Polynucleotide Marker Genes and their Expression, for Diagnosis of Endotoxemia
AssigneeAthlomics Pty Ltd.
InventorsRichard Bruce BRANDON, Mervyn Rees THOMAS
Life of this patent
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Abstract
The invention discloses isolated endotoxemia marker polynucleotides selected from any one of 163 different polynucleotide sequences, or variants thereof. Endotoxemia related conditions are diagnosed in a test subject by aberrant expression of at least one of the endotoxemia markers or variants thereof. Of practical use, is the early diagnosis of disease, determining those animals at risk of developing endotoxemia, monitoring of an animals immune response to the disease and the enablement of better treatments. Of particular interest is the diagnosis of laminitis in hoofed animals, including horses.
Claims31
A method for diagnosing the presence of endotoxemia in a mammalian test subject, comprising comparing expression of at least one endotoxemia marker polynucleotide in a biological sample obtained from the test subject to expression of at least one corresponding endotoxemia marker polynucleotide in a reference sample obtained from one or more normal subjects or from one or more subjects lacking endotoxemia, wherein a difference in the expression between the biological sample and the reference sample indicates presence of endotoxemia, wherein a similarity in the expression between the biological sample and the reference sample indicates absence of endotoxemia, wherein the biological sample comprises leukocytes, and wherein the at least one endotoxemia marker polynucleotide is expressed in leukocytes of animals in which laminitis has been induced by carbohydrate overload and is selected from the group consisting of:
- (a) a polynucleotide comprising a nucleotide sequence that shares at least 90% sequence identity over a comparison window with the sequence set forth in any one of SEQ ID NO:29, 33, 257 or a complement thereof, wherein the comparison window is at least 50 nucleotides in length;
- (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO:12, 30, 34 or 258;
- (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 90% sequence identity with the sequence set forth in any one of SEQ ID NO:12, 30, 34 or 258;
- and (d) a polynucleotide comprising a nucleotide sequence that hybridizes to the sequence of (a), (b), (c) or a complement thereof, under-high stringency conditions.
2-3. (canceled)
A method according to claim 2, wherein the presence, of endotoxemia is determined when the measured level of the or each expression product is 10% higher than the measured level or functional activity of the or each corresponding expression product.
A method according to claim 2, wherein the presence of endotoxemia is determined by detecting an increase in the level of at least one endotoxemia marker polynucleotide selected from (a) a polynucleotide comprising a nucleotide sequence that shares at least 90% sequence identity with the sequence set forth in any one of SEQ ID NO:
- 29, 33 or 257, or a complement thereof;
- (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO:
- 30, 34, or 258;
- (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 90% sequence identity with the sequence set forth in any one of SEQ ID NO:
- or 258;
- and (d) a polynucleotide comprising a nucleotide sequence that hybridizes to the sequence of (a), (b), (c) or a complement thereof, under high stringency conditions.
A method according to claim 2, wherein the presence of endotoxemia is determined when the measured level or functional activity of the or each expression product is 10% lower than the measured level or functional activity of the or each corresponding expression product.
A method according to claim 2, wherein the presence of endotoxemia is determined by detecting a decrease in the level or functional activity of at least one endotoxemia marker polynucleotide selected from (a) a polynucleotide comprising a nucleotide sequence that shares at least 90% sequence identity with the sequence set forth in any one of SEQ ID NO:
- 11 or 269, or a complement thereof;
- (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:12;
- (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 90% sequence identity with the sequence set forth in SEQ ID NO:12;
- and (d) a polynucleotide comprising a nucleotide sequence that hybridizes to the sequence of (a), (b), (c) or a complement thereof, under high stringency conditions.
A method according to claim 2, wherein the absence of endotoxemia is determined when the measured level or functional activity of the or each expression product is the same as or similar to the measured level or functional activity of the or each corresponding expression product.
A method according to claim 2, comprising measuring the level of individual expression products of at least two endotoxemia marker genes.
A method according to claim 2, comprising measuring the level of individual expression products of at least three endotoxemia marker genes.
A method according to claim 2, comprising measuring the level of individual expression products all four endotoxemia marker genes.
14-15. (canceled)
(canceled)
21-22. (canceled)
A method according to claim 2, wherein the expression product or corresponding expression product is a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an endotoxemia marker polynucleotide.
A method according to claim 2, wherein the expression product or corresponding expression product is a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an endotoxemia marker polynucleotide and wherein the measured level or abundance of the target RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy of the reference RNA that is present in the same sample.
A method according to claim 2, wherein the expression product or corresponding expression product is a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an endotoxemia marker polynucleotide and wherein the nucleic acid probe is immobilized on a solid or semi-solid support.
A method according to claim 2, wherein the expression product or corresponding expression product is a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an endotoxemia marker polynucleotide and wherein the nucleic acid probe forms part of a spatial array of nucleic acid probes.
A method according to claim 2, wherein the expression product or corresponding expression product is a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an endotoxemia marker polynucleotide and wherein the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by hybridization.
A method according to claim 2, wherein the expression product or corresponding expression product is a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an endotoxemia marker polynucleotide and wherein the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nucleic acid amplification.
A method according to claim 2, wherein the expression product or corresponding expression product is a target RNA or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least low stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of an endotoxemia marker polynucleotide and wherein the probe for detecting the endotoxemia marker polynucleotide comprises a sequence as set forth in any one of SEQ ID NO:470-480, 800-810, 899-909, 921-931 and 2043-2053.
30-37. (canceled)
A method according to claim 2, wherein a system is used to perform the method, which comprises at least one end station coupled to a base station, wherein the base station is caused (a) to receive subject data from the end station via a communications network, wherein the subject data represents parameter values corresponding to the measured or normalized level of at least one expression product in the biological sample, and (b) to compare the subject data with predetermined data representing the measured or normalized level of at least one corresponding expression product in the reference sample to thereby determine any difference in the level activity of the expression product in the biological sample as compared to the level of the corresponding expression product in the reference sample.
A method according to claim 2, wherein a system is used to perform the method, which comprises at least one end station coupled to a base station, wherein the base station is caused (a) to receive subject data from the end station via a communications network, wherein the subject data represents parameter values corresponding to the measured or normalized level of at least one expression product in the biological sample, and (b) to compare the subject data with predetermined data representing the measured or normalized level of at least one corresponding expression product in the reference sample to thereby determine any difference in the level of the expression product in the biological sample as compared to the level of the corresponding expression product in the reference sample, wherein the base station is further caused to provide a diagnosis for the presence or absence of endotoxemia.
A method according to claim 2, wherein a system is used to perform the method, which comprises at least one end station coupled to a base station, wherein the base station is caused (a) to receive subject data from the end station via a communications network, wherein the subject data represents parameter values corresponding to the measured or normalized level of at least one expression product in the biological sample, and (b) to compare the subject data with predetermined data representing the measured or normalized level of at least one corresponding expression product in the reference sample to thereby determine any difference in the level of the expression product in the biological sample as compared to the level of the corresponding expression product in the reference sample, wherein the base station is further caused to transfer an indication of the diagnosis to the end station via the communications network.
41-54. (canceled)
Dependencies are inferred from the claim text.