Classifications

• • 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

Definitions

• the present invention is related to novel nucleotide and protein sequences that are diagnostic markers for cardiac disease and/or pathological conditions, including cardiac damage, and assays and methods of use thereof.
• 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 (including 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.
• CPK-MB 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.
• cardiac surgery, myocarditis, and electrical cardioversion often result in elevated serum levels ofthe CPK-MB isoenryme. 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 (17kD) protem 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.
• 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 cTnl (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.
• Cardiac troponin's sensitivity is considerably higher than CPK-MB but they suffer from a few disadvantages: 1. They are not early markers - cTnl and cTnT reach peak serum value in about 12 and 48 hours respectively after symptoms onset. 2. Levels of cTnl 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.
• H-FABP Heart Fatty Acid binding protein
• the heart specific variant H-FABP is a low molecular protein (15Kd) 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.
• HFABP 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, H-FABP still misses every 5f 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.
• 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.
• 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.
• 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.
• signalpjimm 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. Localization was also detennined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor.
• T - > C means that the SNP results in a change at the position given in the table from T to C.
• M - > Q 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 (*).
• 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 header of the first column is "SNP position(s) on amino acid sequence", representing a position of a known mutation on amino acid sequence.
• 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.
• microarray 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.
• tissue name was used as the reference to the type of chip for which expression was measured.
• 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.affymeftix.com products/arrays/specific/hgul33.affx; GeneChip Human Genome U133A 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33av2.affx; and Human Genome U133 Plus 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33plus.affx).
• the probe names follow the Affymetrix naming convention.
• ADP adipocyte
• BM bone marrow
• BRS mammary gland
• CAR cartilage
• CNS central nervous system
• E-ADR endocrine_adrenal_gland
• E-PAN endocrine_pancreas
• HN head and neck
• LN lymph node
• PNS peripheral nervous system
• TCELL immune T cells
• THYM thymus
• adipocyte "Al”; “adrenalcortex”, “A2”; “adrenalgland”, “A3”; “amygdala”, “A4"; “appendix”, “A5"; “atrioventricularnode”, “A6”; “bm_cdl 05_endothelial”, “El”; “bm_cd33_myeloid”, “Ml”;
• testisseminiferoustubule , "S6";
• nucleic acid sequences of the present invention refer to portions of nucleic acid sequences that were shown to have one or more properties as described below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below.
• ohgonucleotides which are embodiments of the present invention, for example as amplicons, hybridization units and/or from which primers and/or complementary ohgonucleotides may optionally be derived, and/or for any other use.
• 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 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.
• marker in the context of the present invention lefers 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.
• a cardiac disease such as acute cardiac damage for example
• the phrase “differentially present” refers to differences in the quantity of a marker present in a sample talcen from patients having cardiac disease as compared to a comparable sample taken from patients who do not have cardiac disease.
• 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.
• a marker such as a protein or fragment thereof
• a marker 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.
• 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 detennine such relative levels of the markers; further guidance is provided in the description of each individual marker below.
• 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.
• Diagnosing refers to classifying a disease or a symptom, determining a severity ofthe disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
• the term “detecting” may also optionally encompass any ofthe 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.
• 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.
• the term "level” refers to expression levels of RNA and/or protein or to DNA copy number of a marker ofthe present invention.
• 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 ofthe 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.
• 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.
• 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.
• useful labels include 32 P, 35 S, 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, chromogenic, 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.
• 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. Fahr lander 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 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.
• 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 ofthe 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, and/or quantify the antigen.
• the specified antibodies bind to a particular protein at least two times greater than the background (non-specific 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.
• polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protem 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.
• immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
• solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A
• a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
• a isolated polynucleotide comprising a transcript selected from the group consisting of SEQ ID NO: 1
• an isolated polynucleotide comprising a segment selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
• an isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID NOs: 281, 282, 283 and 284.
• an isolated polynucleotide comprising a transcript selected from the group consisting of SEQ ID NOs: 281, 282, 283 and 284.
• an isolated polynucleotide comprising a segment selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9 and 10 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: 5, 6, 7, 8, 9 and 10 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
• an isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID NOs: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 and 94 .
• an isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID NOs: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 and 94 .
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 12, 13, 14, 15, 16 and 17
• an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112.
• an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 292, 293, 294, 295 and 296
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 18 and 19.
• 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.
• an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 297 and 298.
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 20 and 21.
• an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 123, 124, 125, 126, 127, 128 and 129.
• an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 299 and 300.
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 26, 27, 28, 29 and 30.
• ati isolated polynucleotide comprising a segment SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162 and 163.
• an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 305; 306; 307 and 308
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 31, 32, 33, 34, 35, 36 and 37.
• an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185 and 186
• an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 309, 310, 311 and 312.
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 38, 39, 40 and 41.
• an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 187, 188, 189, 190, 191, 192, 193, 194, 195 and 196.
• ⁇ isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 313, 314, 315 and 316.
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49 and 50.
• an 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.
• an isolated polypeptide comprising a protein variant SELECTED FROM THE GROUP
• an isolated polynucleotide comprising a transcript SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs:51, 52, 53, 54, 55, 56, 57, 58, 59 and 60.
• an isolated polynucleotide comprising a segment SELECTED FROM THE GROUP CONSISTING OF SEQ ID NOs: 209 to 273.
• an isolated polypeptide comprising a protein variant selected from the group consisting of SEQ ID NOs: 326 to 334.
• ai isolated polynucleotide comprising a transcript selected from the group consisting of SEQ ID NOs: 22-25, 353 or 386.
• an isolated polynucleotide comprising a segment selected from the group consisting of SEQ ID NOs: 130-149.
• 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 prefe ⁇ ed embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for SEQ ID NO.
• a first amino acid sequence being at least 90 % homologous to amino acids 1 - 1855 of SEQ ID NO.338, which also co ⁇ esponds to amino acids 1 - 1855 of SEQ ID NO.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.
• 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.
• 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 co ⁇ esponds to amino acids 1 - 1326 of SEQ ID NO.
• 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 co ⁇ esponding 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.
• 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.
• an isolated chimeric polypeptide encoding for SEQ ID NO. 328 comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 - 1508 of SEQ ID NO. 339, which also co ⁇ esponds to amino acids 1 - 1508 of SEQ ID NO.
• 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 co ⁇ esponding 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.
• an isolated chimeric polypeptide encoding for SEQ ID NO. 329 comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 - 1763 of SEQ ID NO. 338, which also co ⁇ esponds to amino acids 1 - 1763 of SEQ ID NO.
• 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 co ⁇ esponding to amino acids 1764 - 1788 of SEQ ID NO. 329, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
• 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%o, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence co ⁇ esponding to amino acids 1 - 22 of SEQ ID NO.
• 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.
• 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 co ⁇ esponds to amino acids 1 - 527 of SEQ ID NO.
• 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 co ⁇ esponding 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.
• polypeptide 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.
• an isolated chimeric polypeptide encoding for SEQ ID NO.332 comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 - 470 of SEQ ID NO.338, which also co ⁇ esponds 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 NO.338, which also co ⁇ esponds to amino acids 471 - 1798 of SEQ ID NO.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 co ⁇ esponding to amino acids 1799 - 1847 of SEQ ID NO.332, wherein said first amino acid sequence, second amino acid sequence and third amino acid sequence are contiguous and in
• an isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO.332, 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 ainino 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.
• an isolated chimeric plypeptide 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 co ⁇ esponds to amino acids 1 - 1775 of SEQ ID NO.333.
• an isolated chimeric polypeptide encoding for SEQ ID NO.334 comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1165 - 1939 of SEQ ID NO.
• 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 co ⁇ esponds to amino acids 1 - 158 of SEQ ID N0.317.
• an 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.
• 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 least about 90% and most preferably at least about 95% homologous to the sequence
• VSVGQECGSG in SEQ ID NO.318.
• 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.
• an isolated polypeptide encoding for a tail of SEQ ID NO.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 NO.319 .
• a first amino acid sequence being at least 90 % homologous to amino acids 1 - 60 of Q96NR4, which also co ⁇ esponds 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 co ⁇ esponding 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.
• 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 co ⁇ esponds to amino acids 1 - 60 of SEQ ID NO.
• a second amino acid sequence bridging amino acid sequence comprising of S and a third amino acid sequence being at least 90 % homologous to co ⁇ 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.
• 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. 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.
• 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 co ⁇ esponding to amino acids 1 - 15 of SEQ ID NO. 321, and a second amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 39 - 110 of SEQ ID NO. 343, which also co ⁇ esponds 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.
• 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. 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
• ai isolated polypeptide encoding for a tail of SEQ ID 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.
• first amino acid sequence being at least 90 % homologous to conesponding to amino acids 1 - 14 of SEQ ID NO. 342, which also co ⁇ esponds 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 co ⁇ esponding to amino acids 62 - 133 of SEQ ID NO. 342, which also co ⁇ esponds 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.
• 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 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.
• 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.
• 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.
• a second amino acid sequence bridging amino acid sequence comprising of S and a third amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 158 - 229 of SEQ ID NO. 341, which also co ⁇ esponds 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.
• 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. 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.
• 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 preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence co ⁇ esponding to amino acids 1 - 23 of SEQ ID NO.
• an isolated chimeric polypeptide encoding for SEQ ID NO. 322. comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 97 - 158 of SEQ ID NO. 341., which also co ⁇ esponds to amino acids 1 - 62 of SEQ ID NO. 322..
• an isolated chimeric polypeptide encoding for SEQ ID NO. 324 comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 - 60 of SEQ ID NO. 342, which also co ⁇ esponds 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 co ⁇ esponding to amino acids 61 - 70 of SEQ ID NO.
• 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.
• 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 co ⁇ esponding to amino acids 1 - 23 of SEQ ID NO. 324, a second amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 - 37 of SEQ ID NO. 343, which also co ⁇ esponds to amino acids 24 - 60 of SEQ ID NO.
• 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 co ⁇ esponding 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.
• 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.
• 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
• an isolated chimeric polypeptide encoding for SEQ ID NO. 324 comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding 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 co ⁇ esponding to amino acids 61 - 70 of SEQ ID NO.
• a isolated polypeptide encoding for a tail of SEQ ID NO. 324 comprising a polypeptide being at least 70%o, 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.
• first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 - 115 of SEQ ID NO. 344, which also co ⁇ esponds to amino acids 1 - 115 of SEQ ID NO. 313, and a second amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 152 - 319 of SEQ ID NO. 344, which also co ⁇ esponds to amino acids 116 - 283 of SEQ ID NO. 313, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
• an isolated chimeric polypeptide encoding for SEQ ID NO. 313, of cluster Z36249 comprising a first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 -
• 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 co ⁇ esponding 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.
• 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+ ((n-2) - x), in which x varies from 0 to n-2.
• first amino acid sequence being at least 90 % homologous to co ⁇ esponding to amino acids 1 - 151 of SEQ ID NO. 344, which also co ⁇ esponds to amino acids 1 - 151 of SEQ ID NO. 315
• 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 co ⁇ esponding to amino acids 152 - 177 of SEQ ID NO. 315, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
• 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. 315.
• 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.
• 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. 315.
• 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 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 n-2.
• 316 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 70 of SEQ ID NO. 345, which also co ⁇ esponds to amino acids 1 - 70 of SEQ ID NO. 316, a bridging amino acid K conesponding to amino acid 71 of SEQ ID NO. 316, a second amino acid sequence being at least 90 % homologous to amino acids 72 - 151 of SEQ ID NO. 345, which also conesponds 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.
• cluster Z36249 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 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 n-2.
• a third amino acid sequence being at least 70%, optionally at least 80%o, preferably at least 85%, more preferably at least 90%> and most preferably at least 95%> homologous to a polypeptide sequence co ⁇ esponding to amino acids
• an 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.
• 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 conesponds to amino acids 1 - 42 of SEQ ID NO. 310, a bridging amino acid N co ⁇ esponding 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 co ⁇ esponds to amino acids 44 - 676 of SEQ ID NO.
• 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 co ⁇ esponding to amino acids 677 - 685 of SEQ ID NO. 310, 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.
• a second amino acid sequence being at least 90 %> homologous to amino acids 44 - 657 of SEQ ID NO. 346, which also conesponds 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 co ⁇ esponding 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.
• 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.
• 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.
• 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 co ⁇ esponds to amino acids 1 - 381 of SEQ ID NO.
• a second amino acid sequence being at least 10%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence co ⁇ esponding 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.
• 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 co ⁇ esponds to amino acids 1 - 338 of SEQ ID NO.
• 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 co ⁇ esponding 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.
• an isolated chimeric polypeptide encoding for SEQ ID NO. 307 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 223 of SEQ ID NO. 347, which also co ⁇ esponds to amino acids 1 - 223 of SEQ ID NO.
• 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% homobgous to a polypeptide sequence co ⁇ esponding to amino acids 117 - 215 of SEQ ID NO. 281, wherein said firstand second amino acid sequences are contiguous and in a sequential order.
• 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.
• 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.
• 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 co ⁇ esponding to amino acids 1 - 116 of FABH HUMAN, which also co ⁇ esponds 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 conesponding 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.
• an isolated polypeptide encoding for a tail of SEQ ID NO. 282 comprismg 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.
• a first amino acid sequence being at least 90 % homologous to amino acids 1 - 116 of AAP35373, which also co ⁇ esponds 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%o homologous to a polypeptide sequence conesponding 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.
• polypeptide being at least 70%
• a polypeptide being at least 70%
• most preferably at least about 95% > homologous to the sequence DVLTAWPSIYRRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG According to prefened embodiments of the present invention, there is provided ai isolated chimeric polypeptide encoding for SEQ ID NO.
• 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 amino acids 1 - 116 of FABHJHUMAN, which also conesponds 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 co ⁇ esponding 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.
• 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.
• 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 co ⁇ esponds to amino acids SEQ ID NO.
• an isolated polypeptide encoding for a tail of SEQ ID NO. 283, comprising a polypeptide being at least 70%o, 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
• a third amino acid sequence being at least 90 % homologous to amino acids 25 - 133 of FABH HUMAN, which also co ⁇ esponds 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.
• 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 co ⁇ esponding 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 co ⁇ esponds 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.
• 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 co ⁇ esponding to amino acids 204 - 240 of SEQ ID NO. 285, wherein said first and second amino acid sequences are contiguous and in a sequential order.
• 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 conesponding to amino 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 co ⁇ esponds 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.
• an 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
• 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 co ⁇ esponds 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 conesponds to amino acids 141 - 337 of SEQ ID NO.
• 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 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.
• an isolated chimeric polypeptide encodmg for SEQ ID NO. 288, comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%o, more preferably at least 90% > and most preferably at least 95% homologous to a polypeptide sequence co ⁇ esponding 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 co ⁇ esponds to amino acids 11 - 99 of SEQ ID NO.
• 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 conesponds to amino acids 101 - 321 of SEQ ID NO. 288, wherein said first, second, third and fourth amino acid sequences are contiguous and in a sequential order.
• polypeptide 288, comprising a polypeptide being at least 10%), 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 ofSEQ ID NO. 288.
• a polypeptide being at least 10%
• 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 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 101 + ((n-2) - x), in which x varies from 0 to n-2.
• 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 co ⁇ esponding to amino acids 1 - 15 of SEQ ID NO. 289, and a second amino acid sequence being at least 90 %> homologous to co ⁇ esponding to amino acids 203 - 399 of SEQ ID NO. 349, which also co ⁇ esponds 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.
• 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% homobgous to the sequence MSSWLSAGSPSSLSV of SEQ ID NO. 289.
• 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 co ⁇ esponding 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 co ⁇ esponds 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.
• a 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
• an 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%o and most preferably at least about 95% homologous to the sequence SRNWTQ in SEQ ID NO. 291.
• 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 co ⁇ esponding 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 co ⁇ esponds 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 co ⁇ esponds to amino acids 263 - 451 of SEQ ID NO.
• 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
• 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.
• a first amino acid sequence being at least 90 % homologous to amino acids 1 - 276 of Q96NF5, which also conesponds 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 co ⁇ esponds 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 co ⁇ esponding to amino acids 402 - 407 of SEQ ID NO.
• 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 conesponds 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 conesponds to amino acids 278 - 374 of SEQ ID NO.
• 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 co ⁇ esponding to amino acids 375 - 390 of SEQ ID NO. 295, wherein said first, A, second and third amino acid sequences are contiguous and in a sequential order.
• a polypeptide being at least 70%
• 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 co ⁇ esponds 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.
• 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 co ⁇ esponds to amino acids 1 - 132 of SEQ ID NO.
• a second amino acid sequence being at least 70%o, optionally at least 80%>, preferably at least 85%, more preferably at least 90%) and most preferably at least 95% homologous to a polypeptide sequence conesponding 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.
• 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 - 109 of Q9NZK3, which also co ⁇ esponds to amino acids 1 - 109 of SEQ ID NO.
• 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 co ⁇ esponding 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.
• 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.
• 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
• 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 Q9NZK3, which also conesponds 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 conesponding to amino acids 108 - 121 of SEQ ID NO.
• 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.
• 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 co ⁇ esponding to amino acids 1 - 8 of SEQ ID NO.
• 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.
• 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 conesponds 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 conesponding 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.
• 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.
• first amino acid sequence being at least 90 %> homologous to amino acids 1 - 8 of TRIC HUMAN, which also conesponds 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 conesponding to amino acids 36 - 209 of TRIC_HUMAN, which also conesponding 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.
• 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.
• 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 TRICJHUMA , which also co ⁇ esponds 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 conesponding 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.
• 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.
• a first amino acid sequence being at least 90 %> homologous to amino acids 1 - 8 of TPJC_HUMAN, which also conesponds 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 co ⁇ esponding 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.
• an antibody capable of specifically binding to an epitope of an amino acid sequence in any one of cluster S67314, N56180, T10377, Z24874, HUMCDDANF, HUMTROPIA, HUMSMCK,
• kits for detecting heart disorders comprising a kit detecting overexpression of a splice variant.
• the kit comprises a NAT-based technology.
• the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence in any one of cluster S67314, N56180, T10377, Z24874, HUMCDDANF, HUMTROPIA, HUMSMCK, H88495, Z36249, FLJ26352, HSACMHCP.
• the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence in any one of cluster S67314, N56180, T10377, Z24874, HUMCDDANF, HUMTROPIA, HUMSMCK, H88495, Z36249, FLJ26352, HSACMHCP.
• kit comprises an antibody as described herein.
• the kit further comprises at least one reagent for performing an ELISA or a Western blot.
• at least one reagent for performing an ELISA or a Western blot comprising detecting overexpression of a splice variant of any of cluster S67314, N56180, T10377, Z24874, HUMCDDANF, HUMTROPIA, HUMSMCK,
• detecting overexpression is performed with a NAT-based technology.
• detecting overexpression is performed with an immunoassay.
• the immunoassay comprises an antibody as described herein.
• 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.
• a method for screening for heart disorders comprising detecting cardiac disease cells or tissue with a biomarker or an antibody.
• a method for diagnosing heart disorders comprising detecting heart cells or tissue with a biomarker or an antibody.
• 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.
• 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.
• 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.
• 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), ohgonucleotides (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.
• 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 ohgonucleotides in various tissues, prob 205738_s_at & prob 214285_at.
• Figure 5A 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 5B 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 libraries/sequences.
• Figure 8 is a histogram showing the actual expression of ohgonucleotides 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
• FIG. 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 Q96NF5 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 ohgonucleotides 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 ohgonucleotides 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 ohgonucleotides in various tissues, prob 205742_at, including heart tissue.
• Figure 21A is a histogram showing specific expression of the above- indicated TRIC_HUMAN Troponin I, cardiac muscle HUMTROPIA transcripts in sequence HUMTROPIA seglO in heart tissue.
• Figure 21 A 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 ofthe 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 ohgonucleotides 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 libraries/sequences
• Figure 26 is a histogram concerning the actual expression of ohgonucleotides 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 ohgonucleotides 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 ohgonucleotides 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, HUMTROPIA_T7, with regard to introducing a mutation to block an additional ORF.
• Figure 34 shows Troponin PCR product after second amplification reaction: Lane 1: 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 pRSET A.
• Figure 37 shows the complete sequence ofthe plasmid shown in Figure 36.
• Figure 38 shows the protein sequence of Troponin variant HUMTROPIA_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 TO; 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: HisTro ⁇ oninT7 pRSETA TO; lane 3: HisTroponinT7 pRSETA T3; lane 4: pRSET empty vector TO (negative control); lane 5: pRSET empty vector T3
• 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 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 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.
• 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.
• hypoxia with or without necrosis
• intracellular proteins that are not 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 conditions.
• BNP brain natriuretic peptide
• ANF atrial natriuretic factor
• 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 co ⁇ elate 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 taken 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 (alorie or in combination), optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
• 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.
• 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 ofthe splice variant is typically highly homologous (often 100% identical) to a portion of the conesponding known protein, while at least a second portion of the variant comprises the tail.
• a “head” refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention.
• 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 ofthe conesponding known protein.
• 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, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two 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.
• the "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 tail/head/insertion and at least one amino acid is from the "known protein" portion of a variant.
• 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.
• bridges are described with regard ⁇ o a sliding window in certain contexts below.
• a bridge between two edges may optionally be described as follows: a bridge portion of CONTIG-NAME_Pl (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_Pl): a sequence starting from any of amino acid numbers 49-x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) -
• 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 co ⁇ esponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
• 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.
• 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.
• this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention.
• this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
• this invention provides 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 co ⁇ esponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction co ⁇ elates with the presence of a splice variant in the biological sample.
• 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 co ⁇ elates with the presence of a splice variant nucleic acid sequence in the biological sample.
• 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.
• any marker 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.
• 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.
• the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
• 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.
• the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence co ⁇ esponding 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, conesponding to a splice variant ofthe 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 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 occu ⁇ ing or artificially induced, either randomly or in a 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 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 occu ⁇ ing 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 of the present invention.
• 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.
• 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 ofthe 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 and/or a composite polynucleotide sequences (e.g., a combination ofthe above).
• cDNA complementary polynucleotide sequence
• genomic polynucleotide sequence e.g., a combination ofthe above.
• composite polynucleotide sequences e.g., a combination ofthe above.
• the phrase "complementary polynucleotide sequence” refers to a 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 in vivo or in vitro using a DNA dependent DNA polymerase.
• genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
• 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.
• Prefe ⁇ ed 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).
• 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, 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).
• 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.
• 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.
• 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 ofthe 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.
• oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.
• Specific examples of prefe ⁇ ed oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
• Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
• Prefe ⁇ ed 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'.
• 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 alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• morpholino linkages formed in part from the sugar portion of a nucleoside
• siloxane backbones sulfide, sulfoxide and sulfone backbones
• fo ⁇ uacetyl and thioformacetyl backbones methylene fonnacetyl and thioformacetyl backbones
• alkene containing backbones sulfamate backbones
• sulfonate and sulfonamide backbones amide backbones
• others having mixed N, O, S and CEfe component parts, as disclosed in U.S. Pat. Nos.
• 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).
• PNA peptide nucleic acid
• 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.
• Oligonucleotides of the present invention may also include base modifications or substitutions.
• "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• 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 alkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5- halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and
• 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 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
• 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2 °C and are presently prefe ⁇ ed base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
• 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.
• 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., di-hexadecyl-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 palmity
• 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.
• 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 further includes at least one cis acting regulatory element.
• cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans 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.
• the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed.
• 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], neuron- specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci.
• the nucleic acid construct 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 transcription therefrom.
• the nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication.
• the nucleic acid construct utilized is a shuttle vector, which can propagate both in E.
• 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.
• suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
• retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, rncludingRetro-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.
• Cu ⁇ ently prefened 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.
• lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
• the most prefe ⁇ ed 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 aheady present in the viral construct.
• a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
• the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
• the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
• 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 non-viral, 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, Cu ⁇ ent Opin. Biotechnol. 10:71-75).
• kits containing probes on a dipstick setup and the like 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.
• 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 10 ⁇ cpm 32 P 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 10 6 cpm 32 P labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.
• a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCI, 1 %> SDS and 5 x 10 ⁇ cpm 32 P labeled probe, at 65 °C
• moderate hybridization is effected using a hybrid
• hybridization of short nucleic acids can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency;
• hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
• 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.
• detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.
• 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 biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
• oligonucleotide probes when fluorescently- labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] 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.
• probes can be labeled according to numerous well known methods.
• radioactive nucleotides can be incorporated into probes of the invention by several methods.
• Non- limiting examples of radioactive labels include 3 H, 14 C, 32 P, and 35 S.
• Probes of the invention can be utilized with naturally occuning 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.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• NAT-based 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 real-time PCR for example).
• a "primer" defines an oligonucleotide which is capable of annealing to
• Amplification of a selected, or target, nucleic acid sequence may be carried 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.
• Non- limiting 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 (Kwoh et al, 1989, Proc. Natl. 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).
• PCR polymerase chain reaction
• LCR ligase chain reaction
• SDA strand displacement amplification
• amplification 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.
• amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
• the oligos are designed to bind to a complementary sequence under selected conditions.
• amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid.
• RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA.
• the amplification of the differentially expressed nucleic acids is ca ⁇ ied 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 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 employed.
• 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.
• 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, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Cu ⁇ ent 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 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 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.
• PCR Polymerase Chain 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 ⁇ /., 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.
• 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.
• Ligase Chain Reaction (LCR or LAR): The ligase chain reaction [LCR; sometimes refe ⁇ ed to as “Ligase Amplification Reaction” (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids.
• 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 are mixed and DNA ligase is added to the mixture.
• ligase will covalently link each set of hybridized molecules.
• 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 Al (1990).
• 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-Sustained Synthetic Reaction (3SR/NASBA) The self- sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a unifo ⁇ n 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.
• 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 ⁇ ) Replicase In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q ⁇ replicase.
• 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.
• a PCR running 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.
• 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.
• 50 % mean efficiency it would take 34 cycles to achieve the million-fold amplification theoretically possible in 20, and at lower efficiencies, the number of cycles required becomes prohibitive.
• any background products that amplify with a better mean efficiency than the intended target will become the dominant products.
• PCR has yet to penetrate the clinical market in a significant way.
• LCR LCR must also be optimized to use different oligonucleotide sequences for each target sequence.
• both methods require expensive equipment, capable of precise temperature cycling.
• 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 polymerase 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 ofthe 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 prevent ligation in the LCR. Any mismatch effectively blocks the action of the the ⁇ nostable ligase, but LCR still has the drawback of target- independent background ligation products initiating the amplification.
• the direct detection method may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
• CPR cycling probe reaction
• branched DNA analysis e.g., a method that does not amplify the signal exponentially is more amenable to quantitative analysis.
• CPR Cycling probe reaction
• 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 canied through sample preparation.
• Branched DNA 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 non-specific binding is similarly increased.
• the detection of at least one sequence change 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 Conformation Polymorphism (SSCP) analysis or Dideoxy fingerprinting (ddF).
• RFLP analysis restriction fragment length polymorphism
• ASO allele specific oligonucleotide
• DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
• SSCP Single-Strand Conformation Polymorphism
• ddF Dideoxy fingerprinting
• test sample e.g., a bacterial isolate
• amplified material e.g., PCR reaction products
• This avoids the time and expense associated with cloning the segment of interest.
• specialized equipment and highly trained personnel are required, and the method is too labor- intense and expensive to be practical and effective in the clinical setting.
• 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 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.
• Restriction fragment length polymorphism RFLP: For detection of single-base differences between like sequences, the requirements ofthe 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.
• a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).
• RFLP restriction fragment length polymorphism
• 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).
• MCC Mismatch Chemical Cleavage
• RFLP analysis suffers from low sensitivity and requires a large amount of sample.
• 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.
• 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.
• Allele specific oligonucleotide 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 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.
• DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
• the fragments to be analyzed are "clamped" at one end by a long stretch of GC base pahs (30-80) to allow complete denaturation ofthe 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.
• DGGE constant denaturant gel electrophoresis
• 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.
• Single-Strand Conformation Polymorphism (SSCP): Another common 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.
• 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 method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
• Dideoxy fingerprinting (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.
• 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).
• all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed.
• 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.
• 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.
• 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.
• the step of searching for any ofthe 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 ⁇ -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.
• any suitable technique including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self-sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing
• 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 immobilized 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.
• the identity ofthe 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.
• polypeptide amino acid sequences and peptides
• polypeptide amino acid sequences and peptides
• polypeptide polypeptide
• peptide amino acid residues
• protein 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 co ⁇ esponding 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.
• polypeptide polypeptide
• polypeptide polypeptide
• 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) 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 Monow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles.
• 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.
• NCBI National Center of Biotechnology Information
• nucleic acid sequence homology 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.
• NBI National Center of Biotechnology Information
• 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 occu ⁇ ing or artificially induced, either randomly or in a targeted fashion.
• 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.
• Trp, Tyr and Phe may be substituted for synthetic non- natural acid such as Phenylglycme, TIC, naphthylelanine (Nol), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o- methyl- Tyr.
• 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. fatty acids, complex carbohydrates etc ) .
• 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.
• amino acid includes bothD- and L- amino acids.
• 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.
• Antibodies 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.
• antibody 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, CHI, CH2 and CH3, but does not include the heavy chain variable region.
• Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
• 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
• (Fab')2 the fragment ofthe 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
• 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
• SCA Single chain antibody
• Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis ofthe 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 5S 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.
• a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
• an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
• Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720].
• the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde.
• the Fv fragments comprise VH and VL chains connected by a peptide linker.
• sFv single- chain antigen binding proteins
• sFv single-chain antigen binding proteins
• 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.
• CDR complementarity- dete ⁇ nining region
• Another form of an antibody fragment is a peptide coding for a single complementarity- dete ⁇ nining 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 non-human (e.g., murine) 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 non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• Fv framework residues of the human immunoglobulin are replaced by co ⁇ esponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions conespond to those of a non-human 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 immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323- 329 (1988); and Presta, Cu ⁇ . Op. Struct. Biol., 2:593-596 (1992)].
• Fc immunoglobulin constant region
• Methods for humanizing non-human antibodies are well known in the art.
• 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 refened to as import residues, 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 co ⁇ esponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the co ⁇ esponding sequence from a non-human species.
• 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, 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.
• human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes ha ⁇ e been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rea ⁇ angement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
• the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention.
• 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.
• 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.
• 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 ofthe antibody bound to the marker in the sample.
• purified protein markers can be used.
• Antibodies that specifically bind to a protein marker 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- linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay see, e.g., U.S. Pat. Nos.
• EIA enzyme immune assay
• ELISA enzyme- linked immunosorbent assay
• RIA radioimmune assay
• Western blot assay e.g., Western blot assay, or a slot blot assay see, e.g., U.S. Pat. Nos.
• a sample obtained from a subject can be contacted with the antibody that specifically binds the marker.
• 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.
• 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.
• 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.
• 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.
• the immunoassay can be used to determine a test amount of a marker in a sample from a subject.
• 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.
• 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.
• antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isofo ⁇ ns 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.
• Prefe ⁇ ed embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled "Antibodies”.
• Radio -immunoassay In one version, this method involves precipitation of the desired substrate and in the methods detailed hereinbelow, with a specific antibody and 125 radiolabelled antibody binding protein (e.g., protein A labeled with f ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
• 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 immunosorbent assay 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 ofthe 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.
• Immunohistochemical 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 FACS: 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 include but are not limited to, positron emission tomography (PET) single photon emission computed tomography (SPECT). Both of these techniques are non- 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, SPECT can optionally be used with two labels simultaneously. SPECT 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 SPECT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
• Display Libraries 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 ofthe present invention.
• display vehicles such as phages, viruses or bacteria
• 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 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.nlm.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.
• 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.
• sequence information particularly of spliced sequences
• chromosomal information particularly of spliced sequences
• 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.
• the potential markers were identified by a computational process mat 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.
• EXAMPLE 1 Identification of differentially 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/gbl36.release.notes) and Genbank 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. 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.
• Protocol of library construction various methods are known in the art for library construction including normalized library construction; non- 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.
• 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
• This ratio was preferably set to be at least about 4, although optionally the ratio could be set to be at least about 2.
• 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 le-5, although optionally it could be set to be less than about le-3.
• RNA preparation - 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, TX 78744 USA, http://www.ambion.com).
• 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 DNasel (Ambion) and purified using RNeasy columns (Qiagen).
• RT PCR - Purified RNA (1 ⁇ g) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 ⁇ M dNTP in a total volume of 15.6 ⁇ l. The mixture was incubated for 5 min at 65 °C and then quickly chilled on ice. Thereafter, 5 ⁇ l of 5X Superscriptll first strand buffer (Invitrogen), 2.4 ⁇ l 0.1M 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.
• 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 no ⁇ nalized to the geometric mean of the relative quantities of several housekeeping (HSKP) genes.
• RT reverse transcription
• HSKP housekeeping
• RPL19 Forward primer TGGCAAGAAGAAGGTCTGGTTAG
• Reverse primer TGATCAGCCCATCTTTGATGAG
• RPL19 -amplicon
• TATA box (GenBank Accession No. NM_003194), TATA box Forward primer : CGGTTTGCTGCGGTAATCAT TATA box Reverse primer: TTTCTTGCTGCCAGTCTGGAC
• Ubiquitin (GenBank Accession No. BC000449)
• Ubiquitin Forward primer ATTTGGGTCGCGGTTCTTG
• Ubiquitin Reverse primer TGCCTTGACATTCTCGATGGT
• Ubiquitin -amplicon
• SDHA (GenBank Accession No. NM_004168) SDHA Forward primer: TGGGAACAAGAGGGCATCTG
• SDHA Reverse primer CCACCACTGCATCAAATTCATG SDHA- amplicon : TGGGAACAAGAGGGCATCTGCTAAAGTTTCAGATTCCATTTCTGCTCAGTATCCAGT AGTGGATCATGAATTTGATGCAGTGGTGG
• Cluster S67314 features 4 transcript(s) and 8 segment(s) 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.
• Protein Fatty acid-binding protein, heart is known or believed to have the following funct ⁇ on(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 NO:348). Known polymorphisms for this sequence are as shown in Table 4.
• Protein Fatty acid-binding protein, heart localization is believed to be Cytoplasmic.
• the following GO Annotation(s) apply to the previously known protein.
• the following annotation(s) were found: negative control of cell proliferation, which are annotation(s) related to Biological Process; and lipid binding, which are annotation(s) 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.nlm.nih.gov/projects/LocusLink/>.
• the heart-selective diagnostic marker prediction engine provided the following results with regard to cluster S67314. 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).
• 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 non-heart 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 l.lOE-25.
• cluster S67314 features 4 transcript(s), which were listed in Table 1 above. These transcript(s) encode for protein(s) which are variant(s) of protein Fatty acid- binding protein, heart.
• Variant protein S67314_PEA_1_P4 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S67314_PEA_1_T4.
• An alignment is given to the known protein (Fatty acid-binding protein, heart) at the end ofthe 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 l O i follows:
• Comparison report between S67314JPEA_1_P4 and FABH_HUMAN l.An isolated chimeric polypeptide encoding for S67314JPEA_1_P4, comprising a first amino acid sequence being at least 70%>, optionally at least 80%, preferably at least 85%o, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
• Comparison report between S67314_PEA_1_P4 and AAP35373 l.An isolated chimeric polypeptide encoding for S67314_PEA_1_P4, comprising a first amino acid sequence being at least 90 % homologous to MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF KNTEISFKLGVEFDETTADDRKVKSrVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL conesponding to amino acids 1 - 116 of AAP35373, which also co ⁇ esponds to amino acids 1 - 116 of S67314_PEA_1_P4, and a second amino acid sequence being at least 70%, optionally at least 80%o, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VRWATLELYLIGYYYCSFSQACSKKPSPP
• 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 TQAGTQILPYRLHDCGQITFSKCNCKTGTNNTNLVGLLGSL in S67314_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 S67314_PEA_1_P4 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 5, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S67314_PEA_1_P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein S67314JPEA_1_P4 is encoded by the following transcript(s): S67314JPEA_1_T4, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript S67314_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 S67314JPEA_1_P4 sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 6 - Nucleic acid SNPs
• Variant protein S67314_PEA_1_P5 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S67314_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 alignments to one or more previously published protein sequences are given at the end of the application.
• polypeptide encoding for a tail of S67314_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 DVLTAWPSIYPvRQVKVLREDEITILPWHLQWSREKATKLLRPTLPSYNNHGWEELRVG KSIV in S67314 PEA 1 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.
• variant protein S67314_PEA_1_P5 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S67314_PEA_1_P5 sequence provides support for the deduced sequence of this variant protein accordmg to the present invention).
• Table 7 - Amino acid mutations Single Nucleotide Polymorphisms
• Variant protein S67314_PEA_1_P5 is encoded by the following transcript(s): S67314_PEA_1_T5, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript S67314_PEA_1_T5 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 in Tabte 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 S67314_PEA_1_P5 sequence provides support for the deduced sequence of this variant protein according to the present invention). Table 8 - Nucleic acid SNPs
• Variant protein S67314_PEA_1_P6 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S67314_PEA_1_T6.
• An alignment is given to the known protein (Fatty acid-binding protein, heart) at the end ofthe 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 S67314_PEA_1_P6 and FABHJHUMAN l.An isolated chimeric polypeptide encoding for S67314_PEA_1_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 MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTF KNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLIL co ⁇ esponding to amino acids 1 - 116 of FABH_HUMAN, which also co ⁇ esponds to amino acids 1 - 116 of S67314_PEA_1_P6, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at
• 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 S67314_PEA_1_P6.
• the location of the variant protein was detennined 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: mtracellularly.
• variant protein S67314_PEA_1_P6 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S67314_PEA_1_P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 9 - Amino acid mutations SNP position(s) on amino acid Alternative amino acid(s) Previously known SNP? sequence 53 K -> R Yes
• Variant protein S67314_PEA_1_P6 is encoded by the following xranscript(s): S67314__PEA_1_T6, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript S67314_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 S67314_PEA_1_P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein S67314_PEA_1_P7 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S67314_PEA_1_T7.
• An alignment is given to the known protein (Fatty acid -binding protein, heart) at the end ofthe 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 S67314_PEA_1_P7 and FABHJHUMAN l.An isolated chimeric polypeptide encoding for S67314_PEA_1_P7, comprising a first amino acid sequence being at least 90 % homologous to MVDAFLGTWKLVDSKNFDDYMKSL co ⁇ esponding to amino acids 1 - 24 of FABH_HUMAN, which also co ⁇ esponds 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 conesponding to amino acids 25 - 35 of S67314_PEA_1_P7, and a third amino acid sequence being at least 90 %> homologous to GVGFATRQVASMTKPTTIIEKNGDILTLKTHSTFKNTE
• 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, co ⁇ esponding to S67314_PEA_1_P7.
• Comparison report between S67314_PEA_1 JP7 and AAP35373 l.An isolated chimeric polypeptide encoding for S67314_PEA_1_P7, comprising a first amino acid sequence being at least 90 % homologous to
• MVDAFLGTWKLVDSKNFDDYMKSL co ⁇ esponding to amino acids 1 - 24 of AAP35373, which also conesponds 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 co ⁇ esponding to amino acids 25 - 35 of S67314_PEA_1_P7, and a third amino acid sequence being at least 90 % homologous to
• VTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTHGTAVCTRTYEKEA co ⁇ esponding to amino acids 25 - 133 of AAP35373, which also co ⁇ esponds to amino acids 36 - 144 of
• S67314_PEA_1_P7 wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
• 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, co ⁇ esponding to S67314_PEA_1_P7.
• the location of the variant protein was detennined 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 S67314_PEA_1_P7 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 11, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S67314_PEA_1_P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 11 - Amino acid mutations Single Nucleotide Polymorphisms
• Variant protein S67314_PEA_1_P7 is encoded by the following transcript(s): S67314_PEA_1_T7, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript S67314_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 nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S67314_PEA_1_P7 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• cluster S67314 features 8 segment(s), which were listed in Table 2 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) 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 S67314_PEA_l_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): S67314_PEA_1_T4, S67314_PEA_1_T5, S67314_PEA_1_T6 and S67314_ PEA_1_T7. Table 13 below describes the starting and ending position of this segment on each transcript. Table 13 - Segment location on transcripts
• Segment cluster S67314_PEA_l_node_l l 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 on transcripts
• Segment cluster S67314_PEA_l_node_13 is supported by 76 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): S67314_PEA_1_T7. Table 15 below describes the starting and ending position of this segment on each transcript. Table 15 - Segment location on transcripts
• Segment cluster S67314_PEA_l_node_15 is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): S67314_PEA_1_T5. Table 16 below describes the starting and ending position of this segment on each transcript. Table 16 - Segment location on transcripts
• Segment cluster S67314_PEA_l_node_17 is supported by 4 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): S67314_PEA_1_T6. Table 17 below describes the starting and ending position of this segment on each transcript. Table 17 - Segment location on transcripts
• Segment cluster S67314JPEA_l_node_4 is supported by 101 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): S67314_PEA_1_T4, S67314_PEA_1_T5, S67314_PEA_1_T6 and S67314_PEA_1_T7. Table 19 below describes the starting and ending position of this segment on each transcript. Table 19 - Segment location on transcripts
• segment cluster S67314_PEA_l_node_10 is supported by 64 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): S67314_PEA_1_T4, S67314_PEA_1_T5, S67314_PEA_1_T6 and S67314_PEA_1_T7. Table 20 below describes the starting and ending position of this segment on each transcript. Table 20 - Segment location on transcripts
• Segment cluster S67314_PEA_l_node_3 is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): S67314_PEA_1_T7. Table 21 below describes the starting and ending position of this segment on each transcript. Table 21 - Segment location on transcripts
• FABH_HUMAN Fatty acid-binding protein transcripts which are detectable by amplicon as depicted in sequence name S67314 specifically in heart tissue. Expression of FABH_HUMAN Fatty acid-binding protein transcripts detectable by or according to segll, S67314 amplicon(s) and S67314 segl lF and S67314 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.
• FABH HUMAN Fatty acid-binding protein transcripts in heart tissue samples as opposed to other tissues.
• 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: S67314 segl lF forward primer; and S67314 segl IR 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: S67314 segl l.
• S67314 segl lF (SEQ ID NO:61): TCCCCTGAGAGCTGTAGAAGCT S67314 segl IR (SEQ ID NO:62): CGGCCTGTGTGAGTCCAAA S67314 segl 1(SEQ ID NO:63):
• FIG. 5B 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 5B, 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. 1-9, 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: S67314 segl5F forward primer; and S67314 segl5R 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: S67314 segl5.
• FIG. 44-46 Table 1, above
• 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: S67314seg4F forward primer; and S67314seg4R 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.
• Cluster N56180 features 7 transcript(s) and 22 segment(s) 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
• 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 40 to 50 moles of calcium.
• 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/proj ects/LocusLink/>.
• 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).
• 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 4.70E-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 non- heart ESTs as described above.
• cluster N56180 features 7 transcript(s), which were listed in Table 1 above. These transcript(s) encode for protein(s) which are variant(s) 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 transcript(s) N56180 T1. 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:
• 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 conesponding to amino acids 1 - 203 of CAQ2 HUMAN, which also co ⁇ esponds 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
• N56180_P2 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 N56180JP2.
• 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_P2 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 7, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) 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).
• SNPs Single Nucleotide Polymorphisms
• Variant protein N56180JP2 is encoded by the following transcript(s): N56180_T1, for which the sequence(s) is/are 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 N56180_P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein N56180_P4 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) N56180 T3. An alignment is given to the l ⁇ iown 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.
• 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 co ⁇ esponding to amino acids 1 - 78 of CAQ2JTUMAN, which also co ⁇ esponds 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 HWQISQWWLHFQTPREEGKMKLLELSESADGAAWKRWGGNSNT
• N56180_P4 An isolated polypeptide encoding for an edge portion of N56180_P4, 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, co ⁇ esponding to N56180_P4.
• the location of the variant protein was detennined 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 cistemae 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 protem 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 position(s) on the amino acid sequence, with the alternative amino acid(s) 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
• Variant protein N56180_P4 is encoded by the following transcript(s): N56180_T3, for which the sequence(s) 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 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_P4 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein N56180_P5 has an amino acid sequence as given at the end ofthe application; it is encoded by transcript(s) N56180 T4.
• 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_P5 and CAQ2 HUMAN: l.An isolated chimeric polypeptide encoding for N56180_P5, comprising a first amino acid sequence being at least 90 % homologous to
• 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 cistemae 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_P5 also has the following non- silent SNPs (Single Nucleotide Polymo ⁇ hisms) as listed in Table 11, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180_P5 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 11 - Amino acid mutations Single Nucleotide Polymo ⁇ hisms
• Variant protein N56180 P5 B encoded by the following transcript(s): N56180_T4, for which the sequence(s) 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_P5 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• 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 transcript(s) 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.
• 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 co ⁇ esponding to amino acids 1 - 10 of N56180_P6, second amino acid sequence being at least 90 % homologous to RAEEGLNFPTYDGKDRWSLSEKNFKQVLKKYDLLCLYYHEPVSSDKVTQKQFQLKEI VLELVAQVLEFIKAJGFVMVDAKKEAKLAKKL co ⁇ esponding to amino acids 18 - 106 of CAQ2_HUMAN, which also co ⁇ esponds to amino acids 11 - 99
• 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.
• 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 Polymo ⁇ hisms) as listed in Table 13, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) 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 -Amino acid mutations Single Nucleotide Polymo ⁇ hisms
• Variant protein N56180_P6 is encoded by the following transcript(s): N56180_T5, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript N56180_T5 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).
• Variant protein N56180_P7 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) N56180 T6.
• 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.
• Comparison report between N56180_P7 and CAQ2JHUMAN 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 co ⁇ esponding to amino acids 1 - 15 of N56180_P7, and a second amino acid sequence being at least 90 % homologous to
• DDDNSDEEDNDDSDDDDDE co ⁇ esponding to amino acids 203 - 399 of CAQ2_HUMAN, which also co ⁇ esponds to amino acids 16 - 212 of N56180_P7, wherein said first and second amino acid sequences are contiguous and in a sequential order.
• 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.
• both signal-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 Polymo ⁇ hisms) as listed in Table 15, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) 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).
• Variant protein N56180_P7 is encoded by the following transcript(s): N56180_T6, for which the sequence(s) 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).
• Variant protein N56180_P8 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) 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:
• N56180JP8 Comparison report between N56180JP8 and CAQ2JHUMAN: 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 conesponding to amino acids 1 - 13 of N56180_P8, and a second amino acid sequence being at least 90 % > homologous to DGYEFLEILKQVARDNTDNPDLSILWIDPDDFPLLVAYWEKTFKIDLFRPQIGVVNVTD ADSVWMEIPDDDDLPTAEELEDWIEDVLSGKLNTEDDDEDDDDDDNSDEEDNDDSDD DDDE co ⁇ esponding to amino acids 280 - 399 of CAQ2JHUMAN, which also co ⁇ esponds to amino acids 14 - 133 of N56
• 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.
• variant protein N56180_P8 is encoded by the following transcript(s): N56180_T7, for which the sequence(s) is/are given at the end ofthe 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 are 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).
• Variant protein N56180_P9 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) 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:
• 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 cistemae 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 protem 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 Polymo ⁇ hisms) as listed in Table 18, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein N56180JP9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 18 -Amino acid mutations Single Nucleotide Polymo ⁇ hisms
• Variant protein N56180_P9 is encoded by the following transcript(s): N56180_T8, for which the sequence(s) 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 l ⁇ iown 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).
• cluster N56180 features 22 segment(s), which were listed in Table 2 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) 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 N56180JT8. Table 20 below describes the starting and ending position of this segment on each transcript. Table 20 - Segment location on transcripts
• Segment cluster N56180_node_20 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, N56180JT3, 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 - Segment location on transcripts
• Segment cluster N56180_node_22 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
• Segment cluster N56180_node_28 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 location on transcripts Transcript name Segment starting position [ Segment ending position N56180 T7 1 136
• Segment cluster N56180_node_34 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_T1, 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
• Segment cluster N56180_node_36 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, N56180JT3, 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 transcripts
• Segment cluster N56180_node_4 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 T5 and N56180_T8. Table 26 below describes the starting and ending position of this segment on each transcript. Table 26 - Segment location on transcripts
• Segment cluster N56180_node_6 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 transcripts
• short segments related to the above cluster are also provided. These segments are up to about 120 bp in length, and so are included in a separate description.
• Segment cluster N56180_node_0 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 on transcripts
• Segment cluster N56180_node_ 10 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 T1, N56180JT3, N56180 T4 and N56180_T8. Table 29 below describes the starting and ending position of this segment on each transcript. Table 29 - Segment location on transcripts
• Segment cluster N56180_node__12 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_T1, N56180JT3 and N56180_T8. Table 30 below describes the starting and ending position of this segment on each transcript. Table 30 - Segment location on transcripts
• Segment cluster N56180_node_14 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_T1, N56180_T3, N56180_T5 and N56180_T8. Table 31 below describes the starting and ending position of this segment on each transcript. Table 31 - Segment location on transcripts
• Segment cluster N56180_node_16 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_T1. Table 32 below describes the starting and ending position of this segment on each transcript. Table 32 - Segment location on transcripts
• Segment cluster N56180_node_18 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
• Segment cluster N56180_node_24 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 T5 and N56180_T6. Table 34 below describes the starting and ending position of this segment on each transcript. Table 34 - Segment location on transcripts
• Segment cluster N56180_node_26 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 - Segment location on transcripts
• Segment cluster N56180_node_29 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_T1, N56180JT3, N56180JT4, N56180 T5, N56180 T6 and N56180 I7. Table 36 below describes the starting and ending position of this segment on each transcript. Table 36 - Segment location on transcripts
• Segment cluster N56180_node_3 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, N56180JT4 and N56180_T8. Table 37 below describes the starting and ending position of this segment on each transcript. Table 37 - Segment location on transcripts
• Segment cluster N56180_node_31 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 - Segment location on transcripts
• Segment cluster N56180_node_33 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, N56180JT5, 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 transcripts
• Segment cluster N56180_node_35 can be found in the following transcript(s): N56180 T1, N56180 T3, N56180 T4, N56180JT5, N56180 T6 and N56180_T7. Table 40 below describes the starting and ending position of this segment on each transcript. Table 40 - Segment location on transcripts
• Segment cluster N56180_node_8 is supported by 25 libraries. The number of libraries was detennined as previously described. This segment can be found in the following transcript(s): N56180_T1, N56180_T3, N56180 T4, N56180_T5 and N56180_T8. Table 41 below describes the starting and ending position of this segment on each transcript. Table 41 - Segment location on transcripts
• Alignment segment 1/1 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: 84.46 Gaps : 1
• Alignment segment 1/1 Quality: 2955.00 Escore: 0 Matching length: 314 Total length: 385 Matching Percent Similarity: 99.04 Matching Percent Identity: 99.04 Total Percent Similarity: 80.78 Total Percent Identity: 80.78 Gaps : 1
• Alignment segment 1/1 Quality: 1959.00 Escore: 0
• cardiac muscle isofo ⁇ n transcripts which are detectable by amplicon as depicted in sequence name N56180 specifically in heart tissue
• cardiac muscle isofo ⁇ n transcripts detectable by or according to seg6, N56180 amplicon(s) and N56180 seg6F and N56180 seg6R primers was measured by real time PCR.
• 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 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 seg6.
• N56180 seg6F (SEQ ID NO:279): ATATCCCAGTGGTGGTTGCATT N56180 seg6R (SEQ ID NO:280): CCCTCCCCAGCGTTTCC N56180 seg6 (SEQ ID NO:335):
• 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.
• FIG. 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.
• 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 NO:337): TCAAGTAGTTGCTACAGACGCCA N56180 seg (SEQ ID NO:361): TTGATACCACTTAGTGTAGCTCCAGCATGGATCAGCAAACTTTTTCTGTAAAGAACA AAATGGTAAATATTTCAGGTTCTGTGGGCCAGATGGCGTCTGTAGCAACTACTTGA
• 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 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 11 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 non-heart 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 check 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.
• cluster T10377 features 6 transcript(s), which were listed in Table 1 above.
• T10377_P2 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) T10377_T1 and T10377_T2.
• transcript(s) 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 protem according to the present invention to each such aligned protein is as follows:
• T10377_P2 Comparison report between T10377_P2 and Q96NF5 (SEQ ID NO:362): 1.
• 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 trans- membrane region prediction programs predicted a trans- membrane region for this protein.
• variant protein T10377_P2 also has the following non-silent SNPs (Single Nucleotide Polymo ⁇ hisms) as listed in Table 5, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) 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).
• SNPs Single Nucleotide Polymo ⁇ hisms
• Variant protein T10377_P2 is encoded by the following transcript(s): T10377_T1 and S T10377_T2, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript T10377_T1 is shown in bold; this coding portion starts at position 166 and ends at position 1518.
• 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 T10377_P2 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• transcript T10377_T2 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 l ⁇ iown 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).
• Variant protein T10377_P5 has an amino acid sequence as given at the end ofthe application; it is encoded by transcript(s) T10377 T5.
• transcript(s) 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:
• T10377_P5 Comparison report between T10377_P5 and Q96NF5: 1.
• the location of the variant protein was detennined 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_P5 also has the following non-silent SNPs (Single Nucleotide Polymo ⁇ hisms) as listed in Table 8, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein T10377_P5 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein T10377_P5 is encoded by the following transcript(s): T10377 T5, for which the sequence(s) is/are given at the end ofthe application.
• the coding portion of transcript T10377_T5 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_P5 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein T10377_P6 has an amino acid sequence as given at the end ofthe application; it is encoded by transcript(s) T10377_T6.
• transcript(s) 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:
• T10377_P6 Comparison report between T10377_P6 and Q96NF5: 1.
• 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 Polymo ⁇ hisms) as listed in Table 10, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) 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).
• Variant protein T10377_P6 is encoded by the following transcript(s): T10377 T6, for which the sequence(s) 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 l ⁇ iown 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 given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is l ⁇ iown or not; the presence of known SNPs in variant protein T10377_P6 sequence provides support for the deduced sequence of this variant protein according
• Variant protein T10377_P7 has an amino acid sequence as given at the end ofthe application; it is encoded by transcript(s) T10377_T7.
• transcript(s) T10377_T7 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 protem according to the present invention to each such aligned protein is as follows:
• T10377_P7 Comparison report between T10377_P7 and Q96NF5: 1.
• 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 protem has a signal peptide..
• Variant protein T10377_P7 also has the following non-silent SNPs (Single Nucleotide Polymo ⁇ hisms) as listed in Table 12, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) 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).
• Variant protein T10377_P7 is encoded by the following transcript(s): T10377_T7, for which the sequence(s) 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 l ⁇ iown 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 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 l ⁇ iown SNPs in variant protein T10377_P7 sequence provides support for the deduced sequence of this variant protein
• Protein T10377_P8 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) T10377_T0.
• transcript(s) 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 follows:
• 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
• YSQYEEKLQEEQRKHSAEKEALLEETNSFLK co ⁇ esponding to amino acids 1 - 261 of Q96NF5, which also co ⁇ esponds 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 SFLEGENNELQSRLDYLTETQAKTEVETREIGVGCDLLPSQTGRTREINMPSRNYTPYTR VLELTMKKTLT co ⁇ esponding to amino acids 263 - 451 of Q96NF5, which also co ⁇ esponds to amino acids 263 - 451 of T10377_P8, wherein said first, second and third
• 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 Polymo ⁇ hisms) as listed in Table 14, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) 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).
• SNPs Single Nucleotide Polymo ⁇ hisms
• Protein T10377_P8 is encoded by the following transcript(s): T10377_T0, for which the sequence(s) is/are given at the end of the application
• the coding portion of transcript T10377_T0 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 sequence, 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).
• cluster T 10377 features 18 segment(s), which were listed in Table 2 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) 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 favention 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 location on transcripts
• Segment cluster T10377_node_ 17 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_T1, 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 - Segment location on transcripts
• Segment cluster T10377_node_19 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_T1, 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 - Segment location on transcripts
• 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 T1, 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
• Segment cluster T10377_node_27 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 on transcripts
• Segment cluster T10377_node_33 is supported by 103 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10377JTO, T10377_T1, T10377_T2, T10377_T5 and T10377_T6. Table 21 below describes the starting and ending position of this segment on each transcript. Table 21 - Segment location on transcripts
• short segments related to the above cluster are also provided. These segments are up to about 120 bp in length, and so are included in a separate description.
• Segment cluster T10377_node_12 is supported by 35 libraries. The number of libraries was detennined as previously described. This segment can be found in the following transcript(s): T10377_T0, T10377_T1, T10377JT2, T10377_T5, T10377_T6 and T10377_T7. Table 22 below describes the starting and ending position of this segment on each transcript. Table 22 - Segment location on transcripts
• Segment cluster T10377_node_14 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, T10377JT1, T10377 T2, T10377JT5, T10377 T6 and T10377_T7. Table 23 below describes the starting and ending position of this segment on each transcript. Table 23 - Segment location on transcripts
• Segment cluster T10377_node_16 can be found in the following transcript(s): T10377_T0, T10377 T1, T10377 T2, T10377JT5, T10377_T6 and T10377_T7.
• Table 24 describes the starting and ending position of this segment on each transcript. Table 24 - Segment location on transcripts
• Segment cluster T10377_node_2 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_T1. Table 25 below describes the starting and ending position of this segment on each trans cript. Table 25 - Segment location on transcripts
• Segment cluster T10377_node_23 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 T1, 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 location on transcripts
• Segment cluster T10377_node_25 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, T10377JT5, T10377_T6 and T10377_T7. Table 27 below describes the starting and ending position of this segment on each transcript. Table 27 - Segment location on transcripts
• Segment cluster T10377_node_29 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 and T10377_T6. Table 28 below describes the starting and ending position of this segment on each transcript. Table 28 - Segment location on transcripts
• Segment cluster T10377_node_3 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
• Segment cluster T10377_node_31 is supported by 52 libraries. The number of libraries was detennined as previously described. This segment can be found in the following transcript(s): T10377_T0, T10377 T1, 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 transcripts
• Segment cluster T10377_node_5 is supported by 30 libraries. The number of libraries was detennined as previously described. This segment can be found in the following transcript(s): T10377_T0, T10377_T1, T10377JT2, T10377 T5, T10377_T6 and T10377_T7. Table 31 below describes the starting and ending position of this segment on each transcript. Table 31 - Segment location on transcripts
• Segment cluster T10377_node_8 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 transcripts
• Segment cluster T10377_node_9 is supported by 35 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): T10377JT0, T10377_T1, T10377 T2, T10377 T5, T10377_T6 and T10377_T7. Table 33 below describes the starting and ending position of this segment on each transcript. Table 33 - Segment location on transcripts
• 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 :
• Q96NF5 transcripts which are detectable by amplicon as depicted in sequence name T10377 specifically in heart tissue. Expression of Q96NF5 transcripts detectable by or according to junc25-31 node(s),
• T10377 amplicon(s) and T10377 junc25-31F and T10377 junc25-31R primers was measured by real time PCR.
• FIG. 12 is a histogram showing specific expression of the above- indicated Q96NF5 transcripts in heart tissue samples as opposed to other tissues. As is evident from Figure 12, 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.
• Primer pahs 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 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 junc25- 31.
• T10377 junc25-31F (SEQ ID NO:363): AGCAGATGGTCGAGGAGAATAATG T10377 junc25-31R (SEQ ID NO:364): ATCTCTCTGGTTTCCACTTCGG T10377 junc25-31 (SEQ ID NO:365):
• FIG. 13 is a histogram showing specific expression of the above-indicated Q96NF5 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.
• Primer pahs 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 non- limiting illustrative example only of a suitable amplicon: T10377 junc29- 33.
• T10377 junc29-33F (SEQ ID NO:366): CTTTCTTAGAAGGAGAGCCAAACAG T10377 junc29-33R (SEQ ID NO:367): CCTAAGTCAGAGTTTTCTTCATGGTTAAC T10377 junc29-33 (SEQ ID NO:368):
• FIG. 14 is a histogram showing specific expression of the above-indicated Q96NF5 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 1, "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 pah; 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 NO:369): CTTCGCATTGTGCATAACACAA T10377 seg2-3R (SEQ ID NO:370): GAAACTCGGATACACAATCTCCAGA T10377 seg2-3 (SEQ ID NO:371): CTTCGCATTGTGCATAACACAAGCCCTGAACCAGCTGCTTTGGGAACCCCTGGGAA TAAAGTGCCCTACCTGCCTTTCAGGCACTGCCAAGCCTGGGGCATCTCTGGAGATTG TGTATCCGAGTTTC
• Cluster Z24874 features 2 transcript(s) and 10 segment(s) 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
• the heart- selective diagnostic marker prediction engine provided the following results with regard to cluster Z24874. 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).
• 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 ofthe 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.
• variant protein Z24874_PEA_2_P5 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s)
• polypeptide encoding for a tail of Z24874_PEA_2_P5, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%o, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence LPGRHEVPRGALP in Z24874_PEA_2_P5.
• Comparison report between Z24874_PEA_2_P5 and Q9NZK3 l.An isolated chimeric polypeptide encoding for Z24874_PEA_2_P5, comprising a first amino acid sequence being at least 90 % homologous to MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLE SLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSYKP co ⁇ esponding to amino acids 1 - 109 of Q9NZK3, which also co ⁇ esponds to amino acids 1 - 109 of Z24874_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
• 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.
• both signal-peptide prediction programs predict that this protein is a non- secreted protein..
• Variant protein Z24874_PEA_2_P5 is encoded by the following transcript(s): Z24874_PEA_2_T10, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript Z24874JPEA_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 Z24874_PEA_2_P5 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Variant protein Z24874_PEA_2_P6 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) Z24874_PEA_2_T11.
• transcript(s) Z24874_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:
• polypeptide encoding for a tail of Z24874_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 Z24874_PEA_2_P6.
• Comparison report between Z24874_PEA_2_P6 and Q9NZK3 l.An isolated chimeric polypeptide encoding for Z24874_PEA_2_P6, comprising a first amino acid sequence being at least 90 % homologous to MKLIVGIGGMTNGGKTTLTNSLLRALPNCCVIHQDDFFKPQDQIAVGEDGFKQWDVLE SLDMEAMLDTVQAWLSSPQKFARAHGVSVQPEASDTHILLLEGFLLYSY conesponding to amino acids 1 - 107 of Q9NZK3, which also co ⁇ esponds to amino acids 1 - 107 of Z24874_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 co ⁇ esponding to amino acids
• nucleic acid sequence encoding for a tail of Z24874_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 Z24874_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.
• Variant protein Z24874_PEA_2_P6 is encoded by the following transcript(s): Z24874_PEA_2_T11, for which the sequence(s) is/are given at the end of the application.
• the coding portion of transcript Z24874_PEA_2_T11 is shown in bold; this coding portion starts at position 292 and ends at position 654.
• the transcript 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 Z24874_PEA_2_P6 sequence provides support for the deduced sequence of this variant protein according to the present invention).
• Table 5 - Nucleic acid SNPs are 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 Z24874_PEA_2_P6 sequence provides support for the deduced sequence of this variant protein according to the present invention.
• cluster Z24874 features 10 segment(s), which were listed in Table 2 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) 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 Z24874_PEA_2_node_21 is supported by 30 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 6 below describes the starting and ending position of this segment on each transcript. Table 6 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_4 is supported by 19 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 7 below describes the starting and ending position of this segment on each transcript. Table 7 - Segment location on transcripts
• short segments related to the above cluster are also provided. These segments are up to about 120 bp in length, and so are included in a separate description.
• Segment cluster Z24874_PEA_2_node_0 is supported by 3 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 8 below describes the starting and ending position of this segment on each transcript. Table 8 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_10 is supported by 25 libraries. The number of libraries was detennined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 9 below describes the starting and ending position of this segment on each transcript. Table 9 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_12 is supported by 26 libraries. The number of libraries was detennined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 10 below describes the starting and ending position of this segment on each transcript. Table 10 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_13 is supported by 21 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 11 below describes the starting and ending position of this segment on each transcript. Table 11 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_14 is supported by 20 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 12 below describes the starting and ending position of this segment on each transcript. Table 12 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_16 is supported by 17 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10. Table 13 below describes the starting and ending position of this segment on each transcript. Table 13 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_3 is supported by 8 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 14 below describes the starting and ending position of this segment on each transcript. Table 14 - Segment location on transcripts
• Segment cluster Z24874_PEA_2_node_6 is supported by 23 libraries. The number of libraries was detennined as previously described. This segment can be found in the following transcript(s): Z24874_PEA_2_T10 and Z24874_PEA_2_T11. Table 15 below describes the starting and ending position of this segment on each transcript. Table 15 - Segment location on transcripts

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