# Patent

## Title
Protein C polymorphisms

## Abstract

The invention provides methods and kits for obtaining a prognosis for a patient having or at risk of developing an inflammatory condition. The method generally comprises determining a protein C promoter genotype of a patient for a polymorphism in the protein C promoter region of the patient, comparing the determined genotype with known genotypes for the polymorphism that correspond with the ability of the patient to recover from the inflammatory condition and identifying patients based on their prognosis. The invention also provides for methods of identifying other polymorphisms that correspond with the ability of the patient to recover from the inflammatory condition.

## Background
RELATED APPLICATION DATA
This application relates to U.S. provisional application No. 60/383,128 filed May 28, 2002, which is incorporated herein by reference.


FIELD OF THE INVENTION
The field of the invention relates to the assessment or treatment of patients with an inflammatory condition.
BACKGROUND OF THE INVENTION
Protein C, when activated to form activated protein C (APC), plays a major role in three biological processes or conditions: coagulation, fibrinolysis and inflammation. Acute inflammatory states decrease levels of the free form of protein S, which decreases APC function because free protein S is an important co-factor for APC. Sepsis, acute inflammation and cytokines decrease thrombomodulin expression on endothelial cells resulting in decreased APC activity or levels. Septic shock also increases circulating levels of thrombomodulin, which is related to increased cleavage of endothelial cell thrombomodulin. Another mechanism for decreased APC function in sepsis is that endotoxin and cytokines, such as TNF-α, down-regulate endothelial cell protein C receptor (EPCR) expression, thereby decreasing action of APC. Severe septic states such as meningococcemia, also result in protein C consumption. Depressed protein C levels correlate with purpura, digital infarction and death in meningococcemia.
Protein C is altered in non-septic patients following cardiopulmonary bypass (CPB). Total protein C, APC and protein S decrease during CPB. Following aortic unclamping (reperfusion at the end of CPB) protein C is further activated so that the proportion of remaining non-activated protein C is greatly decreased. A decrease of protein C during and after CPB increases the risk of thrombosis, disseminated intravascular coagulation (DIC), organ ischemia and inflammation intra- and post-operatively. Patients who have less activated protein C generally have impaired recovery of cardiac function, consistent with the idea that lower levels of protein C increase the risk of microvascular thrombosis and myocardial ischemia. Aprotinin is a competitive inhibitor of APC, and is sometimes used in cardiac surgery and CPB. Aprotinin has been implicated as a cause of post-operative thrombotic complications after deep hypothermic circulatory arrest.
Septic and non-septic stimuli such as bacterial endotoxin and cardiopulmonary bypass (CPB), activate the coagulation system and trigger a systemic inflammatory response syndrome (SIRS). A decrease in protein C levels have been shown in patients with septic shock (GRIFFIN J H. et al. (1982) Blood 60:261-264; TAYLOR F B. et al. (1987) J. Clin. Invest. 79:918-925; HESSELVIK J F. et al. (1991) Thromb. Haemost. 65:126-129; FIJNVANDRAAT K. et al. (1995) Thromb. Haemost. 73(1):15-20), with severe infection (HESSELVIK J F. et al. (1991) Thromb. Haemost. 65:126-129) and after major surgery (BLAMEY S L. et al. (1985) Thromb. Haemost. 54:622-625). It has been suggested that this decrease is caused by a decrease in protein C transcription (SPEK C A. et al. J. Biological Chemistry (1995) 270(41):24216-21 at 24221). It has also been demonstrated that endothelial pathways required for protein C activation are impaired in severe menigococcal sepsis (FAUST S N. et al. New Eng. J. Med. (2001) 345:408-416). Low protein C levels in sepsis patients are related to poor prognosis (YAN S B. and DHAINAUT J-F. Critical Care Medicine (2001) 29(7):S69-S74; FISHER C J. and YAN S B. Critical Care Medicine (2000) 28(9 Suppl):S49-S56; VERVLOET M G. et al. Semin Thromb Hemost. (1998) 24(1):33-44; LORENTE J A. et al. Chest (1993) 103(5):1536-42). Recombinant human activated protein C reduces mortality in patients having severe sepsis or septic shock (BERNARD G R. et al. New Eng. J. Med. (2001) 344:699-709). Thus protein C appears to play an important beneficial role in the systemic inflammatory response syndrome.
The human protein C gene maps to chromosome 2q13-q14 and extends over 11 kb. A representative Homo sapiens protein C gene sequence is listed in GenBank under accession number AF378903. Three single nucleotide polymorphisms (SNPs) have been identified in the 5′ untranslated promoter region of the protein C gene and are characterized as −1654 C/T, −1641 A/G and −1476 A/T (according to the numbering scheme of FOSTER D C. et al. Proc Natl Acad Sci USA (1985) 82(14):4673-4677), or as −153C/T, −140A/G and +26A/T respectively by (MILLAR D S. et al. Hum. Genet. (2000) 106:646-653 at 651).
The genotype homozygous for −1654 C/−1641 G/−1476 T has been associated with reduced rates of transcription of the protein C gene as compared to the −1654 T/−1641 A/ −1476 A homozygous genotype (SCOPES D. et al. Blood Coagul. Fibrinolysis (1995) 6(4):317-321). Patients homozygous for the −1654 C/−1641 G/−1476 T genotype show a decrease of 22% in plasma protein C levels and protein C activity levels as compared to patients homozygous for the −1654 T/−1641 A/−1476 A genotype (SPEK C A. et al. Arteriosclerosis, Thrombosis, and Vascular Biology (1995) 15:214-218). The −1654 C/−1641 G haplotype has been associated with lower protein C concentrations in both homozygotes and heterozygotes as compared to −1654 T/−1641 A (AIACH M. et al. Arterioscler Thromb Vasc Biol. (1999) 19(6):1573-1576).
SUMMARY OF THE INVENTION
This invention is based in part on the surprising discovery that two of the protein C promoter polymorphisms characterized in the scientific literature as being associated with decreased protein C are associated with improved prognosis or patient outcome, in patients with an inflammatory condition. Further, various protein C polymorphisms are useful for patient screening, as an indication of patient outcome, or for prognosis for recovery 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 protein C gene for the patient, wherein said genotype is indicative of an ability of the patient to recover from an inflammatory condition.
The polymorphism site may correspond to position 2418 of SEQ ID NO.: 1 or a polymorphism site linked thereto. Alternatively, the polymorphism site corresponds to position 2418, 1386, 2583 or 3920 in SEQ ID NO: 1.
Genotype may also be determined at a combination of two or more polymorphism sites, the combination being selected from the group of positions corresponds to SEQ ID NO:1 consisting of:

    
    
        5867 and 2405;
        5867 and 4919;
        5867 and 4956;
        5867 and 6187;
        5867 and 9534;
        5867 and 12109;
        4800 and 2405;
        4800 and 4919;
        4800 and 4956;
        4800 and 6187;
        4800 and 9534;
        4800 and 12109;
        9198 and 6379 and 2405;
        9198 and 6379 and 4919;
        9198 and 6379 and 4956;
        9198 and 6379 and 6187;
        9198 and 6379 and 9534; and
        9198 and 6379 and 12109.
    
    


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:

    
    
        1386 T;
        2418 A;
        2583 A;
        3920 T;
        5867 A and 2405 T;
        5867 A and 4919 A;
        5867 A and 4956 T;
        5867 A and 6187 C;
        5867 A and 9534 T;
        5867 A and 12109 T;
        4800 G and 2405 T;
        4800 G and 4919 A;
        4800 G and 4956 T;
        4800 G and 6187 C;
        4800 G and 9534 T;
        4800 G and 12109 T;
        9198 A and 6379 G and 2405 T;
        9198 A and 6379 G and 4919 A;
        9198 A and 6379 G and 4956 T;
        9198 A and 6379 G and 6187 C;
        9198 A and 6379 G and 9534 T; and
        9198 A and 6379 G and 12109 T.
    
    


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:

    
    
        1386 C;
        2418 G;
        2583 T;
        3920 C;
        5867 G and 2405 C;
        5867 G and 4919 G;
        5867 G and 4956 C;
        5867 G and 6187 T;
        5867 G and 9534 C;
        5867 G and 12109 C;
        4800 C and 2405 C;
        4800 C and 4919 G;
        4800 C and 4956 C;
        4800 C and 6187 T;
        4800 C and 9534 C; and
        4800 C and 12109 C.
    
    


In accordance with another aspect of the invention, methods are provided for identifying a polymorphism in a protein C gene sequence that correlates with patient prognosis. Where the method comprises obtaining protein C gene sequence information from a group of patients, identifying a site of at least one polymorphism in the protein C 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 abilities.
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 protein C 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 2418, is adenine (A), the prognosis may be indicative of a decreased likelihood of recovery from an inflammatory condition or of severe cardiovascular or respiratory dysfinction in critically ill patients.
Where the genotype of the patient corresponding to the nucleotide in position 2418, is guanine (G), 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.
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 protein C 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 2418 of SEQ ID NO: 1, position 8 of SEQ ID NO: 2 or to a polymorphism linked thereto. The alternate nucleotides may also correspond to one or more of positions 2418, 1386, 2583, and 3920 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 protein C 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 sreening, comprising the steps of (a) obtaining protein C 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 protein C gene sequence information from the patient and (c) detecting the identity of one or more polymorphisms in the protein C gene, wherein the polymorphism is indicative of the ability of a patient to recover from an inflammatory condition.
The above sequence positions refer to the sense strand of the protein C gene promoter region. 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 higher protein C expression or conversely an indication of less severe patient outcome or prognosis may be correlated with lower protein C expression, which is the opposite of what would be expected.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a comparison of survival rates for patients in shock with those not in shock by genotype of protein C 2418.
FIG. 2 shows a series of graphs plotting Mean Arterial Pressure (mm Hg) over time before and after cardiopulmonary bypass; Cardiac Index (L/m2) over time before and after cardiopulmonary bypass; Systemic Vascular Resistance Index over time before and after cardiopulmonary bypass; and Vasopressor Use over time before and after cardiopulmonary bypass, with each graph comparing AA homozygotes with AG heterozygotes and GG homozygotes of protein C 2418.
FIG. 3 shows a graph plotting percent Arterial Oxygen Saturation over time before and after cardiopulmonary bypass, comparing AA homozygotes with AG heterozygotes and GG homozygotes of protein C 2418.



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.
“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. 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.
The “promoter” region is 5′ or upstream of the translation start site, in this case the translation start site is located at position 4062 of TABLE 1A (SEQ. ID NO: 1) and the transcription start site is located at position 2559 of TABLE 1A (SEQ. ID NO: 1).
Numerous other sites have been identified as polymorphisms in the protein C gene, where those polymorphisms are linked to the polymorphism at position 2418 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 2418 of SEQ. ID NO.: 1:

    
    
        1386;
        2583;
        3920;
        5867 and 2405;
        5867 and 4919;
        5867 and 4956;
        5867 and 6187;
        5867 and 9534;
        5867 and 12109;
        4800 and 2405;
        4800 and 4919;
        4800 and 4956;
        4800 and 6187;
        4800 and 9534;
        4800 and 12109;
        9198 and 6379 and 2405;
        9198 and 6379 and 4919;
        9198 and 6379 and 4956;
        9198 and 6379 and 6187;
        9198 and 6379 and 9534; and
        9198 and 6379 and 12109.
    
    


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 protein C can be created by assessing the SNP's of protein C in normal subjects using a program that has an expectation maximization algorithm (i.e. PHASE). A constructed haplotype of protein C may be used to find combinations of SNP's that are in total linkage disequilibrium (LD) with 2418. Therefore, the haplotype of an individual could be determined by genotyping other SNP's that are in total LD with 2418. 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 indicative of a decreased likelihood of recovery from an inflammatory condition or indicative of severe cardiovascular or respiratory dysfunction in critically ill patients:

    
    
        1386 T;
        2583 A;
        3920 T;
        5867 A and 2405 T;
        5867 A and 4919 A;
        5867 A and 4956 T;
        5867 A and 6187 C;
        5867 A and 9534 T;
        5867 A and 12109 T;
        4800 G and 2405 T;
        4800 G and 4919 A;
        4800 G and 4956 T;
        4800 G and 6187 C;
        4800 G and 9534 T;
        4800 G and 12109 T;
        9198 A and 6379 G and 2405 T;
        9198 A and 6379 G and 4919 A;
        9198 A and 6379 G and 4956 T;
        9198 A and 6379 G and 6187 C;
        9198 A and 6379 G and 9534 T; and
        9198 A and 6379 G and 12109 T.
    
    


Whereas the following genotypes for single polymorphism sites and combined polymorphism sites may indicative of a increased likelihood of recovery from an inflammatory condition or indicative of less severe cardiovascular or respiratory dysfunction in critically ill patients:

    
    
        1386 C;
        2583 T;
        3920 C;
        5867 G and 2405 C;
        5867 G and 4919 G;
        5867 G and 4956 C;
        5867 G and 6187 T;
        5867 G and 9534 C;
        5867 G and 12109; C
        4800 C and 2405 C;
        4800 C and 4919 G;
        4800 C and 4956 C;
        4800 C and 6187 T;
        4800 C and 9534 C; and
        4800 C and 12109 C.
    
    


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 polymorphisms in Foster et al. and Millar et al. above. 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 protein C gene sequence and comprises a sequence as listed in GenBank under accession number AF378903. The SNP's described as −1654 C/T, −1641 A/G and −1476 A/T using the numbering system of Foster et al. correspond to 2405, 2418 and 2583 respectively in TABLE 1A (SEQ ID NO: 1). Polymorphism sites shown below in TABLE 1A are represented by a capital N at the apex of an open triangle. The N is used to indicate that variation in genotype is possible at those positions within a population. The 2418 polymorphism is represented by an N which indicates that the nucleotide at that position may be an a, t, u, g or c. However, the genotype at position 2418 is most commonly an a or g (purine) nucleotide.









TABLE 1A







1
gctctctaac tcacagcgag ctcgctgccc aaagtcctgc




tccgggggct tcctgggtgg


 


61
acctgaccgc gttcgggtgc acgtggggcg actcacacct



gacaagtaaa gcgggtgagg


 


121
ccgcgcctgt gaagggcgcc tggctcctcc gcaggacggt



gcggcgcggc gcccccggct


 


181
ggaaccaggt gtaactgcag agaccctggg atcgcaggaa



cggctggcgg caggactgtc


 


241
cctacctcga gaaggtgacg gggtttcctg cgctgccagc



cgatgaggcg gccgtgacgc


 


301
agcccgccgt gcagagtccc cgtcggccga caggcgtgca



gagctctgca gaggaccctt


 


361
ccgccctctg ggcagcctgc caagccgtgg cacccccaac



ccccagcact gggcacttgg


 


421
gagcattgca gccgccctgg ctcgtaccgg tgccggtgct



ttgggcacct gggctggttt


 


481
ggacatgggt gccccgggca gagtccattt atgcaggtca



gaatcagtgt gtggagcctg


 


541
catagacttg ccctggagcg gctgcctgtg ctggggtggg



gaggagtaga gggcgagaag


 


601
ttggtgggga agggaagcgg cgccaaaaga atacccacaa



catcttgcac ctggaaggca


 


661
aagcagaggg cagtgatctc tgcagacttg cgggggcgac



gcctgaagca aacagggaca


 


721
tacaagctgg tgccttctgt ggttgtgcat ggggtcttca



tgcttcctgt ctgagttccc


 


781
agaagcttgt ctctgctttt ctaggcagct gccacagcct



gtcacaaaca gctcctggtt


 


841
ctccacttct catagtctcg atttcaaaat ccattgcctc



accctccacc tcctctccac


 


901
ctccacccct cctagcacct cctgactgct tgtgttctgt



gtctccccac tgtctcccaa


 


961
cctggggtgg ggttgggggg gatgtctttc ctcctgtctg



ctctttgatg tccagctgaa


 


1021
gtgtcacctc ctacaggcag cctcccctgg ctatgccagc



ttgtactgat tgccctctcc


 


1081
tctgaattct gtaagcattt cctatgtgta cctgcccctg



ggcaaggtgg gcctgacttg


 


1141
ttagagtgtt agagttttac cctgttcctc taggagggcc



tggtaccacc acagcccagc


 


1201
atggtgtggt gcctcagcag gaggcatctg gttacaatca



acacaagctg ttccagccaa


 


1261
tttaaagaaa cttcaggagg aatagggttt taggagggca



tggggaccct cctgcacccg


 


1321
aagccaggat gtgccaccaa tcataaggag gcaggggcct



ccttccgctg ctccctggga


 


1381
ctctcNaggt gtccgtggcc tcagcccccc tctgcacacc



     Δ



tgcatcttcc ttctcatcag


 


1441
cttcctctgc tttaagcgta aacatggatg cccaggacct



ggcctcaatc ttccgagtct


 


1501
ggtacttatg gtgtactgac agtgtgagac cctactcctc



tgatcaatcc cctgggttgg


 


1561
tgacttccct gtgcaatcaa tggaagccag cgaggcaggg



tcacatgccc cgtttagagg


 


1621
tgcagacttg gagaaggaac gtgggcaagt cttcccagga



acaggtaggg cagggaggaa


 


1681
aggggggcat ctctggtgca gcccggttcg gagcaggaag



acgcttaata aatgctgata


 


1741
gactgcagga cacaggcaaa ggtgctgagc tggacccttt



atttctgccc ttctcccttc


 


1801
tggcaccccg gccaggaaat tgctgcagcc tttctggaat



cccgttcatt tttcttactg


 


1861
gtccacaaaa ggggccaaat ggaagcagca agacctgagt



tcaaattaaa tctgccaact


 


1921
accagctcag tgaatctggg cgagtaacac aaaacttgag



tgtccttacc tgaaaaatag


 


1981
aggttagagg gatgctatgt gccattgtgt gtgtgtgttg



ggggtgggga ttgggggtga


 


2041
tttgtgagca attggaggtg agggtggagc ccagtgccca



gcacctatgc actggggacc


 


2101
caaaaaggag catcttctca tgattttatg tatcagaaat



tgggatggca tgtcattggg


 


2161
acagcgtctt ttttcttgta tggtggcaca taaatacatg



tgtcttataa ttaatggtat


 


2221
tttagatttg acgaaatatg gaatattacc tgttgtgctg



atcttgggca aactataata


 


2281
tctctgggca aaaatgtccc catctgaaaa acagggacaa



cgttcctccc tcagccagcc


 


2341
actatggggc taaaatgaga ccacatctgt caagggtttt



gccctcacct ccctccctgc


 


2401
tggaNggcat ccttggtNgg cagaggtggg cttcgggcag



    Δ             Δ



aacaagccgt gctgagctag


 


2461
gaccaggagt gctagtgcca ctgtttgtct atggagaggg



aggcctcagt gctgagggcc


 


2521
aagcaaatat ttgtggttat ggattaactc gaactccagg



ctgtcatggc ggcaggacgg


 


2581
cgNacttgca gtatctccac gacccgcccc tgtgagtccc



  Δ



cctccaggca ggtctatgag


 


2641
gggtgtggag ggagggctgc ccccgggaga agagagctag



gtggtgatga gggctgaatc


 


2701
ctccagccag ggtgctcaac aagcctgagc ttggggtaaa



aggacacaag gccctccaca


 


2761
ggccaggcct ggcagccaca gtctcaggtc cctttgccat



gcgcctccct ctttccaggc


 


2821
caagggtccc cagggcccag ggccattcca acagacagtt



tggagcccag gaccctccat


 


2881
tctccccacc ccacttccac ctttgggggt gtcggatttg



aacaaatctc agaagcggcc


 


2941
tcagagggag tcggcaagaa tggagagcag ggtccggtag



ggtgtgcaga gggccacgtg


 


3001
gcctatccac tggggagggt tccttgatct ctggccacca



gggctatctc tgtggccttt


 


3061
tggagcacct ggtggtttgg ggcaggggtt gaatttccag



gcctaaaacc acacaggcct


 


3121
ggccttgagt cctggctctg cgagtaatgc atggatgtaa



acatggagac ccaggacctt


 


3181
gcctcagtct tccgagtctg gtgcctgcag tgtactgatg



gtgtgagacc ctactcctgg


 


3241
aggatggggg acagaatctg atcgatcccc tgggttggtg



acttccctgt gcaatcaacg


 


3301
gagaccagca agggttggat ttttaataaa ccacttaact



cctccgagtc tcagtttccc


 


3361
cctctatgaa atggggttga cagcattaat aactacctct



tgggtggttg tgagccttaa


 


3421
ctgaagtcat aatatctcat gtttactgag catgagctat



gtgcaaagcc tgttttgaga


 


3481
gctttatgtg gactaactcc tttaattctc acaacaccct



ttaaggcaca gatacaccac


 


3541
gttattccat ccattttaca aatgaggaaa ctgaggcatg



gagcagttaa gcatcttgcc


 


3601
caacattgcc ctccagtaag tgctggagct ggaatttgca



ccgtgcagtc tggcttcatg


 


3661
gcctgccctg tgaatcctgt aaaaattgtt tgaaagacac



catgagtgtc caatcaacgt


 


3721
tagctaatat tctcagccca gtcatcagac cggcagaggc



agccacccca ctgtccccag


 


3781
ggaggacaca aacatcctgg caccctctcc actgcattct



ggagctgctt tctaggcagg


 


3841
cagtgtgagc tcagccccac gtagagcggg cagccgaggc



cttctgaggc tatgtctcta


 


3901
gcgaacaagg accctcaatN ccagcttccg ccctgacggc



                    Δ



cagcacacag ggacagccct


 


3961
ttcattccgc ttccacctgg gggtgcaggc agagcagcag



cgggggtagg cactgcccgg


 


4021
agctcagaag tcctcctcag acaggtgcca gtgcctccag



aatgtggcag ctcacaagcc


 


4081
tcctgctgtt cgtggccacc tggggaattt ccggcacacc



agctcctctt ggtaaggcca


 


4141
ccccacccct accccgggac ccttgtggcc tctacaaggc



ctggtggcat ctgcccaggc


 


4201
cttcacagct tccaccatct ctctgagccc tgggtgaggt



gaggggcaga tgggaatggc


 


4261
aggaatcaac tgacaagtcc caggtaggcc agctgccaga



gtgccacaca ggggctgcca


 


4321
gggcaggcat gcgtgatggc agggagcccc gcgatgacct



cctaaagctc cctcctccac


 


4381
acggggatgg tcacagagtc ccctgggcct tccctctcca



cccactcact ccctcaactg


 


4441
tgaagacccc aggcccaggc taccgtccac actatccagc



acagcctccc ctactcaaat


 


4501
gcacactggc ctcacggctg ccctgcccca acccctttcc



tggtctccac agccaacggg


 


4561
aggaggccat gattcttggg gaggtccgca ggacacatgg



gcccctaaag ccacaccagg


 


4621
ctgttggttt catttgtgcc tttatagagc tgtttatctg



cttgggacct gcacctccac


 


4681
cctttcccaa ggtgccctca gctcaggcat accctcctct



aggatgcctt ttcccccatc


 


4741
ccttcttgct cacaccccca acttgatctc tccctcctaa



ctgtgccctg cacccaagaN



                    Δ


 


4801
agacacttca cagagcccag gagacacctg gggacccttc



ctgggtgata ggtctgtcta


 


4861
tcctccaggt gtccctgccc aaggggagaa gcatggggaa



tacttggttg ggggaggaNa



                   Δ


 


4921
ggaagactgg ggggatgtgt caagatgggg ctgcaNgtgg



                                      Δ



tgtactggca gaagagtgag


 


4981
aggatttaac ttggcagcct ttacagcagc agccagggct



tgagtactta tctctgggcc


 


5041
agggactgta ttggatgttt tacatgacgg tctcatcccc



atgtttttgg atgagtaaat


 


5101
tgaaccttag aaaggtaaag acactggctc aaggtcacac



agagatcggg gtggggttca


 


5161
cagggaggcc tgtccatctc agagcaaggc ttcgtcctcc



aactgccatc tgcttcctgg


 


5221
ggaggaaaag agcagaggac ccctgcgcca agccatgacc



tagaattaga atgagtcttg


 


5281
agggggcgga gacaagacct tcccaggctc tcccagctct



gcttcctcag accccctcat


 


5341
ggccccagcc cctcttaggc ccctccacca aggtgagctc



cccctccctc caaaaccaga


 


5401
ctcagtgttc tccagcagcg agcgtgccca ccaggtgctg



cggatccgca aacgtgccaa


 


5461
ctccttcctg gaggagctcc gtcacagcag cctggagcgg



gagtgcatag aggagatctg


 


5521
tgacttcgag gaggccaagg aaattttcca aaatgtggat



gacacagtaa ggccaccatg


 


5581
ggtccagagg atgaggctca ggggcgagct ggtaaccagc



aggggcctcg aggagcaggt


 


5641
ggggactcaa tgctgaggcc ctcttaggag ttgtgggggt



ggctgagtgg agcgattagg


 


5701
atgctggccc tatgatgtcg gccaggcaca tgtgactgca



agaaacagaa ttcaggaaga


 


5761
agctccagga aagagtgtgg ggtgacccta ggtggggact



cccaccagcc acagtgtagg


 


5821
tggttcagtc caccctccag ccactgctga gcaccactgc



ctccccNtcc cacctcacaa



      Δ


 


5881
agaggggacc taaagaccac cctgcttcca cccatgcctc



tgctgatcag ggtgtgtgtg


 


5941
tgaccgaaac tcacttctgt ccacataaaa tcgctcactc



tgtgcctcac atcaaaggga


 


6001
gaaaatctga ttgttcaggg ggtcggaaga cagggtctgt



gtcctatttg tctaagggtc


 


6061
agagtccttt ggagccccca gagtcctgtg gacgtggccc



taggtagtag ggtgagcttg


 


6121
gtaacggggc tggcttcctg agacaaggct cagacccgct



ctgtccctgg ggatcgcttc


 


6181
agccacNagg acctgaaaat tgtgcacggc ctgggccccc



      Δ



ttccaaggca tccagggatg


 


6241
ctttccagtg gaggctttca gggcaggaga ccctctggcc



tgcaccctct cttgccctca


 


6301
gcctccacct ccttgactgg acccccatct ggacctccat



ccccaccacc tctttcccca


 


6361
gtggcctccc tggcagacNc cacagtgact ttctgcaggc



                   Δ



acatatctga tcacatcaag


 


6421
tccccaccgt gctcccacct cacccatggt ctctcagccc



cagcaggcct tggctggcct


 


6481
ctctgatgga gcaggcatca ggcacaggcc gtgggtctca



acgtgggctg ggtggtcctg


 


6541
gaccagcagc agccgccgca gcagcaaccc tggtacctgg



ttaggaacgc agaccctctg


 


6601
cccccatcct cccaactctg aaaaacactg gcttagggaa



aggcgcgatg ctcaggggtc


 


6661
ccccaaagcc cgcaggcaga gggagtgatg ggactggaag



gaggccgagt gacttggtga


 


6721
gggattcggg tcccttgcat gccagaggct gctgtgggag



cggacagtcg cgagagcagc


 


6781
actgcagctg catggggaga gggtgttgct ccagggacgt



gggatggagg ctgggcgcgg


 


6841
gcgggtggcg ctggagggcg ggggaggggc agggagcacc



agctcctagc agccaacgac


 


6901
catcgggcgt cgatccctgt ttgtctggaa gccctcccct



cccctgcccg ctcacccgct


 


6961
gccctgcccc acccgggcgc gccccctccg cacaccggct



gcaggagcct gacgctgccc


 


7021
gctctctccg cagctggcct tctggtccaa gcacgtcggt



gagtgcgttc tagatccccg


 


7081
gctggactac cggcgcccgc gcccctcggg atctctggcc



gctgaccccc taccccgcct


 


7141
tgtgtcgcag acggtgacca gtgcttggtc ttgcccttgg



agcacccgtg cgccagcctg


 


7201
tgctgcgggc acggcacgtg catcgacggc atcggcagct



tcagctgcga ctgccgcagc


 


7261
ggctgggagg gccgcttctg ccagcgcggt gagggggaga



ggtggatgct ggcgggcggc


 


7321
ggggcggggc tggggccggg ttgggggcgc ggcaccagca



ccagctgccc gcgccctccc


 


7381
ctgcccgcag aggtgagctt cctcaattgc tcgctggaca



acggcggctg cacgcattac


 


7441
tgcctagagg aggtgggctg gcggcgctgt agctgtgcgc



ctggctacaa gctgggggac


 


7501
gacctcctgc agtgtcaccc cgcaggtgag aagcccccaa



tacatcgccc aggaatcacg


 


7561
ctgggtgcgg ggtgggcagg cccctgacgg ggcgcggcgc



ggggggctca ggagggtttc


 


7621
tagggaggga gcgaggaaca gagttgagcc ttggggcagc



ggcagacgcg ccccaacacc


 


7681
ggggccactg ttagcgcaat cagcccggga gctgggcgcg



ccctccgctt tccctgcttc


 


7741
ctttcttcct ggcgtccccg ccttcctccg ggcgccccct



gcgcacctgg ggccacctcc


 


7801
tggagcgcaa gcccagtggt ggctccgctc cccagtctga



gcgtatctgg ggcgaggcgt


 


7861
gcagcgtcct cctccatgta gcctggctgc gtttttctct



gacgttgtcc ggcgtgcatc


 


7921
gcatttccct ctttaccccc ttgcttcctt gaggagagaa



cagaatcccg attctgcctt


 


7981
cttctatatt ttccttttta tgcattttaa tcaaatttat



atatgtatga aactttaaaa


 


8041
atcagagttt tacaactctt acatttcagc atgctgttcc



ttggcatggg tccttttttc


 


8101
attcattttc attaaaaggt ggaccctttt aatgtggaaa



ttcctatctt ctgcctctag


 


8161
ggacatttat cacttatttc ttctacaatc tcccctttac



ttcctctatt ttctctttct


 


8221
ggacctccca ttattcagac ctctttcctc tagttttatt



gtctcttcta tttcccatct


 


8281
ctttgacttt gtgttttctt tcagggaact ttcttttttt



tctttttttt tgagatggag


 


8341
tttcactctt gttgtcccag gctggagtgc aatgacgtga



tctcagctca ccacaacctc


 


8401
cgcctcctgg attcaagcga ttctcctgcc gcagcctccc



gagtagctgg gattacaggc


 


8461
atgcgccacc acgcccagct aattttgtgt ttttagtaga



gaaggggttt ctccgtgttg


 


8521
gtcaagctgg tcttgaactc ctgacctcag gtgatccacc



tgccttggcc tcctaaagtg


 


8581
ctgggattac aggcgtgagc caccgcgccc agcctctttc



agggaacttt ctacaacttt


 


8641
ataattcaat tcttctgcag aaaaaaattt ttggccaggc



tcagtagctc agaccaataa


 


8701
ttccagcact ttgagaggct gaggtgggag gattgcttga



gcttgggagt ttgagactag


 


8761
cctgggcaac acagtgagac cctgtctcta tttttaaaaa



aagtaaaaaa agatctaaaa


 


8821
atttaacttt ttattttgaa ataattagat atttccagga



agctgcaaag aaatgcctgg


 


8881
tgggcctgtt ggcctgtggg tttcctgcaa ggccgtggga



aggccctgtc attggcagaa


 


8941
ccccagatcg tgagggcttt ccttttaggc tgctttctaa



gaggactcct ccaagctctt


 


9001
ggaggatgga agacgctcac ccatggtgtt cggcccctca



gagcagggtg gggcagggga


 


9061
gctggtgcct gtgcaggctg tggacatttg catgactccc



tgtggtcagc taagagcacc


 


9121
actccttcct gaagcggggc ctgaagtccc tagtcagagc



ctctggttca ccttctgcag


 


9181
gcagggagag gggagtcNag tcagtgagga gggctttcgc



                  Δ



agtttctctt acaaactctc


 


9241
aacatgccct cccacctgca ctgccttcct ggaagcccca



cagcctccta tggttccgtg


 


9301
gtccagtcct tcagcttctg ggcgccccca tcacgggctg



agatttttgc tttccagtct


 


9361
gccaagtcag ttactgtgtc catccatctg ctgtcagctt



ctggaattgt tgctgttgtg


 


9421
ccctttccat tcttttgtta tgatgcagct cccctgctga



cgacgtccca ttgctctttt


 


9481
aagtctagat atctggactg ggcattcaag gcccattttg



agcagagtcg ggcNgacctt



              Δ


 


9541
tcagccctca gttctccatg gagtatgcgc tctcttcttg



gcagggaggc ctcacaaaca


 


9601
tgccatgcct attgtaggag ctctccaaga atgctcacct



ccttctccct gtaattcctt


 


9661
tcctctgtga ggagctcagc agcatcccat tatgagacct



tactaatccc agggatcacc


 


9721
cccaacagcc ctggggtaca atgagctttt aagaagttta



accacctatg taaggagaca


 


9781
caggcagtgg gcgatgctgc ctggcctgac tcttgccatt



gggtggtact gtttgttgac


 


9841
tgactgactg actgactgga gggggtttgt aatttgtatc



tcagggatta cccccaacag


 


9901
ccctggggta caatgagcct tcaagaagtt taacaaccta



tgtaaggaca cacagccagt


 


9961
gggtgatgct gcctggtctg actcttgcca ttcagtggca



ctgtttgttg actgactgac


 


10021
tgactgactg gctgactgga gggggttcat agctaatatt



aatggagtgg tctaagtatc


 


10081
attggttcct tgaaccctgc actgtggcaa agtggcccac



aggctggagg aggaccaaga


 


10141
caggagggca gtctcgggag gagtgcctgg caggcccctc



accacctctg cctacctcag


 


10201
tgaagttccc ttgtgggagg ccctggaagc ggatggagaa



gaagcgcagt cacctgaaac


 


10261
gagacacaga agaccaagaa gaccaagtag atccgcggct



cattgatggg aagatgacca


 


10321
ggcggggaga cagcccctgg caggtgggag gcgaggcagc



accggctgct cacgtgctgg


 


10381
gtccgggatc actgagtcca tcctggcagc tatgctcagg



gtgcagaaac cgagagggaa


 


10441
gcgctgccat tgcgtttggg ggatgatgaa ggtgggggat



gcttcaggga aagatggacg


 


10501
caacctgagg ggagaggagc agccagggtg ggtgagggga



ggggcatggg ggcatggagg


 


10561
ggtctgcagg agggagggtt acagtttcta aaaagagctg



gaaagacact gctctgctgg


 


10621
cgggatttta ggcagaagcc ctgctgatgg gagagggcta



ggagggaggg ccgggcctga


 


10681
gtacccctcc agcctccaca tgggaactga cacttactgg



gttcccctct ctgccaggca


 


10741
tgggggagat aggaaccaac aagtgggagt atttgccctg



gggactcaga ctctgcaagg


 


10801
gtcaggaccc caaagacccg gcagcccagt gggaccacag



ccaggacggc ccttcaagat


 


10861
aggggctgag ggaggcccaa ggggaacatc caggcagcct



gggggccaca aagtcttcct


 


10921
ggaagacaca aggcctggcc aagcctctaa ggatgagagg



agctcgctgg gcgatgttgg


 


10981
gtgtggctga gggtgactga aacagtatga acagtgcagg



aacagcatgg gcaaaggcag


 


11041
gaagacaccc tgggacaggc tgacactgta aaatgggcaa



aaatagaaaa cgccagaaag


 


11101
ggcctaagcc tatgcccata tgaccaggga acccaggaaa



gtgcatatga aacccaggtg


 


11161
ccctggactg gaggctgtca ggaggcagcc ctgtgatgtc



atcatcccac cccattccag


 


11221
gtggtcctgc tggactcaaa gaagaagctg gcctgcgggg



cagtgctcat ccacccctcc


 


11281
tgggtgctga cagcggccca ctgcatggat gagtccaaga



agctccttgt caggcttggt


 


11341
atgggctgga gccaggcaga agggggctgc cagaggcctg



ggtaggggga ccaggcaggc


 


11401
tgttcaggtt tgggggaccc cgctccccag gtgcttaagc



aagaggcttc ttgagctcca


 


11461
cagaaggtgt ttggggggaa gaggcctatg tgcccccacc



ctgcccaccc atgtacaccc


 


11521
agtattttgc agtagggggt tctctggtgc cctcttcgaa



tctgggcaca ggtacctgca


 


11581
cacacatgtt tgtgaggggc tacacagacc ttcacctctc



cactcccact catgaggagc


 


11641
aggctgtgtg ggcctcagca cccttgggtg cagagaccag



caaggcctgg cctcagggct


 


11701
gtgcctccca cagactgaca gggatggagc tgtacagagg



gagccctagc atctgccaaa


 


11761
gccacaagct gcttccctag caggctgggg gcacctatgc



attggccccg atctatggca


 


11821
atttctggag ggggggtctg gctcaactct ttatgccaaa



aagaaggcaa agcatattga


 


11881
gaaaggccaa attcacattt cctacagcat aatctatggc



cagtggcccc ccgtggggct


 


11941
tggcttagaa ttcccaggtg ctcttcccag ggaaccatca



gtctggactg agaggacctt


 


12001
ctctctcagg tgggacccgg ccctgtcctc cctggcagtg



ccgtgttctg ggggtcctcc


 


12061
tctctgggtc tcactgcccc tggggtctct ccagctacct



ttgctccaNg ttcctttgtg



        Δ


 


12121
gctctggtct gtgtctgggg tttccagggg tctcgggctt



ccctgctgcc cattccttct


 


12181
ctggtctcac ggctccgtga ctcctgaaaa ccaaccagca



tcctacccct ttgggattga


 


12241
cacctgttgg ccactccttc tggcaggaaa agtcaccgtt



gatagggttc cacggcatag


 


12301
acaggtggct ccgcgccagt gcctgggacg tgtgggtgca



cagtctccgg gtgaaccttc


 


12361
ttcaggccct ctgcccaggc ctgcaggggc acagcagtgg



gtgggcctca ggaaagtgcc


 


12421
actggggaga ggctccccgc agcccactct gactgtgccc



tctgccctgc aggagagtat


 


12481
gacctgcggc gctgggagaa gtgggagctg gacctggaca



tcaaggaggt cttcgtccac


 


12541
cccaactaca gcaagagcac caccgacaat gacatcgcac



tgctgcacct ggcccagccc


 


12601
gccaccctct cgcagaccat agtgcccatc tgcctcccgg



acagcggcct tgcagagcgc


 


12661
gagctcaatc aggccggcca ggagaccctc gtgacgggct



ggggctacca cagcagccga


 


12721
gagaaggagg ccaagagaaa ccgcaccttc gtcctcaact



tcatcaagat tcccgtggtc


 


12781
ccgcacaatg agtgcagcga ggtcatgagc aacatggtgt



ctgagaacat gctgtgtgcg


 


12841
ggcatcctcg gggaccggca ggatgcctgc gagggcgaca



gtggggggcc catggtcgcc


 


12901
tccttccacg gcacctggtt cctggtgggc ctggtgagct



ggggtgaggg ctgtgggctc


 


12961
cttcacaact acggcgttta caccaaagtc agccgctacc



tcgactggat ccatgggcac


 


13021
atcagagaca aggaagcccc ccagaagagc tgggcacctt



agcgaccctc cctgcagggc


 


13081
tgggcttttg catggcaatg gatgggacat taaagggaca



tgtaacaagc acaccggcct


 


13141
gctgttctgt ccttccatcc ctcttttggg ctcttctgga



gggaagtaac atttactgag


 


13201
cacctgttgt atgtcacatg ccttatgaat agaatcttaa



ctcctagagc aactctgtgg


 


13261
ggtggggagg agcagatcca agttttgcgg ggtctaaagc



tgtgtgtgtt gagggggata


 


13321
ctctgtttat gaaaaagaat aaaaaacaca accacgaagc



cactagagcc ttttccaggg


 


13381
ctttgggaag agcctgtgca agccggggat gctgaaggtg



aggcttgacc agctttccag


 


13441
ctagcccagc tatgaggtag acatgtttag ctcatatcac



agaggaggaa actgaggggt


 


13501
ctgaaaggtt tacatggtgg agccaggatt caaatctagg



tctgactcca aaacccaggt


 


13561
gcttttttct gttctccact gtcctggagg acagctgttt



cgacggtgct cagtgtggag


 


13621
gccactatta gctctgtagg gaagcagcca gagacccaga



aagtgttggt tcagcccaga


 


13681
atgagctcac agtgtcgcgg gggaagctgt ttaagaacaa



tgttacacca tcatgaacag


 


13741
cagtaagaaa gaggctctgg cttaacctgg cctgataggc



ctaattgaat gagacagaaa


 


13801
taagtcaagg atgctctgat ttgaaatcat gaagtacctg



atgaaaagaa atggtggtga


 


13861
gataaagctg









Table 1B

The sequences shown in TABLE 1B, are sequence fragments taken from the Protein C sequence shown in TABLE 1A above. Furthermore, SEQ ID NO.: 2 corresponds to the sequence underlined in TABLE 1A above. The nucleotide N, at position 8 in SEQ ID NO.: 2 corresponds to the nucleotide found at position 2418 of SEQ ID NO.: 1. In all of the Sequences found in TABLE 2B below the polymorphism represented by an N may substituted by an a, t, u, g or c. Furthermore, bold and underlined nucleotides represented by N in SEQ ID NOs.: 3-11 in TABLE 2B, all correspond to the nucleotide found at position 2418 of SEQ ID NO.: 1. Due to the potential variability in protein C sequence, the sequence motifs below may be useful in identifying protein C sequences from a patient that are suitable for 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 protein C sequence and that the bold and underlined N corresponds to the polymorphism at position 2418 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.











SEQ ID. NO.
SEQUENCE

















SEQ ID. NO. 2
ccttggtNgg cagaggtggg



 


SEQ ID. NO. 3
tggaNggcat ccttggtNgg


 


SEQ ID. NO. 4
Nggcagaggt gggcttcggg


 


SEQ ID. NO. 5
Nggcagaggt gggcttcggg cagaacaagc


 


SEQ ID. NO. 6
gctggaNggc atccttggtN


 


SEQ ID. NO. 7
ctccctccct gctggaNggc atccttggtN


 


SEQ ID. NO. 8
ttgccctcac ctccctccct gctggaNggc



atccttggtN


 


SEQ ID. NO. 9
caagggtttt gccctcacct ccctccctgc



tggaNggcat ccttggtNgg cagaggtggg



cttcgggcag aacaagccgt gctgagctag


 


SEQ ID. NO. 10
ccttggtNgg cagagg


 


SEQ ID. NO. 11
cttggtNggc ag









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 of a nucleotide or an insertion 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.
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. (Vincent J L. Ferreira F. Moreno R. 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 et al., 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 2 or greater (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 dysfinction 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.







TABLE 1C







Brussels multiple organ dysfunction (MOD) score









ABNORMAL



CLINICALLY SIGNIFICANT



ORGAN DYSFUNCTION









Organ Failure Score













Normal
Mild
Moderate
Severe
Extreme


ORGANS
0
1
2
3
4















Cardiovascular
>90
=90
=90
=90 plus
=90 plus


Systolic BP

Responsive
Unresponsive
pH = 7.3
pH = 7.2


(mmHg)

to fluid
to fluid


Pulmonary
>400
400-301
300-201
200-101
 =100


Pao2/FIo2


Acute lung
ARDS
Severe


(mmHg)


injury

ARDS


CNS
15
14-13
12-10
9-6
=5 


(Glascow


Score)


Coagulation
>120
120-81 
80-51
50-21
=20


Platelets


(×105/mm3)


Renal
<1.5
1.5-1.9
2.0-3.4
3.5-4.9
 =5.0


Creatinine


(mg/d)


Hepatic
<1.2
1.2-1.9
2.0-5.9
 6.0-11.9
=12


Bilirubin


(mg/d)





Round Table Conference on Clinical Trials for the Treatment of Sepsis Brussels, Mar. 12-14, 1994 and Russell J A, Singer J, Bernard G R, Drummond A J, Walley K R, and The Ibuprofen in Sepsis Study Group. Changing pattern of organ dysfunction in early human sepsis is related to mortality. Critical Care Medicine 2000; 28: 3405-3411.






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.
2. General Methods
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.
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, Frederick 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, D. D. L. (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 protein C gene. Provided that the genetic material obtained, contains the sequence of interest. Particularly, a person may be interested in determining the protein C promoter genotype of a patient of interest, where the genotype includes a nucleotide corresponding to position 2418 or SEQ ID NO.: 1 or position 8 of SEQ ID NO.: 2. The sequence of interest may also include other protein C 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. (Sanger et al. Proc. Natl. Acad. Sci. USA (1977) 74(12):5463-5467). The Sangermethodrelies 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. Similary, 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. Nati. 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;
        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; Romppanen 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. Nucleic 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; 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 at the position) of the polymorphism of interest. In the present invention, polymorphisms in the protein C promoter region or other protein C gene polymorphisms, are used to obtain a prognosis or to determine patient outcome. 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:

    
    
        (a) use of anti-inflammatory therapy;
        (b) use of steroids;
        (c) use of antibodies to tumor necrosis factor (TNF) or even antibody to endotoxin;
        (d) use of tumor necrosis factor receptor (TNF);
        (e) use of activated Protein C (Xigris from Lilly);
        (f) use of tissue factor pathway inhibitors (tifacogin alpha from Chiron);
        (g) use of platelet activating factor hydrolase (PAFase from ICOS); and
        (h) use of modulators of the coagulation cascade (such as various versions of heparin).
    
    


Alternatively, similar methods may be employed to identify new polymorphisms in protein C 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 protein C gene. Also, as previously described the sequence identity of one or more single nucleotide polymorphisms in the protein C 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.
3. EXAMPLE 1
Patient Outcome or Prognosis in Two Populations using the 2418 Polymorphism
(a) Population 1 Sepsis SIRS
Inclusion Criteria
All patients admitted to the Intensive Care Unit (ICU) between November 2000 and May 2001 were eligible for entry into this study. Patients were excluded if blood could not be obtained for genotype analysis.
Data Collection
Data was recorded for 28 days or until hospital discharge. Raw clinical and laboratory variables were recorded using the worst or most abnormal variable for each 24 hour period with the exception of Glasgow Coma Score, where the best possible score for each 24 hour period was recorded. Missing data on the date of admission was assigned a normal value and missing data after the day one was substituted by carrying forward the previous day's value. Demographic and microbiologic data were recorded. When data collection for each patient was complete, all patient identifiers were removed from all records and the patient file was assigned a unique random number that was cross referenced with the blood samples. The completed raw data file was converted to calculated descriptive and severity of illness scores using standard definitions (i.e. APACHE II and Days alive and free of organ dysfunction calculated using the Brussels criteria).
(b) Population 2 Non-Septic SIRS
Inclusion Criteria
Caucasian patients booked for new elective coronary artery bypass grafting requiring cardiopulmonary bypass (CPB) were included. Patients undergoing urgent or emergency CPB surgery were not included because these patients may have already been exhibiting an inflammatory response to other triggers such as shock. We did not include patients undergoing valve surgery or repeat cardiac surgery because these patients have different pre-operative pathophysiology and often have longer total surgical and CPB times.
After induction of anesthesia and placement of systemic and pulmonary artery catheter (these are routinely inserted for clinical purposes at our institution), blood was obtained prior to CPB for genotyping and for baseline TNF-α, IL-6, IL-8, and IL-10 measurements. In addition, hemodynamic measurements including mean arterial pressure, thermodiluation cardiac outcome, and right arterial pressure as well as height and weight were recorded to calculate systemic vascular resistance index. Systemic Vascular Resistance Index (SVRI) was calculated as the difference between mean arterial pressure and right arterial pressure divided by cardiac index. Blood sampling was repeated at 4 (representing peak response) and 24 hours (to determine if the response is sustained) post-operatively. Hemodynamics to calculate SVRI were measured at zero, 4 and 24 hours post-operatively.
Common Methods—Both Populations
Blood Collection and Processing
Discarded whole blood samples from both populations above, stored at 4° C., were collected from the hospital laboratory. The Buffy coat was extracted and the samples were transferred to 1.5 ml cryotubes, barcoded and cross-referenced with the unique patient number and stored at −80° C. DNA was extracted from the Buffy coat using a QIA amp DNA maxi kitTM (QIAGEN). Patients were genotyped at −1654(2405) and at −1641 (2418) using an RFLP strategy as described by Spek et al. (Blood Coagulation and Fibrinolysis, 5:309-311, 1994). The first PCR strategy used here introduces a BstXI restriction enzyme cut site in the PCR product when a T is present at position −1654 (2405) so that the 246 bp PCR product is cut by BstXI into fragments of 205 and 41 bp. The second PCR strategy also introduces a BstXI restriction enzyme cut site in the PCR product when a G is present at position −1641 (2418) so that the 233 bp PCR product is cut by BstXI into fragments of 193 and 40 bp. After incubation of the PCR amplified DNA with BstXI, the reaction products were then separated using gel electrophoresis.
Statistical Analysis
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.
Population 1 Septic SIRS—Results
Eighty-one consecutive Caucasian patients admitted to our ICU with SIRS were included in this study. 46.9% of patients were AA homozygotes, 38.3% of patients were AG heterozygotes, and 14.8% of patients were GG homozygotes. The frequency of the A allele was 66% and the frequency of the G allele was 34% and these alleles were in Hardy Weinberg equilibrium in our population. Table 2 shows that there were no significant differences in baseline characteristics between AA, AG, and GG groups. Patients were of similar age, similar sex distribution, had similar admitting APACHE II. Approximately 40% of these patients had sepsis on admission and 10% of these patients had septic shock on admission. Eight percent of these patients developed sepsis at some time during their ICU stay and 45% of these patients developed septic shock at some time during their hospital stay.







TABLE 2







Sepsis SIRS patients baseline characteristics


Baseline Characteristics














Genotype

Sex

Sepsis on
S Shock
Sepsis
S Shock


−1641 (2418)
Age
% Male
Apache II
Admission
Admission
Anytime
Anytime





A A
58 ± 17
55%
19 ± 9 
45%
11%
84%
46%


A G
56 ± 15
62%
17 ± 7 
35%
 3%
81%
34%


G G
52 ± 16
67%
20 ± 11
42%
17%
75%
50%


p
0.34
0.75
0.47
0.49
0.58
0.98
0.64


(AA vs


AG + GG)









Measurements of days alive and free of SIRS and organ failure suggested a co-dominant effect of allele A. Patients with the A allele demonstrated fewer days alive and free of SIRS (Table 3), DAF acute lung injury and DAF cardiovascular failure (Table 3). Interestingly there was also a significant difference in DAF of the use of steroids. The use of steroids is made on a case by case basis by physicians in our intensive care unit in general and are employed more frequently in patients deemed to have severe sepsis and septic shock. In addition we also noted that the A allele was associated with significantly fewer DAF vasopressors. In addition trends towards adverse outcome or prognosis associated with the A allele were noticed in DAF hepatic failure, DAF renal failure, DAF CNS failure, and DAF International Normalized Ratio (INR)>1.5 (Table 4).







TABLE 3







Sepsis SIRS population: DAF SIRS and Key Organ Failure


Key


Differences













Genotype
DAF
DAF
DAF
DAF
DAF
DAF


−1641 (2418)
SIRS 4/4
SIRS 3/4
Steroids
ALI
CVS
Pressors





A A
17.6 ± 10.8
13.6 ± 11.3
12.1 ± 11.9
16.8 ± 12.5
17.9 ± 11.8
16.5 ± 11.5


A G
22.0 ± 9.8 
17.6 ± 10.5
19.0 ± 11.5
20.4 ± 10.6
21.8 ± 9.8 
21.1 ± 10.2


G G
26.1 ± 3.0 
22.1 ± 7.4 
23.8 ± 9.8 
25.5 ± 4.3 
26.8 ± 1.4 
25.2 ± 2.7 


p


(AA vs
0.013
0.027
0.002
0.044
0.022
0.014


AG + GG)
















TABLE 4







Sepsis SIRS Population: DAF Other Organ Failures


Other Results











Genotype
DAF
DAF
DAF
DAF


−1641 (2418)
Hepatic
Renal
CNS
INR > 1.5





A A
17.3 ± 12.0
17.1 ± 11.9
18.5 ± 11.6
18.7 ± 11.5


A G
20.9 ± 9.6 
19.3 ± 11.6
21.3 ± 11.2
19.8 ± 10.7


G G
24.3 ± 7.8 
20.3 ± 10.0
25.6 ± 5.7 
22.8 ± 9.4 


p
0.056
0.337
0.102
0.424


(AA vs


AG + GG









Most significantly, the A allele was associated with decreased survival (FIG. 1). Patients with the AA genotype had a survival of 58%, those with the AG genotype had a 74% survival, and those with a GG genotype had a 100% survival rate (P<0.017). Thus the protein C −1641 (2418) A allele was associated with decreased survival, more SIRS, worse cardiovascular and respiratory failure and trends to worse failure in other organ systems.
Population 2 Non-Septic SIRS—Results
To confirm these observations and to test for evidence of biological plausibility of the hypothesis that protein C −1641 (2418) A allele is associated with worse SIRS we turned to an independent population. We chose to study 61 Caucasian patients following cardiopulmonary bypass (CPB) surgery. CPB is associated with an inflammatory response that fulfills the definition of SIRS and is correlated with increased inflammatory cytokine expression post-CPB. In this population of 61 Caucasians we found 24 patients of AA genotype, 28 patients of a GG genotype, and 9 patients with GG genotype resulting in an A allele frequency of 62% and G allele frequency of 38%. This population was also in Hardy Weinberg equilibrium. At the preoperative baseline there were no significant differences in age, sex distribution, smokers, diabetes, presence of hypertension, preoperative ejection fraction, bypass time, cross-clamp time, and Aprotinin use (Table 5).







TABLE 5







CPB SIRS: Baseline Characteristics


Baseline Characteristics












AG and




AA
GG
p
















Age
66.9 ± 12.1
 65.5 ± 8.6
0.60



Sex (% Male)
79%
70%
0.45



Smokers
17%
22%
0.38



Diabetes
21%
22%
0.94



Hypertension
50%
57%
0.61



Pre-op EF
   56 ± 13%
   53 ± 15%
0.44



Bypass time
 109 ± 43
 106 ± 39
0.81



X clamp time
82 ± 36
79 ± 38
0.76



Aprotinin use
13%
11%
0.84










Post-operatively 64% of patients with the AA genotype developed an SVRI less than 1500 at least once during first 24 hours while only 50% of other patients developed an SVRI less than 1500. The presence of two consecutive SVRI measurements less than 1500 within the first 24 hours occurred in 32% of patients with the AA genotype and only 19% of other patients (p<0.03). SVRI at 1 hour post CVB was reduced in patients with the AA genotype due to a greater reduction in mean arterial pressure (p<0.05) and greater increase in cardiac index at 1 hour post CPB (FIG. 2). The additional observation of a significantly greater use of vasopressors in patients with the AA genotype at one hour post CPB further amplifies the clinical significance of the excessive vasodilation in patients with the AA genotype post CPB. In addition, arterial oxygen saturation was significantly reduced in patients with the AA genotype over the first 24 hours post CPB (FIG. 3).
Patients with the AA genotype had significantly greater serum IL-6 concentrations at 4 and 24 hours post CPB (Table 6). This was associated with trends towards greater increases in TNF-α, IL-8 and especially IL-10 at 4 and 24 hours post CPB. Thus, the protein C −1641 (2418) A allele was associated with more SIRS as indicated by a lower SVRI, increased pro-inflammatory cytokine response, and worse cardiovascular and respiratory failure post CPB, analogous to those findings in the critically ill SIRS patients.







TABLE 6







CPB SIRS: Post CPB Cytokine Expression


Cytokines (pg/mL, Mean ± SE)












AG and




AA
GG
p

















TNFα
 4 h
118 ± 45 
78 ± 26
0.41



TNFα
24 h
118 ± 45 
79 ± 24
0.43



IL-6
 4 h
1901 ± 795 
713 ± 148
0.08



IL-6
24 h
675 ± 154
360 ± 58 
0.04



IL-8
 4 h
133 ± 47 
121 ± 35 
0.84



IL-8
24 h
119 ± 52 
87 ± 29
0.57



IL-10
 4 h
119 ± 53 
42 ± 15
0.10



IL-10
24 h
108 ± 54 
19 ± 7 
0.06










Example Summary
Protein C −1641 (2418) A 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 AA 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. 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 AA genotype in this population is provided by the observation of greater reduction in SVRI and mean arterial pressure (MAP) and greater vasopressor use at 1 hour post CPB as well as increased inflammatory cytokine expression. The increased inflammatory cytokine expression also provides evidence of biological plausibility in that these cytokines were chosen to be representative of an acute inflammatory response, TNF-α, and integrated inflammatory response (IL-6), chemokine expression associated with lung injury (IL-8), and the counter regulatory anti-inflammatory response (IL-10).
Critically ill patients with the −1641 (2418) A allele had significantly worse outcomes as indicated by lower survival, more SIRS, more severe cardiovascular and respiratory failure, and trends to more severe hepatic renal (p=0.056), neurologic, and coagulation dysfunction. The poor clinical phenotype of the patients who had the −1641 (2418) A allele was also associated with greater use of corticosteroids. It is suspected that the reason for increased use of corticosteroids, is that the clinicians judged that there was a greater need for steroid treatment for severe shock and possibly prolonged respiratory failure. The markedly decreased survival in patients who had the −1641 (2418) A allele is more pronounced than the associated survival associations of most other polymorphisms studied to date in the critically ill.
In summary, the −1641 (2418) A allele is associated with more severe outcomes in the critically ill for both population 1-Septic SIRS and population 2—Non-Septic SIRS, as compared to the −1641 (2418) G allele. Patients with the −1641 (2418) A allele generally showed lower survival, more severe SIRS, and more severe cardiovascular and respiratory failure, more severe organ dysfunction, as compared to the −1641 (2418) G allele patients. Therefore, the −1641 (2418) protein C promoter polymorphism has diagnostic and prognostic use in the critically ill and in patients who are selected for elective CPB and other major surgeries.
4. EXAMPLE 2
Patient Outcome or Prognosis in Two Populations Using the 2405 Polymorphism
Similarly, patients in the above populations were also genotyped at position−1654 (2405) using the RFLP strategy described above. The −1654 (2405) C and T alleles were found not to be associated with patient prognosis or outcome in either the critically ill patients in population 1-Septic SIRS or in population 2-Non-Septic SIRS, as compared to the −1641 (2418) alleles. CC genotype had a survival of 63%, those with the CT genotype had a 71% survival, and those with a TT genotype had a 61% survival rate (P<NS). Therefore, the −1654 (2405) protein C promoter polymorphism does not appear to have diagnostic and prognostic use in the critically ill and in patients who are selected for elective CPB and other major surgeries.
5. EXAMPLE 3
2583 and 2322 Polymorphisms
Similarly the 2322 polymorphism was tested and was found to have no association of genotype with survival; genotype AA had 64% 28 day survival, AG had 72% 28 day survival, and GG had 63% 28 day survival (p NS). In addition, although the 2583 polymorphism was not tested as above, this polymorphism is in total linkage disequilibrium with 2418 as well as with polymorphisms at 1386, 3920, and other combinations of SNPs. For example, the combinations of polymorphisms at 5867+2405 and polymorphisms at 5867+4956 are also linked to 2418. Because these polymorphisms are in linkage disequilibrium with 2418, they show association of genotype with survival, organ dysfinction and a patients ability to respond to subsequent treatment, for example with steroids or vasopressors.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings of this invention that changes and modification may be made thereto without departing from the spirit or scope of the appended claims. All patents, patent applications and publications referred to herein are hereby incorporated by reference.

## Claims

1. A method for determining a prognosis for a human subject having, or at risk of developing, an inflammatory condition, the method comprising determining a genotype of said human subject at a polymorphic site in the subject's protein C gene at position 2418 of SEQ ID NO:1, wherein said genotype is indicative of the subject's ability to recover from the inflammatory condition which is SIRS, sepsis or septic shock, wherein the relationship between the nucleotide at said position 2418, the inflammatory condition and the prognosis is as follows:
(a) for SIRS, sepsis or septic shock:
(i) sense strand 2418AA homozygosity, or antisense strand 2418TT homozygosity, is prognostic of a decreased ability to recover; and
(ii) sense strand 2418GG homozygosity, or antisense strand 2418CC homozygosity, is prognostic of an increased ability to recover; and

(b) for septic SIRS:
sense strand 2418AG heterozygosity or antisense strand 2418TC heterozygosity is prognostic of an increased ability to recover compared to that of 2418AA homozygosity and a decreased ability to recover compared to that of 2418GG homozygosity.

2. The method of claim 1, further comprising determining the sequence of protein C of the subject.

3. The method of claim 1, wherein the genotype is determined using a nucleic acid sample from the subject.

4. The method of claim 3, which further comprises obtaining the nucleic acid sample from the subject.

5. The method of claim 1, wherein said genotype is determined using one or more of the following techniques:
(a) restriction fragment length analysis;
(b) sequencing;
(c) hybridization;
(d) oligonucleotide ligation assay;
(e) ligation rolling circle amplification;
(f) 5′ nuclease assay;
(g) polymerase proofreading;
(h) allele specific PCR; and
(i) reading sequence data.

6. The method of claim 1, wherein the subject is critically ill, and has a genotype prognostic of decreased ability to recover from the inflammatory condition and from severe cardiovascular or respiratory dysfunction present in said inflammatory condition.

7. The method of claim 1, wherein the subject is critically ill, and has a genotype prognostic of increased ability to recover from the inflammatory condition and from mild cardiovascular or respiratory dysfunction present in said inflammatory condition.

8. The method of claim 1, wherein the inflammatory condition is SIRS.