Patent Application
20110104683
The present invention relates to the field of assessment and treatment of patients and more specifically, to the determination of a patients genotype for one or more polymorphisms to determine the patient's prognosis and to determine therapy.
Systemic inflammatory response syndrome (SIRS) is characterized by increased inflammation (anti-inflammatory processes), increased coagulation (anti-coagulant processes), and decreased fibrinolysis (Suffredini A F et al., New Engl J Med (1989) 320(18):1165-72; Hesselvik J F et al., Critical Care Medicine (1989) 17(8):724-33; Philippe J et al., Thrombos Haemosta (1991) 65(3):291-5; Lorente J A et al., Chest (1993) 103(5):1536-42; Fourrier F et al., Chest (1992) 101(3):816-23; Mesters R M et al., (1996) 88(3):881-6.). Septic and non-septic stimuli such as bacterial endotoxin and cardiopulmonary bypass (CPB), are implicated in the activation of the coagulation system and trigger a systemic inflammatory response syndrome (SIRS). Plasminogen-activator-inhibitor-1 (PAI-1) is associated with decreased fibrinolysis (Binder B R et al., News in Physiological Sciences (2002) 17:56-61). PAI-1 levels are reported increased in patients with sepsis (Mesters R M et al., Thrombos. Haemostas (1996) 75(6):902-7; Pralong G et al., Thrombos. Haemostas (1989) 61(3):459-62), in meningococcemia (Hermans P W et al., Lancet. (1999) 354(9178):556-60), and in trauma (Menges T et al., Lancet (2001) 357(9262):1096-7). Furthermore, increased PAI-1 levels are associated with decreased survival and increased organ dysfunction (Mesters R M et al., (1996); Pralong G et al., (1989)).
The human PAI-1 gene maps to chromosome 7 q21-q22 and extends over about 12 kb. A representative Homo sapiens PAI-1 gene sequence is listed in GenBank under accession number AF386492 (shown herein as SEQ ID NO:1). Dawson et al., J. Biol. Chem. (1993) 268(15):10739-45, identified an insertion/deletion polymorphism (4G/5G) at position −675 of the PAI-1 promoter sequence, which corresponds to position 837 of SEQ ID NO:1. The 4G allele is a single base pair deletion promoter polymorphism of the PAI-1 gene and is associated with increased protein levels of PAI-1 (Dawson S J et al., supra; Dawson S J et al., Arterioscler. Thromb. (1991) 11(1):183-90). The 4G allele of this single nucleotide polymorphism (SNP) is associated with increased risk of deep venous thrombosis (Segui R et al., Brit J. Haematol. (2000) 111(1):122-8), stroke (Hindorff L A et al., J Cardiovasc Risk (2002) 9(2):131-7), acute myocardial infarction (Boekholdt S M et al., Circulation (2001) 104(25):3063-8; Eriksson P et al., Proc Natl Acad Sci USA (1995) 92(6):1851-5.), late lumen loss after coronary artery stent placement (ORTLEPPG J R et al., Clin Cardiol (2001) 24(9):585-91), and sudden cardiac death (Anvari A et al., Thrombos Res (2001) 103(2):103-7; Mikkelsson J et al., Thrombos. Haemostas. (2000) 84(1):78-82). In the critically ill, the 4G allele is also associated with decreased survival in patients who have had severe trauma (Menges T et al., Lancet, supra) and patients who had meningococcemia (Hermans P W et al., supra) as well as increased risk of shock in patients who had meningococcemia (Westendorp R G et al., Lancet (1999) 354:561-3). The PAI-1 4G genotype has also been associated with adverse patient outcomes ((Menges et al., supra HERMANS et al., supra; Westendorp R G et al., supra; Endler G et al., Brit J. Haematol. (2000) 110(2):469-71; Gardemann A et al., Thrombos. Haemostas. (1999) 82(3):1121-6; Hooper W C et al., Thrombos Res (2000) 99(3):223-30; JONES K et al., Eur J Vasc Endovasc Surg (2002) 23(5):421-5; Haralambous E. et al., Crit. Care Med (2003) 31(12):2788-93; and Roest M et al., Circulation (2000) 101(1):67-70). The 4G/4G genotype of PAI-1 was associated with PAI-1 levels in patients suffering from acute lung injury (Russell J A Crit. Care Med. (2003) 31(4):5243-5247).
This invention is based in part on the surprising discovery that the Plasminogen-Activator-Inhibitor-1 (PAI-1) 4G/5G promoter polymorphism is not as accurate a predictor of patient outcome as the SNP at position 12580 of SEQ ID NO:1. Furthermore, various other PAI-1 polymorphisms in total linkage disequilibrium (LD) with SNP 12580 are identified as being useful for patient screening, as an indication of patient outcome, or for prognosis for recovery from an inflammatory condition or to tailor therapy for patients with inflammatory conditions or for the selection of therapies for patients based on their PAI-1 12580 genotype and haplotype. Surprisingly, the SNPs identified herein as predictors of patient outcome also correspond with lower PAI-1 levels.
In accordance with one aspect of the invention, methods are provided for obtaining a prognosis for a patient having or at risk of developing an inflammatory condition, the method comprising determining a genotype including one or more polymorphism sites in the PAI-1 gene for the patient, wherein said genotype is indicative of an ability of the patient to recover from an inflammatory condition and provided the genotype selected does not correspond to only the 837 position of SEQ ID NO:1.
In accordance with another aspect of the invention, methods are provided for obtaining a prognosis for a patient having or at risk of developing an inflammatory condition, the method comprising determining a genotype of one or more polymorphism sites in the PAI-1 gene for the patient selected from the polymorphism sites corresponding to positions 664, 2037, 2362, 2852, 5834, 5878, 7343, 13605, 7365, 7729, 7771, 12750, 4588, 5404, 5686, 5984, 11312, 2846, 6821, 9759, 10381, 7365, 7729, 7771, 12750, 4588, 5404, 5686, 5984, 11312, 5645, 7121, 7437, 8070, 8406, 9463, 9466, 12219, 12580, 13889, 14440, wherein said genotype is indicative of an ability of the patient to recover from an inflammatory condition.
In accordance with one aspect of the invention, methods are provided for obtaining a prognosis for a patient having or at risk of developing an inflammatory condition, the method comprising determining a genotype including one or more polymorphism sites in the PAI-1 gene for the patient, wherein said genotype is indicative of an ability of the patient to recover from an inflammatory condition and the polymorphism is selected from the region defined by positions 1581 to 14544 of SEQ ID NO:1.
The polymorphism site may correspond to position 12580 of SEQ ID NO:1 or a polymorphism site linked thereto. Alternatively, the polymorphism site may correspond to positions 5645, 7121, 7437, 8070, 8406, 9463, 9466, 12219, 13889 or 14440 in SEQ ID NO:1.
Genotype may also be determined at a combination of multiple polymorphism sites, the combination being selected from the group of positions corresponds to SEQ ID NO:1 consisting of:
Alternatively, genotypes may be determined for polymorphism located within the 1581-14544 of SEQ ID NO:1, to exclude polymorphisms within the promoter region. Preferably, genotypes would be determined for polymorphisms selected from the group consisting of 5645, 7121, 7437, 8070, 8406, 9463, 9466, 12219, 12580, 13889, or 14440 or SEQ ID NO:1.
In accordance with another aspect of the invention, methods are provided for further comparing the genotype so determined with known genotypes, which are indicative of a prognosis for recovery from the same inflammatory condition as for the patient or another inflammatory condition.
The genotype of the patient may be indicative of a decreased likelihood of recovery from an inflammatory condition or indicative of a prognosis of severe cardiovascular or respiratory dysfunction in critically ill patients. Furthermore, such a genotype may be selected from the group of single polymorphism sites and combined polymorphism sites consisting of:
5645 T;
7121 G;
7437 T;
8070 A;
8406 C;
9463 G;
9466 T;
12219 C;
12580 G;
13889 C;
14440 A;
664 A and 2037 T;
664 A and 2362-;
664 A and 2852 A;
664 A and 5834 A;
837- and 2037 T;
837- and 2362-;
837- and 2852 A; and
837- and 5834 A.
The genotype of the patient may be indicative of an increased likelihood of recovery from an inflammatory condition or indicative of a prognosis of less severe cardiovascular or respiratory dysfunction in critically ill patients. Furthermore, such a genotype may be selected from the group of single polymorphism sites and combined polymorphism sites consisting of:
In accordance with another aspect of the invention, methods are provided for identifying a polymorphism in a PAI-1 gene sequence that correlates with patient prognosis. Where the method comprises obtaining PAI-1 gene sequence information from a group of patients, identifying a site of at least one polymorphism in the PAI-1 gene, determining genotypes at the site for individual patients in the group, determining an ability of individual patients in the group to recover from the inflammatory condition and correlating genotypes determined with patient with potential therapies.
The correlation procedure may be repeated on a patient population of sufficient size to achieve a statistically significant correlation.
The methods may further comprise steps of obtaining PAI-1 gene sequence of the patient or obtaining a nucleic acid sample from the patient. The determining of genotype may be performed on a nucleic acid sample from the patient.
Where the genotype of the patient corresponding to the nucleotide in position 12580, is G, the prognosis may be indicative of a decreased likelihood of recovery from an inflammatory condition or of severe cardiovascular or respiratory dysfunction in critically ill patients.
Where the genotype of the patient corresponding to the nucleotide in position 12580, is T, the prognosis may be indicative of a increased likelihood of recovery from an inflammatory condition or of less severe cardiovascular or respiratory dysfunction in critically ill patients.
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, 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 patients undergoing major surgery or dialysis, patients who are immunocompromised, patients on immunosuppressive agents, patients with HIV/AIDS, patients with suspected endocarditis, patients with fever, patients with fever of unknown origin, patients with cystic fibrosis, patients with diabetes mellitus, patients with chronic renal failure, patients with bronchiectasis, patients with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, patients with febrile neutropenia, patients with meningitis, patients with septic arthritis, patients with urinary tract infection, patients with necrotizing fasciitis, patients with other suspected Group A streptococcus infection, patients who have had a splenectomy, patients 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, epiglotittis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhaigic fever, pelvic inflammatory disease, Legionella, Lyme disease, Influenza A, Epstein-Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, 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 determining of a genotype may comprise one or more of: restriction fragment length analysis; sequencing; hybridization; oligonucleotide ligation assay; ligation rolling circle amplification; 5′ nuclease assay; polymerase proofreading methods; allele specific PCR; and reading sequence data.
In accordance with another aspect of the invention, there is provided a kit for determining a genotype at a defined nucleotide position within a polymorphism site in a PAI-1 gene sequence from a patient to provide a prognosis of the patient's ability to recover from an inflammatory condition, the kit comprising, in a package a restriction enzyme capable of distinguishing alternate nucleotides at the polymorphism site or a labeled oligonucleotide having sufficient complementary to the polymorphism site and capable of distinguishing said alternate nucleotides.
The alternate nucleotides may correspond to position 12580 of SEQ ID NO:1, position 10 of SEQ ID NO:2 or to a polymorphism linked thereto. The alternate nucleotides may also correspond to one or more of positions 5645, 7121, 7437, 8070, 8406, 9463, 9466, 12219, 13889 or 14440 of SEQ ID NO:1.
The kit comprising a restriction enzyme may also comprise an oligonucleotide or a set of oligonucleotides suitable to amplify a region surrounding the polymorphism site, a polymerization agent and instructions for using the kit to determine genotype.
In accordance with another aspect of the invention, methods are provided for determining patient prognosis in a patient having or at risk of developing an inflammatory condition, the method comprising detecting the identity of one or more polymorphisms in the PAI-1 promoter region, wherein the identity of said one or more polymorphisms is indicative of the ability of the patient to recover from the inflammatory condition.
In accordance with another aspect of the invention, methods are provided for patient screening, comprising the steps of (a) obtaining PAI-1 gene sequence information from a patient, and (b) determining the identity of one or more polymorphisms in the promoter region, wherein the one or more polymorphisms may be indicative of the ability of a patient to recover from an inflammatory condition.
In accordance with another aspect of the invention methods are provided for patient screening whereby the method includes the steps of (a) selecting a patient based on risk of developing an inflammatory condition or having an inflammatory condition, (b) obtaining PAI-1 gene sequence information from the patient and (c) detecting the identity of one or more polymorphisms in the PAI-1 gene, wherein the polymorphism is indicative of the ability of a patient to recover from an inflammatory condition.
In accordance with another aspect of the invention methods are provided for selecting a group of patients to determine 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 for one or more polymorphism sites in the plasminogen-activator-inhibitor-1 gene for each patient, wherein said genotype is indicative of the patient's ability to recover from the inflammatory condition and sorting patients based on their genotype, provided that the polymorphism site is not solely a polymorphism at a site corresponding to position 837 of SEQ ID NO:1.
The method may include the additional step of comparing patient response to the candidate drug based on genotype of the patient. Response to the candidate drug may be decided by determining each patient's ability to recover from the inflammatory condition.
The above sequence positions refer to the sense strand of the PAI-1 gene. It will be obvious to a person skilled in the art that analysis could be conducted on the anti-sense strand to determine patient outcome.
More severe patient outcome or prognosis may be correlated with lower PAI-1 expression or conversely an indication of less severe patient outcome or prognosis may be correlated with higher PAI-1 expression, which is the opposite of what would be expected.
In the description that follows, a number of terms are used extensively, the following definitions are provided to facilitate understanding of the invention.
“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). “Nucleotides” are 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 “polymorphic site” or “polymorphism site” or “polymorphism” or “single nucleotide polymorphism site” (SNP site) 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. Polymorphism 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, upstream promoters) of genes.
A “clade” is a group of haplotypes that are closely related phylogenetically. For example, if haplotypes are displayed on an phylogentic (evolutionary) tree a clade includes all haplotypes contained within the same branch.
As used herein “haplotype” is a set of alleles of closely linked loci on a chromosome that tend to be inherited together; commonly used in reference to the linked genes of the major histocompatibility complex.
As used herein “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.
Numerous other sites have been identified as polymorphisms in the PAI-1 gene, where those polymorphisms are linked to the polymorphism at position 12580 of SEQ. ID NO: 1 and may therefore be indicative of patient prognosis. The following single polymorphism sites and combined polymorphism sites are linked to 12580 of SEQ. ID NO.: 1:
5645;
7121;
7437;
8070;
8406;
9463;
9466;
12219;
13889;
14440;
664 and 2037;
664 and 2362;
664 and 2852;
664 and 5834;
837 and 2037;
837 and 2362;
837 and 2852; and
837 and 5834.
It will be appreciated by a person of skill in the art that further linked single polymorphism sites and combined polymorphism sites could be determined. The haplotype of PAI-1 can be created by assessing the SNP's of PAI-1 in normal subjects using a program that has an expectation maximization algorithm (i.e. PHASE). A constructed haplotype of PAI-1 may be used to find combinations of SNP's that are in total linkage disequilibrium (LD) with 12580. Therefore, the haplotype of an individual could be determined by genotyping other SNP's that are in total LD with 12580. Linked single polymorphism sites or combined polymorphism sites may also be genotyped for assessing patient prognosis.
The following genotypes for single polymorphism sites and combined polymorphism sites may be indicative of a decreased likelihood of recovery from an inflammatory condition or indicative of severe cardiovascular or respiratory dysfunction in critically ill patients:
5645 T;
7121 G;
7437 T;
8070 A;
8406 C;
9463 G;
9466 T;
12219 C;
12580 G;
13889 C;
14440 A;
664 A and 2037 T;
664 A and 2362-;
664 A and 2852 A;
664 A and 5834 A;
837- and 2037 T;
837- and 2362-;
837- and 2852 A; and
837- and 5834 A.
Whereas the following genotypes for single polymorphism sites and combined polymorphism sites may be indicative of a increased likelihood of recovery from an inflammatory condition or indicative of less severe cardiovascular or respiratory dysfunction in critically ill patients:
(wherein a “+” represents an insertion and a “−” represents a deletion)
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 equivalent polymorphism in Dawson et al., (1993, supra). 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 polymorphism site.
TABLE 1A below is representative of a Homo sapiens Plasminogen Activator Inhibitor-1 (PAI-1) or Serine-cysteine Proteinase Inhibitor clade E member 1 (SERPINE1)
gene sequence and comprises a sequence as listed in GenBank under accession number AF386492. The 4G/5G polymorphism of PAI-1 corresponds to the SNP described as −675 G/-using the numbering system of DAWSON et al., (1993), which corresponds to position 837 in TABLE 1A (SEQ ID NO:1). Polymorphism sites shown below in TABLE 1A are bolded and underlined. The polymorphism sites are represented by their major allele (dark cells in
at position 5445 the most common nucleotide (major allele) is g and the minor allele is a;
at position 837 the most common nucleotide (major allele) is g and the minor allele is −;
at position 5445 the most common nucleotide (major allele) is c and the minor allele is t;
at position 7121 the most common nucleotide (major allele) is a and the minor allele is g;
at position 7437 the most common nucleotide (major allele) is c and the minor allele is t;
at position 8070 the most common nucleotide (major allele) is g and the minor allele is a;
at position 8406 the most common nucleotide (major allele) is t and the minor allele is c;
at position 9463 the most common nucleotide (major allele) is a and the minor allele is g;
at position 9466 the most common nucleotide (major allele) is c and the minor allele is t;
at position 12219 the most common nucleotide (major allele) is t and the minor allele is c;
at position 12580 the most common nucleotide (major allele) is t and the minor allele is g;
at position 13889 the most common nucleotide (major allele) is t and the minor allele is c;
at position 14440 the most common nucleotide (major allele) is g and the minor allele is a.
The sequences shown in TABLE 1B, are sequence fragments taken from the PAI-1 sequence shown in TABLE 1A above. Furthermore, SEQ ID NO:2 corresponds to positions 12571-12590 in TABLE 1A above. The nucleotide t, at position 10 in SEQ ID NO:2 corresponds to the nucleotide found at position 12580 of SEQ ID NO:1.
In all of the sequences found in TABLE 1B below the polymorphism represented by the bold and underlined t may be substituted by a g. In SEQ ID NOs.: 3-11 in TABLE 2B, the bold and underlined t corresponds to the nucleotide found at position 12580 of SEQ ID NO:1. Due to the potential variability in PAI-1 sequence, the sequence motifs below may be useful in identifying PAI-1 sequences from a patient for use in genotype determination. For Example, patient sequences that form alignments with the below motifs (SEQ ID NO:3-11) may indicate that the patient sequence is a PAI-1 sequence and that nucleotide found at the position occupied by the bold and underlined t corresponds to the polymorphism at position 12580 of SEQ ID NO:1 and is therefore suitable for genotype determination. A similar strategy may be applied to the other polymorphism sites identified herein.
t
gtcagatgc gtgcccagct
An “allele” is defined as any one or more alternative forms of a given gene. In a diploid cell or organism 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 identical the cell or organism is said to be “homozygous”, but if genetically different the cell or organism is said to be “heterozygous” with respect to the particular gene.
A “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. Such non-coding sequences may contain regulatory sequences needed for transcription and translation of the sequence or introns etc.
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 characters of an organism.
A “single nucleotide polymorphism” (SNP) occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). A single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site. A “transition” is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A “transversion” is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymorphisms can also arise from a deletion (represented by “−” or “del”) of a nucleotide or an insertion (represented by “+” or “ins”) of a nucleotide relative to a reference allele. Furthermore, it would be appreciated by a person of skill in the art, that an insertion or deletion within a given sequence could alter the relative position and therefore the position number of another polymorphism within the sequence.
A “systemic inflammatory response syndrome” or (SIRS) is defined as including both septic (i.e. 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 presence of two or more of A) temperature>38° C. or <36° C., B) heart rate>90 beats per minute, C) respiratory rate>20 breaths per minute, and D) white blood cell count>12,000 per mm3 or <4,000 mm3. In the following description, the presence of two, three, or four of the “SIRS” criteria were scored each day over the 28 day observation period.
“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 failure by Brussels criteria plus need for vasopressor medication.
Patient outcome or prognosis as used herein refers the ability of a patient to recover from an inflammatory condition. An 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, 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 patients undergoing major surgery or dialysis, patients who are immunocompromised, patients on immunosuppressive agents, patients with HIV/AIDS, patients with suspected endocarditis, patients with fever, patients with fever of unknown origin, patients with cystic fibrosis, patients with diabetes mellitus, patients with chronic renal failure, patients with bronchiectasis, patients with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, patients with febrile neutropenia, patients with meningitis, patients with septic arthritis, patients with urinary tract infection, patients with necrotizing fasciitis, patients with other suspected Group A streptococcus infection, patients who have had a splenectomy, patients 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, epiglotittis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhaigic fever, pelvic inflammatory disease, Legionella, Lyme disease, Influenza A, Epstein-Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, 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.
Assessing patient outcome or prognosis may be accomplished by various methods. For Example, an “APACHE II” score is defined as Acute Physiology And Chronic Health Evaluation and herein was calculated on a daily basis from raw clinical and laboratory variables. (VINCENT et al., Scoring systems for assessing organ dysfunction and survival. Critical Care Clinics. 16:353-366, 2000) summarize APACHE score as follows “First developed in 1981 by KNAUS W A et al., (Chest (1991) 100(6):1619-36), the APACHE score has become the most commonly used survival prediction model in ICUs worldwide. The APACHE II score, a revised and simplified version of the original prototype, uses a point score based on initial values of 12 routine physiologic measures, age, and previous health status to provide a general measure of severity of disease. The values recorded are the worst values taken during the patient's first 24 hours in the ICU. The score is applied to one of 34 admission diagnoses to estimate a disease-specific probability of mortality (APACHE II predicted risk of death). The maximum possible APACHE II score is 71, and high scores have been well correlated with mortality. The APACHE II score has been widely used to stratify and compare various groups of critically ill patients, including patients with sepsis, by severity of illness on entry into clinical trials.”
A “Brussels score” score is a method for evaluating organ dysfunction as compared to a baseline. If the Brussels score is 0 (ie. moderate, severe, or extreme), then organ failure was recorded as present on that particular day (see TABLE 1C below). In the following description, to correct for deaths during the observation period, days alive and free of organ failure (DAF) were calculated as previously described. For example, acute lung injury was calculated as follows. Acute lung injury is defined as present when a patient meets all of these four criteria. 1) Need for mechanical ventilation, 2) Bilateral pulmonary infiltrates on chest X-ray consistent with acute lung injury, 3) PaO2/FiO2 ratio is less than 300, 4) No clinical evidence of congestive heart failure or if a pulmonary artery catheter is in place for clinical purposes, a pulmonary capillary wedge pressure less than 18 mm Hg (1). The severity of acute lung injury is assessed by measuring days alive and free of acute lung injury over a 28 day observation period. Acute lung injury is recorded as present on each day that the person has moderate, severe or extreme dysfunction as defined in the Brussels score. Days alive and free of acute lung injury is calculated as the number of days after onset of acute lung injury that a patient is alive and free of acute lung injury over a defined observation period (28 days). Thus, a lower score for days alive and free of acute lung injury indicates more severe acute lung injury. The reason that days alive and free of acute lung injury is preferable to simply presence or absence of acute lung injury, is that acute lung injury has a high acute mortality and early death (within 28 days) precludes calculation of the presence or absence of acute lung injury in dead patients. The cardiovascular, renal, neurologic, hepatic and coagulation dysfunction were similarly defined as present on each day that the person had moderate, severe or extreme dysfunction as defined by the Brussels score. Days alive and free of steroids are days that a person is alive and is not being treated with exogenous corticosteroids (e.g. hydrocortisone, prednisone, methylprednisolone). Days alive and free of pressors are days that a person is alive and not being treated with intravenous vasopressors (e.g. dopamine, norepinephrine, epinephrine, phenylephrine). Days alive and free of an International Normalized Ratio (INR)>1.5 are days that a person is alive and does not have an INR>1.5.
Analysis of variance (ANOVA) is a standard statistical approach to test for statistically significant differences between sets of measurements.
The Fisher exact test is a standard statistical approach to test for statistically significant differences between rates and proportions of characteristics measured in different groups.
One aspect of the invention may involve the identification of patients or the selection of patients that are either at risk of developing and inflammatory condition or the identification of patients who already have an inflammatory condition. For example, patients who have undergone major surgery or scheduled for or contemplating major surgery may be considered as being at risk of developing an inflammatory condition. Furthermore, patients may be determined as having an inflammatory condition using diagnostic methods and clinical evaluations known in the medical arts. An 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, 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 patients undergoing major surgery or dialysis, patients who are immunocompromised, patients on immunosuppressive agents, patients with HIV/AIDS, patients with suspected endocarditis, patients with fever, patients with fever of unknown origin, patients with cystic fibrosis, patients with diabetes mellitus, patients with chronic renal failure, patients with bronchiectasis, patients with chronic obstructive lung disease, chronic bronchitis, emphysema, or asthma, patients with febrile neutropenia, patients with meningitis, patients with septic arthritis, patients with urinary tract infection, patients with necrotizing fasciitis, patients with other suspected Group A streptococcus infection, patients who have had a splenectomy, patients 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, epiglotittis, E. coli 0157:H7, malaria, gas gangrene, toxic shock syndrome, mycobacterial tuberculosis, Pneumocystic carinii, pneumonia, Leishmaniasis, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, Dengue hemorrhaigic fever, pelvic inflammatory disease, Legionella, Lyme disease, Influenza A, Epstein-Barr virus, encephalitis, inflammatory diseases and autoimmunity including Rheumatoid arthritis, osteoarthritis, 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.
Once a patient is identified as being at risk for developing or having an inflammatory condition, then genetic sequence information may be obtained from the patient. Or alternatively genetic sequence information may already have been obtained from the patient. For example, a patient may have already provided a biological sample for other purposes or may have even had their genetic sequence determined in whole or in part and stored for future use. Genetic sequence information may be obtained in numerous different ways and may involve the collection of a biological sample that contains genetic material. Particularly, genetic material, containing the sequence or sequences of interest. Many methods are known in the art for collecting bodily samples and extracting genetic material from those samples. Genetic material can be extracted from blood, tissue and hair and other samples. There are many known methods for the separate isolation of DNA and RNA from biological material. Typically, DNA may be isolated from a biological sample when first the sample is lysed and then the DNA is isolated from the lysate according to any one of a variety of multi-step protocols, which can take varying lengths of time. DNA isolation methods may involve the use of phenol (SAMBROOK, J. et al., “Molecular Cloning”, Vol. 2, pp. 9.14-9.23, Cold Spring Harbor Laboratory Press (1989) and Ausubel, F M. et al., Current Protocols in Molecular Biology, Vol. 1, pp. 2.2.1-2.4.5, John Wiley & Sons, Inc. (1994)). Typically, a biological sample is lysed in a detergent solution and the protein component of the lysate is digested with proteinase for 12-18 hours. Next, the lysate is extracted with phenol to remove most of 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. No. 5,130,423), non-corrosive phenol derivatives are used for the isolation of nucleic acids. The resulting preparation is a mix of RNA and DNA.
Other methods for DNA isolation utilize non-corrosive chaotropic agents. These methods, which are based on the use of guanidine salts, urea and sodium iodide, involve lysis of a biological sample in a chaotropic aqueous solution and subsequent precipitation of the crude DNA fraction with a lower alcohol. The final purification of the precipitated, crude DNA fraction can be achieved by any one of several methods, including column chromatography (Analects, (1994) Vol 22, No. 4, Pharmacia Biotech), or exposure of the crude DNA to a polyanion-containing protein as described in Koller (U.S. Pat. No. 5,128,247).
Yet another method of DNA isolation, which is described by Botwell DDL (Anal Biochem (1987) 162:463-465) involves lysing cells in 6M guanidine hydrochloride, precipitating DNA from the lysate at acid pH by adding 2.5 volumes of ethanol, and washing the DNA with ethanol.
Numerous other methods are known in the art to isolate both RNA and DNA, such as the one described by Chomczynski (U.S. Pat. No. 5,945,515), whereby genetic material can be extracted efficiently in as little as twenty minutes. EVANS and HUGH (U.S. Pat. No. 5,989,431) describe methods for isolating DNA using a hollow membrane filter.
Once a patient's genetic sequence information has been obtained from the patient it may then be further analyzed to detect or determine the identity or genotype of one or more polymorphisms in the PAI-1 gene. Provided that the genetic material obtained, contains the sequence of interest. Particularly, a person may be interested in determining the PAI-1 promoter genotype of a patient of interest, where the genotype includes a nucleotide corresponding to position 12580 or SEQ ID NO:1 or position 8 of SEQ ID NO:2. The sequence of interest may also include other PAI-1 gene polymorphisms or may also contain some of the sequence surrounding the polymorphism of interest. Detection or determination of a nucleotide identity or the genotype of the single nucleotide polymorphism(s) or other polymorphism, may be accomplished by any one of a number methods or assays known in the art, including but not limited to the following:
Restriction Fragment Length Polymorphism (RFLP) strategy—An RFLP gel-based analysis can be used to distinguish between alleles at polymorphic sites within a gene. Briefly, a short segment of DNA (typically several hundred base pairs) is amplified by PCR. Where possible, a specific restriction endonuclease is chosen that cuts the short DNA segment when one variant allele is present but does not cut the short DNA segment when the other allele variant is present. After incubation of the PCR amplified DNA with this restriction endonuclease, the reaction products are then separated using gel electrophoresis. Thus, when the gel is examined the appearance of two lower molecular weight bands (lower molecular weight molecules travel farther down the gel during electrophoresis) indicates that the initial DNA sample had the allele which could be cut by the chosen restriction endonuclease. In contrast, if only one higher molecular weight band is observed (at the molecular weight of the PCR product) then the initial DNA sample had the allele variant that could not be cut by the chosen restriction endonuclease. Finally, if both the higher molecular weight band and the two lower molecular weight bands are visible then the initial DNA sample contained both alleles, and therefore the patient was heterozygous for this single nucleotide polymorphism;
Sequencing—For example the Maxam-Gilbert technique for sequencing (Maxam A M. and Gilbert W., Proc. Natl. Acad. Sci. USA (1977) 74(4):560-564) involves the specific chemical cleavage of terminally labelled DNA. In this technique four samples of the same labeled DNA are each subjected to a different chemical reaction to effect preferential cleavage of the DNA molecule at one or two nucleotides of a specific base identity. The conditions are adjusted to obtain only partial cleavage, DNA fragments are thus generated in each sample whose lengths are dependent upon the position within the DNA base sequence of the nucleotide(s) which are subject to such cleavage. After partial cleavage is performed, each sample contains DNA fragments of different lengths, each of which ends with the same one or two of the four nucleotides. In particular, in one sample each fragment ends with a C, in another sample each fragment ends with a C or a T, in a third sample each ends with a G, and in a fourth sample each ends with an A or a G. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. This technique permits the sequencing of at least 100 bases from the point of labeling. Another method is the dideoxy method of sequencing was published by Sanger et al., (Proc. Natl. Acad. Sci. USA (1977) 74(12):5463-5467). The Sanger method relies on enzymatic activity of a DNA polymerase to synthesize sequence-dependent fragments of various lengths. The lengths of the fragments are determined by the random incorporation of dideoxynucleotide base-specific terminators. These fragments can then be separated in a gel as in the Maxam-Gilbert procedure, visualized, and the sequence determined. Numerous improvements have been made to refine the above methods and to automate the sequencing procedures. Similarly, RNA sequencing methods are also known. For example, reverse transcriptase with dideoxy-nucleotides have been used to sequence encephalomyocarditis virus RNA (Zimmern D. and Kaesberg P., Proc. Natl. Acad. Sci. USA (1978) 75(9):4257-4261). Mills D R. and Kramer F R. (Proc. Natl. Acad. Sci. USA (1979) 76(5):2232-2235) describe the use of Q.beta. replicase and the nucleotide analog inosine for sequencing RNA in a chain-termination mechanism. Direct chemical methods for sequencing RNA are also known (Peattie D A., Proc. Natl. Acad. Sci. USA (1979) 76(4):1760-1764). Other methods include those of Donis-Keller et al., (1977, Nucl. Acids Res. 4:2527-2538), Simoncsits A. et al., (Nature (1977) 269(5631):833-836), Axelrod V D. et al., Nucl. Acids Res. (1978) 5(10):3549-3563), and Kramer F R. and Mills D R. (Proc. Natl. Acad. Sci. USA (1978) 75(11):5334-5338, which are incorporated herein by reference). Nucleic acid sequences can also be read by stimulating the natural fluoresce of a cleaved nucleotide with a laser while the single nucleotide is contained in a fluorescence enhancing matrix (U.S. Pat. No. 5,674,743);
Hybridization methods for the identification of SNPs using hydridization techniques are described in the U.S. Pat. Nos. 6,270,961 & 6,025,136;
A template-directed dye-terminator incorporation with fluorscent polarization-detection (TDI-FP) method is described by Freeman B D. et al., (J Mol Diagnost (2002) 4(4):209-215) is described for large scale screening;
Oligonucleotide ligation assay (OLA)—is based on ligation of probe and detector oligonucleotides annealed to a polymerase chain reaction amplicon strand with detection by an enzyme immunoassay (Villahermosa M L. J Hum Virol (2001) 4(5):238-48; Rumppanen E L. Scand J Clin Lab Invest (2001) 61(2):123-9; Iannone M A. et al., Cytometry (2000) 39(2):131-40);
Ligation-Rolling Circle Amplification (L-RCA) has also been successfully used for genotyping single nucleotide polymorphisms as described in Qi, X. et al., Nucl Acids Res (2001) 29(22):E116;
5′ nuclease assay has also been successfully used for genotyping single nucleotide polymorphisms (Aydin A. et al., Biotechniques (2001) (4):920-2, 924, 926-8.);
Polymerase proofreading methods are used to determine SNPs identities, as described in WO 0181631;
Detection of single base pair DNA mutations by enzyme-amplified electronic transduction is described in Patolsky F et al., Nat. Biotech. (2001) 19(3):253-257; or
Allele specific PCR methods have also been successfully used for genotyping single nucleotide polymorphisms (Hawkins J R. et al., Hum Mutat (2002) 19(5):543-553).
Alternatively, if a patient's sequence data is already known, then obtaining may involve retrieval of the patients nucleic acid sequence data from a database, followed by determining or detecting the identity of a nucleic acid or genotype at a polymorphism site by reading the patient's nucleic acid sequence at the polymorphic site.
Once the identity of a polymorphism(s) is determined or detected an indication may be obtained as to patient outcome or prognosis based on the genotype (the nucleotide identity at a position) of the polymorphism of interest. In the present invention, polymorphisms in the PAI-1 gene, are used to obtain a prognosis or to determine patient outcome or selection of therapies. Methods for obtaining patient outcome or prognosis or for patient screening may be useful to determine the ability of a patient to recover from an inflammatory condition. Alternatively, single polymorphism sites or combined polymorphism sites may be used as an indication of a patient's ability to recover from an inflammatory condition, if they are linked to a polymorphism determined to be indicative of a patient's ability to recover from an inflammatory condition.
Once patient outcome or a prognosis is determined, such information may be of interest to physicians and surgeons to assist in deciding between potential treatment options, to help determine the degree to which patients are monitored and the frequency with which such monitoring occurs. Ultimately, treatment decisions may be made in response to factors, both specific to the patient and based on the experience of the physician or surgeon responsible for a patient's care. Treatment options that a physician or surgeon may consider in treating a patient with an inflammatory condition may include, but are not limited to the following:
Alternative treatments currently in development and potentially useful in the treatment of an inflammatory condition may include, but are not limited to the following: antibodies to tumor necrosis factor (TNF) or even antibody to endotoxin (i.e. lipopolysaccharide, LPS); tumor necrosis factor receptor (TNF); tissue factor pathway inhibitors (Tifacogin™ alpha from Chiron); platelet activating factor hydrolase (PAFase™ from ICOS); antibodies to IL-6; antibodies, antagonists or inhibitors to high mobility group box 1 (HMGB-1 or HMG-1 tissue plasminogen activator; bradykinin antagonists; antibody to CD-14; interleukin-10; Recombinant soluble tumor necrosis factor receptor-immunoglobulin G1(Roche); Procysteine; Elastase Inhibitor; and human recombinant interleukin 1 receptor antagonist (IL-1 RA).
Alternatively, similar methods may be employed to identify new polymorphisms in PAI-1 sequence that correlate with patient outcome or prognosis.
As described above genetic sequence information or genotype information may be obtained from a patient wherein the sequence information contains one or more single nucleotide polymorphism sites in the PAI-1 gene. Also, as previously described the sequence identity of one or more single nucleotide polymorphisms in the PAI-1 gene of one or more patients may then be detected or determined. Furthermore, patient outcome or prognosis may be assessed as described above, for example the APACHE II scoring system or the Brussels score may be used to assess patient outcome or prognosis by comparing patient scores before and after treatment. Once patient outcome or prognosis has been assessed, patient outcome or prognosis may be correlated with the sequence identity of a single nucleotide polymorphism(s). The correlation of patient outcome or prognosis may further include statistical analysis of patient outcome scores and polymorphism(s) for a number of patients.
The primary outcome variable was survival to hospital discharge. Secondary outcome variables were days alive and free of cardiovascular, respiratory, renal, hepatic, hematologic, and neurologic organ system failure as well as days alive and free of SIRS (Systemic Inflammatory Response Syndrome), occurrence of sepsis, and occurrence of septic shock. SIRS was considered present when patients met at least two of four SIRS criteria. The SIRS criteria were 1) fever (>38° C.) or hypothermia (<35.5° C.), 2) tachycardia (>100 beats/min in the absence of beta blockers, 3) tachypnea (>20 breaths/min) or need for mechanical ventilation, and 4) leukocytosis (total leukocyte count>11,000/μL) (Anonymous. Critical Care Medicine (1992) 20(6):864-74). Patients were included in this cohort on the calendar day on which the SIRS criteria were met.
A patients' baseline demographics that were recorded included age, gender, whether medical or surgical diagnosis for admission (according to APACHE III diagnostic codes (Knaus W A et al., Chest (1991) 100(6):1619-36)), and admission APACHE II score. The following additional data were recorded for each 24 hour period (8 am to 8 am) for 28 days to evaluate organ dysfunction, SIRS, sepsis, and septic shock.
Clinically significant organ dysfunction for each organ system was defined as present during a 24 hour period if there was evidence of at least moderate organ dysfunction using the Brussels criteria (Table 1C) (Russell J A et al., Crit. Care Med (2000) 28(10):3405-11). Because data were not always available during each 24 hour period for each organ dysfunction variable, we used the “carry forward” assumption as defined previously (Anonymous. New Engl J Med (2000) 342(18):1301-8). Briefly, for any 24 hour period in which there was no measurement of a variable, we carried forward the “present” or “absent” criteria from the previous 24 hour period. If any variable was never measured, it was assumed to be normal.
To further evaluate cardiovascular, respiratory, and renal function we also recorded, during each 24 hour period, vasopressor support, mechanical ventilation, and renal support, respectively. Vasopressor use was defined as dopamine>5 μg/kg/min or any dose of norepinephrine, epinephrine, vasopressin, or phenylephrine. Mechanical ventilation was defined as need for intubation and positive airway pressure (i.e. T—piece and mask ventilation were not considered ventilation). Renal support was defined as hemodialysis, peritoneal dialysis, or any continuous renal support mode (e.g. continuous veno-venous hemodialysis).
To assess duration of organ dysfunction and to correct organ dysfunction scoring for deaths in the 28-day observation period, calculations were made of days alive and free of organ dysfunction (DAF) as previously reported (Bernard G R et al., New Engl J Med (1997) 336(13):912-8). Briefly, during each 24-hour period for each variable, DAF was scored as 1 if the patient was alive and free of organ dysfunction (normal or mild organ dysfunction, Table 1). DAF was scored as 0 if the patient had organ dysfunction (moderate, severe, or extreme) or was not alive during that 24-hour period. Each of the 28 days after ICU admission was scored in each patient in this fashion. Thus, the lowest score possible for each variable was zero and the highest score possible was 28. A low score is indicative of more organ dysfunction as there would be fewer days alive and free of organ dysfunction.
Similarly, days alive and free of SIRS (DAF SIRS) were calculated. Each of the four SIRS criteria were recorded as present or absent during each 24 hour period. Presence of SIRS during each 24 hour period was defined by having at least 2 of the 4 SIRS criteria. Sepsis was defined as present during a 24 hour period by having at least two of four SIRS criteria and having a known or suspected infection during the 24 hour period (Anonymous. Critical Care Medicine (1992) 20(6):864-74). Cultures that were judged to be positive due to contamination or colonization were excluded. Septic shock was defined as presence of sepsis plus presence of hypotension (systolic blood pressure<90 mmHg or need for vasopressor agents) during the same 24 hour period.
Haplotypes and Selection of htSNPs
Using unphased Caucasian genotypic data (from pga.mbt.washington.edu (Rieder M J et al., Accession AF386492. Vol. 2001: SeattleSNPs. NHLBI Program for Genomic Applications, UW-FHCRC, Seattle, Wash. (2001)) haplotypes were inferred using PHASE software (
DNA was extracted from peripheral blood samples using a QIAamp DNA Blood Maxi Kit (Qiagen). The 4G/5G polymorphism was genotyped using a restriction fragment length strategy. The htSNPs were genotyped using Masscode tagging (Qiagen Genomics, Inc) (Kokoris M et al., Molec Diag (2000) 5(4):329-40). The genotyping success rate was >99% for all SNPs.
Data were collected for 235 consecutive patients who were admitted to the Intensive Care Unit (ICU) of St. Paul's Hospital. This ICU is a mixed medical—surgical ICU in a tertiary care, university-affiliated teaching hospital of the University of British Columbia. Results are reported for only the Caucasian patients (78.6% of all admissions) who were successfully genotyped (n=223) in order to decrease the potential confounding influence of population admixture secondary to ethnic diversity, on associations between genotype and phenotype. These 235 patients make up the cohort of this study.
First haplotypes of the PAI-1 gene (
An unrooted phylogenetic tree of PAI-1 haplotype clades identified in
As shown in
When only the 4G/5G polymorphism was examined, intermediate results reflecting the addition of the increased survival of the haplotype clade, containing 13985 T allele, to the decreased survival of the haplotype clade, containing the 12580 G allele, were found. The frequency distribution of the 4G/4G, 4G/5G and 5G/5G genotypes were 26.6%, 52.1% and 20.1% which is similar to the frequencies reported in other critically ill Caucasians (MENGES T et al., Lancet (2001) 357(9262):1096-7). Hospital survival rates were 54% for 4G/4G, 61% for 4G/5G, and 74% for 5G/5G groups, which were not statistically significantly different (p=0.09). However, the 4G allele remained associated with significantly fewer days alive and free of respiratory dysfunction (p<0.05), hepatic dysfunction (p<0.05), and neurologic dysfunction (p<0.05) (Table 3). Cardiovascular, renal, and coagulation DAF scores were not significantly different between 4G/5G genotypes (Table 3). These results were supported by the findings of no difference in DAF vasopressors (additional measurement of cardiovascular function) but significantly fewer DAF ventilator (additional measurement of respiratory function) in patients who had the 4G allele (Table 3). Those patients who had the 4G allele were found to have a significantly increased duration of SIRS (DAF SIRS: 19.8 out of 28 for 4G/4G, 20.8 for 4G/5G, 23.4 for 5G/5G), a trend to increased occurrence of sepsis (94% for 4G/4G, 99% for 4G/5G, 74% for 5G/5G, p=0.08) and a significant increase in occurrence of septic shock (94% for 4G/4G, 99% for 4G/5G, 74% for 5G/5G, p=0.04) during the 28 day study period.
To assess duration of organ dysfunction and to correct organ dysfunction scoring for deaths in the 28-day observation period, days alive and free of organ dysfunction (DAF) were calculated. During each 24-hour period for each variable, DAF was scored as “1” if the patient was alive and free of organ dysfunction (normal or mild organ dysfunction—TABLE 1C). DAF was scored as “0” if the patient had organ dysfunction (moderate, severe, or extreme—TABLE 1C) or was not alive during that 24-hour period. Each of the 28 days after ICU admission was scored in each patient in this fashion. Thus, the lowest score possible for each variable was zero and the highest score possible was 28. A low score is indicative of more organ dysfunction as there would be fewer days alive and free of organ dysfunction. There were no significant differences for the PAI-1 12580 genotypes between groups (either GG/GT vs TT or GG, GT and TT separately as shown in TABLES 4A and 4B below) with respect to age, sex, APACHE II score and percent of patients admitted with surgical diagnosis. Similary, there were no significant differences for the PAI-1 4G/5G genotypes between groups (either 5G/5G vs 5G/4G and 4G/4G or any 4G separately as shown in TABLES 5A and 5B below).
Patients having haplotypes containing the 12580 G allele had marked organ dysfunction of respiratory (p<0.05), renal (p<0.05), hepatic (p<0.05), and neurologic (p<0.05) organ systems over the 28 day study period (
It was found that there was no statistically significant difference or trend in DAF when grouped by PAI-1 4G/5G (837) genotype (
In
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Plasminogen activator inhibitor-1 (PAI-1) levels were measured in 67 Caucasian cardiovascular surgery patients who had cardiopulmonary bypass. Serum levels were measured by enzyme-linked immunosorbent assay (ELISA) (Immulyse PAI-1, BioPool International) and presented based on PAI-1 12580 genotype (FIGS. 13A/B, 15A/B, 16 and 17A/B). No significant differences were observed between PAI-1 12580 genotype groups based on age or sex (as shown in TABLES 6A and 6B below).
Similarly, PAI-1 levels were also measured in 55 Caucasian cardiovascular surgery patients who had cardiopulmonary bypass and for comparison purposes serum levels were presented based on PAI-1 4G/5G (837) genotype (FIGS. 14A/B and 18A/B). A significant difference in proportion of female patients was observed (shown in Tables 7A and 7B below), and this was accounted for in later analyses.
Surprisingly, it was found that there was no statistically significant difference (or even a trend) in PAI-1 serum levels and systemic vascular resistance index (SVRI) in patients 4 hours post-operatively after cardiopulmonary bypass surgery when grouped by PAI-1 4G/5G (837) genotype. However, when patients were grouped by PAI-1 12580 genotype a statistically significant decrease in PAI-1 serum levels was associated with increased numbers of the 12580G allele, both post and pre operatively. Similarly, the PAI-1 12580 genotype showed a statistically significant decrease in SVRI associated with the 12580G allele post-operatively. Thus, the PAI-1 12580 G allele was associated with more vasodilation (as shown by lower SVRI), and lower PAI-1 levels after cardiac surgery than PAI-1 12580 T. This represents independent confirmation in an independent cohort of patients who had cardiac surgery of the findings of PAI-1 12580 G in the ICU cohort in that PAI-1 12580 G was associated with worse survival and organ dysfunction in the ICU cohort and lower SVRI and lower PAI-1 levels in the cardiac surgery cohort.
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We compared measures of disease severity using dominant and co-dominant models. We tested for differences between genotype groups using ANOVA for continuous data and a Fisher exact test for discrete data. When appropriate Student's t-test and linear regression were also used.
The sample size described herein (260), compares with sample sizes in trauma of 61 (MENGES T et al., Lancet (2001) 357(9262):1096-7), in pediatric meningococcemia of 175 (DAWSON et al., (1993)), and adult meningococcal septic shock of 50 (WESTENDORP R G et al., Lancet (1999) 354(9178):561-3). Another possible source of error in association studies is population admixture (CARDON LR and BELL JI Nature Reviews Genetics (2001) 2:91-9). This issue was addressed by limiting the analysis to Caucasians and also because this group comprised the majority of our patients available for study.
The PAI-1 12580 G allele is associated with greater evidence of SIRS and severe cardiovascular and respiratory dysfunction in a critically ill SIRS population and in a post CPB SIRS population. The critically ill SIRS population demonstrates that severe SIRS in the patients with the GG genotype was associated with more severe SIRS and more cardiovascular and respiratory failure (including more acute lung injury, more use of vasopressors, more use of steroids), but also in trends to additional organ system dysfunction and importantly, to decreased survival. These observations were confirmed in an analogous but completely independent SIRS population of critically ill patients who had CPB. In the CPB population SIRS was induced by cardiac surgery and the cardiopulmonary bypass procedure itself without evidence of infection. Evidence for increased SIRS in those patients having the GG genotype in this population is provided by the observation of greater reduction in systemic vascular resistance index (SVRI).
Critically ill patients with the 12580 G allele had significantly worse outcomes as indicated by lower survival, more SIRS, more severe cardiovascular and respiratory failure, and strong trends to more severe hepatic renal, neurologic, and coagulation dysfunction. The poor clinical phenotype of the patients who had the 12580 G allele was also associated with greater use of corticosteroids. It is suspected that the reason for increased use of corticosteroids (see
Described herein are haplotypes that are novel in their ability to predict patient outcome due to inflammation, and haplotypes that are much more functional in describing patient risk than the previously described 4G/5G polymorphism. As a result, provided herein is a better strategy of predicting which patients are at greatest risk of an adverse outcome.
The PAI-1 4G allele is shown here to be divided into two distinct haplotype clades as a result of a remote recombination event. Inspection indicates that a recombinant event occurred between the 4G/5G site (position 837) in the promoter region and position 4588 (see
From our results the 4G allele of PAI-1 is not associated with poor prognosis. As described herein a SNP within the 12580 G haplotype clade (clade F) is a better candidate for predicting patient outcome, because the 12580 G haplotype clade was strongly associated with adverse outcomes. In the haplotype map (
In a cohort of critically ill patients, 95% having SIRS, analysis based on the haplotype structure of the PAI-1 gene suggested that the 4G/5G polymorphism was not associated with adverse outcome. Rather, a polymorphism contained within the haplotype clade defined by the minor G allele of the 12580 T/G polymorphism, was a better candidate. The 12580 G haplotype clade was associated with decreased survival and increased organ dysfunction, severity of SIRS, and occurrence of sepsis and septic shock. The association of the 12580 G haplotype clade with adverse outcome is important because it shows that the very commonly reported 4G/5G SNP is not the best predictor of patient outcome. This has implications for critically ill patients who have SIRS and possibly for patients having other diseases (e.g. stroke, myocardial infarction) in which 4G/5G has been reported as associated with adverse outcomes.
The PAI-1 12580 G allele was associated with lower serum PAI-1 levels (FIGS. 13A/B and 15A/B) and with fewer days alive and free of organ dysfunction (respiratory, acute lung injury), support (mechanical ventilation and steroid use) and is associated with significantly lower systemic vascular resistance index in cardiac surgical patients. Therefore, the 12580 G allele is a novel indicator of poor outcome (survival and days alive and free of organ dysfunction as described above) and lower serum PAI-1 levels.
In conclusion, the 4G/5G polymorphism of PAI-1 is not as good a predictor of outcome in patients with SIRS, but rather, a polymorphism contained within the haplotype clade defined by the minor G allele of the 12580 T/G polymorphism, is a better candidate.
This application relates to U.S. provisional application No. 60/455,550 filed Mar. 19, 2003, which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60455550 | Mar 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10549804 | Mar 2006 | US |
| Child | 12900387 | US |
