CA2554585A1 - Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of cardiac disease - Google Patents

Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of cardiac disease Download PDF

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CA2554585A1
CA2554585A1 CA002554585A CA2554585A CA2554585A1 CA 2554585 A1 CA2554585 A1 CA 2554585A1 CA 002554585 A CA002554585 A CA 002554585A CA 2554585 A CA2554585 A CA 2554585A CA 2554585 A1 CA2554585 A1 CA 2554585A1
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seq
amino acid
amino acids
sequence
homologous
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Yossi Cohen
Alexander Diber
Amir Toporik
Sarah Pollock
Zurit Levine
Michal Ayalon-Soffer
Gad S. Cojocaru
Amit Novik
Guy Kol
Osnat Sella-Tavor
Shira Walach
Shirley Sameah-Greenwald
Dvir Dahary
Ronen Shemesh
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Compugen Ltd
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Priority claimed from US11/043,788 external-priority patent/US20060014166A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

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Abstract

Novel markers for cardiac disease that are both sensitive and accurate. These markers are differentially and/or specifically expressed in cardiac tissue, as opposed to other types of tissues, optionally and preferably including muscle tissue. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of cardiac disease, including pathology and/or damage, including acute and/or chronic damage. The markets of the present invention, alone or in combination, show a high degree of differential detection between cardiac disease states and non-cardiac disease states.

Description

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JUMBO APPLICATIONS / PATENTS
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THAN ONE VOLUME.

NOTE: For additional volumes please contact the Canadian Patent O~ce.

NOVEL NUCLEOTIDE AND AMINO ACID SEQUENCES, AND ASSAYS AND
METHODS OF USE THEREOF FOR DIAGNOSIS OF CARDIAC DISEASE
FIELD OF THE INVENTION
The present invention is related to novel nucleotide and protein sequences that are diagnostic markers for cardiac disease andlor pathological conditions, including cardiac damage, and assays and methods of use thereof.
BACKGROUND OF THE INVENTION
Cardiovascular diseases are an important cause of mortality and morbidity.
Amongst all age groups considered, IHD is the most common cause of death not only in men but also in women. Coronary atherosclerosis is a chronic progressing process, associated with angina type symptoms and frequently result in Acute Myocardial Infarction (AMI). The diagnosis is achieved with a combination of patient physical examination, ECG since 1950's molecular markers play the most important role in the differential diagnosis of AMI from other conditions with similar symptoms. Early diagnosis is mandatory of the establishment of early treatment (inc hiding blood diluting agents, thrombolysis, catheterization and surgery).
Early molecular markers for AMI were SGOT and LDH were proved to be of very low specificity and are hardly being used at present. These markers were replaced by CPK, and later on by the heart specific CPK-MB variant. Its specificity is better than for SGOT and LDH, it is still limited both in specificity and sensitivity which reach only 67% when used together with electrocardiogram. In addition, cardiac surgery, myocarditis, and electrical cardioversion often result in elevated serum levels of the CPK-MB isoenzyme. Small infarct with minor myocardial cell necrosis often do not increase serum CPK-MB to a detected level.
Myoglobin is another heart damage low molecular (l7kD) protein but is even less specific to heart muscle compared with CPK-MB. Its advantage over CPK-MB is a rapid rise from the onset of symptoms -usually between 3-6 hours. It is considered one of the earliest indicators (together with H-FABP) but it lacks specificity due to significant expression in skeletal muscle - its concentration is approximately two-fold lower in cardiac than skeletal muscle and the leads to seriously diminished specificity.
Cardiac troponins are currently the routine serum cardiac markers used for the diagnosis of AMI. Troponin-I and Troponin-T have amino acid sequences different from those of the skeletal muscle called cTnT and cTnI (cardiac Troponin-T and I recpectively).
Cardiac troponins are not found in the serum of healthy individuals and rise to up to 20 times above a predefined cut-off level, therefore are very useful and sensitive in the detection of cardiac damage. They are capable of detecting very small cardiac damage - micro-infarction, it is associated with a very adverse longer term prognosis. Cardiac troponin's sensitivity is considerably higher than CPK-MB but they suffer from a few disadvantages: 1.
They are not early markers - cTnI and cTnT reach peak serum value in about 12 and 48 hours respectively after symptoms onset. 2. Le vels of cTnI and cTnT remain elevated for up to 10 days and 14 days respectively after AMI, therefore cannot be used for the detection of re-infarction. 3. Other heart diseases such as Congestive Heart Failure and Myocarditis can increase troponins concentrations in the serum. The lack of specificity for AMI is an advantage when there are other supporting clinical evidence directing the doctor towards another diagnosis. Troponins might have a diagnostic value in assessing myocardial damage after coronary artery perfusion, monitoring progression and prognosis of unstable angina, in the detection and prognosis of cardiac contusion after blunt trauma, detecting myocarditis.
The heart specific variant H-FABP (Heart Fatty Acid binding protein) is a low molecular protein (lSKd) soluble non-enzyme protein. H-FABP concentration in the heart muscle is greater than that in skeletal muscle, and its normal baseline concentration is several fold lower than myoglobin. In addition, it reaches peak value in the urine and blood early, within 2-3 hours from AMI. Within a period of 30-210 minutes after symptoms started, ITFABP has higher sensitivity - up to 80% - when compared with other cardiac markers (CPK-MB and the troponins sensitivity were reported to be 64% in the first 6 hours after AMI).
Yet, I~FABP still misses every S~h patient in this time scale. H-FABP has other limitations as well, including 1.
rising in the plasma after exercise 2. released from muscle in skeletal damage during the course of AMI (like from intramuscular injections) 3. reduced clearance in renal failure situations.
The search for novel cardiac damage markers is ongoing. Other proteins are under trials for that purpose including glycogen phosphorylase BB, HIF and VEGF 21.

SUMMARY OF THE INVENTION
Markers for the cardiac disease and/or cardiac pathology, including but not limited to cardiac damage in the prior art are not sufficiently sensitive and/or accurate, alone or in combination.
The present invention overcomes these deficiencies of the background art by providing novel markers for cardiac disease and/or cardiac pathology, including but not limited to cardiac damage that are both sensitive and accurate. Optionally and preferably, these markers are detected in a biological sample.
According b preferred embodiments of the present invention, cardiac disease and/or pathology and/or condition and/or disorder may comprise one or more of Myocardial infarct, acute coronary syndrome, angina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure or any type of heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, assessing the size of infarct in Myocardial infarct, the differential diagnosis of heart related conditions from lung related conditions (as pulmonary embolism), the differential diagnosis of Dyspnea, and cardiac valves related conditions.
According to preferred embodiments of the present invention, examples of suitable biological samples include but are not limited to blood, serum, plasma, blood cells, urine, sputum, saliva, stool, spinal fluid, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, neuronal tissue, and any human organ or tissue.
In a preferred embodiment, the biological sample comprises cardiac tissue and/or a serum sample and/or a urine sample and/or any other tissue or liquid sample. The sample can optionally be diluted with a suitable eluant before contacting the sample to the antibody.
Information given in the text with regard to cellular localization was determined according to four different software programs: (i) tirihmm (from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://www.cbs.dtu.dk/services/TMHMM/TMHMM2.Ob.guide.php) or (ii) tmpred (from EMBnet, maintained by the ISREC Bionformatics group and the LICR Information Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinformatics, http://www.ch.embnet.org/software/TMPRED form.html) for transmembrane region prediction; (iii) signalp hmm or (iv) signalp nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http:l/www.cbs.dtu.dk/services/SignalP/background/prediction.php) for signal peptide prediction. The terms "signalp hmm" and "signalp nn" refer to two modes of operation for the program SignalP: hmm refers to Hidden Markov Model, while nn refers to neural networks.
Localizatio n was also determined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor.
In some cases for the manual inspection of cellular localization prediction inventors used the ProLoc computational platform [Einat Hazkani-Covo, Erez Levanon, Galit Rotman, Dan Graur and Amit Novik;
(2004) "Evolution of multicellularity in metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis."
Cell Biology International 2004;28(3):171-8.], which predicts protein localization based on various parameters including, protein domains (e.g., prediction of trans-membranous regions and localization thereof within the protein), pI, protein length amino acid composition, homology to pre-annotated proteins, recognition of sequence patterns which direct the protein to a certain organelle (such as, nuclear localization signal, NLS, mitochondria localization signal), signal peptide and anchor modeling and using unique domains from Pfam that are specific to a single compartment.
Information is given iil the text with regard to SNPs (single nucleotide polymorphisms).
A description of the abbreviations is as follows. "T - > C", for example, means that the SNP
results in a change at the position given in the table from T to C. Similarly, "M - > Q", for example, means that the SNP has caused a change in the corresponding amino acid sequence, from methionine (M) to glutamine (Q). If, in place of a letter at the right hand side for the nucleotide sequence SNP, there is a space, it indicates that a frameshift has occurred. A
frameshift may also be indicated with a hyphen ( ). A stop codon is indicated with an asterisk at the right hand side (*). As part of the description of an SNP, a comment may be found in parentheses after the above description of the SNP itself. This comment may include an FTId, which is an identifier to a SwissProt entry that was created with the indicated SNP. An FTId is a unique and stable feature identifier, which allows construction of links directly from position-specific annotation in the feature table to specialized protein-related databases. The FTId is always the last component of a feature in the description field, as follows:
FTId=XXX number, in which XXX is the 3-letter code for the specific feature lcey, separated by an underscore from a 6-digit number. In the table of the amino acid mutations of the wild type proteins of the selected splice variants of the invention, the header of the first colmnn is "SNP positions) on amino acid sequence", representing a position of a known mutation on amino acid sequence.
5 SNPs may optionally be used as diagnostic markers according to the present invention, alone or in combination with one or more other SNPs and/or any other diagnostic marker.
Preferred embodiments of the present invention comprise such SNPs, including but not limited to novel SNPs on the known (WT or wild type) protein sequences given below, as well as novel nucleic acid and/or amino acid sequences formed through such SNPs, and/or any SNP on a variant amino acid and/or nucleic acid sequence described herein.
Information given in the text with regard to the Homology to the known proteins was determined by SmitlrWaterman version 5.1.2 using special (non default) parameters as follows:
-model=sw.model -GAPEXT=0 -GAPOP=100.0 -MATRIX=blosum 100 Information is given with regard to overexpression of a cluster in cancer based on microarrays. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured.
There are two types of microamay results: those from microarrays prepared according to a design by the present inventors, for which the microarray fabrication procedure is described in detail in Materials and Experimental Procedures section herein; and those results from microarrays using Affymetrix technology. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured. For microarrays prepared according to a design by the present inventors, the probe name begins with the name of the cluster (gene), followed by an identifying number. Oligonucleotide microarray results taken from Affymetrix data were from chips available from Affymetrix Inc, Santa Clara, CA, USA (see for example data regarding the Human Genome U133 (HG-U133) Set at www.affymetrix.com/productslarrays/specific/hgu133.affx; GeneChip Human Genome 2.0 Array at www.affymetrix.com/products/arrays/specific/hgu133av2.affi~; and Human Genome U133 Plus 2.0 Array at www.affymetrix.com/products/arrays/specific/hgu133plus.affx). The probe names follow the Affymetrix naming convention. The data is available from NCBI Gene Expression Omnibus (see www.ncbi.nlm.nih.gov/projects/geo/ and Edgar et aI, Nucleic Acids Research, 2002, Vol.
30, No. 1 207-210). The dataset (including results) is available from www.ncbi.nlrn.nih.gov/geo/query/acc.cgi?acc=GSE1133 for the Series GSE1133 database (published on March 2004); a reference to these results is as follows: Su et al (Pros Natl Acad Sci U S A. 2004 Apr 20;I01(16):6062-7. Epub 2004 Apr 09).
Oligonucleotide probes for use with arrays designed by the present inventors:
>567314 0_0 741 (SEQ ID NO 392) CACAGAGCCAGGATGTTCTTCTGACCTCAGTATCTACTCCAGCTCCAGCT
>S673I4 0 0 744 (SEQ ID NO 393) TGGCATGCTGGAACATGGACTCTAGCTAGCAAGAAGGGCTCAAGGAGGTG
In the heart specific clusters, a first set of abbreviations is used for the first histogram ADP = adipocyte BLD = blood BLDR = bladder BRN = brain BONE = bone BM = bone marrow BRS = mammary gland CAR = cartilage CNS = central nervous system COL = colon E-ADR = endocrine adrenal_gland E-PAN = endocrine~ancreas E-PT = endocrine~arathyroid thyroid ENDO = endocrine unchar EPID = epididymis GI = gastrointestinal tract GU = genitourinary HN = head and neck HRT = heart KD = kidney LI = liver LUNG = lung LN = lymph node MUS = muscle OV = ovary PNS = peripheral nervous system PRO = prostate SKIN = skin SPL = spleen SYN = synovial membrane TCELL = immune T cells THYM = thymus TST = testes UTER = cervix-uterus VAS = vascular In the second histograms) of the heart paragraph, the oligo-probe names are abbreviated/enumerated as follows:
"adipocyte", "Al";

"adrenalcortex", "A2";

"adrenalgland", "A3";

~~~Yg~la", ..A4..;

"appendix" "AS"' > >

"atrioventricularnode","A6";

"bm cd105 endothelial", "E1' ;

"bm cd33'myeloid", "M1";

"bm cd34 ", "B1' ;

"bm cd7l earlyerythroid","El";

"bonemarrow", "B2";

"bronchialepithelialcells","B3' ;

"cardiacmyocytes", "C1";

"caudatenucleus", "C2";

"cerebellum", "C3";

"cerebellumpeduncles","C4";

"ciliaryganglion", "CS";

"cingulatecortex", "C6' ;

"globuspallidus", "G1";

"heart" "H1"~
> >

"hypothalamus", "H2";

"kidney", "K1 ";

"liver" "L1 > "' >

~~1~ .. ..L2...
g> >

. "lymphnode", "L3' ;

"medullaoblongata", "Ml";

"occipitallobe", "O1";

"olactorybulb", "02";

.. ~~~3~,.
> >

"pancreas" "Pl"~
> >

"pancreaticislets", "P2";

"parietallobe", "P3";

"pb bdca4 dentritic "P4' cells", ;

"pb'cdl4 monocytes", "PS";

"pb cdl9 bcells", "P6' ;

"pb'cd4 tcells", "P7' ;

"pb cd56 nkcells", "P8";

"pb'cd8 tcells", "P9' ;
"pituitary", "Pa";

"placenta", "Pb";

,.pons" ~,Pc".
a a "prefrontalcortex", "Pd";

"prostate", "Pe";

"salivarygland", "S

";

"skeletalmuscle", "S2";

"skin" "S3".
a a "smoothmuscle", "S4";

"spinalcord", "SS";

"subthalamicnucleus", "S6";

"superiorcervicalganglion","S7";

"temporallobe", "TI
";

"testis" "T2"' a a I "testisgermcell", "T3";
S

"testisinterstitial", "T4";

"testisleydigcell", "TS' ;

"testisseminiferoustubule","S6";

"thalamus", "T7' ;

"thymus", "T8";

"~yroid", "T9";

"tonsil" "Ta"~
a a "trachea" "Tb"~
a a "trigeminalganglion", "Tc";

"uterus" "U1"~
a a "uteruscorpus", "U2";

"wholeblood", "W
1' ;

"wholebrain", "W2";
It should be noted that the terms "segment", "seg" and "node" are used interchangeably in reference to nucleic acid sequences of the present invention; they refer to portions of nucleic acid sequences that were shown to have one or more properties as described below. They are also the building blocles that were used to construct complete nucleic acid sequences as described in greater detail below. Optionally and preferably, they are examples of oligonucleotides which are embodiments of the present invention, for example as amplicons, 5 hybridization units and/or from which primers and/or complementary oligonucleotides may optionally be derived, and/or for any other use.
As used herein the phrase "cardiac disease" includes any type of cardiac pathology and/or disorder and/or damage, including both chronic and acute damage, as well as progression from acute to chronic damage of the heart, and also propagation of one acute event to another 10 acute event. An example of the latter may occur when an infarct is followed by another infarct in a relatively short period of time, such as within 24 hours for example. An infarct may also lead to acute heart failure immediately after the infarct, as another example.
These non-limiting examples are intended to demonstrate that cardiac disease may also comprise a plurality of acute events.
The term "marker" in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients having a cardiac disease, such as acute cardiac damage for example, as compared to a comparable sample taken from subjects who do not have cardiac disease.
As used herein the phrase "differentially present" refers to differences in the quantity of a marker present in a sample taken from patients having cardiac disease as compared to a comparable sample taken from patients who do no t have cardiac disease. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
For example, in the case of acute cardiac damage, it is possible that a marker (such as a protein or fragment thereof) could optionally be present in a blood sample from the patient, indicating the presence of damage; Lack of presence of such a marker (and/or presence at a low Level) would therefore optionally and preferably indicate a lack of such damage.
Alternatively, chronically damaged heart might cause a low level of the marker to be present in the blood sample, while acute damage would cause a high level to be present. One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided in the description of each individual marker below.
As used herein the phrase "diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive.
While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term "detecting" may also optionally encompass any of the above.
Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease. It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.
As used herein, the term "level" refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.
Typically the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual (examples of biological samples are described herein).
Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the Level of DNA, RNA
and/or polypeptide of the variant of interest in the subject.
Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the Level of the variant can be determined and a diagnosis can thus be made.
Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
A "test amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of cardiac disease. A test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
A "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a patient with cardiac disease or a person without cardiac disease. A
control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
"Detect" refers to identifying the presence, absence or amount of the object to be detected.
A "label" includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32p, 355, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
The label often generates a measurable signal, such as a radioactive, chrornogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample. The label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin. The label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly. For example, the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA
molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P.
D. Fahrlander and A. Klausner, Bio/Technology 6:1165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
Exemplary detectable labels, optionally and preferably for use with innnunoassays, include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads.
Alternatively, the marker in the r sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
"Immunoassay" is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, andlor quantify the antigen.
The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide (or other epitope), refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times greater than the background (nonspecific signal) and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or l4 human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A
Laboratory Manual ( 1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than IO to 100 times background.
According to preferred embodiments of the present invention, there is provided ai isolated polynucleotide comprising a transcript selected from the group consisting of SEQ ID
NOs: 1, 2, 3 and 4.
According to preferred embodiments of the present invention, there is provided arz isolated polynucleotide comprising a segment selected from the group consisting of SEQ ID
NOs: 65, 66, 67, 68, 69, 70, 71 and 72.
According to preferred embodiments of the present invention, there is provided as isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID
NOs: 281, 282, 283 and 284.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a transcript selected from the group consisting of SEQ ID
NOs: 5, 6, 7, 8, 9 and I O
According to preferred embodiments of the present invention, there is provided ari isolated polynucleotide comprising a segment selected from the goup consisting of SEQ ID
NOs: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9I, 92, 93 and 94 .
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID
NOs: 285, 286, 287, 288, 289, 290 and 291 According to preferred embodiments of the present invention, there is provided ai isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP

CONSISTING OF SEQ ID NOs: 12, 13, 14, 15, 16 and I7 According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP
CONSISTING
OF SEQ ID NOs: 95, 96, 97, 98, 99, I00, 101, I02, 103, 104, 105, 106, 107, 108, 109, 110, 11 l, 5 112.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 292, 293, 294, 295 and 296 According to preferred embodiments of the present invention, there is provided an 10 isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 18 and 19.
According to preferred embodiments of the present invention, there is provided ai isolated polynucleotide comprising a segment SELECTED FROM THE GROUP
CONSISTING
OF SEQ ID NOs: 113, 114, 115, 116, 117, 118, 119, 120, 121 and 122.
15 According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a protein variant SELECTED FROM . THE GROUP
CONSISTING OF SEQ ID NOs: 297 and 298.
According to preferred embodiments of the present invention, there is provided ari isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 20 and 21.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP
CONSISTING
OF SEQ ID NOs: 123, 124, 125, 126, 127, I28 and 129.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 299 and 300.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 26, 27, 28, 29 and 30.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP
CONSISTING

OF SEQ ID NOs: I50, 151, 152, 153, 154, 155, I56, 157, I58, I59, 160, I61, 162 and 163.
According to preferred embodiments of the present invention, there is provided are isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 305; 306; 307 and 308 According to preferred embodiments of the present is provided invention, there an isolated polynucleotide comprising a transcript SELECTEDTHE GROUP
FROM

CONSISTING OF SEQ ID NOs: 31, 32, 33, 34, 35, 36 and 37.

According to preferred embodiments of the present invention,is provided there an isolated polynucleotide comprising a segment SELECTED
FROM THE GROUP CONSISTING

OF SEQ ID NOs: 164, 165, 166, 167, 168, 169, 170, , 176, 171, 172, 173, 174, 175 177, 178, 179, 180, 181, 182, 183, 184, 185 and 186 According to preferred embodiments of the present invention,is provided there an isolated polypeptide comprising a protein variant SELECTEDTHE GROUP
FROM

CONSISTING OF SEQ ID NOs: 309, 310, 311 and 312.

According to preferred embodiments of the present is provided invention, there an isolated polynucleotide comprising a transcript SELECTEDTHE GROUP
FROM

CONSISTING OF SEQ ID NOs: 38, 39, 40 and 41.

According to preferred embodiments of the present invention,is provided there an isolated polynucleotide comprising a segment SELECTED
FROM THE GROUP CONSISTING

OF SEQ ID NOs: 187, 188, I89, 190, I91, 192, 193, 194, 195 and I96.

According to preferred embodiments of the present invention, there is provided ai isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 313, 314, 315 and 316.
According to preferred embodiments of the present invention, there is provided a1 isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49 and 50.
According to preferred embodiments of the present invention, there is provided al isolated polynucleotide comprising a segment SELECTED FROM THE GROUP
CONSISTING
OF SEQ ID NOs: 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 and 208.

According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs: 317, 318, 319, 320, 321, 322, 323, 324 and 325.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP
CONSISTING OF SEQ ID NOs:5l, 52, 53, 54, 55, 56, 57, 58, 59 and 60.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP
CONSISTING
OF SEQ ID NOs: 209 to 273.
I0 According to prefeiTed embodiments of the present invention, there is provided an isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID
NOs: 326 to 334.
According to preferred embodiments of the present invention, there is provided al isolated polynucleotide comprising a transcript selected from the group consisting of SEQ ID
NOs: 22-2S, 353 or 386.
According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a segment selected from the group consisting of SEQ ID
NOs: 130-149.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID
NOs: 301-304, 325, 354-356 or 387.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 326, comprising a first amino acid sequence being at Ieast 90 % homologous to amino acids 1 - 1855 of SEQ ID
N0.338, which also corresponds to amino acids 1 - 1855 of SEQ ID N0.326, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1856 - 1904 of SEQ ID NO. 326, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an za isolated polypeptide encoding for a tail of SEQ ID NO. 326, composing a polypeptide being at Least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRRTPDTGSRCGSFFSGPTAPPSQGSSHLLLEMLLVDLTFFSRSAVSLT in SEQ ID NO.
326.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 327, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 1326 of SEQ ID NO.
339, which also corresponds to amino acids 1 - 1326 of SEQ ID NO. 327, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1327 - 1336 of SEQ ID NO. 327, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai isolated polypeptide encoding for a tail of SEQ ID NO. 327, comprising a polypeptide being at least 70%, optionally at Least about 80%, preferably at Least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRPSGEGGQA in SEQ ID NO. 327.
According to preferred embodiments of the present invention, there is provided al isolated chimeric polypeptide encoding for SEQ ID NO. 328, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 -1508 of SEQ ID
NO. 339, which also corresponds to amino acids 1 - 1508 of SEQ ID NO. 328, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at Least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1509 - 1534 of SEQ ID NO. 328, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai isolated polypeptide encoding for a tail of SEQ ID NO. 328, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at Least about 90% and most preferably at least about 95% homologous to the sequence GVLGVQEARDELVGGRAMQGQGEHRL in SEQ ID NO. 328.

According to prefewed embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 329, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 -1763 of SEQ ID
NO. 338, which also corresponds to amino acids 1 - 1763 of SEQ ID NO. 329, and a second amino acid sequence being at Least 70%, optionally at Least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1764 - 1788 of SEQ ID NO. 329, whexein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai .
isolated polypeptide encoding for a tail of SEQ ID NO. 329, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSDRPPSASPKDRNKALGPGQATVL in SEQ ID NO. 329.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 330, comprising a first amino acid '.
sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 22 of SEQ ID NO. 330, and a second amino acid sequence being at least 90 % homologous to amino acids 528 - 1939 of SEQ ID NO. 340, which also corresponds to amino acids 23 - 1434 of SEQ ID NO. 330, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, thexe is provided an isolated polypeptide encoding for a head of SEQ ID NO. 330, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MGLWKPGSVLSDSLFASSPCPQ of SEQ ID NO. 330.
According to preferred embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for SEQ ID NO. 331, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 527 of SEQ ID NO.
339, which also corresponds to amino acids I - 527 of SEQ ID NO. 33I, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at Least 90% and most preferably at Least 95% homologous to a polypeptide sequence corresponding to amino acids 528 - 555 of SEQ ID NO. 331, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
5 According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 331,comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VPPWPHHLCPLLCHPDKVVAESLLHPRN in SEQ ID NO. 331.
10 According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID N0.332, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 -470 of SEQ ID
N0.338, which also corresponds to amino acids 1 - 470 of SEQ ID NO.332, a second amino acid sequence being at least 90 % homologous to amino acids 528 - 1855 of SEQ
ID N0.338, 15 which also corresponds to amino acids 471 - 1798 of SEQ ID N0.332, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1799 - 1847 of SEQ ID N0.332, wherein said fixst amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a 20 sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of SEQ ID N0.332, comprising a polypeptide having a length "n", whe rein n is at Least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise DP, having a structure as follows: a sequence starting from any of amino acid numbers 470-x to 470; and ending at any of amino acid numbers 471+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided ai isolated polypeptide encoding for a tail of SEQ ID NO. 332, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRRTPDTGSRCGSFFSGPTAPPSQGSSHLLLEMLLVDLTFFSRSAVSLT in SEQ ID
N0.332.
According to preferred embodiments of the present invention, there is provided an isolated chimeric ~lypeptide encoding for SEQ ID N0.333, comprising a first amino acid sequence being at least 90 % homologous to amino acids 165 - 1939 of SEQ ID
NO. 340, which also corresponds to amino acids 1 - 1775 of SEQ ID N0.333.
According to prefen-ed embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID N0.334, comprising a first amino acid I O sequence being at least 90 % homologous to corresponding to amino acids 1165 - 1939 of SEQ
ID NO. 340, which also corresponds to amino acids 1 - 775 of SEQ ID N0.334.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID N0.317, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 158 of SEQ ID NO.
341, which also corresponds to amino acids 1 - 1 S 8 of SEQ ID N0.317.
According to preferred embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for SEQ ID N0.318, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 156 of SEQ ID NO.
341, which also corresponds to amino acids 1 - 156 of SEQ ID NO.318, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 157 - 166 of SEQ ID N0.318, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO.318, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at Ieast about 90% and most preferably at least about 95% homologous to the sequence VSVGQECGSG in SEQ ID N0.318.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID N0.319, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 156 of SEQ ID NO.
341, which also con-esponds to amino acids 1 - 156 of SEQ ID NO.319, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 157 - 210 of SEQ ID N0.319, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID N0.319, comprising a polypeptide being at Least 70%, optionally at least about 80%, preferably at Least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DGISSLCYSSLSKSLLSQPLRETSSAINDISLLQALMPLLGWTSHWTCITVGLY in SEQ ID
N0.319 .
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 320, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 60 of Q96NR4, which also corresponds to amino acids 1 - 60 of SEQ ID NO. 320, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 61 - 114 of SEQ ID NO. 320, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 320, comprising a polypeptide being at least 70%, optionally at Ieast about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DGISSLCYSSLSKSLLSQPLRETSSAINDISLLQALMPLLGWTSHWTCITVGLY in SEQ ID
NO. 320.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 320, comprising a first amino acid sequence being at least 90 % homologous to amino acids 97 - 156 of SEQ ID NO.
341, which also corresponds to amino acids 1 - 60 of SEQ ID NO. 320, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 61 - 114 of SEQ ID NO. 320, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided al isolated chimeric polypeptide encoding for SEQ ID NO. 321, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 14 of SEQ ID NO.
342, which also corresponds to amino acids 1 - 14 of SEQ ID NO. 321, a second amino acid sequence bridging amino acid sequence comprising of S, and a third amino acid sequence being at Ieast 90 homologous to con-esponding to amino acids 62 - 133 of SEQ ID NO. 342, which also corresponds to amino acids 16 - 87 of SEQ ID NO. 321, wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for an edge portion of SEQ ID NO. 321, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids iii length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise VSI having a structure as follows (numbering according to SEQ ID NO. 321): a sequence starting from any of amino acid numbers 14-x to 14; and ending at any of amino acid numbers 16 + ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 321, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 15 of SEQ ID NO. 321, and a second amino acid sequence being at Ieast 90 % homologous to corresponding to amino acids 39 - 110 of SEQ
ID NO. 343, which also corresponds to amino acids 16 - 87 of SEQ ID NO. 321, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 321, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MRGEHNSTSYDSAVS of SEQ ID NO. 321.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 321, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 97 -110 of SEQ ID
NO. 341, Which also corresponds to amino acids 1 - 14 of SEQ ID NO. 321, a second amino acid sequence bridging amino acid sequence comprising of S, and a third amino acid sequence being at least 90 % homologous to corresponding to amino acids 158 - 229 of SEQ ID NO. 341, which also corresponds to amino acids 16 - 87 of SEQ ID NO. 321, wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for an edge portion of SEQ ID NO. 321, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise VSI having a structure as follows (numbering according to SEQ ID NO. 32I): a sequence starting from any of amino acid numbers 14-x to 14; and ending at any of amino acid numbers 16 + ((n-2) - x), in which x varies from 0 to rr2.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ TD NO. 320, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DGISSLCYSSLSKSLLSQPLRETSSAINDISLLQALMPLLGWTSHWTCITVGLY in SEQ ID
NO. 320.
According to preferred embodiments of the present invention, there is provided al isolated chimeric polypeptide encoding for SEQ ID NO. 321, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 14 of SEQ ID
NO. 342, which also corresponds to amino acids 1 - 14 of SEQ ID NO. 321, a second amino acid sequence bridging amino acid sequence comprising of S, and a third amino acid sequence being at least 90 % homologous to corresponding to amino acids 62 - 133 of SEQ
ID NO. 342, which also corresponds to amino acids 16 - 87 of SEQ ID NO. 321, wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in 5 a sequential order.
According to preferred embodiments of the present invention, there is provided al isolated polypeptide encoding for an edge portion of SEQ ID NO. 321, comprising a polypeptide having a length "n", wherein n is at Least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more 10 preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise VSI having a structure as follows (numbering according to SEQ ID NO. 32I : a sequence starting from any of amino acid numbers 14-x to 14; and ending at any of amino acid numbers 16 + ((~r2) - x), in which x varies from 0 to n-2. ;, 15 According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 321, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - IS of SEQ ID NO. 321, and a second amino acid sequence 20 being at least 90 % homologous to corresponding to amino acids 39 - 110 of SEQ ID NO. 343, which also corresponds to amino acids 16 - 87 of SEQ ID NO. 321, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 321, comprising a polypeptide being at 25 least 70%, optionally at least about 80%, preferably at Least about 85%, more preferably at least about 90% and mo st preferably at Least about 95% homologous to the sequence MRGEHNSTSYDSAVS of SEQ ID NO. 321.
According to preferred embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for SEQ ID NO. 321, comprising a first amino acid sequence being at Least 90 % homologous to corresponding to amino acids 97 -110 of SEQ ID
NO. 341, which also corresponds to amino acids 1 - 14 of SEQ ID NO. 321, a second amino acid sequence bridging amino acid sequence comprising of S, and a third amino acid sequence being at least 90 % homologous to corresponding to amino acids I58 - 229 of SEQ ID NO. 34I, which also corresponds to amino acids 16 - 87 of SEQ ID NO. 321, wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there 'is provided ai isolated polypeptide encoding for an edge portion of SEQ ID NO. 321, comprising a polypeptide having a length "n", wherein n is at least about I O amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least three amino acids comprise VSI having a structure as follows (numbering according to SEQ ID NO. 321): a sequence starting from any of amino acid numbers 14-x to I4; and ending at any of amino acid numbers 16 + ((rr2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 322, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 62 of SEQ ID
NO. 342, which also corresponds to amino acids 1 - 62 of SEQ ID NO. 322.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 322., comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more pxeferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 23 of SEQ ID NO. 322., and a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 39 of SEQ
ID NO. 343., which also corresponds to amino acids 24 - 62 of SEQ ID NO. 322., wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 322., comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MRGEIiNSTSYDSAVIYRGFWAVL of SEQ ID NO. 322..

According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding fox SEQ ID NO. 322., comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 97 -158 of SEQ ID
NO. 341., which also corresponds to amino acids 1 - 62 of SEQ ID NO. 322..
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 324, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 60 of SEQ ID
NO. 342, which also corresponds to amino acids 1 - 60 of SEQ ID NO. 324, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 61 - 70 of SEQ ID NO. 324, wherein said first amino acid sequence and second amino acid sequence axe contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 324, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at Ieast about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSVGQECGSG in SEQ ID NO. 324.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 324, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 23 of SEQ ID NO. 324, a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 37 of SEQ ID NO.
343, which also corresponds to amino acids 24 - 60 of SEQ TD NO. 324, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 61 - 70 of SEQ ID NO. 324, wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided m isolated polypeptide encoding for a head of SEQ ID NO. 324, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MRGEHNSTSYDSAVIYRGFWAVL of SEQ ID NO. 324.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 324, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSVGQECGSG in SEQ ID NO. 324.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 324, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 97 -156 of SEQ ID
NO. 341, which also corresponds to amino acids 1 - 60 of SEQ ID NO. 324, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 61 - 70 of SEQ ID NO. 324, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai isolated polypeptide encoding for a tail of SEQ ID NO. 324, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VSVGQECGSG in SEQ ID NO. 324.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 313, comprising a first amino acid sequence being at Least 90 % homologous to corresponding to amino acids 1 -115 of SEQ ID
NO. 344, which also corresponds to amino acids 1 - 115 of SEQ ID NO. 313, and a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 152 319 of SEQ ID NO. 344, which also corresponds to amino acids 116 - 283 of SEQ
ID NO. 3I3, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 313, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise IY, having a structure as follows: a sequence starting from any of amino acid numbers 115-x to 115; and ending at any of amino acid numbers 116+ ((rr2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 313, of cluster 236249 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 -70 of SEQ ID NO. 345, which also corresponds to amino acids 1 - 70 of SEQ ID
NO. 313, a bridging amino acid K corresponding to amino acid 71 of SEQ ID NO. 313, a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 72 - 115 of SEQ
ID NO. 345, which also corresponds to amino acids 72 - I 15 of SEQ ID NO. 313, and a third amino acid sequence being at least 90 % homologous to corresponding to amino acids 152 -319 of SEQ ID NO. 345, which also corresponds to amino acids 116 - 283 of SEQ
ID NO. 313, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 314, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids I -184 of SEQ ID
NO. 344, which also corresponds to amino acids 1 - 184 of SEQ ID NO. 314, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 185 - 197 of SEQ ID NO. 314, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 314, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VNIFLCLGMSQKK in SEQ ID NO. 314.
According to preferred embodiments of the present invention, there is provided a1 isolated chimeric polypeptide encoding for SEQ ff~ NO. 314, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 70 of SEQ ID
NO. 345, which also corresponds to amino acids 1 - 70 of SEQ ID NO. 314, a bridging amino acid K corresponding to amino acid 71 of SEQ ID NO. 314, a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 72 - 184 of SEQ
ID NO. 345, 5 which also corresponds to amino acids 72 - 184 of SEQ ID NO. 314, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 185 - 197 of SEQ ID NO. 3I4, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are 10 contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 314, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence 15 VNIFLCLGMSQKK in SEQ ID NO. 314.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 313, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more 20 preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise IY, having a structure as follows: a sequence starting from any of amino acid numbers 115-x to 115; and ending at any of amino acid numbers 116+ ((rr2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an 25 isolated chimeric polypeptide encoding for SEQ ID NO. 315, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 -ISI of SEQ ID
NO. 344, which also corresponds to amino acids 1 - 151 of SEQ ID NO. 315, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence 30 corresponding to amino acids 152 - I77 of SEQ ID NO. 315, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 31 S, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 8S%, more preferably at least about 90% and most preferably at least about 9S% homologous to the sequence S VRLMQSTAKSSSLILCFLCFTPVLLI in SEQ ID NO. 315.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 315, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 70 of SEQ ID NO.
345, which also corresponds to amino acids 1 - 70 of SEQ ID NO. 315, a bridging amino acid K
corresponding to amino acid 71 of SEQ ID NO. 31 S, a second amino acid sequence being at least 90 homologous to amino acids 72 - 151 of SEQ ID NO. 345, which also corresponds to amino acids 72 - 1 S 1 of SEQ ID NO. 31 S, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 S2 - 177 of SEQ ID NO. 31 S, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 315, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRLMQSTAKSSSLILCFLCFTPVLLI in SEQ ID NO. 31 S.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 316, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 1S1 of SEQ ID NO.
344, which also corresponds to amino acids 1 - 1 S 1 of SEQ ID NO. 3 I6, and a second amino acid sequence being at least 90 % homologous to amino acids 185 - 319 of SEQ ID NO. 344, which also corresponds to amino acids 1S2 - 286 of SEQ ID NO. 316, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 316, comprising a polypeptide having a length "n", wherein n is at least about I O amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EL, having a structure as follows: a sequence starting from any of amino acid numbers I51-x to 151; and ending at any of amino acid numbers I52+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 316, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 70 of SEQ ID NO.
345, which also corresponds to amino acids 1 - 70 of SEQ ID NO. 316, a bridging amino acid K
corresponding to amino acid 71 of SEQ ID NO. 316, a second amino acid sequence being at least 90 homologous to amino acids 72 - 15I of SEQ ID NO. 345, which also corresponds to amino acids 72 - 151 of SEQ ID NO. 316, and a third amino acid sequence being at least 90 homologous to amino acids 185 - 319 of SEQ ID NO. 345, which also corresponds to amino acids 152 - 286 of SEQ ID NO. 316, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ari isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 316, of cluster 236249 comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at Ieast two amino acids comprise EL, having a structure as follows: a sequence starting from any of amino acid numbers 151-x to 151; and ending at any of amino acid numbers 152+ ((n-2) - x), in which x varies from 0 to rr2.
According to preferred embodiments of the present invention, there is provided a1 isolated chimeric polypeptide encoding for SEQ ID NO. 309, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 42 of SEQ ID NO.
346, which also corresponds to amino acids 1 - 42 of SEQ ID NO. 309, a bridging amino acid N
corresponding to amino acid 43 of SEQ ID NO. 309, a second amino acid sequence being at least 90 homologous to amino acids 44 - 657 of SEQ ID NO. 346, which also corresponds to amino acids 44 - 657 of SEQ ID NO. 309, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 658 - 708 of SEQ ID NO. 309, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai isolated polypeptide encoding for a tail of SEQ ID NO. 309, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRPHLTLKAPLGLRMHRDPLRTPSPKSWPLTQPLTPDATLTPQAILTPTLT in SEQ ID
NO. 309.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 310, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 42 of SEQ ID NO.
346, which also corresponds to amino acids 1 - 42 of SEQ ID NO. 310, a bridging amino acid N
corresponding to amino acid 43 of SEQ ID NO. 310, a second amino acid sequence being at least 90 homologous to amino acids 44 - 676 of SEQ ID NO. 346, which also corresponds to amino acids 44 - 676 of SEQ ID NO. 310, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 677 - 685 of SEQ ID NO. 310, whexein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 310, comprising a polypeptide being at Ieast 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence EHGRGPGI~T
in SEQ ID NO. 310.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 3I l, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 42 of SEQ ID NO.
346, which also corresponds to amino acids 1 - 42 of SEQ ID NO. 311, a bridging amino acid N
corresponding to amino acid 43 of SEQ ID NO. 311, a second amino acid sequence being at least 90 homologous to amino acids 44 - 657 of SEQ ID NO. 346, which also corresponds to amino acids 44 - 657 of SEQ ID NO. 311, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 658 - 696 of SEQ ID NO. 311, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 311, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence GPGRHAGNAGTLTQSLDCDAGVPPPAFQPLSTSYIYFSE in SEQ ID NO. 311.
According to preferred embodiments of the present invention, there is provided an ".
isolated chimeric polypeptide encoding for SEQ ID NO. 312, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 42 of SEQ ID NO.
346, which also corresponds to amino acids 1 - 42 of SEQ ID NO. 312, a bridging amino acid N
corresponding to amino acid 43 of SEQ ID NO. 312, a second amino acid sequence being at least 90 homologous to amino acids 44 - 610 of SEQ ID NO. 346, which also corresponds to amino acids 44 - 610 of SEQ ID NO. 312, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence AMH
corresponding to amino acids 611 - 613 of SEQ ID NO. 312, wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 305, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 381 of SEQ ID NO.
347, which also corresponds to amino acids 1- 381 of SEQ ID NO. 305, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 382 - 387 of SEQ ID NO. 305, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
5 According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 305, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TSLSLS in SEQ
ID NO. 305.
10 According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 306, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 338 of SEQ ID NO.
347, which also corresponds to amino acids 1- 338 of SEQ ID NO. 306, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 15 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 339 - 346 of SEQ ID NO. 306, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 306, comprising a polypeptide being at 20 least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VLLCAQWP in SEQ ID NO. 306.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 307, comprising a first amino acid 25 sequence being at least 90 % homologous to amino acids 1 - 223 of SEQ ID
NO. 347, which also corresponds to amino acids 1- 223 of SEQ ID NO. 307, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence A
corresponding to amino acids 224 - 224 of SEQ ID NO. 307, wherein said first amino acid 30 sequence and second amino acid sequence are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ari isolated chimeric polypeptide encoding for SEQ ID NO. 308, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 294 of SEQ ID NO.
347, which also corresponds to amino acids 1 - 294 of SEQ ID NO. 308, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 295 - 304 of SEQ ID NO. 308, wherein said first amino acid sequence and second amino acid segue nce are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 308, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence RCYLRFLDIY
in SEQ ID NO. 308.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 281, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 8S%, more preferably at least 90% and most preferably at least 95% homologous to a amino acids 1 - 116 of FABH HUMAN, which also corresponds to amino acids 1 - 116 of SEQ ID NO. 281, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 117 - 215 of SEQ ID NO. 281, wherein said firstand second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 281, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRWATLELYLIGYYYCSFSQACSKKPSPPLRAVEAGTREWLWVRWSGGNFLCSGFGL
TQAGTQILPYRLHDCGQITFSKCNCKTGINNTNLVGLLGSL in SEQ ID NO. 281.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 281, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 116 of AAP35373, which also corresponds to amino acids 1 - 116 of SEQ ID NO. 281, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 117 - 215 of SEQ ID NO. 281, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 281, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRWATLELYLIGYYYCSFSQACSKKPSPPLRAVEAGTREWLWVRVVSGGNFLCSGFGL
TQAGTQILPYRLHDCGQITFSKCNCKTG1IVNTNLVGLLGSL in SEQ ID NO. 281.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 282, comprising a fixst amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 116 of FABH HUMAN, which also corresponds to amino acids 1 - 116 of SEQ ID NO. 282, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at Ieast 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 117 - 178 of SEQ ID NO. 282, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 282, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DVLTAWPSIYRRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG
KSIV in SEQ ID NO. 282.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 282, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 116 of AAP35373, which also corresponds to amino acids 1 - 116 of SEQ ID NO. 282, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 117 - 178 of SEQ ID NO. 282, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to prefen-ed embodiments of the present invention, there is provided a1 isolated polypeptide encoding for a tail of SEQ ID NO. 282, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence DVLTAWPSIYRRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG
KSIV in SEQ ID NO. 282.
According to preferred embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for SEQ ID NO. 283, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 8S%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence amino acids 1 - 116 of FABH'HUMAN, which also corresponds to amino acids 1 -116 of SEQ
ID NO. 283, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide sequence corresponding to amino acids 117 - 126 of SEQ ID NO.
283, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided a~
isolated polypeptide encoding for a tail of SEQ ID NO. 283, comprising a polypeptide being at Least 70%, optionally at Least about 80%, preferably at Least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MEI~LQLRNVK in SEQ ID NO. 283.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 283, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 116 of AAP35373, which also corresponds to amino acids SEQ ID NO. 283, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 8S%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 117 - 126 of SEQ ID NO. 283, wherein said first and second amino acid sequences are contiguous and in a sequential order.

According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 283, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at Least about 95% homologous to the sequence MEKLQLRNVK in SEQ ID NO. 283.
According to preferred embodiments of ale present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 284, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 24 of FABH HUMAN, which also corresponds to amino acids 1 - 24 of SEQ ID NO. 284, second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 25 - 35 of SEQ ID NO. 284, and a third amino acid sequence being at least 90 homologous to amino acids 25 - 133 of FABH HUMAN, which also corresponds to amino acids 36 - 144 of SEQ ID NO. 284, wherein said first, second, third and fourth amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for an edge portion of SEQ ID NO. 284, comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence encoding for AHILITFPLPS, corresponding to SEQ ID NO. 284.
According to preferred embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for SEQ ID NO. 284, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 24 of AAP35373, which also corresponds to amino acids 1 - 24 of SEQ ID NO. 284, second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 25 - 35 of SEQ ID NO. 284, and a third amino acid sequence being at least 90 homologous to amino acids 25 - 133 of AAP35373, which also corresponds to amino acids 36 -144 of SEQ ID NO. 284, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.

According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for an edge portion of SEQ ID NO. 284, comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the 5 sequence encoding for AHILITFPLPS, corresponding to SEQ ID NO. 284.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 285, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 203 of SEQ ID NO.
349, which also corresponds to amino acids 1 - 203 of SEQ ID NO. 285, and a second amino acid sequence 10 being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 204 - 240 of SEQ ID NO. 285, wherein said Erst and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai 15 isolated polypeptide encoding for a tail of SEQ ID NO. 285, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LWLTPVIPTLWEADGGGLHEPWSWRPAWATWLQRNYL in SEQ ID NO. 285.
According to preferred embodiments of the present invention, there is provided an 20 isolated chimeric polypeptide encoding for SEQ ID NO. 286, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 78 of SEQ ID NO.
349, which also corresponds to amino acids 1 - 78 of SEQ TD NO. 286, second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino 25 acids 79 - 125 of SEQ ID NO. 286, and a third amino acid sequence being at least 90 homologous to amino acids 79 - 399 of SEQ ID NO. 349, which also corresponds to amino acids 126 - 446 of SEQ ID NO. 286, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an 30 isolated polypeptide encoding for an edge portion of SEQ ID NO. 286, comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence encoding for HWQISQWWLHFQTPREEGKMKLLELSESADGAAWKRWGGNSNTHRIQ, corresponding to SEQ ID NO. 286.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 287, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 140 of SEQ ID NO.
349, which also corresponds to amino acids 1 - 140 of SEQ ID NO. 287, and a second amino acid sequence being at least 90 % homologous to amino acids 203 - 399 of SEQ ID NO. 349, which also corresponds to amino acids 141 - 337 of SEQ ID NO. 287, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 287, comprising a polypeptide having a length "n", wherein "n" is at least about 10 amino acids in length, optionally at Ieast about 20 amino acids in length, preferably at Ieast about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise DV, having a structure as follows: a sequence starting from any of amino acid numbers 140-x to140; and ending at any of amino acid numbers 141+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 288, comprising a first amino acid sequence being at Ieast 70%, optionally at Ieast 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 10 of SEQ ID NO. 288, second amino acid sequence being at least 90 % homologous to amino acids 18 - 106 of SEQ ID
NO. 349, which also corresponds to amino acids 11 - 99 of SEQ ID NO. 288, a third (bridging) amino acid sequence comprising D, and a fourth amino acid sequence being at least 90 % homologous to amino acids 179 - 399 of SEQ ID NO. 349, which also corresponds to amino acids 101 - 321 of SEQ ID NO. 288, wherein said first, second, third and fourth amino acid sequences axe contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 288, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NETEAEQSYV
of SEQ ID NO. 288.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for an edge portion of SEQ ID NO. 288, comprising a polypeptide having a length "n", wherein "n" is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein ~ least two amino acids comprise LDY
having a structure as follows (numbering according to SEQ ID NO. 288): a sequence starting from any of amino acid numbers 99-x to 99; and ending at any of amino acid numbers I01 +
((rr2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided al isolated chimeric polypeptide encoding for SEQ ID NO. 289, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 15 of SEQ ID NO. 289, and a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 203 - 399 of SEQ ID NO. 349, which also corresponds to amino acids 16 - 212 of SEQ ID NO. 289, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 289, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MSSWLSAGSPSSLSV of SEQ ID NO. 289.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 290, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 13 of SEQ ID NO. 290, and a second amino acid sequence being at least 90 % homologous to amino acids 280 - 399 of SEQ ID NO. 349, which also corresponds to amino acids 14 - 133 of SEQ ID NO. 290, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 290, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MCRGYSTLLNPVS of SEQ ID NO. 290.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 291, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 246 of SEQ ID NO.
349, which also corresponds to amino acids 1 - 246 of SEQ ID NO. 291, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 2.47 - 252 of SEQ ID NO. 291, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai isolated polypeptide encoding for a tail of SEQ ID NO. 291, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SRNWTQ in SEQ ID NO. 291.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 292, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 10 of SEQ ID NO. 292, second amino acid sequence being at least 90 % homologous to amino acids 26 - 276 of Q96NF5, which also corresponds to amino acids 11 - 261 of SEQ ID NO. 292, followed by A, and a third amino acid sequence being at least 90 % homologous to amino acids 278 - 466 of Q96NF5, which also corresponds to amino acids 263 - 451 of SEQ ID NO. 292, wherein said first, second, A, and third amino acid sequences are contiguous and in a sequential order.

According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 292, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MEISLVKCSE
of SEQ ID NO. 292 According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 293, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 276 of Q96NF5, which also corresponds to amino acids 1 - 276 of SEQ ID NO. 293, followed by A, a second amino acid sequence being at least 90 % homologous to amino acids 278 - 372 of Q96NF5, which also corresponds to amino acids 278 - 372 of SEQ ID NO. 293, and a third amino acid sequence being at least 90 % homologous to amino acids 401 - 466 of Q96NF5, which also corresponds to amino acids 373 - 438 of SEQ ID NO. 293, wherein said first, A, second, and third amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided a~
isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 293, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EE, having a structure as follows: a sequence starting from any of amino acid numbers 372-x to 372; and ending at any of amino acid numbers 373+ ((n-2) - x), in which x varies from 0 to n-2.
According to preferred embodiments of the present invention, there is provided au isolated chimeric polypeptide encoding for SEQ ID NO. 294, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 276 of Q96NF5, which also corresponds to amino acids 1 - 276 of SEQ ID NO. 294, followed by A, a second amino acid sequence being at least 90 % homologous to amino acids 278 - 401 of Q96NF5, which also corresponds to amino acids 278 - 401 of SEQ ID NO. 294, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 402 - 407 of SEQ ID NO. 294, wherein said first, A, second and third amino acid sequences are contiguous and in a sequential order.
According to prefen-ed embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 294, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least 5 about 90% and most preferably at least about 95% homologous to the sequence PNRQDS in SEQ ID NO. 294.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 295, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 276 of Q96NF5, which also 10 corresponds to amino acids 1 - 276 of SEQ ID NO. 295, followed by A, a second amino acid sequence being at least 90 % homologous to amino acids 278 - 374 of Q96NF5, which also corresponds to amino acids 278 - 374 of SEQ ID NO. 295, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to 15 amino acids 375 - 390 of SEQ ID NO. 295, wherein skid first, A, second and third amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ari isolated polypeptide encoding for a tail of SEQ ID NO. 295, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least 20 about 90% and most preferably at least about 95% homologous to the sequence MSHELFSRFSLRLFGR in SEQ ID NO. 295.
According to preferred embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for SEQ ID NO. 296, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 261 of Q96NF5, which also 25 corresponds to amino acids 1 - 261 of SEQ ID NO. 296, a second amino acid sequence comprising A, and a third amino acid sequence being at least 90 % homologous to amino acids 263 - 451 of Q96NF5, which also corresponds to amino acids 263 - 451 of SEQ ID
NO. 296, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
30 According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 297, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 132 of Q9NPI5, which also corresponds to amino acids 1 - 132 of SEQ ID NO. 297, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 133 - 145 of SEQ ID NO. 297, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 297, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LPGRHEVPRGALP in SEQ ID NO. 297.
According to preferred embodiments of the present invention, there is provided al isolated chimeric polypeptide encoding for SEQ ID NO. 297, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 109 of Q9NZK3, which also corresponds to amino acids 1 - 109 of SEQ ID NO. 297, and a second amino acid sequence ;
being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 110 - 145 of SEQ ID NO. 297, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 297, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at Least about 90% and most preferably at least about 95% homologous to the sequence LVDLYSRRYFLTVPYEECKWRRSLPGRHEVPRGALP in SEQ ID NO. 297.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 298, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 107 of Q9NPI5, which also corresponds to amino acids 1 - 107 of SEQ ID NO. 298, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 108 - 121 of SEQ ID NO. 298, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 298, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NLPGRHEVPRGALP in SEQ ID NO. 298.
According to preferred embodiments of the present invention, there is provided aci isolated chimeric polypeptide encoding for SEQ ID NO. 298, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 107 of Q9NZK3, which also corresponds to amino acids 1 - 107 of SEQ ID NO. 298, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 108 - 121 of SEQ ID NO. 298, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 298, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NLPGRHEVPRGALP in SEQ ID NO. 298.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 299, comprising a first amino acid sequence being at least 90 % homologous to amino acids 51 - 151 of SEQ ID NO.
350, which also corresponds to amino acids 1 - 101 of SEQ ID NO. 299.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 300, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MSSFSTTT corresponding to amino acids 1 - 8 of SEQ ID NO. 300, and a second amino acid sequence being at least 90 % homologous to amino acids 42 - 151 of SEQ ID NO.
350, which also corresponds to amino acids 9 - 118 of SEQ ID NO. 300, wherein said first and second amino acid sequences are contiguous and in a sequential order.

According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a head of SEQ ID NO. 300, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MSSFSTTT of SEQ ID NO. 300.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 301, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 124 of TRIC HUMAN, which also corresponds to amino acids 1 - 124 of SEQ ID NO. 301, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 125- 137 of SEQ ID NO. 301, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided an isolated polypeptide encoding for a tail of SEQ ID NO. 301, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more.preferably at least about 90% and most preferably at least about 95% homologous to the sequence VGRMGSSGTFGVG in SEQ ID NO. 301.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 302, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 8 of TRIC HUMAN, which also corresponds to amino acids 1 - 8 of SEQ ID NO. 302, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 36 - 209 of TRIC HUMAN, which also corresponding to amino acids 9 - 182 of SEQ ID
NO. 302, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 302, comprising a polypeptide having a length "n", wherein "n" is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise AK, having a structure as follows: a sequence starting from any of amino acid numbers 8-x to 8; and ending at any of amino acid numbers 9+ ((n-2) - x), in which x varies from 0 to rr2.
According to preferred embodiments of the present invention, there is provided al isolated chimeric polypeptide encoding for SEQ ID NO. 303, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 36 of TRIC HUMAN, which also corresponds to amino acids 1 - 36 of SEQ ID NO. 303, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 37- 86 of SEQ ID NO. 303, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided ari isolated polypeptide encoding for a tail of SEQ ID NO. 303, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VGRGFLGAEYRRRRDPRPWEWGEEPGLRRGRGLRGGASGAEFCRGSCSDW in SEQ ID
NO. 303.
According to preferred embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO. 304, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 8 of TRIO HUMAN, which also corresponds to amino acids 1 - 8 of SEQ ID NO. 304, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 9- 13 of SEQ ID NO. 304, wherein said first and second amino acid sequences are contiguous and in a sequential order.
According to preferred embodiments of the present invention, there is provided as isolated polypeptide encoding for a tail of SEQ ID NO. 304, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRAAG in SEQ ID NO. 304.

According to preferred embodiments of the present invention, there is provided an antibody capable of specifically binding to an epitope of an amino acid sequence in any one of cluster 567314, N56180, T10377, 224874, HUMCDDANF, HUMTROPIA, HUMSMCK, H88495, 236249, FLJ26352, HSACMHCP. Preferably, the amino acid sequence corresponds to 5 any insertion, including a bridge, edge portion, tail, or head as described herein.
Preferably, the antibody is capable of differentiating between a splice variant having the epitope and a corresponding known protein.
According to preferred embodiments of the present invention, there is provided a kit for detecting heart disorders, comprising a kit detecting overexpression of a splice variant.
10 Optionally, the kit comprises a NAT based technology. Preferably, the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence in any one of cluster 567314, N56180, T10377, 224874, HUMCDDANF, HUMTROPIA, HUMSMCK, H88495, 236249, FLJ26352, HSACMHCP.
Optionally, the kit further comprises at least one oligonucleotide capable of selectively 15 hybridizing to a nucleic acid sequence in any one of cluster 567314, N56180, T10377, 224874, 1 HUMCDDANF, HUMTROPIA, HUMSMCK, H$8495, 236249, FLJ26352, HSACMHCP.
Optionally, kit comprises an antibody as described herein. Preferably, the kit further comprises at least one reagent for performing an ELISA or a Western blot.
According to preferred embodiments of the present invention, there is provided a method 20 for detecting heart disorders, comprising detecting overexpression of a splice variant of any of cluster 567314, N56180, T10377, 224874, HUMCDDANF, HUMTROPIA, HUMSMCK, H88495, 236249, FLJ26352, HSACMHCP. Optionally, detecting overexpression is performed with a NAT-based technology.
Also optionally, detecting overexpression is performed with an immunoassay.
25 Preferably, the immunoassay comprises an antibody as described herein.
According to preferred embodiments of the present invention, there is provided a biomarker capable of detecting heart disorders, comprising any of the above nucleic acid sequences or a fragment thereof, or amino acid sequences or a fragment thereof.
According to preferred embodiments of the present invention, there is provided a method 30 for screening for heart disorders, comprising detecting cardiac disease cells or tissue with a biomarker or an antibody.

According to preferred embodiments of the present invention, there is provided a method for diagnosing heart disorders, comprising detecting heart cells or tissue with a biomarker or an antibody.
According to preferred embodiments of the present invention, there is provided a method for monitoring disease progression, or treatment efficacy, or relapse of heart disorders, or any combination thereof, comprising detecting heart cells or tissue with a biomarker or an antibody or a method or assay as described herein.
According to preferred embodiments of the present invention, there is provided a method of selecting a therapy for heart disorders, comprising detecting heart disorder cells with a biomarker or an antibody or a method or assay as described herein and selecting a therapy according to the detection.
A heart disorder and/or cardiac disease and/or cardiac pathology optionally comprises at least one of Myocardial infarct, ungina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, assessing the size of infarct in Myocardial infarct.
According to preferred embodiments of the present invention, preferably any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
All nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.

1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988);
The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). All of these are hereby incorporated by reference as if fully set forth herein. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows a schematic summary of quantitative real-time PCR analysis.
Figure 2 is a histogram showing expression of ESTs in each category, as "parts per million".
Figures 3 & 4 are histograms showing expression of oligonucleotides in various tissues, prob 205738 s at & prob 214285 at.
Figure SA is a histogram showing specific expression of variant FABH HUMAN
Fatty acid-binding protein transcripts in heart tissue samples as opposed to other tissues.
Figure SB is a histogram showing specific expression of variant FABH HUMAN
protein transcripts.
Figure 6 is a histrogram showing expression of FABH HUMAN known protein transcripts.
Figure 7 is a histogram showing expression of the number of heart tissue-specific clones in librarieslsequences.
Figure 8 is a histogram showing the actual expression of oligonucleotides in various tissues, including heart tissue, prob 207317 s at.
Figure 9 is a histogram showing specific expression of the above-indicated Calsequestrin, cardiac muscle isoform transcripts in sequence N56180, heart tissue samples.
Figure 10 is a histogram showing specific expression of the above-indicated Calsequestrin, cardiac muscle isoform transcripts in heart tissue samples as opposed to other tissues.
Figure 11 is a histogram showing expression of concerning the number of heart tissue-specific clones in libraries/sequences.
Figure 12 is a histogram showing specific expression of Q96NF5 transcripts in sequence T10377 in heart tissue samples.

Figure 13 is a histogram showing specific expression of the Q96NF5 transcripts in sequence T10377 junc29-33 heart tissue samples.
Figure 14 is a histogram showing specific expression of the above-indicated transcripts T10377 seg2-3 in heart tissue samples.
Figure 15 is a histogram concerning the expression of the number of heart specific clones in libraries/sequences.
Figure 16 is a histogram concerning the actual expression of oligonucleotides in various tissues, prob 221051 s at, including heart.
Figure 17A is a histogram concerning the expressions of ESTs in number of heart tissue-specific clones in libraries/sequences;
Figure 17B is a histogram concerning the actual expression of oligonucleotides in various tissues, prob 209957 s-at, including heart tissue.
Figure 18 is a histogram showing expression of known protein transcript for HUMCDDANF T4.
Figure 19 is a histogram concerning expression of ESTs, the number of heart tissue-specific clones in libraries/sequences Figure 20 is a histogram concerning the actual expression of oligonucleotides in various tissues, prob 205742 at, including heart tissue.
Figure 21A is a histogram showing specific expression of the above-indicated TRIO HUMAN Troponin I, cardiac muscle HUMTROPIA transcripts in sequence HUMTROPIA segl0 in heart tissue.
Figure 21A _is a histogram showing specific expression of the TRIC_HUMAN
Troponin I, cardiac muscle HUMTROPIA transcripts in sequence HUMTROPIA seg22 in heart tissue.
Figure 22 is a histogram showing specific expression of the HUMTROPIA known protein sequence in heart tissue.
Figure 23 is a histogram showing ESTs concerning the number of heart tissue-specific clones in libraries/sequences Figure 24 is a histogram concerning the actual expression of oligonucleotides in various tissues, pob 205295 at, including heart tissue.
Figure 25 is a histogram showing ESTs concerning the number of heart tissue -specific clones in librarieslsequences Figure 26 is a histogram concerning the actual expression of oligonucleotides in various tissues, prob 207066 at, including heart tissue.
Figure 27 is a histogram showing ESTs concerning the number of heart specific clones in libraries/sequences.
Figure 28 is a histogram concerning the actual expression of oligonucleotides in various tissues, prob 206029 at, including heart tissue.
Figure 29 is a histogram concerning expression of ESTs in the number of heart tissue -specific clones in libraries/sequences.
Figure 30 is a histogram concerning the expression of ESTs in number of heart tissue-specific clones in libraries/sequences;
Figure 31 is a histogram concerning the actual expression of oligonucleotides in various tissues, prob 204737 s at, including heart tissue.
Figure 32 is a histogram concerning the actual expression of oligonucleotides in various tissues, prob 216265 x at, including heart tissue.
Figure 33 shows a diagram of a troponin I variant, HLTMTROPIA T7, with regard to introducing a mutation to block an additional ORF.
Figure 34 shows Troponin PCR product after second amplification reaction: Lane l: 1Kb MW marker (GibcoBRL Cat# 15615-016) and Lane 2: PCR product.
Figure 35 shows Troponin PCR product sequence.
Figure 36: plasmid map of His Troponin T7 pRSETA.
Figure 37 shows the complete sequence of the plasmid shown in Figure 36.
Figure 38 shows the protein sequence of Troponin variant HL1MTROPIA PEA 2 T7, with the HIS-tag marked.
Figure 39a shows Coomassie staining analysis of SDS-PAGE containing recombinant HisTroponin; lane 1: Molecular weight marker (ProSieve color, Cambrex, Cat #50550); lane 2:
HisTroponinT7 pRSETA T0; lane 3: pRSET A T3; lane 4: pRSET empty vector TO
(negative control); lane 5: pRSET empty vector T3 (negative control).
Figure 39b shows a Western blot analysis of recombinant HisTroponin: lane 1:
His positive control protein; lane 2: HisTroponinT7 pRSETA T0; lane 3:
HisTroponinT7 pRSETA
T3; lane 4: pRSET empty vector TO (negative control); lane 5: pRSET empty vector T3 (negative control) and lane 6: molecular weight marker (ProSieve color, Cambrex, Cat #50550).

DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention is of novel markers for cardiac disease that are both sensitive and accurate. Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using 5 the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
These markers are specifically released to the bloodstream under conditions of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and/or are 10 otherwise expressed at a much higher level and/or specifically expressed in heart. The method of the present invention identifies clusters (genes) which are characterized in that the transcripts are differentially expressed in heart muscle tissue compared with other normal tissues, preferably in comparison to skeletal muscle tissue. In acute conditions under which heart muscle tissue experiences hypoxia (with or without necrosis), intracellular proteins that are not 15 normally secreted can leak through the cell membrane to the extracellular space. Therefore, heart muscle tissue differentially expressed proteins, as through analysis of EST expression, are potential acute heart damage markers.
Leakage of intracellular content can also occur in chronic damage to the heart muscle, therefore proteins selected according to this method are potential markers for chronic heart 20 conditions. When a protein that is differentially expressed in heart muscle is secreted, it is even more likely to be useful as a chronic heart damage marker, since secretion implies that the protein has a physiological role exterior to the cell, and therefore may be used by the heart muscle to respond to the chronic damage. This rationale is empirically supported by the non-limiting examples of the proteins BNP (brain natriuretic peptide) and ANF
(atrial natriuretic 25 factor), which are differentially expressed heart muscle proteins that are secreted and which were shown to be markers for congestive heart failure. In addition, BNP and ANF are not only differentially expressed in heart tissue, they are also overexpressed dramatically (hundreds of times greater expression) when heart failure occurs. Other heart specific secreted proteins might present similar overexpression in chronic damage.
30 Optionally and preferably, the markers described herein are overexpressed in heart as opposed to muscle, as described in greater detail below. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage.
The present invention therefore also relates to diagnostic assays for cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and methods of use of such markers for detection of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage (alone or in combination), optionally and preferably in a sample taleen from a subject (patient), which is more preferably some type of blood sample.
The present invention therefore also relates to diagnostic assays for cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and methods of use of such markers for detection of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage (alone or in combination), optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
In another embodiment, the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides. Such bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.
As used herein a "tail" refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention.
Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
As used herein a "head" refers to a peptide ~quence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.
As used herein "an edge portion" refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein. An edge may optionally arise due to a join between the above "known protein" portion of a variant and the tail, for example, andlor may occur if an internal portion of the wild type sequence is no longer present, such that tvvo portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein. A "bridge" may optionally be an edge portion as described above, but may also include a join between a head and a "known protein" portion of a variant, or a join between a tail and a "known protein"
portion of a variant, or a join between an insertion and a "known protein" portion of a variant.
Optionally and preferably, a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant, comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more 'preferably at least about 40 amino acids, in which at least one amino acid is from the taillhead/insertion and at least one amino acid is from the "known protein"
portion of a variant.
Also optionally, the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 11, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between).
It should be noted that a bridge cannot be extended beyond the length of the sequence in either direction, and it should be assumed that every bridge description is to be read in such manner that the bridge length does not extend beyond the sequence itself.
Furthermore, bridges are described with regard ~to a sliding window in certain contexts below. For example, certain descriptions of the bridges feature the following format: a bridge between two edges (in which a portion of the known protein is not present in the variant) may optionally be described as follows: a bridge portion of CONTIG-NAME P1 (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-NAME P1):
a sequence starting from any of amino acid numbers 49-x to 49 (for example); and ending at any of amino acid numbers 50 + ((rr2) - x) (for example), in which x varies from 0 to n-2.
In this example, it should also be read as including bridges in which n is any number of amino acids between 10-50 amino acids in length. Furthermore, the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49-x (for example) is not less than 1, nor 50 +
((rr2) - x) (for example) greater than the total sequence length.

In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention.
Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
In another embodiment, this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention. In another embodiment, this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
In another embodiment, this invention provid es a method for detecting a splice variant according to the present invention in a biological sample, comprising:
contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variants) in the Examples below), and detecting said interaction; wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample.
In another embodiment, this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
According to the present invention, the splice variants described herein are non-limiting examples of markers for diagnosing cardiac disease and/or cardiac pathology, including but not limited to cardiac damage. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage.
According to optional but preferred embodiments of the present invention, any marleer according to the present invention may optionally be used alone or combination. Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker. Furthermore, such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker. With regard to such a ratio between any marker described herein (or a combination thereof) and a known marker, more preferably the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
According to other preferred embodiments of the present invention, a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof, may be featured as a biomarker for detecting cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, such that a biomarker may optionally comprise any of the above. According to still other preferred embodiments, the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein. Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optionally also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or' bridges. The present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
The present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.

Non-limiting examples of methods or assays are described below.
The present invention also relates to kits based upon such diagnostic methods or assays.
Nucleic acid sequences and Oligonucleotides 5 Various embodiments of the present invention encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a 10 targeted fashion.
The present invention encompasses nucleic acid sequences described herein;
fragments thereof, sequences hybridizable therewith, sequences homologous thereto [e.g., at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % identical to the nucleic acid sequences set forth below], sequences 15 encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either natm-ally occurring or man induced, either randomly or in a targeted fashion. The present invention also encompasses homologous nucleic acid sequences (i. e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides 20 of the present invention.
In cases where the polynucleotide sequences of the present invention encode previously unidentified polypeptides, the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove.
25 A "nucleic acid fragment" or an "oligonucleotide" or a "polynucleotide" are used herein interchangeably to refer to a polymer of nucleic acids. A polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence andlor a composite polynucleotide sequences (e.g., a 30 combination of the above).
As used herein the phrase "complementary polynucleotide sequence" refers to a m sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified izz vivo or izz vitro using a DNA dependent DNA
polymerase.
As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is composed of genomic and cDNA sequences. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
Preferred embodiments of the present invention encompass oligonucleotide probes.
An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
Alternatively, an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, andlor the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989);
"Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed.
(1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New Yorlc (1988) and "Oligonucleotide Synthesis" Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC.
Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases.
Preferably, the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the bio markers of the present invention.
The oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
Preferably used oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.
Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or norrnatural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat. NOs: 4,469,863;
4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;
5,321,131;
5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466, 677; 5,476,925; 5,519,126;
5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.
Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms can also be used.
Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and allcyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones; fonnacetyl and thioformacetyl backbones;
methylene fonnacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CHI component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;
5,216,141; 5,235,033;
5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;
5,541,307;
5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.
Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic, includes peptide nucleic acid (PNA). United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374.
Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (LT).
Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2 aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thio allcyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly ~bromo, ~trifluoromethyl and other 5-substituted uracils and cytosines, 7 methylguanine and 7 methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and ~propynylcytosine.
5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., drhexadecyl-rac-glycerol or triethylammonium 1,2-di O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, as disclosed in IJ.S. Pat. No: 6,303,374.
It is not necessary for all positions in a given oligonucleotide molecule to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
It will be appreciated that oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.
To enable cellular expression of the polynucleotides of the present invention, a nucleic acid construct according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and fiuther includes at least one cis acting regulatory element. As used herein, the phrase "cis acting regulatory element"
refers to a polynucleotide sequence, preferably a promoter, which binds a traps acting regulator and regulates the transcription of a coding sequence located downstream thereto.
Any suitable promoter sequence can be used by the nucleic acid construct of the present invention.
Preferably, the promoter utilized by the nucleic acid construct of the present invention is 5 active in the specific cell population transformed. Examples of cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific, lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;
[Banerji et al.
(1983) Cell 33729-740], neurorrspecific promoters such as the neurofilament promoter [Byrne 10 et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
The nucleic acid cons tract of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the 15 transcription therefrom.
The nucleic acid construct of the present invention preferably further includes an appropriate selectable marker andlor an origin of replication. Preferably, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for 20 propagation in cells, or integration in a gene and a tissue of choice. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.l (+/-), pGL3, PzeoSV2 (+/ ), pDisplay, pEF/myc/cyto, pCMV/myclcyto each of which is 25 commercially available from Invitrogen Co. (www.invitrogen.com). Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif , includingRetro-X
vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
30 Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA
synthesis, and a 3' LTR
or a portion thereof. Other vectors can be used that are norrviral, such as cationic lipids, polylysine, and dendrimers.
Hybridization assays Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-limiting examples of probes according to the present invention were previously described).
Traditional hybridization assays include PCR, RT-PCR, Real-time PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA
detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al.
1999, Current Opin. Biotechnol. 10:71-75). Other detection methods include kits containing probes on a dipstick setup and the like.
Hybridization based assays which allow the detection of a variant of interest (i.e., DNA
or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.
Thus, the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.
Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 3zP
labeled probe, at 65 °C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65°C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCI, 1 % SDS and 5 x 106 cpm 3zP
labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.
More generally, hybridization of short nucleic acids (below 200 by in length, e.g. 17-40 by in length) can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency; (i) hybridization solution of 6 x SSC and 1 SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 %
SDS, 100 ~g/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the Tm, final wash solution of 3 M TMACI, 0.01 M
sodium phosphate (pH
6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm; (ii) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM
EDTA
(pH 7.6), 0.5 % SDS, 100 ~,g/ml denatured salinon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm, final wash solution of 6 x SSC, and final wash at 22 °C; (iii) hybridization solution of 6 x SSC
and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH
7.6), 0.5 SDS, 100 ~.glml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature.
The detection of hybrid duplexes can be carned out by a number of methods.
Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.
Probes can be labeled according to numerous well known methods. Non-limiting examples of radioactive labels include 3H, 14C, 32P, and 35S. Norrlimiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
For example, oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotic to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
Alternatively, when fluorescently-labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, CyS, Cy5.5, Cy7, FluorX
(Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, CalifJ can be attached to the oligonucleotides.
Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods.
As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples of radioactive labels include 3H, 14C' 3zP, and 355.
Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays.
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
NAT Assays Detection of a nucleic acid of interest in a biological sample may also optionally be effected by NAT-based assays, which involve nucleic acid amplification technology, such as PCR for example (or variations thereof such as reap time PCR for example).
As used herein, a "primer" defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
Amplification of a selected, or target, nucleic acid sequence may be carned out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol.
Lab. 8:14 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Norrliiniting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replicase system and NASBA
(I~woh et al., 1989, Proc. NatI. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTeclmology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260;
and Sambrook et al., 1989, supra).
The terminology "amplification pair" (or "primer pair") refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.
In one particular embodiment, amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially 5 expressed nucleic acid. In one preferred embodiment, RT PCR is earned out on an mRNA
sample from a patient under conditions which favor the amplification of the most abundant mRNA. In another preferred embodiment, the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of 10 differentially expressed nucleic acid sequences.
The nucleic acid (i.e. DNA or RNA) for practicing the present invention may be obtained according to well known methods.
Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes 15 employed. Optionally, the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al., 1989, Molecular Cloning -A
Laboratory Manual, 20 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
It will be appreciated that antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre-mRNA level.
Essentially the ability to quantitate transcription from a splice site of interest can be effected 25 based on splice site accessibility. Oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.
The polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non-limiting examples of these reactions are described in greater detail 30 below). The pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 °C, preferably less than 5 °C, more preferably less than 4 °C, most preferably less than 3 °C, ideally between 3 °C and 0 °C.
Polyrnef-ase Chaita Reaction (PCR): The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al., is a method of increasing the concentration of a segment of target sequence in a mixture of genomic DNA
without cloning or purification. This technology provides one approach to the problems of low target sequence concentration. PCR can be used to directly increase the concentration of the target to an easily detectable level. This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
The length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR
amplified."
Ligase Chain Reaction (LCR or LAR): The ligase chain reaction [LCR; sometimes referred to as "Ligase Amplification Reaction" (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand axe mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, and ligation amplify a short segment of DNA.
LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes: see for example Segev, PCT Publication No. W09001069 A1 (1990).
However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
Self-SZtstaifzed Synthetie Reactiof-a (3SRlNASBA): The self sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection. In this method, an oligonucleotide primer is used to add a phage RNA
polymerase promoter to the 5' end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA
polymerase and ribo-and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).
Q-Beta (Q(3) Replicase: In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q~i replicase. A
previously., identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step. However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C.). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
A successful diagnostic method must be very specific. A straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Q~3 systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e., >
55 degrees C). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR currently dominate the research field in detection technologies.
The basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle. The final yield of any such doubling system can be expressed as:
(1+X)n=y, where "X" is the mean efficiency (percent copied in each cycle), "n"
is the number of cycles, and "y" is the overall efficiency, or yield of the reaction. If every copy of a target DNA is utilized as a template in every cycle of a polymerase chain reaction, then the mean efficiency is 100 %. If 20 cycles of PCR are performed, then the yield will be 220, or 1,048,576 copies of the starting material. If the reaction conditions reduce the mean efficiency to 85 %, then the yield in those 20 cycles will be only 1.8520, or 220,513 copies of the starting material. In other words, a PCR rumiing at 85 % efficiency will yield only 21 % as much final product, compared to a reaction running at 100 % efficiency. A reaction that is reduced to 50 % mean efficiency will yield less than 1 % of the possible product.
In practice, routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield. At 50 % mean efficiency, it would take 34 cycles to achieve the millio~rfold amplification theoretically poss>ble in 20, and at lower efficiencies, the number of cycles required becomes prohibitive. In addition, any background products that amplify with a better mean efficiency than the intended target will become the dominant products.
Also, many variables can influence the mean efficiency of PCR, including target DNA
length and secondary structure, primer length and design, primer and dNTP
concentrations, and buffer composition, to name but a few. Contamination of the reaction with exogenous DNA
(e.g., DNA spilled onto lab surfaces) or cross-contamination is also a major consideration.
Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator.
The laboriousness of this process, including numerous technical considerations and other factors, presents a significant drawback to using PCR in the clinical setting. Indeed, PCR has yet to penetrate the clinical market in a significant way. The same concerns arise with LCR, as LCR
must also be optimized to use different oligonucleotide sequences for each target sequence. In addition, both methods require expensive equipment, capable of precise temperature cycling.
Many applications of nucleic acid detection technologies, such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences. One method of the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Taq polymerise to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer. An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence.
This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.
A similar 3'-mismatch strategy is used with greater effect to pre~nt ligation in the LCR.
Any mismatch effectively blocks the action of the thennostable ligase, but LCR
still has the drawback of target-independent background ligation products initiating the amplification.
Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory.
The direct detection method according to various preferred embodiments of the present invention may be, for example a cycling probe reaction (CPR) or a branched DNA
analysis.
When a sufficient amount of a nucleic acid to be detected is available, there are advantages to detecting that sequence directly, instead of making more copies of that target, (e.g., as in PCR and LCR). Most notably, a method that does not amplify the signal exponentially is more amenable to quantitative analysis. Even if the signal is enhanced by attaching multiple dyes to a single oligonucleotide, the correlation between the final signal intensity and amount of target is direct. Such a system has an additional advantage that the products of the reaction will not themselves promote further reaction, so contamination of lab surfaces by the products is not as much of a concern. Recently devised techniques have sought to eliminate the use of radioactivity and/or improve the sensitivity in automatable formats. Two examples are the "Cycling Probe Reaction" (CPR), and "Branched DNA" (bDNA).
Cycling probe reaction (CPR): The cycling probe reaction (CPR), uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H
causes the RNA portion to be digested. This destabilizes the remaining DNA
portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA
portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
Bf~anched DNA: Branched DNA (bDNA), involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from 5 noirspecific binding is similarly increased.
The detection of at least one sequence change according to various preferred embodiments of the present invention may be accomplished by, for example restriction fragment length polymorphism (RFLP analysis), allele specific oligonucleotide (ASO) analysis, Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single-Strand 10 Conformation Polymorphism (SSCP) analysis or Dideoxy fingerprinting (ddF).
The demand for tests which allow the detection of specific nucleic acid sequences and sequence changes is growing rapidly in clinical diagnostics. As nucleic acid sequence data for genes from humans and pathogenic organisms accumulates, the demand for fast, cost effective, and easy to-use tests for as yet mutations within specific sequences is rapidly increasing.
15 A handful of methods have been devised to scan nucleic acid segments for mutations.
One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate).
For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest. However, specialized equipment and highly trained personnel are 20 required, and the method is too labor-intense and expensive to be practical and effective in the clinical setting.
In view of the difficulties associated with sequencing, a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A
25 more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain terminating nucleotide analogs.
30 Restriction fragment length polymorphism (RFLP): For detection of single-base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).
Single point mutations have been also detected by the creation or destruction of RFLPs.
Mutations are detected and localized by the presence and size of the RNA
fragments generated by cleavage at the mismatches. Single nucleotide mismatches in DNA
heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC).
However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.
RFLP analysis suffers from low sensitivity and requires a large amount of sample. When RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA
manipulations.
Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites.
' A handful of rare-cutting restriction enzymes with 8 base-pair specificities have been isolated and these are widely used in genetic mapping, but these enzymes are few in number, are limited to the recognition of G+C-rich sequences, and cleave at sites that tend to be highly clustered. Recently, endonucleases encoded by group I introns have been discovered that might have greater than 12 base-pair specificity, but again, these are few in number.
Allele specific oligonucleotide (ASO): If the change is not in a recognition sequence, then allele-specific oligonucleotides (ASOs), can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis-match. Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific point mutations. The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles. The ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.
With either of the techniques described above (i.e., RFLP and ASO), the precise location of the suspected mutation must be known in advance of the test. That is to say, they are inapplicable when one needs to detect the presence of a mutation within a gene or sequence of interest.
Denatuf°inglTenaperature Gradierat Gel Electf~ophoresis (DGGElTGGE):
Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed "Denaturing Gradient Gel Electrophoresis" (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel. In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of mutations in the target sequences ' because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are "clamped" at one end by a long stretch of G-C base pairs (30-~0) to allow complete denaturation of the sequence of interest without complete dissociation of the strands. The attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature.
Modifications of the technique have been developed, using temperature gradients, and the method can be also applied to RNA:RNA duplexes.
Limitations on the utility of DGGE include the requirement that the denaturing conditions must be optimized for each type of DNA to be tested. Furthermore, the method requires specialized equipment to prepare the gels and maintain the needed high temperatures during electrophoresis. The expense associated with the synthesis of the clamping tail on one oligonucleotide for each sequence to be tested is also a major consideration.
In addition, long running times are required for DGGE. The long running time of DGGE was shortened in a modification of DGGE called constant denaturant gel electrophoresis (CDGE).
CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of mutations.
A technique analogous to DGGE, termed temperature gradient gel electrophoresis (TGGE), uses a thermal gradient rather than a chemical denaturant gradient.
TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA
therefore scanning of large gene segments requires the use of multiple PCR
products prior to running the gel.
Sifzgle-Strand Confo~°mation Polyr~zorphism (SSCP): Another corninon method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.
The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A
serious limitation of this metlbd is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
Dideoxy fifagerprifztirag (ddF): The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations. The ddF technique combines components of Sanger dideoxy sequencing with SSCP. A dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).

In addition to the above limitations, all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed. For the direct sequencing approach, sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub-cloning or primer walking, in order to cover the entire fragment.
SSCP and DGGE
have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments. Although SSCP
is reportedly able to detect 90 % of single-base substitutions within a 200 base-pair fragment, the detection drops to less than 50 % for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs. The ddF technique, as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA
that can be screened.
According to a presently preferred embodiment of the present invention the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self sustained synthetic reaction, Q~3-Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis and dideoxy fingerprinting.
Detection may also optionally be performed with a chip or other such device.
The nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group. This reporter group can be a fluorescent group such as phycoerythrin. The labeled nucleic acid is then incubated with the probes irrnnobilized on the chip using a fluidics station. describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates.
Once the reaction is completed, the chip is inserted into a scanner and patterns of hybridization are detected. The hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
It will be appreciated that when utilized along with automated equipment, the above described detection methods can be used to screen multiple samples for a disease and/or pathological condition both rapidly and easily.
Amino acid sequences and peptides 5 The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms 10 "polypeptide," "peptide" and "protein" include glycoproteins, as well as non-glycoproteins.
Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid - phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) 15 and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
20 Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH
Freeman and Co. N.Y.], after which their composition can be confirmed via amino acid sequencing.
In cases where large amounts of a polypeptide are desired, it can be generated using 25 recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al.
(1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.
3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell.
Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, 30 Section VIII, pp 421-463.
The present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein. The present invention also encompasses homologues of these polypeptides, such homologues can be at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 homologous to the amino acid sequences set forth below, as can be determined using BlastP
software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50. Optionally and preferably, nucleic acid sequence homology (identity) is determined using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters, which preferably include using the DUST filter program, and also preferably include having an E value of 10, filtering low complexity sequences and a word size of 11. Finally, the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
It will be appreciated that peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N
terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomilnetic compounds are well known in the art and are specified. Further details in this respect are provided hereinunder.
Peptide bonds ECO-NH-) within the peptide may be substituted, for example, by I~
methylated bonds ( N(CH3)-CO-), ester bonds ( C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH2-), oc-aza bonds ( NH-N(R)-CO-), wherein R is any allcyl, e.g., methyl, carba bonds CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefmic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fariy acids, complex carbohydrates etc).
As used herein in the specification and in the claims section below the term "amino acid"
or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
Furthermore, the term "amino acid" includes both D- and Iramino acids.
Table 1 norrconventional or modified amino acids which can be used with the present invention.
Table 1 Non-conventional Code Non-conventional Code amino amino acid acid a-aminobutyric Abu LrN-rnethylalanine Nmala acid a-amino-a-methylbutyrateMgabu LrN-methylarginine Nmarg aminocyclopropane-Cpro LrN-methylasparagineNmasn Carboxylate L-N-methylaspartic Nmasp acid aminoisobutyric Aib LrN-methylcysteine Nmcys acid aminonorbornyl- Norb LrN-methylglutamine Nmgin Carboxylate L-N-methylglutamic Nmglu acid Cyclohexylalanine Chexa LrN-methylhistidine Nmhis CyclopentylalanineCpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylinethionineNmmet D-cysteine Dcys IrN-methylnorleucineNmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu LrN-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanineNmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu LrN-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet IrN-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe IrN-methylvaline Nmval D-proline Dpro L-N-methylethylglycineNmetg D-serine Dser L-N-methyl t-butylglycineNmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp Irnorvaline Nva D-tyrosine Dtyr a-methyl-aminoisobutyrateMaib D-valine Dval a-methyl-y-aminobutyrateMgabu D-a-methylalanine Dmala a-methylcyclohexylalanineMchexa D-a-methylarginineDmarg a-methylcyclopentylalanineMcpen D-a-methylasparagineDmasn a-methyl a-napthylalanineManap D-a-methylaspartateDmasp a- methylpenicillamineMpen D-a-methylcysteineDmcys N-(4-aminobutyl)glycineNglu D-a-methylglutamineDmgln N-(2-aminoethyl)glycineNaeg D-a-methylhistidineDmhis N-(3-aminopropyl)glycineNorn D-a-methylisoleucineDmile N- amino-a-methylbutyrateNmaabu D-a-methylleucine Dmleu a-napthylalanine Anap D-a-methyllysine Dmlys N-benzylglycine Nphe D-a-methylmethionineDmmet N-(2-carbamylethyl)glycineNgln D-a-methylornithineDmorn N-(carbamylmethyl)glycineNasn D-a-methylphenylalanineDmphe N-(2-carboxyethyl)glycineNglu D-a-methylproline Dmpro N-(carboxymethyl)glycineNasp D-a-methylserine Dmser N-cyclobutylglycineNcbut D-a-methylthreonineDmthr N-cycloheptylglycineNchep D-a-methyltryptophanDmtrp N-cyclohexylglycineNchex D-a-methyltyrosine Dmty N-cyclodecylglycineNcdec D-a-methylvaline Dmval N-cyclododeclglycineNcdod D-a-methylalnine Dnmala N-cyclooctylglycineNcoct D-a-methylarginine Dmnarg N-cyclopropylglycineNcpro D-a-methylasparagineDnmasn N-cycloundecylglycineNcund D-a-methylasparatateDnmasp N-(2,2-diphenylethyl)glycineNbhm D-a-methylcysteine Dnmcys N-(3,3- Nbhe diphenylpropyl)glycine D-N-methylleucine Dnmleu N-(3-indolylyethyl)Nhtrp glycine D-N-methyllysine Dnmlys N-methyl Y-aminobutyrateNmgabu N_ Nmchexa D-N-methylinethionineDnmmet methylcyclohexylalanine D-N-methylornithineDnmorn N-methylcyclopentylalanineNmcpen N-methylglycine Nala D-N-methylphenylalanineDnmphe N-methylaminoisobutyrateNmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNleu D-N-methylthreonineDnmthr D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycineNva D-N-methyltyrosineDnmtyr N-methyla-napthylalanineNmanap D-N-methylvaline Dnmval N-methylpenicillamineNmpen 'y-aminobutyric Gabu N-(p-hydroxyphenyl)glycineNhtyr acid L-t-butylglycine Tbug N-(thiomethyl)glycineNcys L-ethylglycine Etg Penicillamine Pen L-homophenylalanineHphe L-a-methylalanine Mala Ira-methylarginineMarg L-a,-methylasparagineMasn L-a-methylaspartateMasp Lroc-methyl-t-butylglycineMtbug L-a-methylcysteineMcys L-methylethylglycineMetg L-a-methylglutamineMgln Ira-methylglutamate Mglu Ira-methylhistidineMhis Lra-methylhomo Mhphe phenylalanine L-a-methylisoleucineMile N-(2-methylthioethyl)glycineNmet ;

D-N-methylglutamineDnmgln N-(3- Narg guanidinopropyl)glycine D-N-methylglutamateDnmglu N-(1-hydroxyethyl)glycineNthr D-N-methylhistidineDnmhis N-(hydroxyethyl)glycineNser D-N-methylisoleucineDnmile N-(imidazolylethyl)glycineNhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycineNhtrp D-N-methyllysine Dnmlys N-methyl-'y-aminobutyrateNmgabu N- Nmchexa D-N-methylmethionineDnmmet methylcyclohexylalanine D-N-methylornithineDnmorn N-methylcyclopentylalanineNmcpen N-methylglycine Nala D-N-methylphenylalanineDnmphe N-methylaminoisobutyrateNmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycineNile D-N-methylserine Dnmser N-(2-methylpropyl)glycineNleu D-N-methylthreonine Dnmthr D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycineNval D-N-methyltyrosineDnmtyr N-methyla-napthylalanineNmanap D-N-methylvaline Dnmval N-methylpenicillamineNmpen y-aminobutyric acidGabu N-(p-hydroxyphenyl)glycineNhtyr L-t-butylglycine Tbug N-(thiomethyl)glycineNcys Il-ethylglycine Etg Penicillamine Pen LrhomophenylalanineHphe L-a-methylalanine Mala Lroc-methylarginineMarg L-cc-methylasparagineMasn L-a,-methylaspartateMasp L-a-methyl-t-butylglycineMtbug L-a-methylcysteine Mcys L-methylethylglycineMetg Ira-methylglutamineMgln Ira-methylglutamateMglu Ira-methylhistidineMhis Ira- Mhphe methylhomophenylalanine Ira,-methylisoleucineMile N-(2-methylthioethyl)glycineNmet Ira-methylleucine Mleu L-a-methyllysine Mlys Lr-a,-methylmethionineMmet Lra,-methylnorleucineMnle Ira-methylnorvalineMnva L-a-methylornithineMorn Iroc-methylphenylalanineMphe L-a-methylproline Mpro Lroc-methylserine mser Iroc-methylthreonineMthr Irot-methylvaline Mtrp L-a-methyltyrosine Mtyr L-a-methylleucine Mval LrN- Nmhphe Nnbhm methylhomophenylalanine N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycineNnbhm carbamylinethyl(1)glycineNnbhe 1-carboxy 1-(2,2-diphenylNmbc ethylamino)cyclopropane Table 1 Cont.
Since the peptides of the present invention are preferably utilized in diagnostics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.

The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
The peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing.
In cases where large amounts of the peptides of the present invention are desired, the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Talcamatsu et al.
(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach &
Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 and also as described above.
Antibodies "Antibody" refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad-immunoglobulin variable region genes.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. 'This includes, e.g., Fab' and F(ab)'2 fragments.
The term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA
methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CH1, CH2 and CH3, but does not include the heavy chain variable region.
The functional fragments of antibodies, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages, are described as follows: (1) Fab, the fragment which contains a monovalent antigerrbinding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New Yorlc, 1988, incorporated herein by reference).
Antibody fragments according to the present inversion can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a SS fragment denoted F(ab')2.
This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety.
See also Porter, R.
R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in mbar et al. [Proc. Nat'1 Acad. Sci. USA 69:2659-62 (19720].
Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL
chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL
domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2:
97-105 (1991); Bird etal., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
Another form of an antibody fragment is a peptide coding for a single complementarity-detennining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
Humanized forms of norrhuman (e.g., marine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a norrhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding norrhuman residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized 5 antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a noirhuman immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human 10 immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
Methods for humanizing norrhuman antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, 15 which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S.
20 Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR
residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, 25 including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human 30 immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425;
5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994);
Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14:
826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
Preferably, the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
Optionally, a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site.
An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination. One or more unique sequence portions may optionally combine with one or more other non-contiguous portions of the variant (including a portion which may have high homology to a portion of the known protein) to form an epitope.
Immunoassays In another embodiment of the present invention, an immunoassay can be used to qualitatively or quantitatively detect and analyze markers in a sample. This method comprises:
providing an antibody that specifically binds to a marker; contacting a sample with the antibody;
and detecting the presence of a complex of the antibody bound to the marker in the sample.

To prepare an antibody that specifically binds to a marker, purified protein markers can be used. Antibodies that specifically bind to a protein marlcer can be prepared using any suitable methods known in the art.
After the antibody is provided, a marker can be detected and/or quantified using any of a number of well recognized immunological binding assays. Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linlced immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay see, e.g., U.S. Pat. Nos.
4,366,241; 4,376,110; 4,517,288; and 4,837,168). Generally, a sample obtained from a subject can be contacted with the antibody that specifically binds the marker.
Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include but are not limited to glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead. Antibodies can also be attached to a solid support.
After incubating the sample with antibodies, the mixture is washed and the antibody marker complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent. Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, marker, volume of solution, concentrations and the like. Usually the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10 °C to 40 °C.
The immunoassay can be used to determine a test amount of a marker in a sample from a subject. First, a test amount of a marker in a sample can be detected using the immunoassay methods described above. If a marker is present in the sample, it will form an antibody marker complex with an antibody that specifically binds the marker under suitable incubation conditions described above. The amount of an antibody marker complex can optionally be determined by comparing to a standard. As noted above, the test amount of marker need not be measured in absolute units, as long as the unit of measurement can be compared to a control amount and/or signal.
Preferably used are antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isofonns thereof, for example. Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below. Preferred embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled "Antibodies".
Radio-immunoassay (RIA): In one version, this method involves precipitation of the des>Ied substrate and in the methods detailed hereinbelow, with a specific antibody and radiolabelled antibody binding protein (e.g., protein A labeled with ~25) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
In an alternate version of the RIA, a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
Enzyme linked irnrnufzosor-berat assay (ELISA): This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
Western blot: This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF).
Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabelled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
Inznutnohistochemical analysis: This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies. The substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required.
Fluorescence activated cell sorting (FRCS): This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam.
This method may employ two or more antibodies simultaneously.
Radio-imaging Methods These methods include but are not limited to, positron emission tomography (PET) single photon emission computed tomography (SPELT). Both of these techniques are norr invasive, and can be used to detect and/or measure a wide variety of tissue events and/or functions, such as detecting cancerous cells for example. Unlike PET, SPELT
can optionally be used with two labels simultaneously. SPELT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, US
Patent No.
6,696,686 describes the use of SPELT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
Display Libra ries According to still another aspect of the present invention there is provided a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12-17, 15-20, 15-30 or 20-50 consecutive amino acids derived from the polypeptide sequences of the present invention.
Methods of constructing such display libraries are well known in the art. Such methods are described in, for example, Young AC, et al., "The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neofonnans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes" J Mol Biol 1997 Dec 12;274(4):622-34; Giebel LB et al. "Screening of cyclic peptide phage libraries 5 identifies ligands that bind streptavidin with high affinities" Biochemistry 1995 Nov 28;34(47):15430-5; Davies EL et al., "Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes" J Immunol Methods 1995 Oct 12;186(1):125-35; Jones C RT al. "Current trends in molecular recognition and bioseparation" J
Chromatogr A 1995 Jul 14;707(1):3-22; Deng SJ et al. "Basis for selection of improved 10 carbohydrate-binding single-chain antibodies from synthetic gene libraries"
Proc Natl Acad Sci U S A 1995 May 23;92(11):4992-6; and Deng SJ et al. "Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display" J Biol Chem 1994 Apr 1;269(13):9533-8, which are incorporated herein by reference.
15 The following sections relate to Candidate Marker Examples (first section) and to Experimental Data for these Marker Examples (second section). It should be noted that Table numbering is restarted within each section.
CANDIDATE MARKER EXAMPLES SECTION
20 This section relates to examples of sequences according to the present invention, including illustrative methods of selection thereof.
Description of the methodology undertaken to uncover the biomolecular sequences of the present invention Human ESTs and cDNAs were obtained from GenBank versions 136 (June 15, 2003 25 ftp.ncbi.nih.gov/genbank/release.notes/gbl36.release.notes); NCBI genome assembly of April 2003; RefSeq sequences from June 2003; Genbank version 139 (December 2003);
Human Genome from NCBI (Build 34) (from Oct 2003); and RefSeq sequences from December 2003.
With regard to GenBank sequences, the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPRI) section were used; also the human 30 nucleotide RefSeq mRNA sequences were used (see for example www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html and for a reference to the EST section, see www.ncbi.nhn.nih.gov/dbEST/; a general reference to dbEST, the EST
database in GenBank, may be found in Boguski et al, Nat Genet. 1993 Aug;4(4):332-3; all of which are hereby incorporated by reference as if fully set forth herein).
Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R., Ast, G. & Graur, D. Alu-containing exons are alternatively spliced.
Genome Res 12, 1060-7 (2002); US patent No: 6,625,545; and U.S. Pat. Appl. No.
10/426,002, published as US20040101876 on May 27 2004; all of which are hereby incorporated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins. It then aligns the expressed sequences to the genome taking alternatively splicing into account and clusters overlapping expressed sequences into "clusters" that represent genes or partial genes.
These were annotated using the GeneCarta (Compugen, Tel-Aviv, Israel) platform. The GeneCarta platform includes a rich pool of annotations, sequence information (particularly of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.
A brief explanation is provided with regard to the method of selecting the candidates.
However, it should be noted that this explanation is provided for descriptive purposes only, and is not intended to be limiting in any way. The potential markers were identified by a computational process that was designed to find genes and/or their splice variants that are specifically expressed in cardiac tissue, as opposed to other types of tissues and also particularly as opposed to muscle tissue, by using databases of expressed sequences.
Various parameters related to the information in the EST libraries, determined according to classification by library annotation, were used to assist in locating genes and/or splice variants thereof that are specifically andlor differentially expressed in heart tissues. The detailed description of the selection method and of these parameters is presented in Example 1 below.

Identification of d~erentially expressed gene products -Algorithm In order to distinguish between differentially expressed gene products and constitutively expressed genes (i.e., house keeping genes), an algorithm based on an analysis of frequencies was configured. A specific algorithm for identification of transcripts specifically expressed in heart tissue is described hereinbelow.
EST analysis ESTs were taken from the following main sources: libraries contained in Genbank version 136 (June 15, 2003 ftp.ncbi.nih.gov/genbank/release.notes/gb136.release.notes) and Genbanlc version 139 (December 2003); and from the LifeSeq library of Incyte Corporation (ESTs only; Wilmington, DE, USA). With regard to GenBank sequences, the human EST
sequences from the EST (GBEST) section were used.
I O Library annotation - EST libraries were manually classified according to:
1. Tissue origin 2. Biological source - Examples of frequently used biological sources for construction of EST libraries include cancer cell-lines; normal tissues;
cancer tissues; foetal tissues; and others such as normal cell lines and pools of normal cell-lines, cancer cell-lines and combinations thereof. A specific description of abbreviations used below with regard to these tissues/cell lines etc is given above.
3. Protocol of library construction - various methods are known in the art for library construction including normalized library construction; norr normalized library construction; subtracted libraries; ORESTES and others (described in the annotation available in Genbank). It will be appreciated that at times the protocol of library construction is not indicated in the information available about that library.
The following rules were followed:
EST libraries originating from identical biological samples were considered as a single library.
EST libraries which included above-average levels of contamination, such as DNA
contamination for example, were eliminated. The presence of such contamination was determined as follows. For each library, the number of unspliced ESTs that are not fully contained within other spliced sequences was counted. If the percentage of such sequences (as compared to all other sequences) was at least 4 standard deviations above the average for all libraries being analyzed, this library was tagged as being contaminated and was eliminated from further consideration in the below analysis (see also Sorek, R. & Safer, H.M. A novel algorithm for computational identification of contaminated EST libraries. Nucleic Acids Res 31, 1067-74 (2003)for further details).
Clusters (genes) having at least five sequences including at least two sequences from the tissue of interest were analyzed. Splice variants were identified by using the LEADS software package as described above.

Identification of heart tissue specific genes For detection of heart tissue specific clusters, heart tissue libraries/sequences were compared to the total number of libraries/sequences in the cluster and in Genebank, and to the relevant numbers for muscle tissue libraries/sequences. Statistical tools were employed to identify clusters that were heart tissue specific, both as compared to all other tissues and also in comparison to muscle tissue.
The algorithm - for each tested tissue T and for each tested cluster the following were examined:
1. Each cluster includes at least 2 libraries from the tissue T. At least 3 clones (weighed - as described above) from tissue T in the cluster;
2. The following equation was then used to determine heart tissue-specific expression as compared to expression in all tissue types for a particular cluster: t/ ~ t T~Z in which n Tl N-T-M
is the total number of ESTs available for a cluster, while N is the total number of ESTs available in all of the libraries considered in the analysis (effectively all ESTs in Genbank, except for those that were rejected as belonging to contaminated libraries). This ratio was preferably set to be at least about 8, although optionally the ratio could be set to be at least about 5.
3. The following equation was then used to determine heart tissue-specific expression ~.
t expression in skeletal muscle tissue for a particular cluster: ~ m/M in which t represents the number of heart tissue-specific ESTs for the cluster, while T is the number of all heart tissue specific ESTs in the analysis; m is the number of skeletal muscle tissue-specific ESTs for the cluster, while M is the number of all skeletal muscle tissue-specific ESTs in the analysis. This ratio was preferably set to be at least about 4, although optionally the ratio could be set to be at least about 2.
4. Fisher exact test P-values were computed for weighted clone counts to check that the counts are statistically significant according to the following function:
F(t,T,n,N) which is the probability of a cluster actually being overexpressed in heart tissue, as compared to its overall level of expression. The P-value was preferably set to be less than about 1e-5, although optionally it could be set to be less than about 1 e-3.
The results obtained are explained in greater detail for each marker below.
Actual Marker Examples The following examples relate to specific actual marker examples. It should be noted that Table numbering is restarted within each example related to a particular Cluster, as indicated by the titles below.
EXAMPLES SECTION
This Section relates to Examples of sequences according to the present invention, including experiments involving these sequences, and illustrative, non-limiting examples of methods, assays and uses thereof. The materials and experimental procedures are exp lamed first, as all experiments used them as a basis for the work that was performed.
The marlcers of the present invention were tested with regard to their expression in various heart and non-heart tissue samples. Unless otherwise noted, all experimental data relates to variants of the present invention, named according to the segment being tested (as expression was tested through RT PCR as described). A description of the samples used in the panel is provided in Table 1 below. Tests were then performed as described in the Examples below.
Table 1: Tissue samples in testing panel Lot no. SourceTissue PathologySex/Age 1-ArrrColon 071P10B AmbionColon PM F/43 (C71) 2-B-Colon (C69)A411078 BiochainColon PM-Pool M&F
of 10 3-Cl-Colon (C70)1110101 ClontechColon PM-Pool M&F
of 3 4-AnrSmallIntestine091P0201AmbionSmall IntestinePM M/75 5-B-Small IntestineA501158 BiochainSmall IntestinePM M/63 6-B-Rectum A605138 BiochainRectum PM M/25 7-B-Rectum A610297 BiochainRectum PM M/24 8-B-Rectum A610298 BiochainRectum PM M/27 9-ArrrStomach 110P04A AmbionStomach PM M/16 10-B-Stomach A501159 BiochainStomach PM M/24 11-B-Esophagus A603814 BiochainEsophagus PM M/26 12-B-Esophagus A603813 BiochainEsophagus PM M/41 13-Am-Pancreas 071P25C AmbionPancreas PM M/25 14-CG-Pancreas CG-255-2IchilovPancreas PM M175 15-B-Lung A409363 BiochainLung PM F/26 16-Am-Lung (L93)111P0103AmbionLung PM F/61 17-B-Lung (L92)A503204 BiochainLung PM M/28 18-Am-Ovary 061P43A AmbionOvary PM F/16 (047) 19-B-Ovary (048)A504087 BiochainOvary PM F/51 20-B-Ovary (046)A504086 BiochainOvary PM F141 21-Am-Cervix 101P0101AmbionCervix PM F/40 22-B-Cervix A408211 BiochainCervix PM F/36 23-B-Cervix A504089 BiochainCervix PM-Pool M&F
of 5 24-B-Uterus A411074 BiochainUterus PM-Pool M&F
of 10 25-B-Uterus A409248 BiochainUterus PM F/43 26-B-Uterus A504090 BiochainUterus PM-Pool M&F
of 5 27-B-Bladder A501157 BiochainBladder PM M/29 28-Am-Bladder 071P02C AmbionBladder PM M/20 29-B-Bladder A504088 BiochainBladder PM-Pool M&F
of 5 30-Am-Placenta 021P33AAmbion Placenta PB F/33 31-B-Placenta A410165BiochainPlacenta PB F/26 32-B-Placenta A411073BiochainPlacenta PB-Pool M&F
of 5 33-B-Breast A607155BiochainBreast PM F/36 (B59) 34-Am-Breast 26486 Ambion Breast PM F/43 (B63) 35-Am-Breast 23036 Ambion Breast PM F/57 (B64) 36-C1 Prostate 1070317ClontechProstate PB-Pool M&F
(P53) of47 37-Am-Prostate 061P04AAmbion Prostate PM M/47 (P42) 38-Am-Prostate 25955 Ambion Prostate PM M/62 (P59) 39-Am-Testis 111P0104Ambion Testis PM M/25 40-B-Testis A411147BiochainTestis PM M/74 41-Cl Testis 1110320ClontechTestis PB-Pool M&F
of 45 42-CG-Adrenal CG-184-lOIchilovAdrenal PM F/81 43-B-Adrenal A610374BiochainAdrenal PM F/83 44-B-Heart A411077BiochainHeart PB-Pool M&F
of 5 45-CG-Heart CG-255-9IchilovHeart PM M/75 46-CG-Heart CG-227-1IchilovHeart PM F/36 47-AnrLiver 081P0101Ambion Liver PM M/64 48-CG-Liver CG-93-3IchilovLiver PM F/19 49-CG-Liver CG-124-4IchilovLiver PM F/34 50-Cl BM 1110932ClontechBone MarrowPM-Pool M&F
of 8 51-CGEN-Blood WBC#5 CGEN Blood M
52-CGEN-Blood WBC#4 CGEN Blood M
53-CGEN-Blood WBC#3 CGEN Blood M
54-CG-Spleen CG-267 IchilovSpleen PM F/25 55-CG-Spleen 111P0106BAmbion Spleen PM M/25 56-CG-Spleen A409246BiochainSpleen PM F/12 56-CG-Thymus CG-98-7IchilovThymus PM F/28 58-Am-Thymus 101P0101Ambion Thymus PM M/14 59-B-Thymus A409278 BiochainThymus PM M/28 60-B-Thyroid A610287 BiochainThyroid PM M/27 61-B-Thyroid A610286 BiochainThyroid PM M/24 62-CG-Thyroid CG-119-2IchilovThyroid PM F/66 63-C1 Salivary 1070319 ClontechSalivary PM-Pool M&F
Gland Gland of24 64-ArrrKidney 111 PO AmbionKidney PM-Pool M&F
101 of 14 B

65-Cl Kidney 1110970 ClontechKidney PM-Pool M&F
of 14 66-B-Kidney A411080 BiochainKidney PM-Pool M&F
of 5 67-CG-CerebellumCG-183-5IchilovCerebellumPM M/74 68-CG-CerebellumCG-212-5IchilovCerebellumPM M/54 69-B-Brain A411322 BiochainBrain PM M/28 70-Cl Brain 1120022 ClontechBrain PM-Pool M&F
of 2 71-B-Brain A411079 BiochainBrain PM-Pool M&F
of 2 72-CG-Brain CG-151-1IchilovBrain PM F/86 73-Am-Skeletal l O1P013AAmbionSkeletal PM F/28 Muscle Muscle 74-Cl Skeletal 1061038 ClontechSkeletal PM-Pool M&F
Muscle Muscle of 2 Materials and Experifraefztal Procedures RNA pf°epay~ation - RNA was obtained from Clontech (Franklin Lakes, NJ
USA 07417, www.clontech.com), BioChain Inst. Inc. (Hayward, CA 94545 USA
www.biochain.com), ABS
(Wilmington, DE 19801, USA, http://www.absbioreagents.com) or Ambion (Austin, USA, http://www.ambion.com). Alternatively, RNA was generated from tissue samples using TRI-Reagent (Molecular Research Center), according to Manufacturer's instructions. Tissue and RNA samples were obtained from patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion) and purified using RNeasy columns (Qiagen).
RT PCR - Purified RNA (1 ~.g) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 p.M dNTP in a total volume of 15.6 p.1. The mixture was incubated for 5 min at 65 °C and then quickly chilled on ice. Thereafter, 5 p,1 of SX
SuperscriptII first strand buffer (Invitrogen), 2.4p.1 O.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 min at 25 °C, followed by further incubation at 42 °C for 2 min.

Then, 1 p,1 (200units) of SuperscriptII (Invitrogen) was added and the reaction (final volume of 25,1) was incubated for 50 min at 42 °C and then inactivated at 70 °C for l5min. The resulting cDNA was diluted 1:20 in TE buffer (10 mM Tris pH=8, 1 mM EDTA pH=8).
Real-Tin2e RT-PCR anal~rsis- cDNA (5~.1), prepared as described above, was used as a template in Real-Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers and UNG Enzyme (Eurogentech or ABI or Roche). The amplification was effected as follows: 50 °C for 2 min, 95 °C for 10 min, and then 40 cycles of 95 °C for l5sec, followed by 60 °C for 1 min. Detection was performed by using the PE
Applied Biosystem SDS
7000. The cycle in which the reactions achieved a threshold level (Ct) of fluorescence was registered and was used to calculate the relative transcript quantity in the RT reactions. The relative quantity was calculated using the equation Q=efficiency~-~'. The efficiency of the PCR
reaction was calculated from a standard curve, created by using serial dilutions of several reverse transcription (RT) reactions To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to the geometric mean of the relative quantities of several housekeeping (HSI~P) genes. Schematic summary of quantitative real-time PCR
analysis is presented in Figure 1. As shown, the x-axis shows the cycle number. The CT =
Threshold Cycle point, which is the cycle that the amplification curve crosses the fluorescence threshold that was set in the experiment. This point is a calculated cycle number in which PCR
products signal is above the background level (passive dye ROX) and still in the Geometric/Exponential phase (as shown, once the level of fluorescence crosses the measurement threshold, it has a geometrically increasing phase, during which measurements are most accurate, followed by a linear phase and a plateau phase; for quantitative measurements, the latter two phases do not provide accurate measurements). The y-axis shows the normalized reporter fluorescence. It should be noted that this type of analysis provides relative quantification.
The sequences of the housekeeping genes measured in all the examples on normal tissue samples panel were as follows:
RPL19 (GenBank Accession No. NM 000981), RPLl9 Forward primer: TGGCAAGAAGAAGGTCTGGTTAG

RPL19 Reverse primer: TGATCAGCCCATCTTTGATGAG
RPL19 -amplicon:
TGGCAAGAAGAAGGTCTGGTTAGACCCCAATGAGACCAATGAAATCGCCAATGCCA
ACTCCCGTCAGCAGATCCGGAAGCTCATCAAAGATGGGCTGATCA
TATA box (GenBank Accession No. NM 003194), TATA box Forward primer : CGGTTTGCTGCGGTAATCAT
TATA box Reverse primer: TTTCTTGCTGCCAGTCTGGAC
TATA box - -amplicon:
CGGTTTGCTGCGGTAATCATGAGGATAAGAGAGCCACGAACCACGGCACTGATTTT
CAGTTCTGGGAAAATGGTGTGCACAGGAGCCAAGAGTGAAGAACAGTCCAGACTG
GCAGCAAGAAA
Ubiquitin (GenBank Accession No. BC000449) Ubiquitin Forward primer: ATTTGGGTCGCGGTTCTTG
Ubiquitin Reverse primer: TGCCTTGACATTCTCGATGGT
Ubiquitin -amplicon:
ATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGACAATGCAGAT
CTTCGTGAAGACTCTGACTGGTAAGACCATCACCCTCGAGG
TTGAGCCCAGTGACACCATCGAGAATGTCAAGGCA
SDHA (GenBank Accession No. NM 004168) SDHA Forward primer:
TGGGAACAAGAGGGCATCTG
SDHA Reverse primer: CCACCACTGCATCAAATTCATG
SDHA-amplicon TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATCCAGT
AGTGGATCATGAATTTGATGCAGTGGTGG

Cluster 567314 features 4 transcripts) and 8 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - Transcripts of interest Transcript SEQ ID NO ..
Name Table 2 - Segments of ifaterest S~egrrzent ~ ' SEQ TD NCJ
Naz~ie node0 65 PEA node PEA node PEA node PEA node PEA node PEA node PEA node Table 3 -1'f-otei~s o, f inter°est Pxatein ~ ~E~ ~ NO
Name PEA

PEA

PEA

PEA

These sequences are variants of the known protein Fatty acid-binding protein, heart (SwissProt accession identifier FABH HUMAN; known also according to the synonyms H-FABP; Muscle fatty acid-binding protein; M-FABP; Mammary derived growth inhibitor;
MDGI), referred to herein as the previously known protein.
Protein Fatty acid-binding protein, heart is known or believed to have the following function(s): FABP are thought to play a role in the intracellular transport of long-chain fatty acids and their acyl-CoA esters. The sequence for protein Fatty acid-binding protein, heart is given at the end of the application, as "Fatty acid-binding protein, heart amino acid sequence"
(SEQ ID N0:348). Known polymorphisms for this sequence are as shown in Table 4.
Table 4 - Amino acid mutati~fzs foY Kraowra Protein SNP _-: positions)Com~n~nt , o~ ~

aTr11t10 aCld Sf:qllenG~;

1 V->A

104 L -> K

124 C -> S

129 E -> Q

Protein Fatty acid-binding protein, heart localization is believed to be Cytoplasmic.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: negative control of cell proliferation, which are annotations) related to Biological Process; and lipid binding, which are annotations) related to Molecular Function.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http://www.expasy.ch/sprot/>; or Locuslink, available from <http://www.ncbi.nhn.nih.gov/projects/LocusLink/>.
'The heart selective diagnostic marker prediction engine provided the following results with regard to cluster 567314. Predictions were made for selective expression of transcripts of this cluster in heart tissue, according to the previously described methods.
The numbers on the y-axis of Figure 2 refer to weighted expression of ESTs in each category, as "parts per million"
(ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).

Overall, the following results were obtained as shown with regard to the histogram in Figure 2, concerning the number of heart-specific clones in libraries/sequences; as well as with regard to the histogram in Figures 3 - 4, concerning the actual expression of oligonucleotides in various tissues, including heart.
This cluster was found to be selectively expressed in heart for the following reasons: in a comparison of the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in norrheart ESTs, which was found to be 13.8; the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in muscle-specific ESTs which was found to be 2.6; and fisher exact test P-values were computed both for library and weighted clone counts to check that the counts are statistically significant, and were found to be 1.10E-25.
One particularly important measure of specificity of expression of a cluster in heart tissue is the previously described comparison of the ratio of expression of the cluster in heart as opposed to muscle. This cluster was found to be specifically expressed in heart as opposed to non-heart ESTs as described above. However, many proteins have been shown to be generally expressed at a higher level in both heart and muscle, which is less desirable.
For this cluster, as described above, the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in muscle-specific ESTs which was found to be 2.6, which clearly supports specific expression in heart tissue.
As noted above, cluster 567314 features 4 transcript(s), which were listed in Table 1 above. These transcripts) encode for proteins) which are variants) of protein Fatty acid-binding protein, heart. A description of each variant protein according to the present invention is now provided.
Variant protein S67314 PEA 1 P4 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) 567314 PEA 1 T4. An alignment is given to the lenown protein (Fatty acid-binding protein, heart) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:

Comparison report between 567314 PEA_1 P4 and FABH HUMAN:
l.An isolated chimeric polypeptide encoding for 567314 PEAT P4, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF
KNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL
corresponding to amino acids 1 - 116 of FABH HUMAN, which also corresponds to amino acids 1 - 116 of 567314 PEA 1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence VRWATLELYLIGYYYCSFSQACSKKPSPPLRAVEAGTREWLWVRVVSGGNFLCSGFGL
TQAGTQILPYRLHDCGQITFSKCNCKTGINNTNLVGLLGSL corresponding to amino acids 117 - 215 of 567314 PEA 1 P4, wherein said firstand second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of S67314 PEA 1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VRWATLELYLIGYYYCSFSQACSKKPSPPLRAVEAGTREWLWVRVVSGGNFLCSGFGL
TQAGTQILPYRLHDCGQITFSKCNCKTGINNTNLVGLLGSL in 567314 PEA_1 P4.
Comparison report between 567314 PEA 1 P4 and AAP35373:
l.An isolated chimeric polypeptide encoding for 567314 PEA 1 P4, comprising a first amino acid sequence being at least 90 % homologous to MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF
KNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL
corresponding to amino acids 1 - 116 of AAP35373, which also corresponds to amino acids 1 -116 of 567314 PEA 1 P4, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRWATLELYLIGYYYCSFSQACSKKPSPPLRAVEAGTREWLWVRVVSGGNFLCSGFGL
TQAGTQILPYRLHDCGQITFSKCNCKTGINNTNLVGLLGSL corresponding to amino acids 117 - 215 of 567314 PEA 1 P4, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 567314 PEA 1 P4, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VRWATLELYLIGYYYCSFSQACSKKPSPPLRAVEAGTREWLWVRVVSGGNFLCSGFGL
TQAGTQILPYRLHDCGQITFSKCNCKTGINNTNLVGLLGSL in 567314 PEA 1 P4.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein bcalization is believed to be intracellular because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein..
Variant protein 567314 PEA 1 P4 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 5, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 567314 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 5 -Amino acid mutations SNF positions) on Alternative amino ' Previously known aminb acid acids) SNP?

sequence 53 K -> R Yes zlo Variant protein S67314 PEA 1 P4 is encoded by the following transcript(s):
567314 PEA 1 T4, for which the sequences) is/are given at the end of the application. The coding portion of transcript 567314 PEA 1 T4 is shown in bold; this coding portion starts at position 925 and ends at position 1569. The transcript also has the following SNPs as listed in Table 6 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 567314 PEA-1 P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 6 - Nucleic acid SNPs SNP '.posiCiah: ari ~ Alternative nucleic'acid.' F Previously knawn nucleotide SNP

sequ~iica :~ . ' i:
j .. w 580 T _> C ~ Yes 1082 A -> G Yes 1670 A _> C Yes Variant protein S67314 PEA 1 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) 567314 PEA 1 T5. An alignment is given to the known protein (Fatty acid-binding protein, heart) at the end of the application. One or more alignmer~s to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 567314 PEA 1 PS and FABH HUMAN:
l.An isolated chimeric polypeptide encoding for 567314 PEA 1 P5, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF
KNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL

izz corresponding to amino acids 1 - 116 of FABH'HUMAN, which also corresponds to amino acids 1 - 116 of S67314 PEA-1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence DVLTAWPSIYRRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG
KSIV corresponding to amino acids 117 - 178 of S67314 PEA 1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 567314 PEA 1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DVLTAWPSIYRRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG
KSIV in 567314 PEA 1 P5.
Comparison report between 567314 PEA 1 PS and AAP35373:
l.An isolated chimeric polypeptide encoding for 567314 PEA 1 P5, comprising a first amino acid sequence being at least 90 % homologots to MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF
KNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL
corresponding to amino acids 1 - 116 of AAP35373, which also corresponds to amino acids 1 -116 of 567314 PEA 1 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence DVLTAWPSIYRRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG
KSIV corresponding to amino acids 117 - 178 of 567314 PEA 1 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 567314 PEA 1 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence DVLTAWPSIYRRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG
KSIV in 567314 PEA 1 P5.

m The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein..
Variant protein 567314 PEA 1 PS also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 567314 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 -Amino acid mutations SNP os~~~n s on aminr~Alte~atzve a~i~irio Previ6uslyWo~h SNP~-acid ac~d(s).. .
(), sequence ; . _ , .'y -'_, , _:, v ;; .. ' ; , 53 K -> R Yes Variant protein 567314 PEA 1 PS is encoded by the following transcript(s):
567314 PEA 1 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript 567314 PEA 1 TS is shown in bold; this coding portion starts at position 925 and ends at position 1458. The transcript also has the following SNPs as listed iii Table 8 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 567314 PEA 1 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 8 - Nucleic acid SNPs SNP position an nucleotide ~ Alternative nucleic acid I Previously l~iiown SNP?
sequence ~ p 580 T -> C Yes 1082 A -> G Yes 1326 A -> G Yes Variant protein 567314 PEA 1 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) S67314 PEA 1 T6. An alignment is given to the known protein (Fatty acid-binding protein, heart) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 567314 PEA 1 P6 and FABH HUMAN:
l.An isolated chimeric polypeptide encoding for S67314 PEA 1 P6, comprising a first a amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF
KNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL
corresponding to amino acids 1 - 116 of FABH HUMAN, which also corresponds to amino acids 1 - 116 of 567314 PEA_1 P6, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence MEKLQLRNVK
corresponding to amino acids 117 - 126 of 567314 PEA 1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 567314 PEA 1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MEKLQLRNVK in 567314 PEA_l P6.

Comparison report between S67314 PEA 1 P6 and AAP35373:
l.An isolated chimeric polypeptide encoding for S67314 PEA 1 P6, comprising a first amino acid sequence being at least 90 % homologous to MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF
KNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL
corresponding to amino acids 1 - 116 of AAP35373, which also corresponds to amino acids 1 -116 of 567314 PEA_1 P6, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MEKLQLRNVK corresponding to amino acids 117 - 126 of 567314 PEA 1 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of S67314 PEA 1 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MEKLQLRNVK in 567314 PEA 1 P6.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the traps-membrane region prediction programs predicted a traps-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein..
Variant protein 567314 PEA 1 P6 also has the following norrsilent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 567314 PEA_l P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 - Amino acid mutations SNP positions) on . Alternative amiha Previously known SNP'?
amino acid acids) sequence 53 K -> R Yes Variant protein 567314 PEA I P6 is encoded by the following ti-anscript(s):
567314 PEA 1 T6, for which the sequences) is/are given at the end of the application. The coding portion of transcript 567314 PEA_1 T6 is shown in bold; this coding portion starts at position 925 and ends at position 1302. The transcript also has the following SNPs as listed in Table 10 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 567314 PEA_l P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 10 - Nucleic aeid S'NPs 5~1'P ~qsitzou opt Alte~~ative nuclext<vxevio~sly,la~ow~.
nucleotide. aczd ,- . SNI'?
s~(~ite~LCe ' , ' ~ Y
, 580 T -> C Yes 1082 A -> G Yes 1444 T -> C Yes Variant protein 567314 PEA 1 P7 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) 567314 PEA 1 T7. An alignment is given to the known protein (Fatty acid-binding protein, heart) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 567314 PEA 1 P7 and FABH HUMAN:
l.An isolated chiineric polypeptide encoding for S67314 PEA 1 P7, comprising a first amino acid sequence being at least 90 % homologous to MVDAFLGTWKLVDSKNFDDYMKSL corresponding to amino acids 1 - 24 of FABH HUMAN, which also corresponds to amino acids 1 - 24 of S67314 PEA-1 P7, second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence AHILITFPLPS corresponding to amino acids 25 - 35 of S67314 PEA 1 P7, and a third amino acid sequence being at least 90 % homologous to GVGFATRQVASMTKPTTIIEKNGDILTLKTHSTFKNTEISFKLGVEFDETTADDRKVKSI
VTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTHGTAVCTRTYEKEA corresponding to amino acids 25 - 133 of FABH HUMAN, which also corresponds to amino acids 36 - 144 of S67314 PEA 1 P7, wherein said first, second, third and fourth amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for an edge portion of 567314 PEA-1 P7, comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence encoding for AHILITFPLPS, corresponding to S67314 PEA 1 P7.
Comparison report between 567314 PEA-1 P7 and AAP35373:
l.An isolated chimeric polypeptide encoding for 567314 PEA 1 P7, comprising a first amino acid sequence being at least 90 % homologous to MVDAFLGTWKLVDSKNFDDYMKSL corresponding to amino acids 1 - 24 of AAP35373, which also corresponds to amino acids 1 - 24 of 567314 PEA 1 P7, second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence AHILITFPLPS corresponding to amino acids 25 - 35 of 567314 PEA 1 P7, and a third amino acid sequence being at least 90 % homologous to GVGFATRQVASMTKPTTIIEKNGDILTLKTHSTFKNTEISFKLGVEFDETTADDRKVKSI
VTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTHGTAVCTRTYEKEA corresponding to amino acids 25 - 133 of AAP35373, which also corresponds to amino acids 36 -144 of 567314 PEA 1 P7, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.

2.An _isolated polypeptide encoding for an edge portion of S67314 PEA_1 P7, comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence encoding for AHILITFPLPS, corresponding to S67314 PEA 1 P7.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant gotein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein..
Variant protein 567314 PEA 1 P7 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 11, (given according b their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 567314 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table l l - Amino acid rnutatioyas SN'P positze~ri(s) Alt~rnativ~ amino Previously lcizown on mina acid acids) SNP's sequence 64 K -> R Yes Variant protein 567314 PEA 1 P7 is encoded by the following transcript(s):
567314 PEA 1 T7, for which the sequences) is/are given at the end of the application. The coding portion of transcript 567314 PEA 1 T7 is shown in bold; this coding portion starts at position 925 and ends at position 1356. The transcript also has the following SNPs as listed in Table 12 (given according to their position on the nucleotide sequence, with the alternative me nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S67314 PEA_l P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 12 - Nucleic acid SNPs SNP position on nucleotide=.Alteiar~ative nucleicPfeviously known SNP'?
sequence acid -580 ' T -> C Yes 1115 A -> G Yes 2772 G -> A Yes 2896 C -> A Yes 2918 G -> C Yes 3003 A -> G Yes 3074 T -> G Yes 1344 T -> C Yes 1522 -> T No 1540 -> A No 1540 -> T No 1578 - G -> A Yes 1652 G -> A Yes 2263 G -> A Yes 2605 T -> C Yes As noted abo~, cluster 567314 features 8 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application. These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster 567314 PEA 1 node 0 according to the present invention is supported by 90 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 567314 PEA_1 T4, 567314 PEA 1 T5, 567314 PEA_1 T6 and 567314 PEA_1 T7. Table 13 below describes the starting and ending position of this segment on each transcript.
Table 13 - Segment location on transcripts Transcript name. Segin~ent starting Seg~~.eht endu~,g posxt.~on position ' .

Segment cluster 567314 PEA 1 node_11 according to the present invention is supported by 6 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): S67314 PEA 1 T4. Table 14 below describes the starting and ending position of this segment on each transcript.
Table 14 - Segment location ofa tf-anscf°ipts Transcript name Segme~tt star~i~,g.laositio~Segrrient ending pos~tzon Segment cluster 567314 PEA 1 node 13 according to the present invention is supported by 76 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 567314 PEA 1 T7. Table 15 below describes the starting and ending position of this segment on each transcript.
Table I S - Segment location on tr°anscripts Transcript name ._ Segment starting ' Segment ending pasition position Zzo Segment cluster 567314 PEA-1 node_15 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 567314 PEA-1_T5. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segznerzt location on tnarzscripts Transcript name Segment starting pasitionSegirient ending position Segment cluster S67314 PEA 1 node 17 according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 567314 PEA_l T6. Table 17 below describes the starting and ending position of this segment on each transcript.
Table 17 - Segment location orz transcripts Traixscript ~raz~e Segment starling 'egment ez~di~g Position posltian ' . .
j ; _- _ . t, . ~_. ,.
" ' ~ .
_ , . .

Segment cluster S67314 PEA_1 node 4 according to the present invention is supported by 101 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 567314 PEA_1 T4, 567314 PEA_1 T5, 567314 PEA 1 T6 and 567314 PEA_1 T7. Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on tz-anscripts Transcript name Segment starting pos~tzo~; S~gmenC endzng position 567314 PEA 1 T7 1031 ~ 1203 According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster 567314 PEA 1 node_10 according to the present invention is supported by 64 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 567314 PEA_1 T4, 567314 PEA_l T5, S67314 PEA 1 T6 and 567314 PEA 1_T7. Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segment location on transcripts Transcript name Segrrie~t stating . Segment ending pc~sition,s posxt~c~n ::

Segment cluster 567314 PEA 1 node 3 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 567314 PEA_1 T7. Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21- Segment location on transcripts TraaascriPt sae ~~ Se~z~,ez~t startauposzt~on Segrmer~~ e~di~Position ~ -567314 PEA 1 T7 99~ 1030 Variant protein alignment to the previously known protein:
Sequence name: /tmp/EQOnMn6tqU/R73CUVICUk5:FABH HUMAN
Sequence documentation:

Alignment of: 567314_PEA_1_P4 x FABH HUMAN
Alignment segment 1/1:
Quality: 1095.00 Escore: 0 Matching length: 115 Total length: 115 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 1~ Gaps: 0 Alignment:

IIIIllllllllllllllllllllllllllllllllllllllllllllll Illlllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIII

Sequence name: /tmp/EQOnMn6tqU/R73CUVKUk5:AAP35373 Sequence documentation:
3o Alignment of: S67314 PEA-1 P4 x AAP35373 ..
Alignment segment 1/1:
Quality: 1107.00 Escore: 0 Matching length: 116 Total length: 116 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IO 51 TLKTHSTFKNTEISFKLGVEFDETTADDRKVKSIVTLDGGKhVHLQKWDG 100 IIIIIIIIIIIIIIII

IS
Sequence name: /tmp/ql9YPIBbdQ/SeofJfCmJW:FABH,HUMAN
Sequence documentation:
Alignment of: 567319 PEA'1 P5 x FABH HUMAN ..
Alignment segment 1/1:
Quality: 1095.00 Escore: 0 2$ Matching length: 115 Total length: 115 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 3O Alignment:

IIIIIIIIilllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIilllllllllllllllllllllllllllllllllllll IIIllllllllllll Sequence name: /tmp/ql4YPIBbdQ/SeofJfCmJW:AAP35373 Sequence documentation:
1~ Alignment of: 567314_PEA_1 P5 x AAP35373 ..
Alignment segment 1/1:
Quality: 1107.00 Escore: 0 Matching length: 116 Total length: 116 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllll -IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIII

Sequence name: /tmp/PXra2DxLlv/QBGTrzNMVX:FABH HUMAN
Sequence documentation:

Alignment of: S67314_PEA~1_P6 x FABH HUMAN ..
Alignment segment 1/1:
S Quality: 1095.00 Escore: 0 Matching length: 115 Total length: 115 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~IIIIIIIII~II~III

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~I

IIIIIIIIIIIIIII

2S Sequence name: /tmp/PXra2DxLlv/QBGTrzNMVX:AAP35373 Sequence documentation:
Alignment of: 567314-PEA_l P6 x AAP35373 ..
Alignment segment 1/1:
Quality: 1107.00 Escore: 0 Matching length: 116 Total length: 116 3S Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

llllllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIlllllllllllll 1S Sequence name: /tmp/xYzWyViDom/twDu3T69pd:FABH HUMAN
Sequence documentation:
Alignment of: 567314_PEA 1 P7 x FABH HUMAN ..
Alignment segment 1/1:
Quality: 1160.00 Escore: 0 Matching length: 132 Total length: 143 2S Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 92.31 Total Percent Identity: 92.31 Gaps: 1 Alignment:

lllllllllllllllllllllll IIIIllllllllllll 1 VDAFLGTWKLVDSKNFDDYMKSL...........GVGFATRQVASMTKPT 39 llllllllllllillllllllllllllllllllllllllllllllillll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

S Sequence name: /tmp/xYzWyViDom/twDu3T69pd:AAP35373 Sequence documentation:
Alignment of: 567314 PEA l P7 x AAP35373 ..
Alignment segment 1/1:
Quality: 1172.00 Escore: 0 Matching length: 133 Total length: 144 1S Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 92.36 Total Percent Identity: 92.36 Gaps: 1 Alignment:

IIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIII
1 MVDAFLGTWKLVDSKNFDDYMKSL...........GVGFATRQVASMTKP 39 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Expression of FABH HUMAN Fatty acid-binding protein transcripts which are detectable by amplicon as depicted in sequence name 567314 specifically in heart tissue.
3S Expression of FABH HUMAN Fatty acid-binding protein transcripts detectable by or according to segl l, 567314 amplicon(s) and 567314 segl 1F aryl 567314 segllR
primers was measured by real time PCR. In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM 000981; RPL19 amplicon), TATA box (GenBank Accession No.
NM 003194; TATA amplicon), Ubiquitin (GenBank Accession No. BC000449; amplicon -Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM 004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the houselceeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos. 44, 45, 46, Table l, "Tissue samples in testing panel", above), to obtain a value of fold up-regulation for each sample relative to median of the heart.
Figure SA is a histogram showing specific expression of the above-indicated FABH HUMAN Fatty acid-binding protein transcripts in heart tissue samples as opposed to other tissues.
As is evident from Figure SA, the expression of FABH HUMAN Fatty acid-binding protein transcripts detectable by the above amplicon(s) in heart tissue samples was significantly higher than in most other samples (non heart tissue sample Nos. 1-11,13-21,23-26,28-43, 47-74, Table 1 above, "Tissue samples in testing panel").
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non limiting illustrative example only of a suitable primer pair: 567314 segl 1F
forward primer; and 567314 segllR reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: 567314 segl 1.
567314 segl 1F (SEQ ID N0:61): TCCCCTGAGAGCTGTAGAAGCT
567314 segllR (SEQ ID N0:62): CGGCCTGTGTGAGTCCAAA
567314 segl 1(SEQ ID N0:63):
TCCCCTGAGAGCTGTAGAAGCTGGGACAAGAGAGTGGTTGTGGGTCAGGGTGGTAT
CAGGTGGGAATTTTCTGTGTAGTGGCTTTGGACTCACACAGGCCG
Expression of FABH HUMAN Fatty acid-binding protein 567314 transcripts, which are detectable by amplicon as depicted in sequence name 567314 segl5 specifically in heart tissue Expression of FABH HUMAN Fatty acid-binding protein transcripts detectable by or according to segl5 node(s), 567314 segl5 amplicon(s) and 567314 seglSF and S67314 seglSR
primers was measured by real time PCR. In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM 000981; RPL19 amplicon), TATA box (GenBank Accession No. NM 003194; TATA amplicon), Ubiquitin (GenBank Accession No.
BC000449;
amplicon -- Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM 004168;
amplicon -SDHA-amplicon), was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes.
The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos. 44-46, Table l, above "Tissue samples in testing panel"), to obtain a value of fold up-regulation for each sample relative to median of the heart.
Figure SB is a histogram showing specific expression of the above-indicated FABH HUMAN Fatty acid-binding protein transcripts in heart tissue samples as opposed to other tissues.
As is evident from Figure SB, the expression of FABH HUMAN Fatty acid-binding protein transcripts detectable by the above amp licon(s) in heart tissue samples was significantly higher than in most other samples (non-heart tissue sample Nos. 1-9, 11-21, 23-26, 28-43, 47-74 Table 1 above, "Tissue samples in testing panel").
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: 567314 seglSF
forward primer; and S67314 seglSR reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: 567314 segl5.
567314 seglSF (SEQ ID NO:64) Forward primer: TTCCTTGGCATCTCCAATGG
567314 seglSR (SEQ ID N0:274) Reverse primer: GCCAACTCTCAGCTCCTCCC
567314 segl5 (SEQ ID NO:275) Amplicon:
TTCCTTGGCATCTCCAATGGAGTAGAGAGAAGGCAACAAAGCTTCTCAGACCCACA
TTACCGAGCTATAACAACCATGGCTGGGAGGAGCTGAGAGTTGGC
Expression of FABH HUMAN Fatty acid-binding protein 567314 transcripts which are zso detectable by amplicon as depicted in sequence name S67314seg4 specifically in heart tissue Expression of FABH HUMAN Fatty acid-binding protein transcripts detectable by or according to seg4 node(s), 567314 seg4 amplicon(s) and primers ,567314seg4F
and 567314seg4R was measured by real time PCR (this transcript corresponds to the known or WT
protein). In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM 000981; RPL19 -amplicon), TATA box (GenBank Accession No. NM 003194;
TATA
amplicon), Ubiquitin (GenBank Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM_004168; amplicon - SDHA-amplicon), was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos.
44-46, Table 1, above), to obtain a value of relative expression for each sample relative to median of the heart samples.
Figure 6 is a histogram showing relative expression of the above-indicated FABH HUMAN Fatty acid-binding protein transcripts in heart tissue samples as opposed to other tissues.
As is evident from Figure 6, the expression of FABH HUMAN Fatty acid-binding protein transcripts detectable by the above amplicon(s) in heart tissue samples was significantly higher than in the other samples (Sample Nos. 44-46 Table 1, "Tissue samples in testing panel") Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: 567314seg4F
forward primer; and 567314seg4R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: S67314seg4.
Forward primer 567314seg4F (SEQ ID N0:276): CCAAGCCTACCACAATCATCG
Reverse primer 567314seg4R (SEQ ID N0:277): CTCCACCCCCAACTTAAAGCT
Amplicon 567314seg4 (SEQ ID N0:278):

CCAAGCCTACCACAATCATCGAAAAGAATGGGGACATTCTCACCCTAAAAACACAC
AGCACCTTCAAGAACACAGAGATCAGCTTTAAGTTGGGGGTGGAG

Cluster N56180 features 7 transcripts) and 22 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - Transcripts of interest Transcript Name -- ; Seq ID Na Table 2 - Segmeyzts of interest Seg~.~.ent Naxne ; Seq 'fD No.

N56180 node 2 73 N56180 node 20 74 N56180 node 22 75 N56180 node 28 76 N56180 node 34 77 N56180 node 36 78 N56180 node 4 79 N56180 node 6 80 N56180 node 0 81 N56180 node 10 82 »~
N56180node12 83 N56180node14 84 N56180node16 85 N56180node18 86 N56180node24 87 N56180node26 88 N56180node29 89 N56180node3 90 N56180node31 91 N56180node33 92 N56180node35 93 N56180node~8 ~ 94 Table 3 - Pr-oteifas of ifzterest Protein Nana~ : ~~q ZD No , .. . . ..

'These sequences are variants of the known protein Calsequestrin, cardiac muscle isoform precursor (SwissProt accession identifier CAQ2 HUMAN; known also according to the synonyms Calsequestrin 2), referred to herein as the previously known protein.
Protein Calsequestrin, cardiac muscle isoform precursor is known or believed to have the following function(s): Calsequestrin is a high-capacity, moderate affinity, calcium-binding protein and thus acts as an internal calcium store in muscle. The release of calcium bound to calsequestrin through a calcium release channel triggers muscle contraction.
The protein binds iss 40 to 50 moles of calcium. The sequence for protein Calsequestrin, cardiac muscle isoform precursor is given at the end of the application, as "Calsequestrin, cardiac muscle isoform precursor amino acid sequence" (SEQ ID N0:349). Known polymorphisms for this sequence are as shown in Table 4.
Table 4 - Amirao acid mutations for Known Ps°otein SNP pcsition(s)Comment an, amine acid sequence 307 D -> H~(in VTSIP). /FTId=VAR 016075.

67 Q -> P

Protein Calsequestrin, cardiac muscle isoform precursor localization is believed to be in the sarcoplasmic reticulum's terminal cisternae luminal spaces of cardiac and slow skeletal muscle cells.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: striated muscle contraction; heart development;
muscle development, which are annotations) related to Biological Process; calcium storage, which are annotations) related to Molecular Function; and smooth endoplasmic reticulum, which are annotations) related to Cellular Component.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http://www.expasy.ch/sprot/>; or Locuslink, available from <http:/lwww.ncbi.nhn.nih.gov/projectslLocusLink/>.
The heart selective diagnostic marker prediction engine provided the following results with regard to cluster N56180. Predictions were made for selective expression of transcripts of this cluster in heart tissue, according to the previously described methods.
The numbers on the y axis of Figure 7 refer to weighted expression of ESTs in each category, as "parts per million"
(ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histogram in Figure 7, concerning the number of heart-specific clones in libraries/sequences; as well as with regard to the histogram in Figure 8, concerning the actual expression of oligonucleotides in various tissues, including heart.
This cluster was found to be selectively expressed in heart for the following reasons: in a comparison of the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in non-heart ESTs was found to be 11; the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in muscle-specific ESTs was found to be 2.4; and fisher exact test P-values were computed both for library and weighted clone counts to check that the counts are statistically significant, and were found to be 4.70& 14.
One particularly important measure of specificity of expression of a cluster in heart tissue is the previously described comparison of the ratio of expression of the cluster in heart as opposed to muscle. This cluster was found to be specifically expressed in heart as opposed to norrheart ESTs as described above. However, many proteins have been shown to be generally expressed at a higher level in both heart and muscle, which is less desirable.
For this cluster, as described above, the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in muscle-specific ESTs was found to be 2.4, which clearly supports specific expression in heart tissue.
As noted above, cluster N56180 features 7 transcript(s), which were listed in Table 1 above. These transcripts) encode for proteins) which are variants) of protein Calsequestrin, cardiac muscle isoform precursor. A description of each variant protein according to the present invention is now provided.
Variant protein N56180 P2 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) N56180 T1. An alignment is given to the known protein (Calsequestrin, cardiac muscle isoform precursor) at the end of the application. One or more aligmnents to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between N56180 P2 and CAQ2 HUMAN:
l.An isolated chimeric polypeptide encoding for N56180 P2, comprising a first amino acid sequence being at least 90 % homologous to MKRTHLFIVGIYFLSSCRAEEGLNFPTYDGKDRVVSLSEKNFKQVLKKYDLLCLYYHEP
VSSDKVTQKQFQLKEIVLELVAQVLEHKAIGFVMVDAKKEAKLAKKLGFDEEGSLYIL
KGDRTIEFDGEFAADVLVEFLLDLIEDPVEIISSKLEVQAFERIEDYIKLIGFFKSEDSEYY
KAFEEAAEHFQPYIKFFATFDKGV corresponding to amino acids 1 - 203 of CAQ2 HUMAN, which also corresponds to amino acids 1 - 203 of N56180 P2, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LWLTPVIPTLWEADGGGLHEPWSWRPAWATWLQRNYL corresponding to amino acids 204 - 240 of N56180 P2, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An -isolated polypeptide encoding for a tail of N56180 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LWLTPVIPTLWEADGGGLHEPWSWRPAWATWLQRNYL in N56180 P2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted or localized in the sarcoplasmic reticulum's terminal cisternae luminal spaces of cardiac and slow skeletal muscle cells like the WT protein. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither traps-membrane region prediction program predicts that this protein has a traps-membrane region..
Variant protein N56180_P2 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 -Anaino acid mutations SNP positions) on=aminoAlternative amino Previously known SNP?
acid acids) sequence 66 T -> A Yes 76 V -> M Yes Variant protein N56180 P2 is encoded by the following transcript(s): N56180 T1, for which the sequences) islare given at the end of the application. The coding portion of transcript N56180 T1 is shown in bold; this coding portion starts at position 242 and ends at position 961.
The transcript also has the following SNPs as listed in Table 8 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table ~ - Nucleic acid SNPs Sue, :position 'on A~fier~native nucleic' Previously kxitawn rtuclei~tid'e aid SNP .' .
sequence _ .: ~ _ ~ ' ' r ,: _ . .-74 T -> No 105 T -> C Yes 2168 C -> G ' Yes 2289 G -> T No 2489 A -> C No 2545 A -> Yes 263 8 A -> T Yes 206 G -> A Yes 221 G -> A Yes 228 A -> C Yes 437 A -> G Yes 467 G -> A Yes 1021 A -> No 1521 C -> T Yes 2018 C -> T Yes Variant protein N56180 P4 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) N56180 T3. An aligmnent is given to the ltnown protein (Calsequestrin, cardiac muscle isoform precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between N56180 P4 and CAQ2 HUMAN:
l.An isolated chimeric polypeptide encoding for N56180 P4, comprising a first amino acid sequence being at least 90 % homologous to MKRTHLFIVGIYFLSSCRAEEGLNFPTYDGKDRVVSLSEKNFKQVLKKYDLLCLYYHEP
VSSDKVTQKQFQLKEIVLE corresponding to amino acids 1 - 78 of CAQ2 HUMAN, which also corresponds to amino acids 1 - 78 of N56180 P4, second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence HWQISQWWLHFQTPREEGKMKLLELSESADGAAWKRWGGNSNTHRIQ corresponding to amino acids 79 - 125 of N56180 P4, and a third amino acid sequence being at least 90 homologous to LVAQVLEHKAIGFVMVDAKKEAKLAKKLGFDEEGSLYILKGDRTIEFDGEFAADVLVE
FLLDLIEDPVEIISSKLEVQAFERIEDYIKLIGFFKSEDSEYYKAFEEAAEHFQPYIKFFATF
DKGVAKKLSLKMNEVDFYEPFMDEPIAIPNKPYTEEELVEFVKEHQRPTLRRLRPEEMF
ETWEDDLNGIHIVAFAEKSDPDGYEFLEILKQVARDNTDNPDLSILWIDPDDFPLLVAY
WEKTFKIDLFRPQIGWNVTDADSVWMEIPDDDDLPTAEELEDWIEDVLSGKINTEDDD
EDDDDDDNSDEEDNDDSDDDDDE corresponding to amino acids 79 - 399 of CAQ2 HUMAN, which also corresponds to amino acids 126 - 446 of N56180 P4, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for an edge portion of N56180 P4, comprising an z3a amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence encoding for HWQISQWWLHFQTPREEGKMKLLELSESADGAAWKRWGGNSNTHRIQ, corresponding to N56180 P4.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted or localized in the sarcoplasmic reticulum's terminal cisternae luminal spaces of cardiac and slow skeletal muscle cells like the WT protein. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein N56180 P4 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 -Amino acid mutations SNl' positions) ~i~z Altexnative amino F Previaiisly l~nown aaaxuso acid a.cid(s) SNP?, sequence 115 W -> R Yes 276 . N -> No 66 T -> A Yes 76 V -> M Yes Variant protein N56180 P4 is encoded by the following transcript(s): N56180 T3, for which the sequences) is/are given at the end of the application. The coding portion of transcript N56180 T3 is shown in bold; this coding portion starts at position 242 and ends at position 1579. The transcript also has the following SNPs as listed in Table 10 (given according to their position on the nuc leotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 10 - Nucleic acid SNPs SNP' positipn oxt Alterxtative nucleicPreviciusly known riucieotide acid SNP'?
sequence 74 T -> No 1 OS T -> C Yes 2064 C -> T Yes 2214 C -> G Yes 2335 G -> T No 2535 A -> C No 2591 A -> Yes 2684 A -> T Yes 206 G -> A Yes 221 G -> A Yes 228 A -> C Yes 437 A -> G Yes 467 G -> A Yes 584 T -> C Yes 1067 A -> No 1567 C -> T Yes Variant protein N56180 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) N56180 T4. An alignment is given to the known protein (Calsequestrin, cardiac muscle isoform precursor) at the z4o end of the application. One or more aligmnents to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between N56180 PS and CAQ2 HUMAN:
l.An isolated chimeric polypeptide encoding for N56180 P5, comprising a first amino acid sequence being at least 90 % homologous to MKRTHLFIVGIYFLSSCRAEEGLNFPTYDGKDRVVSLSEKNFKQVLKKYDLLCLYYHEP
VSSDKVTQKQFQLKEIVLELVAQVLEHKAIGFVMVDAKKEAKLAKKLGFDEEGSLYIL
KGDRTIEFDGEFAADVLVEFLLD corresponding to amino acids 1 - 140 of CAQ2 HUMAN, which also corresponds to amino acids 1 - 140 of N56180 P5, and a second amino acid sequence being at least 90 % homologous to VAKKLSLKMNEVDFYEPFMDEPIAIPNKPYTEEELVEFVKEHQRPTLRRLRPEEMFETW
EDDLNGIHIVAFAEKSDPDGYEFLEILKQVARDNTDNPDLSILWIDPDDFPLLVAYWEKT
FKIDLFRPQIGVVNVTDADSVWMEIPDDDDLPTAEELEDWIEDVLSGKINTEDDDEDDD
DDDNSDEEDNDDSDDDDDE corresponding to amino acids 203 - 399 of CAQ2~HUMAN, which also corresponds to amino acids 141 - 337 of N56180 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated chimeric polypeptide encoding for an edge portion of N56180 P5, comprising a polypeptide having a length "n", wherein "n" is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise DV, having a structure as follows: a sequence starting from any of amino acid numbers 140-x to 140; and ending at any of amino acid numbers 141+ ((n-2) - x), in which x varies from 0 to n-2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted or localized in the sarcoplasmic reticulum's terminal cisternae luminal spaces of cardiac and slow skeletal muscle cells like the WT protein. The protein localization is believed to be secreted because both signal peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.
Variant protein N56180 PS also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 11, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 1l - AmifZO acid niutatiofZs SNP,positzon(s'~ ;tin,Alternative amino Previously known SNP.' amino acid acids) .- _- ~.Y. ', .
sequence. ;. ' ~ ,,f 167 N -> No 66 T -> A Yes 76 V -> M Yes Variant protein N56180 PS 's encoded by the following transcript(s): N56180 T4, for which the sequences) is/are given at the end of the application. The coding portion of transcript N56180_T4 is shown in bold; this coding portion starts at position 242 and ends at position 1252. The transcript also has the following SNPs as listed in Table 12 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 12 - Nucleic acid SNPs SNP positzon on nucleotideAlternative nucleic Previously l~nt~wn acid SNP?

sequence 74 T -> No 105 T -> C Yes 1887 C -> G Yes 2008 G -> T No 2208 A -> C No 2264 A -> Yes 2357 A -> T Yes 206 G -> A Yes 221 G -> A Yes 228 A -> C Yes 437 A -> G Yes 467 G -> A Yes 740 A -> No 1240 C -> T Yes 1737 C -> T Yes Variant protein N56180 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) N56180 T5. An alignment is given to the known protein (Calsequestrin, cardiac muscle isoform precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between N56180 P6 and CAQ2 HUMAN:
l.An isolated chimeric polypeptide encoding for N56180 P6, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence NETEAEQSYV corresponding to amino acids 1 - 10 of N56180 P6, second amino acid sequence being at least 90 % homologous to RAEEGLNFPTYDGKDRWSLSEKNFKQVLKKYDLLCLYYHEPVSSDKVTQKQFQLKEI
VLELVAQVLEHKAIGFVMVDAKKEAKLAKKL corresponding to amino acids 18 - 106 of CAQ2 HUMAN, which also corresponds to amino acids 11 - 99 of N56180 P6, a third (bridging) amino acid sequence comprising D, and a fourth amino acid sequence being at least 90 % homologous to YKAFEEAAEHFQPYIKFFATFDKGVAKKLSLKMNEVDFYEPFMDEPIAIPNKPYTEEEL
VEFVKEHQRPTLRRLRPEEMFETWEDDLNGIHIVAFAEKSDPDGYEFLEILKQVARDNT
DNPDLSILWIDPDDFPLLVAYWEKTFKIDLFRPQIGVVNVTDADSVWMEIPDDDDLPTA
EELEDWIEDVLSGKINTEDDDEDDDDDDNSDEEDNDDSDDDDDE corresponding to amino acids 179 - 399 of CAQ2 HUMAN, which also corresponds to amino acids 101 - 321 of N56180 P6, wherein said first, second, third and fourth amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a head of N56180 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence NETEAEQSYV of N56180 P6.
3.An isolated polypeptide encoding for an edge portion of N56180 P6, comprising a polypeptide having a length "n", wherein "n" is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise LDY
having a structure as follows (numbering according to N56180 P6): a sequence starting from any of amino acid numbers 99-x to 99; and ending at any of amino acid numbers 101 +
((n-2) - x), in which x varies from 0 to n-2.
Variant protein N56180 P6 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 13, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 13 -Anai~o acid mutations SNP Positxon(~) on A~tez~ative amino Previously kr~o~wn az~a.zno acid acids) SNP'?

seqtaence 151 N -> No 59 T -> A Yes 69 V -> M Yes Variant protein N56180 P6 is encoded by the following transcript(s): N56180 T5, for which the sequences) is/are given at the end of the application. The coding portion of transcript N56180 TS is shown in bold; this coding portion starts at position 1 and ends at position 964.
The transcript also has the following SNPs as listed in Table 14 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 14 - Nucleic acid SNPs SNP, position , on- Alternative nucleic ~ ~'xeviously mown nucleotide acid SNP?~.
seq~ieuce ~; . ' ,, ~ ~ _ y = ; ,. , . , .

176 A -> G Yes 206 G -> A Yes 452 A -> No 952 C -> T Yes 1449 C -> T Yes 1599 C -> G Yes 1720 G -> T No 1920 A -> C No 1976 A -> Yes 2069 A -> T Yes Variant protein N56180 P7 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) N56180 T6. An alignment is given to the known protein (Calsequesti-in, cardiac muscle isoform precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between N56180 P7 and CAQ2 HUMAN:
l.An isolated chimeric polypeptide encoding for N56180 P7, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MSSWLSAGSPSSLSV corresponding to amino acids 1 - 15 of N56180 P7, and a second amino acid sequence being at least 90 % homologous to VAKKLSLKMNEVDFYEPFMDEPIAIPNKPYTEEELVEFVKEHQRPTLRRLRPEEMFETW
EDDLNGIHIVAFAEKSDPDGYEFLEILKQVARDNTDNPDLSILWIDPDDFPLLVAYWEKT
FI~IDLFRPQIGWNVTDADSVWMEIPDDDDLPTAEELEDWIEDVLSGKINTEDDDEDDD
DDDNSDEEDNDDSDDDDDE corresponding to amino acids 203 - 399 of CAQ2 HUMAN, which also corresponds to amino acids 16 - 212 of N56180 P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a head of N56180 P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MSSWLSAGSPSSLSV of N56180 P7.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signa~peptide prediction programs predict that this protein is a non-secreted protein..
Variant protein N56180 P7 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 15, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table I S - Amino acid mutations SNP positian(s)'vn Alternative amino gr~viously ki.~o'wri aznina' acztl acids) SNP? _.

sequence , . " ,. f ' . : ~.., '' . ._, 42 N -> No Variant protein N56180 P7 is encoded by the following transcript(s): N56180 T6, for which the sequences) is/are given at the end of the application. The coding portion of transcript N56180 T6 is shown in bold; this coding portion starts at position 71 and ends at position 706.
The transcript also has the following SNPs as listed in Table 16 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 16 - Nucleic acid SNPs SNP pcisitzon .on Altc~tiv~ nuo~oic:acidPxev7ausly known nucleotide NPR"
s~queizce 194 A -> No 694 C -> T Yes 1191 C -> T Yes 1341 C -> G Yes 1462 G -> T No 1662 A -> C No 1718 A -> Yes 1811 A -> T Yes Variant protein N56180 P8 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) N56180 T7. An alignment is given to the known protein (Calsequestrin, cardiac muscle isofonn precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between N56180 P8 and GAQ2 HUMAN:
l.An isolated chimeric polypeptide encoding for N56180 P8, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MCRGYSTLLNPVS corresponding to amino acids 1 - 13 of N56180 P8, and a second amino acid sequence being at least 90 % homologous to DGYEFLEILKQVARDNTDNPDLSILWIDPDDFPLLVAYWEKTFKIDLFRPQIGVVNVTD
ADSVWMEIPDDDDLPTAEELEDWIEDVLSGKINTEDDDEDDDDDDNSDEEDNDDSDD
DDDE corresponding to amino acids 280 - 399 of CAQ2 HUMAN, which also corresponds to amino acids 14 - 133 of N56180 P8, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a head of N56180 P8, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MCRGYSTLLNPVS of N56180 P8.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as fillows with regard to the cell:
intracellularly. The protein localization is believed to be intracellularly because neither of the traps-membrane region prediction programs predicted a traps-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein.
Variant protein N56180 P8 is encoded by the following transcript(s): N56180 T7, for which the sequences) islare given at the end of the application. The coding portion of transcript N56180 T7 is shown in bold; this coding portion starts at position 97 and ends at position 495.
The transcript also has the following SNPs as listed in Table 17 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P8 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 17 - Nucleic acid SNPs ~~ position ozi iiuc~eotzdeAlternative nuclexc ~xeviox~sly ImovGm sequence x .~ , acid. SNP2.
~ : . . : Q
.

483 C -> T Yes 980 C -> T Yes 1130 C -> G Yes 1251 G -> T No 1451 A -> C No .

1507 A -> Yes 1600 A -> T Yes Variant protein N56I80 P9 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) N56180 T8. An alignment is given to the known protein (Calsequestrin, cardiac muscle isoform precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between N56180 P9 and CAQ2 HUMAN:
l.An isolated chimeric polypeptide encoding for N56180 P9, comprising a first amino acid sequence being at least 90 % homologous to MKRTHLFIVGIYFLSSCRAEEGLNFPTYDGKDRWSLSEKNFKQVLKKYDLLCLYYHEP
VSSDKVTQKQFQLKEIVLELVAQVLEHKAIGFVMVDAKKEAKLAKKLGFDEEGSLYIL

KGDRTIEFDGEFAADVLVEFLLDLIEDPVEIISSKLEVQAFERIEDYIKLIGFFKSEDSEYY
KAFEEAAEHFQPYIKFFATFDKGVAKKLSLKMNEVDFYEPFMDEPIAIPNKPYTEEELVE
FVKEHQR corresponding to amino acids 1 - 246 of CAQ2 HUMAN, which also corresponds to amino acids 1 - 246 of N56180 P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence SRNWTQ
corresponding to amino acids 247 - 252 of N56180 P9, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of N56180 P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SRNWTQ in N56180 P9.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted or localized in the sarcoplasmic reticulum's terminal cisternae luminal spaces of cardiac and slow skeletal muscle cells like the WT protein. The protein localization is believed to be secreted because both signakpeptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region..
Variant protein N56180 P9 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 18, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 1 ~ - Anairzo acid mutations SNP positions) a~ Alternative anr~ino ~ Previously known am~n~ acid acids) SNP'?

l sequence 229 N -> No z5o 66 T -> A Yes 76 V -> M Yes Variant protein N56180 P9 is encoded by the following transcript(s): N56180 T8, for which the sequences) is/are given at the end of the application. The coding portion of transcript N56180 T8 is shown in bold; this coding portion starts at position 242 and ends at position 997.
The transcript also has the following SNPs as listed in Table 19 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180 P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 19 - Nucleic acid SNPs SNP . pc~sit~on : ,Alternative nucleicPreviously lenowy o~. nucleotide acid: SNT'? __._ sequenced . -. _ . ~ ~, "...

74 T -> No 105 T -> C Yes 1153 G -> A Yes 1170 G -> A Yes 206 G -> A Yes 221 G -> A Yes 228 A -> C Yes 437 A -> G Yes 467 G -> A Yes 926 A -> No 1095 A -> No 1095 A -> T No As noted above, cluster N56180 features 22 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster N56180 node'2 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4 and N56180 T8.
Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segf~zent locati~n osz transcripts Transcript name Segment starting positionsegment ending positioy Segment cluster N56180 node 20 according to the present invention is supported by 30 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4, N56180 T5, N56180 T6 and N56180 T8. Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21 - Segrr~ent location on trafZSCripts Transcript nape Segment starting ' Segin~nt ending position position 7.52 Segment cluster N56180 node 22 according to the present invention is supported by 3 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T8. Table 22 below describes the starting and ending position of this segment on each transcript.
Table 22 - Segment location on transcripts Transcript'naine~ Segment starting.positican ' Segment.endi~g position' Segment cluster N56180 node 28 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T7. Table 23 below describes the starting and ending position of this segment on each transcript.
Table 23 - Segment locati~n on transcripts Traa~sct~pt r~ae Segnaept starting ~-~egme~.t endiriig fiositiota-. position Segment cluster N56180 node 34 according to the present invention is supported by 37 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 Tl, N56180 T3, N56180 T4, N56180 T5, N56180 T6 and N56180 T7. Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - Segment location on transcripts Transcript name Segment stating I Segment ending position pasitian N56180 T3 1443 ~ 1690 Segment cluster N56180 node 36 according to the present invention is supported by 77 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4, N56180 T5, N56180 T6 and N56180 T7. Table 25 below describes the starting and ending position of this segment on each transcript.
Table 25 - Segment location on ti°ansci°ipts Transcript ~a~.e Segnr~ent staring Seg~~.ent e~du~,g positit~u pasitio~

N56180 Tl 1655 ~ 2778 N56180- T3- i70i. _ 2824 N56180 T7 ~ 617 -. ~ 1740 Segment cluster N56180 node 4 according to the present invention is supported by 34 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4, N56180 TS and N56180 T8. Table 26 below describes the starting and ending position of this segment on each transcript.
Table 26 - Segment location on tYanscripts Transcript dame Segment stating position. Segment ending position N56180 T8 ~ 295 ~ 475 Segment cluster N56180 node 6 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T3. Table 27 below describes the starting and ending position of this segment on each transcript.
Table 27 - Segment location on tr-ansc~ipts ~'ranscript name 'Seganent starting :~~egnrient exiling - po~it~t~n position N56180 T3 476 ~~ ~ 616 According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster N56180 node 0 according to the gesent invention is supported by 1 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T5. Table 28 below describes the starting and ending position of this segment on each transcript.
Table 28 - Segment location ora transcripts Transcript name ~~~gme~t starting ' Se~mex~t ending position position Segment cluster N56180 node_10 according to the present invention is supported by 24 libraries. The number of libraries was determined as previously described.
'This segment can be found in the following transcript(s): N56180 Tl, N56180 T3, N56180 T4 and N56180 T8.
Table 29 below describes the starting and ending position of this segment on each transcript.
Table 29 - Segment location ozz transcripts Transcript name Segment starting Segment ending position position,:

N56180 Tl 561 661 N56180 T8 561 ~ 661 Segment cluster N56180 node 12 according to the present invention is supported by 27 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 Tl, N56180 T3 and N56180 T8.
Table 30 below describes the starting and ending position of this segment on each transcript.
Table 30 - Segment locatiozz orz transcripts 'T'ranscript name Segment starting ~~ Se~ment:ending positici~ . positron ~

Segment cluster N56180 node 14 according to the present invention is supported by 26 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 Tl, N56180 T3, N56180 TS and N56180 T8.
Table 31 below describes the starting and ending position of this segment on each transcript.
Table 31 - Segznent location orz trazzsez-ipts Transcript name Seg~ent starting Segment ending position position N56180 T1 774 ~~847 l56 N56180'T8 ~ 774 ~ 847 Segment cluster N56180 node 16 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 Tl. Table 32 below describes the starting and ending position of this segment on each transcript.
Table 32 - Segment location on tf°anscripts Tra~is~ript,~ame ~"Segr~ient startingegme~ ending.pcisi~ian ' positiozi- :, .

Segment cluster N56180 node 18 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T6. Table 33 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segment location on transcripts 'fxan cript name Segm~r~t starting egm~rit evading pc~sitzon pasitaon . .

Segment cluster N56180 node 24 according to the present invention is supported by 25 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4, N56180 TS and N56180 T6. Table 34 below describes the starting and ending position of this segment on each transcript.
Table 34 - Segment location on transcf°ipts Transcript name Segment starting position Segment ending position Segment cluster N56180 node 26 according to the present invention is supported by 28 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4, N56180 T5 and N56180 T6. Table 35 below describes the starting and ending position of this segment on each transcript.
Table 35 - Segrrrent location osz tf°anscripts Transcript n~n~ro ~Seg~nt stating posltaon:' S~~aanezat.endingfptrsition ~ ~.~

N56180 Tl 1120 1174 Segment cluster N56180 node 29 according to the present invention is supported by 32 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 Tl, N56180 T3, N56180 T4, N56180 T5, N56180 T6 and N56180 T7. Table 36 below describes the starting and ending position of this segment on each transcript.
Table 36 - Segrnesrt location oh transcripts Transcript name Segment starting positionSegment ending position N56180 Tl 1175 1275 Segment cluster N56180 node 3 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 Tl, N56180 T3, N56180 T4 and N56180 T8.
Table 37 below describes the starting and ending position of this segment on each transcript.
Table 37 - Segment location on transcripts Transcript name Segment sfartmg positionSegment ending position .~

N56180 Tl 238 294 N56180 T3 238 294 __ _ Segment cluster N56180 node 31 according to the present invention is supported by 30 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4, N56180 T5, N56180 T6 and N56180 T7. Table 38 below describes the starting and ending position of this segment on each transcript.
Table 38 - Segrnent locati~n on transcripts Transcript name Seg~ne~.t start.g Segment ending position position N56180 TS 707 ~ 781 N56180 T7 ~ 238 ( 312 Segment cluster N56180 node'33 according to the present invention is supported by 30 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180 Tl, N56180 T3, N56180 T4, N56180 T5, N56180 T6 and N56180 T7. Table 39 below describes the starting and ending position of this segment on each transcript.
Table 39 - Segment location on tf-anscf°ipts ~'~'aTISCrip~ TlBrrle,.S,eglllent Starting' SegITlent enC1111g ~lloSJ.~1012 pClSltlOn , .

N56180 T4 1070 - i 115 N56180 T6 - 524-. 569_ Segment cluster N56180 node 35 according to the present invention can be found in the following transcript(s): N56180 Tl, N56180 T3, N56180 T4, N56180 T5, N56180 T6 and N56180 T7. Table 40 below describes the starting and ending position of this segment on each transcript.
Table 40 - Segment location ora tr~a~rscripts Tra~ascrzpt name Seg~e.~t starting Segment eroding position position iFn N56180 T7 ~ 607 ~ 616 Segment cluster N56180 node 8 according to the present invention is supported by 25 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): N56180_Tl, N56180 T3, N56180 T4, N56180 TS and N56180 T8. Table 41 below describes the starting and ending position of this segment on each transcript.
Table 41 - Segment locatiosZ on tr°ansca~ipts Trazzscript name Segmezit~starting-pasitio~y~~ment encliiig position N56180 T8 476 ~ 560 Variant protein alignment to the previously known protein:
Sequence name: /tmp/QH4bp760jk/sAp7DyaTKD:CAQ2 HUMAN
Sequence documentation:
Alignment of: N56180 P2 x CAQ2 HUMAN ..
Alignment segment 1/1:
Quality: 1955.00 Escore: 0 Matching length: 203 Total length: 203 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIII

MKRTHLFIVGIYFLSSCRAEEGLNFPTYDGKDRVVSLSEKNFKQVLKKYD

IO 5l LLCLYYHEPVSSDKVTQKQFQLKEIVLELVAQVLEHKAIGFVMVDAKKEA100 IIIIIIIIIIIIIIIIIIIII
IIIIIII
I
III

I
II
I
LLCLYYHEPVSSDKVTQKQFQLKEIVLELVAQVLEHKAIGFVMVDAKKEA

Illlllllllllllllllllllllllllllllllllllllllllllllll Sequence name: /tmp/VtcMdCiEuz/FlmsgLbcq4:CAQ2'HUMAN
Sequence documentation:
3O Alignment of: N56180-P4 x CAQ2 HUMAN ..
Alignment segment 1/1:
Quality: 3806.00 Escore: 0 Matching length: 399 Total length: 446 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 89.46 Total Percent Identity: 89.46 Gaps: 1 Alignment:

Illlllllllllllllllllllllllllllllllllllllllllllllll IIIlllllllllllllllllllllllll 1O 51 LLCLYYHEPVSSDKVTQKQFQLKEIVLE...................... 78 101 LELSESADGAAWKRWGGNSNTHRTQLVAQVLEHKAIGFVMVDAKKEAKLA l50 IIIIIIIIIIIIIIIIIIIIIIIII
79 .........................LVAQVLEHKAIGFVMVDAKKEAKLA 103 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

llllllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Sequence name: /tmp/lRixkfCRfD/JDL7BwYPJs:CAQ2_HUMAN
Sequence documentation:
Alignment of: N56180 P5 x CAQ2 HUMAN ..
Alignment segment 1/1:
to Quality: 3202.00 Escore: 0 Matching length: 337 Total length: 399 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 84.46 Total Percent Identity: 84.96 Gaps: 1 Alignment:

lllllllllllllll l 2O 1 l 50 lllllllllllllllllllllllllllllllll MKRTHLFIVGIYFLSSCRAEEGLNFPTYDGKDRVVSLSEKNFKQVLKKYD

IIIIIIIIIIIIIIIIIIIIIIIIIIII

LLCLYYHEPVSSDKVTQKQFQLKETVLELVAQVLEHKAIGFVMVDAKKEA

.

101 KLAKKLGFDEEGSLYILKGDRTIEFDGEFAADVLVEFLLD..........140 IIIIIII
III

IIIIIIIIIIIIIIIIIIIIIIIIIIIII
KLAKKLGFDEEGSLYILKGDRTIEFDGEFAADVLVEFLLDLIEDPVEIIS

140 ..................................................140 141 ..VAKKLSLKMNEVDFYEPFMDEPIAIPNKPYTEEELVEFVKEHQRPTLR 188 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

lllllllllllllllllllllllllllllllllllllllllllllillll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Sequence name: /tmp/rs5xPc26iA/XlzfpEDJF7:CAQ2~HUMAN
Sequence documentation:
IS
Alignment of: N56180_P6 x CAQ2 HUMAN ..
Alignment segment 1/1:
2O Quality: 2955.00 Escore: 0 Matching length: 3l4 Total length: 385 Matching Percent Similarity: 99.04 Matching Percent Identity: 99.04 Total Percent Similarity: 80.78 Total Percent Tdentity: 80.78 Gaps: 1 Alignment:

I IIIIIIIIIIIIIIIIIIIIIIillllllllllllllllllllllll 58 VTQKQFQLKETVLELVAQVLEHKAIGFVMVDAKKEAKLAKKLD....... 100 IIIIIIIIIIIIIIIIIIIIIIIIIIilllllllllllllll . . . . -100 .................................................. 100 101 ..............YKAFEEAAEHFQPYIKFFATFDKGVAKKLSLKMNEV 136 IIIIIIIIIIIIIIIIIIIIilllllllllllllll lllllllll l 215 11.I 264 llllllllllllllllllillllllllllllllllll DFYEPFMDEPTAIPNKPYTEEELVEFVKEHQRPTLRRLRPEEMFETWEDD

IIIIIIIIIIIIIIIIIIII
I

II
LNGIHTVAFAEKSDPDGYEFLEILKQVARDNTDNPDLSILWIDPDDFPLL

15 Illllllllllllllllillllllllllilllllllllllllllllllll Illllllllllllllllilllllllllllllllll Sequence name: /tmp/YOj6jtvAt2/UVZXGVRVOx:CAQ2 HUMAN
Sequence documentation:
Alignment of: N56180_P7 x CAQ2 HUMAN ..
Alignment segment l/1:
3~ Quality: 1959.00 Escore: 0 Matching length: 197 Total length: l97 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IS
Sequence name: /tmp/kmYMCJIGuB/no5BP02sjR:CAQ2 HUMAN
Sequence documentation:
ZO Alignment of: N56180_P8 x CAQ2 HUMAN ..
Alignment segment 1/1:
Quality: 1187.00Escore: 0 25 Matching length: 120 Total length: 120 Matching Percent Similarity:100.00 Matching Percent Identity:100.00 Total Percent Similarity: 100.00 Total Percent Identity:100.00 Gaps: 0 30 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIilllllillllllllllllllll 35 . . . . .

IIIIIIIIIIIIIIIIIIIIIillllllllllllllllllllllllllll llllllllllllllllllll Sequence name: /tmp/JIYFiyiYEk/c42Jok7Lfq:CAQ2~HUMAN
Sequence documentation:
l~ Alignment of: N56180'P9 x CAQ2 HUMAN ..
Alignment segment 1/1:
Quality: 2388.00 Escore: 0 Matching length: 246 Total length: 246 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiI

. . - . .

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllll IIIIIIIIIIIIIIIIIIIillllllllllllllllllllllllll Expression of Calsequestrin, cardiac muscle isofonn transcripts which are detectable by amplicon as depicted in sequence name N56180 specifically in heart tissue Expression of Calsequestrin, cardiac muscle isofonn transcripts detectable by or according to seg6, N56180 amplicon(s) and N56180 seg6F and N56180 seg6R primers was measured by real time PCR. In parallel the expression of four housekeeping genes- RPL19 (GenBank Accession No. NM 000981; RPL19 amplicon), TATA box (GenBank Accession No. NM_003194;
TATA
amplicon), Ubiquitin (GenBank Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA (GenBanlc Accession No. NM 004168; amplicon - SDHA-amplicon) was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos.
44, 45, 46, Table 1, above, "Tissue samples in testing panel"), to obtain a value of fold up-regulation for each sample relative to median of the heart.
Figure 9 is a histogram showing specific expression of the above-indicated Calsequestrin, cardiac muscle isoform transcripts in heart tissue samples as opposed to other tissues. As is evident from Figure 9, the expression of Calsequestrin, cardiac muscle isoform transcripts detectable by the above amplicon(s) in heart tissue samples was significantly higher than in most other samples (non-heart tissue sample Nos. 1-21,23-26,28,30-43 47-74 Table 1 above, "Tissue samples in testing panel").

Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: N56180 seg6F
forward primer; and N56180 seg6R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a norr limiting illustrative example only of a suitable amplicon: N56180 seg6.
N56180 seg6F (SEQ ID N0:279): ATATCCCAGTGGTGGTTGCATT
N56180 seg6R (SEQ ID NO:280): CCCTCCCCAGCGTTTCC
N56180 seg6 (SEQ ID N0:335):
ATATCCCAGTGGTGGTTGCATTTCCAAACCCCAAGAGAGGAAGGCAAAATGAAGTT
GCTGGAGTTGAGTGAATCTGCAGATGGAGCTGCGTGGAAACGCTGGGGAGGG
Expression of Calsequestrin, cardiac muscle isoform transcripts detectable by or according to seg node(s), N56180 amplicon(s) and N56180 seg F and N56180 seg R primers was measured by real time PCR. In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM 000981; RPL19 amplicon), TATA box (GenBank Accession No.
NM 003194; TATA amplicon), LTbiquitin (GenBank Accession No. BC000449;
amplicon -_Ubiquitiiramplicon) and SDHA (GenBank Accession No. NM 004168; amplicon -SDHA
amplicon), was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos. 44, 45, 46, Table 1, above, "Tissue samples in testing panel"), to obtain a value of fold up-regulation for each sample relative to median of the heart.
Figure 10 is a histogram showing specific expression of the above-indicated Calsequestrin, cardiac muscle isoform transcripts in heart tissue samples as opposed to other tissues.As is evident from Figure 10, the expression of Calsequestrin, cardiac muscle isoform transcripts detectable by the above amplicon(s) in heart tissue samples was significantly higher than in most of the other samples (non-heart tissue sample Nos. 1-21, 23-26, 28-43, 47-74 Table 1, "Tissue samples in testing panel").

Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: N56180 seg F
forward primer; and N56180 seg R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: N56180 seg N56180 seg F (SEQ ID NO:336): TTGATACCACTTAGTGTAGCTCCAGC
N56180 seg R (SEQ ID N0:337): TCAAGTAGTTGCTACAGACGCCA
N56180 seg (SEQ ID N0:361):
TTGATACCACTTAGTGTAGCTCCAGCATGGATCAGCAAACTTTTTCTGTAAAGAACA
AAATGGTAAATATTTCAGGTTCTGTGGGCCAGATGGCGTCTGTAGCAACTACTTGA

Cluster T10377 features 6 transcripts) and 18 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - Trarascripts of interest T~ar~saript Name ~ee~ ID No.

Table 2 - Segments of irrterest Segment Name ~ Seq ID No.

T10377node0 95 T10377node17 96 T10377node19 97 T10377node21 98 T10377node27 99 T10377node33 100 T10377node12 101 T10377node14 102 T10377node16 103 T10377node2 104 T10377node23 105 T10377node25 106 T10377node29 107 T10377node3 108 T10377node31 109 T10377node5 110 T10377node8 111 T10377node9 112 Table 3 - Pf-oteiras of interest Protein Name Seq fD ~o.

T10377 P8 ~ 296 The heart selective diagnostic marker prediction engine provided the following results with regard to cluster T10377. Predictions were made for selective expression of transcripts of ma this cluster in heart tissue, according to the previously described methods.
The numbers on the y-axis of Figure 11 refer to weighted expression of ESTs in each category, as "parts per million"
(ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histogram in Figure 1 l, concerning the number of heart-specific clones in libraries/sequences.
This cluster was found to be selectively expressed in heart for the following reasons: in a comparison of the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in noirheart ESTs, which was found to be 10.9. The expression level of this gene in muscle was negligible; and fisher exact test P-values were computed both for library and weighted clone counts to checle that the counts are statistically significant, and were found to be 8.60E-15.
One particularly important measure of specificity of expression of a cluster in heart tissue is the previously described comparison of the ratio of expression of the cluster in heart as opposed to muscle. This cluster was found to be specifically expressed in heart as opposed to norrheart ESTs as described above. However, many proteins have been shown to be generally expressed at a higher level in both heart and muscle, which is less desirable.
For this cluster, as described above, the expression level of this gene in muscle was negligible, which clearly supports specific expression in heart tissue.
As noted above, cluster T10377 features 6 transcript(s), which were listed in Table 1 above. A description of each variant protein according to the present invention is now provided.
Variant protein T10377 P2 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10377 T1 and T10377 T2. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10377 P2 and Q96NF5 (SEQ ID N0:362):
1. An isolated chimeric polypeptide encoding for T10377 P2, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MEISLVKCSE corresponding to amino acids 1 -of T10377 P2, second amino acid sequence being at least 90 % homologous to ANVCRLRLTVPPESPVPEQCEKKIERKEQLLDLSNGEPTRKLPQGVVYGVVR
RSDQNQQKEMVVYGWSTSQLKEEMNYIKDVRATLEKVRKRMYGDYDEMR

VDVTLENSNIKDQIRNLQQTYEASMDKLREKQRQLEVAQVENQLLKMKVES
SQEANAEVMREMTKKLYSQYEEKLQEEQRKHSAEKEALLEETNSFLK
corresponding to amino acids 26 - 276 of Q96NF5, which also corresponds to amino acids 11 - 261 of T10377 P2, followed by A, and a third amino acid sequence being 10 at least 90 % homologous to IEEANKKMQAAEISLEEKDQRIGELDRLIERMEKERHQLQLQLLEHETEMSG
ELTDSDKERYQQLEEASASLRERIRHLDDMVHCQQKKVKQMVEEIES LKKK
LQQKQLLILQLLEKISFLEGENNELQSRLDYLTETQAI~TEVETREIGVGCDLL
PSQTGRTREIVMPSRNYTPYTRVLELTMKKTLT corresponding to amino acids 278 - 466 of Q96NF5, which also corresponds to amino acids 263 - 451 of T10377 P2, wherein said first, second, A, and third amino acid sequences are contiguous and in a sequential order.
2. An isolated polypeptide encoding for a head of T10377 P2, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence MEISLVKCSE of T10377 P2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the traps-membrane region prediction programs predicted a traps-membrane region for this protein.
In addition both signappeptide prediction programs predict that this protein is a norrsecreted protein..
Variant protein T10377 P2 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 5, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377_P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 5 - Amino acid mutations SNI' pusition(s~ on Alternativeyamino ' Pxev~oiisly known amino acid' acitl(s) SNP , , sequence ,, - ~. ,. ' , ,.

262 A -> V No 30 C -> S No 323 R -> G No 36 R -> K No 439 T -> No Variant protein T10377 P2 is encoded by the following transcript(s): T10377 T,l and _, T10377 T2, for which the sequences) is/are given at the end of the application. ' The coding portion of transcript T10377 Tl is shown iii bold; this coding portion starts at position 166 and ends at position 1518. The transcript also has the following SNPs as lis~~ted in Table 6 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377 P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 6 - Nucleic acid SNPs SN.P ~ositxon art Alterztatzv~ ,nucleic~ Pre~i.ously known nuclentidc acid S:NPy.
sequence 152 A -> T Yes 253 T -> A No 272 G -> A No 624 A -> G Yes 786 G -> A No 950 C -> T No 1077 A -> G No 1132 A -> G No 1482 A -> No The coding portion of transcript T10377 T2 is shown in bold; this coding portion starts at position 270 and ends at position 1622. The transcript also has the following SNPs as listed in Table 7 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377 P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 - Nucleic acid SNPs SN'P pos~taon on. Alternative n~xc~~~c prevxciusly l~cia nuc~~atide=~ acid SNP?
sequence t ,1 -- ~ . ;F . = , , w - y 13 G -> T Yes 26 G -> A Yes 890 G -> A No 1054 C -> T No 1181 A -> G No 1236 A -> G No 1586 A -> No 88 C -> T Yes 115 G -> A Yes 126 A -> G Yes 212 A -> G No 256 A -> T Yes 357 T -> A No 376 G -> A No 728 A -> G Yes i~~
Variant protein T I 0377 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10377_T5.
One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10377_PS and Q96NF5:
1. An isolated chimeric polypeptide encoding for T10377 P5, comprising a first amino acid sequence being at least 90 % homologous to MLRSTSTVTLLSGGAARTPGAPSRRANVCRLRLTVPPESPVPEQCEKKIERKE
QLLDLSNGEPTRKLPQGVVYGVVRRSDQNQQKEMVVYGWSTSQLKEEMN
YIKDVRATLEKVRKRMYGDYDEMRQKIRQLTQELSVSHAQQEYLENHIQTQ
SSALDRFNAMNSALASDSIGLQKTLVDVTLENSNIKDQIRNLQQTYEASMDK
LREKQRQLEVAQVENQLLKMKVESSQEANAEVMREMTKKLYSQYEEKLQE
EQRKHSAEKEALLEETNSFLK corresponding to amino acids 1 - 276 of Q96NF5, which also corresponds to amino acids 1 - 276 of T10377 P5, followed by A, a second amino acid sequence being at least 90 % homologous to IEEANKKMQAAEISLEEKDQRIGELDRLIERMEKERHQLQLQLLEHETEMSG
ELTDSDKERYQQLEEASASLRERIRHLDDMVHCQQKKVKQMVE
corresponding to amino acids 278 - 372 of Q96NF5, which also coiTesponds to amino acids 278 - 372 of T10377 P5, and a third amino acid sequence being at least 90 % homologous to ENNELQSRLDYLTETQAKTEVETREIGVGCDLLPSQTGRTREIVMPSRNYTPY
TRVLELTMKKTLT corresponding to amino acids 401 - 466 of Q96NF5, which also corresponds to amino acids 373 - 438 of T10377 P5, wherein said first, A, second, and third amino acid sequences are contiguous and in a sequential order.
2. An isolated chimeric polypeptide encoding for an edge portion of T10377 P5, comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EE, having a structure as follows: a sequence starting from any of amino acid numbers 372-x to 372; and ending at any of amino acid numbers 373+ ((rr2) - x), in which x varies from 0 to rr2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because one of the two signal-peptide prediction programs (HMM:Signal peptide,NN:NO) predicts that this protein has a signal peptide..
Variant protein T10377 PS also has the following norrsilent SNPs (Single Nucleotide Polymorphisms) as listed in Table 8, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 8 - Anaino acid rnu2atioras ititin s on ah~izao Alternaazve ari~ino Previously lcna~vn.SNP~
acid acxd~s) :- .
SNP pc~s ( ) .:

y . ~: ;
~ ice - -se e. , ' ~ ~ ~ -' . R _> G No 25.

277 A -> V No 338 R-> G No 426 T -> No 45 C -> S No 51 R->K No Variant protein T10377 _P5 is encoded by the following transcript(s): T10377 T5, for which the sequences) is/axe given at the end of the application. The coding portion of transcript T10377 TS is shown in bold; this coding portion starts at position 140 and ends at position 1453. The transcript also has the following SNPs as listed in Table 9 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 - Nucleic acid SNPs ' SNP position tin Alternative nucleic Previously known SNP?
nuclet~tide' acid.
set~r~e~ce 13 G -> T Yes 26 G -> A Yes 969 C -> T No 1096 A -> G No 1151 A -> G No 1417 A -> No 88 C -> T Yes 115 G -> A Yes 126 A -> G Yes 212 A -> G No 272 T -> A No 291 G -> A No 643 A -> G Yes 805 G -> A No Variant protein T10377 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10377 T6.
One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10377 P6 and Q96NF5:
1. An isolated chimeric polypeptide encoding for T10377 P6, comprising a first amino acid sequence being at least 90 % homologous to MLRSTSTVTLLSGGAARTPGAPSRRANVCRLRLTVPPESPVPEQCEKKIERKE
QLLDLSNGEPTRKLPQGVVYGVVRRSDQNQQKEMVVYGWSTSQLKEEMN
YIKDVRATLEKVRKRMYGDYDEMRQKIRQLTQELSVSHAQQEYLENHIQTQ
SSALDRFNAMNSALASDSIGLQKTLVDVTLENSNIKDQIRNLQQTYEASMDK
LREKQRQLEVAQVENQLLKMKVESSQEANAEVMREMTKKLYSQYEEKLQE
EQRKHSAEKEALLEETNSFLK corresponding to amino acids 1 - 276 of Q96NF5, which also corresponds to amino acids 1 - 276 of T10377 P6, followed by A, a second amino acid sequence being at least 90 % homologous to IEEANKKMQAAEISLEEKDQRIGELDRLIERMEKERHQLQLQLLEHETEMSG
ELTDSDKERYQQLEEASASLRERIRHLDDMVHCQQKKVKQMVEEIESLKKK
LQQKQLLILQLLEKISFLEGE corresponding to amino acids 278 - 401 of Q96NF5, which also corresponds to amino acids 278 - 401 of T10377 P6, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence PNRQDS corresponding to amino acids 402 - 407 of T10377 P6, wherein said first, A, second and third amino acid sequences are contiguous and in a sequential order.
2. An isolated polypeptide encoding for a tail of T10377 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at Least about 95%
homologous to the sequence PNRQDS in T10377 P6.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because one of the two signal-peptide prediction programs (HMM:Signal peptide,NN:NO) predicts that this protein has a signal peptide..
Variant protein T10377 P6 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 10, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377_P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 1 D - Amirxo acid mutations SNP positxora(s) ' Alternative amine Previously known SNP?
on ~yina acid acids) ~ . .
se uenGe..-,~,. ~~ ~ ~ . .
r. <

25 R -> G No 277 A -> V No 338 R-> G No 45 C -> S No 51 R -> K No Variant protein T10377 _P6 is encoded by the following transcript(s): T10377 T6, for which the sequences) is/are given at the end of the application. The coding portion of transcript T10377 T6 is shown in bold; this coding portion starts at position 140 and ends at position 1360. The transcript also has the following SNPs as listed in Table 11 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is lrnown or not; the presence of known SNPs in variant protein T10377 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 11 - Nucleic acid SNPs SNP posh J.4n Qn A~.tG~at~"t>e ~'1;'LlG~eIGPrG'V'~OllSly ~CLOWn nLlGleOtlC~~ aCiCl. ~N~'~
Bef~tlenGe 13 G -> T Yes 26 G -> A Yes 969 -- C -> T No 1096 A -> G No 1151 A -> G No 1400 A -> No ~R1 88 C -> T Yes 115 G -> A Yes 126 A -> G Yes 212 A -> G No 272 T -> A No 291 G -> A No 643 A -> G Yes 805 - G -> A No Variant protein T10377 P7 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10377 T7.
One or more aligmnents to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between T10377 P7 and Q96NF5:
1. An isolated chimeric polypeptide encoding for T10377 P7, comprising a first amino acid sequence being at least 90 % homologous to MLRSTSTVTLLSGGAARTPGAPSRRANVCRLRLTVPPESPVPEQCEKKIERKF
QLLDLSNGEPTRKLPQGVWGWRRSDQNQQKEMVWGWSTSQLKEEMN
YIKDVRATLEKVRKRMYGDYDEMRQKIRQLTQELSVSHAQQEYLENHIQTQ
SSALDRFNAMNSALASDSIGLQKTLVDVTLENSNIKDQIRNLQQTYEASMDK
LREKQRQLEVAQVENQLLKMKVESSQEANAEVMREMTKKLYSQYEEKLQE
EQRKHSAEKEALLEETNSFLK corresponding to amino acids 1 - 276 of Q96NF5, which also corresponds to amino acids 1 - 276 of T10377 P7, followed by A, a second amino acid sequence being at least 90 % homologous to IEEANKKMQAAEISLEEKDQRIGELDRLIERMEKERHQLQLQLLEHETEMSG
ELTDSDKERYQQLEEASASLRERIRHLDDMVHCQQKKVKQMVEEI
corresponding to amino acids 278 - 374 of Q96NF5, which also corresponds to amino acids 278 - 374 of T10377 P7, and a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90%
and most preferably at least 95% homologous to a polypeptide having the sequence MSHELFSRFSLRLFGR corresponding to amino acids 375 - 390 of T10377 P7, wherein said first, A, second and third amino acid sequences are contiguous and in a sequential order.
2. An isolated polypeptide encoding for a tail of T10377 P7, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MSHELFSRFSLRLFGR in T10377 P7.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
secreted. The protein localization is believed to be secreted because one of the two signal-peptide prediction programs (HMM:Signal peptide,NN:NO) predicts that this protein has a signal peptide..
Variant protein T10377 P7 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 12, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377 P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 12 -Arnino acid fnutations SNP positions) on Alternative amino P~eviaitsly lc~aovvn amino aaid a,cxd(s) SNP?
sec~uenc~

R -> G No 277 A -> V No 338 R-> G No 45 C -> S No 51 R-> K No Variant protein T10377 P7 is encoded by the following transcript(s): T10377 T7, for which the sequences) is/are given at the end of the application. The coding portion of transcript T10377 T7 is shown in bold; this coding portion starts at position 140 and ends at position 1309. The transcript also has the following SNPs as listed in Table 13 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed;
the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377 P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 13 - Nucleic acid SNPs ~P position Qn . nucleotideAlternative nucleic.Previously l~notvn sequence y,. r ; acid SI~TP? .
7= ~ :' , . _,':y , ., 13 G -> T Yes 26 G -> A Yes 969 C -> T No 1096 A -> G No 1151 A -> G No 88 C -> T Yes 115 G -> A Yes 126 A -> G Yes 212 A -> G No 272 T -> A No 291 G -> A No -643 A -> G Yes 805 G -> A No Protein T10377 P8 has an amino acid sequence as given at the end of the application; it is encoded by transcripts) T10377 T0. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follov~:
Comparison report between T10377 P8 and Q96NF5:
An isolated chimeric polypeptide encoding for T10377 P8, comprising a first amino acid sequence being at least 90 % homologous to MEISLVKCSEANVCRLRLTVPPESPVPEQCEKKIERKEQLLDLSNGEPTRKLPQGVVYG
VVRRSDQNQQKEMWYGWSTSQLKEEMNYIKDVRATLEKVRKRMYGDYDEMRQKIR
QLTQELSVSHAQQEYLENHIQTQSSALDRFNAMNSALASDSIGLQKTLVDVTLENSNIK
DQIRNLQQTYEASMDKLREKQRQLEVAQVENQLLKMKVESSQEANAEVMREMTKKL
YSQYEEKLQEEQRKHSAEKEALLEETNSFLK corresponding to amino acids 1 - 261 of Q96NF5, which also corresponds to amino acids 1 - 261 of T10377 P8, a second amino acid sequence comprising A, and a third amino acid sequence being at least 90 %
homologous to IEEANKKMQAAEISLEEKDQRIGELDRLIERMEKERHQLQLQLLEHETEMSGELTDSDK
ERYQQLEEASASLRERIRHLDDMVHCQQKKVKQMVEEIESLKKKLQQKQLLILQLLEKI
SFLEGENNELQSRLDYLTETQAKTEVETREIGVGCDLLPSQTGRTREIVMPSRNYTPYTR
VLELTMKKTLT corresponding to amino acids 263 - 451 of Q96NF5, which also corresponds to amino acids 263 - 451 of T10377 P8, wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
The location of the protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because one of the two signal-peptide prediction programs (HMM:Signal peptide,NN:NO) predicts that this protein has a signal peptide..
Protein T10377 P8 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 14, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in protein T10377 P8 sequence provides support for the deduced sequence of this protein according to the present invention).
Table 14 - Arnino acid nzutations SNP positions) on -Alrez~iative ammo Pi-evioztsly known arxiirzoacid acid(s),: SNP?
sequence 25 R -> G No 277 V -> A No 338 R -> G No 45 C -> S No 454 T -> No 51 R -> K No Protein T10377 P8 is encoded by the following transcript(s): T10377 T0, for which the sequences) is/are given at the end of the application The coding portion of transcript T10377 TO is shown in bold; this coding portion starts at position 140 and ends at position 1537. The transcript also has the following SNPs as listed in Table 15 (given according to their position on the nucleotide segue nce, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in protein T10377 P8 sequence provides support for the deduced sequence of this protein according to the present invention).
Table I S - Nucleic acid SNPs SNP'. posi.tion on Al.teriiatzve nucleic. Previously known:SNP2 nucl.~otide acid ~
.
sequence f 13 G -> T Yes 26 G -> A Yes 969 C -> T No 1096 A -> G No 1151 A -> G No 1501 A -> No 88 C -> T Yes 115 . G -> A Yes 126 A -> G Yes 212 A -> G No 272 T -> A No 291 G -> A No 643 A -> G Yes 805 G -> A No As noted above, cluster T10377 features 18 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster T10377 node 0 according to the present nvention is supported by 25 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 T2, T10377_T5, T10377 T6 and T10377 T7. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segment locatiofz oh transcripts Transcript z~a~ne Segment starting S~gme~t ending pcisition . , pcisition ~

Segment cluster T10377 node 17 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 Tl, T10377 T2, T10377_T5, T10377 T6 and T10377_T7. Table 17 below describes the starting and ending position of this segment on each transcript.
Table 17 - Segrnertt location on trafZSCf-ipts Transcript name Segmen t starting. Segrxient ending position position ;

T10377 T7 685 ~ 817 Segment cluster T10377_node 19 according to the present invention is supported by 38 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377_Tl, T10377 T2, T10377 T5, T10377 T6 and T10377_T7. Table 18 below describes the starting and ending position of this segment on each transcript.
Table 18 - Segrraent location on t~arzscripts Transcript Laurie Segment staring pasit~a~~ e~naent ending pasitxo , ~ ~. , T10377 Tl 799 924 Segment cluster T10377 node 21 according to the present invention 's supported by 42 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 Tl, T10377_T2, T10377 T5, T10377_T6 and T10377_T7. Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts Tra~iscx~.pt x~a~.e :Segment sta~iin'~ Segxue~.t ending position positzo~

T10377 Tl 925 1053 Segment cluster T10377 node 27 according to the present invention is supported by 1 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T7. Table 20 below describes the starting and ending position of this segment on each transcript.
Table 20 - Segment location orz transcripts Transcript ~i~arne Segment sta7rting position ' l ~eg~nent ending position Segment cluster T10377 node 33 according to the present invention is supported by 103 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 Tl, T10377_T2, T10377_TS and T10377 T6. Table 21 below describes the starting and ending position of this segment on each transcript.
Table ~1 - Segment location on transcripts Transcript name ~ Segment starting position I, Segment ending position According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster T10377 node 12 according to the present invention is supported by 35 libraries. The number of libraries was detemnined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 Tl, T10377 T2, T10377_T5, T10377 T6 and T10377 T7. Table 22 below describes the starting and ending position of this segment on each transcript.
Table 22 - S'egnaent locatiora on transcripts '~.'ransc~~~t name segment starting-pasrtzan; e~~e~t ending pasitian -_.__ ':

Segment cluster T10377 node 14 according to the present invention is supported by 28 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 T1, T10377 T2, T10377 T5, T10377 T6 and T10377_T7. Table 23 below describes the starting and ending position of this segment on each transcript.
Table 23 - Segment locatiozz ozz ti°anscripts Trai~scz~pt .name- Segment sta~ting~ Seg~ne~:~. ending a posxt~cix~ :~ position :a .~ . ' . , T10377 T7 551 -. L S64 Segment cluster T10377 node_16 according to the present invention can be found in the following transcript(s): T10377 T0, T10377_T1, T10377 T2, T10377 T5, T10377 T6 and T10377 T7. Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - Segment location ozz transcripts Tz~ariscript name Segment starting '' lenient ending p~sitia~ position Segment cluster T10377 node 2 according to the present invention is supported by 3 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 Tl. Table 25 below describes the starting and ending position of this segment on each trans cript.

Table 25 - SegmesTt locatiofZ ofz tnasascripts Transcript name Segment starting Segixlent ending position position T10377 Tl 1 110 Segment cluster T10377 node 23 according to the present invention is supported by 44 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377_Tl, T10377 T2, T10377 T5, T10377 T6 and T10377_T7. Table 26 below describes the starting and ending position of this segment on each transcript.
Table 26 - Segment locatiora ofa t~°afascf°ipts Tz~ansci~pt name Segzraent startizig Segment ending posxtzcin position T10377 Tl 1054 1133 Segment cluster T10377 node 25 according to the present invention is supported by 50 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 T1, T10377 T2, T10377 T5, T10377_T6 and T10377 T7. Table 27 below describes the starting and ending position of this segment on each transcript.
Table 27 - Segrrae~t locatiofa on transcYipts Transcript name Segment starting ; Segment ending laosit~on position Segment cluster T10377 node 29 according to the present invention is supported by 50 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377_T0, T10377 TI, T10377 T2 and T10377 T6.
Table 28 below describes the starting and ending position of this segment on each transcript.
Table 28 - Segment location on transcripts TrauSCrlpt I1~1110 ~'~~118nt staTtlng ~ :r~~.'egm~;llt ~11d7T1g .-._ pOSItlOrl pE)S7t1Q11 , T103?7 T1 1240 1323 Segment cluster T10377 node 3 according to the present invention is supported by 4 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T1 and T10377 T2. Table 29 below describes the starting and ending position of this segment on each transcript.
Table 29 - Segment location on transcripts Tiansc~ipt name Sognient starting Segment ending position:
position Segment cluster T10377 node 31 according to the present invention is supported by 52 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 Tl, T10377 T2 and T10377 T5.
Table 30 below describes the starting and ending position of this segment on each transcript. .
Table 30 - Segment location on tf°ansct°ipts ",l"ranscxalit ,name Segiraenit starting Seez~i erad3~~ posit~iip position Segment cluster T10377 node S according to the present invention is supported by 30 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377_T0, T10377_T1, T10377_T2, T10377 T5, T10377_T6 and T10377 T7. Table 31 below describes the starting and ending position of this segment on each transcript.
Table 31 - Segfnefit location on transcripts Transcript name Segment staring position-Segruient ending p~isitioii T10377 TO 215 ~ 301 Segment cluster T10377 node 8 according to the present invention is supported by 35 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377 T1, T10377 T2, T10377 T5, T10377~T6 and T10377'T7. Table 32 below describes the starting and ending position of this segment on each transcript.
Table 32 - Segment location on ty-ansc~ipts Transcript name . Segrrient startingSegment ending ppsition position Segment cluster T10377 node 9 according to the present invention is supported by 35 libraries. The number of libraries was determined as previously described.
This segment can be found in the following transcript(s): T10377 T0, T10377_Tl, T10377 T2, T10377 T5, T10377 T6 and T103?7 T7. Table 33 below describes the starting and ending position of this segment on each transcript.
Table 33 - Segment location on transcripts Tra~s~ript' ~aari~~ Segmen~fi starting ~ eg~:ent end~g position :. position . .
' ' T10377 Tl 389 438 IS
Alignment of: T10377 P2 ac Q96NF5 ..
Alignment segment 1/1:

Quality: 4288.00 Escore: 0 Matching length: 491 Total length: 441 Matching Percent Similarity: 99.77 Matching Percent Identity: 99.77 Total Percent Similarity: 99.77 Total Percent Identity: 99.77 Gaps: 0 Alignment:

llllillllllllllllllllllllllllllllfllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

111 RQKTRQLTQELSVSHAQQEYLENHIQTQSSALDRFNAMNSALASDSTGLQ l60 IIIIIIIIIIIIilllllllllllllllllllllllllllllllllllll l6l KTLVDVTLENSNIKDQTRNLQQTYEASMDKLREKQRQLEVAQVENQLLKM 210 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Illllllllllllllllllllllllllllllllllllllllilllillli Illllllllllllilllllllllllllllllllllllllll Alignment of: T10377_P5 x Q96NF5 ..
Alignment segment 1/1:
Quality: 9159.00 Escore: 0 Matching length: 438 Total length: 466 Matching Percent Similarity: 99.77 Matching Percent Identity: 99.77 IS Total Percent Similarity: 93.78 Total Percent Identity: 93.78 Gaps: 1 Alignment:
ZO l MLRSTSTVTLLSGGAARTPGAPSRRANVCRLRLTVPPESPVPEQCEKKTE 50 Illllllllllllllllllllllllllllillllllllllllllllliil IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
3O 101 MNYIKDVRATLEKVRKRMYGDYDEMRQKIRQLTQELSVSHAQQEYLENHI l50 IIIIIilillllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIillllllillllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIII IIIllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

1O 351 ERIRHLDDMVHCQQKKVKQMVE............................ 372 IIIlllllllllllllllllll Illlllllllllllll Alignment of: T10377_P6 x Q96NF5 ..
Alignment segment 1/1:
Quality: 3896.00 Escore: 0 Matching length: 403 Total length: 403 Matching Percent Similarity: 99.50 Matching Percent Identity: 99.50 Total Percent Similarity: 99.50 Total Percent Identity: 99.50 3~ Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

II

Alignment of: T10377 P7 x Q96NF5 ..
Alignment segment 1/1:
Quality: 3642.00 Escore: 0 Matching length: 376 Total length: 376 Matching Percent Similarity: 99.47 Matching Percent Identity: 99.47 Total Percent Similarity: 99.47 Total Percent Identity: 99.47 Gaps: 0 Alignment:
$ 1 MLRSTSTVTLLSGGAARTPGAPSRRANVCRLRLTVPPESPVPEQCEKKIE 50 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

101 MNYIKDVRATLEKVRKRMYGDYDEMRQKIRQLTQELSVSHAQQEYLENHI l50 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllllllllllllllll IS l01 MNYIKDVRATLEKVRKRMYGDYDEMRQKIRQLTQELSVSHAQQEYLENHI 150 15l QTQSSALDRFNAMNSALASDSIGLQKTLVDVTLENSNIKDQIRNLQQTYE 200 llllllllllllllllllllllllllllllllllllllllllllllllll I

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

ASMDKLREKQRQLEVAQVENQLLKMKVESSQEANAEVMREMTKKLYSQYE

II IIIIIIIIIIIIIIIIIIIIIII
III
IIII
IIII
I
I

II
II
II
I
II
EKLQEEQRKHSAEKEALLEETNSFLKVIEEANKKMQAAEISLEEKDQRIG

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllllll 30l ELDRLIERMEKERHQLQLQLLEHETEMSGELTDSDKERYQQLEEASASLR350 IIIIIIIIIIIIIIIIIIIIIIII I

Alignment of: T10377 P8 x Q96NF5 ..

Alignment segment 1/1:

Quality: 9519.00 Escore: 0 Matching length: 965 Total length: 966 S Matching Percent Similarity: 99 Matching Percent Identity: 99 Total Percent Similarity: 99 Total Percent Identity: 99 Gaps: 0 Alignment:

I

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIlllllllllllllllllllllllllllllllllllllllllllllil Illlllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIilllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIII

Expression of Q96NF5 transcripts which are detectable by amplicon as depicted irr sequence name TI0377 specifically in heart tissue.
1 S Expression of Q96NFS transcripts detectable by or according to junc2S-31 node(s), T10377 amplicon(s) and T10377 junc2S-31F and T10377 junc2S-31R primers was measured by real time PCR. In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM 000981; RPL19 amplicon), TATA box (GenBanlc Accession No.
NM 003194; TATA amplicon), Ubiquitin (GenBank Accession No. BC000449; amplicon -Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM 004168; amplicon - SDHA-amplicon), was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos. 44, 4S, 46, Table l, above "Tissue samples in testing panel"), to 2S obtain a value of fold up-regulation for each sample relative to median of the heart.
Figure 12 is a histogram showing specific expression of the above-indicated (~96NFS
transcripts in heart tissue samples as opposed to other tissues.
As is evident from Figure 12, the expression of Q96NFS transcripts detectable by the above amplicon(s) in heart tissue samples was significantly higher than in most other samples (norrheart tissue sample Nos. 1-26,28-43 47-74 Table l, above "Tissue samples in testing panel").
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: T10377 junc25-31F forward primer;
and T10377 junc25-31R reverse primer.
The present invention also preferably encompasses any amplicon obtained tlu-ough the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a norr limiting illustrative example only of a suitable amplicon: T10377 junc25-31.
T10377 junc25-31F (SEQ ID N0:363): AGCAGATGGTCGAGGAGAATAATG
T10377 junc25-31R (SEQ ID NO:364): ATCTCTCTGGTTTCCACTTCGG
T10377 junc25-31 (SEQ ID N0:365):
AGCAGATGGTCGAGGAGAATAATGAACTACAAAGCAGGTTGGACTATTTAACAGA
AACCCAGGCCAAGACCGAAGTGGAAACCAGAGAGAT
Expression of Q96NF5 transcripts detectable by or according to junc29-33 node(s), T10377 amplicon(s) and T10377 junc29-33F and T10377 junc29-33R primers was measured by real time PCR. In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM 000981; RPL19 amplicon), TATA box (GenBank Accession No.
NM 003194; TATA amplicon), LTbiquitin (GenBank Accession No. BC000449;
amplicon -Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM 004168; amplicon - SDHA-amplicon), was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos. 44, 45, 46, Table 1, above "Tissue samples in testing panel"), to obtain a value of fold up-regulation for each sample relative to median of the heart.
Figure 13 is a histogram showing specific expression of the above-indicated transcripts in heart tissue samples as opposed to other tissues.
As is evident from Figure 13, the expression of Q96NF5 transcripts detectable by the above amplicon(s) in heart tissue samples was significantly higher than in most other samples (non-heart tissue sample Nos. 126, 28-43, 47-74 Table 1 above "Tissue samples in testing panel").
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: T10377 junc29-33F forward primer;
and T10377 junc29-33R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a norrlimiting illustrative example only of a suitable amplicon: T10377 junc29-33.
T10377 junc29-33F (SEQ ID NO:366): CTTTCTTAGAAGGAGAGCCAAACAG
T10377 junc29-33R (SEQ ID N0:367): CCTAAGTCAGAGTTTTCTTCATGGTTAAC
T10377 junc29-33 (SEQ ID N0:368):
CTTTCTTAGAAGGAGAGCCAAACAGGCAGGACTCGTGAAATTGTGATGCCTTCTAG
GAACTACACCCCATACACAAGAGTCCTGGAGTTAACCATGAAGAAAACTCTGACTT
AGG
Expression of Q96NF5 transcripts detectable by or according to seg2-3 node(s), amplicon(s) and T10377 seg2-3F and T10377 seg2-3R primers was measured by real time PCR.
In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No.
NM 000981; RPL19 amplicon), TATA box (GenBank Accession No. NM 003194; TATA
amplicon), Ubiquitin (GenBanle Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA (GenBank Accession No. NM 004168; amplicon - SDHA-amplicon), was measured similarly. For each RT sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the median of the quantities of the heart samples (Sample Nos.
44, 45, 46, Table l, above "Tissue samples in testing panel"), to obtain a value of fold up-regulation for each sample relative to median of the heart.
Figure 14 is a histogram showing specific expression of the above-indicated transcripts in heart tissue samples as opposed to other tissues.
As is evident from Figure 14, the expression of Q96NF5 transcripts detectable by the above amplicon(s) in heart tissue samples was significantly higher than in the skeletal muscle (non-heart tissue sample Nos. 1-9,13-26,28-43,47-74 Table l, "Tissue samples in testing samples").

Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: T10377 seg2-3F
forward primer; and T10377 seg2-3R reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon: T10377 seg2-3.
T10377 seg2-3F (SEQ ID N0:369): CTTCGCATTGTGCATAACACAA
T10377 seg2-3R (SEQ ID N0:370): GAAACTCGGATACACAATCTCCAGA
T10377 seg2-3 (SEQ ID N0:371):
CTTCGCATTGTGCATAACACAAGCCCTGAACCAGCTGCTTTGGGAACCCCTGGGAA
TAAAGTGCCCTACCTGCCTTTCAGGCACTGCCAAGCCTGGGGCATCTCTGGAGATTG
TGTATCCGAGTTTC

Cluster 224874 features 2 transcripts) and 10 segments) of interest, the names for which are given in Tables l and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - Ti°azzscz°ipts of izztez-est Transcript Name : ; Seq ID No.

224874 PEA~~ 2 T10 ~ 18 Table 2 - Segzzzents of interest Segment Name --. , S~q ID No.

224874 PEA 2 node 21 113 node 4 node node node node node node node 224874 PEA 2 6 ~ 122 node Table 3 - Proteins of ihteYest P.rotelu. Nai~a~e Seq ~.O ~o:

224874 PEA 2 P6 ~ 298 The heart selective diagnostic marker prediction engine provided the following results with regard to cluster 224874. Predictions were made for selective expression of transcripts of this cluster in heart tissue, according to the previously described methods.
The numbers on the y-axis of Figure 15 refer to weighted expression of ESTs in each category, as "parts per million"
(ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histogram in Figure 15, concerning the number of heart-specific clones in libraries/sequences; as well as with regard to the histogram in Figure 16, concerning the actual expression of oligonucleotides in various tissues, including heart.
This cluster was found to be selectively expressed in heart for the following reasons: in a comparison of the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in non-heart ESTs, which was found to be 16.7; the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in muscle-specific ESTs which was found to be 2.1; and fisher exact test P-values were computed both for library and weighted clone counts to check that the counts are statistically significant, and were found to be 3.20E-09.
One particularly important measure of specificity of expression of a cluster in heart tissue is the previously described comparison of the ratio of expression of the cluster in heart as opposed to muscle. This cluster was found to be specifically expressed in heart as opposed to non-heart ESTs as described above. However, many proteins have been shown to be generally expressed at a higher level in both heart and muscle, which is less desirable.
For this cluster, as described above, the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in muscle-specific ESTs which was found to be 2.1, which clearly supports specific expression in heart tissue.
As noted above, cluster 224874 natures 2 transcript(s), which were listed in Table 1 above. A description of each variant protein according to the present invention is now provided.
Variant protein 224874 PEA 2 PS according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) 224874 PEA 2 T10. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 224874 PEA 2 PS and Q9NPI5:
l.An isolated chimeric polypeptide encoding for 224874 PEA 2 P5, comprising a first amino acid sequence being at least 90 % homologous to MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLE
SLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSYKPLVDLYSR
RYFLTVPYEECKWRRS corresponding to amino acids 1 - 132 of Q9NPI5, which also corresponds to amino acids 1 - 132 of 224874 PEA 2 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence LPGRHEVPRGALP corresponding to amino acids 133 - 145 of 224874 PEA 2 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 224874 PEA 2 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence LPGRHEVPRGALP in 224874 PEA 2 P5.
Comparison report between 224874 PEA 2'P5 and Q9NZK3:
l.An isolated chimeric polypeptide encoding for 224874 PEA 2 P5, comprising a first amino acid sequence being at least 90 % homologous to MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLE
SLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSYKP
corresponding to amino acids 1 - 109 of Q9NZK3, which also corresponds to amino acids 1 -109 of 224874 PEA 2 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least ..
95% homologous to a polypeptide having the sequence LVDLYSRRYFLTVPYEECKWRRSLPGRHEVPRGALP corresponding to amino acids 110 -145 of 224874 PEA 2 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 224874 PEA 2 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence LVDLYSRRYFLTVPYEECKWRRSLPGRHEVPRGALP in 224874 PEA 2 P5.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellularly because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a norrsecreted protein..
Variant protein 224874 PEA 2 PS is encoded by the following transcript(s):

224874 PEA 2 T10, for which the sequences) is/are given at the end of the application. The coding portion of transcript 224874 PEA 2 T10 is shown in bold; this coding portion starts at position 292 and ends at position 726. The transcript also has the following SNPs as listed in Table 4 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 224874 PEA 2 PS sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 4 - Nucleie acid SNPs SNP position oi~ Alternat~~e n~zceic Previously lfnowii nucleotide acid SNP?
equence 1 G -> C No 70 G -> A Yes 504 C -> T No 645 C -> T Yes 954 C -> T Yes Variant protein 224874 PEA 2 P6 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) 224874 PEA 2 T11. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between 224874 PEA 2 P6 and Q9NPI5:
l.An isolated chimeric polypeptide encoding for 224874 PEA 2 P6, comprising a first amino acid sequence being at least 90 % homologous to MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLE
SLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSY corresponding to amino acids 1 - 107 of Q9NPI5, which also corresponds to amino acids 1 -107 of 224874 PEA 2 P6, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence NLPGRHEVPRGALP corresponding to amino acids 108 - 121 of 224874 PEA 2 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 224874 PEA~2 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence NLPGRHEVPRGALP in 224874 PEA 2 P6.
Comparison report between 224874 PEA 2 P6 and Q9NZK3:
l.An isolated chimeric polypeptide encoding for 224874 PEA 2 P6, comprising a first amino acid sequence being at least 90 % homologous to MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLE
SLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSY corresponding to amino acids 1 - 107 of Q9NZK3, which also corresponds to amino acids 1 -107 of 224874 PEA 2 P6, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95%
homologous to a polypeptide having the sequence NLPGRHEVPRGALP corresponding to amino acids 108 - 121 of 224874 PEA 2 P6, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of 224874 PEA 2 P6, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence NLPGRHEVPRGALP in 224874 PEA 2 P6.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellularly because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein..

Variant protein 224874 PEA 2 P6 is encoded by the following transcript(s):
224874 PEA 2 Tll, for which the sequences) is/are given at the end of the application. The coding portion of transcript 224874 PEA 2 T11 is shown in bold; this coding portion starts at position 292 and ends at position 654. The tl-anscript also has the following SNPs as listed in Table 5 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein 224874 PEA 2 P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 5 - Nucleic acid SNPs SNF posit'x~iii on Altertiativc nuicleicF previously. knciwn nucleotide acid ,SNP?
sequenco ~. _ 1 ;

1 G -> C No 70 G -> A Yes 504 C -> T No 882 C -> T Yes As noted above, cluster 224874 features 10 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster 224874 PEA 2 node 21 according to the present invention is supported by 30 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 6 below describes the starting and ending position of this segment on each transcript.
Table 6 - Segmefzt location on transcYipts 2l1 Transcript name Segment starting Segirient ending position position ~~

Segment cluster 224874 PEA 2 node 4 according to the present invention is supported by 19 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 7 below describes the starting and ending position of this segment on each transcript.
Table 7 - Segment location ofz tf°ansc~ipts Transcript name Segment starting S.egrxidrit endiri~
- posWon. ~~ position ~

According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster 224874 PEA 2 node 0 according to the present invention is supported by 3 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 8 below describes the starting and ending position of this segment on each transcript.
Table 8 - Segment location ora transcripts Transcript name cement starting positionSegment eudi?ag position 224874 PEA 2 T10 1 ~~ ~77 ~~

~Z24874 PEA 2 T11 1 77 Segment cluster 224874 PEA'2'node'10 according to the present invention is supported by 25 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 9 below describes the starting and ending position of this segment on each transcript.
Table 9 - Segment locatioiz on 'transcripts Transcript name ~~ent starting po~itiori :.~~ ent endi~
; l~'pasitian Segment cluster 224874 PEA 2 node-12 according to the present invention is supported by 26 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 10 below describes the starting and ending position of this segment on each transcript.
Table 10 - Segment location ora ty-afascripts Trar~scnpt niaz~r~ ~egamenr sla~'zngposxtao~ , ~g.~,t e,~du~.laosi~.o~
, ..

Segment cluster 224874 PEA 2 node 13 according to the present invention is supported by 21 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 11 below describes the starting and ending position of this segment on each transcript.
Table 11- Segment location on transcy-ipts T~c~xlat .~azne Seg~rnent starting Segment endurg pasi~.o~
position Segment cluster 224874 PEA 2 node~14 according to the present invention is supported by 20 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 12 below describes the starting and ending position of this segment on each transcript.
Table 12 - Segment location on tYanscripts Transcript came Segment starting Segrrient ending pQSition ~ posltlori , Segment cluster 224874 PEA 2 node-16 according to the present invention is supported by 17 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10. Table 13 below describes the starting and ending position of this segment on each transcript.
Table 13 - Segnaerat location on tf~afzscripts _Transcript i~arne Segment startitag ' egmen~t ending positioia position 224874 PEA 2 T10 615 686 ~~

Segment cluster 224874 PEA 2 node 3 according to the present invention is supported by 8 libraries. The number of libraries was determined as previously describ ed. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 14 below describes the starting and ending position of this segment on each transcript.
Table 14 - Segment location orc transcYipts T.~aasc~.pt name S~~ent staxting Segm.eut ending posittan.
posxtio~a z14 Segment cluster 224874 PEA 2 node 6 according to the present invention is supported by 23 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): 224874 PEA 2 T10 and 224874 PEA 2 T11.
Table 15 below describes the starting and ending position of this segment on each transcript.
Table I S - Segfneszt location on transcripts Tra~scnpt.~name regiment starting ~ Segment ending position position ,-:

Variant protein alignment to the previously known protein:
Sequence name: /tmp/Ro5LG30hE3/oQvcWauNWJ:Q9NPI5 Sequence documentation:
Alignment of: 224874 PEA 2 P5 x Q9NPI5 ..
Alignment segment 1/1:
Quality: 1307.00Escore: 0 Matching length: 132 Total length: 132 Matching Percent Similarity:100.00 Matching Percent Identity:100.00 Total Percent Similarity: 100.00 Total Percent Identity:100.00 Gaps: 0 Alignment:
. . . , .

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Sequence name: /tmp/Ro5LG30hE3/oQvcWauNWJ:Q9NZK3 Sequence documentation:
Alignment of: Z24874_PEA 2_P5 x Q9NZK3 ..
Alignment segment 1/1:
IS Quality: 1070.00 Escore: 0 Matching length: 109 Total length: 109 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIII~II~IIIIIIIIIIIIIIII

5l FKQWDVLESLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLE 100 ~IIII~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

I)IIIIIII

3S Sequence name: /tmp/TxcCIAWX3r/LIzBcJOujT:Q9NPI5 Sequence documentation:

Alignment of: Z24874_PEA 2_P6 x Q9NPI5 ..
Alignment segment 1/1:
S Quality: 1048.00 Escore: 0 Matching length: 107 Total length: 107 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

llllllllllllllllllllllllllllllllllllllllllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-Illlllllllllllil 2S Sequence name: /tmp/TxcCIAWX3r/LIzBcJOujT:Q9NZK3 Sequence documentation:
Alignment of: Z24874_PEA 2-P6 x Q9NZK3 ..
Alignment segment 1/1:
Quality: 1048.00Escore: 0 Matching length: 107 Total length: 107 3S Matching Percent Similarity:100.00 Matching Percent Identity:100.00 Total Percent Similarity: 100.00 Total Percent Identity:100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
5l FKQWDVLESLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLE 100 DESCRIPTION FOR CLUSTER HUMCDDANF
Cluster HUMCDDANF features 2 transcripts) and 7 segments) of interest, the names for which are given in Tables l and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table 1 - Tra~scf-ipts of ifaterest Transcnpt Name ~eq ILD No..~
f , Table 2 - Segrnerats of interest Segu.~ent N~~.e Seq ID 'loo.
~~

HUMCDDANF node 0 123 HLTMCDDANF node 10 124 HLTMCDDANF node 2 125 HLIMCDDANF node 5 126 HUMCDDANF node 8 127 HLTMCDDANF node 11 128 HUMCDDANF node 12 129 Table 3 - Proteifzs of interest Pr4t~in Name ~ ~~q II3 No. , These sequences are variants of the known protein Atrial natriuretic factor precursor (SwissProt accession identifier ANF HUMAN; known also according to the synonyms ANF;
Atrial natriuretic peptide; ANP; Prepronatriodilatin), referred to herein as the previously known protein; it contains Cardiodilatin-related peptide (CDP).
Protein Atrial natriuretic factor precursor is known or believed to have the following function(s): Atrial natriuretic factor (ANF) is a potent vasoactive substance synthesized in mammalian atria and is thought to play a key role in cardiovascular homeostasis; has a cGMP-stimulating activity. The sequence for protein Atrial natriuretic factor precursor is given at the end of the application, as "Atrial natriuretic factor precursor amino acid sequence" (SEQ ID
N0:350). Known polymorphisms for this sequence are as shown in Table 4.
Table 4 - Arnino acid mutations foY Known Protein SNP -posilion(s)Co~nent- , an ~mirno acid s~qtxence 32 V -> M (in dbSNP:5063). /FTId=VAR 014579.

152 - 153 Missing (in isoform 2). /FTId=VAR 000594.

65 E -> D

Protein Atrial natriuretic factor precursor localization is believed to be Secreted.
It has been investigated for clinical/therapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows:
Aldosterone antagonist;
Diuretic; Electrolyte absorption agonist. A therapeutic role for a protein represented by the cluster has been predicted. The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication:
Antihypertensive, diuretic;
Antiasthma; Urological; Cardiostimulant, Antianaemic, Cardiovascular, Neuroprotective, Fertility enhancer, Male contraceptive, Hypolipaemic/Antiatherosclerosis, Hepatoprotective and renal failure.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: physiological processes; blood pressure regulation, which are annotations) related to Biological Process; hormone activity, which are annotations) related to Molecular Function; and extracellular, which are annotations) related to Cellular Component.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http://www.expasy.ch/sprot/>; or Locuslink, available from <http:l/www.ncbi.nhn.nih.gov/projects/LocusLink/>.
The heart selective diagnostic marker prediction engine provided the following results with regard to cluster HUMCDDANF. Predictions were made for selective expression of transcripts of this cluster in heart tissue, according to the previously described methods. The numbers on the y-axis of Figure 17A refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histogram in Figure 17A, concerning the number of heart specific clones in libraries/sequences; as well as with regard to the histogram in Figure 17B, concerning the actual expression of oligonucleotides in various tissues, including heart.
This cluster was found to be selectively expressed in heart for the following reasons: a comparison of the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in non-heart ESTs was found to be 56.3; The expression levels of this gene in muscle was negligible; and fisher exact test P-values were computed both for library and weighted clone counts to check that the counts are statistically significant, and were found to be 1.20E-249.
One particularly important measure of specificity of expression of a cluster in heart tissue is the previously described comparison of the ratio of expression of the cluster in heart as opposed to muscle. This cluster was found to be specifically expressed in heart as opposed to non-heart ESTs as described above. However, many proteins have been shown to be generally expressed at a higher level in both heart and muscle, which is less desirable.
For this cluster, as described above, the expression levels of this gene in muscle was negligible, which clearly supports specific expression in heart tissue.
As noted above, cluster HUMCDDANF features 2 transcript(s), which were listed in Table 1 above. These transcripts) encode for proteins) which are variants) of protein Atrial natriuretic factor precursor. A description of each variant protein according to the present invention is now provided.
Variant protein HUMCDDANF P2 according to the preser~ invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) HUMCDDANf-T3. An alignment is given to the known protein (Atrial natriuretic factor precursor) at the end of the application. One or more alignme~s to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMCDDANF P2 and ANF HUMAN:
l.An isolated chimeric polypeptide encoding for HUMCDDANF P2, comprising a first amino acid sequence being at least 90 % homologous to MPLEDEVVPPQVLSEPNEEAGAALSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRS
ALLKSKLRALLTAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY corresponding to amino acids 51 - 151 of ANF HUMAN, which also corresponds to amino acids 1 - 101 of HUMCDDANF P2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the traps-membrane region prediction programs predicted a traps-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a norrsecreted protein..
Variant protein HLTMCDDANF P2 also has the following norrsilent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their positions) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 7 - Arnino acid mutations SNP position(syoxi Alternative amino ~ Previouslyknciwn amino acid acids) - :: SNP , sequexxce, ,, ., ; - . . . . r . , ~ . . _ , Q; ,.
, A -> V Yes 27 L -> F Yes 74 S -> No 76 R -> Q Yes 15 Variant protein HUMCDDANF P2 is encoded by the following transcript(s):
HUMCDDANF T3, for which the sequences) is/are given at the end of the application. The coding portion of transcript HUMCDDANF-T3 is shown in bold; this coding portion starts at position 381 and ends at position 683. The transcript also has the following SNPs as listed in Table 8 (given according to their position on the nucleotide sequence, with the alternative 20 nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMCDDANF P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table ~ - Nucleic acid SNPs SNP positiosi ~ on Alterailative nucleicPreviously l~iown nucleotide acid SNP:
sequence 199 C -> T Yes 374 A -> G No 771 T -> C Yes 778 T -> C Yes 809 C -> T Yes 887 C -> G No 968 A -> C Yes 439 C -> T Yes 458 C -> T No 459 C -> T Yes 602 C -> No 607 G -> A Yes 684 T -> C Yes (short/long variant) 711 A -> G No 757 G -> T Yes Variant protein HUMCDDANF P3 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) HUMCDDANF T4. An alignment is given to the known protein (Atrial natriuretic factor precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMCDDANF P3 and ANF HUMAN:
l.An isolated chimeric polypeptide encoding for HUMCDDANF P3, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence MSSFSTTT corresponding to amino aids 1 - 8 of HUMCDDANF P3, and a second amino acid sequence being at Ieast 90 % homologous to NLLDHLEEKMPLEDEVVPPQVLSEPNEEAGAALSPLPEVPPWTGEVSPAQRDGGALGR
GPWDSSDRSALLKSKLRALLTAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY
corresponding to amino acids 42 - 151 of ANF HCTNIAN, which also corresponds to amino acids 9 - 118 of HUMCDDANF P3, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a head of HUMCDDANF P3, comprising a polypeptide being at )cast 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence MSSFSTTT of HUMCDDANF P3 The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellularly because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein..
Variant protein HUMCDDANF P3 also has the following norrsilent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their pasition(s) on the amino acid sequence, with the alternative amino acids) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 9 -Amino acid mutations SNP pasitia.n(s) Alternative amino ' Previously ~a~;vn are amiua acid acids) SNP?

seclue~ce 37 A -> V Yes 44 -- L _> F Yes 91 S -> No 93 R -> Q Yes Variant protein HUMCDDANF P3 is encoded by the following transcript(s):
HUMCDDANF T4, for which the sequences) is/are gven at the end of the application. The coding portion of transcript HUMCDDANF T4 is shown in bold; this coding portion starts at position 104 and ends at position 457. The transcript also has the following SNPs as listed in Table 10 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMCDDANF P3 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 10 - Nucleic acid SNPs SNP position 'an nucleotideAlternative nucleic ~- Previously knuv~ih.
sequence ., y~ acid ~NP~
_ _ , ; , ; , ,, .; : r a-.; .
, , 148 A -> G No 213 C -> T Yes 552 T -> C Yes 583 C -> T Yes 661 C -> G No 742 A -> C Yes 232 C -> T No 233 C -> T Yes 376 C -> No 3 81 G -> A Yes 458 T -> C Yes (short/long isoform) 485 A -> G No 531 G -> T Yes 545 T -> C Yes 44 L -> F Yes 91 S -> No 93 R -> Q Yes Variant protein HUMCDDANF P3 is encoded by the following transcript(s):
HUMCDDANF T4, for which the sequences) is/are even at the end of the application. The coding portion of transcript HUMCDDANF T4 is shown in bold; this coding portion starts at position 104 and ends at position 457. The transcript also has the following SNPs as listed in Table 10 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HUMCDDANF P3 sequence provides support for the deduced sequence of this variant protein according to the present invention).
Table 10 - Nucleic acid SNPs ~.~..:°~~
a , a -~ ,~. ,~~sm ,~r~' _.. ~ . a ~ _.s ~ a S P ~ . osxtron.. o~ . uel~otide Alteriiativ ~.nuelerc~~actd ~ ~ ~~
~Prevrously~known S'. ° ?.,-°;
sequences 3 ~,~~, ~~~ ' '~ .~lr~ ~ ~ ~ ~~ ~~ A~ ~ ,~ ~~:, ~~.~- ~-3a"
~~~:, 148 A -> G No 213 C -> T Yes 552 T -> C Yes 583 C -> T Yes 661 C -> G No 742 A -> C Yes 232 C -> T No 233 C -> T Yes 376 C -> No 381 G -> A Yes 458 T -> C Yes (short/long isoform) 485 A -> G No 531 G -> T Yes 545 T -> C Yes As noted above, cluster 1-IUMCDDANF features 7 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster HUMCDDANF node 0 according to the present invention is supported by 5 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMCDDANF T3. Table 11 below describes the starting and ending position of this segment on each transcript.
Table Il - Segment location on transcripts r Transcn - t name Se en stairin ~ . ;NSe ent:.endln r;
.~ ~-F~a ositio - ~ osition p d ~ ~ g~ ~ ..g F~
~

Segment cluster HUMCDDANF mde-10 according to the present invention is supported by 49 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMCDDANF T3 and HUMCDDANF T4.
Table 12 below describes the starting and ending position of this segment on each transcript.
Segment cluster HUMCDDANF node 2 according to the present invention is supported by 41 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMCDDANF T4. Table 13 below describes the starting and ending position of this segment on each transcript.
Table 12 - Segment location on transcripts Table l3 - Segment location on transcripts 't -.
ranscn t name. . Se ent staivtin _osi-Se merit eridin osition p yon ~~ g p ' ~
g p ,.. ~~ . ~- ~ = ;_ ~._g. ' ..
- __ . -- ~ z.
_~.aM. ~

Segment cluster HUMCDDANF node-5 according to the present invention is supported by 62 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMCDDANF T3 and HUMCDDANF T4.
Table 14 below describes the starting and ending position of this segment on each transcript.
Table 14 - Segment location on transcripts t ~i ~~ltlon.~ ~. " ment~endm W os _~. ~~ :anscn t_name ~, on A~ ~: ~~Segrrtent s artFng Seg . g_po~~t~
~,w~~ p~ -..; pos ~. o rv~~ : ~ ti ~
~ ~ ~ ~ . ~ ~ -~~-:

HUMCDDANF'_T4 128 f 454 Segment cluster HUMCDDANF'-node 8 according to the present invention is supported by 56 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMCDDANF T3 and HUMCDDANF T4.
Table below describes the starting and ending position of this segment on each transcript.
15 Table 15 - Segment location on transcripts According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.

22'7 Segment cluster HUMCDDANF node-l l according to the present invention can be found in the following transcript(s): HUMCDDANF T3 and HUMCDDANF T4. Table 16 below describes the starting and ending position of this segment on each transcript.
Table 16 - Segment locatioh on transcripts ,. , w ~. ~~ . w~ . .:~. ~_ ~ ,1 t i. ._ .-Se --.~.ent slant Se. .~e~ a d n ositio Transert - t name n os t otj' ~ , ~ nr t~ ~ nz:
~:fP~~-. ~' g g F -p ~?' ~

~ x r ~ ; ~ ~~ ~~ - ~~
- ' ~~
-~

_--w- _ _.-.-~ _ _ . ~W_ ~ ______. : v ...
~ ~ ,_~. ~ ~ ._ _ :~_ , . ._ ~ : , -~~~ . m -Segment cluster HUMCDDANF node-12 according to the present invention is supported by 36 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMCDDANF T3 and HUMCDDANF T4.
Table 17 below describes the starting and ending position of this segment on each transcript.
Table 17 - Segment location on transcripts ~-~ .
i~ri t name . ~~nt~~ta ~ in os.t q : :S.e men .hendln ran p,, o :-, osi:tiorin ,p ~ ; ; ~,~ Segrza. g~.. . ~~ ..H~
~~ ,... ~; , ~. ~t ~"rY .~,B ,.
~;~, ~
:

HUMCDDANF T3 952 u HUMCDDANF_T4 726 r766 Variant protein alignment to the previously known protein:
Sequence name: /tmp/3GyiZQyJBL/jYng3zFfcE:ANF_HUMAN
Sequence documentation:
Alignment of: HUMCDDANF-P2 x ANF_HUMAN ..
Alignment segment 1/1:
Quality: 988.00 Escore: 0 Matching length: 101 Total length: 101 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 2$ Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

I

IS
Sequence name: /tmp/mnb70PVCPP/oTrSwgJLyB:ANF-HUMAN
Sequence documentation:
ZO Alignment of: HUMCDDANF-P3 x ANF-HUMAN ..
Alignment segment 1/1:
Quality: 1076.00 Escore: 0 25 Matching length: 110 Total length: 110 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00 Gaps: 0 30 Alignment:

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Expression of Human cardiodilatin-atrial natriuretic factor (CDD-ANF) HUMCDDANF
transcripts which are detectable by amplicon as depicted in sequence name HUMCDDANFjunc2-SF2R2 specifically in heart tissue Expression of Human cardiodilatin-atrial natriuretic factor (CDD-ANF) transcripts detectable by or according to junc2-5 node(s), HUMCDDANFjunc2-SF2R2 amplicon and primers HUMCDDANFjunc2-SF2 HUMCDDANFjunc2-SR2 was measured by real time PCR
(this transcript relates to the known or WT protein). In parallel the expression of four housekeeping genes - RPL19 (GenBank Accession No. NM 000981; RPL19 amplicon), TATA
box (GenBank Accession No. NM 003194; TATA amplicon), Ubiquitin (GenBank Accession No. BC000449; amplicon - Ubiquitin-amplicon) and SDHA (GenBank Accession No.
NM 004168; amplicon- SDHA-amplicon) was measured similarly. For each RT
sample, the expression of the above amplicons was normalized to the geometric mean of the quantities of the housekeeping genes. The normalized quantity of each RT sample was then divided by the quantity of heart sample no. 45 (Table 1, above), to obtain a value of relative expression for each sample relative to this heart sample.
As is evident from Figure 18, the expression of Human cardiodilatin-atrial natriuretic factor (CDD-ANF) transcripts detectable by the above amplicon(s) in one of the heart tissue samples (Sample Nos. 45, Table 1, "Tissue samples in testing panel") was significantly higher than in the other samples, including other two heart samples. Sample 45 is from fibrotic heart, as opposed to heart samples 44 and 46 that are from normal hearts. (Note - the product in samples 10 and 11 was found to be a non-specific product by inspecting the dissociation curve that was created in the real-time PCR experiment).
Primer pairs are also optionally and preferably encompassed within the present invention; for example, for the above experiment, the following primer pair was used as a non-limiting illustrative example only of a suitable primer pair: HUMCDDANFjunc2-SF2 forward primer; and HUMCDDANFjunc2-SR2 reverse primer.
The present invention also preferably encompasses any amplicon obtained through the use of any suitable primer pair; for example, for the above experiment, the following amplicon was obtained as a non-limiting illustrative example only of a suitable amplicon:
HUMCDDANFjunc2-SF2R2.
Forward primer I-IUMCDDANFjunc2-SF2 (SEQ ID N0:374):
CTTCTCCACCACCACCAATTTG
Reverse primer HUMCDDANFjunc2-SR2 (SEQ 1D N0:375):
GAGAGCAGCCCCCGCT
Amplicon HUMCDDANFjunc2-SF2R2 (SEQ ID N0:376):
CTTCTCCACCACCACCAATTTGCTGGACCATTTGGAAGAAAAGATGCCTTTAGAAG
ATGAGGTCGTGCCCCCACAAGTGCTCAGTGAGCCGAATGAAGAAGCGGGGGCTGCT
CTC
DESCRIPTION FOR CLUSTER HUMTROPIA
Cluster HUMTROPIA features 4 transcripts) and 20 segments) of interest, the names for which are given in Tables 1 and 2, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 3.
Table I - Transcripts of interest ~~.",F&..dm~~ 2 6. ~;~ ~ . ' . ~", I ~, '~.. ~F ~, 41~ ~' ~i~~ ~~'~~ a '~~'~'' r t J ~ t~'~ ~ ~ k , ~~q ~ a' ~ ~= r _.::~ ~: _;._. W . ... _~ .. . r_W.
..

Table 2 - Segments of interest i ~i ~ :~grt,~ ~.n '~ ~aa~n ~ ~LIIi s i ~~o ,~i ~~ I I ylll I ' i~ ~ ~t~

i' __ HUMTROPIA PEA 2 node 0 130 HUMTROPIA PEA 2 node 10 131 HUMTROPIA PEA 2 node 22 132 node node node node HUMTROPIA PEA' node16 137 node node node node node node node node node node node node Table 3 - Proteins of interest ~,otem ~l' . Se ~ IinD

These sequences are variants of the known protein Troponin I, cardiac muscle (SwissProt accession identifier TRIC HUMAN), referred to herein as the previously known protein and shown as SEQ ID NO: 351.
Protein Troponin I, cardiac muscle is known or believed to have the following function(s): Troponin I is the inhibitory subunit of troponin, the thin filament regulatory complex which confers calcium-sensitivity to striated muscle actomyosin ATPase activity.
Troponin I, cardiac muscle Binds to actin and tropomyosin. Defects in Troponin I, cardiac muscle are the cause of familial hypertrophic cardiomyopathy type 7 (CMH7) [YlIM:191044];
also known as FHC type 7. CMH7 is an autosomal dominant disorder characterized by increased myocardial mass with myocyte and myofibrillar disarray. Defects in Troponin I, cardiac muscle are the cause of familial restrictive cardiomyopathy (RCM) ~VIIM:115210].
RCM is a heart muscle disorder characterized by impaired filling of the ventricles with reduced volume in the presence of normal or near normal wall thickness and systolic function. The disease may be associated with systemic disease but is most often idiopathic.
The sequence for protein Troponin I, cardiac muscle is given at the end of the application, as "Troponin I, cardiac muscle amino acid sequence" (SEQ ID N0:351). Known polymorphisms for this sequence are as shown in Table 4.
Table 4 - Amino acid mutations for Known Protein - ~~,~ s''~a~~
SNP po ~Ltio - ,'S~ il~, ~omriierl '~ J~ " ,~ '~~' ~ ~ ~~
--~~~:z' ~M~-~ ~ _~~ ~.
mino aci S Gj - . G - _ ,~ .a...,.
81 x P -> S (in CMH7). /FTId=VAR 016078.
143 L -> Q (in RCM). /FTId=VAR 016079.
144 R -> G (in CMH7). /FTId=VAR 007603.
144 R -> W (in RCM). /FTId=VAR O l 6080.
170 A -> T (in RCM). /FTId=VAR 016081.
177 K -> E (in RCM). /FTId=VAR 016082.
189 D -> H (in CMH7 and RCM). /FTId=VAR 016083.
191 R -> H (in RCM). /FTId=VAR O I 6084.
195 D -> N (in CMH7). /FTId=VAR 016085.
205 K -> Q (in CMH7). /FTId=VAR 007604.
In addition to the above known polymorphisms, the present inventors have uncovered two new additional SNPs (shown with regard to SEQ ID N0:352 for the resultant amino acid sequence, and SEQ ID N0:353 for the nucleic acid sequence). This SNP is C->
(missing nucleotide "C"; will affect amino acid residues from 167 onwards). This will create a frame shift. A new protein will be formed. However, this SNP was located in a stretch of cytosine residues, which are known to be prone to errors in sequencing.
The previously known protein also has the following indications) and/or potential therapeutic use(s): Cancer, lung, norrsmall cell; Cancer, breast; Cancer, sarcoma. It has been investigated for clinical/therapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein are as follows: Angiogenesis inhibitor; Epidermal growth factor antagonist; Fibroblast growth factor receptor antagonist. A
therapeutic role for a protein represented by the cluster has been predicted. The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication:
Ophthalmological; Anticancer.
The following GO Annotations) apply to the previously known protein. The following annotations) were found: control of heart, which are annotations) related to Biological Process;
and troponin complex, which are annotations) related to Cellular Component.
The GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from <http://www.expasy.ch/sprot/>; or Locuslink, available from <http://www.ncbi.nlm.nih.gov/projects/LocusLink/>
The heart selective diagnostic marker prediction engine provided the following results with regard to cluster HUMTROPIA. Predictions were made for selective expression of transcripts of this cluster in heart tissue, according to the previously described methods. The numbers on the taxis of Figure 19 refer to weighted expression of ESTs in each category, as "parts per million" (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
Overall, the following results were obtained as shown with regard to the histogram in Figure 19, concerning the number of heart-specific clones in libraries/sequences; as well as with regard to the histogram in Figure 20, concerning the actual expression of oligonucleotides in various tissues, including heart.

This cluster was found to be selectively expressed in heart for the following reasons: in a comparison of the ratio of expression of the cluster in heart specific ESTs to the overall expression of the cluster in norrheart ESTs, which was found to be 27.5. The expression level of this gene in muscle was negligible; and fisher exact test P-values were computed both for library and weighted clone counts to check that the counts are statistically significant, and were found to be 2.10E-88.
One particularly important measure of specificity of expression of a cluster in heart tissue is the previously described comparison of the ratio of expression of the cluster in heart as opposed to muscle. This cluster was found to be specifically expressed in heart as opposed to nocrheart ESTs as described above. However, many proteins have been shown to be generally expressed at a higher level in both heart and muscle, which is less desirable.
For this cluster, as described above, the expression level of this gene in muscle was negligible which clearly supports specific expression in heart tissue.
As noted above, cluster HUMTROPIA features 4 transcript(s), which were listed in Table 1 above. These transcripts) encode for proteins) which are variants) of protein Troponin 1, cardiac muscle. A description of each variant protein according to the present invention is now provided.
Variant protein HUMTROPIA PEA 2 PS according b the present invention has an amino acid sequence as given at the end of the application; it is encoded by transeript(s) HUMTROPIA PEA 2 T3. An alignment is given to the known protein (Troponin I, cardiac muscle) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMTROPIA PEA 2 PS and TRIC HUMAN:
l.An isolated chimeric polypeptide encoding for HUMTROPIA PEA 2 P5, comprising a first amino acid sequence being at least 90 % homologous to MADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLKTLLLQIAKQ
ELEREAEERRGEKGRALSTRCQPLELAGLGFAELQDLCRQLHARVDKVDEERYDIEAK

VTKNITE corresponding to amino acids 1 - 124 of TRIC HUMAN, which also corresponds to amino acids 1 - 124 of HUMTROPIA PEA 2 P5, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VGRMGSSGTFGVG corresponding to amino acids 125- 137 of HUMTROPIA PEA 2 P5, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMTROPIA PEA 2 P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VGRMGSSGTFGVG in HUMTROPIA PEA 2 P5.
The cellular location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: intracellularly. The protein localization is believed to be intracellular because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a non-secreted protein..
Variant protein HUMTROPIA PEA 2 PS is encoded by the following transcript(s):
HUMTROPIA PEA 2 T3, for which the sequences) is/are given at the end of the application.
The coding portion of transcript HUMTROPIA PEA 2 T3 is shown in bold; this coding portion starts at position 148 and ends at position 558.
Variant protein HUMTROPIA PEA 2 P12 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) HUMTROPIA PEA 2 TIS. An alignment is given to the known protein (Troponin I, cardiac muscle) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMTROPIA PEA 2 P12 and TRIC_HUMAN:

I.An isolated chimeric polypeptide encoding for HUMTROPIA-PEA-2 P12, comprising a first amino acid sequence being at least 90 % homologous to MADGSSDA
corresponding to amino acids 1 - 8 of TRIC-HUMAN, which also corresponds to amino acids 1 - 8 of HUMTROPIA PEA 2 P 12, and a second amino acid sequence being at least 70%, optionally S at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at bast 95% homologous to a polypeptide having the sequence KKSKISASRKLQLKTLLLQIAKQELEREAEERRGEKGRALSTRCQPLELAGL

RISADAMMQALLGARAKESLDLRAHLKQVKKEDTEKENREVGDWRKNIDALSGMEG
RKKKFES corresponding to amino acids 36 - 209 of TRIC HUMAN, which also corresponding to amino acids 9 - 182 of HUMTROPIA PEA 2 P 12, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated chimeric polypeptide encoding for an edge portion of HUMTROPIA PEA 2 P12, comprising a polypeptide having a length "n", wherein "n"
is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise AK, having a structure as follows: a sequence starting from any of amino acid numbers 8-x to 8; and ending at any of amino acid numbers 9+ ((rr2) - x), in which x varies from 0 to rr2.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the traps-membrane region prediction programs predicted a traps-membrane region for this protein.
In addition both signahpeptide prediction programs predict that this protein is a non-secreted protein..
Variant protein HUMTROPIA PEA 2 P12 is encoded by the following transcript(s):
HUMTROPIA PEA 2 T15, for which the sequences) is/are given at the end of the application. The coding portion of transcript HUMTROPIA PEA 2 T1S is shown in bold; this coding portion starts at position 148 and ends at position 693.
Variant protein HUMTROPIA PEA 2 P 17 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) S HUMTROPIA PEA 2 T7. An alignment is given to the known protein (Troponin I, cardiac muscle) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMTROPIA PEA 2 P 17 and TRIC HUMAN:
l.An isolated chimeric polypeptide encoding for HUMTROPIA PEA 2 P17, comprising a first amino acid sequence being at least 90 % homologous to MADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAK corresponding to amino acids 1 - 36 of TRIC HUMAN, which also corresponds to amino acids 1 - 36 of HUMTROPIA PEA 2 P17, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 8S%, more preferably at least 90% and most preferably at least 9S% homologous to a polypeptide having the sequence VGRGFLGAEYRRRRDPRPWEWGEEPGLRRGRGLRGGASGAEFCRGSCSDW
corresponding to amino acids 37- 86 of HUMTROPIA PEA 2 P17, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMTROPIA PEA 2 P17, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the 2S sequence VGRGFLGAEYRRRRDPRPWEWGEEPGLRRGRGLRGGASGAEFCRGSCSDW
in HUMTROPIA PEA 2 P 17.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellular because neither of the traps-membrane region prediction programs predicted a traps-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a norrsecreted protein..
Variant protein HUMTROPIA PEA 2 P17 is encoded by the following transcript(s):
HUMTROPIA PEA 2 T7, for which the sequences) is/are given at the end of the application.
The coding portion of transcript HUMTROPIA PEA 2 T7 is shown in bold; this coding portion starts at position 148 and ends at position 405.
Variant protein HUMTROPIA PEA 2 P18 according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcripts) HUMTROPIA PEA 2 T10. An alignment is given to the known protein (Troponin I, cardiac muscle) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
Comparison report between HUMTROPIA PEA 2 P18 and TRIC HUMAN:
l.An isolated chimeric polypeptide encoding for HUMTROPIA PEA 2 PIB, comprising a first amino acid sequence being at least 90 % homologous to MADGSSDA
corresponding to amino acids 1 - 8 of TRIC HUMAN, which also corresponds to amino acids 1 - 8 of HUMTROPIA PEA 2 P 18, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRAAG corresponding to amino acids 9- 13 of HUMTROPIA PEA 2 P18, wherein said first and second amino acid sequences are contiguous and in a sequential order.
2.An isolated polypeptide encoding for a tail of HUMTROPIA PEA 2 P 18, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VRAAG in HUMTROPIA PEA 2 P18.
The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell:
intracellularly. The protein localization is believed to be intracellularly because neither of the trans-membrane region prediction programs predicted a trans-membrane region for this protein.
In addition both signal-peptide prediction programs predict that this protein is a norrsecreted protein..
Variant protein HUMTROPIA PEA 2 P18 is encoded by the following transcript(s):
HUMTROPIA PEA 2 T10, for which the sequences) is/are given at the end of the application. The coding portion of transcript HUMTROPIA PEA 2 T10 is shown in bold; this coding portion starts at position 148 and ends at position 186.
As noted above, cluster HUMTROPIA features 20 segment(s), which were listed in Table 2 above and for which the sequences) are given at the end of the application.
These segments) are portions of nucleic acid sequences) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
Segment cluster HUMTROPIA PEA 2 node 0 according to the present invention is supported by 29 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 TIO, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 7 below describes the starting and ending position of this segment on each transcript.
Table 7 - Segment location on transcripts ! ~V3~~~~~ilr~~~1 anscrip , , a ' a ~ E ; ~ . : egt~ne en ~ f ~g o e~.t - p~sit~o ~n ; , , p ~
j,~,u~~y~,.

~~~~wM~
HUMTROPIA PEA T10 l 158 Segment cluster HUMTROPIA PEA 2 node-10 according to the present invention is supported by S libraries. The number of libraries was determined as previously described. This segment can be found in the following transcripc(s): HUMTROPIA-PEA 2 T7. Table 8 below describes the starting and ending position of this segment on each transcript.
Table 8 - Segment location on transcripts TranSCrl t:riame.:: =Se ent startin : Se enteiZdin ~ .ositioai : osition..~_ . =-~
p ~ - ~
g ...... .~ . _ _ _.., ~' _ ~-Segment cluster HUMTROPIA PEA 2 node 22 according to the present invention is supported by 8 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T3. Table 9 below describes the starting and ending position of this segment on each transcript.
Table 9 - Segment location on transcripts r,; ~:..,- _. ~ ~. ~ _ mss- .,ssrs.
. ~ ~ ~~
sew . t na ~~' qx,~ :.. ~._~,".. <,~ Se ment.endin ~ ~ . ~ ~Se entWstartm ~ itinn t~sitioii ,ran.y p ~'~~-~ ~ f ~~~ , g _ _~~~g p ~ ~
~

Segment cluster HUMTROPIA PEA 2 node 23 according to the present invention is supported by 49 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 10 below describes the starting and ending position of this segment on each transcript.
Table 10 - Segment location on transcripts ~;;~ ~ ~; ~,f~e.,~~,,~vwl ~ 9~~.t , ~~~il ame T ~ ~r mnt~tartrng~tpt~~t ~~w~~~~:,~u ,n ~egmen i tug . ~,.
~to _.._ According to an optional embodiment of the present invention, short segments related to the above cluster are also provided. These segments are up to about 120 by in length, and so are included in a separate description.
Segment cluster HUMTROPIA PEA 2 node-11 according to the present invention is supported by 28 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 11 below describes the starting and ending position of this segment on each transcript.
I 0 Table Il - Segment location on transcripts -Se men c =un = a invent-~endiii 'T~ranscn t.name e~sihori~ .-~ ositnm - g t start g~ S g g P
o ~ ~P

HUMTROPIA PEA T7 661 ~ 702 Segment cluster HUMTROPIA PEA 2 node-14 according to the present invention is supported by 37 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 12 below describes the starting and ending position of this segment on each transcript.
Table 12 - Segment location on transcripts ~G':wl I~I I ~I ~'~I ~ I~~ ~~j,,,~V~U~f;
cr< t'~n ~r, a ~~ : a < en ~ ='n ~egmen y ~t~an ~f~:QS~..
u, i ~ os~t ~ on <t ~ i ~ ~~~ I i ;~~~ ~, ! _~, ~~~; I ~i~ 9 ~ ~i~

Segment cluster HUMTROPIA PEA 2 node_15 according to the present invention is supported by 42 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 13 below describes the starting and ending position of this segment on each transcript.
Table 13 - Segment location on transcripts ._~- ~ ..
~. -~lWianscr~-t'n a _....._Se .-: ent,srartXnSe .m nt.eridtri .
~.~I?A . ~ ~ . ~ -,~ositioii...... .asition..~~
,. .. _......, . - b~ ~p ~ l~ p~, - -,:-. =~ . ~,._rv- . :~ ,_~ . _x._. ....~~ _.
;,-~- ... _. a ..,..~ _.._.... :
.._~ . _.. .-w HUMTROPIA PEA 2 T7 739 ~ 782 Segment cluster HUMTROPIA PEA 2 node_16 according to the present invention is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA_PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 14 below describes the starting and ending position of this segment on each transcript.
Table 14 - Segment location on transcripts ,. R ~' y~ ~~ ~i~,~~,..'g, a n c~ < name _,; ~ ~ Y~ ~ a ~ raging.. ..~~h~
poi Rio - '' ,. ~/~/t~;/~~ Wu ~' t gel dln'~g~p~a~,,siti ,m~
J

HUMTROPIA PEA T 423 ~

Segment cluster HUMTROPIA PEA 2 node 20 according to the present invention is supported by 44 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 TIS, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 15 below describes the starting and ending position of this segment on each transcript.
Table I S - Segment location on transcripts t ~a- , ;" ~, h ,_~..
H ~ ~ ._ e nt atartin osltion .'Se imeiitemdin Transcnpt,~name '- osirion a -.~"a:ra~_. :~". ~~~,.>.m.,m"...'a.gme g p g g p ._ .",.,-. .-.r_ _....a ' _.:- .._>.. w.rv _,.,........
, . __.5:;. ~ ,: .._...,.. .,.a~~-S . ,-.~m~.~,.'r 5 Segment cluster HUMTROPIA PEA 2 node 21 according to the present invention is supported by 44 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 16 below describes the starting and ending position of this segment on each transcript.
10 Table 16 - Segment location on transcripts cep ~, a S H.gmei~t startin r-a .merit endings o its n tion HUMTROPIA PEA 2 871 ~ 924 Segment cluster HUMTROPIA PEA 2 node 24 according to the present invention can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 17 below describes the starting and ending position of this segment on each transcript.
Table 17 - Segment location on transcripts PEA

Segment cluster HUMTROPIA PEA 2 node 25 according to the present invention can be found in the following transcript(s): HUMTROPIA PEA 2 T 10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 18 below describes the starting and ending position of this segment on each transcript.
Table I8 - Segment location on transcripts eE4Y,S."~ .;,ate Tra~u~sc = - namer~ Se v ent ~startm ~" ~ ~ ositlon~ . , g ~ P
,~ --~ ~ 1; P ~.~ ~Se mentmenduz osWon .

HUMTROPIA PEA 2 T7 1087 ~ 1101 Segment cluster HUMTROPIA PEA 2 node 29 according to the present invention can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 19 below describes the starting and ending position of this segment on each transcript.
Table 19 - Segment location on transcripts a... , .;
T P ~ ; , : egment startyg , eg, . . en a . , p4s1 aiqn , , ,'; .'on HUMTROPIA_PEA 2 T7 1102 ~ 1121 Segment cluster HUMTROPIA PEA-2_node 30 according to the present invention can be found in the following transcript(s): HUMTROPIA PEA 2 T 10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 20 below describes the starting and ending position of this segment on each transcript.
Ta6le 20 - Segment location on transcripts ;,- ~~ ~, e;~ .:~.c.. ~ . w .:
. n ~ iti : . ~'_ _.Transez~ .t name Se ~ ent start os a W eint-= 1: n ~: on S endui~ w osit o HUMTROPIA PEA 2 T7 1122 ~ 1134 Segment cluster HUMTROPIA PEA 2 node 31 according to the present invention can be found in the following transcript(s): HUMTROPIA PEA 2 T 10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 21 below describes the starting and ending position of this segment on each transcript.
Table 21 - Segment location on transcripts :ranscii ~ t name S~gment~st~ osition, Sel; ent a . i_ng ~ ~. x os~it~ an ~ ~~
Hh Segment cluster HUMTROPIA PEA 2 node 32 according to the present invention is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA-PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 22 below describes the starting and ending position of this segment on each transcript.

Table 22 - Segment location on transcripts . raiiscri 't.narne Se '' ei~c startni ~ : osition.. ~ A.
Se merit endin osxtion . ~ p -~ ,,. ' ~_ _~__.. ~ . ',~ ~_p_ ... -, g _ ; -~ ~ . , , . . . _..- ., T :

Segment cluster HUMTROPIA PEA 2 node 4 according to the present invention can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T15, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 23 below describes the starting and ending position of this segment on each transcript.
Table 23 - Segment location on transcripts yran eri ~t atne _P en startm ~ osttion;HSe went ~ndu~ .osition ~~ , . ' ~~~ . 3 ~ ~, ~~~~~ e, .. g.~-~ ~ 2~ g p p , .t y 5~ ro ~, s..3'N~~ ~' ,~ ' & . , y3 - ~
i.;~ ~ , ..' t,"~ aV...~ ~~...;f F

Segment cluster HUMTROPIA PEA 2 node S according to the present invention is supported by 6 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T 10.
Table 24 below describes the starting and ending position of this segment on each transcript.
Table 24 - Segment location on transcripts Segment cluster I-IUMTROPIA PEA 2 node-8 according to the present invention is supported by 27 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, I-IUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 25 below describes the starting and ending position of this segment on each transcript.
Table 25 - Segme~rt location on transcripts -, =~=- -~sfarhn w ositioiiSe erit..endiii ~-Transcript name Segment gp osition ' gm g p ~.._ , -~ ~ -~~ ~v ~r....:'~ ~~~ .. :-.. m _. e~.~..W.
r,..F~,.~Y_:.~ ~=.~-:

Segment cluster HUMTROPIA PEA 2 node 9 according to the present invention is supported by 27 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HUMTROPIA PEA 2 T10, HUMTROPIA PEA 2 T3 and HUMTROPIA PEA 2 T7. Table 26 below describes the starting and ending position of this segment on each transcript.
Table 26 - Segment location on transcripts anscriplnasne ~~; .~~..g ; en = ,a a ent endlng'~os-~~ici 3iw ,;posttip . a~~
~~.~ , ,r i , ~ .. .

y,~ ~ a p . ~w.~~,~

Variant protein alignment to the previously known protein:
Sequence name: /tmp/pSCHmauP3N/NVyK809uFt:TRIC HUMAN
Sequence documentation:
Alignment of: HUMTROPIA-PEA-2_P5 x TRIC_HUMAN ..

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COMPREND PLI1S D'UN TOME.

NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
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NOTE: For additional volumes please contact the Canadian Patent O~'ice.

Claims (37)

1. An isolated polynucleotide comprising a transcript selected from the group consisting of SEQ ID NOs: 22-25, 353 or 386, or a polynucleotide at least about 95%
homologous thereto.
2. An isolated polynucleotide comprising a segment selected from the group consisting of SEQ ID NOs: 130-149, or a polynucleotide at least about 95%
homologous thereto.
3. An isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID NOs: 301-304, 325, 354-356 or 387, or a polypeptide at least about 95%
homologous thereto.
4. An isolated chimeric polypeptide encoding for SEQ ID NO. 301, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 -124 of TRIC_HUMAN, which also corresponds to amino acids 1 - 124 of SEQ ID NO. 301, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 125- 137 of SEQ ID NO. 301, wherein said first and second amino acid sequences are contiguous and in a sequential order.
5. An isolated polypeptide encoding for a tail of SEQ ID NO. 301, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VGRMGSSGTFGVG in SEQ ID NO. 301.
6. An isolated chimeric polypeptide encoding for SEQ ID NO. 302, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 8 of TRIC_HUMAN, which also corresponds to amino acids 1 - 8 of SEQ ID NO. 302, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 36 - 209 of TRIC_HUMAN, which also corresponding to amino acids 9 - 182 of SEQ ID NO. 302, wherein said first and second amino acid sequences are contiguous and in a sequential order.
7. An isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO.
302, comprising a polypeptide having a length "n", wherein "n" is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise AK, having a structure as follows: a sequence starting from any of amino acid numbers 8-x to 8; and ending at any of amino acid numbers 9+ ((n-2) - x), in which x varies from 0 to n-2.
8. An isolated chimeric polypeptide encoding for SEQ ID NO. 303, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 36 of TRIC_HUMAN, which also corresponds ~ amino acids 1 - 36 of SEQ ID NO. 303, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 37- 86 of SEQ ID NO. 303, wherein said first and second amino acid sequences are contiguous and in a sequential order.
9. An isolated polypeptide encoding for a tail of SEQ ID NO. 303, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VGRGFLGAEYRRRRDPRPWEWGEEPGLRRGRGLRGGASGAEFCRGSCSDW
in SEQ ID NO. 303.
10. An isolated chimeric polypeptide encoding for SEQ ID NO. 304, comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 8 of TRIC_HUMAN, which also corresponds to amino acids 1 - 8 of SEQ ID NO. 304, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 9- 13 of SEQ ID NO. 304, wherein said first and second amino acid sequences are contiguous and in a sequential order.
11. An isolated polypeptide encoding for a tail of SEQ ID NO. 304, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95%
homologous to the sequence VRAAG in SEQ ID NO. 304.
12. An isolated oligonucleotide, comprising an amplicon selected from the group consisting of SEQ ID NOs: 379, 382 or 385.
13. A primer pair, comprising a pair of isolated oligonucleotides capable of amplifying said amplicon of claim 12.
14. The primer pair of claim 13, comprising a pair of isolated oligonucleotides selected from the group consisting of: SEQ NOs 377 and 378; 380 and 381; or 383 and 384.
15. An antibody capable of specifically binding to an epitope of an amino acid sequence of any of claims 3-11.
16. The antibody of claim 15, wherein said amino acid sequence comprises one of:
a tail of SEQ ID NO. 301, comprising a polypeptide being at least 70 homologous to the sequence VGRMGSSGTFGVG in SEQ ID NO. 301;
a tail of SEQ ID NO. 303, comprising a polypeptide being at least 70%
homologous to the sequence VGRGFLGAEYRRRRDPRPWEWGEEPGLRRGRGLRGGASGAEFCRGSCSDW in SEQ ID
NO. 303;
a tail of SEQ ID NO. 304, comprising a polypeptide being at least 70%
homologous to the sequence VRAAG in SEQ ID NO. 304; or an edge portion of SEQ ID NO. 302, comprising a polypeptide having a length "n", wherein "n" is at least about 10 amino acids in length, wherein at least two amino acids comprise AK, having a structure as follows: a sequence starting from any of amino acid numbers 8-x to 8; and ending at any of amino acid numbers 9+ ((n2) - x), in which x varies from 0 to n-2.
17. The antibody of claims 15, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein TRIC_HUMAN.
18. A kit for detecting heart disorders, comprising a kit detecting overexpression of a splice variant according to any of claims 1-11.
19. The kit of claim I 8, wherein said kit comprises a NAT-based technology.
20. The kit of claim 19, wherein said kit further comprises at least one primer pair capable of selectively amplifying the nucleic acid sequence.
21. The kit of claim 18, wherein said kit further comprises at least one oligonucleotide capable of selectively hybridizing to the nucleic acid sequence.
22. A kit for detecting heart disorders, comprising a kit an antibody according of claim 15.
23. The kit of claim 22, wherein said kit further comprises at least one reagent for performing an ELISA or a Western blot.
24. A method for detecting heart disorders, comprising detecting overexpression of a splice variant according to any of claims 1-11.
25. The method of claim 24, wherein said detecting overexpression is performed with a NAT-based technology.
26. The method of claim 24, wherein said detecting overexpression is performed with an immunoassay.
27. The method of claim 26, wherein said immunoassay comprises an antibody.
28. A biomarker capable of detecting heart disorders, comprising the nucleic acid sequences or a fragment thereof, or amino acid sequences or a fragment thereof of any of claims 1-12.
29. A method for screening for heart disorders, comprising detecting heart disorder cells using the biomarkers or antibodies of any of claims 1-12.
30. A method for diagnosing heart disorders, comprising detecting heart disorder cells using the biomarkers or antibodies of any of claims 1-12.
31. A method for monitoring disease progression, or treatment efficacy, or relapse of heart disorders, or any combination thereof, comprising detecting heart disorder cells using the biomarkers or antibodies or a method or assay according to any of claims 1-12.
32. A method of selecting a therapy for heart disorders, comprising detecting heart disorder cells with any of the above biomarkers or antibodies or a method or assay according to any of claims 1-12 and selecting a therapy according to said detection.
33. A method according to claim 28, wherein a heart disorder and/or cardiac disease and/or cardiac pathology comprises at least one of: Myocardial infarct, ungina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, or assessing the size of infarct in Myocardial infarct.
34. A method according to claim 29, wherein a heart disorder and/or cardiac disease and/or cardiac pathology comprises at least one of: Myocardial infarct, ungina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, or assessing the size of infarct in Myocardial infarct.
35. A method according to claim 30, wherein a heart disorder and/or cardiac disease and/or cardiac pathology comprises at least one of: Myocardial infarct, ungina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, or assessing the size of infarct in Myocardial infarct.
36. A method according to claim 31, wherein a heart disorder and/or cardiac disease and/or cardiac pathology comprises at least one of: Myocardial infarct, ungina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, or assessing the size of infarct in Myocardial infarct.
37. A method according to claim 32, wherein a heart disorder and/or cardiac disease and/or cardiac pathology comprises at least one o~ Myocardial infarct, ungina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, or assessing the size of infarct in Myocardial infarct.
CA002554585A 2004-01-27 2005-01-27 Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of cardiac disease Abandoned CA2554585A1 (en)

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US53912804P 2004-01-27 2004-01-27
US53912904P 2004-01-27 2004-01-27
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US60/620,916 2004-10-22
US62113104P 2004-10-25 2004-10-25
US60/621,131 2004-10-25
US62232004P 2004-10-27 2004-10-27
US60/622,320 2004-10-27
US62813404P 2004-11-17 2004-11-17
US62812304P 2004-11-17 2004-11-17
US62819004P 2004-11-17 2004-11-17
US60/628,134 2004-11-17
US60/628,190 2004-11-17
US60/628,123 2004-11-17
US63055904P 2004-11-26 2004-11-26
US60/630,559 2004-11-26
PCT/IB2005/001306 WO2005069724A2 (en) 2004-01-27 2005-01-27 Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of cardiac disease
US11/043,788 2005-01-27
US11/043,788 US20060014166A1 (en) 2004-01-27 2005-01-27 Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of endometriosis

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