >5% at 3 months. Myocardial biopsies were taken from the distal right ventricular septum via a percutaneous approach.

[000172] Changes in cardiac function after 3 months and 12 months of therapy were also measured. On average responder status patients improved their ejection fraction by 11.3 EF units at 3 months and 16.2 EF units at 12 months. Over 80% of responder status patients had an improvement in ejection fraction by more than 3 units by 3 months of therapy. The majority of improvement in ejection occurred in the first 3 months of therapy. There were no significant changes in EF response to β-blockers by treatment group (p=.23 by ANOVA).

[000173] Three patient groups were defined:

[000174] a) responder (R, n=30),

[000175] b) non-responder (NR, n=17), and

[000176] c) non-failing controls (NFC, n=4).

[000177] Data were normalized controlling for batch effect, and low variance probe-sets were removed. The remaining 16,383 probe-sets were analyzed initially by ANOVA comparing all 6 groups followed by Tukey pairwise tests to compare R to NR at baseline. A number of (35) significant differences at baseline between R and NR were found to be predictive for beta-blocker response. Upon further analysis, all candidate mRNAs were analyzed (and including NFC), using non-parametric ANOVA (Kruskal-Wallis) followed by non-parametric t-test (Wilcoxon) to compare all the baseline groups and then the same comparing NFC to the 12 month data. Doing the analysis this way on this group of mRNAs selects for the molecules whose expression serves predictively and reproducibly with respect to longitudinal beta-blocker response.

[000178] These results provide the ability to tailor a beta-blocker treatment regimen for an individual patient, and are informative about the biology of the heart failure itself and have the potential to illuminate pathways to completely unrelated and currently non-existent treatments.

[000179] Figure 1 shows biomarkers (using Affymetrix nomenclature from which the relevant sequences can be derived), in order of statistical significance (i.e. K-W p<0.05, Wilcoxon p<0.05, sorted by Wilcoxon p value).

[000180] A nine biomarker signature is shown in Figure 2. [000181] Example 3.

[000182] Collected data was re-analyzed. Following the 16,383 probesets being analyzed by repeated measures ANOVA in responders vs. nonresponders at all three timepoints, Tukey posthox tests were used to determine pairwise differences. mRNAs were assessed for their predictive contributions using logistic regression. Candidate mRNA sets were then consolidated into a single value for each patient and evaluated by receiver operating characteristic (ROC) area under the curve (AUC) analysis.

[000183] The pairwise comparisons reveals 35 probesets that differed significantly (p <0.05) between responders and nonresponders at baseline. The receiver operating characteristic AUC analysis identified four mRNA probesets that yielded high AUC values: hCG 2045206 (SEQ ID NO: 10); 241584 at (SEQ ID NO: 1 1); C4orf49 (SEQ ID NO: 12); and KLRC3 (SEQ ID NO: 13) (FIG. 19A - 19D). From these four mRNA probesets, two particular combinations of three probesets were identified that yielded both high maximum sensitivity and specificity. The group of hCG 2045206 (SEQ ID NO: 10), 241584 at (SEQ ID NO: 1 1), and C4orf49 (SEQ ID NO: 12) resulted in a calculated max sensitivity of 0.94, with a specificity of 0.94 (FIG. 20). The group of hCG 2045206 (SEQ ID NO: 10), 241584 at (SEQ ID NO: 1 1), and KLRC3 (SEQ ID NO: 13) resulted in a calculated max sensitivity of 0.94, with a specificity of 0.88 (FIG. 20).

[000184] AUC values were used as a measure of overall performance (sensitivity and specificity) of a model in predicting β-blocker responder status. AUC values >0.7 are generally considered good in clinical outcomes research, while values >0.8 are excellent, and values >0.9 are exceptional.

[000185] Generalized Linear Model

[000186] The 35 individual mRNAs were systematically tested for predictive power (β-blocker responders vs. non-responder). This demonstrated the predictive power of each mRNA as a single measurement for β-blocker responder status. To determine whether a pair of mRNA probesets were a better predictor of responder status, all pairs of the top 20 were tested. A multivariate generalized linear model was derived for each mRNA pair (glm function (binomial/logit model) in the R

Statistical Package). Predict (R Statistical Package) was then used to calculate probabilities of response to β-blockers for each patient based on the mRNA pair. The performance of the algorithm was evaluated using the ROCR package (R Statistical Package). Prediction (R Statistical Package) was used to calculate probabilities of response for each patient, while performance (R Statistical Package) was used to calculate AUC.

[000187] Similar analysis was applied to sets of three mRNAs, resulting in two sets of three mRNAs (hCG 2045206 (SEQ ID NO: 10), 241584 at (SEQ ID NO: 11), and C4orf49 (SEQ ID NO:

12) ; and hCG 2045206 (SEQ ID NO: 10), 241584 at (SEQ ID NO: 11), and KLRC3 (SEQ ID NO:

13) ) that gave high specificity (>90%) and sensitivity (low error rate, greater than 88%). These two sets of three mRNAs appeared to perform better than any single mRNA or pair of mRNA as a predictor of β-blocker status.

[000188] The absolute range of each mRNA relative to normalizing factor(s) such as a housekeeping gene(s) determines the final coefficients in the mathematical algorithm that estimates likelihood of response to β-blockers.

[000189] Figure 1 shows biomarkers (using Affymetrix nomenclature from which the relevant sequences can be derived), in order of statistical significance (i.e. K-W p<0.05, Wilcoxon p<0.05, sorted by Wilcoxon p value).

[000190] A four biomarker signature is shown in Figure 21.

[000191] Example 4

[000192] Similar analysis can be conducted as described in Example 3. A free-standing diagnostic can be created utilizing the four mRNA probesets identified in Example 3 (hCG 2045206 (SEQ ID NO: 10); 241584 at (SEQ ID NO: 1 1); C4orf49 (SEQ ID NO: 12); and KLRC3 (SEQ ID NO: 13)), along with up to approximately 10 transcripts whose expression varies little among patients. These additional transcripts will allow for internal normalization, and can be identified

mathematically in the complete Affymetrix data. Following normalization, the four mRNA probesets can be used to predict β-blocker status, particularly in the two combinations set out in Example 3, using a generalized linear model.

[000193] A standard form of the generalized linear model (logit) that may be used to determine β-blocker responder status utilizing normalized expression values is:

Probability of Response = intercept + coefficientl *mRNAl + coefficient2*mRNA2 + coefficient3 *mRNA3.

Claims

CLAIMS What is claimed is:

1. A method to identify a human as having β-blocker responder status, comprising:

c) identifying the human as having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 11 and SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 are higher compared to non-failing control mRNA expression levels.

2. A method to identify a human as having β-blocker responder status, comprising:

c) identifying the human as having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are higher compared to non- failing control mRNA expression levels.

3. A method to identify a human as having β-blocker responder status, comprising:

c) identifying the human has having β-blocker responder status if: i) a decision algorithm including the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates the human as having β-blocker responder status; and

ii) a decision algorithm including the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates the human as having β-blocker responder status.

4. The method of claim 1, further comprising obtaining mRNA expression levels in the sample for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 13, and identifying the human as having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 11 and SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are higher compared to non-failing control mRNA expression levels.

5. The method of any one of the claims herein, further comprising identifying the human as not having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 11 and SEQ ID NO: 12 are not lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are not higher compared to non-failing control mRNA expression levels..

6. The method of any one of the claims herein, wherein the sample is obtained from a human having a condition selected from the group consisting of: idiopathic dilated cardiomyopathy; left ventricular ejection fraction less than 40%; one or more biomarkers of heart failure; heart failure; elevated atrial natriuretic peptide; elevated beta-type natriuretic peptide; and elevated cholesteryl ester transfer protein.

7. The method of any one of the claims herein, wherein the mRNA levels are obtained via use of a physical assay selected from the group consisting of: qPCR; competitive hybridization; and microarray.

8. The method of any one of the claims herein, wherein a human identified as having β-blocker responder status has a higher likelihood of an outcome selected from the group consisting of: greater likelihood of shorter duration of heart failure prior to testing than non-responders; more myocyte reverse remodeling compared to non-responders; better prognosis compared to non-responders; more heart tissue repair compared to non-responders; improved left ventricle ejection fraction compared to non-responders; decreased end diastolic volume compared to non-responders; reductions in heart rate compared to non-responders; reductions in blood urea nitrogen compared to non-responders;

reductions in atrial natriuretic peptide compared to non-responders; reductions in increases in body mass index compared to non-responders; and reductions in increases in systolic pressure compared to non-responders.

9. The method of any one of the claims herein, further comprising administering to the human identified as having β-blocker responder status a therapeutic dose of β-blocker.

10. The method of claim 9, wherein the β-blocker is selected from the group consisting of:

metoprolol succinate; metoprolol succinate and doxazosin; and carvedilol.

11. The method of claim 10, wherein the β -blocker is metoprolol succinate and is administered once daily at a dosage range selected from the group consisting of: 5-15 mg; 7.5-15 mg; 12.5 - -25 mg; 15 - 30 mg; 25 - 50 mg; 35 - 60 mg; 45 - 70 mg; 50 - 75 mg; 60 - 85 mg; 75- 100 mg; 85 - 110 mg; 95- 120mg; 100 -125 mg; 110-135mg; 120- 145 mg; 125- 150 mg; 135 - 160 mg; 145- 170 mg; 150- 175 mg; 160- 185 mg; 170- 195 mg; 175- 200 mg; 185 - 210; and 195 - 220 mg.

12. The method of claim 10, wherein the β-blocker is metoprolol succinate and doxazosin and is administered once daily at a metoprolol succinate dosage selected from the group consisting of: 5 - 15 mg; 7.5 - 15 mg; 12.5 - -25 mg; 15 - 30 mg; 25 - 50 mg; 35 - 60 mg; 45 - 70 mg; 50 - 75 mg; 60 - 85 mg; 75- 100 mg; 85-110mg; 95- 120mg; 100 -125 mg; 110- 135 mg; 120- 145 mg; 125- 150 mg; 135- 160 mg; 145- 170 mg; 150- 175 mg; 160- 185 mg; 170- 195 mg; 175- 200 mg;

185 - 210; and 195 - 220 mg, and a doxazosin dosage selected from the group consisting of 0.25 - 1.25 mg; 0.5-1.5 mg; 1-2 mg; 1.25 - 2.25 mg; 1.5-2.5 mg; 2- 3 mg; 2-4 mg; 3-5 mg; 4-6 mg; 4-8 mg; 5 -7 mg; 5-8 mg; 6 - 8mg; 6-10 mg; and 7-10 mg.

13. The method of claim 10, wherein the β-blocker is carvedilol and is administered twice daily at a dosage selected from the group consisting of: 1 - 5 mg; 2-4 mg; 2.5 - 3.5 mg; 3-7 mg; 3.125 - 6.25mg;4-8mg;5-10mg;5-7mg; 7.5-15 mg; 10-15mg; 11.5-13.5 mg; 12.5-25 mg; 15- 30 mg; 15 - 25 mg; 20 -30 mg; 25 - 50 mg; 30 - 50 mg; 40 - 50 mg; 30 - 60 mg; 40 - 60 mg; and 50 - 60 mg.

14. The method of any one of the claims herein, further comprising recommending one or more lifestyle changes selected from the group consisting of: reducing sodium intake to 2,000 - 3,000 mg/day, or less; increase dietary fiber intake; increase potassium intake; moderate fluid intake; achieve and/ or maintain a healthy body weight; begin and/or continue mild to moderate aerobic exercise; .smoking cessation; limit or cease alcohol intake.

15. A method to identify a human as having β-blocker non-responder status, comprising:

c) identifying the human as having β-blocker non-responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 11 ; and SEQ ID NO: 12 are substantially similar to non-failing control mRNA expression levels.

16. A method to identify a human as having β-blocker non-responder status, comprising:

c) identifying the human as having β-blocker non-responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 11 ; and SEQ ID NO: 13 are substantially similar to non-failing control mRNA expression levels.

17. A method to identify a human as having β-blocker non-responder status, comprising:

c) identifying the human has having β-blocker non-responder status if:

ii) a generalized linear model algorithm including the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 indicates the human as having β-blocker non-responder status.

18. The method of any one of the claims, further comprising administering one or more therapeutic agents to the human identified as having β-blocker non-responder status that are not β- blockers.

19. The method of claim 18, wherein the one or more therapeutic agents are selected from the group consisting of: angiotensin-converting enzyme (ACE) inhibitors; angiotensin II receptor blockers; diuretics; aldosterone antagonists; anticoagulants; cardiac glycosides; vasodilators;

antiarrhythmics; human B-type natriuretic peptide; and inotropic agents.

20. The method of any one of the claims herein, wherein a human identified as having non- responder status has a higher likelihood of an outcome selected from the group consisting of: greater likelihood of longer duration of heart failure prior to testing compared to responders; limited or no myocyte reverse remodeling compared to responders; poor prognosis compared to responders; more heart tissue damage compared to responders; longer duration of heart failure compared to responders; higher prevalence of atrial fibrillation compared to responders; wider QRS complex duration compared to responders; and lower renal function compared to responders.

21. A computer-assisted method to generate a report of the responder/non-responder status of a human, comprising:

a) obtaining a myocardial tissue sample from a human;

e) generating a report of the responder/non-responder status of the human.

22. A computer-assisted method to generate a report of the responder/non-responder status of a human, comprising:

a) obtaining a myocardial tissue sample from a human;

e) generating a report of the responder/non-responder status of the human.

23. The method of any one of the claims herein, wherein the decision algorithm comprises a decision selected from the group consisting of:

a) non-responder status if the sample mRNA expression levels are substantially similar to non-failing control levels and/or non-responder control levels; or

24. The method of any one of the claims herein, wherein the decision algorithm is based on a generalized linear model having the form of:

probability of response to β-blockers = intercept + coefficientl*mRNAl +

coefficient2*mRNA2 + coefficient3*mRNA3.

25. The method of any one of the claims herein, further comprising determining overall performance of the decision algorithm, wherein the overall performance is determined by calculating a receiver operating characteristic area under the curve.

26. The method of any one of the claims herein, wherein the receiver operating characteristic area under the curve is >0.7, >0.8, or >0.9.

27. The method of any one of the claims herein, further comprising determining an absolute range of an mRNA level relative to one or more housekeeping genes, thereby determining a coefficient for the mRNA.

28. The method of any one of the claims herein, wherein the physical assay is carried out using an Affymetrix HGU133 plus 2 chip.

29. The method of claim 23, wherein the human is identified as having β-blocker responder status if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 11 and SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are higher compared to non-failing control mRNA expression levels.

30. The method of any one of the claims herein, wherein said method comprises the use of computer software.

31. The method of any one of the claims herein, which is an automated method.

32. A kit comprising:

b) non-failing control RNA or non-responding control RNA.

33. The kit of any one of the claims herein, further comprising one or more additional containers comprising one or more of: wash reagent(s); and one or more detection reagents, capable of detecting presence of a bound nucleic acid.

34. The kit of claim 33, wherein the detection reagent is labeled with a reporter dye selected from the group consisting of: acridine; AMCA; BODIPY; cascade blue; Cy2; Cy3; Cy5; Cy7; dabcyl; edans; eosin; erythrosine; fluorescein; 6-Fam; tet; joe; hex; Oregon green; rhodamine; rhodol green; tamra; rox; and Texas red.

35. The kit of any one of the claims herein, wherein the detection reagent is labeled with a quencher dye.

36. The kit of any one of the claims herein, wherein the detection reagent is labeled with a label selected from the group consisting of: biotin; hapten; oligonucleotide; and luminescent tags.

37. A microarray comprising two or more isolated nucleic acids comprising two or more sequences selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11 ; SEQ ID NO: 12; and SEQ ID NO: 13.

38. A microarray comprising isolated nucleic acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

39. The microarray of any one of the claims herein, wherein the isolated nucleic acids are synthetic oligonucleotides and/or fragments of cDNA.

40. The microarray of any one of the claims herein, further comprising isolated nucleic acids comprising a variant of one or more sequences selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11 ; SEQ ID NO: 12; and SEQ ID NO: 13, wherein the variant of the one or more sequences is a negative control.

41. The microarray of any one of the claims herein, wherein the synthetic oligonucleotides are 6 - 60 nucleotides in length, 15 - 30 nucleotides in length, 20 - 25 nucleotides in length, or 7 -20 nucleotides in length.

42. A method to identify a therapy for the amelioration of heart failure symptoms, comprising: a) administering a test therapy to a human with heart failure due to dilated

cardiomyopathy, wherein the patient is in need of therapy;

b) obtaining at least one myocardial tissue sample from the human;

c) conducting at least one assay of the sample so as to obtain one or more mRNA expression levels in the sample for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; and SEQ ID NO: 13, and

43. The method of claim 42, wherein the therapy for the amelioration of heart failure is the administering to the patient a therapeutic dose of β-blocker if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 11 and SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 are higher compared to non-failing control mRNA expression levels.

44. The method of claim 42, wherein the therapy for the amelioration of heart failure is the administering to the patient a therapeutic dose of β-blocker if the sample mRNA expression levels for mRNAs which are capable of hybridizing to nucleic acids of SEQ ID NO: 12 are lower compared to non-failing control mRNA expression levels and the sample mRNA expression levels for mRNAs which are capable of hybridizing nucleic acids of SEQ ID NO: 10 and SEQ ID NO: 13 are higher compared to non-failing control mRNA expression levels.

45. The method of claim 42, wherein the therapy for the amelioration of heart failure is the administering to the patient a therapeutic dose of β-blocker if the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6 are lower compared to non- failing control mRNA expression levels and/or the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 are higher compared to non-failing control mRNA expression levels.

46. The method of claim 42, wherein the therapy for the amelioration of heart failure comprises administering one or more therapeutic agents selected from the group consisting of: angiotensin- converting enzyme (ACE) inhibitors; angiotensin II receptor blockers; diuretics; aldosterone antagonists; digoxin; and anticoagulants, and/or recommending one or more lifestyle changes selected from the group consisting of: reducing sodium intake to 2,000 - 3,000 mg/day, or less; increase dietary fiber intake; increase potassium intake; moderate fluid intake; achieve and/or maintain a healthy body weight; begin and/or continue mild to moderate aerobic exercise; .smoking cessation; limit or cease alcohol intake, if mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; and SEQ ID NO: 13 are substantially similar to non-failing control mRNA expression levels.

47. The method of claim 42, wherein the therapy for the amelioration of heart failure comprises administering one or more therapeutic agents selected from the group consisting of: angiotensin- converting enzyme (ACE) inhibitors; angiotensin II receptor blockers; diuretics; aldosterone antagonists; digoxin; and anticoagulants, and/or recommending one or more lifestyle changes selected from the group consisting of: reducing sodium intake to 2,000 - 3,000 mg/day, or less; increase dietary fiber intake; increase potassium intake; moderate fluid intake; achieve and/or maintain a healthy body weight; begin and/or continue mild to moderate aerobic exercise; .smoking cessation; limit or cease alcohol intake, if mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 are substantially similar to non-failing control mRNA expression levels.

48. A method to treat idiopathic dilated cardiomyopathy, comprising administering at least one composition selected from the group consisting of: cholesteryl ester transfer protein agonist, or pharmaceutically-acceptable formulation thereof; cholesteryl ester transfer protein mimic, or pharmaceutically-acceptable formulation thereof; cholesteryl ester transfer protein enhancer, or pharmaceutically-acceptable formulation thereof; or cholesteryl ester transfer protein peptide, or pharmaceutically-acceptable formulation thereof.

49. The method of any one of the claims herein, which further comprises repeating the method over time.

50. The method of any one of the claims herein, which further comprises decreasing or increasing the dose of a therapeutic agent based on the human's identified β-blocker responder status.

51. The method herein, which further comprises communicating the data or β-blocker responder status to at least one human.

52. The method herein, wherein the data and/or status is communicated in paper form or computer readable medium form.

53. A method to identify a human as having β-blocker non-responder status, comprising:

b) conducting at least one physical assay of the sample so as to obtain mRNA expression levels in the sample for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9; and c) identifying the human as having β-blocker non-responder status if the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 are substantially similar to non-failing control mRNA expression levels.

54. A computer-assisted method to generate a report of the responder/non-responder status of a human, comprising:

a) obtaining a myocardial tissue sample from a human;

b) conducting at least one assay of the sample so as to obtain mRNA expression levels in the sample for one or more mRNAs capable of hybridizing to a nucleic acid selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9;

e) generating a report of the responder/non-responder status of the human.

55. The method of claim 53, wherein the algorithm comprises a decision selected from the group consisting of:

56. The method of claim 54, wherein the human is identified as having β-blocker responder status if the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6 are lower compared to non-failing control mRNA expression levels and/or the sample mRNA expression levels for one or more mRNAs which are capable of hybridizing to one or more nucleic acids selected from the group consisting of: SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 are higher compared to non-failing control mRNA expression levels.

57. A kit comprising:

a) an isolated nucleic acid comprising one or more sequences selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9, and

b) non-failing control RNA or non-responding control RNA.