WO2007085087A1 - Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects - Google Patents

Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects Download PDF

Info

Publication number
WO2007085087A1
WO2007085087A1 PCT/CA2007/000111 CA2007000111W WO2007085087A1 WO 2007085087 A1 WO2007085087 A1 WO 2007085087A1 CA 2007000111 W CA2007000111 W CA 2007000111W WO 2007085087 A1 WO2007085087 A1 WO 2007085087A1
Authority
WO
WIPO (PCT)
Prior art keywords
subjects
nucleic acid
genotype
vasopressin
subject
Prior art date
Application number
PCT/CA2007/000111
Other languages
French (fr)
Inventor
James A. Russell
Keith R. Walley
Hugh F. Wellman
Nathan J. Markward
Original Assignee
The University Of British Columbia
Sirius Genomics Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of British Columbia, Sirius Genomics Inc. filed Critical The University Of British Columbia
Priority to JP2008551611A priority Critical patent/JP2009523456A/en
Priority to MX2008009654A priority patent/MX2008009654A/en
Priority to CA002638773A priority patent/CA2638773A1/en
Priority to US12/162,066 priority patent/US20090298711A1/en
Priority to AU2007209725A priority patent/AU2007209725A1/en
Priority to EP07719352A priority patent/EP1984546A4/en
Publication of WO2007085087A1 publication Critical patent/WO2007085087A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/095—Oxytocins; Vasopressins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/08—Bronchodilators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Vasopressin Pathway Polymorphisms as Indicators of Subject Outcome in Critically 111 Subjects
  • the field of the invention relates to the assessment and/or treatment of subjects with an inflammatory condition.
  • Arginine vasopressin has both vasoconstrictor and anti-diuretic properties.
  • AVP is synthesized in the hypothalamus and secreted from posterior pituitary gland, secreted into the circulation and binds to several receptors.
  • AVP binds to vasopressin-specific membrane bound receptor AVPRlA on vascular smooth muscle (MOUILLAC B. et al. J Biol Chem (1995) 270: 25771-25777), AVPR2 in the distal convoluted tubule and collecting ducts in the kidney and AVPRlB pituitary receptors that modify adrenocorticotropin hormone (ACTH) production (ORLOFF J. and HANDLER J.
  • ACTH adrenocorticotropin hormone
  • AVP leucyl/cystinyl aminopeptidase
  • AVP does not contribute much to the maintenance of blood pressure (GROLLMAN J Pharm Exper Therap ( 1932) 46:447-460; GRAYBIEL Am Heart J (1941) 21:481-489; and WAGNER, J Clin Invest (1956) 35: 1412-1418).
  • AVP is fundamental to the response to hypotension as AVP is released from the posterior pituitary and causes arterial smooth muscle to contract (vasoconstriction) (WAGNER, J Clin Invest (1956) 35: 1412-1418; AISENBREY J Clin Invest (1981) 67:961-968; and SCHWARTZ Endocrinology (1981) 108: 1778-1780). If AVP is not secreted by the posterior pituitary in response to hypotension, then blood pressure remains low or falls further as a result of inappropriate vasodilation.
  • AVP can be administered to subjects who have septic shock who are not responding adequately. It has been reported that AVP increases blood pressure, decreases need for vasopressors such as norepinephrine, and increases urine output (LANDRY DW et al. Circulation. (1997) 95: 1122-1125; HOLMES CL et al. Int. Care Med. (2001) 27: 1416-1421).
  • Vasopressin is commonly used after cardiac surgery as studies have shown that AVP levels are lower after cardiac surgery compared to baseline.
  • AVP infusion has been demonstrated to increase blood pressure after cardiac surgery (ARGENZIANO J Circulation (1997) 96(9 Suppl):II-286-90; ARGENZIANO J Thorac. Cardiovasc Surg. (1998) 116(6):973-80; CHEN Circulation (1999) 100(19 Suppl):II244-6; and ROSENZWEIG Circulation (1999) 100(19 Suppl):II182-6).
  • Arginine vasopressin also known as antidiuretic hormone or ADH
  • AVP AVP - neurophysin II gene
  • AVP is synthesized in the hypothalamus as a precursor polypeptide (prepro-AVP-NPII) and undergoes post-translational processing to yield three functional peptides: AVP, NPII, and copeptin (Entrez Gene; http://www.ncbi.nlm.nih.gov/entrez).
  • the AVP-NPl 1 complex is transported along nerve axons to the posterior pituitary where it is secreted into the bloodstream or directly into the brain.
  • _AVP acts to maintain fluid homeostasis by signaling through AVPR2 receptors in the collecting ducts of the kidney (BIRNB AUMER M Trends Endocrinol Metab (2000) 10:406-10) and plays a role in pH regulation (TASHEVIA Y et al Plufgers Arch (2001) 442(5):652-61. Furthermore, AVP is thought to be involved in cognition, tolerance, adaptation as well as complex sexual and maternal behavior (YOUNG WS et al Neurosci (2006) 143(4): 1031-9). A representative human AVP mRNA sequence is listed in GenBank under accession numbers NM_00490 (633 bp). NM 00490 contains AVP rs 1410713 but not rs857242.
  • AVPRlA Human arginine vasopressin receptor IA
  • VIaR Via vasopressin receptor
  • SCCL vasopressin subtype Ia receptor
  • Vl-vascular vasopressin receptor Vl-vascular vasopressin receptor
  • antidiuretic hormone receptor IA vascular/hepatic -type arginine vasopressin receptor
  • AVPRlA maps to chromosomal region 12ql4-ql5.
  • the protein encoded by this gene acts as receptor for arginine vasopressin (AVP).
  • AVP arginine vasopressin
  • This receptor belongs to the subfamily of G-protein coupled receptors which also includes AVPRlB, AVPR2 and OXTR.
  • AVPRlA agonist binding increases intracellular calcium concentrations by signaling through the phospholipase C cascade (OMM: 600821).
  • OMM phospholipase C cascade
  • the downstream effects of this signaling cascade include cell contraction and proliferation, platelet aggregation, release of coagulation factors and glycogenolysis.
  • AVPRlA has been investigated for associations with social behaviors, including affiliation and attachment (YOUNG LJ et al Nature (1999) 400(6746):766-8) as well as essential hypertension (THIBONNIER M et al l MoI Cell Cardiol (2000) 32(4):557-564).
  • a representative human AVPRlA mRNA sequence is listed in GenBank under accession number NM_000706 (4154 bp).
  • the NM_000706 sequence contains AVPRlA SNP rs3803107 (and rslO42615), but not rsl495027 or rsl0877970.
  • LNPEP Homo sapiens leucyl/cystinyl aminopeptidase
  • AT (4) receptor angiotensin IV receptor
  • insulin-regulated aminopeptidase insulin-responsive aminopeptidase
  • insulin-responsive aminopeptidase insulin-responsive aminopeptidase
  • otase oxytocinase
  • placental leucine aminopeptidase and vasopressinase.
  • LNPEP maps to chromosomal region 5ql5.
  • the LNPEP gene encodes a metalloproteinase that cleaves polypeptides such as vasopressin, oxytocin, lys-bradykinin, met-enkephalin and dynorphin A (Entrez Gene: www.ncbi.nlm.nih.gov/entrez). LNPEP also catalyzes the conversion of angiotensinogen to angiotensin IV (AT4) and is thought to play a role in memory processing by acting as a receptor for AT4 (LEW RA et al J Neurochem (2003) 86(2):344-50. LNPEP also plays a role in the maintenance of pregnancy (NORMURA S et al Biochim Biophys Acta (2005) 1751(l): 19-25).
  • LNPEP LNPEP mRNA sequence
  • GenBank GenBank under accession number NM_005575 (4470 bp).
  • the NM_005575 sequence does not contain the LNPEP SNP rs 18059.
  • Homo sapiens leukocyte-derived arginine aminopeptidase (LRAP) is also known as endoplasmic reticulum aminopeptidase 2; (ERAP2).
  • LRAP maps to chromosomal region 5ql5, immediately upstream of LNPEP.
  • the longest annotated transcript of LRAP (NM 022350) has 18 exons and is predicted to encode a protein of 915 amino acids (aa).
  • LRAP is localized to the endoplasmic reticulum (ER) of the cell where it functions to cleave antigenic peptides greater than nine aa for presentation to major histocompatibility complex 1 (MHC-I) molecules (TANIOKA T et al J Biol Chem (2003) 278(34):32275-83).
  • MHC-I major histocompatibility complex 1
  • a representative human LRAP mRNA sequence is listed in GenBank under accession number NM_022350 (3356 bp).
  • Genotype has been shown to play a role in the prediction of subject outcome in inflammatory and infectious diseases (MCGUIRE W. et al. Nature (1994) 371 :508-10; NADEL S. et al. Journal of Infectious Diseases (1996) 174:878-80; MIRA JP. et al. JAMA (1999) 282:561-8; MAJETSCHAK M. et al. Ann Surg (1999) 230:207-14; STUBER F. et al. Crit Care Med (1996) 24:381-4; STUBER F. et al. Journal of Inflammation (1996) 46:42-50; and WEITKAMP JH. et al. Infection (2000) 28:92-6).
  • genotype can alter response to therapeutic interventions.
  • Genentech's HERCEPTIN® was not effective in its overall Phase III trial but was shown to be effective in a genetic subset of subjects with human epidermal growth factor receptor 2 (HER2)- positive metastatic breast cancer.
  • Novartis' GLEEVEC® is only indicated for the subset of chronic myeloid leukemia subjects who carry a reciprocal translocation between chromosomes 9 and 22.
  • This invention is based in part on the surprising discovery that vasopressin pathway SNPs from AVP, AVPRlA, LNPEP and LRAP are predictive or indicative of subject outcome, wherein subject outcome is the ability of the subject to recover from an inflammatory condition based on having a particular AVP, AVPRlA, LNPEP or LRAP genotype as compared to a subject not having that genotype.
  • vasopressin pathway SNPs having an association with improved prognosis or subject outcome, in subjects with an inflammatory condition.
  • various vasopressin pathway SNPs are provided which are useful for subject screening, as an indication of subject outcome, or for prognosis for recovery from an inflammatory condition.
  • This invention is also based in part on the identification that the particular nucleotide (allele) or genotype at the site of a given SNP may be associated with a decreased likelihood of recovery from an inflammatory condition ('risk genotype') or an increased likelihood of recovery from an inflammatory condition ('decreased risk genotype'). Furthermore, this invention is in part based on the discovery that the genotype or allele may be predictive of increased responsiveness to the treatment of the inflammatory condition with vasopressin receptor agonist (i.e. "adverse response genotype” (ARG) or "improved response genotype” (IRG)).
  • the vasopressin receptor agonist may be vasopressin.
  • the inflammatory condition may be SIRS, sepsis or septic shock.
  • This invention is also based in part on the surprising discovery that AVP, AVPRlA LNPEP and LRAP SNPs alone or in combination are useful in predicting the response a subject with an inflammatory condition will have to vasopressin receptor agonist treatment or vasopressin treatment. Whereby the subjects having an improved response genotype are more likely to benefit from and have an improved response to vasopressin receptor agonist treatment and subjects having a non-improved response genotype are less likely to benefit from the same treatment.
  • AVP AVPRlA LNPEP
  • LRAP SNPs and SNPs in linkage disequilibrium (LD) thereto which are also useful in predicting the response a subject with an inflammatory condition will have to vasopressin receptor agonist treatment or vasopressin treatment.
  • methods for obtaining a prognosis for a subject having, or at risk of developing, an inflammatory condition, the method including determining a genotype of said subject which includes one or more polymorphic sites in the subject's vasopressin pathway gene sequences or a combination thereof, wherein said genotype is indicative of an ability of the subject to recover from the inflammatory condition.
  • methods for identifying a polymorphism in a vasopressin pathway gene sequence that correlates with prognosis of recovery from an inflammatory condition, the method including: obtaining vasopressin pathway gene sequence information from a group of subjects having an inflammatory condition; identifying at least one polymorphic nucleotide position in the vasopressin pathway gene sequence in the subjects; determining a genotypes at the polymorphic site for individual subjects in the group; determining recovery capabilities of individual subjects in the group from the inflammatory condition; and correlating the genotypes determined in step (c) with the recovery capabilities determined in step (d) thereby identifying said vasopressin pathway gene sequence polymorphisms that correlate with recovery.
  • a kit for determining a genotype at a defined nucleotide position within a polymorphic site in vasopressin pathway gene sequence in a subject to provide a prognosis of the subject's ability to recover from an inflammatory condition, the kit including: a restriction enzyme capable of distinguishing alternate nucleotides at the polymorphic site; or a labeled oligonucleotide having sufficient complementary to the polymorphic site so as to be capable of hybridizing distinctively to said alternate.
  • the kit may further include an oligonucleotide or a set of oligonucleotides operable to amplify a region including the polymorphic site.
  • the kit may further include a polymerization agent.
  • the kit may further include instructions for using the kit to determine genotype.
  • methods for treating an inflammatory condition in a subject in need thereof, the method including administering to the subject a vasopressin receptor agonist, wherein said subject has an improved response genotype in their vasopressin pathway associated gene sequence.
  • methods for treating an inflammatory condition in a subject in need thereof, the method including: selecting a subject having an improved response genotype in their vasopressin pathway associated gene sequence; and administering to said subject one or more vasopressin receptor agonist(s).
  • methods for treating a subject with an inflammatory condition by administering a vasopressin receptor agonist, the method including administering the vasopressin receptor agonist to subjects that have an improved response genotype in their vasopressin pathway associated gene sequence, wherein the improved response genotype is predictive of increased responsiveness to the treatment of the inflammatory condition with a vasopressin receptor agonist.
  • methods for identifying a subject with increased responsiveness to treatment of an inflammatory condition with a vasopressin receptor agonist, including the step of screening a population of subjects to identify those subjects that have an improved response genotype in their vasopressin pathway associated gene sequence, wherein the identification of a subject with an improved response genotype in their vasopressin pathway associated gene sequence is predictive of increased responsiveness to the treatment of the inflammatory condition with the vasopressin receptor agonist.
  • methods are provided for selecting a subject for the treatment of an inflammatory condition with a vasopressin receptor agonist, including the step of identifying a subject having an improved response genotype in their vasopressin pathway associated gene sequence, wherein the identification of a subject with the improved response genotype is predictive of increased responsiveness to the treatment of the inflammatory condition with the vasopressin receptor agonist.
  • methods for treating an inflammatory condition in a subject, the method including administering a vasopressin receptor agonist to the subject, wherein said subject has an improved response genotype in their vasopressin pathway associated gene sequence.
  • methods for treating an inflammatory condition in a subject, the method including: identifying a subject having an improved response genotype in their vasopressin pathway associated gene sequence; and administering a vasopressin receptor agonist to the subject.
  • vasopressin receptor agonist(s) are provided for administering one or more vasopressin receptor agonist(s) to a subject in need thereof, said subject having an improved response genotype in their vasopressin pathway associated gene sequence.
  • methods are provided for treating an inflammatory condition in a subject, the method including: identifying a subject having an adverse response genotype in their vasopressin pathway associated gene sequence; and selectively not administering a vasopressin receptor agonist to the subject.
  • methods are provided for selectively not administering one or more vasopressin receptor agonist(s) to a subject, wherein said subject has an adverse response genotype in their vasopressin pathway associated gene sequence.
  • vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition, wherein the subjects treated have an improved response polymorphism in their vasopressin pathway associated gene sequence.
  • vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition, wherein the subjects treated do not have an adverse response polymorphism in their vasopressin pathway associated gene sequence.
  • vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition in a subset of subjects, wherein the subset of subjects have an improved response polymorphism in their vasopressin pathway associated gene sequence.
  • vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition in a subset of subjects, wherein the subset of subjects do not have an adverse response polymorphism in their vasopressin pathway associated gene sequence.
  • a commercial package containing, as active pharmaceutical ingredient, use of a vasopressin receptor agonist, or a pharmaceutically acceptable salt thereof, together with instructions for its use for the curative or prophylactic treatment of an inflammatory condition in a subject, wherein the subject treated has an improved response polymorphism in their vasopressin pathway associated gene sequence.
  • a commercial package containing, as active pharmaceutical ingredient, use of a vasopressin receptor agonist, or a pharmaceutically acceptable salt thereof, together with instructions for its use for the curative or prophylactic treatment of an inflammatory condition in a subject, wherein the subject treated does not have an adverse response polymorphism in their vasopressin pathway associated gene sequence.
  • the method or use may further include determining the subject's APACHE II score as an assessment of subject risk.
  • the method or use may further include determining the number of organ system failures for the subject as an assessment of subject risk.
  • the subject's APACHE II score may be indicative of an increased risk when > 25. 2 or more organ system failures may be indicative of increased subject risk.
  • the improved response genotype may be found at one or more of the following polymorphic sites: rsl8059; rs2771 1; rsl0051637; rsl410713; rs857240; rs857242; and rsl495027; or a polymorphic site in linkage disequilibrium thereto.
  • the polymorphic site in linkage disequilibrium is selected from one or more of the following: rs2762; rslOO51637; rsl477364; rs7731592; rs7736466; rsl363974; rs2351010; rsl423357; rsl544777; rs2161548; rs38032; rs38034; rs38041 ; rs27436; rs27306; rs27307; rs27397; rs27659; rs27711 ; rs27290; rs38030; rs27294; rs27747; rs39602; rs248215; rs27302; rs2278018; rsl559355; rs3734015; rs4869315; rs2247650; rs2549781
  • the improved response genotype may be selected from one or more of the following: rsl8059CT; rsl8059TT; rs2771 IGG; rsl0051637GA; rsl0051637AA; rsl410713AC; rsl410713AA; rs857240CC; rs857242CC; rsl495027CC; and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto.
  • the adverse response genotype which may be selected from one or more of the following: rsl8059CC; rs2771 IAA; rsl0051637GG; rsl410713CC; rs857240CT; rs857242AC; and rsl495027TT; or a polymorphic site in linkage disequilibrium thereto.
  • the genotype of the polymorphic site in linkage disequilibrium may be selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
  • the subject having one or more improved response genotypes may be selectively administered the vasopressin receptor agonist.
  • the subject having one or more adverse response genotypes may be selectively not administered the vasopressin receptor agonist.
  • methods are provided for selecting a group of subjects for determining the efficacy of a candidate drug known or suspected of being useful for the treatment of an inflammatory condition, the method including determining a genotype at one or more polymorphic sites in a vasopressin pathway gene sequence for each subject, wherein said genotype is indicative of the subject' s ability to recover from the inflammatory condition and sorting subjects based on their genotype.
  • the method may further include, administering the candidate drug to the subjects or a subset of subjects and determining each subject's ability to recover from the inflammatory condition.
  • the method may further include comparing subject response to the candidate drug based on genotype of the subject.
  • the polymorphic site may be selected from one or more of the following: rs 18059; rs27711; rs38041; rsl0051637; rsl410713; rs857240; rs857242; rsl0877970; rs3803107; and rsl495027; or a polymorphic site in linkage disequilibrium thereto.
  • the polymorphic site in linkage disequilibrium may be selected from one or more of the following: rs2762; rslOO51637; rsl477364; rs7731592; rs7736466; rsl363974; rs2351010; rsl423357; rsl544777; rs2161548; rs38O32; rs38034; rs38041; rs27436; rs27306; rs27307; rs27397; rs27659; rs27711 ; rs27290; rs38O3O; rs27294; rs27747; rs39602; rs248215; rs27302; rs2278018; rs 1559355; rs3734015; rs4869315; rs2247
  • the method may further include comparing the genotype determined with known genotypes, which are known to be indicative of a prognosis for recovery from the subject's type of inflammatory condition, or another inflammatory condition.
  • the method may further include obtaining vasopressin pathway gene sequence information for the subject.
  • the genotype may be determined using a nucleic acid sample from the subject.
  • the method may further include obtaining the nucleic acid sample from the subject.
  • the genotype may be determined using one or more of the following techniques: restriction fragment length analysis; sequencing; micro-sequencing assay; hybridization; invader assay; gene chip hybridization assays; oligonucleotide ligation assay; ligation rolling circle amplification; 5' nuclease assay; polymerase proofreading methods; allele specific PCR; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; ligase chain reaction assay; enzyme-amplified electronic transduction; single base pair extension assay; and reading sequence data.
  • the genotype of the subject may be indicative of increased risk of death or organ dysfunction from the inflammatory condition.
  • the subject may be critically ill and the genotype is indicative of a pro
  • the genotype may include at least one of the following risk genotypes: rsl8059CT; rsl8059TT; rs27711GA; rs27711GG; rs38041GA; rs38041GG; rsl0051637GA; rslOO51637GG; rsl410713AA; rs857240CC; rs857242CC; rslO87797OCC; rs3803107TT; and rsl495027TT; or a polymorphic site in linkage disequilibrium thereto.
  • the genotype may include at least one of the following risk alleles: rs3803107T; and rslO87797OC; or a polymorphic site in linkage disequilibrium thereto.
  • the genotype of the subject may be indicative of decreased risk of death or organ dysfunction from the inflammatory condition.
  • the subject may be critically ill and the genotype is indicative of a prognosis of mild cardiovascular or respiratory dysfunction.
  • the genotype may include at least one of the following reduced risk genotypes: rsl8059CC; rs2771 IAA; rs38041AA; rsl0051637AA; rsl410713CC; rsl410713AC; rs857240TT; rs857240CT; rs857242AA; rs857242AC; rsl0877970TT; rslO87797OCT; rs3803107CC; rs3803107CT; rsl495027CC and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto.
  • the genotype may include at least one of the following reduced risk alleles: rs38O31O7C
  • the genotype of the polymorphic site in linkage disequilibrium may be selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
  • the inflammatory condition may be selected from the group consisting of: sepsis, septicemia, pneumonia, septic shock, systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), acute lung injury, aspiration pneumonitis, infection, pancreatitis, bacteremia, peritonitis, abdominal abscess, inflammation due to trauma, inflammation due to surgery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue damage due to chemotherapy or radiotherapy, and reactions to ingested, inhaled, infused, injected, or delivered substances, glomerulonephritis, bowel infection, opportunistic infections, and for subjects undergoing major surgery or dialysis, subjects who are immunocompromised, subjects on immunosuppressive agents, subjects with HIV/ AIDS, subjects with suspected endocarditis, subjects with fever, subjects with fever of unknown origin, subjects with cystic fibrosis, subjects with diabetes mellitus, subjects
  • coli 0157:H7 malaria, gas gangrene, toxic shock syndrome, pre-eclampsia, eclampsia, HELLP syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhagic fever, pelvic inflammatory disease, Legionella, Lyme disease,
  • Influenza A Epstein-Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, progressive systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, systemic vasculitis, Wegener's granulomatosis, transplants including heart, liver, lung kidney bone marrow, graft-versus-host disease, transplant rejection, sickle cell anemia, nephrotic syndrome, toxicity of agents such as OKT3, cytokine therapy, and cirrhosis.
  • the inflammatory condition may be SIRS.
  • the inflammatory condition may be sepsis.
  • the inflammatory condition may be septic shock.
  • the vasopressin receptor agonist may be vasopressin.
  • two or more oligonucleotides or peptide nucleic acids of about 10 to about 400 nucleotides that hybridize specifically to a sequence contained in a human target sequence consisting of a subject's vasopressin pathway associated gene sequence, a complementary sequence of the target sequence or RNA equivalent of the target sequence and wherein the oligonucleotides or peptide nucleic acids are operable in determining the presence or absence of two or more polymorphism(s) or in their vasopressin pathway associated gene sequence selected from of the following polymorphic sites: rsl8059; rs2771 1 ; rs38041; rsl0051637; rsl410713; rs857240; rs857242; rsl0877970; rs3803107; and rsl495027; or one or more polymorphic sites in linkage disequilibrium thereto.
  • two or more oligonucleotides or peptide nucleic acids selected from the group including of: (a) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO: 1 having a T at position 201 but not to a nucleic acid molecule including SEQ ID NO: 1 having a C at position 201 ; (b) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:1 having a C at position 201 but not to a nucleic acid molecule including SEQ ID NO: 1 having a T at position 201 ; (c) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:2 having a G at position 201 but
  • an array of oligonucleotides or peptide nucleic acids attached to a solid support the array including two or more of the oligonucleotides or peptide nucleic acids as set out herein.
  • composition including an addressable collection of two or more oligonucleotides or peptide nucleic acids, the two or more oligonucleotides or peptide nucleic acids selected from the oligonucleotides or peptide nucleic acids as set out herein.
  • composition including an addressable collection of two or more oligonucleotides or peptide nucleic acids, the two or more oligonucleotides or peptide nucleic acids consisting essentially of two or more nucleic acid molecules set out in SEQ ID NO: 1-264 or compliments, fragments, variants, or analogs thereof.
  • ancomposition including an addressable collection of two or more oligonucleotides or peptide nucleic acids, the two or more oligonucleotides or peptide nucleic acids consisting essentially of two or more nucleic acid molecules set out in TABLES 1C and ID or compliments, fragments, variants, or analogs thereof.
  • the oligonucleotides or peptide nucleic acids described herein may further include one or more of the following: a detectable label; a quencher; a mobility modifier; a contiguous non-target sequence situated 5' or 3' to the target sequence or 5' and 3' to the target sequence.
  • a computer readable medium including a plurality of digitally encoded genotype correlations selected from the vasopressin pathway associated gene SNP correlations in TABLE IE, wherein each correlation of the plurality has a value representing an ability to recover from an inflammatory condition and a value representing an indication of responsiveness to treatment with a vasopressin receptor agonist.
  • the oligonucleotides or peptide nucleic acids may further include one or more of the following: a detectable label; a quencher; a mobility modifier; a contiguous non-target sequence situated 5' or 3' to the target sequence or 5' and 3' to the target sequence.
  • the oligonucleotides or peptide nucleic acids may alternatively be of about 10 to about 400 nucleotides, about 15 to about 300 nucleotides.
  • the oligonucleotides or peptide nucleic acids may alternatively be of about 20 to about 200 nucleotides, about 25 to about 100 nucleotides.
  • the oligonucleotides or peptide nucleic acids may alternatively be of about 20 to about 80 nucleotides, about 25 to about 50 nucleotides.
  • the genotype may be determined using a nucleic acid sample from the subject. Genotype may be determined using one or more of the following techniques: restriction fragment length analysis; sequencing; micro-sequencing assay; hybridization; invader assay; gene chip hybridization assays; oligonucleotide ligation assay; ligation rolling circle amplification; 5' nuclease assay; polymerase proofreading methods; allele specific PCR; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; ligase chain reaction assay; enzyme-amplified electronic transduction; single base pair extension assay; and reading sequence data.
  • MALDI-TOF matrix assisted laser desorption ionization time of flight
  • a determination of whether a site is in linkage disequilibrium (LD) with another site may be determined based on an absolute r value or D' value.
  • D' value an absolute value for linkage disequilibrium
  • a high degree of linkage disequilibrium may be represented by an absolute value for D' of > 0.6 or r 2 > 0.6.
  • a higher degree of linkage disequilibrium may be represented by an absolute value for D' of > 0.7 or r 2 > 0.7 or by an absolute value for D' of > 0.8 or r 2 > 0.8.
  • a high degree of linkage disequilibrium may be represented by an absolute value for D' of > 0.85 or r 2 > 0.85 or by an absolute value for D' of > 0.9 or r 2 > 0.9.
  • Two or more oligonucleotides or peptide nucleic acids may include 3 or more; 4 or more; 5 or more; 6 or more; 7 or more; 8 or more; 9 or more; 10 or more; 1 1 or more; 12 or more; 13 or more; 14 or more; 15 or more; 16 or more; 17 or more; 18 or more; 19 or more; or 20 or more.
  • Sequence variations may be assigned to a gene if mapped within 2 kb or more of an mRNA sequence feature.
  • a sequence may extend many kilobases (kb) from a vasopressin pathway gene and into neighbouring genes, where the LD within a region is strong.
  • LNPEP Leucyl aminopeptidase
  • AVPRlA arginine vasopressin receptor
  • AVPRlA arginine vasopressin receptor
  • Vassopressin Receptor Agonist as used herein includes any vasopressin molecule, vasopressin derivative, vasopressin variant, vasopressin analogue, non-peptidyl analogues and any prodrug thereof, metabolite thereof, isomer thereof, combination of isomers thereof, or pharmaceutical composition of any of the preceding.
  • Such agonists may be capable of binding to or interacting with a vasopressin receptor and initiating one or more of the types of responses typically produced by the binding of an endogenous vasopressin molecule to a vasopressin receptor (for example, AVPRlA, AVPRlB, AVPR2 and OXTR).
  • Vasopressin receptor agonists may be used alone or in combination with other vasopressin receptor agonists or other medications. Vasopressin receptor agonists may be synthesized or purified. Examples of vasopressin receptor agonists capable of increasing blood pressure, include, but are not limited to, arginine vasopressin (AVP), lysine vasopressin (LVP), triglycil-lysine vasopressin (also known as Terlipressin or Glycopressin), Octapressin, Ornipressin, Desmopressin, Desmopressin acetate, Lypressin, Felypressin, and
  • Vasopressin analogues may be 1 - 3 amino acids such as AIa-AVP, Ser-Ala-AVP, Thr-Ser-Ala-A VP (KALISZAN R. et al. Pharmacol Res Commun (1988) 20(5):377-381) or 3- beta- (2-thienyl)-L-alanine)-8-lysine-vasopressin and other similar analogues (Smith CW. Acta Pharmacol Toxicol (Copenhag) (1978) 43(3): 190-195). Examples of derivatives, variants, analogues or compositions etc.
  • Vasopressin as used herein includes: Antidiuretic hormone; Argiprestocin; Arginine Vasopressin; Arginine oxytocin; Pitressin tannate; Arginine vasotocin; Vasotocin; Vasopressin, isoleucyl; 3-Isoleucyl vasopressin; l-[[19-amino-13-butan-2-yl-10-(2-carbamoylethyl)-7- (carbamoylmethyl)- 16-[(4-hydroxyphenyl)methyl]-6,9, 12,15,18-pentaoxo- 1 ,2-dithia-5,8, 11,14,17- pentazacycloicos-4-yl]carbonyl]-N-[l-(carbamoylmethylcarbamoyl)-4-guanidino-butyl]- pyrrolidine-2-carboxamide (IUPAC name).
  • Vasopressin is a nine amino acid peptide (Cys-Tyr- Ile-Gln-Asn-Cys-Pro-Arg-Gly, cyclic 1-6 disulfide) secreted from the posterior pituitary and binds to receptors in blood vessels, the brain and distal or collecting tubules of the kidney to promote vasoconstriction or reabsorption of water back into the circulation.
  • Vasopressin receptor targets include AVPRlA, AVPRlB, AVPR2 and OXTR.
  • Vasopressin for example, is sold as PRESSYN ARTM by Ferring Inc., and also sold in various formulations as VASOPRESSIN by Ferring Inc., Sandoz Canada Inc.
  • PITRESSINTM is sold by Warner-Lambert Company, Parke-Davis Division, as a synthetic injectable vasopressin (8- Arginine vasopressin). It is substantially free from the oxytocic principle and is standardized to contain 20 pressor units/mL. The solution contains 0.5% Chlorobutanol (chloroform derivative) as a preservative. Also, DIAPIDTM is sold as a nasal spray by Sandoz Inc.
  • vasopressin is intended for use in the prevention of treatment of post-operative abdominal distension, dispelling of gas shadows in abdominal roentgenography and symptomatic control of diabetes insipidus.
  • Genetic material includes any nucleic acid and can be a deoxyribonucleotide or ribonucleotide polymer in either single or double-stranded form.
  • a “purine” is a heterocyclic organic compound containing fused pyrimidine and imidazole rings, and acts as the parent compound for purine bases, adenine (A) and guanine (G).
  • a "Nucleotide” is generally a purine (R) or pyrimidine (Y) base covalently linked to a pentose, usually ribose or deoxyribose, where the sugar carries one or more phosphate groups.
  • Nucleic acids are generally a polymer of nucleotides joined by 3 '-5' phosphodiester linkages.
  • purine is used to refer to the purine bases, A and G, and more broadly to include the nucleotide monomers, deoxyadenosine-5' -phosphate and deoxyguanosine-5' -phosphate, as components of a polynucleotide chain.
  • a "pyrimidine” is a single-ringed, organic base that forms nucleotide bases, cytosine (C), thymine (T) and uracil (U).
  • pyrimidine is used to refer to the pyrimidine bases, C, T and U, and more broadly to include the pyrimidine nucleotide monomers that along with purine nucleotides are the components of a polynucleotide chain.
  • a nucleotide represented by the symbol M may be either an A or C
  • a nucleotide represented by the symbol W may be either an T/U or A
  • a nucleotide represented by the symbol Y may be either an C or T/U
  • a nucleotide represented by the symbol S may be either an G or C
  • a nucleotide represented by the symbol R may be either an G or A
  • a nucleotide represented by the symbol K may be either an G or TAJ.
  • nucleotide represented by the symbol V may be either A or G or C
  • a nucleotide represented by the symbol D may be either A or G or T
  • a nucleotide represented by the symbol B may be either G or C or T
  • a nucleotide represented by the symbol H may be either A or C or T.
  • a "polymorphic site” or “polymorphism site” or “polymorphism” or “single nucleotide polymorphism site” (SNP site) or single nucleotide polymorphism” (SNP) as used herein is the locus or position with in a given sequence at which divergence occurs.
  • a “polymorphism” is the occurrence of two or more forms of a gene or position within a gene (allele), in a population, in such frequencies that the presence of the rarest of the forms cannot be explained by mutation alone. The implication is that polymorphic alleles confer some selective advantage on the host.
  • Preferred polymorphic sites have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
  • Polymorphic sites may be at known positions within a nucleic acid sequence or may be determined to exist using the methods described herein. Polymorphisms may occur in both the coding regions and the noncoding regions (for example, promoters, introns or untranslated regions) of genes. Polymorphisms may occur at a single nucleotide site (SNPs) or may involve an insertion or deletion as described herein.
  • SNPs single nucleotide site
  • a "risk genotype” as used herein refers to an allelic variant (genotype) at one or more polymorphic sites within the vasopressin pathway gene (i.e. AVP, AVPRlA and LNPEP) sequences described herein as being indicative of a decreased likelihood of recovery from an inflammatory condition or an increased risk of having a poor outcome.
  • the risk genotype may be determined for either the haploid genotype or diploid genotype, provided that at least one copy of a risk allele is present. Risk genotype may be an indication of an increased risk of not recovering from an inflammatory condition.
  • Subjects having one copy (heterozygotes) or two copies (homozygotes) of the risk allele are considered to have the "risk genotype" even though the degree to which the subjects risk of not recovering from an inflammatory condition may increase, depending on whether the subject is a homozygote rather than a heterozygote.
  • Such "risk alleles” or “risk genotypes” may be selected from the following: rsl8059CT; rsl8059TT; rs2771 1GA; rs27711GG; rs38041GA; rs38041GG; rslOO51637GA; rslOO51637GG; rsl410713AA; rs857240CC; rs857242CC; rsl0877970TT; rs38O31O7TT; and rsl495027CC; or a polymorphic site in linkage disequilibrium thereto.
  • a “decreased risk genotype” as used herein refers to an allelic variant (genotype) at one or more polymorphic sites within the vasopressin pathway gene (i.e. AVP, AVPRlA and LNPEP) sequences described herein as being indicative of an increased likelihood of recovery from an inflammatory condition or a decreased risk of having a poor outcome.
  • the decreased risk genotype may be determined for either the haploid genotype or diploid genotype, provided that at least one copy of a risk allele is present.
  • Decreased risk genotype may be an indication of an increased likelihood of recovering from an inflammatory condition.
  • Subjects having one copy (heterozygotes) or two copies (homozygotes) of the decreased risk allele are considered to have the "decreased risk genotype" even though the degree to which the subjects risk of not recovering from an inflammatory condition may increase, depending on whether the subject is a homozygote rather than a heterozygote.
  • Such “decreased risk alleles” or “decreased risk genotypes” or “reduced risk genotypes” may be selected from the following: rsl8059CC; rs27711AA; rs38041AA; rsl0051637AA; rsl410713CC; rsl410713AC; rs857240TT; rs857240CT; rs857242AA; rs857242AC; rsl0877970TT; rslO87797OCT; rs3803107CC; rs3803107CT; rsl495027CC and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto.
  • IRG improved response genotype
  • FRP improved response polymorphic variant
  • AVP arginine vasopressin
  • AVPRlA arginine vasopressin receptor IA
  • LNPEP leucyl/cystinyl aminopeptidase
  • LRAP leukocyte -derived aminopeptidase
  • ARG adverse response genotype
  • AVP arginine vasopressin
  • AVPRlA arginine vasopressin receptor IA
  • LNPEP leucyl/cystinyl aminopeptidase
  • LRAP leukocyte- derived aminopeptidase
  • a "clade” is a group of haplotypes that are closely related phylogenetically. For example, if haplotypes are displayed on a phylogenetic (evolutionary) tree a clade includes all haplotypes contained within the same branch.
  • Haplotype The pattern of a set of markers along a chromosome is referred to as a "Haplotype". Accordingly, groups of alleles on the same small chromosomal segment tend to be transmitted together. Haplotypes along a given segment of a chromosome are generally transmitted to progeny together unless there has been a recombination event. Absence of a recombination event, haplotypes can be treated as alleles at a single highly polymorphic locus for mapping.
  • haplotype is a set of alleles of closely linked loci on a chromosome that tend to be inherited together. Such allele sets occur in patterns, which are called haplotypes. Accordingly, a specific SNP or other polymorphism allele at one SNP site is often associated with a specific SNP or other polymorphism allele at a nearby second SNP site or other polymorphism site. When this occurs, the two SNPs or other polymorphisms are said to be in LD because the two SNPs or other polymorphisms are not just randomly associated (i.e. in linkage equilibrium).
  • the detection of nucleic acids in a sample depends on the technique of specific nucleic acid hybridization in which the oligonucleotide is annealed under conditions of "high stringency" to nucleic acids in the sample, and the successfully annealed oligonucleotides are subsequently detected (see for example Spiegelman, S., Scientific American, Vol. 210, p. 48 (1964)).
  • Hybridization under high stringency conditions primarily depends on the method used for hybridization, the oligonucleotide length, base composition and position of mismatches (if any).
  • High-stringency hybridization is relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high-stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization.
  • these aforementioned techniques are usually performed with relatively short probes (e.g., usually about 16 nucleotides or longer for PCR or sequencing and about 40 nucleotides or longer for in situ hybridization).
  • the high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998.
  • Oligonucleotides as used herein are variable length nucleic acids, which may be useful as probes, primers and in the manufacture of microarrays (arrays) for the detection and/or amplification of specific nucleic acids. Such DNA or RNA strands may be synthesized by the sequential addition (5'-3' or 3'-5') of activated monomers to a growing chain, which may be linked to an insoluble support. Numerous methods are known in the art for synthesizing oligonucleotides for subsequent individual use or as a part of the insoluble support, for example in arrays (BERNFIELD MR. and ROTTMAN FM. J. Biol. Chem.
  • oligonucleotides are synthesized through the stepwise addition of activated and protected monomers under a variety of conditions depending on the method being used. Subsequently, specific protecting groups may be removed to allow for further elongation and subsequently and once synthesis is complete all the protecting groups may be removed and the oligonucleotides removed from their solid supports for purification of the complete chains if so desired.
  • PNA protein nucleic acids
  • PNA protein nucleic acids
  • DNA/RNA DNA/RNA
  • backbone structure of PNA does not inherently have a charge. Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences.
  • PNAs are generally more robust than nucleic acids. PNAs may also be used in arrays and in other hybridization or other reactions as described above and herein for oligonucleotides.
  • an "addressable collection” as used herein is a combination of nucleic acid molecules or peptide nucleic acids capable of being detected by, for example, the use of hybridization techniques or by any other means of detection known to those of ordinary skill in the art.
  • a DNA microarray would be considered an example of an "addressable collection”.
  • linkage refers to the co-inheritance of two or more nonallelic genes or sequences due to the close proximity of the loci on the same chromosome, whereby after meiosis they remain associated more often than the 50% expected for unlinked genes.
  • a physical crossing between individual chromatids may result in recombination.
  • Recombination generally occurs between large segments of DNA, whereby contiguous stretches of DNA and genes are likely to be moved together in the recombination event (crossover).
  • regions of the DNA that are far apart on a given chromosome are more likely to become separated during the process of crossing-over than regions of the DNA that are close together.
  • Polymorphic molecular markers like SNPs, are often useful in tracking meiotic recombination events as positional markers on chromosomes.
  • Linkage Disequilibrium This sort of disequilibrium generally implies that most of the disease chromosomes carry the same mutation and the markers being tested are relatively close to the disease gene(s).
  • SNPs can be useful in association studies for identifying polymorphisms, associated with a pathological condition, such as sepsis. Unlike linkage studies, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. In a SNP association study the frequency of a given allele (i.e. SNP allele) is determined in numerous subjects having the condition of interest and in an appropriate control group. Significant associations between particular SNPs or SNP haplotypes and phenotypic characteristics may then be determined by numerous statistical methods known in the art. Association analysis can either be direct or LD based. In direct association analysis, potentially causative SNPs may be tested as candidates for the pathogenic sequence.
  • SNPs may be chosen at random over a large genomic region or even genome wide, to be tested for SNPs in LD with a pathogenic sequence or pathogenic SNP.
  • candidate sequences associated with a condition of interest may be targeted for SNP identification and association analysis. Such candidate sequences usually are implicated in the pathogenesis of the condition of interest.
  • candidate sequences may be selected from those already implicated in the pathway of the condition or disease of interest. Once identified, SNPs found in or associated with such sequences, may then be tested for statistical association with an individual's prognosis or susceptibility to the condition.
  • VNTRs variable number tandem repeats
  • STRs short tandem repeats
  • linkage disequilibrium is the occurrence in a population of certain combinations of linked alleles in greater proportion than expected from the allele frequencies at the loci. For example, the preferential occurrence of a disease gene in association with specific alleles of linked markers, such as SNPs, or between specific alleles of linked markers, are considered to be in LD.
  • This sort of disequilibrium generally implies that most of the disease chromosomes carry the same mutation and that the markers being tested are relatively close to the disease gene(s). Accordingly, if the genotype of a first locus is in LD with a second locus (or third locus etc.), the determination of the allele at only one locus would necessarily provide the identity of the allele at the other locus. When evaluating loci for LD those sites within a given population having a high degree of linkage disequilibrium (i.e. an absolute value for r 2 > 0.5) are potentially useful in predicting the identity of an allele of interest (i.e. associated with the condition of interest).
  • a high degree of linkage disequilibrium may be represented by an absolute value for r 2 > 0.6.
  • a high degree of linkage disequilibrium may be represented by an absolute value for r 2 > 0.7 or by an absolute value for r 2 > 0.8.
  • a high degree of linkage disequilibrium may be represented by an absolute value for r 2 > 0.85 or by an absolute value for r 2 > 0.9.
  • two SNPs that have a high degree of LD may be equally useful in determining the identity of the allele of interest or disease allele. Therefore, we may assume that knowing the identity of the allele at one SNP may be representative of the allele identity at another SNP in LD. Accordingly, the determination of the genotype of a single locus can provide the identity of the genotype of any locus in LD therewith and the higher the degree of linkage disequilibrium the more likely that two SNPs may be used interchangeably.
  • the determination of the genotype at rs 18059 will provide the identity of the genotype at rs2762 or any other locus in "linkage disequilibrium" therewith. Particularly, where such a locus is has a high degree of linkage disequilibrium thereto.
  • LD is useful for genotype-phenotype association studies. For example, if a specific allele at one SNP site (e.g. "A") is the cause of a specific clinical outcome (e.g. call this clinical outcome "B") in a genetic association study then, by mathematical inference, any SNP (e.g. "C") which is in significant LD with the first SNP, will show some degree of association with the clinical outcome. That is, if A is associated ( ⁇ ) with B, i.e. A-B and C-A then it follows that C-B. Of course, the SNP that will be most closely associated with the specific clinical outcome, B, is the causal SNP - the genetic variation that is mechanistically responsible for the clinical outcome. Thus, the degree of association between any SNP, C, and clinical outcome will depend on LD between A and C.
  • LD helps identify potential candidate causal SNPs and also helps identify a range of SNPs that may be clinically useful for prognosis of clinical outcome or of treatment effect. If one SNP within a gene is found to be associated with a specific clinical outcome, then other SNPs in LD will also have some degree of association and therefore some degree of prognostic usefulness.
  • TABLE IA Numerous sites have been identified as polymorphic sites in the vasopressin pathway associated genes (see TABLE IA). Furthermore, the polymorphisms in TABLE IA are linked to (in LD with) numerous polymorphism as set out in TABLE IB below and may also therefore be indicative of subject prognosis.
  • NA as used above indicates that the LD allele with the information currently available to the inventors could not with any confidence be assigned without further routine analysis, due to the lack of suitable information currently available regarding the corresponding allele designations. However, it would be well within the abilities of a person of skill in the art to make LD allele designations for the NA polymorphisms using routine analysis.
  • a haplotype of vasopressin pathway associated genes can be created by assessing polymorphisms in vasopressin pathway-associated genes in normal subjects using a program that has an expectation maximization algorithm (i.e. PHASE).
  • a constructed haplotype of vasopressin pathway associated genes may be used to find combinations of SNPs that are in LD with the tag SNPs (tSNPs) identified herein. Accordingly, the haplotype of an individual could be determined by genotyping other SNPs or other polymorphisms that are in LD with the tSNPs identified herein.
  • Single polymorphic sites or combined polymorphic sites in LD may also be genotyped for assessing subject response to vasopressin receptor agonist treatment.
  • the numerical designations of the positions of polymorphisms within a sequence are relative to the specific sequence. Also the same positions may be assigned different numerical designations depending on the way in which the sequence is numbered and the sequence chosen, as illustrated by the alternative numbering of the equivalent polymorphism (rs3803107), whereby the same polymorphism identified C/T at position 3536 of the NM_000706.3 (GI:33149325), which corresponds to position 201 of SEQ ED NO:9.
  • sequence variations within the population such as insertions or deletions, may change the relative position and subsequently the numerical designations of particular nucleotides at and around a polymorphic site.
  • Polymorphic sites in SEQ ED NO: 1-10 are identified by their variant designation (i.e. M, W, Y, S, R, K, V, B, D, H or by "-" for a deletion, a "+”or for example "G” etc. for an insertion).
  • Polymorphic sites in SEQ ED NO: 11-264 are identified by their allelic change (i.e. A, C, G, T or by "-" for a deletion, a "+”or for an insertion).
  • the "rs” numbers are the NCBI rsSNP ID form.
  • TABLE ID below shows the flanking sequences for a selection of vasopressin pathway associated gene SNPs in LD with the tagged SNPs in TABLE 1C, providing their rs designations and corresponding SEQ ID NO designations.
  • a SNP in LD is also an htSNP it only occurs in TABLE 1C above.
  • Each SNP is at position 200 of the flanking sequence (unless otherwise indicated) and is underlined.
  • allelic pair i e. the two alleles of a given gene
  • ⁇ "gene” is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product and may include untranslated and untranscribed sequences in proximity to the coding regions (5' and 3' to the coding sequence). Such non-coding sequences may contain regulatory sequences needed for transcription and translation of the sequence or introns etc. or may as yet to have any function attributed to them beyond the occurrence of the SNP of interest.
  • a “genotype” is defined as the genetic constitution of an organism, usually in respect to one gene or a few genes or a region of a gene relevant to a particular context (i.e. the genetic loci responsible for a particular phenotype).
  • a "phenotype” is defined as the observable chaiacters of an organism In gene association studies, the genetic model at a given locus can change depending on the selection pressures (i e , the
  • the model at rs 1410713 changed between the risk of death claims (AA versus AC/CC) and the vasopressin IRP claims (AA/AC veisus C C) This is a case of the same outcome variable (survival) tollow ing a different genetic model in diffeient environments (i e . no vasopressin treatment versus vasopressin treatment)
  • Hemoglobin S results in abnoimal sitkle-shaped ied blood cells w hich lead to anemia and other serious complications including death In the absence of malana.
  • a gene association study w ith the HBB gene would suggest a codominant model (surv ival(BB) > survival (BS) > surv ival (SS))
  • BS survival
  • SS surv ival
  • a gene association study w ith the HBB gene would suggest a heterozygote adv antage model (survival(BB) ⁇ surv ival(BS) >
  • ⁇ single nucleotide polymoiphism " ' (SNP) occurs at a polymorphic site occupied by a single nucleotide, w hich is the site of vai ution between allelic sequences The site is usually pieceded by and followed by highly conserved sequences of the allele (e g , sequences that vary in less than
  • a single nucleotide polymoiphism usually arises due to substitution of one nucleotide for another at the polymorphic site
  • a "transition” is the teplacement of one purine by another pu ⁇ ne oi one pyi imidine by another pynmidine
  • a "tiansvei sion” is the replacement of a pur ne by a pynmidine oi v ice veisa
  • Single nucleotide poly morphisms can also a ⁇ se fiom a deletion (represented by "-" oi "del") of a nucleotide oi an
  • an insertion or deletion w ithin a given sequence could alter the relative poMtion and theiefore the position number of another polymorphism w ithin the sequence
  • an insertion or deletion may by some definitions not qualify as a SNP as it may involve the deletion of or insertion of more than a single nucleotide at a given position, as used heiein such polymorphisms are also called SNPs as the y generall) result from an insertion or deletion at a single site w ithin a given sequence
  • systemic inflammatory response syndrome or (SIRS) is defined as including both septic 1 1 e 5 sepsis or septic shock) and non-septic systemic inflammatory response (i e post operative)
  • SIRS is further defined according to ACCP (American College of Chest Physicians) guidelines as themony of two 01 moie of A) temperature > 38 0 C or ⁇ 36"C, B) heart rate > 90 beats per minute, C) respnatory rate > 20 breaths per minute, or PaCCK ⁇ M mm Hg oi the need for mechanical ventilation, and D) white blood cell count > 12,000 per mm or ⁇ 4.000 mm
  • ACCP American College of Chest Physicians
  • Sepsis is defined as the presence of at least two “SIRS” criteria and known or suspected source of infection Septic shock was defined as sepsis plus one new organ failuie by Brussels c ⁇ tei ia I s plus need foi vasopiessor medication or v asopressin ieceptor agonist
  • Subject outcome or prognosis as used herein refers the ability of a subject to recover from an inflammatoiy condition and may be used o determine the efficacy of a tieatment legimen, foi example the administration of a vasopressin ieceptor agonist
  • An inflammatoiy condition may be
  • renin-up consisting of sepsis, septicemia, pneumonia, septic shock, systemic inflammatoiv iesponse syndrome (SIRS).
  • SIRS systemic inflammatoiv iesponse syndrome
  • ARDS Acute Respiratory Distiess Syndrome
  • aspiiation pneumonitis infection, pancreatitis, bacteremia, pe ⁇ tonitis, abdominal abscess, inflammation due to trauma, inflammation due to suigery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue
  • subjects w ho are immunocompromised subjects on immunosuppiessi ⁇ e agents, subjects w ith HIV/AIDS, subjects w ith suspected endocaiditis, subjects w ith fever, subjects w ith fevei of unknown ongin. sublets w ith cystic fibrosis, subjec ts
  • ATN aciitt tubulai necrosis
  • subjects w ith bronchiectasis subjects w ith chronic obstructive lung disease, chionic bronchitis, emphysema ot asthma, subjects w ith febrile neutropenia, subjects w ith meningitis, subjects w ith septic arthi itis, subjects w ith unnary tract infection, subjects w ith necrotizing fasciitis, subjects w ith other Hispected Group A streptococcus infection, subjects who ha ⁇ e had a splenectomy, subjects w ith recurrent or suspected enteiococcus infection, other medical and suigical conditions associated w ith increased risk of infection, Gram positive sepsis.
  • Gram negative sepsis culture negative sepsis, fungal sepsis, meningococcemia, post-pump syndrome.
  • s cardiac stun syndrome myocardial infarction, stioke, congestive heart failure, hepatitis, epiglottitis.
  • E coli 0157 H7. malaria gas gangiene, toxic shock s> ndrome, pre-eclampsia, eclampsia.
  • HELLP syndiome mycobacte rial tuberculosis, Pneumocystis carina pneumonia, pneumonia.
  • Leishmaniasis hemolytic uremic syndrome/thrombotic thrombocytopenic purpuia, Dengue hemoirhagic fever, pelvic inflammatory disease, Legionella, Lyme disease. Influenza A,
  • Wegener's gianulomatosis tiansplants including heart, liver, lung kidney bone mauow, graft-veisus-host disease, tiansplant rejection, sickle cell anemia,
  • I S nephtotic syndiome toxicity of agents such as OKTl, cytokine theiapy, and ciirhosis
  • APACHE IT' score is defined as Acute Physiology And Chioni; Health Evaluation and herein was calculated on a daily 0 basis from taw clinical and laboiatoiy vai iables Vincent et al (Vincent JL Feireira F Moreno R 2000 Cut Caie Clin 16 153-366) summarize APACHE scoie as follows "Fust developed in 1981 by Knaus c t al .
  • the AP ⁇ CHE scoie has become the most commonly used sutvival prediction model in ICUs worldwide
  • the APACHE II score a rev ised and simplified version of the original prototype, uses a point scoie based on mi ial values of 12 ioutine physiologic measures, age. ind
  • a "Brussels score” score is a method for evaluating oigan dysf unction as compared to a baseline If the Brussels scoie is 0 (i e modeiate, severe, oi extieme). then oigan failure was recoided as present on that particular day (see TABLE 2A below)
  • DAF organ failuie
  • a pulmonary capillary wedge pressure less than 18 mm Hg The severity of acute lung injury is assessed by measuring days alivt and fiee of acute lung injury over a 28-day observation penod Acute lung injury is iecorded as piesent on each day that the person has modeiate, severe or extreme dysfunction as defined in the Brussels score Days alive and fiee of acute lung injury is calculated as the number of days af ter onset of acute lung injuiy that a subject is alive and free of acute lung injuiy over a def ined obsei vation period (28 days) Thus, a low ei score foi days alive and free of acute lung injury indicates more seveie acute lung in)uiy The reason that days alive and free of acute lung injuiy is
  • I 1 S piefeiable to simplyumblence oi absence of acute lung injury is that acute lung injury has a hi gh acute moitality and early death (w ithin 28 days) precludes calculation of the presence oi absence of acute lung injuiy in dead subjects
  • the cardiovasculai, ienal, neuiologic hepatic and coagulation dysfunction were similarly def ined as present on each day that the person had moderate seveie oi extreme dysfunction a> defined by the Brussels score Days alive and fiee of
  • One aspect of the invention may involve the identification of subjects oi the selection ot subjects that aie either at nsk of developing and inflammatory condition or the identif ication of subject ⁇ s who already have an inflammatoiy condition
  • subjects who have undeigone major surgeiy or scheduled foi oi contemplating major surgery may be considered as being at i isk of developing an inflammatoiy condition
  • subjects may be detei mined as ha ⁇ ing an inflammatory condition using diagnostic methods and clinical evaluations known in the medical aits An inflammatory condition.
  • ma> be selected from the gioup consisting of sepsis, septicemia.
  • SIRS systemic inflammatory iesponse syndiome
  • ARDS Acute Respiratory Distiess Syndrome
  • ⁇ failure hepatitis, epiglottitis, E coll 0157 H7. malaria, gas gangrene, toxic shock syndrome, preeclampsia eclampsia, HELLP syndrome, mycobacterial tuberculosis, Pneumocystis cai inii pneumonia, pneumonia.
  • Leishmaniasis hemolytic uremic syndiome/thrombotic thiombocytopenic meme memea. Dengue hemorrhagic fever, pelvic inflammatoiy disease, Legionella, Lyme disease, Influenza ⁇ , Epstem-Barr v n us, encephalitis, inflammatory diseases and autoimmunity including
  • genetic sequence information may be obtained from the subject or alternatively genetic sequence information may already have betn obtained horn the subject For example, a subject may have already provided a biological sample
  • Genetic sequence information may be obtained in numeious diffeient ways and may involve the collection of a biological sample that contains genetic material, particularly, genetic matenal containing the sequence oi sequences of interest Many methods are known in the art for collecting biological samples and ex racting genetic matei ial from those samples Genetic
  • DNA ma> be isolated fiom a biological sample when first the sample is lv, sed and then the DNA is sepaiated horn the lysate according to any one of a variety of multi-step piotocols. which can take varying lengths of time DNA isolation methods may involve the use of phenol (Sambrook. J et al ,
  • Othei methods for DNA isolation utilize non-coirosive chaotropic agents
  • These methods which die based on the use of guanidine salts, uiea and sodium iodide, involve lysis of a biological sample in a chaotropic aqueous solution and subsequent piecipitation of the crude DNA fraction w ith a lowei alcohol
  • the final pot ification of the precipitated, ciude DNA f raction can be achieved by any one of several methods, including column chromatography (Analects, ( 1994) VoI
  • RT-PCR Reverse Tiansc ⁇ ption Polymerase Chain Reaction
  • PCR Polymerase Chain Reaction
  • TMA Tiansc ⁇ ption Mediated Amplification
  • LCR Ligase chain reaction
  • NASBA Nucleic Acid Sequence Based Amplification
  • othei methods know n in the art, and then furthei analyzed to detect or determine the presence or absence of one oi more polymoi phisms or mutations in the sequence of mteiest, provided that the genetic matei ial obtained contains the sequence of interest Particulai ly.
  • a person may be interested in determining theernence oi absence of a mutation in a uisopiessin pathway associated gene sequence, as
  • sequence of interest tnaj also include othei mutations, oi may also contain some of the sequence surrounding the mutation of interest
  • Detection oi determination of a nucleotide identity, or the presence of one or tnoie single nucleotide polymorphism(s) may be accomplished by any one of a number methods or assays known in the ait Many DNA typing methodologies are useful for use in the detection of SNPs
  • the majority of SNP genotyping ieactions or assays can be assigned to one of foui broad groups (sequence-specific hybridization, primer extension, oligonucleotide ligation and invasive clea ⁇ age) Fuithermore, there are numerous methods for analyzing/detecting the products of each 5 type of reaction (for example, fluorescence luminescence, mass measurement, electrophoresis, etc ) Furtheimore. reactions can occur in solution or on a solid support such as a glass slide, a chip, a bead, etc
  • sequence-speciiic hybridization involves a hybridization probe, which is capable of 10 distinguishing between two DNA taigets differing at one nucleotide position by hybndization
  • primer extension ieactions i e mini sequencing, nucleotide-specific extensions, oi simple PCR amplification
  • sequence discrimination ieactions For example, in mini sequencing a pnmer anneals to its taiget DNA immediately upstieam of the SNP and is extended 25 w ith a single nucleotide complementary to lhe polymoiphic site Wheie the nucleotide is not complementaiy, no extension occuis
  • Oligonucleotide ligation assays lequire two sequence-specific piobes and one common ligation piobe per SNP
  • the common ligation probe hybridizes adjacent to a sequence-specific probe and W when there is a peifect match of the appiopnate sequence-specific probe, the ligase joins both the sequence-specific and the common probes Where there is not a perfect match the ligase is unable to join the sequence-specific and common piobes
  • Piobes used in hybndization can include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids Hybndization methods for the identification of single nucleotide polymoiphisms oi othei mutations involving a few nucleotides aie described in the U S Pat 6.270,961 , 6.025, 136, and 6,872.530 Suitable hybridization probes for use in accordance
  • an invasive cleavage method lequnes an oligonucleotide called an InvaderTM piobe and sequence-specific piobes to anneal to the target DNA w ith an overlap of one nucleotide
  • sequence-specific probe is complementary to the polymorphic base
  • oveilaps of the !' end of the invader oligonucleotide foim a facilitatorcture that is lecognized and cleaved by a Flap 0 endonuclease releasing the 5' arm of the allele specific probe
  • 5' exonutlease activity or TaqManTM assay is based on the 5' nuclease activity of Taq polymeiase that displaces and cleaves the oligonucleotide probes hybridized to the taiget DNA generating a fluorescent signal It is necuney to have two probes that differ at the
  • one probe is complementary to the "noimal' sequence and the other to the mutation of interest
  • These piobes have diffeient fluoiescent dyes attached to the 5' end and a quenchei attached to the T end when the piobes aie intact the quencher internets w ith the fluorophor bv fluoiescence resonance eneigy tiansfer (FRET) to quench the fluorescence of tht piobe Din ing the PCR annealing step
  • FRET fluorophor bv fluoiescence resonance eneigy tiansfer
  • the hybridization probes hybridize to target DNA
  • the 5' fluoiescent dye is cleaved by the 5' nuclease activity of Taq polymeiase, leading to an inciease in fluorescence of the ieporter dve Mismatched probes are displaced w ithout fiagmentation
  • Theistnce of a mutation in a sample is determined by measu
  • the Illumina Golden Gate 1 M Assay uses a ⁇ ombined oligonucleotide ligation assay/ allele-speufic hybndization appioach (SHEN R et til Mut.it Res 200 ⁇ 573 70-82)
  • SHEN R et til Mut.it Res 200 ⁇ 573 70-82 The first series of steps inv olve the hybridization of three oligonucleotides to a set of specific target SNPs, two ol these are fluorescently-labelled allele-specihc oligonucleotides (ASOs) and the thud a locus-specific oligonucleotide (LSO) binding 1 -20 bp dow nstieam of the ASOs
  • a second series of steps involve
  • I S Mutation detection methods may include but aie not limited to the following
  • RFLP Restriction Fragment Length Polymoi phism
  • Maxam-Gilbert technique foi sequencing involves the specific chemical cleavage of terminally labelled DNA
  • four samples of the same labeled DNA are each subjected to a diffeient chemical ieaction to effect preferential cleavage of the DNA molecule at one or two nucleotides of a specific base identity
  • the conditions aie adjusted to obtain only partial cleavage.
  • each sample contains DNA fiagments of different lengths, each of which ends w ith the same one 01 two ot the four nucleotides In particular in one sample each fragment ends w ith a C, in another sample each fiagment ends w ith a C or a T, in i third sample each ends w ith a G, and in a fourth sample each ends w ith an A or a G
  • each fragment ends w ith a C ends w ith the same one 01 two ot the four nucleotides
  • each fragment ends w ith a C in another sample each fiagment ends w ith a C or a T, in i third sample each ends w ith a G, and in a fourth sample each ends w ith an A or a G
  • leverse transcriptase w ith dideoxx nucleotides have been used to sequence encephalomyocaiditis v uus RNA (ZIMMERN D and KAESBERG P Pioc Natl Acad Sci USA ( 1978) 75(9) 4257-4261 ) MILLS DR and KRAMER FR (Proc Natl Acad Sci USA ( 1979) 76( 5) 2232-2235) desci ibe the use ol Q ⁇ ieplicase and the nucleotide analog inosine foi sequencing RNA in a chain-termination mechanism Diiect chemical methods for sequencing
  • Othei methods include those of Donis-Keller i t ai ( 1977. Niicl Acids Res 4 2527-2538). SIMONCSITS A i t al (Natuie ( 1977) 269(5611 ) 811-816), AXELROD VD et al (Nucl Acids Res ( 1978 ) 5( 10) 1 ⁇ 49-1561).
  • Nucleic acid sequences can also be read by stimulating the natural i() tluoiesce of a cleaved nucleotide w ith a laser while the single nucleotide is contained in a fluoiescence enhancing matr ix (U S Pat # ⁇ ,674 743), In a mini sequencing reaction, a primer that anneals to taigct DNA adjacent to a SNP is extended by DNA polymerase w ith a single nucleotide that is complementary to the polymoiphic site This method is based on the high accuiacy of nucleotide incorporation by DNA poly merases There are diffeient technologies for analyzing the primer extension products For example, the use of labeled or unlabeled nucleotides, ddNTP combined w ith dNTP or only
  • S Piobes used in hybridization can include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids
  • Hyb ⁇ dization methods for the identification of single nucleotide polymorphisms or othei mutations involving a few nucleotides are desci ibed in the U S Pat 6.270,961 , 6,025, 1 16, and 6.872,530
  • Suitable hybridization probes for use in accoi dance w ith the invention include oligonucleotide ⁇ and PNAs from about 10 to about 400 nucleotides, 10 alternatively from about 20 to about 200 nucleotides, or from about 10 to about 100 nucleotides in length
  • TDI-FP fluoiescent polarization-detection
  • Oligonucleotide ligation assay is based on ligation of probe and detectoi oligonucleotides annealed to a polymerase chain ieaction amplicon strand w ith detection by an enzyme immunoassay (VILLAHERMOSA ML J Hum Vnol (2001 ) 4(5) 238-48, ROMPPANEN EL 20 Scand J Clin Lab Invest (2001 ) 61 (2) 123A IANNONE MA et al Cytometry (2000) 39(2) 131 - 40),
  • Ligation-Rolling Ciicle Amplification has also been successfully used for genotyping single nucleotide polymoi phisms as desciibed in QI X et al Nucleic Acids Res (2001 ) 2s 29(22) E l 16,
  • 5' nuclease assay has also been successfully used for genotyping single nucleotide polymoiphisms (AYDIN A et cil Biotechniques (2001 ) (4) 920-2. 924, 926-8 ),
  • Matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy is also usef ul in the genotyping single nucleotide polymoi phisms through the analysis of crosequencing products (HAFF LA and SMIRNOV IP Nucleic Acids Res ( 1997) 2"S( 18) 3749- 5 O, HAFF LA and SMIRNOV IP Genome Res ( 1997) 7 178-388, SUN X et al Nucleic Acids Res (2000) 28 I O c68, BRAUN A et al Clin Chem ( 1997) 43 1 151 - 1 1 58.
  • Sequence-specific PCR methods have also been successfully used for genotyping single nucleotide I S polymorphisms (HAWKINS JR et al Hum Mutat (2002) 19(5) 543-553)
  • SSCP Single- Stranded Confoi manorial Polymorphism
  • CFLP Cleavase Fragment Length Polymoi phism
  • obtaining may involve retrieval 20 of the subjects nucleic acid sequence data ( foi example horn a database), followed by determining or detecting the identity of a nucleic acid oi genotype at a polymoiphic site by reading the subject ' s nucleic acid sequence at the one oi moie polymorphic sites
  • polymorphisms in vasopressin pathway associated gene sequences are used to pi edict a subject's iesponse to v asopiessin ieceptor agonist tieatment
  • Methods foi predicting a subject's iesponst to vasopressin ieceptor agonist tieatment may be useful in making decisions regaiding the M) administration of vasopiessin ieceptor agonist
  • An improved response may include an improvement subsequent to administration of said therapeutic agent, whereby the subject has an increased likelihood of survival, reduced likelihood of organ damage or organ dysf unction (Brussels scoie), an improved APACHE II scoie, days alive and free of pressors, inotiopes. and ieduced systemic dysfunction (cardiovascular, lespnatory, ⁇ entilation, cential neivous system, coagulation [INR> 1 5
  • sequence information or genotype information may be obtained from a subject w herein the sequence information contains one or more polymorphic sites in a vasopressin pathway associated gene sequence Also, as prev iously desci ibed the sequence identity of one or more poly morphisms in a vasopressin pathway associated gene sequence of one or more subjects
  • subject iesponse to administration of vasopressin ieceptor agonist may be assessed as described above Foi example, the APACHE II scoi ing system or the Brussels score may be used to assess a subject's iesponse to treatment by compai ing subject scores before and after treatment
  • subject response may be correlated w ith the sequence identity of one oi more polv moiphism(s)
  • the correlation of subject iesponse may fuither include statistical analysis of subject outcome scoics and poly moi phism(s) foi a number of subjects
  • An improved iesponse may include an unpiovement subsequent to administiation of said therapeutic agent, w hereby the subject has an inci eased likelihood ofenteval. reduced likelihood of oigan damage or oigan dysfunction (Brussels scoie). an impi oved APACHE II score, days alive and free of picssors, inotiopes. and reduced systemic dysfunction (cardiovasculai, respnatoiy, ventilation,
  • sequence infoi mation oi genotype information may be obtained fiom a subject w heiein the sequence information contains one or more single nucleotide polymorphic sites in AVP.
  • AVPRl A LNPEP or LRAP sequences Also, as previously described the sequence M) identity ot one or more single nucleotide polymoiphisms in the AVP, AVPRl A oi LNPEP sequences ot one or moie subjects may then be detected or detei mined Fuithermoie, subject outcome or piognosis may be assessed as described above, foi example the APACHE II scoring system or the Brussels scoie may be used to assess subject outcome or prognosis by compai ing sub
  • ICU Intensive Care Unit
  • SPH ICU St Paul's Hospital
  • the seventy of cardiovascular dysfunction was assessed by measuring days alive and fiee ot caidiovasculai dysfunction ovei a 28-day observation period Days alive and free of caidiovascular dysfunction was calculated as the numbei of days after inclusion that a patient was alive and fiee of cardiovascular dysfunction over 28 days Thus, a lowei score for days alive and fiee of caidiovascular dysfunction indicates moie caidiov ascular dysfunction The ieason that
  • caidiov asculai dysfunction I ⁇ days alive and hee of caidiov asculai dysfunction is pieferable to simplyfeednce or absence oF caidiov ascular dysfunction is that seveie sepsis has a high acute moitahty so that early death (w ithin 28 days) precludes calculation of thepsychnce or absence of cardiov ascular dysfunction in dead subjects Oigan dysfunction has been evaluated in this way in observ ational studies (Russell J A t t ⁇ l Cut Care Med (2000) 28( 10) 3405 1 1 ) and in landomized controlled trials of new
  • vasopressor support To furthei evaluate cardiovascular, respiratory, and ienal function we also recorded, dunng each 24 hour penod. vasopressor support, mechanical ventilation, and renal support, lespectively
  • IS Vasopressoi use was defined as dopamine > 5 ⁇ g/kg/min or any dose of norepinephrine, epinephrine, v asopressin, oi phenylephrine
  • Mechanical ventilation was defined as need for intubation and positive anway pressure (i e T- piece and mask ventilation were not considered ventilation)
  • Renal support was defined as hemodialysis pei itoneal dialysis, or any continuous renal support mode (e g continuous veno-venous hemodialysis)
  • SIRS ciitei ia As a cumulative measuie of the severity of SIRS the presence of two, three or foui of the SIRS ciitei ia was scoied each day over the 28-da/ obseivation penod SIRS was considered present w hen subjects met at least two of tour SIRS cntei ia
  • Baseline characte ⁇ stics foi the Biological Plausibility cohoit included age in years. 7c males Tsmoket s, r f diabetes, c /c hypertension, ejection fraction, bypass time, clamp time and aprotinin Outcome vai tables measured in the Biological Plausibility cohoit included Granulocyte colony stimulating factor (GCSF), Interleukin 10 (ILl O), Intel leukin receptoi I a (IL I ra), Inerukin 6 (IL6), Intel leukin 8 (IL8) and Monocyte Chemoattiactant Protein 1 (MCPl )
  • GCSF Granulocyte colony stimulating factor
  • ILl O Interleukin 10
  • IL I ra Intel leukin receptoi I a
  • IL6 Intel leukin 6
  • IL8 Intel leukin 8
  • MCPl Monocyte Chemoattiactant Protein 1
  • genotype data was queried from the International HapMap Project t vv vv w hapmap org) and Perlegen Sciences Inc (www perlegen com) to select a set of tag SNF's
  • tSNPs in the LNPEP, AVP and AVPR l A regions each hav ing a minor allele frequency (MAF) greater than 0 05
  • LD pairw ise linkage disequilibrium
  • haplotype SEPHENS M et al Am J Hum Genet (2001 ) 68 978-989. and EXCOFFIER L and SLATKIN M MoI Biol Evol ( 1995) 12(3) 921 -927) and haplotype block (HAWLEY ME and
  • DNA samples were transfe ⁇ ed to 1 5 ⁇ iL cr>otubes, bar coded and ci oss-i eferenced w ith a unique patient numbei and stored at -8O 0 C
  • the AVP, AVPR l A. LNPEP and LRAP genes are central to the action of vasopressin given that ⁇ asopiessin induces vasoconstnction by signaling through the AVPR l A ieceptor and that vasopiessin activ ity is inhibited when cleaved by LNPEP
  • Similar protein homology between LNPEP and LRAP suggest that these two genes aiose through an ancient gene duplication event (DANCHIN E et til , Immunol Re ⁇ (2004) 198 216-332) This homology and the observation o ⁇ an extended linkage disequilibrium (LD) block throughout the LRAP and LNPEP region (HapMap
  • LNPEP Leucyl/Cystinyl Aminopeptidase
  • Table 3 4 and Table 3 5 show 28-day sui vival and oigan dysfunction data by LNPEP is l 8059 genotype for vasopiessin-treated and contiol subjects respectively
  • Table 1 6 show s the dif ieiences in suiv ival and measures of oigan dysfunction between by LNPEP is 18059 genotype
  • LNPEP leucyl/cystinyl aminopeptidase
  • LNPEP leucyl/cystinyl ammopeptidase
  • LNPEP leucv/1/cystinyl aminopeptidase
  • I s i s2771 1 can be used to pi edict iesponse to vasopressin in subjects w ith septic shock using 28-day surviv al and measures of organ dysfunction as outcome vanables Of 103 vasopressin-treated and 103 matched-contiol subjects w ith septic shock.
  • 70 and 81 weie iespectively genotyped tor LNPEP rs2771 1
  • Baseline characteristics for subjects w ith genotypes are show n in Table 3 8 and Table ⁇ 9
  • LNPEP i s2771 1 is in linkage w ith, for example.
  • Baseline chaiacte ⁇ stics ot a group of Caucasian septic-shock contiol subjects by LNPEP rs277 1 1 genotype
  • Tables 3 10, 1 1 1 and 3 12 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for LNPEP rs2771 1
  • vasopressin-treated subjects w ith the LNPEP rs2771 1 AA genotype also had increased organ dysfunction as demonstrated by fewer DAF of organ dysfunction compaied w ith control
  • a response association of leucv, l/cystinyl aminopeptidase is2771 1 in a group of Caucasian ICU septic shock subjects who v ere tieated w ith vasopiessin For all variables besides 28-day suiv lval, data is given as 25 lh percentile [ median
  • LNPEP leucyl/cystinyl aminopeptidase
  • LNPEP leucyl/cystinyl aminopeptidase
  • LNPEP rs 10051617 can be used to pi edict response to ⁇ asoptessin in subjects w ith septic shock using 28- day tov al and measures of organ dysfunction as outcome variables Of 103 vasopressin-treated and 103 matched-control subjects w ith septic shock, 72 and 81 were respectively genotyped toi LNPEP rs 10051637 Baseline character istics for subjects w ith genotypes are show n in Table 3 1 3 and Table 3 14 LNPEP rs 10051637 is in linkage disequilibrium w ith, foi example LNPEP isl 8059 and LNPEP G9419812A. which were also genotyped in this cohoit.
  • Baseline characteristics of a matched-control group of Caucasian septic-shock subjects by leucy 1/cyst my 1 aminopeptidase is 10051637 genotype For age and APACHE II score. data is given as 25 lh percentile
  • Tables 3.15, 3.16 and Tables 3.17 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for LNPEP rsl0051637.
  • Vasopressin-treated subjects w ith the LNPEP rs 10051637 GG genotype were also observed to have more organ dysfunction as demonstrated by fewer DAF of organ dysfunction In contiast. vasopressin-treated subjects w ith the LNPEP i s i 0051637 AG and AA genotypes had incieased survival (26 9c) compaied to controls (20 9c )
  • LNPEP leucyl/cystinyl aminopeptidase
  • LNPEP leucyl/cystinyl ciminopeptidase
  • AVP rsl 410713 can be used to predict response to vasopressin in subjects with septic shock using 28-day survival and measures of organ dysfunction as outcome variables. Of 103 vasopressin-treated and 103 matched-control subjects with septic shock, 72 and 81 were respectively genotyped for AVP rs 1410713. Baseline
  • N number of subjects.
  • vasopressin AVP rs 1410713 genotype.
  • AVP vasopressin
  • Tables 3.20. 3.21 and 3.22 contain 28-day survival and organ dysfunction data for septic- shock subjects genoty ped for AVP rsl410713 Vasopressin -treated subjects w ith the AVP 1S14107 H AA genotype had a dramatically incieased survival (38 c /c ) compared to controls (0 7c) as demonstrated by the positive values in the AVP rs 1410713 AA DELTA column in Table 3 22 Furtheimore, v asopressin-treated subjects w ith the AVP rs 14107 H AA genotype weie observed to have less organ dysfunction as demonstrated by more DAF of organ dyst unction Vasopressin- treated subjects w ith AVP rs 1410713 AC genotype were also observed to have increased 28-day suiv ival (477r ) compared w ith that of control subjects (37%)
  • Difteiencc in response association of arginine vasopiessin (AVP) isl410713 between cases (vasopressm-treated group) (Treat) and controls (vasopressin untreated matched contiol) (Cont) of Caucasian ICU subjects diagnosed with septic shock
  • AVP arginine vasopiessin
  • AVP rs857240 can be used to piedict response to vasopressin in subjects w ith septic shock using 28-day survival and measures of organ dysfunction as respective primary and secondary outcome variables Of 10 " ' vasopiessm-tieated and 103 matched-control subjects w ith septic shock, 73 and 83 were lespectively genotyped for LNPEP i s857240 Baseline characteristics for subjects w ith genotypes
  • Tables 1 25, 1 26 and 1 27 contain 28-day survival and oigan dysfunction data for septic shock subjects genotyped for AVP is857240 Vasopiessin -treated subjects w ith the AVP rs857240 CT genotype had dramatically decreased survival if vasopressin-treated (29 c /c) compaied to contiols (41 c/ c) as demonstrated by the negative values in the AVP is857240 CT DELTA column in Table 1 27 Furthei moie.
  • vasopiessin-treated subjects w ith the AVP rs857240 CT genotype were observed to have moie organ dysfunction than AVP rs857240 CT control subjects as demonstrated by more DAF of organ dysfunction
  • vasopressin-treated subjects w ith the AVP rs857240 CC genotype had increased surv ival (41 c /c ) compared to controls ( 10 c /c ) as
  • AVP arginine vasopiessin
  • Baseline chaiacteristics of a gioup of vasopressin-tieated Caucasian ICU septic shock subjects by genotype ot aiginine vasopressin (AVP) is 857242 Foi age and APACHE II score, data is given as 2?"' peicentile
  • Tables 3.30, 3.31 and 3.32 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for AVP rs857242 Vasopressin -treated subjects w ith the AVP rs857242 AC genotype had a dramatically decieased surv ival (38 7c ) compared to controls (54 7c ) as
  • vasopressin-treated subjects w ith the AVP rs857242 AC genotype were observed to have more organ dysfunction as demonstrated by more DAF of organ dysfunction.
  • v asopressm-treated subjects w ith the AVP rs857242 CC genotype were observed to have increased surv ival (419r) compared w ith controls (307c ).
  • rs857242 CC genotype were obseived to have increased 28-day surv ival (47%) compared w ith that of control subjects 017c ) as demonstrated by the positive values in the AVP rs857242 CC DELTA column in Table 3 32 Furthermore, vasopressin-treated subjects w ith the AVP rs857242 CC genotype were observed to have less organ dysfunction as demonstrated by more DAF of organ dysfunction
  • AVP arginine v asopressin
  • a response association of arginine vasopiessin (AVP) rs857242 in Caucasian septic-shock control subjects For all variables besides 28-day survival, data is given as 25 th percentile
  • 75 th percentile For 28-day survival, data is given as % (N surv ived / N total) N, number of subjects
  • AVPRlA Arginine Vasopressin Receptor Ia
  • AVPRl A isl495027 can be used to piedict response to vasopiessin in subjects w ith septic shock using 28- K) day surviv al and measures of oigan dysfunction as lespective p ⁇ maiy and secondary outcome variables Of 103 vasopressin-tieated and 03 matched-control subjects w ith septic shock. 72 and 79 were respectively genotyped for AVPR l A rs 1495027 Baseline characteristics for subjects w ith genotypes are shown in Table 3 33 and Table 3 34
  • Tables 1 15 1 16 and 1 17 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for AVPRl A rs 1495027 Vasopiessin-treated subjects w ith the AVPRlA i s 1495027 TT had a dramatically decreased sui v ival (21 9c ) compaied to controls (46 9c ) as demonstrated by the negative values in the ⁇ .VPR1 A rs l495027 TT DELTA column in Table 1 17 Fuitheimoie.
  • vasopressin-treated subjects w ith the AVPRl A tsl495Q27 TT genotype vveie obsei ved to have moie oigan dysfunction as demonstrated by fewei DAF of organ dysfunction
  • vasopiessin-tieated subjects w ith the AVPR l A rsl495027 CC genotype weie shown to have incieased suiv ival (507r) over AVPR l A rs 1495027 CC contiols (247r) as demonstiated by the positive values in the AVPRl A rs 1495027 TT DELTA column in Table 1 17
  • vasopressin subjects w ith the AVPR l A i sl495027 CC genotype had less oigan dysfunction as ev idenced by more DAF of organ dysfunction
  • AVP rs857240, AVP rs857242, and AVPR l A rs 1495027 in subjects w ith septic shock can predict response to administration of vasopressin as measured by 28-day survival and/or DAF of organ dysfunction
  • Subjects w ith genotypes including LNPEP rs 18059 CC.
  • LNPEP rs2771 1 AA, LNPEP i s l 0051637 GG, AVP rs 1410713 CC, AVP rs857240 CT, AVP rs857242 AC and AVPR l A rs 1495027 TT should not be administered a vasopressin receptor agonist as this could
  • LNPEP Leucyl/Cystinyl Aminopeptidase 2.1.1 LNPEP rsl8059 2.1.1.1 Systematic Inflammatory Response Syndrome - Caucasians
  • Baseline ehaiacte ⁇ stics ol a cohort of Caucasian Subjects w ith septic shock by allele of leuc> l/c> stinyl aminopeptidase (LNPEP) is 18059 (CC vs CT/TT) For age and APACHE II scoie, data is given as 25 lh percentile / median / 75 th peicentile For all other vanables, data is
  • K Baseline characteristics of a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs38041 (AA vs. GG/AG). For age and APACHE II score, data is given as 25 th percentile / median / 75* percentile. For all olher variables, data is given as c k (N survived / N total). N, number of subjects.
  • LNPEP leucyl/cystinyl aminopeptidase
  • Arginine Vasopressin (AVP) 2.2.1 AVP rs 1410713
  • Baseline characteristics of a cohort of Caucasian Subjects w ith systematic inflammatory response syndrome by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/AC).
  • AVP Arginine Vasopressin
  • FIG. 2 and TABLE 4.14 summarize important SNP-phenotype associations for AVP rs 1410713.
  • Baseline characteristics of a cohort of Caucasian Subjects with sepsis by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/AC).
  • AVP Arginine Vasopressin
  • C A N survived / N total).
  • N number of sub ects.
  • FIG. 3 and TABLE 4.16 summarize important SNP-phenotype associations for AVP rs 1410713.
  • Baseline characteristics of a cohort of Caucasian Subjects w ith septic shock by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/AC).
  • AVP Arginine Vasopressin
  • FIG. 4 and TABLE 4.18 summarize important SNP-phenotype associations for AVP rs 1410713.
  • a Sepsis - Caucasians TABLE 4.19 gives the baseline characteristics (age, gender, APACHE Il score, medical vs. surgical diagnosis, shock upon admission and septic shock anytime) of 573 Caucasian Subjects with sepsis who were successfully genotyped at AVP rs857240. No significant differences were detected between the genotype groups on admission to the ICU.
  • Baseline characteristics of a cohort of Caucasian Subjects with sepsis by genotype of Arginine Vasopressin (AVP) rs857240 (CC vs. CT/TT).
  • AVP Arginine Vasopressin
  • CC vs. CT/TT For age and APACHE II score, data is given as 25* percentile / median / 75 lh percentile.
  • c k N survived / N total). N. number of subjects.
  • Baseline characteristics of a cohort of Caucasian Subjects with septic shock by genotype of Arginine Vasopressin (AVP) rs857240 (CC vs. CT /TT).
  • AVP Arginine Vasopressin
  • CC vs. CT /TT For age and APACHE II score, data is given as 25 lh percentile / median / 75 m percentile. For all other variables, data is given as % (N survived / N total). N, number of sub ects.
  • These findings indicate thai Caucasian subjects with septic shock who had either the TT or CT genotype at AVP rs857240 have less need of vasopressor and inotrope therapy, have less SIRS, and have a lower risk of organ dysfunction (renal and hepatic).
  • FIG. 5 and TABLE 4.24 summarize important SNP-phenotype associations for AVP rs857242.
  • FIG. 6 and TABLE 4.26 summarize important SNP-phenotype associations for AVP rs857242.
  • Baseline characteristics of a cohort of Caucasian Subjects with septic shock by genotype of Arginine Vasopressin (AVP) rs857242 (AC/AA ⁇ s. CC).
  • AVP Arginine Vasopressin
  • FIG. 7 and TABLE 4.28 summarize important SNP-phenotype associations for AVP rs857242.
  • AVPRlA Arginine Vasopressin Receptor Ia
  • TABLE 4.29 gives the baseline characterisi ics (age, gender, APACHE II score, and medical vs. surgical diagnosis) of the 361 Caucasian septic shock subjects who were successfully genotyped at AVPRl A rs 1495027 (CC vs. CITTT). No s gnificant differences were detected between the two genotype groups on admission to the ICU.
  • Baseline characteristics of a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of arginine vasopressin receptor I a (AVPRlA) r.s3803107 (CC/CT vs. TT).
  • AVPRlA arginine vasopressin receptor I a
  • CC/CT vs. TT arginine vasopressin receptor I a
  • FIG. 8 and TABLE 4.32 summarize important SNP-phenotype association results for AVPRl A rs38O3107.
  • AVPR l A arginine vasopressin receptor a
  • RC/CT vs. TT CC/CT vs. TT
  • FIG. 9 and TABLE 4.34 summarize important SNP-phenotype association results for AVPR I A rs3803107.
  • H median / 75 th percentile. For 28-day survival, data is given as % (N survived / N total). N, number of sub ects.
  • AVPR l A arginine vasopressin receptor Ia
  • rs 10877970 CC vs. TT/CT
  • TABLE 4.37 gives the baseline characteristics (age, gender, APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of the 108 Asian systematic inflammatory response syndrome subjects who were successfully genotyped (C vs. T) at AVPR l A rs 10877970. No significant differences, other than a small difference in APACHE II score, were detected between the two allelic groups on admission to the ICLJ.
  • FIG. 10 and TABLE 4.38 summarizes important SNP-phenotype association results for AVPR l A rs 10877970.
  • R A language and environment
  • LNPEP Leucyl/Cystinyl Aminopeptidase
  • LNPEP leucyl/cystinyl aminopeptidase
  • Table 5 2 show s the distribution of vasopressin administration by LNPEP rs 18059 genotype Subjects w ith the LNPEP isl 8059 CC genctype were obseived to have been administered
  • Eiaseline characteristics for septic shock subjects with LNPEP rs2771 1 genotypes are shown in Table 5.3. No significant differences between the genotype groups were detected on admission to the ICU.
  • Table 5.4 shows the distribution of vasopressin administration by LNPEP rs 10051637 genotype. Subjects w ith the GG genotype of LNPEP rs 10051637 were more frequently observed to be administered vasopressin (P ⁇ 0.001 ) compared to subjects who carried the AG or AA genotype of LNPEP rs 10051637 (TABLE 5.6).
  • Table 5.4 shows the distribution of vasopressin administration by AVPR l A rs 1495027 genotype.
  • Subjects with the AVPRl A rs 1495027 CT genotype had significantly increased use of vasopressin (P 0.0240) compared to subjects who carried either the CC or TT genotype of AVPR l A T AVPR 1 A rs 1495027 (TABLE 5.8).
  • Examples 1 -3 show that polymorphisms of the AVP, AVPRl A and LNPEP genes are associated with altered outcome in critically ill subjects.
  • the present example examines subjects w ith non-septic causes of systemic inflammatory response syndrome by analyzing SNP-phenotype interactions in subjects having undergone cardiopulmonary bypass surgery. If an AVP. AVPR 1 A, LNPEP or LRAP gene polymorphism was associated w ith altered survival and organ dysfunction, that polymorphism is also likely to be associated w ith changes in pro-inflammatory proteins such as serum granulocyte colony stimulating factor (GCSF), interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCPl ) 5 METHODS
  • GCSF serum granulocyte colony stimulating factor
  • IL-8 interleukin 8
  • MCPl monocyte chemotactic protein 1
  • LNPEP Leueyl/Cystinyl Aminopeptidase
  • Baseline characteustics of a cohort of non-septic CSICU subjects diagnosed w ith systematic inflammatory response syndrome by genoty pe of leucyl/cystinyl aminopeptidase is l 8059 (CC vs CT vs TT)
  • Baseline characteristics of a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs2771 l (AA vs. GG/AG).
  • LNPEP leucyl/cystinyl aminopeptidase
  • LNPEP rs 100516.37 TABLE 6.5 summarizes the baseline characteristics of 70 non-septic SIRS subjects who were successfully genotyped (AA/ AG vs. GG) ai LNPEP rs 10051637. No significant differences between the genotype groups were detected on admission to the CSICU.
  • Biomarkers are measured in /ml.
  • TABLL 6.9 summa ⁇ zes the baseline chaia ⁇ e ⁇ stics of S7 non-septic SIRS subjects who weie genotvped at AVP rs857242 No significanl differences between the genotype groups weie detected on admission to the CSICU TABLE 6.9
  • Baseline characteristics of a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of Arginine Vaso ressin (AVP) rs857242.
  • I O CC genotype have a decreased chemokine (GCSF) response after cardiopulmonary bypass surgery.
  • GCSF chemokine
  • Vaso ressin (AVP) rs857242 Biomarkers are measured in /ml.
  • Biomarkers are measured in pg/ml.
  • vasopressin AVP rs 1410713. rs857240. rs857242
  • AVPRlA rs 1495027 the arginine vasopressin Al receptor
  • LNPEP rs 18059, rs2771 I , and rs 10051637 gene are associated with response (measured as survival, organ dysfunction and need of life support) to AVP.
  • markers m the ⁇ asopressinase gene (LNPEP rsl 8059, rs2771 1. and rsl0051637) ind the ⁇ asopiessin A l receptor gene (AVPRl A rsl495027) are also markers of increased use ot AVP in a cohort of critically ill subjects who have septic shock Accordingly, clinicians more fiequently administei infused AVP to subjects who have LNPEP genotypes rsl 8059 CC. rs2771 1 AA and 5 rs l 0051617 GG and subjects who have the AVPR l A genotype. isl495027 CT These genotypes also have a significantly decreased chance of surv ival when treated w ith infused AVP compared to comparable subjects who have septic shock but who are not infused w ith AVP (control)
  • LNPEP rs 18059 CC, LNPEP rs2771 1 AA and LNPEP rs 10051617 GG are associated w ith decreased inflammatory ic-ponse (measured as GCSF and IL-8 response) to non- septic causes of systemic inflammatory iesponse syndrome (subjects having cardiopulmonaiy bypass surgeiy )
  • vasopiessin receptor agonist(s) e g V-I ieceptor agonist, e g a V i a ieceptor agonist, e g an AVPRl agonist
  • vasopiessin reeeptoi agon ⁇ st(s) dramatically decieases then sui uvdl and incieases the i isk ot oigan dysfunction
  • vasopressin reeeptoi agonist rsl410711. rs857240 and is857242
  • AV PRlA vasopressin A l receptor
  • LNPEP LNPEP gene (rsl 8059, ⁇ s2771 1 and rsl 0051617) Subjects who have the AVP rs 1410711 AA or AC, rs857240 CC or rs857242 CC genotypes, the AVPR l A rsl 495027 CC genotv pe.
  • vasopressin receptor agonist(s) e g V-I ieceptor agonist, e g a V i a ieceptor agonist, e g an AVPR l agonist
  • v asopressin receptor agonist(s) dramatically increases their tract and decreases the risk of organ is dysf unction
  • cardiac surgery requiring cardiopulmonary bypass cardiac surgery not requiring cardiopulmonary bypass, cardiac transplantation and hypotension
  • dialysis-induced hypotension, autonomic neuropathy, trauma and hypotension are also likely to be administered a vasopressin receptor agonist and should also be genotypes for single nucleotide polymorphisms of the vasopressin (AVP) gene (rs 1410713.
  • AVP vasopressin
  • rs857240, and rs857242 the vasopressin A l receptor (AVPR l A) gene (rs 1495027), and the vasopressinase (LNPEP) gene (rs ! 8059, rs2771 1 and rsl 0051637).
  • AVPR l A vasopressin A l receptor
  • LNPEP vasopressinase
  • vasopressin AVP
  • AVPR l A vasopressin A l receptor
  • LNPEP vasopressinase
  • TABLE 7.2 shows that subjects who have he AVP rs 1410713 CC, AVP rs857240 CT. and AVP rs857242 AC genotypes w ho receive AVP infusion have decreased survival compared to subjects w ho have the AVP rs 1410713 CC, AVP rs857240 CT. and AVP rs857242 AC genotypes who do not receive AVP infusion.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Pulmonology (AREA)
  • Urology & Nephrology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Communicable Diseases (AREA)
  • Zoology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Hematology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Obesity (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Endocrinology (AREA)
  • Pathology (AREA)

Abstract

The invention provides methods, nucleic acids, compositions and kits for predicting a subject's response to treatment with one or more vasopressin receptor agonists to identify subjects having a greater benefit from treatment with vasopressin receptor agonist(s). The method generally comprises determining a vasopressin pathway associated gene polymorphism genotype(s) of a subject for one or more polymorphisms in the these genes, comparing the determined genotype with known genotypes for the polymorphism that correspond with an improved response genotype to identify potential subjects having an inflammatory condition who are more likely to benefit from treatment with a vasopressin receptor agonist and subsequent to treatment recover from the inflammatory condition. The invention also provides for methods of treating such subjects with vasopressin receptor agonists based on the subject's genotype.

Description

Vasopressin Pathway Polymorphisms as Indicators of Subject Outcome in Critically 111 Subjects
FIELD OF THE INVENTION The field of the invention relates to the assessment and/or treatment of subjects with an inflammatory condition.
BACKGROUND OF THE INVENTION
Arginine vasopressin (AVP) has both vasoconstrictor and anti-diuretic properties. AVP is synthesized in the hypothalamus and secreted from posterior pituitary gland, secreted into the circulation and binds to several receptors. AVP binds to vasopressin-specific membrane bound receptor AVPRlA on vascular smooth muscle (MOUILLAC B. et al. J Biol Chem (1995) 270: 25771-25777), AVPR2 in the distal convoluted tubule and collecting ducts in the kidney and AVPRlB pituitary receptors that modify adrenocorticotropin hormone (ACTH) production (ORLOFF J. and HANDLER J. Am J Med (1967) 42: 757-768). Binding to AVPRlA induces vasoconstriction. AVP has a very short half-life and is metabolized by leucyl/cystinyl aminopeptidase (LNPEP).
Under normal physiological conditions, AVP does not contribute much to the maintenance of blood pressure (GROLLMAN J Pharm Exper Therap ( 1932) 46:447-460; GRAYBIEL Am Heart J (1941) 21:481-489; and WAGNER, J Clin Invest (1956) 35: 1412-1418). However, when blood pressure falls, AVP is fundamental to the response to hypotension as AVP is released from the posterior pituitary and causes arterial smooth muscle to contract (vasoconstriction) (WAGNER, J Clin Invest (1956) 35: 1412-1418; AISENBREY J Clin Invest (1981) 67:961-968; and SCHWARTZ Endocrinology (1981) 108: 1778-1780). If AVP is not secreted by the posterior pituitary in response to hypotension, then blood pressure remains low or falls further as a result of inappropriate vasodilation.
Critically ill subjects with septic shock have been shown to have low serum AVP levels (LANDRY Circ. (1997) 95: 1122-1125). Although AVP levels are initially high in septic shock, they fall within hours (GOETZ Proc. Exp. Biol. Med. (1974) 145(l):277-80; WILSON Surg. Gynecol. Obstet. (1981) 153(6):869-72; (MORALES D. et al. Circulation (1999) 100(3): 226-9); and ERRINGTON J Physiol (1971) 217(1): 43P-45P). Indeed, septic shock develops in part because there is a defect in the baro-receptor-mediated increase in AVP secretion (LANDRY Circ. (1997) 95: 1122-1 125). AVP can be administered to subjects who have septic shock who are not responding adequately. It has been reported that AVP increases blood pressure, decreases need for vasopressors such as norepinephrine, and increases urine output (LANDRY DW et al. Circulation. (1997) 95: 1122-1125; HOLMES CL et al. Int. Care Med. (2001) 27: 1416-1421). In a small, proof of concept randomized controlled trial of norepinephrine (NE) versus AVP in subjects with severe septic shock, it has been shown that AVP spared NE use, maintained mean arterial pressure and cardiac index, and improved measures of renal function including increased urine output and creatinine clearance ( PATEL BM et al. Anesthesiology (2002) 96:576-582). Blood AVP levels were also found to be very low (1.3 +/- 0.9pg/ml) (HOLMES CL et al Int. Care Med. (2001) 27: 1416-1421 ; and PATEL BM et al Anesthesiology (2002) 96:576-582). Several other studies have also shown that AVP increases blood pressure in septic shock (LANDRY DW et al. Circulation (1997) 95:1122-5; MALAY MB et al. J Trauma (1999) 47(4): 699-703; GOLD JA et al. Crit. Care Med. (2000) 28(1): 249-52; and MORALES DL. et al. Ann Thorac Surg. (2000) 69(1): 102-6).
Vasopressin is commonly used after cardiac surgery as studies have shown that AVP levels are lower after cardiac surgery compared to baseline. In addition, AVP infusion has been demonstrated to increase blood pressure after cardiac surgery (ARGENZIANO J Circulation (1997) 96(9 Suppl):II-286-90; ARGENZIANO J Thorac. Cardiovasc Surg. (1998) 116(6):973-80; CHEN Circulation (1999) 100(19 Suppl):II244-6; and ROSENZWEIG Circulation (1999) 100(19 Suppl):II182-6).
Arginine vasopressin (also known as antidiuretic hormone or ADH) is encoded by the AVP - neurophysin II gene (AVP) which contains three exons and maps to chromosome 20pl 3. AVP is synthesized in the hypothalamus as a precursor polypeptide (prepro-AVP-NPII) and undergoes post-translational processing to yield three functional peptides: AVP, NPII, and copeptin (Entrez Gene; http://www.ncbi.nlm.nih.gov/entrez). The AVP-NPl 1 complex is transported along nerve axons to the posterior pituitary where it is secreted into the bloodstream or directly into the brain. In addition to its vasoconstrictor properties,_AVP acts to maintain fluid homeostasis by signaling through AVPR2 receptors in the collecting ducts of the kidney (BIRNB AUMER M Trends Endocrinol Metab (2000) 10:406-10) and plays a role in pH regulation (TASHEVIA Y et al Plufgers Arch (2001) 442(5):652-61. Furthermore, AVP is thought to be involved in cognition, tolerance, adaptation as well as complex sexual and maternal behavior (YOUNG WS et al Neurosci (2006) 143(4): 1031-9). A representative human AVP mRNA sequence is listed in GenBank under accession numbers NM_00490 (633 bp). NM 00490 contains AVP rs 1410713 but not rs857242.
Human arginine vasopressin receptor IA (AVPRlA) is also known as the Via vasopressin receptor (VIaR); SCCL vasopressin subtype Ia receptor; Vl-vascular vasopressin receptor; antidiuretic hormone receptor IA; and vascular/hepatic -type arginine vasopressin receptor. AVPRlA maps to chromosomal region 12ql4-ql5. The protein encoded by this gene acts as receptor for arginine vasopressin (AVP). This receptor belongs to the subfamily of G-protein coupled receptors which also includes AVPRlB, AVPR2 and OXTR. AVPRlA agonist binding increases intracellular calcium concentrations by signaling through the phospholipase C cascade (OMM: 600821). The downstream effects of this signaling cascade include cell contraction and proliferation, platelet aggregation, release of coagulation factors and glycogenolysis. AVPRlA has been investigated for associations with social behaviors, including affiliation and attachment (YOUNG LJ et al Nature (1999) 400(6746):766-8) as well as essential hypertension (THIBONNIER M et al l MoI Cell Cardiol (2000) 32(4):557-564).
A representative human AVPRlA mRNA sequence is listed in GenBank under accession number NM_000706 (4154 bp). The NM_000706 sequence contains AVPRlA SNP rs3803107 (and rslO42615), but not rsl495027 or rsl0877970.
Homo sapiens leucyl/cystinyl aminopeptidase (LNPEP) is also known as AT (4) receptor; angiotensin IV receptor; insulin-regulated aminopeptidase; insulin-responsive aminopeptidase; otase; oxytocinase; placental leucine aminopeptidase; and vasopressinase. LNPEP maps to chromosomal region 5ql5. The LNPEP gene encodes a metalloproteinase that cleaves polypeptides such as vasopressin, oxytocin, lys-bradykinin, met-enkephalin and dynorphin A (Entrez Gene: www.ncbi.nlm.nih.gov/entrez). LNPEP also catalyzes the conversion of angiotensinogen to angiotensin IV (AT4) and is thought to play a role in memory processing by acting as a receptor for AT4 (LEW RA et al J Neurochem (2003) 86(2):344-50. LNPEP also plays a role in the maintenance of pregnancy (NORMURA S et al Biochim Biophys Acta (2005) 1751(l): 19-25).
A representative human LNPEP mRNA sequence is listed in GenBank under accession number NM_005575 (4470 bp). The NM_005575 sequence does not contain the LNPEP SNP rs 18059. Homo sapiens leukocyte-derived arginine aminopeptidase (LRAP) is also known as endoplasmic reticulum aminopeptidase 2; (ERAP2). LRAP maps to chromosomal region 5ql5, immediately upstream of LNPEP. The longest annotated transcript of LRAP (NM 022350) has 18 exons and is predicted to encode a protein of 915 amino acids (aa). LRAP is localized to the endoplasmic reticulum (ER) of the cell where it functions to cleave antigenic peptides greater than nine aa for presentation to major histocompatibility complex 1 (MHC-I) molecules (TANIOKA T et al J Biol Chem (2003) 278(34):32275-83).
A representative human LRAP mRNA sequence is listed in GenBank under accession number NM_022350 (3356 bp).
Genotype has been shown to play a role in the prediction of subject outcome in inflammatory and infectious diseases (MCGUIRE W. et al. Nature (1994) 371 :508-10; NADEL S. et al. Journal of Infectious Diseases (1996) 174:878-80; MIRA JP. et al. JAMA (1999) 282:561-8; MAJETSCHAK M. et al. Ann Surg (1999) 230:207-14; STUBER F. et al. Crit Care Med (1996) 24:381-4; STUBER F. et al. Journal of Inflammation (1996) 46:42-50; and WEITKAMP JH. et al. Infection (2000) 28:92-6). Furthermore, genotype can alter response to therapeutic interventions. Genentech's HERCEPTIN® was not effective in its overall Phase III trial but was shown to be effective in a genetic subset of subjects with human epidermal growth factor receptor 2 (HER2)- positive metastatic breast cancer. Similarly, Novartis' GLEEVEC® is only indicated for the subset of chronic myeloid leukemia subjects who carry a reciprocal translocation between chromosomes 9 and 22.
SUMMARY OF THE INVENTION
This invention is based in part on the surprising discovery that vasopressin pathway SNPs from AVP, AVPRlA, LNPEP and LRAP are predictive or indicative of subject outcome, wherein subject outcome is the ability of the subject to recover from an inflammatory condition based on having a particular AVP, AVPRlA, LNPEP or LRAP genotype as compared to a subject not having that genotype.
This invention is also based in part on the surprising discovery of vasopressin pathway SNPs having an association with improved prognosis or subject outcome, in subjects with an inflammatory condition. Furthermore, various vasopressin pathway SNPs are provided which are useful for subject screening, as an indication of subject outcome, or for prognosis for recovery from an inflammatory condition.
This invention is also based in part on the identification that the particular nucleotide (allele) or genotype at the site of a given SNP may be associated with a decreased likelihood of recovery from an inflammatory condition ('risk genotype') or an increased likelihood of recovery from an inflammatory condition ('decreased risk genotype'). Furthermore, this invention is in part based on the discovery that the genotype or allele may be predictive of increased responsiveness to the treatment of the inflammatory condition with vasopressin receptor agonist (i.e. "adverse response genotype" (ARG) or "improved response genotype" (IRG)). The vasopressin receptor agonist may be vasopressin. The inflammatory condition may be SIRS, sepsis or septic shock.
This invention is also based in part on the surprising discovery that AVP, AVPRlA LNPEP and LRAP SNPs alone or in combination are useful in predicting the response a subject with an inflammatory condition will have to vasopressin receptor agonist treatment or vasopressin treatment. Whereby the subjects having an improved response genotype are more likely to benefit from and have an improved response to vasopressin receptor agonist treatment and subjects having a non-improved response genotype are less likely to benefit from the same treatment. Furthermore, there are provided herein AVP, AVPRlA LNPEP and LRAP SNPs and SNPs in linkage disequilibrium (LD) thereto, which are also useful in predicting the response a subject with an inflammatory condition will have to vasopressin receptor agonist treatment or vasopressin treatment.
In accordance with one aspect of the invention, methods are provided for obtaining a prognosis for a subject having, or at risk of developing, an inflammatory condition, the method including determining a genotype of said subject which includes one or more polymorphic sites in the subject's vasopressin pathway gene sequences or a combination thereof, wherein said genotype is indicative of an ability of the subject to recover from the inflammatory condition.
In accordance with a further aspect of the invention, methods are provided for identifying a polymorphism in a vasopressin pathway gene sequence that correlates with prognosis of recovery from an inflammatory condition, the method including: obtaining vasopressin pathway gene sequence information from a group of subjects having an inflammatory condition; identifying at least one polymorphic nucleotide position in the vasopressin pathway gene sequence in the subjects; determining a genotypes at the polymorphic site for individual subjects in the group; determining recovery capabilities of individual subjects in the group from the inflammatory condition; and correlating the genotypes determined in step (c) with the recovery capabilities determined in step (d) thereby identifying said vasopressin pathway gene sequence polymorphisms that correlate with recovery.
In accordance with a further aspect of the invention, a kit is provided for determining a genotype at a defined nucleotide position within a polymorphic site in vasopressin pathway gene sequence in a subject to provide a prognosis of the subject's ability to recover from an inflammatory condition, the kit including: a restriction enzyme capable of distinguishing alternate nucleotides at the polymorphic site; or a labeled oligonucleotide having sufficient complementary to the polymorphic site so as to be capable of hybridizing distinctively to said alternate. The kit may further include an oligonucleotide or a set of oligonucleotides operable to amplify a region including the polymorphic site. The kit may further include a polymerization agent. The kit may further include instructions for using the kit to determine genotype.
In accordance with a further aspect of the invention, methods are provided for treating an inflammatory condition in a subject in need thereof, the method including administering to the subject a vasopressin receptor agonist, wherein said subject has an improved response genotype in their vasopressin pathway associated gene sequence.
In accordance with a further aspect of the invention, methods are provided for treating an inflammatory condition in a subject in need thereof, the method including: selecting a subject having an improved response genotype in their vasopressin pathway associated gene sequence; and administering to said subject one or more vasopressin receptor agonist(s).
In accordance with a further aspect of the invention, methods are provided for treating a subject with an inflammatory condition by administering a vasopressin receptor agonist, the method including administering the vasopressin receptor agonist to subjects that have an improved response genotype in their vasopressin pathway associated gene sequence, wherein the improved response genotype is predictive of increased responsiveness to the treatment of the inflammatory condition with a vasopressin receptor agonist. In accordance with a further aspect of the invention, methods are provided for identifying a subject with increased responsiveness to treatment of an inflammatory condition with a vasopressin receptor agonist, including the step of screening a population of subjects to identify those subjects that have an improved response genotype in their vasopressin pathway associated gene sequence, wherein the identification of a subject with an improved response genotype in their vasopressin pathway associated gene sequence is predictive of increased responsiveness to the treatment of the inflammatory condition with the vasopressin receptor agonist.
In accordance with a further aspect of the invention, methods are provided for selecting a subject for the treatment of an inflammatory condition with a vasopressin receptor agonist, including the step of identifying a subject having an improved response genotype in their vasopressin pathway associated gene sequence, wherein the identification of a subject with the improved response genotype is predictive of increased responsiveness to the treatment of the inflammatory condition with the vasopressin receptor agonist.
In accordance with a further aspect of the invention, methods are provided for treating an inflammatory condition in a subject, the method including administering a vasopressin receptor agonist to the subject, wherein said subject has an improved response genotype in their vasopressin pathway associated gene sequence.
In accordance with a further aspect of the invention, methods are provided for treating an inflammatory condition in a subject, the method including: identifying a subject having an improved response genotype in their vasopressin pathway associated gene sequence; and administering a vasopressin receptor agonist to the subject.
In accordance with a further aspect of the invention, methods are provided for administering one or more vasopressin receptor agonist(s) to a subject in need thereof, said subject having an improved response genotype in their vasopressin pathway associated gene sequence.
In accordance with a further aspect of the invention, methods are provided for treating an inflammatory condition in a subject, the method including: identifying a subject having an adverse response genotype in their vasopressin pathway associated gene sequence; and selectively not administering a vasopressin receptor agonist to the subject. In accordance with a further aspect of the invention, methods are provided for selectively not administering one or more vasopressin receptor agonist(s) to a subject, wherein said subject has an adverse response genotype in their vasopressin pathway associated gene sequence.
In accordance with another aspect of the invention, there is provided a use of a vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition, wherein the subjects treated have an improved response polymorphism in their vasopressin pathway associated gene sequence.
In accordance with another aspect of the invention, there is provided a use of a vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition, wherein the subjects treated do not have an adverse response polymorphism in their vasopressin pathway associated gene sequence.
In accordance with another aspect of the invention, there is provided a use of a vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition in a subset of subjects, wherein the subset of subjects have an improved response polymorphism in their vasopressin pathway associated gene sequence.
In accordance with another aspect of the invention, there is provided a use of a vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition in a subset of subjects, wherein the subset of subjects do not have an adverse response polymorphism in their vasopressin pathway associated gene sequence.
In accordance with another aspect of the invention, there is provided a commercial package containing, as active pharmaceutical ingredient, use of a vasopressin receptor agonist, or a pharmaceutically acceptable salt thereof, together with instructions for its use for the curative or prophylactic treatment of an inflammatory condition in a subject, wherein the subject treated has an improved response polymorphism in their vasopressin pathway associated gene sequence.
In accordance with another aspect of the invention, there is provided a commercial package containing, as active pharmaceutical ingredient, use of a vasopressin receptor agonist, or a pharmaceutically acceptable salt thereof, together with instructions for its use for the curative or prophylactic treatment of an inflammatory condition in a subject, wherein the subject treated does not have an adverse response polymorphism in their vasopressin pathway associated gene sequence.
The method or use may further include determining the subject's APACHE II score as an assessment of subject risk. The method or use may further include determining the number of organ system failures for the subject as an assessment of subject risk. The subject's APACHE II score may be indicative of an increased risk when > 25. 2 or more organ system failures may be indicative of increased subject risk.
The improved response genotype may be found at one or more of the following polymorphic sites: rsl8059; rs2771 1; rsl0051637; rsl410713; rs857240; rs857242; and rsl495027; or a polymorphic site in linkage disequilibrium thereto. The polymorphic site in linkage disequilibrium is selected from one or more of the following: rs2762; rslOO51637; rsl477364; rs7731592; rs7736466; rsl363974; rs2351010; rsl423357; rsl544777; rs2161548; rs38032; rs38034; rs38041 ; rs27436; rs27306; rs27307; rs27397; rs27659; rs27711 ; rs27290; rs38030; rs27294; rs27747; rs39602; rs248215; rs27302; rs2278018; rsl559355; rs3734015; rs4869315; rs2247650; rs2549781; rs2549782; rs2161657; rs251339; rsl87265; rs2548527; rslO56893; rs2548523; rs2255546; rs2255637; rslO195O3; rs251344; rsl981846; rsl0071975; rs7700332; rs38042; rsl8059; rs9127; rs7972829; rslO784339; rs38O31O7; rsl l836346; rs7308008; rsl l835545; rs7959001; rsl l832877; rslO877977; rs2201895; rs7302323; rsl0877986; rs2030106 and rsl8059; rs27296; rs27300; rs27613; rs2771 1 ; rs38033; rs38035; rs38036; rs38041; rs38043; rs716848; rsl216565; rsl23O358; rsl363907; rsl974871; rs2042385; rs21 13050; rs2113189; rs2161658; rs2255633; rs2255634; rs2287988; rs2548524; rs2548529; rs2548530; rs2548532; rs2548533; rs2548536; rs2548538; rs2548539; rs2548540; rs2549783; rs2549784; rs2549790; rs2549791; rs2549794; rs2549795; rs2549796; rs2549797; rs2617447; rs2910686; rs2927609 rs3797796; rs3849749; rs3849750; rs4360063; rs4869314; rs4869316; rs6556942; rs7713127; rs7716222; rs7719705; rs 10044354; rs 10051637; rs 10058476; rs 12516666; and rs 12716486.
The improved response genotype may be selected from one or more of the following: rsl8059CT; rsl8059TT; rs2771 IGG; rsl0051637GA; rsl0051637AA; rsl410713AC; rsl410713AA; rs857240CC; rs857242CC; rsl495027CC; and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto. The adverse response genotype which may be selected from one or more of the following: rsl8059CC; rs2771 IAA; rsl0051637GG; rsl410713CC; rs857240CT; rs857242AC; and rsl495027TT; or a polymorphic site in linkage disequilibrium thereto. The genotype of the polymorphic site in linkage disequilibrium may be selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
The subject having one or more improved response genotypes may be selectively administered the vasopressin receptor agonist. The subject having one or more adverse response genotypes may be selectively not administered the vasopressin receptor agonist.
In accordance with a further aspect of the invention, methods are provided for selecting a group of subjects for determining the efficacy of a candidate drug known or suspected of being useful for the treatment of an inflammatory condition, the method including determining a genotype at one or more polymorphic sites in a vasopressin pathway gene sequence for each subject, wherein said genotype is indicative of the subject' s ability to recover from the inflammatory condition and sorting subjects based on their genotype. The method may further include, administering the candidate drug to the subjects or a subset of subjects and determining each subject's ability to recover from the inflammatory condition. The method may further include comparing subject response to the candidate drug based on genotype of the subject.
The polymorphic site may be selected from one or more of the following: rs 18059; rs27711; rs38041; rsl0051637; rsl410713; rs857240; rs857242; rsl0877970; rs3803107; and rsl495027; or a polymorphic site in linkage disequilibrium thereto. The method of claim 2, wherein the polymorphic site in linkage disequilibrium may be selected from one or more of the following: rs2762; rslOO51637; rsl477364; rs7731592; rs7736466; rsl363974; rs2351010; rsl423357; rsl544777; rs2161548; rs38O32; rs38034; rs38041; rs27436; rs27306; rs27307; rs27397; rs27659; rs27711 ; rs27290; rs38O3O; rs27294; rs27747; rs39602; rs248215; rs27302; rs2278018; rs 1559355; rs3734015; rs4869315; rs2247650; rs2549781 ; rs2549782; rs2161657; rs251339; rsl87265; rs2548527; rslO56893; rs2548523; rs2255546; rs2255637; rslO195O3; rs251344; rsl981846; rslOO71975; rs7700332; rs38042; rsl8059; rs9127; rs7972829; rsl0784339; rs3803107; rsl 1836346; rs7308008; rsl 1835545; rs7959001; rsl 1832877; rsl0877977; rs2201895; rs7302323; rslO877986; rs2030106; rsl495027; rslO877962; rslO42615; rsl6856; rsl8059; rs27296; rs27300; rs27613; rs27711 ; rs38O33; rs38035; rs38036; rs38041; rs38043; rs716848; rsl216565; rsl23O358; rsl363907; rsl974871; rs2042385; rs2113050; rs2113189; rs2161658; rs2255633; rs2255634; rs2287988; rs2548524; rs2548529; rs2548530; rs2548532; rs2548533; rs2548536; rs2548538; rs2548539; rs2548540; rs2549783; rs2549784; rs2549790; rs2549791 ; rs2549794; rs2549795; rs2549796; rs2549797; rs2617447; rs2910686; rs2927609 rs3797796; rs3849749; rs3849750; rs4360063; rs4869314; rs4869316; rs6556942; rs7713127; rs7716222; rs7719705; rsl0044354; rsl0051637; rslOO58476; rsl 2516666; and rsl2716486.
The method may further include comparing the genotype determined with known genotypes, which are known to be indicative of a prognosis for recovery from the subject's type of inflammatory condition, or another inflammatory condition.
The method may further include obtaining vasopressin pathway gene sequence information for the subject. The genotype may be determined using a nucleic acid sample from the subject. The method may further include obtaining the nucleic acid sample from the subject. The genotype may be determined using one or more of the following techniques: restriction fragment length analysis; sequencing; micro-sequencing assay; hybridization; invader assay; gene chip hybridization assays; oligonucleotide ligation assay; ligation rolling circle amplification; 5' nuclease assay; polymerase proofreading methods; allele specific PCR; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; ligase chain reaction assay; enzyme-amplified electronic transduction; single base pair extension assay; and reading sequence data. The genotype of the subject may be indicative of increased risk of death or organ dysfunction from the inflammatory condition. The subject may be critically ill and the genotype is indicative of a prognosis of severe cardiovascular or respiratory dysfunction.
The genotype may include at least one of the following risk genotypes: rsl8059CT; rsl8059TT; rs27711GA; rs27711GG; rs38041GA; rs38041GG; rsl0051637GA; rslOO51637GG; rsl410713AA; rs857240CC; rs857242CC; rslO87797OCC; rs3803107TT; and rsl495027TT; or a polymorphic site in linkage disequilibrium thereto. The genotype may include at least one of the following risk alleles: rs3803107T; and rslO87797OC; or a polymorphic site in linkage disequilibrium thereto.
The genotype of the subject may be indicative of decreased risk of death or organ dysfunction from the inflammatory condition. The subject may be critically ill and the genotype is indicative of a prognosis of mild cardiovascular or respiratory dysfunction. The genotype may include at least one of the following reduced risk genotypes: rsl8059CC; rs2771 IAA; rs38041AA; rsl0051637AA; rsl410713CC; rsl410713AC; rs857240TT; rs857240CT; rs857242AA; rs857242AC; rsl0877970TT; rslO87797OCT; rs3803107CC; rs3803107CT; rsl495027CC and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto. The genotype may include at least one of the following reduced risk alleles: rs38O31O7C; and rslO87797OT; or a polymorphic site in linkage disequilibrium thereto.
Alternatively, the genotype of the polymorphic site in linkage disequilibrium may be selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
The inflammatory condition may be selected from the group consisting of: sepsis, septicemia, pneumonia, septic shock, systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), acute lung injury, aspiration pneumonitis, infection, pancreatitis, bacteremia, peritonitis, abdominal abscess, inflammation due to trauma, inflammation due to surgery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue damage due to chemotherapy or radiotherapy, and reactions to ingested, inhaled, infused, injected, or delivered substances, glomerulonephritis, bowel infection, opportunistic infections, and for subjects undergoing major surgery or dialysis, subjects who are immunocompromised, subjects on immunosuppressive agents, subjects with HIV/ AIDS, subjects with suspected endocarditis, subjects with fever, subjects with fever of unknown origin, subjects with cystic fibrosis, subjects with diabetes mellitus, subjects with chronic renal failure, subjects with acute renal failure, oliguria, subjects with acute renal dysfunction, glomerulo-nephritis, interstitial-nephritis, acute tubular necrosis (ATN), subjects , subjects with bronchiectasis, subjects with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, subjects with febrile neutropenia, subjects with meningitis, subjects with septic arthritis, subjects with urinary tract infection, subjects with necrotizing fasciitis, subjects with other suspected Group A streptococcus infection, subjects who have had a splenectomy, subjects with recurrent or suspected enterococcus infection, other medical and surgical conditions associated with increased risk of infection, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, meningococcemia, post-pump syndrome, cardiac stun syndrome, myocardial infarction, stroke, congestive heart failure, hepatitis, epiglottitis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, pre-eclampsia, eclampsia, HELLP syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhagic fever, pelvic inflammatory disease, Legionella, Lyme disease,
Influenza A, Epstein-Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, progressive systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, systemic vasculitis, Wegener's granulomatosis, transplants including heart, liver, lung kidney bone marrow, graft-versus-host disease, transplant rejection, sickle cell anemia, nephrotic syndrome, toxicity of agents such as OKT3, cytokine therapy, and cirrhosis. The inflammatory condition may be SIRS. The inflammatory condition may be sepsis. The inflammatory condition may be septic shock.
The vasopressin receptor agonist may be vasopressin.
In accordance with another aspect of the invention, there are provided two or more oligonucleotides or peptide nucleic acids of about 10 to about 400 nucleotides that hybridize specifically to a sequence contained in a human target sequence consisting of a subject's vasopressin pathway associated gene sequence, a complementary sequence of the target sequence or RNA equivalent of the target sequence and wherein the oligonucleotides or peptide nucleic acids are operable in determining the presence or absence of two or more polymorphism(s) or in their vasopressin pathway associated gene sequence selected from of the following polymorphic sites: rsl8059; rs2771 1 ; rs38041; rsl0051637; rsl410713; rs857240; rs857242; rsl0877970; rs3803107; and rsl495027; or one or more polymorphic sites in linkage disequilibrium thereto.
In accordance with another aspect of the invention, there are provided two or more oligonucleotides or peptide nucleic acids selected from the group including of: (a) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO: 1 having a T at position 201 but not to a nucleic acid molecule including SEQ ID NO: 1 having a C at position 201 ; (b) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:1 having a C at position 201 but not to a nucleic acid molecule including SEQ ID NO: 1 having a T at position 201 ; (c) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:2 having a G at position 201 but not to a nucleic acid molecule including SEQ ID NO:2 having a A at position 201; (d) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:2 having an A at position 201 but not to a nucleic acid molecule including SEQ ID NO:2 having a G at position 201; (e) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:3 having an A at position 201 but not to a nucleic acid molecule including SEQ ID NO:3 having a G at position 201 ; (f) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO: 3 having a G at position 201 but not to a nucleic acid molecule including SEQ ID NO:3 having an A at position 201; (g) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:4 having a G at position 201 but not to a nucleic acid molecule including SEQ ID NO:4 having an A at position 201; (h) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:4 having an A at position 201 but not to a nucleic acid molecule including SEQ ED NO:4 having a G at position 201; (i) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO.5 having an A at position 201 but not to a nucleic acid molecule including SEQ ID NO:5 having a C at position 201; (j) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ED NO:5 having a C at position 201 but not to a nucleic acid molecule including SEQ ED NO:5 having an A at position 201; (k) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ED NO:6 having an T at position 201 but not to a nucleic acid molecule including SEQ ED NO:6 having a C at position 201 ; (1) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ED NO:6 having a C at position 201 but not to a nucleic acid molecule including SEQ ID NO:6 having an T at position 201; (m) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:7 having an A at position 201 but not to a nucleic acid molecule including SEQ ED NO :7 having a C at position 201; (n) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ED NO.7 having a C at position 201 but not to a nucleic acid molecule including SEQ ED NO:7 having an A at position 201 ; (o) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO:8 having a T at position 201 but not to a nucleic acid molecule including SEQ ED NO:8 having a C at position 201; (p) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ED NO:8 having a C at position 201 but not to a nucleic acid molecule including SEQ ED NO:8 having a T at position 201 ; (q) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ED NO:9 having a C at position 201 but not to a nucleic acid molecule including SEQ ED NO.9 having a T at position 201; (r) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ED NO:9 having a T at position 201 but not to a nucleic acid molecule including SEQ ED NO:9 having a C at position 201; (s) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO: 10 having a T at position 201 but not to a nucleic acid molecule including SEQ ID NO: 10 having a C at position 201; (t) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule including SEQ ID NO: 10 having a C at position 201 but not to a nucleic acid molecule including SEQ ID NO: 10 having a T at position 201 ; (u) an oligonucleotide or peptide nucleic acid capable of hybridizing under high stringency conditions to a nucleic acid molecule including a first allele for a given polymorphism selected from the polymorphisms listed in TABLE ID but not capable of hybridizing under high stringency conditions to a nucleic acid molecule including a second allele for the given polymorphism selected from the polymorphisms listed in TABLE ID; and (v) an oligonucleotide or peptide nucleic acid capable of hybridizing under high stringency conditions to a nucleic acid molecule including the second allele for a given polymorphism selected from the polymorphisms listed in TABLE ID but not capable of hybridizing under high stringency conditions to a nucleic acid molecule including the first allele for the given polymorphism selected from the polymorphisms listed in TABLE ID.
In accordance with another aspect of the invention, there is provided an array of oligonucleotides or peptide nucleic acids attached to a solid support, the array including two or more of the oligonucleotides or peptide nucleic acids as set out herein.
In accordance with another aspect of the invention, there is provided a composition including an addressable collection of two or more oligonucleotides or peptide nucleic acids, the two or more oligonucleotides or peptide nucleic acids selected from the oligonucleotides or peptide nucleic acids as set out herein.
In accordance with another aspect of the invention, there is provided a composition including an addressable collection of two or more oligonucleotides or peptide nucleic acids, the two or more oligonucleotides or peptide nucleic acids consisting essentially of two or more nucleic acid molecules set out in SEQ ID NO: 1-264 or compliments, fragments, variants, or analogs thereof.
In accordance with another aspect of the invention, there is provided ancomposition including an addressable collection of two or more oligonucleotides or peptide nucleic acids, the two or more oligonucleotides or peptide nucleic acids consisting essentially of two or more nucleic acid molecules set out in TABLES 1C and ID or compliments, fragments, variants, or analogs thereof. The oligonucleotides or peptide nucleic acids described herein may further include one or more of the following: a detectable label; a quencher; a mobility modifier; a contiguous non-target sequence situated 5' or 3' to the target sequence or 5' and 3' to the target sequence.
In accordance with another aspect of the invention, there is provided a computer readable medium including a plurality of digitally encoded genotype correlations selected from the vasopressin pathway associated gene SNP correlations in TABLE IE, wherein each correlation of the plurality has a value representing an ability to recover from an inflammatory condition and a value representing an indication of responsiveness to treatment with a vasopressin receptor agonist.
The oligonucleotides or peptide nucleic acids may further include one or more of the following: a detectable label; a quencher; a mobility modifier; a contiguous non-target sequence situated 5' or 3' to the target sequence or 5' and 3' to the target sequence. The oligonucleotides or peptide nucleic acids may alternatively be of about 10 to about 400 nucleotides, about 15 to about 300 nucleotides. The oligonucleotides or peptide nucleic acids may alternatively be of about 20 to about 200 nucleotides, about 25 to about 100 nucleotides. The oligonucleotides or peptide nucleic acids may alternatively be of about 20 to about 80 nucleotides, about 25 to about 50 nucleotides. The genotype may be determined using a nucleic acid sample from the subject. Genotype may be determined using one or more of the following techniques: restriction fragment length analysis; sequencing; micro-sequencing assay; hybridization; invader assay; gene chip hybridization assays; oligonucleotide ligation assay; ligation rolling circle amplification; 5' nuclease assay; polymerase proofreading methods; allele specific PCR; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; ligase chain reaction assay; enzyme-amplified electronic transduction; single base pair extension assay; and reading sequence data. A determination of whether a site is in linkage disequilibrium (LD) with another site may be determined based on an absolute r value or D' value. When evaluating loci for LD those sites within a given population having a high degree of linkage disequilibrium (for example an absolute value for D' of > 0.5 or r2 > 0.5) are potentially useful in predicting the identity of an allele of interest (for example associated with the condition of interest). A high degree of linkage disequilibrium may be represented by an absolute value for D' of > 0.6 or r2 > 0.6. Alternatively, a higher degree of linkage disequilibrium may be represented by an absolute value for D' of > 0.7 or r2 > 0.7 or by an absolute value for D' of > 0.8 or r2 > 0.8. Additionally, a high degree of linkage disequilibrium may be represented by an absolute value for D' of > 0.85 or r2 > 0.85 or by an absolute value for D' of > 0.9 or r2 > 0.9. Two or more oligonucleotides or peptide nucleic acids may include 3 or more; 4 or more; 5 or more; 6 or more; 7 or more; 8 or more; 9 or more; 10 or more; 1 1 or more; 12 or more; 13 or more; 14 or more; 15 or more; 16 or more; 17 or more; 18 or more; 19 or more; or 20 or more.
Sequence variations may be assigned to a gene if mapped within 2 kb or more of an mRNA sequence feature. In particular, such a sequence may extend many kilobases (kb) from a vasopressin pathway gene and into neighbouring genes, where the LD within a region is strong.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows a Kaplan-Meier curve for a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of Leucyl aminopeptidase (LNPEP) rsl8059 (CC = dashed CT/TT = solid).
FIGURE 2 shows Kaplan-Meier survival curves for a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of Arginine Vasopressin (AVP) rs l410713 (AA = dashed CC/AC = solid).
FIGURE 3 shows Kaplan-Meier survival curves for a cohort of Caucasian Subjects with sepsis by genotype of Arginine Vasopressin (AVP) rsl410713 (AA = dashed CC/AC = solid).
FIGURE 4 shows Kaplan-Meier survival curves for a cohort of Caucasian Subjects with septic shock by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA = dashed CC/AC = solid).
FIGURE 5 shows Kaplan-Meier survival curves for a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of Arginine Vasopressin (AVP) rs857242 (AC/AA = solid vs. CC = dashed). FIGURE 6 shows Kaplan-Meier survival curves for a cohort of Caucasian Subjects with sepsis by genotype of Arginine Vasopressin (AVP) rs857242 (AC/AA = solid vs. CC = dashed). FIGURE 7 shows Kaplan-Meier survival curves for a cohort of Caucasian Subjects with septic shock by genotype of Arginine Vasopressin (AVP) rs857242 (AC/AA = solid vs. CC = dashed). FIGURE 8 shows Kaplan-Meier survival curves for a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of arginine vasopressin receptor (AVPRlA) rs3803107 (CC/CT = solid vs. TT = dashed). FIGURE 9 shows a Kaplan Meier survival curve over 28 days for a cohort of Asian Subjects with systematic inflammatory response syndrome by allele of arginine vasopressin receptor (AVPRlA) rs3803107 (C = solid vs. T = dashed).
FIGURE 10 shows a Kaplan Meier survival curve over 28 days for a cohort of Asian Subjects with systematic inflammatory response syndrome by allele of arginine vasopressin receptor (AVPRlA) rsl0877970 (T = dashed vs. C = solid).
DETAILED DESCRIPTION OF THE INVENTION 1. Definitions In the description that follows, a number of terms are used extensively, the following definitions are provided to facilitate understanding of the invention.
"Vasopressin Receptor Agonist" as used herein includes any vasopressin molecule, vasopressin derivative, vasopressin variant, vasopressin analogue, non-peptidyl analogues and any prodrug thereof, metabolite thereof, isomer thereof, combination of isomers thereof, or pharmaceutical composition of any of the preceding. Such agonists may be capable of binding to or interacting with a vasopressin receptor and initiating one or more of the types of responses typically produced by the binding of an endogenous vasopressin molecule to a vasopressin receptor (for example, AVPRlA, AVPRlB, AVPR2 and OXTR). Such activity may be present at the time of or following, administration to a subject. Vasopressin receptor agonists may be used alone or in combination with other vasopressin receptor agonists or other medications. Vasopressin receptor agonists may be synthesized or purified. Examples of vasopressin receptor agonists capable of increasing blood pressure, include, but are not limited to, arginine vasopressin (AVP), lysine vasopressin (LVP), triglycil-lysine vasopressin (also known as Terlipressin or Glycopressin), Octapressin, Ornipressin, Desmopressin, Desmopressin acetate, Lypressin, Felypressin, and
Argipressin. Vasopressin analogues may be 1 - 3 amino acids such as AIa-AVP, Ser-Ala-AVP, Thr-Ser-Ala-A VP (KALISZAN R. et al. Pharmacol Res Commun (1988) 20(5):377-381) or 3- beta- (2-thienyl)-L-alanine)-8-lysine-vasopressin and other similar analogues (Smith CW. Acta Pharmacol Toxicol (Copenhag) (1978) 43(3): 190-195). Examples of derivatives, variants, analogues or compositions etc. may found in US patent applications: 20050075328; 20040229798; 20030134845; 20030021792; 20030018024; 20030008863; 20030004159; 20020198196; 20020198191 ; 20020049194; 20050075328; 20040229798; 20030018024; and 20020198191 and issued US patents: 6,903,091; 6,831 ,079; 6,642,223; 6,620,807; 6,511, 974; 6,344,451; 6,335,327; 6,297,234; 6,268,360; 6,235,900; 6,204,260; 6,194,407; 6,096,736; 6,096,735; 6,090,803; 4,908,475; 4,810,778; 4,760,052; 4,711,877;6,903,091; 6,620,807; 6,344,451 ; 6,297,234; and 6,268,360.
"Vasopressin" as used herein includes: Antidiuretic hormone; Argiprestocin; Arginine Vasopressin; Arginine oxytocin; Pitressin tannate; Arginine vasotocin; Vasotocin; Vasopressin, isoleucyl; 3-Isoleucyl vasopressin; l-[[19-amino-13-butan-2-yl-10-(2-carbamoylethyl)-7- (carbamoylmethyl)- 16-[(4-hydroxyphenyl)methyl]-6,9, 12,15,18-pentaoxo- 1 ,2-dithia-5,8, 11,14,17- pentazacycloicos-4-yl]carbonyl]-N-[l-(carbamoylmethylcarbamoyl)-4-guanidino-butyl]- pyrrolidine-2-carboxamide (IUPAC name). Vasopressin is a nine amino acid peptide (Cys-Tyr- Ile-Gln-Asn-Cys-Pro-Arg-Gly, cyclic 1-6 disulfide) secreted from the posterior pituitary and binds to receptors in blood vessels, the brain and distal or collecting tubules of the kidney to promote vasoconstriction or reabsorption of water back into the circulation. Vasopressin receptor targets, include AVPRlA, AVPRlB, AVPR2 and OXTR. Vasopressin, for example, is sold as PRESSYN AR™ by Ferring Inc., and also sold in various formulations as VASOPRESSIN by Ferring Inc., Sandoz Canada Inc. and Pharmaceutical Partners of Canada Inc. Similarly, PITRESSIN™ is sold by Warner-Lambert Company, Parke-Davis Division, as a synthetic injectable vasopressin (8- Arginine vasopressin). It is substantially free from the oxytocic principle and is standardized to contain 20 pressor units/mL. The solution contains 0.5% Chlorobutanol (chloroform derivative) as a preservative. Also, DIAPID™ is sold as a nasal spray by Sandoz Inc. The current published indications for vasopressin (from the label of Ferring's PRESSYN AR™) are "Vasopressin is intended for use in the prevention of treatment of post-operative abdominal distension, dispelling of gas shadows in abdominal roentgenography and symptomatic control of diabetes insipidus."
"Genetic material" includes any nucleic acid and can be a deoxyribonucleotide or ribonucleotide polymer in either single or double-stranded form.
A "purine" is a heterocyclic organic compound containing fused pyrimidine and imidazole rings, and acts as the parent compound for purine bases, adenine (A) and guanine (G). A "Nucleotide" is generally a purine (R) or pyrimidine (Y) base covalently linked to a pentose, usually ribose or deoxyribose, where the sugar carries one or more phosphate groups. Nucleic acids are generally a polymer of nucleotides joined by 3 '-5' phosphodiester linkages. As used herein "purine" is used to refer to the purine bases, A and G, and more broadly to include the nucleotide monomers, deoxyadenosine-5' -phosphate and deoxyguanosine-5' -phosphate, as components of a polynucleotide chain. A "pyrimidine" is a single-ringed, organic base that forms nucleotide bases, cytosine (C), thymine (T) and uracil (U). As used herein "pyrimidine" is used to refer to the pyrimidine bases, C, T and U, and more broadly to include the pyrimidine nucleotide monomers that along with purine nucleotides are the components of a polynucleotide chain.
A nucleotide represented by the symbol M may be either an A or C, a nucleotide represented by the symbol W may be either an T/U or A, a nucleotide represented by the symbol Y may be either an C or T/U, a nucleotide represented by the symbol S may be either an G or C, while a nucleotide represented by the symbol R may be either an G or A, and a nucleotide represented by the symbol K may be either an G or TAJ. Similarly, a nucleotide represented by the symbol V may be either A or G or C, while a nucleotide represented by the symbol D may be either A or G or T, while a nucleotide represented by the symbol B may be either G or C or T, and a nucleotide represented by the symbol H may be either A or C or T.
A "polymorphic site" or "polymorphism site" or "polymorphism" or "single nucleotide polymorphism site" (SNP site) or single nucleotide polymorphism" (SNP) as used herein is the locus or position with in a given sequence at which divergence occurs. A "polymorphism" is the occurrence of two or more forms of a gene or position within a gene (allele), in a population, in such frequencies that the presence of the rarest of the forms cannot be explained by mutation alone. The implication is that polymorphic alleles confer some selective advantage on the host. Preferred polymorphic sites have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. Polymorphic sites may be at known positions within a nucleic acid sequence or may be determined to exist using the methods described herein. Polymorphisms may occur in both the coding regions and the noncoding regions (for example, promoters, introns or untranslated regions) of genes. Polymorphisms may occur at a single nucleotide site (SNPs) or may involve an insertion or deletion as described herein.
A "risk genotype" as used herein refers to an allelic variant (genotype) at one or more polymorphic sites within the vasopressin pathway gene (i.e. AVP, AVPRlA and LNPEP) sequences described herein as being indicative of a decreased likelihood of recovery from an inflammatory condition or an increased risk of having a poor outcome. The risk genotype may be determined for either the haploid genotype or diploid genotype, provided that at least one copy of a risk allele is present. Risk genotype may be an indication of an increased risk of not recovering from an inflammatory condition. Subjects having one copy (heterozygotes) or two copies (homozygotes) of the risk allele (for example rs 18059 CT, rs 18059 TT) are considered to have the "risk genotype" even though the degree to which the subjects risk of not recovering from an inflammatory condition may increase, depending on whether the subject is a homozygote rather than a heterozygote. Such "risk alleles" or "risk genotypes" may be selected from the following: rsl8059CT; rsl8059TT; rs2771 1GA; rs27711GG; rs38041GA; rs38041GG; rslOO51637GA; rslOO51637GG; rsl410713AA; rs857240CC; rs857242CC; rsl0877970TT; rs38O31O7TT; and rsl495027CC; or a polymorphic site in linkage disequilibrium thereto.
A "decreased risk genotype" as used herein refers to an allelic variant (genotype) at one or more polymorphic sites within the vasopressin pathway gene (i.e. AVP, AVPRlA and LNPEP) sequences described herein as being indicative of an increased likelihood of recovery from an inflammatory condition or a decreased risk of having a poor outcome. The decreased risk genotype may be determined for either the haploid genotype or diploid genotype, provided that at least one copy of a risk allele is present. Decreased risk genotype may be an indication of an increased likelihood of recovering from an inflammatory condition. Subjects having one copy (heterozygotes) or two copies (homozygotes) of the decreased risk allele (for example rs 1410713 CC rs 1410713 AC) are considered to have the "decreased risk genotype" even though the degree to which the subjects risk of not recovering from an inflammatory condition may increase, depending on whether the subject is a homozygote rather than a heterozygote. Such "decreased risk alleles" or "decreased risk genotypes" or "reduced risk genotypes" may be selected from the following: rsl8059CC; rs27711AA; rs38041AA; rsl0051637AA; rsl410713CC; rsl410713AC; rs857240TT; rs857240CT; rs857242AA; rs857242AC; rsl0877970TT; rslO87797OCT; rs3803107CC; rs3803107CT; rsl495027CC and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto.
An "improved response genotype" (IRG) or improved response polymorphic variant (FRP) as used herein refers to an allelic variant or genotype at one or more polymorphic sites within the vasopressin pathway associated polymorphisms selected from arginine vasopressin (AVP), arginine vasopressin receptor IA (AVPRlA) leucyl/cystinyl aminopeptidase (LNPEP) or leukocyte -derived aminopeptidase (LRAP) as described herein as being predictive of a subject's improved survival in response to vasopressin receptor agonist treatment (for example rsl8059TT, rs2771 IGG, rsl0051637AA, rsl410713AA, rs857240CC, rs857242CC or rsl495027CC), or a polymorphic site in linkage disequilibrium thereto.
An "adverse response genotype" (ARG) or adverse response polymorphic variant as used herein refers to an allelic variant or genotype at one or more polymorphic sites within the vasopressin pathway associated polymorphisms selected from arginine vasopressin (AVP), arginine vasopressin receptor IA (AVPRlA), leucyl/cystinyl aminopeptidase (LNPEP) or leukocyte- derived aminopeptidase (LRAP) as described herein as being predictive of a subject's decreased survival in response to vasopressin receptor agonist treatment (for example rsl8059CC, rs27711AA, rsl0051637GG, rsl410713CC, rs857240CT, rs857242AC or rsl495027TT), or a polymorphic site in linkage disequilibrium thereto. Subjects having a ARG are preferably selected for treatments not involving vasopressin receptor agonist administration.
A "clade" is a group of haplotypes that are closely related phylogenetically. For example, if haplotypes are displayed on a phylogenetic (evolutionary) tree a clade includes all haplotypes contained within the same branch.
The pattern of a set of markers along a chromosome is referred to as a "Haplotype". Accordingly, groups of alleles on the same small chromosomal segment tend to be transmitted together. Haplotypes along a given segment of a chromosome are generally transmitted to progeny together unless there has been a recombination event. Absence of a recombination event, haplotypes can be treated as alleles at a single highly polymorphic locus for mapping.
As used herein "haplotype" is a set of alleles of closely linked loci on a chromosome that tend to be inherited together. Such allele sets occur in patterns, which are called haplotypes. Accordingly, a specific SNP or other polymorphism allele at one SNP site is often associated with a specific SNP or other polymorphism allele at a nearby second SNP site or other polymorphism site. When this occurs, the two SNPs or other polymorphisms are said to be in LD because the two SNPs or other polymorphisms are not just randomly associated (i.e. in linkage equilibrium).
In general, the detection of nucleic acids in a sample depends on the technique of specific nucleic acid hybridization in which the oligonucleotide is annealed under conditions of "high stringency" to nucleic acids in the sample, and the successfully annealed oligonucleotides are subsequently detected (see for example Spiegelman, S., Scientific American, Vol. 210, p. 48 (1964)). Hybridization under high stringency conditions primarily depends on the method used for hybridization, the oligonucleotide length, base composition and position of mismatches (if any). High-stringency hybridization is relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high-stringency PCR, DNA sequencing, single strand conformational polymorphism analysis, and in situ hybridization. In contrast to Northern and Southern hybridizations, these aforementioned techniques are usually performed with relatively short probes (e.g., usually about 16 nucleotides or longer for PCR or sequencing and about 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998.
"Oligonucleotides" as used herein are variable length nucleic acids, which may be useful as probes, primers and in the manufacture of microarrays (arrays) for the detection and/or amplification of specific nucleic acids. Such DNA or RNA strands may be synthesized by the sequential addition (5'-3' or 3'-5') of activated monomers to a growing chain, which may be linked to an insoluble support. Numerous methods are known in the art for synthesizing oligonucleotides for subsequent individual use or as a part of the insoluble support, for example in arrays (BERNFIELD MR. and ROTTMAN FM. J. Biol. Chem. (1967) 242( 18) :4134-43; SULSTON J. et al. PNAS (1968) 60(2):409-415; GILLAM S. et al. Nucleic Acid Res.(1975) 2(5):613-624; BONORA GM. et al. Nucleic Acid Res.(1990) 18(11):3155-9; LASHKARI DA. et al. Proc Nat Acad Sci (1995) 92(17):7912-5; MCGALL G. et al. PNAS (1996) 93(24): 13555-60; ALBERT TJ. et al. Nucleic Acid Res.(2003) 31(7):e35; GAO X. et al. Biopolymers (2004) 73(5):579-96; and MOORCROFT MJ. et al. Nucleic Acid Res.(2005) 33(8):e75). In general, oligonucleotides are synthesized through the stepwise addition of activated and protected monomers under a variety of conditions depending on the method being used. Subsequently, specific protecting groups may be removed to allow for further elongation and subsequently and once synthesis is complete all the protecting groups may be removed and the oligonucleotides removed from their solid supports for purification of the complete chains if so desired.
"Peptide nucleic acids" (PNA) as used herein refer to modified nucleic acids in which the sugar phosphate skeleton of a nucleic acid has been converted to an N-(2-aminoethyl)-glycine skeleton. Although the sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge under neutral conditions resulting in electrostatic repulsion between complementary chains, the backbone structure of PNA does not inherently have a charge. Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences. Furthermore, PNAs are generally more robust than nucleic acids. PNAs may also be used in arrays and in other hybridization or other reactions as described above and herein for oligonucleotides.
An "addressable collection" as used herein is a combination of nucleic acid molecules or peptide nucleic acids capable of being detected by, for example, the use of hybridization techniques or by any other means of detection known to those of ordinary skill in the art. A DNA microarray would be considered an example of an "addressable collection".
In general the term "linkage", as used in population genetics, refers to the co-inheritance of two or more nonallelic genes or sequences due to the close proximity of the loci on the same chromosome, whereby after meiosis they remain associated more often than the 50% expected for unlinked genes. However, during meiosis, a physical crossing between individual chromatids may result in recombination. "Recombination" generally occurs between large segments of DNA, whereby contiguous stretches of DNA and genes are likely to be moved together in the recombination event (crossover). Conversely, regions of the DNA that are far apart on a given chromosome are more likely to become separated during the process of crossing-over than regions of the DNA that are close together. Polymorphic molecular markers, like SNPs, are often useful in tracking meiotic recombination events as positional markers on chromosomes.
Furthermore, the preferential occurrence of a disease gene in association with specific alleles of linked markers, such as SNPs or other polymorphisms, is called "Linkage Disequilibrium" (LD). This sort of disequilibrium generally implies that most of the disease chromosomes carry the same mutation and the markers being tested are relatively close to the disease gene(s).
For example, in SNP-based association analysis and LD mapping, SNPs can be useful in association studies for identifying polymorphisms, associated with a pathological condition, such as sepsis. Unlike linkage studies, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. In a SNP association study the frequency of a given allele (i.e. SNP allele) is determined in numerous subjects having the condition of interest and in an appropriate control group. Significant associations between particular SNPs or SNP haplotypes and phenotypic characteristics may then be determined by numerous statistical methods known in the art. Association analysis can either be direct or LD based. In direct association analysis, potentially causative SNPs may be tested as candidates for the pathogenic sequence. In LD based SNP association analysis, SNPs may be chosen at random over a large genomic region or even genome wide, to be tested for SNPs in LD with a pathogenic sequence or pathogenic SNP. Alternatively, candidate sequences associated with a condition of interest may be targeted for SNP identification and association analysis. Such candidate sequences usually are implicated in the pathogenesis of the condition of interest. In identifying SNPs associated with inflammatory conditions, candidate sequences may be selected from those already implicated in the pathway of the condition or disease of interest. Once identified, SNPs found in or associated with such sequences, may then be tested for statistical association with an individual's prognosis or susceptibility to the condition.
For an LD based association analysis, high density SNP maps are useful in positioning random SNPs relative to an unknown pathogenic locus. Furthermore, SNPs tend to occur with great frequency and are often spaced uniformly throughout the genome. Accordingly, SNPs as compared with other types of polymorphisms are more likely to be found in close proximity to a genetic locus of interest. SNPs are also mutationally more stable than variable number tandem repeats (VNTRs) and short tandem repeats (STRs).
In population genetics linkage disequilibrium refers to the "preferential association of a particular allele, for example, a mutant allele for a disease with a specific allele at a nearby locus more frequently than expected by chance" and implies that alleles at separate loci are inherited as a single unit (Gelehrter, T.D., Collins, F.S. (1990). Principles of Medical Genetics. Baltimore: Williams & Wilkens). Accordingly, the alleles at these loci and the haplotypes constructed from their various combinations serve as useful markers of phenotypic variation due to their ability to mark clinically relevant variability at a particular position, such as position 201 of SEQ ID NO:1 (see Akey, J. et al. Eur J Hum Genet (2001) 9:291-300; and Zhang, K. et al. (2002). Am J Hum Genet. 71 : 1386-1394). This viewpoint is further substantiated by Khoury et al. ((1993). Fundamentals of Genetic Epidemiology. New York: Oxford University Press at p. 160) who state, "[w]henever the marker allele is closely linked to the true susceptibility allele and is in [linkage] disequilibrium with it, one can consider that the marker allele can serve as a proxy for the underlying susceptibility allele." As used herein "linkage disequilibrium" (LD) is the occurrence in a population of certain combinations of linked alleles in greater proportion than expected from the allele frequencies at the loci. For example, the preferential occurrence of a disease gene in association with specific alleles of linked markers, such as SNPs, or between specific alleles of linked markers, are considered to be in LD. This sort of disequilibrium generally implies that most of the disease chromosomes carry the same mutation and that the markers being tested are relatively close to the disease gene(s). Accordingly, if the genotype of a first locus is in LD with a second locus (or third locus etc.), the determination of the allele at only one locus would necessarily provide the identity of the allele at the other locus. When evaluating loci for LD those sites within a given population having a high degree of linkage disequilibrium (i.e. an absolute value for r2 > 0.5) are potentially useful in predicting the identity of an allele of interest (i.e. associated with the condition of interest). A high degree of linkage disequilibrium may be represented by an absolute value for r2 > 0.6. Alternatively, a high degree of linkage disequilibrium may be represented by an absolute value for r2 > 0.7 or by an absolute value for r2 > 0.8. Additionally, a high degree of linkage disequilibrium may be represented by an absolute value for r2 > 0.85 or by an absolute value for r2 > 0.9.
Accordingly, two SNPs that have a high degree of LD may be equally useful in determining the identity of the allele of interest or disease allele. Therefore, we may assume that knowing the identity of the allele at one SNP may be representative of the allele identity at another SNP in LD. Accordingly, the determination of the genotype of a single locus can provide the identity of the genotype of any locus in LD therewith and the higher the degree of linkage disequilibrium the more likely that two SNPs may be used interchangeably. For example, in the population from which the tagged SNPs were identified from the SNP identified by rs 18059 is in "linkage disequilibrium" with the SNP identified by rs2762, whereby when the genotype of rs 18059 is T the genotype of rs2762 is G. Similarly, when the genotype of rs 18059 is C the genotype of rs2762 is A. Accordingly, the determination of the genotype at rs 18059 will provide the identity of the genotype at rs2762 or any other locus in "linkage disequilibrium" therewith. Particularly, where such a locus is has a high degree of linkage disequilibrium thereto.
LD is useful for genotype-phenotype association studies. For example, if a specific allele at one SNP site (e.g. "A") is the cause of a specific clinical outcome (e.g. call this clinical outcome "B") in a genetic association study then, by mathematical inference, any SNP (e.g. "C") which is in significant LD with the first SNP, will show some degree of association with the clinical outcome. That is, if A is associated (~) with B, i.e. A-B and C-A then it follows that C-B. Of course, the SNP that will be most closely associated with the specific clinical outcome, B, is the causal SNP - the genetic variation that is mechanistically responsible for the clinical outcome. Thus, the degree of association between any SNP, C, and clinical outcome will depend on LD between A and C.
Until the mechanism underlying the genetic contribution to a specific clinical outcome is fully understood, LD helps identify potential candidate causal SNPs and also helps identify a range of SNPs that may be clinically useful for prognosis of clinical outcome or of treatment effect. If one SNP within a gene is found to be associated with a specific clinical outcome, then other SNPs in LD will also have some degree of association and therefore some degree of prognostic usefulness.
By way of prophetic example, if multiple polymorphisms were tested for individual association with an improved response to vasopressin receptor agonist administration in our SIRS/sepsis/septic shock cohort of ICU subjects, wherein the multiple polymorphisms had a range of LD with LNPEP rs 18059 and it was assumed that rs 18059 was the causal polymorphism, and we were to order the polymorphisms by the degree of LD with rs 18059, we would expect to find that polymorphisms with high degrees of LD with rs 18059 would also have a high degree of association with this specific clinical outcome. As LD decreased, we would expect the degree of association of the polymorphism with an improved response vasopressin receptor agonist administration to also decrease. It follows that any polymorphism, whether already discovered or as yet undiscovered, that is in LD with one of the improved response genotypes described herein will likely be a predictor of the same clinical outcomes that rsl 8059 is a predictor of. The similarity in prediction between this known or unknown polymorphism and rsl 8059 would depend on the degree of LD between such a polymorphism and rsl 8059.
Numerous sites have been identified as polymorphic sites in the vasopressin pathway associated genes (see TABLE IA). Furthermore, the polymorphisms in TABLE IA are linked to (in LD with) numerous polymorphism as set out in TABLE IB below and may also therefore be indicative of subject prognosis.
TABLE IA. Polymorphisms in the vasopressin pathway associated genes genotyped in a cohort of critically ill Subjects with severe sepsis. Minor Allele Frequencies (MAFs) for Caucasians were taken from Hapmap.org (Thorisson GA. et al. The International HapMap Project Website. Genome Research (2005) 15 : 1591-1593).
Figure imgf000029_0001
Figure imgf000030_0001
TABLE IB. Polymorphisms in linkage disequilibrium with those listed in TABLE IA above, as identified using the Haploview program (BARRETT JC. et al. Bioinformatics (2005) 21(2):263-5 (http://www.broad.mit.edu/mpg/haploview/)) and the LD function in the Genetics Package in R (R Core Development Group, 2005 - R Development Core Team (www.R-project.org). Linkage Disequilibrium between markers was defined using r2 whereby all SNPs available on Hapmap.org (phase II) (cohort H), all SNPs genotyped internally using the Illumina Goldengate assay (cohort I) and all SNPs sequenced using the Sequenom Iplex Platform (cohort S) in our genes of interest were included. A minimum r2 of 0.5 was used as the cutoff to identify LD SNPs. The genes are identified, along with the alleles, rs designation and the chromosomal position (March 2006 Build 36). An LD allele was only predicted for those cohorts that had sufficient power and NA designations indicate that the sample sizes were insufficient to make an allele designation with confidence at the time of filing. However, the assignment of allele designations for NA designated LD alleles is a routine procedure. A '*' indicates that there is more than one RSID assigned to a single SNP.
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
NA as used above indicates that the LD allele with the information currently available to the inventors could not with any confidence be assigned without further routine analysis, due to the lack of suitable information currently available regarding the corresponding allele designations. However, it would be well within the abilities of a person of skill in the art to make LD allele designations for the NA polymorphisms using routine analysis.
It will be appreciated by a person of skill in the art that further linked polymorphic sites and combined polymorphic sites may be determined. A haplotype of vasopressin pathway associated genes can be created by assessing polymorphisms in vasopressin pathway-associated genes in normal subjects using a program that has an expectation maximization algorithm (i.e. PHASE). A constructed haplotype of vasopressin pathway associated genes may be used to find combinations of SNPs that are in LD with the tag SNPs (tSNPs) identified herein. Accordingly, the haplotype of an individual could be determined by genotyping other SNPs or other polymorphisms that are in LD with the tSNPs identified herein. Single polymorphic sites or combined polymorphic sites in LD may also be genotyped for assessing subject response to vasopressin receptor agonist treatment. It will be appreciated by a person of skill in the art that the numerical designations of the positions of polymorphisms within a sequence are relative to the specific sequence. Also the same positions may be assigned different numerical designations depending on the way in which the sequence is numbered and the sequence chosen, as illustrated by the alternative numbering of the equivalent polymorphism (rs3803107), whereby the same polymorphism identified C/T at position 3536 of the NM_000706.3 (GI:33149325), which corresponds to position 201 of SEQ ED NO:9. Furthermore, sequence variations within the population, such as insertions or deletions, may change the relative position and subsequently the numerical designations of particular nucleotides at and around a polymorphic site.
Polymorphic sites in SEQ ED NO: 1-10 are identified by their variant designation (i.e. M, W, Y, S, R, K, V, B, D, H or by "-" for a deletion, a "+"or for example "G" etc. for an insertion).
Polymorphic sites in SEQ ED NO: 11-264 are identified by their allelic change (i.e. A, C, G, T or by "-" for a deletion, a "+"or for an insertion).
An "rs" prefix designates a SNP in the database is found at the NCBI SNP database (http://www.ncbi.nlm.nih.gov/entrez/query .fcgi?db=Snp). The "rs" numbers are the NCBI rsSNP ID form.
TABLE 1C below shows the flanking sequences for a selection of vasopressin pathway associated gene SNPs providing their rs designations and corresponding SEQ ED NO designations. Each polymorphism is at position 201 within the flanking sequence, and identified in bold and underlined.
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
The Sequences given in TABLE 1C (SEQ ID NOr l-10) above and in TABLE ID (SEQ ID NO: 1 1- 264) would be useful to a person of skill in the art in the design of primers and probes or other oligonucleotides for the identification of vasopressin pathway associated gene SNP alleles and or genotypes as described herein.
TABLE ID below shows the flanking sequences for a selection of vasopressin pathway associated gene SNPs in LD with the tagged SNPs in TABLE 1C, providing their rs designations and corresponding SEQ ID NO designations. However, where a SNP in LD is also an htSNP it only occurs in TABLE 1C above. Each SNP is at position 200 of the flanking sequence (unless otherwise indicated) and is underlined.
Figure imgf000055_0002
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
An "allele" is defined as any one or more alternative forms of a given gene In a diploid cell or oiganism the members of an allelic pair ( i e. the two alleles of a given gene) occupy corresponding positions ( loci) on a pair of homologous chromosomes and if these alleles are genetically idem ical the cell or organism is said to be "homozygous", but if genetically different the cell or organism is said to be "heterozygous" w ith respect to the particular gene.
\ "gene" is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product and may include untranslated and untranscribed sequences in proximity to the coding regions (5' and 3' to the coding sequence). Such non-coding sequences may contain regulatory sequences needed for transcription and translation of the sequence or introns etc. or may as yet to have any function attributed to them beyond the occurrence of the SNP of interest.
A "genotype" is defined as the genetic constitution of an organism, usually in respect to one gene or a few genes or a region of a gene relevant to a particular context (i.e. the genetic loci responsible for a particular phenotype).
I O TABLE IE. Genotype correlations for SNPs in vasopressin pathway associated genes w ith va lues representing an ability to recover from an inflammatory condition and an indication of res onsiveness to treatment of an inflammatory condition with a vasopressin receptor agonist.
Figure imgf000098_0001
iood = 2; poor = 1 . ' Responsive (R), Poor Response (PR)
A "phenotype" is defined as the observable chaiacters of an organism In gene association studies, the genetic model at a given locus can change depending on the selection pressures (i e , the
S en\ ironment), the population studied, or the outcome variable (i e , the phenotype) For example, the model at rs 1410713 changed between the risk of death claims (AA versus AC/CC) and the vasopressin IRP claims (AA/AC veisus C C) This is a case of the same outcome variable (survival) tollow ing a different genetic model in diffeient environments (i e . no vasopressin treatment versus vasopressin treatment)
K)
\ similai observation would be seen in a gene association study w ith the hemoblobin, beta gene (HBB) w ith moitality as the pπmaiy outcome vanable A mutation in the HBB gene, which noi mally pioduces the beta chain subunit of hemoglobin (B allele), results in an abnoimal beta chain called hemoglobin S (S allele, Allison A ( 1955) Cold Spring Haibor Symp Quant Biol
I 5 20 219-255 ) Hemoglobin S results in abnoimal sitkle-shaped ied blood cells w hich lead to anemia and other serious complications including death In the absence of malana. a gene association study w ith the HBB gene would suggest a codominant model (surv ival(BB) > survival (BS) > surv ival (SS)) However, in the piesence of mailana, a gene association study w ith the HBB gene would suggest a heterozygote adv antage model (survival(BB) < surv ival(BS) >
20 suiv ival( SS))
Λ "single nucleotide polymoiphism"' (SNP) occurs at a polymorphic site occupied by a single nucleotide, w hich is the site of vai ution between allelic sequences The site is usually pieceded by and followed by highly conserved sequences of the allele (e g , sequences that vary in less than
25 1/ 100 oi 1/1000 membei s ot the populations) A single nucleotide polymoiphism usually arises due to substitution of one nucleotide for another at the polymorphic site A "transition" is the teplacement of one purine by another puπne oi one pyi imidine by another pynmidine A "tiansvei sion" is the replacement of a pur ne by a pynmidine oi v ice veisa Single nucleotide poly morphisms can also aπse fiom a deletion (represented by "-" oi "del") of a nucleotide oi an
M) insertion (lepresented by "+" oi "ins" or "I") of a nucleotide relative to a reference allele
Fiuthermore. a person of skill in the art would appreciate that an insertion or deletion w ithin a given sequence could alter the relative poMtion and theiefore the position number of another polymorphism w ithin the sequence Furthermore, although an insertion or deletion may by some definitions not qualify as a SNP as it may involve the deletion of or insertion of more than a single nucleotide at a given position, as used heiein such polymorphisms are also called SNPs as the y generall) result from an insertion or deletion at a single site w ithin a given sequence
\ "systemic inflammatory response syndrome" or (SIRS) is defined as including both septic 1 1 e 5 sepsis or septic shock) and non-septic systemic inflammatory response (i e post operative)
"SIRS" is further defined according to ACCP (American College of Chest Physicians) guidelines as the piesence of two 01 moie of A) temperature > 380C or < 36"C, B) heart rate > 90 beats per minute, C) respnatory rate > 20 breaths per minute, or PaCCK < M mm Hg oi the need for mechanical ventilation, and D) white blood cell count > 12,000 per mm or < 4.000 mm In ihe 10 follow ing description, the presence of two, three, or tour of the "SIRS" cπteπa were seoied each day o\ei the 28 day obsei vation peπod
" Sepsis" is defined as the presence of at least two "SIRS" criteria and known or suspected source of infection Septic shock was defined as sepsis plus one new organ failuie by Brussels cπtei ia I s plus need foi vasopiessor medication or v asopressin ieceptor agonist
Subject outcome or prognosis as used herein refers the ability of a subject to recover from an inflammatoiy condition and may be used o determine the efficacy of a tieatment legimen, foi example the administration of a vasopressin ieceptor agonist An inflammatoiy condition, may be
20 selected liom the gioup consisting of sepsis, septicemia, pneumonia, septic shock, systemic inflammatoiv iesponse syndrome (SIRS). Acute Respiratory Distiess Syndrome (ARDS) acute lung lnjui) . aspiiation pneumonitis, infection, pancreatitis, bacteremia, peπtonitis, abdominal abscess, inflammation due to trauma, inflammation due to suigery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue
25 damage due to chemotheiapy oi radiotherapy, and reactions to ingested, inhaled, infused, injected, or delivered substances, glomerulonepht itis. bowel infection, oppoitunistic infections, and for subjects undergoing major suigery or dialysis, subjects w ho are immunocompromised subjects on immunosuppiessi\e agents, subjects w ith HIV/AIDS, subjects w ith suspected endocaiditis, subjects w ith fever, subjects w ith fevei of unknown ongin. sublets w ith cystic fibrosis, subjec ts
M) w ith diabetes melhtus. subjects w ith chion ic ienal failure, subjects w ith acute renal failuie oligui ia. subjects w ith acute renal dysfunction, glomerulo-nephπtis. interstitial-nephritis, aciitt tubulai necrosis (ATN). subjects w ith bronchiectasis, subjects w ith chronic obstructive lung disease, chionic bronchitis, emphysema ot asthma, subjects w ith febrile neutropenia, subjects w ith meningitis, subjects w ith septic arthi itis, subjects w ith unnary tract infection, subjects w ith necrotizing fasciitis, subjects w ith other Hispected Group A streptococcus infection, subjects who ha\e had a splenectomy, subjects w ith recurrent or suspected enteiococcus infection, other medical and suigical conditions associated w ith increased risk of infection, Gram positive sepsis. Gram negative sepsis, culture negative sepsis, fungal sepsis, meningococcemia, post-pump syndrome. s cardiac stun syndrome, myocardial infarction, stioke, congestive heart failure, hepatitis, epiglottitis. E coli 0157 H7. malaria, gas gangiene, toxic shock s> ndrome, pre-eclampsia, eclampsia. HELLP syndiome, mycobacte rial tuberculosis, Pneumocystis carina pneumonia, pneumonia. Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpuia, Dengue hemoirhagic fever, pelvic inflammatory disease, Legionella, Lyme disease. Influenza A,
10 Epstein-Barr v irus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid aithπtis osteoaithπtis, progressive systemic sclerosis, systemic lupus erythematosus, inflammatoiy bowel disease, idiopathic pulmonary f ibrosis, sarcoidosis, hypersensitivity pneumonitis, systemic vasculitis. Wegener's gianulomatosis, tiansplants including heart, liver, lung kidney bone mauow, graft-veisus-host disease, tiansplant rejection, sickle cell anemia,
I S nephtotic syndiome, toxicity of agents such as OKTl, cytokine theiapy, and ciirhosis
Assessing subject outcome, prognosis, or response of a subject to vasopressin ieceptor agonist administration may be accomplished by vanous methods For Example, an "APACHE IT' score is defined as Acute Physiology And Chioni; Health Evaluation and herein was calculated on a daily 0 basis from taw clinical and laboiatoiy vai iables Vincent et al (Vincent JL Feireira F Moreno R 2000 Cut Caie Clin 16 153-366) summarize APACHE scoie as follows "Fust developed in 1981 by Knaus c t al . the APΛCHE scoie has become the most commonly used sutvival prediction model in ICUs worldwide The APACHE II score, a rev ised and simplified version of the original prototype, uses a point scoie based on mi ial values of 12 ioutine physiologic measures, age. ind
2s prev ious health status to piovide a geneial measure of seventy of disease The values iecorded are the woi st values taken duπng the subject\ first 24 hours in the ICU The scoie is applied to one of 34 admission diagnoses to estimate a disease-specific probability of mortality (APACHE II predicted nsk of death) The maximum possible APACHE II score is 71 , and high scores have been well coπelated w ith mortality The APACHE II score has been w idely used to stiatify and
M) compare various groups of critically ill subjects, including subjects w ith sepsis, by severity of illness on entiy into clinical trials"
A "Brussels score" score is a method for evaluating oigan dysf unction as compared to a baseline If the Brussels scoie is 0 (i e modeiate, severe, oi extieme). then oigan failure was recoided as present on that particular day (see TABLE 2A below) In the following description, to correct for deaths during the observation period, days alive and free of organ failuie (DAF) weie calculated as pre\ iously described For example, acute lung injury was calculated as follows Acute lung injury is defined as present w hen a subject meets all of these foui criteria 1 ) Need for mechanical ventilation. 2) Bilateral pulmonary infiltrates on chest X iay consistent w ith acute lung injuiy 1) PaOi/FiOi ratio is less than ^OOmmHg, 4) No clinical evidence of congestive heart failure or if a pulmonaiy aitery catheter is in place for clinical puψoses. a pulmonary capillary wedge pressure less than 18 mm Hg ( 1 ) The severity of acute lung injury is assessed by measuring days alivt and fiee of acute lung injury over a 28-day observation penod Acute lung injury is iecorded as piesent on each day that the person has modeiate, severe or extreme dysfunction as defined in the Brussels score Days alive and fiee of acute lung injury is calculated as the number of days af ter onset of acute lung injuiy that a subject is alive and free of acute lung injuiy over a def ined obsei vation period (28 days) Thus, a low ei score foi days alive and free of acute lung injury indicates more seveie acute lung in)uiy The reason that days alive and free of acute lung injuiy is
I 1S piefeiable to simply piesence oi absence of acute lung injury, is that acute lung injury has a hi gh acute moitality and early death (w ithin 28 days) precludes calculation of the presence oi absence of acute lung injuiy in dead subjects The cardiovasculai, ienal, neuiologic hepatic and coagulation dysfunction were similarly def ined as present on each day that the person had moderate seveie oi extreme dysfunction a> defined by the Brussels score Days alive and fiee of
20 ^•teioids are days that a person is alive and is not being tieated w ith exogenous corticosteioids (e g hydiocoitisone, prednisone, methylprednisolone) Days alive and free of piessors aie days thai a pei son is alive and not being tieated w ith intiavenous vasopressors (e g dopamine, noiepinephnne. epinephi me or phenylephnne) Days alive and free of an International Normalized Ratio (INR) > 1 ^ are days that a person is alive and does not have an INR > 1 5
TABLE 2A Brussels Organ Dysfunction Scoring System
Figure imgf000102_0001
Figure imgf000103_0001
2. General Methods
One aspect of the invention may involve the identification of subjects oi the selection ot subjects that aie either at nsk of developing and inflammatory condition or the identif ication of subject^ s who already have an inflammatoiy condition Foi example, subjects who have undeigone major surgeiy or scheduled foi oi contemplating major surgery may be considered as being at i isk of developing an inflammatoiy condition Furthermoie, subjects may be detei mined as ha\ ing an inflammatory condition using diagnostic methods and clinical evaluations known in the medical aits An inflammatory condition. ma> be selected from the gioup consisting of sepsis, septicemia.
K) pneumonia, septic shock, systemic inflammatory iesponse syndiome (SIRS), Acute Respiratory Distiess Syndrome (ARDS). acute lung lnjuiy. aspπation pneumonitis, infection, pancreatitis, bacteiemia, pentonitis. abdominal abscess, inflammation due to trauma, inflammation due to surgery, chronic inflammatoiy disease, ischemia, ischemia-reperfusion injury of an organ oi tissue, tissue damage due to disease, tissue damage due to chemotherapy or radiotherapy, and ieactions to
I s ingested, inhaled, infused, injected, or dehv eied substances, glomeiulonephi itis, bowel infection, opportunistic infections, and for subjects undergoing major suigery or dialysis, subjects w ho ai e immunocompiomised. subjects on immunosuppiessive agents, subjects w ith HIV/AIDS, subjec ts w ith suspected endocaiditis. subjects w ith tevei , subjects w ith fever of unknown oπgin, subjects w ith cystic f ibrosis, subjects w ith diabetes melhtus, subjects w ith chronic renal failuie, subject^
20 w ith acute ienal failuie. oliguria, subjects w ith acute renal dysfunction, glomerulonephi itis. interstitial-nephritis, acute tubular necrosis (ATN). subjects w ith bronchiectasis, subjects w ith chronic obstructive lung disease, chronic bionchitis. emphysema, oi asthma, subjects w ith febrile neutiopenia, subjects w ith meningitis, subjects w ith septic arthritis, subjects w ith ui inai ) tract infection, subjects w ith necrotizing fasciitis subjects w ith other suspected Group A stieptococcus infection, subjects who have had a splenec omy. subjects w ith iecurrent or suspected enterococ cus infection, other medical and surgical conditions associated w ith increased πsk of infection. Gram positive sepsis. Giam negative sepsis, culture negative sepsis, fungal sepsis, meningococcemia, post-pump sv ndiome, cardiac stun syndrome, myocardial infarction, stroke, congestive heart
^ failure, hepatitis, epiglottitis, E coll 0157 H7. malaria, gas gangrene, toxic shock syndrome, preeclampsia eclampsia, HELLP syndrome, mycobacterial tuberculosis, Pneumocystis cai inii pneumonia, pneumonia. Leishmaniasis, hemolytic uremic syndiome/thrombotic thiombocytopenic pui puia. Dengue hemorrhagic fever, pelvic inflammatoiy disease, Legionella, Lyme disease, Influenza Λ, Epstem-Barr v n us, encephalitis, inflammatory diseases and autoimmunity including
K) rheumatoid arthritis, osteoaithπtis. progressive systemic scleiosis. systemic lupus erythematosus, inflammatoiy bowel disease, idiopathic pulmonaiy fibiosis, saicoidosis. hypersensitiv ity pneumonitis, systemic vasculitis, Wegenei's gianulomatosis, tiansplants including heart, liver, lung kidney bone mairow. graft-versus-host disease, tiansplant rejection, sickle cell anemia, nephrotic syndiome. toxicity of agents such as OKTl. cytokine therapy, and cirrhosis i s
Once a subject is identified as being at nsk for developing or having an inflammatoi y condition 01 is to be administered vasopressin receptor agonist, then genetic sequence information may be obtained from the subject Or alternatively genetic sequence information may already have betn obtained horn the subject For example, a subject may have already provided a biological sample
20 for othei pin poses or ma> have even had their genetic sequence detei mined in whole oi in part and stored toi lutuie use Genetic sequence information may be obtained in numeious diffeient ways and may involve the collection of a biological sample that contains genetic material, particularly, genetic matenal containing the sequence oi sequences of interest Many methods are known in the art for collecting biological samples and ex racting genetic matei ial from those samples Genetic
2^ material can be extracted fiom blood, tissue , hair and othei biological matenal There are many methods known to isolate DNA and RNA from biological matenal Typically. DNA ma> be isolated fiom a biological sample when first the sample is lv, sed and then the DNA is sepaiated horn the lysate according to any one of a variety of multi-step piotocols. which can take varying lengths of time DNA isolation methods may involve the use of phenol (Sambrook. J et al ,
M) "Molecular Cloning ', VoI 2, pp 9 14 9 21 Cold Spring Harboi Laboratoiy Press ( 1989) and Ausubel. Frederick M et al . 'Cuirent Protocols in Molecular Biology", VoI l , pp 2 2 1 2 4 5, John W iley & Sons, Inc ( 1994)) Typically, a biological sample is lysed in a detergent solution and the protein component of the lysate is digested w ith pioteinase for 12- 18 houis Next, the lysate is extracted w ith phenol to lemove most ot the cellular components, and the remaining aqueous phase is processed further to isolate DNA In another method, described in Van Ness et al (U S Pat # 5.130.423), non-coirosive phenol derivatives aie used for the isolation of nucleic acids The resulting preparation is a mix of RNA and DNA
s Othei methods for DNA isolation utilize non-coirosive chaotropic agents These methods, which die based on the use of guanidine salts, uiea and sodium iodide, involve lysis of a biological sample in a chaotropic aqueous solution and subsequent piecipitation of the crude DNA fraction w ith a lowei alcohol The final pui ification of the precipitated, ciude DNA f raction can be achieved by any one of several methods, including column chromatography (Analects, ( 1994) VoI
K) 22, No 4. Pharmacia Biotech), or exposute of the crude DNA to a polyanion-containing piotein as desci ibed in Koller (U S Pat # 5.128.247)
Yet anothei method of DNA isolation, which is described by Botwell. D D L (Anal Biochem ( 1987) 162 463-465) involves lysing cells in 6M guanidine hydiochloπde, precipitating DNA from I S the lysate at acid pH by adding 2 5 volumes of ethanol, and washing the DNA w ith ethanol
Numerous other methods aie known in the art to isolate both RNA and DNA. such as the one described b> CHOMCZYNSKI (U S Pat # 5,945,515), whereby genetic material can be extracted efficiently in as little as twenty minutes E VANS and HUGH (U S Pat # 5.989.431 ) describe 20 methods foi isolating DNA using a hollow membrane filter
Once a subject's genetic material has been obtained from the subject it may then be further be amplified by Reverse Tianscπption Polymerase Chain Reaction (RT-PCR). Polymerase Chain Reaction (PCR), Tianscπption Mediated Amplification (TMA). Ligase chain reaction (LCR).
2s Nucleic Acid Sequence Based Amplification (NASBA) or othei methods know n in the art, and then furthei analyzed to detect or determine the presence or absence of one oi more polymoi phisms or mutations in the sequence of mteiest, provided that the genetic matei ial obtained contains the sequence of interest Particulai ly. a person may be interested in determining the piesence oi absence of a mutation in a uisopiessin pathway associated gene sequence, as
M) described in TABLES I A-D The sequence of interest tnaj also include othei mutations, oi may also contain some of the sequence surrounding the mutation of interest
Detection oi determination of a nucleotide identity, or the presence of one or tnoie single nucleotide polymorphism(s) (SNP typing), may be accomplished by any one of a number methods or assays known in the ait Many DNA typing methodologies are useful for use in the detection of SNPs The majority of SNP genotyping ieactions or assays can be assigned to one of foui broad groups (sequence-specific hybridization, primer extension, oligonucleotide ligation and invasive clea\ age) Fuithermore, there are numerous methods for analyzing/detecting the products of each 5 type of reaction (for example, fluorescence luminescence, mass measurement, electrophoresis, etc ) Furtheimore. reactions can occur in solution or on a solid support such as a glass slide, a chip, a bead, etc
In geneial. sequence-speciiic hybridization involves a hybridization probe, which is capable of 10 distinguishing between two DNA taigets differing at one nucleotide position by hybndization
Usually pi obes are designed w ith the polymorphic base in a cential position in the probe sequence, whereby under optimized assay conditions only the perfectly matched probe target hybrids are stable and hybnds w ith a one base mismatch are unstable A strategy which couples detection uid sequence discrimination is the use of a "moleculai beacon"', whereby the hybridization probe I s (moleculai beacon) has V and 5' ieporter and quencher molecules and T and 5' sequences which are complementary such that absent an adequate binding taiget for the inteivening sequence tht piobe w ill form a haii pin loop The hairpin loop keeps the iepoitei and quencher in close proximit) iesulting in quenching of the fluoiophor (iepoitei ) which ieduces fluoiescence emissions Howevei . w hen the molecular beacon hybndizes to the taiget the fluorophoi and the 20 quencher aie sufficiently sepaiated to allow fluoiescence to be emitted fiom the fluorophor
Similarly, primer extension ieactions (i e mini sequencing, nucleotide-specific extensions, oi simple PCR amplification) are useful in sequence discrimination ieactions For example, in mini sequencing a pnmer anneals to its taiget DNA immediately upstieam of the SNP and is extended 25 w ith a single nucleotide complementary to lhe polymoiphic site Wheie the nucleotide is not complementaiy, no extension occuis
Oligonucleotide ligation assays lequire two sequence-specific piobes and one common ligation piobe per SNP The common ligation probe hybridizes adjacent to a sequence-specific probe and W when there is a peifect match of the appiopnate sequence-specific probe, the ligase joins both the sequence-specific and the common probes Where there is not a perfect match the ligase is unable to join the sequence-specific and common piobes Piobes used in hybndization can include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids Hybndization methods for the identification of single nucleotide polymoiphisms oi othei mutations involving a few nucleotides aie described in the U S Pat 6.270,961 , 6.025, 136, and 6,872.530 Suitable hybridization probes for use in accordance w ith the invention include oligonucleotides and PNAs from about 10 i o about 400 nucleotides, alternatively from about 20 to about 200 nucleotides, or from about 30 to about 100 nucleotides in length
Alternatively, an invasive cleavage method lequnes an oligonucleotide called an Invader™ piobe and sequence-specific piobes to anneal to the target DNA w ith an overlap of one nucleotide When the sequence-specific probe is complementary to the polymorphic base, oveilaps of the !' end of the invader oligonucleotide foim a stiucture that is lecognized and cleaved by a Flap 0 endonuclease releasing the 5' arm of the allele specific probe
5' exonutlease activity or TaqMan™ assay (Applied Biosystems) is based on the 5' nuclease activity of Taq polymeiase that displaces and cleaves the oligonucleotide probes hybridized to the taiget DNA generating a fluorescent signal It is necessaiy to have two probes that differ at the
15 polymoiphic site w herein one probe is complementary to the "noimal' sequence and the other to the mutation of interest These piobes have diffeient fluoiescent dyes attached to the 5' end and a quenchei attached to the T end when the piobes aie intact the quencher internets w ith the fluorophor bv fluoiescence resonance eneigy tiansfer (FRET) to quench the fluorescence of tht piobe Din ing the PCR annealing step the hybridization probes hybridize to target DNA In the 0 extension step the 5' fluoiescent dye is cleaved by the 5' nuclease activity of Taq polymeiase, leading to an inciease in fluorescence of the ieporter dve Mismatched probes are displaced w ithout fiagmentation The piesence of a mutation in a sample is determined by measui ing the signal intensity of the two different dyes
^ The Illumina Golden Gate1 M Assay uses a < ombined oligonucleotide ligation assay/ allele-speufic hybndization appioach (SHEN R et til Mut.it Res 200^573 70-82) The first series of steps inv olve the hybridization of three oligonucleotides to a set of specific target SNPs, two ol these are fluorescently-labelled allele-specihc oligonucleotides (ASOs) and the thud a locus-specific oligonucleotide (LSO) binding 1 -20 bp dow nstieam of the ASOs A second series of steps involve
M) the use of a stringent polymerase w ith high T specif icity that extends only oligonucleotides specifically matching an allele at a taiget S VP The polymerase extends until it reaches the LSO Locus-specif icity is ensuied by requiring the hybridization of both the ASO and LSO in oider that extension can proceed After PCR amplification w ith univeisal primers, these allele specific oligonucleotide extension products are hybi ldized to an an ay which has multiple disci etely tagged addresses ( in this case 1536 addresses) which match an address embedded in each LSO Fluorescent signals produced by each hybridization product are detected by a bead array leader from which genotypes at each SNP locus may be ascertained
s It w ill be appreciated that numeious other methods foi sequence discrimination and detection are known in the art and some of which are described in further detail below It will also be appieciated that reactions such as arrayed primer extension mini sequencing, tag microairays and sequence-specific extension could be performed on a microarray One such array based genotyping platform is the miciosphere ba^ed tag-it high throughput genotyping array I O (BORTOLIN S et al Clinical Chemistry ( 2004) S()( 1 1 ) 2028-36) This method amplifies genomic DNA by PCR followed by sequence-specific primer extension w ith universally tagged genotyping pi imers The products aie then sorted on a Tag-It array and detected using the Luminex xMAP sWem
I S Mutation detection methods may include but aie not limited to the following
Restriction Fragment Length Polymoi phism (RFLP) stiategy - An RFLP gel-based analysis can be used to indicate the presence or absence of a specific mutation at polymoiphic sites w ithin a gene Brief ly a short segment of DNA (typically se\eial hundred base pairs) is amplified by PCR Where possible, a specific iestriction endonuclease is chosen that cuts the short DNA segment
20 w hen one polymoiphism is piesent but does not cut the short DNA segment when the polymoiphism is not piesent. or v ice veisa After incubation of the PCR amplified DNA w ith 1 his iestriction endonuclease, the ieaction products aie then sepaiated using gel electrophoiesis Thus, w hen the gel is examined the appeal ance of two lowei molecular weight bands (lower molecul ir weight molecules tiavel farther down the gel dui ing electrophoiesis) indicates that the DNA
2S sample had a polymoiphism was present that peimitted cleavage by the specific restiiction endonuclease In contiast, if only one higher molecular weight band is observed (at the molecular weight of the PCR pioduct) then the initial DNA sample had the polymorphism that could not be cleaved bv the chosen restiiction endonuclease Finally, if both the higher molecular weight band and the two lowei moleculai weight bands aie v isible then the DNA sample contained both
M) polymoiphisms, and therefoie the DNA sample, and by extension the subject prov iding the DNA sample, was heteiozygous for this pol> morphism.
For example the Maxam-Gilbert technique foi sequencing (MAXAM A.M and GILBERT W Pioc Natl Acad Sci USA ( 1977) 74(4) 5o0-%4) involves the specific chemical cleavage of terminally labelled DNA In this technique four samples of the same labeled DNA are each subjected to a diffeient chemical ieaction to effect preferential cleavage of the DNA molecule at one or two nucleotides of a specific base identity The conditions aie adjusted to obtain only partial cleavage. DNA fragments aie thus Lenerated in each sample whose lengths are dependent ^ upon the position w ithin the DNA base sequence of the nucleotide^ ) which are subject to such cleavage After partial cleavage is performed, each sample contains DNA fiagments of different lengths, each of which ends w ith the same one 01 two ot the four nucleotides In particular in one sample each fragment ends w ith a C, in another sample each fiagment ends w ith a C or a T, in i third sample each ends w ith a G, and in a fourth sample each ends w ith an A or a G When the
K) products ot these four reactions are lesolved by size, by electiophoresis on a polyacrylamide gt l, the DNA sequence can be iead from the pattern of radioactive bands This technique permits the sequencing of at least 100 bases fiom the point of labeling Another method is the dideoxy mei hod ot sequencing was published by SANGER et til (Proc Natl Acad Set USA ( 1977) 74( 12) 5461- 5467) The Sanger method relies on enzymatic activ ity of a DNA polymerase to synthesize
I s sequence dependent fiagments of vai ions lengths The lengths ol the fiagments are determined by the random incorporation of dideoxy nucleotide base-specific teiminatois These fiagments can then be sepaiated in a gel as in the Maxam-Gilbeit procedure, visualized, and the sequence determined Numeious improvements have been made to refine the above methods and to automate the sequencing piocedures Similarly, RNA sequencing methods aie also known For
20 example, leverse transcriptase w ith dideoxx nucleotides have been used to sequence encephalomyocaiditis v uus RNA (ZIMMERN D and KAESBERG P Pioc Natl Acad Sci USA ( 1978) 75(9) 4257-4261 ) MILLS DR and KRAMER FR (Proc Natl Acad Sci USA ( 1979) 76( 5) 2232-2235) desci ibe the use ol Qβ ieplicase and the nucleotide analog inosine foi sequencing RNA in a chain-termination mechanism Diiect chemical methods for sequencing
2s RNA aie also known (PEATTIE DA Proc Natl Acad Sci USA ( 1979) 76(4) 1760- 1764) Othei methods include those of Donis-Keller i t ai ( 1977. Niicl Acids Res 4 2527-2538). SIMONCSITS A i t al (Natuie ( 1977) 269(5611 ) 811-816), AXELROD VD et al (Nucl Acids Res ( 1978 ) 5( 10) 1^49-1561). and KRAME R FR and MILLS DR (Pioc Natl Acad Sci USA ( 1978) 75( 1 1 ) 5114-5118) Nucleic acid sequences can also be read by stimulating the natural i() tluoiesce of a cleaved nucleotide w ith a laser while the single nucleotide is contained in a fluoiescence enhancing matr ix (U S Pat # ^,674 743), In a mini sequencing reaction, a primer that anneals to taigct DNA adjacent to a SNP is extended by DNA polymerase w ith a single nucleotide that is complementary to the polymoiphic site This method is based on the high accuiacy of nucleotide incorporation by DNA poly merases There are diffeient technologies for analyzing the primer extension products For example, the use of labeled or unlabeled nucleotides, ddNTP combined w ith dNTP or only ddNTP in tht mini sequencing reaction depends on the method chosen for detecting the products,
S Piobes used in hybridization can include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids Hybπdization methods for the identification of single nucleotide polymorphisms or othei mutations involving a few nucleotides are desci ibed in the U S Pat 6.270,961 , 6,025, 1 16, and 6.872,530 Suitable hybridization probes for use in accoi dance w ith the invention include oligonucleotide^ and PNAs from about 10 to about 400 nucleotides, 10 alternatively from about 20 to about 200 nucleotides, or from about 10 to about 100 nucleotides in length
A template-diiected dye-terminator incoiporation w ith fluoiescent polarization-detection (TDI-FP) method is described by FREEMAN BD et al (J MoI Diagnostics (2002) 4(4) 209-215) for large I S scale screening.
Oligonucleotide ligation assay (OLA) is based on ligation of probe and detectoi oligonucleotides annealed to a polymerase chain ieaction amplicon strand w ith detection by an enzyme immunoassay (VILLAHERMOSA ML J Hum Vnol (2001 ) 4(5) 238-48, ROMPPANEN EL 20 Scand J Clin Lab Invest (2001 ) 61 (2) 123A IANNONE MA et al Cytometry (2000) 39(2) 131 - 40),
Ligation-Rolling Ciicle Amplification (L-RCA) has also been successfully used for genotyping single nucleotide polymoi phisms as desciibed in QI X et al Nucleic Acids Res (2001 ) 2s 29(22) E l 16,
5' nuclease assay has also been successfully used for genotyping single nucleotide polymoiphisms (AYDIN A et cil Biotechniques (2001 ) (4) 920-2. 924, 926-8 ),
M) Polymei ase proofieading methods are used to deteimine SNPs identities, as described in WO 0181631.
Detection of single base pair DNA mutations by enzyme-ampltfied electronic tiansduction is described in PATOLSKY F et al Nat Biotech (2001 ) 19(3) 253-257. Gene chip technologies are also known for single nucleotide polymorphism disci imination whereby numerous polymorphisms may be tested for simultaneously on a single array (EP 1 120646 and GILLES PN et al Nat Biotechnology ( 1999) 17(4) 365-70),
Matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy is also usef ul in the genotyping single nucleotide polymoi phisms through the analysis of miciosequencing products (HAFF LA and SMIRNOV IP Nucleic Acids Res ( 1997) 2"S( 18) 3749-5O, HAFF LA and SMIRNOV IP Genome Res ( 1997) 7 178-388, SUN X et al Nucleic Acids Res (2000) 28 I O c68, BRAUN A et al Clin Chem ( 1997) 43 1 151 - 1 1 58. LITTLE DP et al Eur J Clin Chem Clin Biochem ( 1997) 35 545-548, FEI Z et al Nucleic Acids Res (2000) 26 2827-2828. and BLONDAL T et al Nucleic Acids Res (2003) 31 (24) el 55)
Sequence-specific PCR methods have also been successfully used for genotyping single nucleotide I S polymorphisms (HAWKINS JR et al Hum Mutat (2002) 19(5) 543-553) Alternatively a Single- Stranded Confoi manorial Polymorphism (SSCP) assay or a Cleavase Fragment Length Polymoi phism (CFLP) assay may be used to detect mutations as desci ibed herein
Alternatively, if a subject's sequence data i> alieady known, then obtaining may involve retrieval 20 of the subjects nucleic acid sequence data ( foi example horn a database), followed by determining or detecting the identity of a nucleic acid oi genotype at a polymoiphic site by reading the subject's nucleic acid sequence at the one oi moie polymorphic sites
Once the identity of a polymoiphism(s) is determined or detected an indication may be obtained as 2s to subject iesponse to vasopiessin rcceptoi agonist administiation based on the genotype (the nucleotide at the position) of the polymorphism of interest In the present invention, polymorphisms in vasopressin pathway associated gene sequences, are used to pi edict a subject's iesponse to v asopiessin ieceptor agonist tieatment Methods foi predicting a subject's iesponst to vasopressin ieceptor agonist tieatment may be useful in making decisions regaiding the M) administration of vasopiessin ieceptor agonist
Methods of tieatment of an inflammatoiy condition in a subject hav ing an improved response genotype in a vasopressin pathway associated gene are desci ibed herein An improved response may include an improvement subsequent to administration of said therapeutic agent, whereby the subject has an increased likelihood of survival, reduced likelihood of organ damage or organ dysf unction (Brussels scoie), an improved APACHE II scoie, days alive and free of pressors, inotiopes. and ieduced systemic dysfunction (cardiovascular, lespnatory, \entilation, cential neivous system, coagulation [INR> 1 5|, renal and/or hepatic)
As described above genetic sequence information or genotype information may be obtained from a subject w herein the sequence information contains one or more polymorphic sites in a vasopressin pathway associated gene sequence Also, as prev iously desci ibed the sequence identity of one or more poly morphisms in a vasopressin pathway associated gene sequence of one or more subjects
10 may then be detected oi determined Furthermore, subject iesponse to administration of vasopressin ieceptor agonist may be assessed as described above Foi example, the APACHE II scoi ing system or the Brussels score may be used to assess a subject's iesponse to treatment by compai ing subject scores before and after treatment Once subject response has been assessed, subject response may be correlated w ith the sequence identity of one oi more polv moiphism(s)
I S The correlation of subject iesponse may fuither include statistical analysis of subject outcome scoics and poly moi phism(s) foi a number of subjects
Methods of treatment of an inflammatory condition in a subject hav ing one or moie of the t isk genotypes in AVP, AVPR l A LNPEP or LRAP (or a SNP in linkage disequihbi ium thereto)
20 associated w ith impioved iesponse to a theiapeutic agent are described heiein An improved iesponse may include an unpiovement subsequent to administiation of said therapeutic agent, w hereby the subject has an inci eased likelihood of suivival. reduced likelihood of oigan damage or oigan dysfunction (Brussels scoie). an impi oved APACHE II score, days alive and free of picssors, inotiopes. and reduced systemic dysfunction (cardiovasculai, respnatoiy, ventilation,
2S cential nei vous system, coagulation |INR> 1 5], ienal and/or hepatic)
λs desci ibed above genetic sequence infoi mation oi genotype information may be obtained fiom a subject w heiein the sequence information contains one or more single nucleotide polymorphic sites in AVP. AVPRl A LNPEP or LRAP sequences Also, as previously described the sequence M) identity ot one or more single nucleotide polymoiphisms in the AVP, AVPRl A oi LNPEP sequences ot one or moie subjects may then be detected or detei mined Fuithermoie, subject outcome or piognosis may be assessed as described above, foi example the APACHE II scoring system or the Brussels scoie may be used to assess subject outcome or prognosis by compai ing sub|ect scoi es before and after treatment Once subject outcome or prognosis has been assessed, subject outcome or prognosis may be correlated w ith the sequence identity of one or more single nucleotide polymorphism(s) The correlation of subject outcome or prognosis may further include statistical analysis of subject outcome scores and polymorphism(s) for a number of subjects
s 3. Analytical Methods Patient Cohort Selection
a. Intensive Care Unit (ICU) Cohort Inclusion Criteria
All subjects admitted to the ICU of St Paul's Hospital (SPH) weie screened for study inclusion 10 SPH ICU is a mixed medical-surgical ICU in a tertiary care, university-affiliated teaching hospital Subjects weie included in the study if they met at least two out of four SIRS criteria 1 ) fever ( > 18 C) or hypothermia (<16 1 C). 2) tachycardia (>90 beats/minute), 1) tachypnea (>20 breaths/minute), PaCOi < M mm Hg, or need tor mechanical ventilation, and 4) leukocytosis (total leukocyte count > 12,000 mm") or leukopenia (< 4.000 mm1) Subjects weie included in the I s analysis il they met the diagnostic ci iteπa loi septic shock (sepsis and caidiovascular dysfunction (as defined by Biussels scoring system) and one other otgan dysfunction) on admission to the ICU Subjects weie excluded if blood could not be obtained for genotype analysis Baseline chaiacteπstics (age. gender, admission APACHE II score ( KNAUS WA et til Cπt Care Med ( 198*1) I l 818-829), togethei w ith medical vs suigical diagnosis KNAUS WA et al Chest ( l c>91 ) 20 100 1619- 1636 ) weie iecoided on admission to the ICU The full cohort meeting these ciiteπa included 1072 Caucasian subjects and 1 Η Asian subjects
The Institutional Rev iew Board at Piov idence Health Caie and the University of British Columbia appiov ed this study 2s b. Biological Plausibility (BP) Cohort Inclusion Criteria
An independent cohort of Caucasian subjects (N = 102) scheduled foi first time elective coronary artery bypass grafting that lequired cardiopulmonary bypass is iefened to as the "Biological Plausibility" (BP) cohort Significant SNP-biomarker associations identified in this cohort may M) piovide insight into biological processes undeilying SNP-phenotype associations observed in the ICU cohort or subsets of the ICU cohort
Foi the BP cohort, individuals weie included in the analysis if they weie met diagnostic criteria for sy stemic inflammatory iesponse syndrome (SIRS) Subjects were excluded fiom the study if they had undergone 1 ) urgent or emergency cardiopulmonary bypass surgery or 2) valve or repeat cardiac suigery Subjects w ith urgent or emergency cardiopulmonary bypass surgery were excluded because they may have had an inflammatory iesponse due to other triggers (i e shock) Subjects w ith valve surgery or repeat surgt iy weie excluded because they could have had different S pre-operative pathophysiology or longer total surgical and cardiopulmonary bypass time than subjects having elective cardiopulmonary bypass surgery
The Institutional Review Boaid at Providence Health Care and the University of Bi itish Columbia approved this study 10
Clinical Phenotype
The pnmaiy outcome v ariable evaluated in this study was 28-day mortality Various organ dysf unctions were considered as secondary outcome variables Baseline demographics iecordtd were age, gendei , admission APACHE II scoie (KNAUS WA c t al Crit Care Med ( 198S) 1 1 818- I s 829) and medical or suigical diagnosis on admission to the ICU (based on the APACHE III diagnostic codes) (KNAUS WA et al Chest ( 1991 ) 100 1619- 1616) (TABLE 2B)
T4BLE 2B Baseline chaiacteiistics key
Figure imgf000114_0001
20 Altei meeting the inclusion cπteπa. data were tecoided for each 24-houi peuod (8 am to 8 am) tor 28-days after ICU admission or until hospital discharge to evaluate organ dysfunction, the intei sity of SIRS (Systemic Inflammatory Response Syndiome) and sepsis Raw clinical and laboiatoiy vaπables weie iecoided using the wotst or nost abnormal \auable for each 24-hour period w ith the exception of Glasgow Coma Scoie, for w hich the best possible scoie for each 24-houi period
IS was iecoided Missing data on the date of admission was assigned a normal value and missing data after day one was substituted by caitying forward the value from the previous day When data collection for each patient was complere, all patient identifiers were lemoved from all iecords and the patient file was assigned a unique iandom number linked w ith the blood samples The completed raw data file was used to calcuUte descriptive and seventy of illness scores using standard definitions as described below
Organ dysfunction was first evaluated at baseline and then daily using the Brussels score s (SIBBALD WJ and VINCENT JL Chest 1995) 107(2) 522-7) (see TABLE 2A in General
Methods Section) If the Brussels score was moderate, severe, or extreme dysfunction then organ dysfunction was recoided as present on that day To correct for deaths during the observation penod, we calculated the days alive and fit e of organ dysfunction (RUSSELL JA et αl Crit Care Med (2000) 28( 10) 3405-1 1 and BERNARD GR et αl Chest ( 1997) 1 12( 1 ) 164 72) (TABLE
10 2C) Foi example, the seventy of cardiovascular dysfunction was assessed by measuring days alive and fiee ot caidiovasculai dysfunction ovei a 28-day observation period Days alive and free of caidiovascular dysfunction was calculated as the numbei of days after inclusion that a patient was alive and fiee of cardiovascular dysfunction over 28 days Thus, a lowei score for days alive and fiee of caidiovascular dysfunction indicates moie caidiov ascular dysfunction The ieason that
I ^ days alive and hee of caidiov asculai dysfunction is pieferable to simply piesence or absence oF caidiov ascular dysfunction is that seveie sepsis has a high acute moitahty so that early death (w ithin 28 days) precludes calculation of the piesence or absence of cardiov ascular dysfunction in dead subjects Oigan dysfunction has been evaluated in this way in observ ational studies (Russell J A t t αl Cut Care Med (2000) 28( 10) 3405 1 1 ) and in landomized controlled trials of new
20 theiapy in sepsis, acute iespiratory distress syndiome (BERNARD GR et αl N Engl J Med ( 1 C(97) 336( H) 912-8) and in ciitical care (HEBERT PC α αl N Engl J Med ( 1999) 340(6) 409-17)
To furthei evaluate cardiovascular, respiratory, and ienal function we also recorded, dunng each 24 hour penod. vasopressor support, mechanical ventilation, and renal support, lespectively
IS Vasopressoi use was defined as dopamine > 5 μg/kg/min or any dose of norepinephrine, epinephrine, v asopressin, oi phenylephrine Mechanical ventilation was defined as need for intubation and positive anway pressure (i e T- piece and mask ventilation were not considered ventilation) Renal support was defined as hemodialysis pei itoneal dialysis, or any continuous renal support mode (e g continuous veno-venous hemodialysis)
M)
As a cumulative measuie of the severity of SIRS the presence of two, three or foui of the SIRS ciitei ia was scoied each day over the 28-da/ obseivation penod SIRS was considered present w hen subjects met at least two of tour SIRS cntei ia The SIRS cπtena weie 1 ) fever (>38 0C) or hypothermia (<36 'C) 2) tachycaidia (>90 beats/min in the absence of beta-blockei s, 3 ) tachypnea 20 breaths/min) or need for mechanical ventilation, and 4) leukocytosis (total leukocyte count > ,000/μL or <4,000/μL).
TABLE 2C. Primary and secondary outcome variables foi the ICU cohort and subsets
Figure imgf000117_0001
Baseline characteπstics foi the Biological Plausibility cohoit included age in years. 7c males Tsmoket s, rf diabetes, c/c hypertension, ejection fraction, bypass time, clamp time and aprotinin Outcome vai tables measured in the Biological Plausibility cohoit included Granulocyte colony stimulating factor (GCSF), Interleukin 10 (ILl O), Intel leukin receptoi I a (IL I ra), Inteileukin 6 (IL6), Intel leukin 8 (IL8) and Monocyte Chemoattiactant Protein 1 (MCPl ) A key foi the vanables evaluated in the Biological Plausibility cohort is provided in TABLE 2D
TABLE 2D. Biological plausibility key
Figure imgf000117_0002
15
Figure imgf000118_0001
Selection of SNPs for Genotyping
Publicly available genotype data was queried from the International HapMap Project t vv vv w hapmap org) and Perlegen Sciences Inc (www perlegen com) to select a set of tag SNF's
S (tSNPs) in the LNPEP, AVP and AVPR l A regions each hav ing a minor allele frequency (MAF) greater than 0 05 These tSNPs were chosen using several statistical methods, including pairw ise linkage disequilibrium (LD) measures (DEVLIN B and RISCH N Genomics ( 1995) 29 31 1 -322). haplotype (STEPHENS M et al Am J Hum Genet (2001 ) 68 978-989. and EXCOFFIER L and SLATKIN M MoI Biol Evol ( 1995) 12(3) 921 -927) and haplotype block (HAWLEY ME and
IO KIDD KK J Heredity ( 1995) 86 409-41 1 ) patterns, as well as phylogenetic (cladistic) distance metπcs (HAWLEY ME and KIDD KK ( 1995)) When these methods did not yield a paisimonious conclusion, as was the case for AVP, SNPs closest in physical distance to the given gene ot interest weie selected Each polymorphism was genotyped in the ICU Cohort and the Biological Plausibility Cohort i s
Sample Analysis
Sample Piepaiation
Discji ded w hole blood samples, stoied at 40C, were collected fiom the hospital laboratory DNA was extracted fiom buffy coat using the Ql λamp DNA Midi kit (Qiagen, Mississauga. ON,
20 Canada) After extraction, the DNA samples were transfeπed to 1 5 πiL cr>otubes, bar coded and ci oss-i eferenced w ith a unique patient numbei and stored at -8O0C
\BI Genotyping
Single nucleotide polymoiphisms in AVP. LNPEP and AVPR l \ weie genotyped using the 5' 21S nuclease. Taqman™ (Applied Biosystems, Fostei City, CA) polymerase chain reaction (PCR) method TABLE 2E prov ides a complete list of the I O SNPs genotyped for this study
TABLE 2E: List of tSNPs genotyped in ICU and Biological Plausibility Cohorts
16
Figure imgf000119_0001
Illumina Genotyping
Single nucleotide polymoiphisms in AVP, LNPEP and AVPRl A were genotyped using the Illumina Golden GateIM assay from 250 ng of DNA extracted from buffy coat A list of these 1S SNPs can be found labeled as cohort T in TABLE I B found in the General Methods section
Sequencing of LNPEP region
Sequencing of a 157 1 kb iegion including the LNPEP and LRAP genes was undertaken using DNA extracted from six CEPH (i e , Centre d'Etudes du Polymoiphisme Humain) individuals 10 obtained thiough the Coπell Institute for Medical Research using the Applied Biosystems 3730 platfoim Ascertained polymoi phisms weie investigated for NCBI is Id annotation using the LJCSC genome biowser (http //genome iii sc edu ) If a polymorphism was found to not have an is Id assigned, it was given a numeric id prefixed by 'sinus' (i e siπusx)
Linkage Disequilibrium Analysis
Included in this patent are SNPs found to be associated w ith 28-day surv ival or response to vasopiessin as well as SNPs detei mined to be in LD w ith the former LD SNPs weie ascertained using either Haploview (BARRETT JC et til Bioinformatics (2005) 21 (2) 263-5 ( hup //w w w bioad mit edu/mpg/haplov iew ')) oi the LD function in the Genetics Package in R (R 0 Core Development Group. 2005 - R Development Core Team (w w w R-piojei t oi g) A R2 thieshold of 0 5 was lequned in oider that a SNP be considered in LD w ith those claimed herein All LD SNPs are shown in table 1 B
The AVP, AVPR l A. LNPEP and LRAP genes are central to the action of vasopressin given that \ asopiessin induces vasoconstnction by signaling through the AVPR l A ieceptor and that vasopiessin activ ity is inhibited when cleaved by LNPEP Similar protein homology between LNPEP and LRAP suggest that these two genes aiose through an ancient gene duplication event (DANCHIN E et til , Immunol Re\ (2004) 198 216-332) This homology and the observation oϊ an extended linkage disequilibrium (LD) block throughout the LRAP and LNPEP region (HapMap
M) Phase II data, www hapmap org) supports the inclusion of LRAP in the vasopiessin pathway Furthermore, vai lability in response to infused (/ e , administered) \asopressin most likely occurs as a result of polymorphisms in the AVP. AVPRl A. LNPEP and LRAP genes because the proteins that these genes encode aie central to the actions of native and infused vasopressin (AVP)
s Statistical Analysis
A description of the statistical analysis used is piovided for each example in the follow ing sections
I O EXAMPLES
EXAMPLE 1: RESPONSE TO VASOPRESSIN IN SEPTIC SHOCK
METHODS
I S Cohort Selection
To investigate whether genotype predicts iesponse to v asopressin, a subset of Caucasian subjec ts w ith septic shock and treated w ith vasopressin (N = ICH) were compaied to a contiol group of Caucasian subjects w ith septic shock who had not been admiπisteied vasopressin (N = 101) Vasopressin-treated and control subjects w jre matched based on age, gendei, admission APACHE 20 II scoie, medical veisus suigical diagnosis and days alive and fiee of 1 of 4 systematic inflammatoty iesponse syndrome (SIRS) cπteπa The baseline characteristics o! these groups are presented in Table 1 1
TABLE 3.1 is Baseline characteristics oi cases (Caucasian ICU septic shock subjects treated w ith vasopressin) and contiols (Caucasian ICU subjects w ith septic shock, matched (see text for details) and not treated w ith v asopiessin) For age and APACHE II scoie. data is given as 25th peicentile | median 7^'h ercentile For all other variables, data is iven as Vc (N /N total) N, number of subjects
Figure imgf000120_0001
K) Data Analysis
All data analysis was earned out using statistical packages available in R (R Coie Development Group. 2005 R Development Core Team (www R-pioject or.g) R A language and envπonment for statistical computing Vienna, Austria 2005) Chi-square and Kruskal-Wallis ( KW) test statistics were used in conjunction w ith Cox proportional hazards (CPH) regression to identify significant SNP-phenotype associations, as well as to identify significantly different baseline characteristics (age. gender, admitting APACHE II score, and medical vs surgical admitting 1S diagnosis) requiring post-hoc, multivariate adjustment The contiol population was selected by matching, using the Matchlt package in R, by age, gender. APACHE II scoie, medical v s surg ical diagnosis, and days aliv e and free of 3 of 4 SIRS ci itcπa There were no differences in baseline characteristics between vasopressin-treated cases and controls
I O Using 28-day survival as the outcome v aπable and a chi-squared test of significance, SNP- phenotype compai isons were undertaken w ithin and between treatment groups We considered a by-genot\ pe effect to be significant when tw o ci iteria were fulfilled First, we expected an increase in 28-day surv ival foi vasopressin-treated subjects compaied to contiols Second, we l equπed a p-value < 0 1 for this difference in 28-day survival When both criteria were met, we
15 consideied the allele oi genotype piedictin? increased 28-day sui v iv al w ith v asopressin treatment to be an "improv ed iesponse genotype"' ( IF G ) Only IRG polymoiphisms were evaluated for oigan dysfunction results and weie compaied between v asopressin-tieated subjects and matched contiols using a Kruskal-Walhs test
20 Results
1.1 Leucyl/Cystinyl Aminopeptidase (LNPEP)
1.1.1 \dverse Response to Vasopressin Treatment of subjects who have the CC Genolype of LNPEP rs 18059 and Improved Response to Vasopressin Treatment of subjects 25 who have the TT Genotype of LNPEP rsl8059
it was unknown w hether SNPs w ithin the L NPEP gene and those iegions immediately upstieam and dow nstieam would be associated w ith I he response to v asopressin It w as found that LNPEP i s l 80S9 can be used to predict response (28-day sui v ival ) to vasopressin in subjects w ith septic M) shock Of 101 v asopressin-treated and 103 matched-control subjects w ith septic shock, 73 and 81 weie lespectively genotyped tor LNPEP rs 18059 Baseline characteristics for subjects w ith genotypes are shown in Table 3 2 and Table 3 3
TABLE 3.2 Baseline characteristics of a group or vasopressin-treated Caucasian septic-shock subjects by genotype of leucyl/cystinyl aminopeptidass (LNPEP) rsl 8059 For age and APACHE II score, data is given as 25th percentile | median | 75lh percentile For all other variables, data is given as 7c
Figure imgf000122_0001
Figure imgf000122_0003
TABLE 3.3
Baseline characteristics of a vasopressin untreated matched control group of Caucasian ICU septic shock subjects by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs 18059 For age and APACHE II score, data is given as 25th percentile | median | 75th percentile For all other variables,
10 data is iven as 7c (N /N total) N. number of sub ects
Figure imgf000122_0004
Table 3 4 and Table 3 5 show 28-day sui vival and oigan dysfunction data by LNPEP is l 8059 genotype for vasopiessin-treated and contiol subjects respectively Table 1 6 show s the dif ieiences in suiv ival and measures of oigan dysfunction between by LNPEP is 18059 genotype
I > between vasopiessin-tieated and contiol subjects
In geneial. Table λ 6 show s that vasopressin-treated subjects w ith LNPEP i s 18059 CC had lower surv ival and moie organ dysfunction than contiols as evidenced by negative values for the LNPEP i si 8059 CC subjects in the DELTA column In contiast, vasopressin-treated subjects w ith the LNPEP rs 18059 TT genotype had inci eased suiv ival and lmpioved oigan f unction (shown by 0 gieatei DAF) compared to controls as demonsti ated by the geneially positive values in DELTA column Theie was a small increase in survival of subjects w ith the LNPEP is l 8059 CT genotype in vasopiessin-tieated subjects ( 16 Vc ) compared to controls (28 7c)
TABLE 3.4
25S A iesponse association of leucyl/cystinyl aminopeptidase (LNPEP) rs 18059 in a group of
Caucasian ICU septic shock subjects treated w ith vasopressin For 28-day surv ival, data is given data
Figure imgf000122_0005
Figure imgf000122_0002
Figure imgf000123_0002
TABLE 3.5
A iesponse association of leucyl/cystinyl ammopeptidase (LNPEP) i s 18059 in a matched control gioup of Caucasian ICU septic shock subjects not treated w ith vasopressin For 28-day survival, 28-day
Figure imgf000123_0001
Figure imgf000123_0003
Figure imgf000124_0001
TABLE 3.6
Difference in response association of leucv/1/cystinyl aminopeptidase (LNPEP) rsl 8059 between cases (vasopressin-tieated group) (Treat) and controls (vasopressin untreated matched contiol) (Cont) ot Caucasian ICU subjects diagnosed w ith septic shock For all variables besides 28-day suivival, data is presented as medians For 28-day survival, data is presented as Vc(N surv ived / N
Figure imgf000124_0002
A logistic regression approach was used to test for a statistically significant interaction between genotype and vasopicssin use as predicted by 28-day survival TABLE 3.7 shows that theie is a statistically significant interaction between LNPEP i s 180^9 genotype, vasopressin treatment and survival (P = 0 0191 ). confiiming that treatment w ith vasopressin decieases 28-day sui v ival in LNPEP I s I SO1SQ CC subjects In contiast. 28-day surv ival for vasopressin-trcated subjects w ith the LNPEP i s 18059 TT genotype is impioved compared w ith controls Follow ing adjustment for aste. admission APACHE U scoie, gendei . medical, suigical diagnosis and 1 ot 4 systematic inflammatory response syndrome (SERS) criteria, there was still a statistically significant interaction of the LNPEP rs 18059 genotype, tieatment w ith vasopressin and survival (P = 0 0555)
TABLE 3.7
Interaction between vasopressin use vs no vasopressin use (controls) and CC or CT genotype Λ
Figure imgf000125_0001
1.1.2 Adverse Response to Vasopressin 1 reatment of Subjects Who Have the AA Genotype I O of LNPEP rs27711 and Improved Response to Vasopressin Treatment of Subjects Who Have the GG Genotvpe of LNPEP rs27711
It was unknown w hether SNPs w ithin the I NPEP gene and those iegions immediately upstream and downstieam aie associated w ith the ie^ponse to vasopiessin It was found that LNPEP
I s i s2771 1 can be used to pi edict iesponse to vasopressin in subjects w ith septic shock using 28-day surviv al and measures of organ dysfunction as outcome vanables Of 103 vasopressin-treated and 103 matched-contiol subjects w ith septic shock. 70 and 81 weie iespectively genotyped tor LNPEP rs2771 1 Baseline characteristics for subjects w ith genotypes are show n in Table 3 8 and Table λ 9 LNPEP i s2771 1 is in linkage
Figure imgf000125_0002
w ith, for example. LNPEP rs 18059 and
20 LNPEP ιs lOO51617. which were also genotyped in this cohort
TABLE 3.8
Baseline characteristics ot vasopressin-tiea ed Caucasian septic-shock subjects by LNPEP i s2771 1 genotype For age and APACHE II scoie. data is given as 25lh percentile | median | 75* percenlile For all other variables, data is iven as Vc (N /N total) N. number ot sub ects
Figure imgf000125_0003
TABI.E 3.9
Baseline chaiacteπstics ot a group of Caucasian septic-shock contiol subjects by LNPEP rs277 1 1 genotype For age and ΛPACHE II score, data is given as 25th percentile | median | 75'h percentile
M) For all other variables, data is iven as c/c (N /N total) N. number of sub ects
Figure imgf000125_0004
Figure imgf000126_0002
Tables 3 10, 1 1 1 and 3 12 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for LNPEP rs2771 1 In general, vasopressin-treated subjects w ith the LNPEP i s2771 1 AA genotype had a dramatically decreased surv ival (43 r/f ) compared to controls (60 ΓA ) as demonstrated by the negative \ alues in the LNPEP rs2771 1 AA DELTA column in Table 1 12 In geneial, vasopressin-treated subjects w ith the LNPEP rs2771 1 AA genotype also had increased organ dysfunction as demonstrated by fewer DAF of organ dysfunction compaied w ith control In contrast, vasopiessin -treated subjects w ith the LNPEP is2771 1 GG genotype had an increased survival ( 33 r/c) compaied to contiols ( 19 ck ) as demonstrated by the positive values in the LNPEP rs2771 1 GG DELTA column in Table 3 12
TABLE 3.10
A response association of leucv, l/cystinyl aminopeptidase (LNPEP) is2771 1 in a group of Caucasian ICU septic shock subjects who v ere tieated w ith vasopiessin For all variables besides 28-day suiv lval, data is given as 25lh percentile [ median | VSth percentile For 28-day sin vival. data is iven as CA (N survived / N total) N, number of subjects
Figure imgf000126_0001
\N\ HEP DAF I 7 24 1 14 | 24 75 4 28 9 24 75 F=O 77 d t =2 67 P=O 466
TABLE 3.11
A iesponse association of leucyl/cystinyl aminopeptidase (LNPEP) ι s2771 1 in a matched control group of Caucasian ICU septic shock subjects who were not treated w ith vasopressin For all vaπables besides 28-day survival, data is given as 25lh percentile | median | 75lh percentile For 28- da survival, data is iven as Vc (N survived / N total ) N. number of sub ects
Figure imgf000127_0001
TABLE 3.12
Diffeicnce in response association of leucyl/cystinyl aminopeptidase (LNPEP) rs2771 1 between cases (vasopiessin-tieated group) (Treat) and controls (vasopressin untreated matched control) (Cont) of Caucasian ICU subjects diagnosed w ith septic shock For all variables besides 28-day surv ival, data is presented as medians For 28-day suiv ival, data is presented as Vc(N survived / N total) N. number of subjects
Figure imgf000127_0002
Figure imgf000128_0001
1.1.3 Adverse Response to Vasopressin Treatment of subjects who have the GG Genotype of LNPEP rsl0051637
It was unknown whether SNPs w ithin the LNPEP gene and those legions immediately upstream and downstream aie associated w ith the response to vasopiessin It was found that LNPEP rs 10051617 can be used to pi edict response to \asoptessin in subjects w ith septic shock using 28- day suiviv al and measures of organ dysfunction as outcome variables Of 103 vasopressin-treated and 103 matched-control subjects w ith septic shock, 72 and 81 were respectively genotyped toi LNPEP rs 10051637 Baseline character istics for subjects w ith genotypes are show n in Table 3 1 3 and Table 3 14 LNPEP rs 10051637 is in linkage disequilibrium w ith, foi example LNPEP isl 8059 and LNPEP G9419812A. which were also genotyped in this cohoit.
Figure imgf000128_0002
TABLE 3.14
Baseline characteristics of a matched-control group of Caucasian septic-shock subjects by leucy 1/cyst my 1 aminopeptidase (LNPEP) is 10051637 genotype For age and APACHE II score. data is given as 25lh percentile | median | 75th percentile For all other variables, data is given is 9c (N /N total) N, number of sub ects
Figure imgf000129_0002
Tables 3.15, 3.16 and Tables 3.17 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for LNPEP rsl0051637. Vasopressin-treated subjects w ith the LNPEP i s i 0051637 GG genotype had a dramatically decreased survival (46 9c ) compared to controls (60 9c ) as demonstrated by the negative values in the LNPEP ts lOO51637 GG DELTA column in Table 3 17 Vasopressin-treated subjects w ith the LNPEP rs 10051637 GG genotype were also observed to have more organ dysfunction as demonstrated by fewer DAF of organ dysfunction In contiast. vasopressin-treated subjects w ith the LNPEP i s i 0051637 AG and AA genotypes had incieased survival (26 9c) compaied to controls (20 9c )
TABLE 3.15
A iesponse association of leucyl/cystinyl aminopeptidase (LNPEP) rs 10051637 and use of vasopiessin in a group of vasopressin-treated Caucasian ICU septic-shock subjects For all variables besides 28-day survival, data is given as 25 1 percentile | median 75 ' percentile Foi 28 day suiv iv al. data is given as ck (N suivived / N total) N. number of subjects
Figure imgf000129_0001
Figure imgf000130_0001
TABLE 3.16
A response association of leucyl/cystinyl ciminopeptidase (LNPEP) rs 100516^7 and use of vasopressin in a matehed control group of Caucasian ICU septic shock subjects who were not treated w ith vasopressin For all variables besides 28-day survival, data is given as 25th percentile | median | 75lh percentile For 28-day survival, data is given as % (N survived / N total) N, number
Figure imgf000130_0002
TABLE 3.17 Difference in iesponse association of leucyl/cystinyl aminopeptidase (LNPEP) i s 10051637 and use of vasopiessin between cases (vasopressin-treated group) and controls (vasopressin untieated
Figure imgf000130_0003
28
Figure imgf000131_0001
1.2 Arginine Vasopressin (AVP)
1.2.1 Improved Response to Vasopressin Treatment of subjects who have the AA or AC
Genotype of AVP rsl4107l3
It is unknown whether SNPs within the AVP gene and those regions immediately upstream and downstream are associated with the response to vasopressin. AVP rsl 410713 can be used to predict response to vasopressin in subjects with septic shock using 28-day survival and measures of organ dysfunction as outcome variables. Of 103 vasopressin-treated and 103 matched-control subjects with septic shock, 72 and 81 were respectively genotyped for AVP rs 1410713. Baseline
I O characteristics for subjects with genotypes are shown in Table 3.18 and Table 3.19.
TABLE 3.18
Baseline characteristics of a group of vasopressin-treated Caucasian septic-shock subjects by arginine vasopressin (AVP) rs 1410713 genotype. For age and APACHE II score, data is given as
1 ? 25th percentile | median | 75th percentile. For all other variables, data is given as % (N /N total). N = number of subjects.
Figure imgf000131_0002
TABLE 3.19
Baseline characteristics of a group of Caucasian septic-shock control subjects by arginine
20 vasopressin (AVP) rs 1410713 genotype. For age and APACHE II score, data is given as 25th percentile | median | 75th percentile. For all other variables, data is given as % (N /N total). N. number of subjects.
CONTROL AA AC CC Combined Test
Figure imgf000132_0001
Tables 3.20. 3.21 and 3.22 contain 28-day survival and organ dysfunction data for septic- shock subjects genoty ped for AVP rsl410713 Vasopressin -treated subjects w ith the AVP 1S14107 H AA genotype had a dramatically incieased survival (38 c/c ) compared to controls (0 7c) as demonstrated by the positive values in the AVP rs 1410713 AA DELTA column in Table 3 22 Furtheimore, v asopressin-treated subjects w ith the AVP rs 14107 H AA genotype weie observed to have less organ dysfunction as demonstrated by more DAF of organ dyst unction Vasopressin- treated subjects w ith AVP rs 1410713 AC genotype were also observed to have increased 28-day suiv ival (477r ) compared w ith that of control subjects (37%)
TABLE 3.20
A response association of arginine vasopressin (AVP) i s 1410713 in a group of Caucasian ICU septic shock subjects who weie treated w ith vasopressin For all vanables besides 28-day survival, data is given as 25th percentile | median | 7'i"1 percentile For 28-day survival, data is given as (f
Figure imgf000132_0002
TABLE 3.21 A iesponse association of arginine vasopressin (AVP) rs 1410713 in a matched control group of Caucasian ICU septic shock subjects who were not treated with vasopressin For
Figure imgf000133_0001
TABLE 3.22
Difteiencc in response association of arginine vasopiessin (AVP) isl410713 between cases (vasopressm-treated group) (Treat) and controls (vasopressin untreated matched contiol) (Cont) of Caucasian ICU subjects diagnosed with septic shock For all variables besides 28-day suivival data is piesented as medians For 28-day suivival, data is presented as %(N survived / N total) N, number ot subjects
Figure imgf000133_0002
Figure imgf000134_0001
1.2.2 Adverse Response to Vasopressin Treatment of subjects who have the CT Genotype of AVP rs857240 and Improved Response to Vasopressin Treatment of subjects who have the CC Genoty pe of AVP rs857240
It w as unknow n whether SNPs w ithin the AVP gene and those regions immediately upstream and downstream are associated w ith the response to vasopressin It was found that AVP rs857240 can be used to piedict response to vasopressin in subjects w ith septic shock using 28-day survival and measures of organ dysfunction as respective primary and secondary outcome variables Of 10"' vasopiessm-tieated and 103 matched-control subjects w ith septic shock, 73 and 83 were lespectively genotyped for LNPEP i s857240 Baseline characteristics for subjects w ith genotypes
K) are shown in Table 3 23 and Table 3 24
TABLE 3.23
Baseline characteristics of a group of \asopressin-treated Caucasian septic shock subjects by aigmine vasopressin (AVP) rs857240 genolype. For age and APACHE II scoie, data is given as 25lh percentile | median | 75lh percentile For all other variables, data is given as c/c (N /N total) N, number of subjects
Figure imgf000134_0002
TABLE 3.24
Baseline charactei istics of Caucasian septic shock control subjects by arginine vasopressin (AVP)
20 ι s857240 genotype For age and APACHE II score, data is given as 25th percentile | median | 7?th peicentile For all other variables, data is given as % (N /N total) N, number of subjects
C ONTROL C C C T Combined Test
( N =69) ( \= I4) ( N=XI) SutisUc
AGF 44 I 33 68 i6 75 I 5 5 71 44 56 I 7 1 5 F=O 12 d t = 1 H l P=O 7
Figure imgf000135_0002
Tables 1 25, 1 26 and 1 27 contain 28-day survival and oigan dysfunction data for septic shock subjects genotyped for AVP is857240 Vasopiessin -treated subjects w ith the AVP rs857240 CT genotype had dramatically decreased survival if vasopressin-treated (29 c/c) compaied to contiols (41 c/c) as demonstrated by the negative values in the AVP is857240 CT DELTA column in Table 1 27 Furthei moie. vasopiessin-treated subjects w ith the AVP rs857240 CT genotype were observed to have moie organ dysfunction than AVP rs857240 CT control subjects as demonstrated by more DAF of organ dysfunction In contrast, vasopressin-treated subjects w ith the AVP rs857240 CC genotype had increased surv ival (41 c/c ) compared to controls ( 10 c/c ) as
K) demonstrated by the positive values in the AVP rs857240 CC DELTA column in Table 1 27 Fuitheimoie, vasopiessin-tieated subjects WP rs857240 CC subjects were obseived to have less organ dysfunction than AVP rs857240 CC control subjects
TABLE 3.25 15S A iesponse association of arginine vasopressin (AVP) i s857240 in a group of Caucasian ICU septic shock subjects w ho were treated w ith v asopressin For all v ariables besides 28-day surv ival. data is given as 25th percentile | median | 7s>lh peicentile For 28-day sui v ival, data is given as r'c
Figure imgf000135_0001
AW HbP DAF 2 j I l 3 27 23 1 1 1 5 I 2 9 J 25 F= I 7 d t =l 71 P=O 197
TABLE 3.26
A response association of arginine vasopiessin (AVP) rs857240 a matched control group of Caucasian ICU septic shock subjects who were not treated w ith vasopressin For all variables besides 28-day survival, data is given as 25th percentile | median | 75th percentile For 28-day survival, data is given as Vc (N survived / N total) N, number of subjects Note TT genotype frequency = 0
Figure imgf000136_0001
TABLE 3.27
Difference in iesponse association ol arginine vasopressin (AVP) rs857240 between cases (vasopiessin tieated gioup) (Tieat) and controls (vasopressin untreated matched control) (Cont) of Caucasian ICU subjects diagnosed w ith septic shock For all variables besides 28-day suiv ival, data is piesented as medians For 28-day sui v lval. data is presented as vT(N suiv i\ed / N total) N, number of subjects Note TT enoty e he uencv, = 0
Figure imgf000136_0002
Figure imgf000137_0001
1.2.3 Response to Vasopressin Treatment of subjects who have the AC Genotype of AVP rs857242 and Improved Response fo Vasopressin Treatment of subjects who have the CC Genotype of AVP rs857242
It was unknown whether SNPs w ithin the AVP gene and those regions immediately upstream and downstream are associated w ith the iesponse to \ asoριessin It was found that λVP rs857242 can be used to pi edict iesponse to vasopressin in subjects w ith septic shock using 28-day suiv ival and measures ot organ dysfunction as lespettive primary and secondary outcome variables Of 101 vasopressin-tieated and 1(B matched-contiol subjects w ith septic shock, 75 and 81 weie lespectively genotyped for AVP ιs857242 Baseline characteristics for subjects w ith genotype*- ate shown in Table 3 28 and Table 3 29
TABLE 3.28
Baseline chaiacteristics of a gioup of vasopressin-tieated Caucasian ICU septic shock subjects by genotype ot aiginine vasopressin (AVP) is 857242 Foi age and APACHE II score, data is given as 2?"' peicentile | median | 75th percentile Foi all other vai tables, data is given as Vf (N /N total) N, number ot sub ects
Figure imgf000137_0002
TABLE 3.29
Baseline characteristics of a vasopressin untieated matched control group of Caucasian ICU sepi ic shock subjects by genotype of arginine vasopressin (AVP) rs 857242 For age and APACHE II score, data is given as 251'1 percentile | median | 75th peicentile For all othei variables, data is given as c/c (N /N total) N, number of subjects
Figure imgf000138_0002
Tables 3.30, 3.31 and 3.32 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for AVP rs857242 Vasopressin -treated subjects w ith the AVP rs857242 AC genotype had a dramatically decieased surv ival (38 7c ) compared to controls (54 7c ) as
S demonstrated by the negative values in the AVP rs857242 AC DELTA column in Table 3.32
Furthermoie, vasopressin-treated subjects w ith the AVP rs857242 AC genotype were observed to have more organ dysfunction as demonstrated by more DAF of organ dysfunction. In contrast, v asopressm-treated subjects w ith the AVP rs857242 CC genotype were observed to have increased surv ival (419r) compared w ith controls (307c ). As well, vasopressin-treated subjects w ith AVP
K) rs857242 CC genotype were obseived to have increased 28-day surv ival (47%) compared w ith that of control subjects 017c ) as demonstrated by the positive values in the AVP rs857242 CC DELTA column in Table 3 32 Furthermore, vasopressin-treated subjects w ith the AVP rs857242 CC genotype were observed to have less organ dysfunction as demonstrated by more DAF of organ dysfunction
15
TABLE 3.30
A iesponse association of arginine v asopressin (AVP) rs857242 in a group of Caucasian ICU septic shock subjects who were treated w ith vasopressin. For all variables besides 28-day sui v i val, giv en as 7c 20
Figure imgf000138_0001
Figure imgf000138_0003
Figure imgf000139_0001
TABLE 3.31
A response association of arginine vasopiessin (AVP) rs857242 in Caucasian septic-shock control subjects For all variables besides 28-day survival, data is given as 25th percentile | median | 75th percentile For 28-day survival, data is given as % (N surv ived / N total) N, number of subjects =
Figure imgf000139_0002
TABLE 3.32
Dif feience in iesponse association of ai gin me vasopressin (AVP) is857242 between cases (v asopressin-treated group) (Treat) and controls (vasopressin untreated matched control) (Cont) of Caucasian ICU subjects diagnosed w ith septic shock. For all variables besides 28-day survival, data is presented as medians For 28-day survival, data is presented as 9c(N survived / N total) N, number of sub ects Note' AA enot e fre uenc - 0
Figure imgf000139_0003
Figure imgf000140_0001
1.3 Arginine Vasopressin Receptor Ia (AVPRlA)
1.3.1 Adverse Response to Vasopressin Treatment of subjects who have the TT Genotype of AVPRlA rsl495027 and Improved Response to Vasopressin Treatment of subjects who have S the CC Genotype of AVPR 1 A rs 1495027
It was unknown whethei SNPs w ithin the AVPR l A gene and those iegions immediately upstream and downstream aie associated w ith the ie^ponse to vasopressin It w as found that AVPRl A isl495027 can be used to piedict response to vasopiessin in subjects w ith septic shock using 28- K) day surviv al and measures of oigan dysfunction as lespective pπmaiy and secondary outcome variables Of 103 vasopressin-tieated and 03 matched-control subjects w ith septic shock. 72 and 79 were respectively genotyped for AVPR l A rs 1495027 Baseline characteristics for subjects w ith genotypes are shown in Table 3 33 and Table 3 34
TABLE 33i
Baseline chaiacteπstics of a group of vasopressin-treated Caucasian ICU septic shock subjects by genotype of aiginine vasopressin receptor Ia (AVPRl A) rsl495027. For age and APACHE II score, data is given as 25lh percentile | median | 75lh percentile For all other vaπables, data is r
Figure imgf000140_0002
TABLE 3.34
Baseline charactei istics of a vasopressin untreated matched control group of Caucasian ICU septic shock subjects by genotype of arginine vasopressin receptor I a (AVPR l A) rs 1495027 Foi age and APACHE II score, data is given as 25th percentile | median | 75th percentile For all other variables, data is given as % (N /N total) N. number of subjects
Figure imgf000141_0002
Tables 1 15 1 16 and 1 17 contain 28-day survival and organ dysfunction data for septic-shock subjects genotyped for AVPRl A rs 1495027 Vasopiessin-treated subjects w ith the AVPRlA i s 1495027 TT had a dramatically decreased sui v ival (21 9c ) compaied to controls (46 9c ) as demonstrated by the negative values in the Λ.VPR1 A rs l495027 TT DELTA column in Table 1 17 Fuitheimoie. vasopressin-treated subjects w ith the AVPRl A tsl495Q27 TT genotype vveie obsei ved to have moie oigan dysfunction as demonstrated by fewei DAF of organ dysfunction In contrast, vasopiessin-tieated subjects w ith the AVPR l A rsl495027 CC genotype weie shown to have incieased suiv ival (507r) over AVPR l A rs 1495027 CC contiols (247r) as demonstiated by the positive values in the AVPRl A rs 1495027 TT DELTA column in Table 1 17 In addition, vasopressin subjects w ith the AVPR l A i sl495027 CC genotype had less oigan dysfunction as ev idenced by more DAF of organ dysfunction
TABLE 3.35
A iesponse association of AVPR l A i s 149^027 in v asopiessin-tieated Caucasian septic-shock subjects For all variables besides 28-day Hirvival. data is given as 25 percentile median | 75 -ih percentile For 28-day survival, data is given as 9c (N survived / N total) N. number
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
A logistic i egression approach was used to test tor a statistically signihcant inteiacUon between genotype and vasopressin use as predicted by 28-day surv ival TABLE 3.38 show s that theie was a statistically significant interaction between AVPRl A rs 1495027 genotype, vasopressin tieatment and surv ival, confir ming vasopressin tieatment deueases 28 day suiv ival in AVPR l A rs 1495027 TT genotvpe subjects w hile vasopressin treatment increases 28 day surv ival in AVPR l A is 1495027 CC subjects compared to controls (P = 0 04662) Follow ing adjustment tor age, admission APACHE II scoie, gendei. medical, suigical diagnosis and days alive and free of 3 of 4 systematic inflammatory iesponse syndiome (SIRS) cr iteria, there was still a statistically significant interaction of the AVPRI A isl495027 genotype and tieatment w ith vasopressin (P = 0 0Η9)
TABLE 3.38
Interaction between genotype and vasopressin use vs no vasopressin (Controls) and CC or CT genotype \s TT genotype of argmine vasopressin receptor I a (AVPR l A) r sl495027 on 28 da> surv ival
Figure imgf000143_0002
Example 1 Summary
Genotyping of SNPs LNPEP i s 18059, LNPEP r s2771 1. LNPEP rs 10051637, A VP i s 14107 π,
AVP rs857240, AVP rs857242, and AVPR l A rs 1495027 in subjects w ith septic shock can predict response to administration of vasopressin as measured by 28-day survival and/or DAF of organ dysfunction Subjects w ith genotypes including LNPEP rs 18059 CC. LNPEP rs2771 1 AA, LNPEP i s l 0051637 GG, AVP rs 1410713 CC, AVP rs857240 CT, AVP rs857242 AC and AVPR l A rs 1495027 TT should not be administered a vasopressin receptor agonist as this could
S potentially decrease survival and increase risk of organ dysfunction In contrast, subjects w ith LNPEP rs 18059 TT, LNPEP rs2771 1 GG, LNPEP rs 10051637 AA, AVP rs 1410713 AA and rs 1410713 AC. AVP i s857240 CC. AVP rs857242 CC and A VPR 1 A i s 1495027 CC genotypes should be administered a vasopressin receptor agonist as such treatment has the potential to increase surv ival and decrease πsk of organ dysfunction.
I O
EXAMPLE 2: RISK OF DEATH AND ORGAN DYSFUNCTION
Methods Cohort Selection
I S To investigate whethei genotype predicts πsk or death and organ dysfunction, selected subsets ot the ICU cohort weie used for this study. All patients who were treated w ith vasoptessin for septic shock weie excluded The four study gioups were ICU Caucasians w ith SIRS upon admission (n=874), ICU Caucasians w ith sepsis upon admission (n=690). ICU Caucasians w ith septic shock upon admission (n=440) and ICU Asians v ith SIRS upon admission (n=108)
20
Data Analysis
<\ll data analysis was carried out using statistical packages available in R (R Core Development Gi oup, 2005 - R Development Core Team i w ww R-project org) R A language and env ironment roi statistical computing Vienna, Austria. 2005) Chi-square and Kruskal-Wallis (KW) test
25J statistics were used in conjunction w ith Co\ proportional hazards (CPH) regression to identify significant SNP-phenotype associations, as well as to identify baseline characteristics (age, gender, admitting APACHE II scote, and medical vs surgical admitting diagnosis) requiring post-hoc, multivaπate adjustment Genetically heterogenous populations were subsetted prior to analysis to avoid confounding from potential population stratification
M)
Results
2.1 Leucyl/Cystinyl Aminopeptidase (LNPEP) 2.1.1 LNPEP rsl8059 2.1.1.1 Systematic Inflammatory Response Syndrome - Caucasians
TABLE 4.1 gives the baseline characteristics of 710 Caucasian SIRS subjects who were successf ully genotyped (CC vs. CTVTT) at LNPEP rs 18059 No significant differences were detected between the two genotype groups on admission to the ICU
TABLE 4.1
Baseline characteristics of a cohort of Caucasian Subjects w ith systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs 18059 (CC \s CT/TT) For age and <\PACHE II score, data is given as 25lh percentile / median / 75th percentile For all other variables, data is iven as ck (N survived / N total). N. number of sub ects.
Figure imgf000145_0001
Figure 1 and TABLE 4.2 summarize important SNP-phenotype associations Subjects w ith LNPEP rs l 8059 CC genotype showed a significantly gteater suiv ival (P = 0 0331 ) and had significantly moie days alive (P = O 0144) and days alive and free of vasopressors (P = 0 0088), da>s alive and free oi vasopressors at doses of more than 2 ug/mιn(P=0.0101 ). 5 ug/min (P=O 0037) and 15 ug/min (P=O 0157). inoi ropes (P = O 0252). coagulation dysfunction (P = ϋ 0030). any ienal dysfunction (P = 0 0088), renal support (P = 0 0145), acute hepatic dysfunction (P = O 0335) and any hepatic dysfunction (P = 0 0456) Subjects w ho carried the LNPEP rs l 8059 CC genoty pe also showed a strong tiend for more days alive and free of neuiological d>sfunction (P = 0 071 ) These findings indicate that these patients who have w ho cany the LNPEP i s l 805c> CC genotype at LNPEP rsl 8059 CC have less need of inotrope and vasopressor therapy and have a lowei πsk of organ dysfunction (coagulation, ienal. hepatic and neurological)
TABLE 4.2
Days alive and free of organ dysfunction (DAF) by allele of leucyl/cystinyl ammopeptidase (LNPEP) i s 18059 (CC vs CT/TT) in a cohort of Caucasian subjects w ith systematic inflammatory iesponse syndrome For all variables besides 28-day suivival. data is given as 25th peicentile / median / 75* percentile For 28-day survival, data is given as ck (N surv ived / N total) N, number of subjects
Figure imgf000145_0002
Figure imgf000146_0001
TABLE 4.4
Days alive and free of oigan dysfunction (DAF) by allele of leucyl/cystinyl aminopeptidase (LNPEP) is 18059 (CC vs CTfTT) in a cohort of Caucasian subjects w ith sepsis Data is given as 25lh ercentile / median / 75lh ercentile N, number of sub ects
Figure imgf000147_0002
2.1.1.3 Septic Shock - Caucasians
TABLE 4.5 gives the baseline chaiacteiMics (age, gender, APACHE II score and medical vs sui gical diagnosis) of 366 Caucasian septic shock subjects who were successfully genotyped (CC vs CT/TT) at LNPEP isl 8059 No significant differences were detected between the two genotype gtoups on admission to the ICU
TABLE 4.5
Baseline ehaiacteπstics ol a cohort of Caucasian Subjects w ith septic shock by allele of leuc> l/c> stinyl aminopeptidase (LNPEP) is 18059 (CC vs CT/TT) For age and APACHE II scoie, data is given as 25lh percentile / median / 75th peicentile For all other vanables, data is
Figure imgf000147_0001
TABLE 4.6 summai izes important SNP-phenotype associations Subjects w ith the LNPEP i s 18059 CC genotype showed a strong tiend tor greater suiv ival (P = 0 0862) and significantly more days alive (P = 0 0353) and days alive and fiee of vasopressors (P = 0 0404), days alive and hee of vasopressois at doses of more than 2 ug/min (P = O 0372), 5 ug/min (P = 0 01 32) and 1 5 ug/min (P = O 0371). coagulation dysfunction (P = O 0079). any ienal dysfunction (P = O 0394) and renal support (P = O 0364) LNPEP i s 18059 CC individuals also showed a stiong trend for more days alive and fiee of inotiopes (P = 0 0646) and acute ienal dysfunction (P = O 0593) These findings indicate that Caucasian septic shock subjects who carry the CC genotype at LNPEP rsl 8059 have less need of inotrope and vasopressoi theiapy and ate have a lower risk of oigan dysfunction (coagulation and renal)
TABLE 4.6 Days alive and free of organ dysfunction (DAF) by allele of leucyl/cystinyl aminopeptidase (LNPEP) rs 18059 (CC vs. CT7TT) in a cohort of Caucasian subjects with septic shock. For all variables besides 28-day survival, data is given as 25lh percentile / median / 75th percentile. For 28-day survival, data is given as % (N survived / N total). N, number of subjects.
Figure imgf000148_0001
2.1.2 LNPEP rs27711
2.1.2.2 Systematic Inflammatory Response Syndrome - Caucasians
TABLE 4.7 summarizes the baseline characteristics (age. gender, APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of 717 Caucasian systematic inflammatory response syndrome subjects who were successfully genotyped (AA vs. GG/AG) at LNPEP rs2771 1. No significant differences were detected between the two genotype groups on admission to the ICU.
TABLE 4.7
Baseline characteristics of a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs2771 1 (GG/AG vs. AA). For age and APACHE II score, data is given as 25th percentile / median / 75lh percentile. For all other variables, data is given as c/c (N survived / N total). N, number of subjects.
Figure imgf000148_0002
Figure imgf000149_0001
TABLE 4.8 summarizes important SNP-phenotype associations Subjects w ith the LNPEP rs2771 1 AA genotype showed significantly more days alive and free of vasopressors (P = 0 0330). days alive and free of vasopressors at doses of more than 2 ug/min(P=0 0362), 5 ug/min (P=O 0222) and 15 ug/min (P=O 0961 ) Subjects w ith the LNPEP rs2771 1 AA genotype also had a stiong trend for moie days alive and fiee of steroids (P = 0 0871 ) These findings indicate thai Caucasian subjects who have SIRS and have the AA genotype at LNPEP rs2771 1 have less need for vasopressor therapy and steroid therapy
TABLE 4.8
Days alive and fiee of organ dysfunction (DAF) by allele of leucyl/cystinyl aminopeptidase (LNPEP) rs2771 1 (GG/ AG vs AA) in a cohort of Caucasian subjects w ith systematic inflammatory response syndrome Data l1- given as 25th percentile / median / 75lh percentile N. number ot sub ects.
Figure imgf000149_0002
I S
2.1.3 LNPEP rs 10051637
2.1.3.1 Systematic Inflammatory Response Syndrome - Caucasians
TABLE 4.9 summarizes the baseline characteiistics (age, gender, APACHE II score, medical v s
20 surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of 710 Caucasian SIRS sublets who were successfully genotyped (AA vs AG/GG) at LNPEP is 10051637 No significant baseline differences were detected between the two genotype groups on admission to the ICU although the AG/GG group is more likely to be diagnosed w ith sepsis throughout an ICU stay
"1S TABLE 4.9
Baseline characteristics of a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs 10051637 (AA vs. AG/GG). For age and APACHE II score, data is given as 25Ih percentile / median / 75lh percentile. For all other variables, data is given as % (N survived / N total). N, number of sub ects.
Figure imgf000150_0001
TABLE 4.10 summarizes important SNP-phenotype associations. Subjects with the LNPEP rs 10051637 AG or GG genotype showed significantly more days alive and free of inotropes (F1 = 0.0357) and 2 of 4 SIRS criteria (P = 0.0226). These findings indicate that Caucasian subjects Λho have SIRS who carry either the AG or GG genotype at LNPEP rs 10051637 have less need of inotrope therapy and less SIRS.
TABLE 4.10
Days alive and free of organ dysfunction (DAF) by allele of leucyl/cystinyl aminopeptidase (LNPEP) rs 10051637 (AA vs. AG/GG) in a cohort of Caucasian subjects with systematic inflammatory response syndrome. Data is given as 25Ih percentile / median / 75th percentile. N, number of subjects.
AG /
AA GG Combined Test
(N=474
(N=236) ) (N=710) Statistic
15 / 28 / 1 1.3 / 28 /
INO.DAF 7 / 28 / 28 28 28 F=4.43 d f = 1.708 P=().O357
MSIRS2. DAF 0 /2 / 20 0 /6 / 21 0 /5 / 21 F=5.22 d .f.= 1.708 P=O.O226
CSIRS2.D AF 0 /3 / 20 0 /5 / 20 0 /5 / 20 F=3.23 d .f.= 1.708 P=O.0726
2.1.4 LNPEP rs38041 2.1.4.1 Systematic Inflammatory Response Syndrome - Caucasians
TABLE 4.11 summarizes the baseline characteristics (age, gender, APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of 717 Caucasian SIRS subjects who were successfully genotyped (AA vs. GG/ AG) at LNPEP rs38041 . No significant differences were detected between the two genotype groups on admission to the ICU.
TABLE 4.11
K) Baseline characteristics of a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs38041 (AA vs. GG/AG). For age and APACHE II score, data is given as 25th percentile / median / 75* percentile. For all olher variables, data is given as ck (N survived / N total). N, number of subjects.
Figure imgf000151_0001
15 TABLE 4.12 summarizes important SNP-phenotype associations for LNPEP rs38041. Subjects with the LNPEP rs38041 AA genotype showed significantly more days alive and free of vasopressors at doses of more than 5 ug/min (0.0278) and 15 ug/min (0.0384) and any renal dysfunction (P = 0.0475). Subjects with the LNPEP rs38041 AA genotype also showed a strong trend for more days alive and free of vasopressors (P = 0.067) and days alive and free of
20 vasopressors at a dose of more than 2 ug/min (0.0751 ). These findings indicate that Caucasian subjects who have SIRS and have the AA genotype at LNPEP rs38041 have less need of vasopressor therapy and are a lower risk of organ dysfunction (renal).
TABLE 4.12
">S Days alive and free of organ dysfunction (DAF) by allele of leucyl/cystinyl aminopeptidase (LNPEP) rs38041 (GG/AG vs. AA) in a cohort of Caucasian subjects with systematic inflammatory response syndrome. Data is given as 25th percentile / median / 75th percentile. N. number of sub ects.
Figure imgf000152_0001
Arginine Vasopressin (AVP) 2.2.1 AVP rs 1410713
2.2.1.1 Systematic Inflammatory Response Syndrome - Caucasians
TABLE 4.13 summarizes the baseline characteristics (age. gender. APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of 717 Caucasian SIRS subjects who were successfully genotyped at AVP rs 1410713. No significant differences were detected between the genotype groups on admission to the ICU.
TABLE 4.13
Baseline characteristics of a cohort of Caucasian Subjects w ith systematic inflammatory response syndrome by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/AC). For age and APACHE II score, data is given as 25th percentile / median / 75th percentile. For all other variables, data is given as 9c (N survived / N total). N, number of subjects.
Figure imgf000152_0002
Figure 2 and TABLE 4.14 summarize important SNP-phenotype associations for AVP rs 1410713. Subjects in the AVP rs 1410713 CC/AC genotype group had significantly increased survival (P = 0.0140), significantly more days alive (P = 0.0149) and significantly more days alive and free of neurological dysfunction (P = 0.0482), coagulation dysfunction (P = 0.0167), INR > 1.5 (P=OO l 08), acute renal dysfunction (P = 0.0414), acute hepatic dysfunction (P = 0.0218) and any hepatic dysfunction (P = 0.0175). The AVP rs 1410713 AA group also showed a strong trend for fewer days alive and free of inotropes (P=0.0709). These findings indicate that Caucasian subjects with SIRS and either the AVP rs 1410713 CC or AC genotype have a lower risk of organ dysfunction (neurological, coagulation, renal and hepatic).
TABLE 4.14
Days alive and free of organ dysfunction (DAF) by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/AC) in a cohort of Caucasian subjects with systematic inflammatory response syndrome. For all variables besides 28-day survival, data is given as 251'1 percentile / median / 75lh percentile. For 28-day survival, data is given as ck (N survived / N total). N, number of sub ects.
Figure imgf000153_0001
2.2.1.2. Sepsis - Caucasians
TABLE 4.15 summarizes the baseline characteristics (age. gender, APACHE II score, medical vs. surgical diagnosis and shock upon admission and septic shock anytime) of 564 Caucasian sepsis subjects w ho were successfully genotyped at AVP rs 1410713. No significant differences, other than a small gender difference, were detected between the genotype groups on admission to the ICU.
TABLE 4.15
Baseline characteristics of a cohort of Caucasian Subjects with sepsis by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/AC). For age and APACHE II score, data is given as 25lh percentile / median / 75th percentile. For all other variables, data is given as CA (N survived / N total). N, number of sub ects.
Figure imgf000153_0002
SS.ΛNY 60% ( 24/40) 69% (362/524) 68% (386/564) XΛ2=1.42 d.f.= I P=0.233
Figure 3 and TABLE 4.16 summarize important SNP-phenotype associations for AVP rs 1410713. Subjects with either the AVP is 1410713 CC or AC genotype had significantly increased survival (P = 0.0325), significantly more days alive (P = 0.0314) and significantly more days alive and free of acute renal dysfunction (P = 0.0388). Subjects with either the AVP rs 1410713 CC or AC genotype also had a strong trend for more days alive and free of coagulation dysfunction (P = 0.0706), acute hepatic dysfunction (P = 0.0783) and any hepatic dysfunction i P = 0.0627). These findings indicate that Caucasian sepsis subjects who have either the CC or AC genotype at AVP rs 1410713 have a lower risk of organ dysfunction (coagulation, renal and hepatic).
TABLE 4.16
Days alive and free of organ dysfunction (DAF) by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/ AC) in a cohort of Caucasian subjects with sepsis. For all variables besides 28-day survival, data is given as 25lh percentile / median / 75th percentile. For 28-day survival, data is iven as c/c (N survived / N total). N, number of sub ects.
Figure imgf000154_0001
2.2.1.3 Septic Shock - Caucasians
TABLE 4.17 summarizes the baseline characteristics (age, gender. APACHE II score and med ical vs. surgical diagnosis) of 366 Caucasian septic shock subjects who were successfully genotyped at AVP rs 1410713. No significant differences were detected between the genotype groups on admission to the ICU.
TABLE 4.17
Baseline characteristics of a cohort of Caucasian Subjects w ith septic shock by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/AC). For age and APACHE II score, data is given as 25lh percentile / median / 75lh percentile. For all other variables, data is given as 7c (N survived / N total). N, number of subjects.
Figure imgf000155_0001
Figure 4 and TABLE 4.18 summarize important SNP-phenotype associations for AVP rs 1410713. Subjects with either the AVP rs 1410713 CC or AC genotype had significantly increased survival (P = 0.0269), significantly more days alive (P = 0.0402) and significantly more days alive and free of 4 of 4 SIRS criteria (P = 0.0445), acute renal dysfunction (P = 0.0373) and INR>1 .5 (P=0.00816). Subjects with either the AVP rs 1410713 CC or AC genotype also had a strong trend for more days alive and free of vasopressors at doses of more than 2 ug/min(P = 0.0982) and 5 ug/min (P = 0.0982). inotropes (P = 0.0962). coagulation dysfunction (P = 0.0931 ), any renal dysfunction (P = 0.0744) and any hepatic dysfunction (P = 0.0619). These findings
K) indicate that Caucasian septic shock subjects, who have either the CC or AC genotype at AVP rs 1410713 have less need of vasopressor, and inotrope therapy, have less severe SIRS and have a lower risk of organ dysfunction (coagulation, renal and hepatic).
TABLE 4.18
15 Days alive and free of organ dysfunction (DAF) by genotype of Arginine Vasopressin (AVP) rs 1410713 (AA vs. CC/ AC) in a cohort of Caucasian subjects with septic shock. For all variables besides 28-day survival, data is given as 25lh percentile / median / 75th percentile. For 28-day survival, data is iven as 7c (N survived / N total). N. number of sub ects.
Figure imgf000155_0002
20 2.2.2 AVP rs857240
2.2.2 A Sepsis - Caucasians TABLE 4.19 gives the baseline characteristics (age, gender, APACHE Il score, medical vs. surgical diagnosis, shock upon admission and septic shock anytime) of 573 Caucasian Subjects with sepsis who were successfully genotyped at AVP rs857240. No significant differences were detected between the genotype groups on admission to the ICU.
TABLE 4.19
Baseline characteristics of a cohort of Caucasian Subjects with sepsis by genotype of Arginine Vasopressin (AVP) rs857240 (CC vs. CT/TT). For age and APACHE II score, data is given as 25* percentile / median / 75lh percentile. For all other variables, data is given as ck (N survived / N total). N. number of subjects.
Figure imgf000156_0001
TABLE 4.20 summarizes important SNP-phenotype associations for AVP rs857240. Subjects with either the AVP rs857240 TT or CT genotype had a trend for increased survival (P = 0.0697). significantly more days alive (P = 0.0398), significantly more days alive and free of inotropes (P = 0.04.57). coagulation dysfunction (P = 0.0382). INR> 1 .5 (P=0.036). acute renal dysfunction (P = 0.0238 ). any renal dysfunction (P = 0.0087). renal support (P = 0.0126), acute hepatic dysfunction (P = 0.0292) and any hepatic dysfunction (P = 0.0251 ). Subjects with either the AVP rs857240 TT or CT genotype also had a strong trend for more days alive and free of 4 of 4 SIRS criteria (P = 0.0555). These findings indicate that Caucasian subjects w ho have sepsis who carry either the AVP rs857240 TT or CT genotype at AVP rs857240 have less need of inotrope therapy, have less severe SIRS, and have a lower risk of organ dysfunction (coagulation, renal and hepatic).
TABLE 4.20
Days alive and free of organ dysfunction (DAF) by genotype of Arginine Vasopressin (AVP) rs857240 (CC vs. CT/TT) in a cohort of Caucasian subjects with sepsis. For all variables besides 28-day survival, data is given as 25lh percentile / median / 75lh percentile. For 28-day survival, data is iven as % (N survived / N total). N, number of sub ects.
Figure imgf000156_0002
Figure imgf000157_0001
2.2.2.2 Septic Shock - Caucasians
TABLE 4.21 summarizes the baseline characteristics (age. gender. APACHE II score and medical vs. surgical diagnosis) of 373 Caucasian septic shock subjects who were successfully genotyped at AVP rs857240. No significant differences were detected between the genotype groups on admission to the ICU.
TABLE 4.21
Baseline characteristics of a cohort of Caucasian Subjects with septic shock by genotype of Arginine Vasopressin (AVP) rs857240 (CC vs. CT /TT). For age and APACHE II score, data is given as 25lh percentile / median / 75m percentile. For all other variables, data is given as % (N survived / N total). N, number of sub ects.
Figure imgf000157_0002
TABLE 4.22 summarizes important SNP-phenotype associations for AVP rs857240. Subjects with either the AVP rs857240 TT or CT genotype had a trend for increased survival (P = 0.091 ι. significantly more days alive (P = 0.0467). significantly more days alive and free of inotropes (P = 0.0416), acute renal dysfunction (P = 0.01 14). any renal dysfunction (P = 0.0052). renal support (P = 0.0266), acute hepatic dysfunction (P = 0.0190) and any hepatic dysfunction (P = 0.01 15). Subjects with either the AVP rs857240 TT or CT genotype also had a strong trend for fewer days alive and free of vasopressors at doses of more than 5 ιιg/min(P = 0.0895) and 15 ug/min (P = 0.0747) and days alive and free of 4 of 4 SIRS criteria (P = 0.0771 ). These findings indicate thai Caucasian subjects with septic shock who had either the TT or CT genotype at AVP rs857240 have less need of vasopressor and inotrope therapy, have less SIRS, and have a lower risk of organ dysfunction (renal and hepatic).
TABLE 4.22
Days alive and free of organ dysfunction (DAF) by genotype of Arginine Vasopressin (AVP) rs857240 (CC vs. CT/TT) in a cohort of Caucasian subjects with septic shock. For all variables besides 28-day survival, data is given as 2!5th percentile / median / 75th percentile. For 28-day survival, data is given as c/c (N survived / N total). N, number of subjects.
Figure imgf000158_0001
2.2.3 AVP rs857242
2.2.3.1 Systematic Inflammatory Response Syndrome - Caucasians
TABLE 4.23 summarizes the baseline characteristics (age, gender, APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of 722 Caucasian systematic inflammatory response syndrome subjects who were successfully genotyped at AVP rs857242. Significant differences were detected between the genotype groups on admission to the ICU ( APACHE II).
TABLE 4.23 Baseline characteristics of a cohort of Caucasian Subjects w ith systematic inflammatory response syndrome by genotype of Arginine Vasopressin (AVP) rs857242 (AC/ AA vs. CC). For age and APACHE II score, data is given as 25lh percentile / median / 75th percentile. For all other variables, data is iven as Vr (N survived / N total). N. number of sub ects.
Figure imgf000158_0002
SEP.ANY 74% ( 1 14/ 154) 82% (465/568) 80 %- (579/722) XΛ2=4.69 d.f.= l P=O.0304
SS.ADMIT 49% ( 76/ 154) 5 1 % (292/568) 5 1 % ( 368/722) XΛ2=0.21 d.f.= P=O .65
SS.ANY 53'4 ( 82/154) 55^ ( M . './5681 55' 'r (394/722) XΛ2=0.14 d f.=l P=0.7 l
Figure 5 and TABLE 4.24 summarize important SNP-phenotype associations for AVP rs857242. Subjects with either the AVP rs857242 AC or AA genotype had significantly increased survival (P = 0.0108), significantly more days alive (P = 0.0032) and significantly more days alive and free of vasopressors at doses of more than 5 ug/min (P = 0.0361 ) and 15 ug/min (P = 0.0026), days alive and free of inotropes (P = 0.0394), 3 of 4 SIRS criteria (P=OO l 70), 4 of 4 SIRS criteria (P = 0.0043 ), neurological dysfunction (P = 0.033), coagulation dysfunction (P < 0.001 ). acute renal dysfunction (P = 0.0341 ), any renal dysfunction (P = 0.0127), renal support (P = 0.0017). acme hepatic dysfunction (P = 0.0013 ) and any hepatic dysfunction (P = 0.0021 ). The AVP rs8572-2 AC or AA individuals also showed a strong trend for days alive and free of vasopressors (P=O.0752). days alive and free of vasopressors at a dose of more than 2 ug/min (P=0.0524), 2 of 4 SIRS criteria (P=0.059). INR> 1 .5 (P=0.0679). These findings indicate that Caucasian subjects w ith SIRS w ho had either the AC or AA genotype at AVP rs857242 have less need of vasopressor and inotrope therapy, have less severe SIRS and have a lower risk of organ dysfunction (neurological, coagulation, renal and hepatic).
TABLE 4.24
Days alive and free of organ dysfunction ( DAF) by genotype of Arginine Vasopressin (AVP) rs857242 (AC/AA vs. CC) in a cohort of Caucasian subjects w ith systematic inflammatory response syndrome. For all variables besides 28-day survival, data is given as 25th percentile / median / 75 percentile. For 28-day survival, data is given as % (N survived / N total). N, number of sub ects.
Figure imgf000159_0001
Figure imgf000160_0001
2.2.3.2 Sepsis - Caucasians
TABLE 4.25 gives the baseline characteristics (age, gender, APACHE II score, medical vs. surgical diagnosis, shock upon admission and septic shock anytime) of 567 Caucasian sepsis subjects who were successfully genotyped at AVP rs857242. No significant differences were detected between the genotype groups on admission to the ICU.
TABLE 4.25
Baseline characteristics of a cohort of Caucasian Subjects with sepsis by genotype of Arginine Vasopressin (AVP) rs857242 (AC/AA vs. CC). For age and APACHE II score, data is given as 25lh percentile / median / 75Ih percentile. For all other variables, data is given as rA (N survived / N total). N. number of sub ects.
Figure imgf000160_0002
Figure 6 and TABLE 4.26 summarize important SNP-phenotype associations for AVP rs857242. Subjects with either the AVP rs857242 AC or AA genotype had significantly increased survival (P = 0.0220). significantly more days alive (P = 0.0059) and significantly days alive and free of vasopressors at a dose of more than 15 ug/min (P = 0.0078), 3 of 4 SIRS criteria (P=0.0219), 4 of 4 SIRS criteria (P = 0.0058). coagulation dysfunction (P = 0.0012). acute renal dysfunction (P --= 0.01 16). any renal dysfunction (P = 0.0089), renal support (P = 0.0104). acute hepatic dysfunction ( P = 0.0013) and any hepatic dysfunction (P = 0.0014). Subjects with either the AVP rs857242 AC or AA genotype also had a strong trend for more days alive and free of inotropes (P = 0.0646) INR>] .5 (P=0.0636) and neurological dysfunction (P = 0.0803). These findings indicate that Caucasian subjects with sepsis who had either the AVP rs857242 AC or AA genotype at AVP rs857242 have less need of vasopressor and inotrope therapy, have less severe SIRS and are have a lower risk of organ dysfunction (neurological, coagulation, renal and hepatic).
TABLE 4.26 Days alive and free of organ dysfunction (DAF) by genotype of Arginine Vasopressin (AVP i rs857242 (AC/AA vs. CC) in a cohort of Caucasian subjects with sepsis. For all variables besides 28-day survival, data is given as 25lh percentile / median / 75lh percentile. For 28-day survival, data is iven as ck (N survived / N total). N, number of sub ects.
Figure imgf000161_0001
2.2.3.3 Septic Shock - Caucasians
TABLE 4.27 summarizes the baseline characteristics (age, gender, APACHE II score and medical vs. surgical diagnosis) of 368 Caucasian septic shock subjects who were successfully genotyped at AVP rs857242. No significant differences were detected between the genotype groups on
K) admission to the ICU.
TABLE 4.27
Baseline characteristics of a cohort of Caucasian Subjects with septic shock by genotype of Arginine Vasopressin (AVP) rs857242 (AC/AA \ s. CC). For age and APACHE II score, data is given as 25lh percentile / median / 75Ih percentile. For all other variables, data is given as 7c (N survived / N total). N. number of sub ects.
Figure imgf000161_0002
Figure 7 and TABLE 4.28 summarize important SNP-phenotype associations for AVP rs857242. Subjects w ith either the AVP rs857242 AC or AA genotype had significantly increased surviva' (P 20 = 0.0466), significantly more days alive (P ■= 0.0129) and significantly more days alive and free of vasopressors at a dose of more than 15 ug/min (P = 0.0032) , 4 of 4 SIRS criteria (P = 0.0146). neurological dysfunction (P = 0.0365) coagulation dysfunction (P = 0.0027). acute renal dysfunction (P = 0.0103). any renal dysfunction (P = 0.0063), renal support (P = 0.0165), acute hepatic dysfunction (P = 0.0013) and any hepatic dysfunction (P < 0.001 ). Subjects with either the AVP rs857242 AC or AA genotype also had a strong trend for days alive and free of vasopressors at doses of more than 2 ug/min (P = 0.0839) and 5 ug/min (P = 0.054), INR>1.5 (P=0.0549) a nd inotropes (P = 0.0592). These findings indicate that Caucasian subjects with septic shock who had either the AC or AA genotype at AVP rs857242 had less need of vasopressor, and inotrope therapy, had more sever SIRS and had a lower risk of organ dysfunction (neurological, coagulation, renal and hepatic).
TABLE 4.28
Days alive and free of organ dysfunction (DAF) by genotype of Arginine Vasopressin (AVP) rs857242 ( AC/AA vs. CC) in a cohort of Caucasian subjects with septic shock. For all variables besides 28-day survival, data is given as 25th percentile / median / 75th percentile. For 28-day
Figure imgf000162_0001
Arginine Vasopressin Receptor Ia (AVPRlA) 2.3.1 AVPRlA rs 1495027
2.3.1.1 Septic Shock - Caucasians
TABLE 4.29 gives the baseline characterisi ics (age, gender, APACHE II score, and medical vs. surgical diagnosis) of the 361 Caucasian septic shock subjects who were successfully genotyped at AVPRl A rs 1495027 (CC vs. CITTT). No s gnificant differences were detected between the two genotype groups on admission to the ICU. TABLE 4.29
Baseline characteristics of a cohort of Caucasian Subjects with septic shock by genotype of arginine vasopressin receptor I a (AVPRl A) rs 1495027 (CC vs. CT/TT). For age and APACHE II score, data is given as 25lh percentile / median / 75lh percentile. For all other variables, data is given as CA (N survived / N total). N, number of sub ects.
Figure imgf000163_0001
TABLE 4.30 summarizes important SNP phenotype associations for AVPRlA rs 1493027. Subjects with either the AVPR I A rs 1495027 CT or TT genotype had significantly more days alive and free of renal support (P = 0.0325 ). Subjects with either the AVPR 1 A rs 1495027 CT or TT genotype also had a strong trend for more days alive and free of vasopressors at a dose of 5 uε/min (P = 0.0832) and 2 of 4 SIRS criteria (P = 0.0958). These findings indicate that Caucasian subjects with septic shock with the CT or TT genotype at AVPR l A rs 1495027 have less need of vasopressors and have decreased risk of Si1RS and organ dysfunction (renal).
TABLE 4.30
Days alive and free of organ dysfunction ( DAF) by genotype of arginine vasopressin receptor I a (AVPR I A) rsl495027 (CC vs. CT/TT) in a cohort of Caucasian subjects with septic shock. Data is given as 25th percentile / median / 75lh percentile. N. number of subjects.
Figure imgf000163_0002
2.3.2 AVPRlA rs3803107
2.3.2.1 Systematic inflammatory response syndrome - Caucasians TABLE 4.31 gives the baseline characteristics (age, gender, APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of the 729 Caucasian SIRS subjects who were successfully genotyped (CT/TT vs. CC) at AVPR l A rs38O3107. No significant differences were detected between the two genotype groups on admission to the ICU. TABLE 4.31
Baseline characteristics of a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of arginine vasopressin receptor I a (AVPRlA) r.s3803107 (CC/CT vs. TT). For age and APACHE II score, data is given as 25Ih percentile / median / 75th percentile. For all other variables, data is given as 9f (N survived / N total). N, number of subjects.
Figure imgf000164_0001
Figure 8 and TABLE 4.32 summarize important SNP-phenotype association results for AVPRl A rs38O3107. Subjects with either the AVPR IA rs38O3107 CC or CT genotype had a strong trend for increased 28-day survival (P = 0.0709) and significantly more days alive (P = 0.0468) and significantly more days alive and free of vasopressors (P = 0.0270), more days alive and free of vasopressors at doses of 2 iig/min (P = 0.0286) and 5 ug/min (P = 0.0163), cardiovascular dysfunction (P = 0.0304) and respiratory dysfunction (P = 0.0476). Subjects with either the AVPRl A rs38O31O7 CC or CT genotype also had a strong trend for more days alive and free of inotropes (P = 0.0966), 4 of 4 SIRS criteria (P = 0.0621 ), mechanical ventilation (P = 0.0763) and acute hepatic dysfunction (P = 0.0871 ). These findings indicate that. Caucasian subjects with SIRS who had either the CC or CT genotype at AVPR l A rs38O3107 have less need of vasopressors therapy and have decreased risk of SIRS and organ dysfunction (cardiovascular, respiratory, and hepatic).
TABLE 4.32
Days alive and free of organ dysfunction (DAF) by genotype of arginine vasopressin receptor a (AVPR l A) rs38O31O7 (CC/CT vs. TT) in a cohort of Caucasian subjects with systematic inflammatory response syndrome. For all variables besides 28-day survival, data is given as 25lh percentile / median / 15ih percentile. For 28-day survival, data is given as ck (N survived / N total). N, number of subjects.
Figure imgf000164_0002
Figure imgf000165_0001
2.3.2.2 Systematic inflammatory response syndrome - Asians
TABLE 4.33 summarizes the baseline characteristics (age. gender. APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of the 108 Asian SIRS subjects who were successfully genotyped (C vs. T) at AVPR l A rs38O31O7. No significant differences were detected between the two allelic groups on admission to the ICU.
TABLE 4.33
Baseline characteristics of a cohort of Asian Subjects with systematic inflammatory response syndrome by allele of arginine vasopressin receptor Ia (AVPRlA) rs3803 107 (C vs. T). For age and APACHE II score, data is given as 25lh percentile / median / 75lh percentile. For all other variables, data is iven as ck (N survived / N total). N, number of sub ects.
Figure imgf000165_0002
Figure 9 and TABLE 4.34 summarize important SNP-phenotype association results for AVPR I A rs3803107. Subjects with the C allele had a significantly increased 28-day survival (P = 0.0377) and significantly more days alive (P = 0.0206) and significantly more days alive and free of vasopressors (P = 0.0386), more days alive and free of vasopressors at doses of 2 ug/min (P = 0.02=286), 5 (P = 0.0296) and 15 ug/min (P = 0.0132), inotropes (P = 0.0379), 4 of 4 SIRS criteria (P = 0.0494). cardiovascular dysfunction (P = 0.0365), respiratory dysfunction (P = 0.0214) mechanical ventilation (P = 0.041 1 ), neurological dysfunction (P = 0.0488) and INR>1.5 (P=0.0296). Subjects with the AVPR l A rs3803107 C allele also had a strong trend for more days alive and free of any hepatic dysfunction (P = 0.0894). These findings indicate that, Asian subjects with SIRS who had the C allele at AVPRl A rs38O31O7 have less need of vasopressors and are at decreased risk of SIRS and organ dysfunction (cardiovascular, respiratory, neurological and hepatic).
TABLE 4.34
Days alive and free of organ dysfunction i DAF) by allele of arginine vasopressin receptor 1 a (AVPR l A) rs3803107 (C vs. T) in a cohort of Asian subjects with systematic inflammatory response syndrome. For all variables besides 28-day survival, data is given as 25lh percentile /
H) median / 75th percentile. For 28-day survival, data is given as % (N survived / N total). N, number of sub ects.
Figure imgf000166_0001
2.3.3 A VPR 1 A rs 10877970
2.3.3.1 Systematic inflammatory response syndrome - Caucasians
15 TABLE 4.35 gives the baseline characteristics (age. gender, APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and sepϋc shock anytime) of 725 Caucasian SIRS subjects who were successfully genotyped (CC vs. TT/CT) at AVPRl A rs 10877970. No significant differences were detected between the two genotype groups on admission to the ICL .
TABLE 4.35
Baseline characteristics of a cohort of Caucasian Subjects with systematic inflammatory response syndrome by genotype of arginine vasopressin receptor I a (AVPR l A) rs 10877970 (CC vs. TT/CT). For age and APACHE II score, data is given as 25th percentile / median / 75th percentile. For all other variables, data is iven as 7c (N survived / N total). N. number of sub ects.
Figure imgf000167_0001
TABLE 4.36 summarizes important SNP-phenotype associations for AVPRl A rs 10877970. Subjects with either the TT or CT genotype had significantly more days alive and free of acute lung injury (P = 0.0331 ), respiratory dysfunction (P = 0.0134) and mechanical ventilation (P = 0.0276). Subjects with either the AVPRl A rs 10877970 TT or CT genotype also had a strong trend for more days alive and free of vasopressors (P = 0.0183), and more days alive and free of vasopressors at doses of 2 ug/min (P = 0.0638) and 5 ug/min (0.0575). These findings indicate that Caucasian subjects with SIRS with the TT or CT genotype at AVPR l A rs 10877970 have less need of vasopressors, are at decreased risk of acute lung injury and organ dysfunction (respiratory).
TABLE 4.36
Days alive and free of organ dysfunction (DAF) by genotype of arginine vasopressin receptor Ia (AVPR l A) rs 10877970 (CC vs. TT/CT) in a cohort of Caucasian subjects with systematic inflammatory response syndrome. Data is given as 25Ih percentile / median / 75th percentile. N. number of subjects.
Figure imgf000167_0002
Figure imgf000168_0001
2.3.3.2 Systematic inflammatory response syndrome - Asians
TABLE 4.37 gives the baseline characteristics (age, gender, APACHE II score, medical vs. surgical diagnosis, sepsis upon admission, sepsis anytime, septic shock upon admission and septic shock anytime) of the 108 Asian systematic inflammatory response syndrome subjects who were successfully genotyped (C vs. T) at AVPR l A rs 10877970. No significant differences, other than a small difference in APACHE II score, were detected between the two allelic groups on admission to the ICLJ.
10
TABLE 4.37
Baseline characteristics of a cohort of Asian Subjects with systematic inflammatory response syndrome by allele of arginine vasopressin receptor Ia (AVPRlA) rs 10877970 (C vs. T). For age and APACHE II score, data is given as 25'1' percentile / median / 75th percentile. For all other
15 variables, data is iven as ck (N survived / V total). N. number of subjects.
Figure imgf000168_0002
Figure 10 and TABLE 4.38 summarizes important SNP-phenotype association results for AVPR l A rs 10877970. Subjects with the AVPRI A rs 10877970 T allele had a strong trend for increased 28-day survival (P = 0.0586) and significantly more days alive (P = 0.0349) and
20 significantly more days alive and free of vasopressors at doses of 5 ug/min (P = 0.0417) and 15 ug/min (P = 0.0175). inotropes (P = 0.0423) and respiratory dysfunction (P = 0.0427). Subjects with the AVPR I A rs 10877970 T allele also showed a strong trend for more days alive and free of 4 of 4 SIRS criteria (P = 0.0655) cardiovascular dysfunction (P = 0.079), ventilation (P = 0.057). neurological dysfunction (P = 0.064) and any hepatic dysfunction (P = 0.0827). These findings
->*, indicate that Asian subjects with SIRS who had the T allele at AVPR l A rs 10877970 have less need of vasopressors and are at a decreased risk of severe SIRS and organ dysfunction (cardiovascular, respiratory, neurological and hepatic).
TABLE 4.38
Days alive and free of organ dysfunction (DAF) by allele of arginine vasopressin receptor I a (AVPR l A) rs 10877970 (C vs. T) in a cohort of Asian subjects with systematic inflammatory response syndrome. For all variables besides 28-day survival, data is given as 25lh percentile / median / 75lh percentile. For 28-day survival, data is given as % (N survived / N total). N, number of sub ects.
Figure imgf000169_0001
K)
EXAMPLE 3: INCREASED USE OF VASOPRESSIN
Methods
Cohort Selection
To investigate whether genotype predicts increased use of vasopressin, a subset of Caucasian
15 subjects with septic shock was selected for this analysis (N=543).
Data Analysis
All data analysis was carried out using statistical packages available in R (R Core Development Group, 2005 - R Development Core Team (www.R-proiect.org). R: A language and environment
20 for statistical computing. Vienna, Austria. 2005). Chi-square tests were used to identify significant associations between SNP and increased u>e of vasopressin as well as to identify baseline chaiacteiistics (age. gender, admitting APACHE II score, and medical vs surgical admitting diagnosis) requiring post-hoc, multivariate adjustment
5 Results
3.1 Leucyl/Cystinyl Aminopeptidase (LNPEP)
3.1.1 Association of CC genotype of LNPEP rs 18059 with Use of Vasopressin
It was unknown whether SNPs w ithin the LNPEP gene and those regions immediately upstream and downstream aie associated w ith the use of vasopressin It was found that LNPEP rs 18059 is associated w ith the use of vasopiessin by comparing LNPEP rs 18059 genotypes ior \asopressin- treated subjects (N=7λ) w ith control subjects who did not receive vasopressin at any time during then ICU stay (N=166) Baseline tharacteiistics for septic shock subjects w ith LNPEP rs 18059 genotypes ate shown in Lable 5 1 No sign ificant differences between the genotype groups were
I S detected on admission to the ICU
TABLE 5.1
Baseline characteristics of Caucasian ICU septic-shock subjects by leucyl/cystinyl aminopeptidase (LNPEP) is l 8059 genotype Fot age and APACHE II score, data is given as 25th percentile | 0 median 75 ' peicentile For a other variables, data is given as ck (N /N total) N, number of sub ects
Figure imgf000170_0001
Table 5 2 show s the distribution of vasopressin administration by LNPEP rs 18059 genotype Subjects w ith the LNPEP isl 8059 CC genctype were obseived to have been administered
25 vasoptessin more frequently, than controls compared w ith subjects who were LNPEP rsl 8059 CT oi TT (P = 0 0257)
TABLE 5.2
Measure of vasopiessin treatment of Caucasian ICU septic shock subjects by genotype of 30 leuc l/c stin l amino e tidase (LNPEP) rs 18059
Figure imgf000170_0002
Figure imgf000171_0001
3.1.2 Association of AA genotype of LNFEP rs27711 with Use of Vasopressin
It was unknown whether SNPs within the LNPEP gene and those regions immediately upstream and downstream are associated with the use of vasopressin. It was found that LNPEP rs2771 1 is associated with the use of vasopressin by comparing the frequency of LNPEP rs2771 1 genotypes for vasopressin-treated subjects (N=70) and control subjects who did not receive vasopressin at any time during their ICU stay (N=368). Eiaseline characteristics for septic shock subjects with LNPEP rs2771 1 genotypes are shown in Table 5.3. No significant differences between the genotype groups were detected on admission to the ICU.
K)
TABLE 5.3
Baseline characteristics of Caucasian ICU septic shock subjects by leucyl/cystinyl aminopepticlase (LNPEP) rs2771 1 genotype. For age and APACHE II score, data is given as 25th percentile | median | 75th percentile. For all other variables, data is given as ck (N /N total). N, number of
15 sub ects.
Figure imgf000171_0002
Table 5.4 shows the distribution of vasopressin administration by LNPEP rs2771 1 genotype. Subjects with the LNPEP rs2771 1 AA genotype were more frequently observed to be administered vasopressin compared to subjects with LNPEP rs2771 1 AG or GG genotypes (P = 0.0033).
20
TABLE 5.4
Measure of vasopressin treatment of Caucasian ICU septic shock subjects by genotype of leuc l/cystinyl aminopeptidase (LNPEP) rs2771
Figure imgf000171_0003
25 3.1.3 Association of GG genotype of LNPEP rsl0051637 with Use of Vasopressin It was unknown whether SNPs within the LNPEP gene and those regions immediately upstream and downstream are associated with the use of vasopressin. It was found that LNPEP rs 10051637 is associated with the use of vasopressin by comparing the frequency of LNPEP rs 10051637 genotypes for vasopressin-treated subjects (N=72) with control subjects (N=36 I ) who did not receive vasopressin at any time during their ICU stay. Baseline characteristics for septic shock subjects with LNPEP rs 10051637 genotypes are shown in Table 5.5. No significant differences between the genotype groups were detected on admission to the ICU.
TABLE 5.5
10 Baseline characteristics of Caucasian ICU septic shock subjects by leucyl/cystinyl aminopeptidase (LNPEP) rs 10051637 genotype. For age and APACHE II score, data is given as 25lh percentile | median | 75th percentile. For all other variables, data is given as 7c (N /N total). N, number of subjects.
AA AG GG Combined Test
(N=133) <N=223) (N=77) (N=433) Statistic
49 63
AGE 72 48 63 72 43 58 71 48 61 72 F=2.05 d.f. =2.430 P=O.130
62% ( 66% 66% ( 657c
GENDER 82/ 133) ( 147/223) 5 1/77) (280/433 ) XΛ2=0.76 d.f.=2 P=0.682
21 26
APACHE II 31 21 26 30 19 25 M 20 26 31 F=0.04 d.f. =2,430 P=().%
23% ( 29% ( 29% ( 27%
SURGICAL 30/133) 64/223) 22/77) ( i 16/433 ) XΛ2=1 .75 d.t =2 P=0.416
15 Table 5.4 shows the distribution of vasopressin administration by LNPEP rs 10051637 genotype. Subjects w ith the GG genotype of LNPEP rs 10051637 were more frequently observed to be administered vasopressin (P < 0.001 ) compared to subjects who carried the AG or AA genotype of LNPEP rs 10051637 (TABLE 5.6).
TABLE 5.6
20 Measure of vasopressin treatment of Caucasian ICU septic shock subjects by genotype of leuc l/c stin l amino e tidase (LNPEP) rs 10051637.
Figure imgf000172_0001
3.2 Arginine Vasopressin Receptor Ia (AVPRlA)
3.2.1 Association of CT genotype of AVPRlA rsl495027 with Use of Vasopressin
">S
It was unknown whether SNPs within the AVPR l A gene and those regions immediately upstream and downstream are associated with the use of vasopressin in subjects with septic shock. It was found that AVPR l A rs 1495027 is associated with the use of vasopressin by comparing the frequency of AVPRlA rs 1495027 genotypes for vasopressin-treated subjects (N=72) with corrxol subjects (N=361 ) who did not receive vasopressin at any time during their ICU stay.
Baseline characteristics for septic shock subjects with AVPR l A rs 1495027 genotypes are shown in Table 5.7. No significant differences between the genotype groups were detected on admission to the ICU.
TABLE 5.7
Baseline characteristics of Caucasian ICU >eptic shock subjects by genotype of arginine vasopressin receptor I a (AVPR l A) rs 1495027. For age and APACHE II score, data is given as 25"' percentile | median | 75lh percentile. For all other variables, data is given as 7c (N /N total). N, number of sub ects.
Figure imgf000173_0001
Table 5.4 shows the distribution of vasopressin administration by AVPR l A rs 1495027 genotype. Subjects with the AVPRl A rs 1495027 CT genotype had significantly increased use of vasopressin (P = 0.0240) compared to subjects who carried either the CC or TT genotype of AVPR l A T AVPR 1 A rs 1495027 (TABLE 5.8).
TABLE 5.8
Measure of vasopressin treatment of Caucasian ICU septic shock subjects by genotype of vaso ressin rece tor I a (AVPR l A) rs 1495027.
Figure imgf000173_0002
EXAMPLE 4: BIOLOGICAL PLAUSIBILITY
Examples 1 -3 show that polymorphisms of the AVP, AVPRl A and LNPEP genes are associated with altered outcome in critically ill subjects. To further explore the relationship between inflammation and infection, the present example examines subjects w ith non-septic causes of systemic inflammatory response syndrome by analyzing SNP-phenotype interactions in subjects having undergone cardiopulmonary bypass surgery. If an AVP. AVPR 1 A, LNPEP or LRAP gene polymorphism was associated w ith altered survival and organ dysfunction, that polymorphism is also likely to be associated w ith changes in pro-inflammatory proteins such as serum granulocyte colony stimulating factor (GCSF), interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCPl ) 5 METHODS
Cohort Selection
The Biological Plausibility cohort was used for this study
Measurement of Chemokine and Cytokines 0 Attei induction of anesthesia and placement of systemic and pulmonary artery catheters that weie ioiitinely inseited tor clinical puiposes at SPH, blood was obtained at baseline and at 3 hours postoperatively toi serum GCSF, MCPl and IL-8 measurements were made using ELISA
Data Analysis
15 The primary outcome variables for the Biological Plausibility cohort were change in GCSF, MCPl and IL-8 concentiations from baseline to three hours after surgery All data analysis was earned out using statistical packages available in R (R Coie Development Gioup, 2005 - R Development Core Team (www R-proιect org) Vienna Austria 2005) Chi-squaied and Kruskal-Wallis test statistics were used to identify significant SNP-phenotype and associations, as well as to look at 0 baseline chaiacteπstics
Results
4.1 Leueyl/Cystinyl Aminopeptidase (LNPEP) 4.1.1 LNPEP rs 18059
TABLE 6.1 summarizes the baseline chaiacteπstics of 69 non-septic SIRS subjects who were successfully genotyped (LNPEP rsl8059 CC (N=20) vs CT (N=36) vs TT (N=H)) at LNPEP i sl 8059 No significant ditfeiences weie detected between the three genotype groups on admission to the CSICU
M)
TABLE 6.1
Baseline characteustics of a cohort of non-septic CSICU subjects diagnosed w ith systematic inflammatory response syndrome by genoty pe of leucyl/cystinyl aminopeptidase (LNPEP) is l 8059 (CC vs CT vs TT)
Figure imgf000174_0001
P=0.545
XΛ2= 1.86 d.f.=2
SMOKER 257c 5 ) 197c 7 ) 387c 5 ) 257c ( 17) P=0.394
XΛ2=0.49 ±f.=2
DIABETES 157c 3) 227c 8) 237c 3) 20% 14) P=0.782
XΛ2=0.62 .J.t".=2
H.TENSE 6()7r ( 12) 567c 20) 467c 6) 557c ( 38) P=O.734
0.37 I 0.50 I 0.50 I 0.50 I F=0.56 d.f.=2.64
EJECFRAC 0.60 0.60 0.46 I 0.58 I 0.60 0.50 I 0.50 I 0.60 P=0.575
1 .48 1 .65 1 .1 3 1 .57 | F=0.56 d.f.=2.66
BYPASS 2.02 2.00 1 .33 1 .73 2.45 1.31 I 1 .65 2.05 P=0.575
1 .04 1 .32 0.83 1.19 | F=0.2 d.f.=2.66
CLAMP 1 .57 1 .69 0.93 1 .43 1 .78 0.92 I 1.29 1 .70 P=0.822
XΛ2=0.22 d.f.=2
APROTININ 5% ( 1 ) 87c ( 3) 87c ( 1 ) 77c ( 5 ) P=O.897
TABLE 6.2 summarizes important SNP-biomarker associations. Subjects with the CC genotype had significantly smaller increase in serum GCSF levels (P = 0.0135) post-cardiopulmonary bypass surgery. These findings suggest that non-septic SIRS Subjects with the CC genotype at LNPEP rs l 8059 are more likely to experience a less intense chemokine (GCSF) response after cardiopulmonary bypass surgery.
TABLE 6.2
Biological plausibility of leucyl/cystinyl aminopeptidase association using biomarkers in a cohort of non-septic CSICU subjects diagnosed w ith systematic inflammatory response syndrome by enoty e of leucyl/cystinyl aminopeptidase (LNPEP) rs l 8059 Biomarkers are measured in pg/ml.
Figure imgf000175_0001
4.1.2 LNPEP rs27711
TABLE 6.3 summarizes the baseline characteristics of 69 non-septic SIRS subjects who were successfully genotyped (AA (N= 14) vs. AG/GG (N=55)) at LNPEP rs2771 1 . No significant differences between the genotype groups were detected on admission to the CSICU.
TABLE 6.3
Baseline characteristics of a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs2771 l (AA vs. GG/AG).
Figure imgf000175_0002
Figure imgf000176_0001
TABLE 6.4 summarizes important SNP-biomarkcr associations observed for LNPEP rs2771 1. Subjects with the LNPEP rs2771 1 AA genotype showed a smaller change in GCSF levels from baseline to 3 hours post-surgery (P < 0.001 ) and had lower preoperative interleukin 8 (IL8) levels (P = 0.05 ) than subjects with LNPEP rs2771 1 AG or GG genotypes . These findings suggest that non-septic SIRS Subjects with the AA genotype at LNPEP rs277 I 1 are more likely to experience a less intense chemokine (GCSF) response after cardiopulmonary bypass and are more likely to have hi she r baseline levels of IL-8.
TABLE 6.4
Biological plausibility of leucyl/cystinyl aminopeptidase association using biomarkers in a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by enot e of leuc l/c stin l amino e tidase rs2771 I (AA vs. GG/ AG).
Figure imgf000176_0002
4.1.3 LNPEP rs 100516.37 TABLE 6.5 summarizes the baseline characteristics of 70 non-septic SIRS subjects who were successfully genotyped (AA/ AG vs. GG) ai LNPEP rs 10051637. No significant differences between the genotype groups were detected on admission to the CSICU.
TABLE 6.5
Baseline characteristics of a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs l OO51637 (GG VS. AA/AG)
Figure imgf000176_0003
Figure imgf000177_0001
TABLE 6.6 summarizes important SNP-biomarker associations. Subjects with the LNPEP rs 10051637 GG genotype showed a smaller change in serum GCSF levels from baseline to 3 hours post-surgery than subjects with the LNPEP rs 10051637 AG or AA genotypes (P < 0.001 ). Furthermore. LNPEP rs 10051637 AA subjects were observed to have lower baseline interleukin-8 (IL8) levels (P = 0.0443 ) 3 hours post- surgery. These findings suggest that non-septic SIRS subjects with the LNPEP rs 10051637 GG genotype have a decreased chemokine (GCSF) and proinflammatory (IL-8) response after cardiopulmonary bypass.
TABLE 6.6
Biological plausibility of leucyl/cystinyl aminopeptidase association using biomarkers in a cohort of non-septic CSICLJ subjects diagnosed with systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs 10051637 (GG vs. A A/ AG). Biomarkers are measured in /ml.
Figure imgf000177_0002
4.1.4 LNPEP rs38041
TABLE 6.7 summarizes the baseline characteristics of 70 non-septic SIRS subjects who were successfully genotyped (GG/AG vs. AA) at LNPEP rs38041. No significant differences between the two genotype groups were detected on admission to the CSICU.
TABLE 6.7 Baseline characteristics of a cohort ot non septic CSICU subjects diagnosed w ith systematic inflammatory response syndrome by genotype of leucyl/cystinyl aminopeptidase (LNPEP) rs!8041 (AA vs GG/AG)
Figure imgf000178_0002
TABLE 6.8 summaπzes important SNP b iomai ker associations Subjects w ith the AA genoty pe had a sign ificantly smaller change in sei um GCSF levels fi om baseline to three hours post cai diopulmonary bypass (P = 0 00226) and significantly lower baseline serum interleukin 8 ( IL8) lev els ( P = 0 04 1 7 ) compared to subjects w ith LNPEP t si 8041 AG oi GG These findings suggest that non-septic SIRS subjects w ith LNPEP rs!8041 AA hav e a decreased chemokine (GCSF) i esponse artei cai diopulmonary bypass anc lowei basel ine sei um IL 8 levels
TABLE 6.8
Biological plausibility of leucyl/cystinyl aminopeptidase association using biomaikers in a cornrt ot non septic CSICU subjects diagnosed w ith systematic inflammatory iesponse syndiome by genotype of leucyl/cystinyl aminopeptidise rs!8041 (AA \ s GG/AG) Biomarkei s aie measuied
Figure imgf000178_0001
Figure imgf000178_0003
4.2 Arginine Vasopressin (AVP 4.2.1. AVP rs857242
TABLL 6.9 summaπzes the baseline chaiaαeπstics of S7 non-septic SIRS subjects who weie genotvped at AVP rs857242 No significanl differences between the genotype groups weie detected on admission to the CSICU TABLE 6.9
Baseline characteristics of a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of Arginine Vaso ressin (AVP) rs857242.
Figure imgf000179_0001
TABLE 6.10 summarizes important SNP-biomarker associations for AVP rs857242. Subjects w ith the AVP rs857242 CC genotype showed a strong trend towards a smaller change in GCSF levels at three hours post-cardiopulmonary bypass than subjects with the AVP rs857242 AC genotype (p=0.0978 ). These findings suggest that non-septic SIRS subjects with the AVP position rs857242
I O CC genotype have a decreased chemokine (GCSF) response after cardiopulmonary bypass surgery.
TABLE 6.10
Biological plausibility of Factor V association using biomarkers in a cohort of non-septic CSIC U subjects diagnosed with systematic inflammatory response syndrome by genotype of Arginine
15 Vaso ressin (AVP) rs857242 . Biomarkers are measured in /ml.
Figure imgf000179_0002
4.3 Arginine Vasopressin Receptor Ia (AVPRlA) 4.3.1 AVPRlA rsl495027
TABLE 6.11 summarizes the baseline characteristics of 69 non-septic SIRS subjects w ho were
20 successfully genotyped (CT/TT \ s. CC) at AVPR l A rs 1495027. Subjects with the CC genotype had shorter clamp time (P = 0.03) than subjects with the CT/TT genotypes. There were no other significant differences prior to cardiopulmonary bypass surgery.
TABLE 6.11 Baseline characteristics of a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of arginine vasopressin receptor 1 a (AVPRl A I rs! 495027 (CC vs. CT/TT).
Figure imgf000180_0001
TABLE 6.12 summarizes important SNP-biomarker associations for AVPR l A rs 1495027. Subjects with the AVPRl A rs 1495027 CC genotype were observed to have lower interleukin 8 ( IL8) levels at baseline (p=0.046) and at three hours post cardiopulmonary bypass (p=0.0231 ) and had a strong trend towards smaller change in IL8 levels post-cardiopulmonary bypass surgery when compared to AVPRl A rs 1495027 CT or TT subjects (P = 0.0664). These findings suggest that non-septic SIRS Subjects with the AVPRl A rs 1495027 CC genotype have a decreased proinflammatory cytokine (IL8) response at baseline and after cardiopulmonary bypass surgery. A trend towards lower MCP l levels at baseline was also observed for subjects with the CC genotype compared with AVPRlA rs 1495027 subjects with AVPRl A rs 1495027 CT/TT genotypes P = 0.09).
TABLE 6.12
Biological plausibility of arginine vasopressin receptor Ia association using biomarkers in a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of arginine vasopressin receptor I a (AVPR l A) rs 1495027 (CC vs. CT/TT). Biomarkers are measured in pg/ml.
Figure imgf000180_0002
4.3.2 AVPRlA rs3803107 TABLE 6.13 summarizes the baseline ch.uacteπstics of the 70 non-septic SIRS subjects who were successfully genotyped (CT/TT vs CC) ai AVP position rs!803107 No significant differences were detected between the two genotype groups pπor to cardiopulmonary bypass surgery
TABLE 6.13
Baseline characteristics of a cohort of non septic CSICU subjects diagnosed w ith systematic inflammatory response syndrome by genotype of arginine vasopressin ieceptor I a (AVPR l A) rs180Η 07 (CT/TT vs CC)
Figure imgf000181_0001
TABLE 6.14 summarizes important SNP biomai ker associations tor AVPR l A rs38O31 O7 Subjects w ith the AVPR l A rs38OΗ O7 CC genotype had significantly highei seium MCP l concentiatioπs at baseline compared to those w ith AVPR l A i sl803 107 CT or TT(P = 0 0288) This finding suggests that the non septic SIRS subjects w ith the AVPRl A i s!80Η 07 CC genotype had highei MCP I levels at baseline
TABLE 6.14
Biological plausibility of aigirune vasopies>in receptor I a association using biomaikers in a cohoit of non septic CSICU subjects diagnosed w ith systematic inflammatory response syndrome by genotype of arginine vasopressin receptor Ia (AVPRl A) rs3801107 (CT/TT vs CC) Biomaikers are measuied in /tnl
Figure imgf000181_0002
4.3.3 AVPRlA rs 10877970
TABLE 6.15 summarizes the baseline characteristics of the 69 non septic SIRS subjects who were successf ully genotyped (CC/CT vs TT) at AVPR l A is 10877970 No significant differences were detected between the two genotype gioups prior to cardiopulmonary bypass surgery TABLE 6.15
Baseline characteristics of a cohort of non-septic CSICU subjects diagnosed with systematic inflammatory response syndrome by genotype of arginine vasopressin receptor Ia (AVPR l A) rs l0877970 (CC/CT vs. TT).
Figure imgf000182_0001
TABLE 6.16 summarizes important SNP-biomarker associations for AVPRlA rslO87797O. Subjects w ith the AVPR l A rs 10877970 TT genotype showed a trend towards higher serum MCP levels (P = 0.0865) at baseline compared to subjects with AVPR l A rs 10877970 CT or CC. This
! () finding suggests that non-septic SIRS subjects who carry either the AVPRl A rsl0877970 CT or CC genotypes had lower MCPl levels at baseline.
TABLE 6.16
Biological plausibility of arginine vasopressin receptor Ia association using biomarkers in a cohort
15 of non-septic CSICU subjects diagnosed w ith systematic inflammatory response syndrome by genotype of arginine vasopressin receptor I a (AVPRl A) rsl0877970 (CC/CT vs. TT). Biomarkers are measured in pg/ml.
Figure imgf000182_0002
SUMMARY
20 Numerous discoveries described herein show that single nucleotide polymorphisms of the vasopressin (AVP rs 1410713. rs857240. rs857242) gene, the arginine vasopressin Al receptor (AVPRlA rs 1495027) gene, and the leucyl/cystinyl aminopeptidatase (LNPEP rs 18059, rs2771 I , and rs 10051637) gene are associated with response (measured as survival, organ dysfunction and need of life support) to AVP.
23 Furthermore, markers m the \asopressinase gene (LNPEP rsl 8059, rs2771 1. and rsl0051637) ind the \asopiessin A l receptor gene (AVPRl A rsl495027) are also markers of increased use ot AVP in a cohort of critically ill subjects who have septic shock Accordingly, clinicians more fiequently administei infused AVP to subjects who have LNPEP genotypes rsl 8059 CC. rs2771 1 AA and 5 rs l 0051617 GG and subjects who have the AVPR l A genotype. isl495027 CT These genotypes also have a significantly decreased chance of surv ival when treated w ith infused AVP compared to comparable subjects who have septic shock but who are not infused w ith AVP (control)
In a separate study of an independent cohoit of subjects w ith caidiopulmonary bypass surgery, we 10 hav e also found that LNPEP rs 18059 CC, LNPEP rs2771 1 AA and LNPEP rs 10051617 GG are associated w ith decreased inflammatory ic-ponse (measured as GCSF and IL-8 response) to non- septic causes of systemic inflammatory iesponse syndrome (subjects having cardiopulmonaiy bypass surgeiy )
I s The clinical utility of these discoveries is that before subjects w ho have SIRS, sepsis or septic shock and othei inflammatoiy conditions listed below aie considered for tieatment w ith a \asopiessin ieceptor agonist, they may be genotyped for single nucleotide polymoi phisms of the vasopressin (AVP) gene (rs l41071 1, i s85724O, and i s857242), the vasopressin A l receptor (AVPR l A) gene (i s l495027), and the vasooiessinase (LNPEP) gene (rsl 8059, rs2771 1 and 0 i s 10051637) Subjects w ho have AVP i s857240 CT or rs857242 AC genotypes, the AVPR 1 A i s 1495027 TT genotype, oi the LNPEP rs l 8059 CC. rs2771 1 AA or i s 10051617 GG genotypes should not receiv e vasopiessin receptor agonist(s) (e g V-I ieceptor agonist, e g a V i a ieceptor agonist, e g an AVPRl agonist) because vasopiessin reeeptoi agonιst(s) dramatically decieases then sui uvdl and incieases the i isk ot oigan dysfunction
25
Similaily, befoie subjects who have SIRS, ^epsis oi septic shock and the conditions listed below are consideied lor tieatment w ith any vasopressin reeeptoi agonist(s). they should be genotyped for single nucleotide polymorphisms of the vasopressin (AVP) gene (rsl410711. rs857240 and is857242), the vasopressin A l receptor (AV PRlA) gene (isl495027). and the vasopiessinase
K) (LNPEP) gene (rsl 8059, ιs2771 1 and rsl 0051617) Subjects who have the AVP rs 1410711 AA or AC, rs857240 CC or rs857242 CC genotypes, the AVPR l A rsl 495027 CC genotv pe. and the LNPEP i sl 8059 TT or i s2771 1 GG genotypes should leceive vasopiessin ieceptor agonist(s) (e g V-I ieceptor agonist, e g a V i a ieceptor agonist, e g an AVPR l agonist) because v asopressin receptor agonist(s) dramatically increases their suivival and decreases the risk of organ is dysf unction Furthermore, subjects undergoing or having cardiac surgery (of all types in all ages and hypotensions), cardiac surgery requiring cardiopulmonary bypass, cardiac surgery not requiring cardiopulmonary bypass, cardiac transplantation and hypotension, dialysis-induced hypotension, autonomic neuropathy, trauma and hypotension are also likely to be administered a vasopressin receptor agonist and should also be genotypes for single nucleotide polymorphisms of the vasopressin (AVP) gene (rs 1410713. rs857240, and rs857242), the vasopressin A l receptor (AVPR l A) gene (rs 1495027), and the vasopressinase (LNPEP) gene (rs ! 8059, rs2771 1 and rsl 0051637).
K)
Similarly, before subjects who have pregnancy-associated diuresis, diabetes insipidus and are considered for treatment w ith vasopressin, they should be genotyped for single nucleotide polymorphisms of the vasopressin (AVP) gene ( rs 1410713 , rs857240, and rs857242), the vasopressin A l receptor (AVPR l A) gene ( rs 1495027), and the vasopressinase (LNPEP) gene
15 (rs l 8059. rs277 l 1 and rs! 0051637).
TABLE 7.1 shows that subjects who have he LNPEP rs 18059 CC. rs2771 1 AA or rs 10051637 GG genotypes (P = 0.0398 interaction statistic of LNPEP rs 18059 TT and AVP infusion and survival) who receive AVP infusion have decreased survival compared to subjects who have the
20 LNPEP rs l 8059 CC, rs2771 1 AA or rsl0051637 GG genotypes who do not receive AVP infusion.
Furthermore. TABLE 7.1 shows that subjects who cany the LNPEP rs 18059 CC genotype have a significantly increased chance of receiving AVP infusion than subjects who do not carry the LNPEP rs 18059 CC genotype (p = 0.0257) Furthermore, subjects who carry the LNPEP rs2771 1
25 AA genotype have a significantly increased chance of receiving AVP infusion than subjects who do not carry the LNPEP rs2771 1 AA genotype (p = 0.0033). Furthermore, subjects who carry the LNPEP rs 10051637 GG genotype have a significantly increased chance of receiving AVP infuMon than subjects who do not carry the LNPEP rs 10051637 GG genotype (p < 0.001 ).
30 TABLE 7.1
S u mmary of Key Results of SNPs. Alleles and Genotypes of the Vasopressinase Gene (LNPEP)
Figure imgf000184_0001
Figure imgf000185_0001
In addition, subjects who have the LNPEP rs l 8059 CC genotype have a less pronounced rise in GCSF after cardiac surgery (p = 0.003) In addition, subjects who carry the LNPEP rs2771 1 AA genotype have a less pronounced rise in GCSF (p = 0.001 ) and IL-8 (p = 0.05) after cardiac surgery. In addition, subjects who have the LNPEP rs 10051637 GG genotype have a less pronounced rise in GCSF (p = 0.001 ) and IL-8 (p = 0.04) after cardiac surgery.
TABLE 7.2 shows that subjects who have he AVP rs 1410713 CC, AVP rs857240 CT. and AVP rs857242 AC genotypes w ho receive AVP infusion have decreased survival compared to subjects w ho have the AVP rs 1410713 CC, AVP rs857240 CT. and AVP rs857242 AC genotypes who do not receive AVP infusion.
TABLE 7.2
Summary of Key Results of SNPs, Alleles and Genotypes of the Vasopressin Gene (AVP).
Figure imgf000185_0002
Subjects who have the AVP rs857242 AC genotype have a greater rise in GCSF (p = 0 07) after cardiac surgery than subjects who do have the AVP rs857242 CC genotype. TABLE 7.3 shows that subjects who have the AVPRl A rs 1495027 TT genotype (P = O 0466 intei action statistic of AVPRlA rs 1495027 TT and AVP infusion and survival) who receive AVP infusion have decreased survival compared to subjects who have the AVPRl A rs 1495027 TT genotype who do not receive AVP infusion
TABLE 7.3
Summary of Key Results of SNPs. Alleles and Genotypes of the AVPRl Gene
Figure imgf000186_0001
Furthermoie. TABLE 7.3 show s that subjects w ho cany the AVPRl A rs 1495027 CT genotyp; IO have a significantly increased chance of receiv ing AVP infusion than subjects who do not carry the AVPR l A rs 1495027 CT genotype (p = 0 C 240)
Subjects who have the AVPR l A i s 1495027 CT/TT genotypes have a greater use in IL-8 (p = 0 06) aftei caidiac surgeiy than subjects who do have the AVPRl A is 1495027 CC genotype
I S
Although the foiegoing invention has been descπbed in some detail by way of illustration and example foi purposes of claiity of undeista iding. it will be ieadily appaient to those of skill in fhe ait in light of the teachings of this invention that changes and modification may be made thereto w ithout departing fiom the spirit or scope of the appended claims

Claims

CLAIMS What is Claimed is:
1. A method for obtaining a prognosis for a subject having, or at risk of developing, an inflammatory condition, the method comprising determining a genotype of said subject which includes one or more polymorphic sites in the subject's vasopressin pathway gene sequences or a combination thereof, wherein said genotype is indicative of an ability of the subject to recover from the inflammatory condition.
2. The method of claim 1, wherein the polymorphic site is selected from one or more of the following: rsl8059; rs27711; rs38041; rslOO51637; rsl410713; rs857240; rs857242; rslO87797O; rs3803107; and rsl495027; or a polymorphic site in linkage disequilibrium thereto.
3. The method of claim 2, wherein the polymorphic site in linkage disequilibrium is selected from one or more of the following: rs2762; rsl0051637; rsl477364; rs7731592; rs7736466; rsl363974; rs2351010; rsl423357; rsl544777; rs2161548; rs38O32; rs38034; rs38041; rs27436; rs27306; rs27307; rs27397; rs27659; rs277l l; rs27290; rs38O3O; rs27294; rs27747; rs39602; rs248215; rs27302; rs2278018; rsl559355; rs3734015; rs4869315; rs2247650; «2549781; rs2549782; rs2161657; rs251339; rsl87265; rs2548527; rslO56893; rs2548523; rs2255546; rs2255637; rslO195O3; rs251344; rsl981846; rsl0071975; rs7700332; rs38042; rsl8059; rs9127; rs7972829; rsl0784339; rs3803107; rsl 1836346; rs7308008; rsl 1835545; rs7959001; rsl l832877; rslO877977; rs2201895; rs7302323; rslO877986; rs2030106; rsl495027; rslO877962; rslO42615; rsl6856; rsl8059; rs27296; rs27300; rs27613; rs27711; rs38033; rs38O35; rs38036; rs38041; rs38043; rs716848; rsl216565; rsl230358; rsl363907; rs 1974871; rs2042385; rs2113050; rs2113189; rs2161658; rs2255633; rs2255634; rs2287988; rs2548524; rs2548529; rs2548530; rs2548532; rs2548533; rs2548536; rs2548538; rs2548539; rs2548540; rs2549783; rs2549784; rs2549790; rs2549791 ; rs2549794; rs2549795; rs2549796; rs2549797; rs2617447; rs2910686; rs2927609 rs3797796; rs3849749; rs3849750; rs4360063; rs4869314; rs4869316; rs6556942; rs7713127; rs7716222; rs7719705; rslOO44354; rslOO51637; rsl0058476; rsl2516666; and rsl 2716486.
4. The method of any one of claims 1 -3, further comprising comparing the genotype so determined with known genotypes which are known to be indicative of a prognosis for recovery from:
(i) the subject's type of inflammatory condition; or (ii) another inflammatory condition.
5. The method of any one of claims 1 -4, further comprising obtaining vasopressin pathway gene sequence information for the subject.
6. The method of any one of claims 1 -5, wherein the genotype is determined using a nucleic acid sample from the subject.
7. The method of claim 6, further comprising obtaining the nucleic acid sample from the subject.
8. The method of any one of claims 1-7, wherein said genotype is determined using one or more of the following techniques:
(a) restriction fragment length analysis;
(b) sequencing;
(c) micro-sequencing assay; (d) hybridization;
(e) invader assay;
(f) gene chip hybridization assays;
(g) oligonucleotide ligation assay;
(h) ligation rolling circle amplification; (i) 5' nuclease assay;
(j) polymerase proofreading methods;
(k) allele specific PCR;
(1) matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; (m) ligase chain reaction assay;
(n) enzyme-amplified electronic transduction;
(o) single base pair extension assay; and
(p) reading sequence data.
9. The method of any one of claims 1-8, wherein the genotype of the subject is indicative of increased risk of death or organ dysfunction from the inflammatory condition.
10. The method of claim 9, wherein the subject is critically ill and the genotype is indicative of a prognosis of severe cardiovascular or respiratory dysfunction.
11. The method of claim 9 or 10, wherein the genotype comprises at least one of the following risk genotypes: rsl8059CT; rsl8059TT; rs27711GA; rs27711GG; rs38041GA; rs38041GG; rslOO51637GA; rsl0051637GG; rsl410713AA; rs857240CC; rs857242CC; rsl0877970CC; rs3803107TT; and rsl495027TT; or a polymorphic site in linkage disequilibrium thereto.
12. The method of claim 9 or 10, wherein the genotype comprises at least one of the following risk alleles: rs38O31O7T; and rslO87797OC; or a polymorphic site in linkage disequilibrium thereto.
13. The method of any one of claims 1-8, wherein the genotype of the subject is indicative of decreased risk of death or organ dysfunction from the inflammatory condition.
14. The method of claim 13, wherein the subject is critically ill and the genotype is indicative of a prognosis of mild cardiovascular or respiratory dysfunction.
15. The method of claim 13 or 14, wherein the genotype comprises at least one of the following reduced risk genotypes: rsl8059CC; rs2771 IAA; rs38041AA; rsl0051637AA; rsl410713CC; rsl410713AC; rs857240TT; rs857240CT; rs857242AA; rs857242AC; rslO87797OTT; rsl0877970CT; rs3803107CC; rs3803107CT; rsl495027CC and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto.
16. The method of claim 13 or 14, wherein the genotype comprises at least one of the following reduced risk alleles: rs3803107C; and rslO87797OT; or a polymorphic site in linkage disequilibrium thereto.
17. The method of claim 11 or 12, wherein the genotype of the polymorphic site in linkage disequilibrium is selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
18. The method of claim 15 or 16, wherein the genotype of the polymorphic site in linkage disequilibrium is selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
19. The method of any one of claims 1-18, wherein the inflammatory condition is selected from the group consisting of: sepsis, septicemia, pneumonia, septic shock, systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), acute lung injury, aspiration pneumonitis, infection, pancreatitis, bacteremia, peritonitis, abdominal abscess, inflammation due to trauma, inflammation due to surgery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue damage due to chemotherapy or radiotherapy, and reactions to ingested, inhaled, infused, injected, or delivered substances, glomerulonephritis, bowel infection, opportunistic infections, and for subjects undergoing major surgery or dialysis, subjects who are immunocompromised, subjects on immunosuppressive agents, subjects with HIV/ AIDS, subjects with suspected endocarditis, subjects with fever, subjects with fever of unknown origin, subjects with cystic fibrosis, subjects with diabetes mellitus, subjects with chronic renal failure, subjects with acute renal failure, oliguria, subjects with acute renal dysfunction, glomerulo-nephritis, interstitial-nephritis, acute tubular necrosis (ATN), subjects , subjects with bronchiectasis, subjects with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, subjects with febrile neutropenia, subjects with meningitis, subjects with septic arthritis, subjects with urinary tract infection, subjects with necrotizing fasciitis, subjects with other suspected Group A streptococcus infection, subjects who have had a splenectomy, subjects with recurrent or suspected enterococcus infection, other medical and surgical conditions associated with increased risk of infection, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, meningococcemia, post-pump syndrome, cardiac stun syndrome, myocardial infarction, stroke, congestive heart failure, hepatitis, epiglottitis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, pre-eclampsia, eclampsia, HELLP syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhagic fever, pelvic inflammatory disease, Legionella, Lyme disease, Influenza A, Epstein- Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, progressive systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, systemic vasculitis, Wegener's granulomatosis, transplants including heart, liver, lung kidney bone marrow, graft-versus-host disease, transplant rejection, sickle cell anemia, nephrotic syndrome, toxicity of agents such as OKT3, cytokine therapy, and cirrhosis.
20. The method of any one of claims 1-19, wherein the inflammatory condition is selected from one or more of the following: SIRS, sepsis and septic shock.
21. A method for selecting a group of subjects for determining the efficacy of a candidate drug known or suspected of being useful for the treatment of an inflammatory condition, the method comprising determining a genotype at one or more polymorphic sites in a vasopressin pathway gene sequence for each subject, wherein said genotype is indicative of the subject's ability to recover from the inflammatory condition and sorting subjects based on their genotype.
22. The method of claim 21 further comprising, administering the candidate drug to the subjects or a subset of subjects and determining each subject's ability to recover from the inflammatory condition.
23. The method of claim 22, further comprising comparing subject response to the candidate drug based on genotype of the subject.
24. A method of treating an inflammatory condition in a subject in need thereof, the method comprising administering to the subject a vasopressin receptor agonist, wherein said subject has an improved response genotype in their vasopressin pathway associated gene sequence.
25. A method of treating an inflammatory condition in a subject in need thereof, the method comprising selectively not administering a vasopressin receptor agonist to the subject, wherein said subject has an adverse response genotype in their vasopressin pathway associated gene sequence.
26. A method of selecting a subject for the treatment of an inflammatory condition with a vasopressin receptor agonist, comprising the step of identifying a subject having an improved response genotype in their vasopressin pathway associated gene sequence, wherein the identification of a subject with the improved response genotype is predictive of increased responsiveness to the treatment of the inflammatory condition with the vasopressin receptor agonist.
27. A method of selecting a subject for the treatment of an inflammatory condition, comprising the step of identifying a subject having an adverse response genotype in their vasopressin pathway associated gene sequence and selectively not treating with a vasopressin receptor agonist, wherein the identification of a subject with the adverse response genotype is predictive of decreased responsiveness to the treatment of the inflammatory condition with the vasopressin receptor agonist.
28. A use of a vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition, wherein the subjects treated have an improved response genotype in their vasopressin pathway associated gene sequence.
29. A use of a vasopressin receptor agonist in the manufacture of a medicament for the treatment of an inflammatory condition, wherein the subjects treated do not have an adverse response genotype in their vasopressin pathway associated gene sequence.
30. A use of a vasopressin receptor agonist for administration to a subject in need thereof, said subject having an improved response genotype in their vasopressin pathway associated gene sequence.
31. The method or use of any one of claims 24 to 30, further comprising determining the number of organ system failures for the subject as an assessment of subject risk.
32. The method of claim 31 , wherein 2 or more organ system failures are indicative of increased subject risk.
33. The method or use of any one of claims 24-32, wherein the inflammatory condition is selected from the group consisting of: sepsis, septicemia, pneumonia, septic shock, systemic inflammatory response syndrome (SIRS), Acute Respiratory Distress Syndrome (ARDS), acute lung injury, aspiration pneumonitis, infection, pancreatitis, bacteremia, peritonitis, abdominal abscess, inflammation due to trauma, inflammation due to surgery, chronic inflammatory disease, ischemia, ischemia-reperfusion injury of an organ or tissue, tissue damage due to disease, tissue damage due to chemotherapy or radiotherapy, and reactions to ingested, inhaled, infused, injected, or delivered substances, glomerulonephritis, bowel infection, opportunistic infections, and for subjects undergoing major surgery or dialysis, subjects who are immunocompromised, subjects on immunosuppressive agents, subjects with HIV/ AIDS, subjects with suspected endocarditis, subjects with fever, subjects with fever of unknown origin, subjects with cystic fibrosis, subjects with diabetes mellitus, subjects with chronic renal failure, subjects with acute renal failure, oliguria, subjects with acute renal dysfunction, glomerulo-nephritis, interstitial-nephritis, acute tubular necrosis (ATN), subjects with bronchiectasis, subjects with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, subjects with febrile neutropenia, subjects with meningitis, subjects with septic arthritis, subjects with urinary tract infection, subjects with necrotizing fasciitis, subjects with other suspected Group A streptococcus infection, subjects who have had a splenectomy, subjects with recurrent or suspected enterococcus infection, other medical and surgical conditions associated with increased risk of infection, Gram positive sepsis, Gram negative sepsis, culture negative sepsis, fungal sepsis, meningococcemia, post-pump syndrome, cardiac stun syndrome, myocardial infarction, stroke, congestive heart failure, hepatitis, epiglottitis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, pre-eclampsia, eclampsia, HELLP syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhagic fever, pelvic inflammatory disease, Legionella, Lyme disease, Influenza A, Epstein- Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, progressive systemic sclerosis, systemic lupus erythematosus, inflammatory bowel disease, idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, systemic vasculitis, Wegener's granulomatosis, transplants including heart, liver, lung kidney bone marrow, graft-versus-host disease, transplant rejection, sickle cell anemia, nephrotic syndrome, toxicity of agents such as OKT3, cytokine therapy, and cirrhosis.
34. The method or use of any one of claims 24-33, wherein the inflammatory condition is selected from one or more of the following: SIRS, sepsis and septic shock.
35. The method or use of any one of claims 24-34, wherein the improved response genotype is found at one or more of the following polymorphic sites: rsl8059; rs27711; rslOO51637; rs 1410713; rs857240; rs857242; and rs 1495027; or a polymorphic site in linkage disequilibrium thereto.
36. The method or use of claim 35, wherein the polymorphic site in linkage disequilibrium is selected from one or more of the following: rs2762; rslOO51637; rsl477364; rs7731592; rs7736466; rsl363974; rs2351010; rsl423357; rsl544777; rs2161548; rs38032; rs38034; rs38041; rs27436; rs27306; rs27307; rs27397; rs27659; rs27711; rs27290; rs38O3O; rs27294; rs27747; rs39602; rs248215; rs27302; rs2278018; rsl559355; rs3734015; rs4869315; rs2247650; rs2549781; rs2549782; rs2161657; rs251339; rsl87265; rs2548527; rslO56893; rs2548523; rs2255546; rs2255637; rsl019503; rs251344; rsl981846; rsl0071975; rs7700332; rs38042; rsl8059; rs9127; rs7972829; rsl0784339; rs3803107; rsl 1836346; rs73O8OO8; rsl 1835545; rs7959001; rsl 1832877; rsl0877977; rs2201895; rs7302323; rsl0877986; rs2030106 and rsl8059; rs27296; rs27300; rs27613; rs27711; rs38O33; rs38O35; rs38036; rs38041; rs38043; rs716848; rsl216565; rsl230358; rsl363907; rsl974871; rs2042385; rs2113050; rs2113189; rs2161658; rs2255633; rs2255634; rs2287988; rs2548524; rs2548529; rs2548530; rs2548532; rs2548533; rs2548536; rs2548538; rs2548539; rs2548540; rs2549783; rs2549784; rs2549790; rs2549791; rs2549794; rs2549795; rs2549796; rs2549797; rs2617447; rs2910686; rs2927609 rs3797796; rs3849749; rs3849750; rs4360063; rs4869314; rs4869316; rs6556942; rs7713127; rs7716222; rs7719705; rsl0044354; rslOO51637; rsl0058476; rsl2516666; and rsl 2716486.
37. The method or use of claim 35, wherein the improved response genotype is selected from one or more of the following: rsl8059CT; rsl8059TT; rs2771 IGG; rsl0051637GA; rslOO51637AA; rsl410713AC; rsl410713AA; rs857240CC; rs857242CC; rsl495027CC; and rsl495027CT; or a polymorphic site in linkage disequilibrium thereto.
38. The method or use of claim 37, wherein the genotype of the polymorphic site in linkage disequilibrium is selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
39. The method or use of claim 37 or 38, wherein the subject having one or more improved response genotypes is selectively administered the vasopressin receptor agonist.
40. The method or use of claim 37, wherein the subject has an adverse response genotype which is selected from one or more of the following: rsl8059CC; rs27711AA; rsl0051637GG; rsl410713CC; rs857240CT; rs857242AC; and rsl495027TT; or a polymorphic site in linkage disequilibrium thereto.
41. The method or use of claim 40, wherein the genotype of the polymorphic site in linkage disequilibrium is selected from one or more of the polymorphic sites and corresponding genotypes set out in TABLES IB and ID.
42. The method or use of claim 40 or 41, wherein the subject having one or more adverse response genotypes is selectively not administered the vasopressin receptor agonist.
43. The method or use of any one of claims 24-42, wherein the vasopressin receptor agonist is vasopressin.
44. Two or more oligonucleotides or peptide nucleic acids of about 10 to about 400 nucleotides that hybridize specifically to a sequence contained in a human target sequence consisting of a subject's vasopressin pathway associated gene sequence, a complementary sequence of the target sequence or RNA equivalent of the target sequence and wherein the oligonucleotides or peptide nucleic acids are operable in determining the presence or absence of two or more polymorphism(s) or in their vasopressin pathway associated gene sequence selected from of the following polymorphic sites: rsl8059; rs27711; rs38041; rslOO51637; rsl410713; rs857240; rs857242; rslO87797O; rs3803107; and rsl495027; or one or more polymorphic sites in linkage disequilibrium thereto.
45. The oligonucleotides or peptide nucleic acids of claim 44, wherein the one or more polymorphic sites in linkage disequilibrium thereto is selected from one or more of the following polymorphic sites: rs2762; rslOO51637; rsl477364; rs7731592; rs7736466; rsl363974; rs2351010; rsl423357; rsl544777; rs2161548; rs38032; rs38034; rs38O41; rs27436; rs27306; rs27307; rs27397; rs27659; rs27711; rs27290; rs38O3O; rs27294; rs27747; rs39602; rs248215; rs27302; rs2278018; rsl559355; rs3734015; rs4869315; rs2247650; rs2549781; rs2549782; rs2161657; rs251339; rsl87265; rs2548527; rslO56893; rs2548523; rs2255546; rs2255637; rsl019503; rs251344; rsl981846; rsl0071975; rs7700332; rs38042; rsl8059; rs9127; rs7972829; rslO784339; rs38O3lO7; rsl 1836346; rs7308008; rsl 1835545; rs7959001; rsl 1832877; rslO877977; rs2201895; rs7302323; rslO877986; rs2030106; rsl495027; rsl0877962; rslO42615; rsl6856; rsl8059; rs27296; rs27300; rs27613; rs27711; rs38O33; rs38O35; rs38036; rs38041; rs38043; rs716848; rsl216565; rsl230358; rsl363907; rsl974871; rs2042385; rs2113050; rs2113189; rs2161658; rs2255633; rs2255634; rs2287988; rs2548524; rs2548529; rs2548530; rs2548532; rs2548533; rs2548536; rs2548538; rs2548539; rs2548540; rs2549783; rs2549784; rs2549790; rs2549791 ; rs2549794; rs2549795; rs2549796; rs2549797; rs2617447; rs2910686; rs2927609 rs3797796; rs 3849749; rs3849750; rs4360063; rs4869314; rs4869316; rs6556942; rs7713127; rs7716222; rs7719705; rslOO44354; rslOO51637; rsl0058476; rsl2516666; and rsl2716486.
46. Two or more oligonucleotides or peptide nucleic acids selected from the group consisting of. (a) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:1 having a T at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:1 having a C at position 201;
(b) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:1 having a C at position 201 but not to a nucleic acid molecule comprising SEQ ID NO: 1 having a T at position 201 ;
(c) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:2 having a G at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:2 having a A at position 201; (d) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ED NO.2 having an A at position 201 but not to a nucleic acid molecule comprising SEQ BD NO:2 having a G at position 201;
(e) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:3 having an A at position 201 but not to a nucleic acid molecule comprising SEQ ED NO:3 having a G at position 201;
(f) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ED NO.3 having a G at position 201 but not to a nucleic acid molecule comprising SEQ ED NO:3 having an A at position 201; (g) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:4 having a G at position 201 but not to a nucleic acid molecule comprising SEQ ED NO:4 having an A at position 201;
(h) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ DD NO:4 having an A at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:4 having a G at position 201 ;
(i) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ED NO:5 having an A at position 201 but not to a nucleic acid molecule comprising SEQ ED NO:5 having a C at position 201;
(j) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ED NO:5 having a C at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:5 having an A at position 201;
(k) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:6 having an T at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:6 having a C at position 201 ; (1) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ED NO:6 having a C at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:6 having an T at position 201;
(m) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:7 having an A at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:7 having a C at position 201 ;
(n) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:7 having a C at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:7 having an A at position 201; (o) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:8 having a T at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:8 having a C at position 201;
(p) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:8 having a C at position 201 but not to a nucleic acid molecule comprising SEQ ED NO:8 having a T at position 201;
(q) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:9 having a C at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:9 having a T at position 201 ; (r) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO:9 having a T at position 201 but not to a nucleic acid molecule comprising SEQ ID NO:9 having a C at position 201;
(s) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ID NO: 10 having a T at position 201 but not to a nucleic acid molecule comprising SEQ ED NO: 10 having a C at position 201;
(t) an oligonucleotide or peptide nucleic acid that hybridizes under high stringency conditions to a nucleic acid molecule comprising SEQ ED NO: 10 having a C at position 201 but not to a nucleic acid molecule comprising SEQ ED NO: 10 having a T at position 201;
(u) an oligonucleotide or peptide nucleic acid capable of hybridizing under high stringency conditions to a nucleic acid molecule comprising a first allele for a given polymorphism selected from the polymorphisms listed in TABLE 1 D but not capable of hybridizing under high stringency conditions to a nucleic acid molecule comprising a second allele for the given polymorphism selected from the polymorphisms listed in TABLE ID; and
(v) an oligonucleotide or peptide nucleic acid capable of hybridizing under high stringency conditions to a nucleic acid molecule comprising the second allele for a given polymorphism selected from the polymorphisms listed in TABLE ID but not capable of hybridizing under high stringency conditions to a nucleic acid molecule comprising the first allele for the given polymorphism selected from the polymorphisms listed in TABLE ID.
47. An array of oligonucleotides or peptide nucleic acids attached to a solid support, the array comprising two or more of the oligonucleotides or peptide nucleic acids set out in any one of claims 44-46.
48. A composition comprising an addressable collection of two or more oligonucleotides or peptide nucleic acids, the two or more oligonucleotides or peptide nucleic acids consisting essentially of two or more nucleic acid molecules set out in SEQ ED NO: 1-264 or compliments, fragments, variants, or analogs thereof.
49. The oligonucleotides or peptide nucleic acids of any one of claims 44 to 48, further comprising one or more of the following: a detectable label; a quencher; a mobility modifier; a contiguous non-target sequence situated 5' or 3' to the target sequence or 5' and 3' to the target sequence.
PCT/CA2007/000111 2006-01-24 2007-01-24 Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects WO2007085087A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP2008551611A JP2009523456A (en) 2006-01-24 2007-01-24 Vasopressin pathway polymorphism as an indicator of subject outcome in severe subjects
MX2008009654A MX2008009654A (en) 2006-01-24 2007-01-24 Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects.
CA002638773A CA2638773A1 (en) 2006-01-24 2007-01-24 Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects
US12/162,066 US20090298711A1 (en) 2006-01-24 2007-01-24 Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects
AU2007209725A AU2007209725A1 (en) 2006-01-24 2007-01-24 Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects
EP07719352A EP1984546A4 (en) 2006-01-24 2007-01-24 Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US76132806P 2006-01-24 2006-01-24
US60/761,328 2006-01-24
US79515706P 2006-04-27 2006-04-27
US60/795,157 2006-04-27

Publications (1)

Publication Number Publication Date
WO2007085087A1 true WO2007085087A1 (en) 2007-08-02

Family

ID=38308805

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2007/000111 WO2007085087A1 (en) 2006-01-24 2007-01-24 Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects

Country Status (6)

Country Link
EP (1) EP1984546A4 (en)
JP (1) JP2009523456A (en)
AU (1) AU2007209725A1 (en)
CA (1) CA2638773A1 (en)
MX (1) MX2008009654A (en)
WO (1) WO2007085087A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033820A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009033783A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009033782A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009039981A2 (en) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009043458A2 (en) * 2007-09-11 2009-04-09 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
EP2789689A1 (en) 2009-06-29 2014-10-15 Luminex Corporation Chimeric primers with hairpin conformations and methods of using same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989011548A1 (en) * 1988-05-20 1989-11-30 Cetus Corporation Immobilized sequence-specific probes
CA2498509A1 (en) * 2002-05-17 2003-11-27 Riken Method of detecting gene polymorphism
US20050244834A1 (en) * 1999-09-10 2005-11-03 Whitehead Institute For Biomedical Research Single nucleotide polymorphisms in genes

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040033582A1 (en) * 2002-06-03 2004-02-19 Manling-Ma Edmonds Human single nucleotide polymorphisms
CA2519362A1 (en) * 2003-03-19 2004-09-30 The University Of British Columbia Plasminogen activator inhibitor-1 (pai-1) haplotypes useful as indicators of patient outcome
CA2542823C (en) * 2003-10-21 2015-05-26 Suntory Limited Method for prognostic evaluation of carcinoma using anti-p-lap antibody
JP2007527718A (en) * 2004-03-04 2007-10-04 ザ ユニバーシティ オブ ブリティッシュ コロンビア Toll-like receptor 2 (TLR-2) haplotype predicts patient outcomes (OutcomeofPatients)
JP2007525980A (en) * 2004-03-04 2007-09-13 ザ ユニバーシティ オブ ブリティッシュ コロンビア Thrombomodulin (THBD) haplotype to predict patient outcome
CA2479968A1 (en) * 2004-09-01 2006-03-01 Keith R. Walley Protein c and endothelial protein c receptor polymorphisms as indicators of patient outcome

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989011548A1 (en) * 1988-05-20 1989-11-30 Cetus Corporation Immobilized sequence-specific probes
US20050244834A1 (en) * 1999-09-10 2005-11-03 Whitehead Institute For Biomedical Research Single nucleotide polymorphisms in genes
CA2498509A1 (en) * 2002-05-17 2003-11-27 Riken Method of detecting gene polymorphism

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DELMAS A. ET AL.: "Clinical review: Vasopressin and terlipressin in septic shock patients", CRITICAL CARE, vol. 9, no. 2, April 2005 (2005-04-01), pages 212 - 222, XP003016404 *
SAITO S. ET AL.: "Catalog of 178 variations in the Japanse population among eight human genes encoding G protein-coupled receptors (GPCRs)", J. HUMAN GENETICS, vol. 48, no. 9, September 2003 (2003-09-01), pages 461 - 468, XP003016403 *
See also references of EP1984546A4 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009033820A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009033783A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009033782A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009039981A2 (en) * 2007-09-11 2009-04-02 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009043458A2 (en) * 2007-09-11 2009-04-09 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009043457A2 (en) * 2007-09-11 2009-04-09 Mondobiotech Laboratories Ag Use of a peptide as a therapeutic agent
WO2009033783A3 (en) * 2007-09-11 2009-11-26 Mondobiotech Laboratories Ag Use of carbetocin to treat of eg aids or idiopathic pulmonary fibrosis
WO2009043457A3 (en) * 2007-09-11 2009-11-26 Mondobiotech Laboratories Ag Use of human hemokinin-1 and optionally oxytocin to treat diseases involving excessive angiogenesis
WO2009039980A3 (en) * 2007-09-11 2009-11-26 Mondobiotech Laboratories Ag Use of bfgf 1-24 and optionally (arg 8) vasopressin to treat eg s. pneumoniae infection
WO2009039981A3 (en) * 2007-09-11 2009-11-26 Mondobiotech Laboratories Ag Use of (arg 8) vasopressin to treat eg s. pneumoniae infection
WO2009043458A3 (en) * 2007-09-11 2009-11-26 Mondobiotech Laboratories Ag Use of oxytocin to treat many diseases
WO2009033820A3 (en) * 2007-09-11 2009-11-26 Mondobiotech Laboratories Ag Use of saralasin and carbetocin in combination to treat of eg aids or idiopathic pulmonary fibrosis
WO2009033782A3 (en) * 2007-09-11 2009-11-26 Mondobiotech Laboratories Ag Use of a teprotide and optionally carbetocin to treat eg aids or idiopathic pulmonay fibrosis
EP2789689A1 (en) 2009-06-29 2014-10-15 Luminex Corporation Chimeric primers with hairpin conformations and methods of using same

Also Published As

Publication number Publication date
AU2007209725A1 (en) 2007-08-02
JP2009523456A (en) 2009-06-25
MX2008009654A (en) 2009-01-15
EP1984546A1 (en) 2008-10-29
EP1984546A4 (en) 2009-09-23
CA2638773A1 (en) 2007-08-02

Similar Documents

Publication Publication Date Title
US20070281300A1 (en) Thrombomodulin (Thbd) Haplotypes Predict Outcome
US20090176206A1 (en) Toll-like receptor 2 (tlr-2) haplotypes predict outcome of patients
US20100041600A1 (en) Interferon gamma polymorphisms as indicators of subject outcome in critically ill subjects
US20090298711A1 (en) Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects
US20080026371A1 (en) Protein C And Endothelial Protein C Receptor Polymorphisms As Indicators Of Subject Outcome
EP1984546A1 (en) Vasopressin pathway polymorphisms as indicators of subject outcome in critically ill subjects
US20110110930A1 (en) Mitogen-Activated Protein Kinase Kinase Kinase 14 (MAP3K14) Polymorphisms As Indicators of Subject Outcome in Critically Ill Subjects
US20100209413A1 (en) Serpine1 polymorphisms are predictive of response to activated protein c administration and risk of death
US20110027184A1 (en) Protein c pathway associated polymorphisms as response predictors to activated protein c or protein c-like compound administration
US20090148458A1 (en) Coagulation factor iii polymorphisms associated with prediction of subject outcome and response to therapy
US20110171200A1 (en) Protein c rs2069915 as a response predictor to survival and administration of activated protein c or protein c-like compound

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2638773

Country of ref document: CA

Ref document number: 2008551611

Country of ref document: JP

Ref document number: MX/A/2008/009654

Country of ref document: MX

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2007209725

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2007719352

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2007209725

Country of ref document: AU

Date of ref document: 20070124

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 12162066

Country of ref document: US