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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/172—Haplotypes

Definitions

• the present invention relates, to methods of identifying individuals at risk of perioperative bleeding, perioperative renal dysfunction and/or perioperative stroke.
• CPB cardiopulmonary bypass
• CPB-induced alterations in the hemostatic system are multifactorial, pertaining to excessive activation of coagulation and fibrinolytic pathways with interplay of cellular and soluble hemostatic and inflammatory systems; hypothermia and hemodilution further complicate the situation (Despotis et al, Annals of Thoracic Surgery 72:S1821-1831 (2001)).
• Coagulopathy following CPB represents one extreme on a continuum of coagulation function, with perioperative prothrombotic outcomes (e.g. coronary graft thrombosis, myocardial infarction, stroke and pulmonary embolism) at the other end of the spectrum (Spiess and Chandler, Best Practice & Research: Clinical Anaesthesiology 15:195-211 (2001)).
• prothrombotic genetic polymorphisms are known to exist.
• the present invention results, at least in part, from studies designed to investigate the impact of multi-locus genetic influences on the incidence and severity of perioperative bleeding after CABG surgery.
• the invention provides, in one embodiment, a method of identifying patients with prothrombotic gene polymorphisms, which polymorphisms are associated with increased postoperative bleeding.
• Acute renal dysfunction evidenced by rapid decline in glomerular filtration rate and accumulation of nitrogenous waste products (blood urea nitrogen and creatinine), is a major medical problem occurring in 5% of all patients admitted to the hospital and 30% of those admitted to an intensive care unit (Hou et al, Am. J. Med.
• kidneys receive more blood flow per gram of tissue than any other major organ, they are also the most vulnerable to ischemic injury.
• Metabolic demands from active tubular reabsorption and the oxygen diffusion shunt characteristic of renal circulation contribute to the precarious physiology of renal perfusion including low medullary pO2 (10- 20mmHg) (Brezis and Rosen, N. Engl. J. Med. 332(10):647-655 (1995)).
• Key to regulation of renal blood flow are paracrine systems (e.g., renin-angiotensin system [RAS], nitric oxide[NO]) that modulate microvascular function and oxygen delivery in the renal medulla (Navar et al, Physiol. Rev.
• cytokines e.g., tumor necrosis factor alpha [TNF ⁇ ], interleukin 6 [IL-6]
• TNF ⁇ tumor necrosis factor alpha
• IL-6 interleukin 6
• the present invention further results, at least in part, from studies designed to investigate the association between genetic variants of inflammatory and paracrine pathways at multiple loci and susceptibility to perioperative acute renal injury.
• significant neurologic morbidity continues to occur (Wolman et al, Stroke 30(3):514-522 (1999), Roach et al, N. Engl. J. Med. 335(25):185701863 (1996), Newman et al, N. Engl. J. Med. 344:395-402 (2001), Bucerius et al, Ann. Thorac.
• the present invention relates generally to perioperative bleeding, renal dysfunction and stroke. More specifically, the invention relates to methods of identifying individuals at risk of perioperative bleeding, renal dysfunction and stroke, and to kits suitable for use in such methods. Objects and advantages of the present invention will be clear from the description that follows.
• CTD denotes the predicted chest tube drainage for all the 128 possible allele combinations of the 7 polymorphisms, for the first 12 postoperative hours.
• Black bars mark allele combinations actually observed in the study population; the gray bars represent hypothetical allele combinations.
• the shaded cells in the table mark the prothrombotic allele and illustrate clustering of the prothrombotic alleles in the combinations associated with increased bleeding.
• the minor prothrombin 2021 OA allele was rare and did not appear in any of the examples.
• Figures 2 A and 2B Results of the model predicting peak postoperative serum creatinine rise (% ⁇ Cr) after aortocoronary surgery for all pairwise gene polymo ⁇ hism combinations identified in multi variable genetic predictive models for Caucasian (Fig. 2A) and African American (Fig. 2B) patients. Six polymorphisms in Caucasians and 4 in African Americans result in sixty-four (2 6 ) and sixteen (2 4 ) different possible combinations, respectively. Combinations observed in the study population are shown as black bars. The dashed line represents 2-fold (100%) increase in predicted postoperative serum creatinine rise, roughly equivalent to the loss of one kidney.
• Figures 3A and 3B Differences in predicted peak postoperative serum creatinine rise after coronary bypass surgery for two gene polymo ⁇ hism interactions identified in the multivariable genetic predictive models.
• the dashed line represents 2-fold (100%) increase in predicted renal injury, roughly equivalent to the loss of one kidney.
• FIGS 4 A-4G Graphic representation of individual two-way gene polymo ⁇ hism interactions in the multivariable genetic only models for predicted peak postoperative serum creatinine rise (% ⁇ Cr) after aortocoronary surgery.
• the dashed line represents a 2-fold (100%) increase in predicted renal injury.
• the present invention results from studies designed to prospectively examine specific genetic variants involved in bleeding pathways and how they influence postoperative bleeding.
• Polymo ⁇ hisms found to be significantly associated with postoperative bleeding include integrin -52C/T and 807C/T dimo ⁇ hisms, GPIb 524C/T, tissue factor - 603 A/G, prothrombin 20210G/A, tissue factor pathway inhibitor -399C/T polymo ⁇ hisms, and the angiotensin converting enzyme insertion/deletion polymo ⁇ hism.
• the present invention provides, in part, definitive association between these genetic variants and clinical postoperative bleeding in the perioperative and intensive care unit setting.
• This aspect of the invention is exemplified by reference to cardiac surgery patients but includes all perioperative, periprocedure (endoscopy, bronchoscopy, cardiac catheterization, angioplasty, etc.), and intensive care unit settings.
• the presence of one or more of the above-referenced polymo ⁇ hisms present in a sample can be determined using any of a variety of genotyping techniques known in the art. Examples of such techniques include the use of polymerase chain reaction and extension primers (see too Example 1 below).
• Suitable techniques also include the use of RFLP analysis and mass spectrometry (see also Ye et al, Hum. Mutat. 17(4):305 (2001), Chen et al, Genome Res. 10:549 (2000).)
• SNPs genetic variants
• the genetic variants (SNPs) described above and in Example 1 can be used, for example, to predict predict postoperative and ICU bleeding.
• screening for genetic variants of the invention is also relevant for other invasive procedures including but not limited to endoscopy, bronchoscopy, cardiac catheterization, and angioplasty. Preoperative screening for genetic variants enables clinicians to better stratify a given patient for therapeutic inteivention, either with drug therapy or with other modalities to alter homeostatic pathways.
• non-specific therapies to reduce thrombin activation and bleeding such as aprotinin, prostaglandins (Kozek-Langenecker et al, Anesthesia & Analgesia 87:985-988 (1998)) or more aggressive heparin dosing (Despotis et al, Thrombosis & Haemostasis 76:902-908 (1996)), can be employed for at- risk patients.
• a biphasic anti-thrombotic therapeutic approach can have merit, initially to halt sub-clinical thrombosis, then postoperatively to prevent thrombotic complications.
• Optimum timing and dosing of any such intervention can be established by one skilled in the art. It will be appreciated from a reading of this disclosure that certain of0 the particular genetic variants are important independently and others interact to become significant in combination with other genes relative to postoperative bleeding.
• the invention also relates to kits suitable for use in testing for the presence of the polymo ⁇ hisms identified above.
• kits can include, for5 example, reagents (e.g., probes or primers) necessary to identify the presence of one or more of the above-referenced polymo ⁇ hisms associated with perioperative bleeding.
• reagents e.g., probes or primers
• the present invention results from studies designed to examine the association between specific genetic variants and o perioperative renal dysfunction. When twelve candidate polymo ⁇ hisms are assessed, two demonstrate strong association with renal injury (>50% decrease renal filtration); these genes include interleukin 6 G572C and angiotensinogen T842C in Caucasians (when present together pO.OOOl).
• the presence of one or more of the above-referenced polymo ⁇ hisms present in a sample can be determined using any of a variety of genotyping techniques known in the art. Examples of such techniques include the use of polymerase chain reaction and extension primers (see too Example 2). Suitable techniques also include the use of RFLP analysis and mass spectrometry (see also Ye et al, Hum. Mutat. 17(4):305 (2001), Chen et al, Genome Res. 10:549 (2000).)
• SNPs genetic variants described above and in Example 2 can be used, for example, to predict predict postoperative and ICU renal dysfunction.
• screening for genetic variants of the invention is also relevant for other invasive procedures including but not limited to endoscopy, bronchoscopy, cardiac catheterization, and angioplasty.
• Preoperative screening for genetic variants enables clinicians to better stratify a given patient for therapeutic intervention, either with drug therapy or with other modalities.
• knowledge of genetic variants allows patients to choose, in a more informed way in consultation with their physician, medical versus procedural therapy. Identifying these genetic variants in patients who are already experiencing renal injury can result in the alteration or modification of the therapeutic strategy.
• preoperative genotype testing can refine risk stratification and improve patient outcome.
• drugs already available and used to delay progression of chronic renal disease may be useful in reducing renal damage in acute settings such as cardiac surgery.
• Identification of the genetic markers described herein may facilitate individually tailored medical therapy (personalized medicine) designed to reduce acute renal injury and associated morbidity and mortality. It will be appreciated from a reading of this disclosure that certain of the particular genetic variants are important independently and others interact to become significant in combination with other genes relative to postoperative renal dysfunction.
• the invention also relates to kits suitable for use in testing for the presence of the polymo ⁇ hisms identified above.
• kits can include, for example, reagents (e.g., probes or primers) necessary to identify the presence of one or more of the above-referenced polymo ⁇ hisms associated with perioperative renal dysfunction.
• reagents e.g., probes or primers
• the present invention results from studies designed to examine the association between specific genetic polymo ⁇ hisms and stroke risk after surgery (e.g., cardiac surgery). These studies demonstrate that specific genetic variants contribute to the risk of postoperative stroke and suggest that inflammation plays a pivotal role. It will be appreciated from a reading of this disclosure that a SNP pair representing individuals having minor alleles for both of CRP (TUTR 1846C/T) and LL6 (-174G/C) significantly associated with stroke.
• SNPs single nucleotide polymo ⁇ hisms
• the present invention provides definitive association between these genetic variants and clinical postoperative stroke in the perioperative setting.
• the invention is exemplified by reference to cardiac surgery patients but includes all perioperative, periprocedure (endoscopy, bronchoscopy, cardiac catheterization, angioplasty, etc.), and intensive care unit settings.
• the presence of one or more of the above-referenced polymo ⁇ hisms present in a sample can be determined using any of a variety of genotyping techniques known in the art. Examples of such techniques include the use of polymerase chain reaction and extension primers (see too Example 3). Suitable techniques also include the use of RFLP analysis and mass spectrometry (see also Ye et al, Hum. Mutat. 17(4):305 (2001), Chen et al, Genome Res. 10:549 (2000)). The genetic variants (SNPs) described above and in Example 3 can be used, for example, to predict postoperative and ICU stroke risk.
• SNPs genetic variants
• screening for genetic variants of the invention is also relevant for other invasive procedures including but not limited to endoscopy, bronchoscopy, cardiac catheterization, and angioplasty.
• Preoperative screening for genetic variants enables clinicians to better stratify a given patient for therapeutic intervention, either with drug therapy or with other modalities.
• knowledge of genetic variants allows patients to choose, in a more informed way in consultation with their physician, medical versus procedural therapy. Identifying these genetic variants in patients who decide to undergo surgery or other invasive procedure enables health care providers to design altered therapeutic strategies aimed at preventing the incidence of stroke in the subset of patients with enhanced risk. In addition, identifying these genetic variants in patients who have already experienced a stroke might also lead to alteration or modification in the therapeutic strategy.
• preoperative genotype testing can refine risk stratification and improve patient outcome.
• drugs already available and used to minimize the risk of stroke and/or damage associated therewith e.g., Apoprotinin
• Identification of the genetic markers described herein can facilitate individually tailored medical therapy (personalized medicine) designed to reduce stroke risk and associated morbidity and mortality.
• Perioperative screening can facilitate alterations in the usual course of the surgical procedure with institution of procedures designed to additionally reduce this risk (e.g., cardiac surgery without aortic cross-clamping).
• the invention also relates to kits suitable for use in testing for the presence of the polymo ⁇ hisms identified above.
• kits can include, for example, reagents (e.g., probes or primers) necessary to identify the presence of one or more of the above-referenced polymo ⁇ hisms associated with perioperative stroke.
• reagents e.g., probes or primers
• the present invention relates to methods of identifying compounds suitable for use in minimizing the risk of stroke. These methods can comprise screening compounds for their ability to modulate (e.g., inhibit) inflammation (e.g., perioperative inflammation). Such methods are made possible by the presently described interaction of two specific pathways. Certain aspects of the invention can be described in greater detail in the non-limiting Examples that follow.
• POGO PeriOperative Genetics and Outcomes
• Patient data collection and definition ofphenotypes Blood was collected prior to anesthetic induction for isolation of genomic DNA. Co-variate data included age, sex, race, height, weight, smoking, diabetes, preoperative hemoglobin level and platelet count, preoperative aspirin or intravenous heparin therapy, duration of cardiopulmonary bypass and number of bypass grafts performed, heparin dose, protamine dose, intraoperative blood product usage. Cardiopulmonary bypass was conducted using mild to moderate hypothermia and a target activated clotting time of 480 seconds, with a crystalloid primed circuit and cold blood/crystalloid cardioplegia.
• Selection strategies included polymo ⁇ hisms with previously demonstrated high likelihood (based on location and type) of functionally significant effects on the gene product itself (nonsense or misssense non-synonymous variants or frame shift insertions/deletions in the coding sequence), or in the response of the gene to the product of other genes (i.e., transcription factors) through mutations in the promoter or control sequence of a gene (Tabor et al, Nat. Rev. Genet. 3:391-397 (2002)).
• Structure gene polymorphism selection 58 unlinked marker gene polymo ⁇ hisms were chosen to assess/control for population admixture (genomic control).
• DNA extraction was performed using the PuregeneTM system (Gentra Systems, Minneapolis, MN), quantitated and stored at 4°C under chloroform.
• the genotyping assays were conducted at Agencourt Bioscience Co ⁇ oration (Beverly, MA) by Matrix Assisted Laser Deso ⁇ tion/Ionization, Time-Of- Flight (MALDI-TOF) mass spectrometry, using the SequenomTM MassARRAYTM system (Sequenom, San Diego, CA) (Sun et al, Nucleic Acids Research 28:E68 (2000)). Polymerase chain reaction and extension primers for each polymo ⁇ hism are presented in Tables 5 and 6 along with the details of the polymo ⁇ hisms.
• the genotyping accuracy of the SequenomTM MassARRAYTM system was estimated at 99.6% (Gabriel et al, Science 296:2225-2229 (2002)). Polymo ⁇ hisms with error rates >2%, with genotype call rates ⁇ 75%, or departing from Hardy- Weinberg equilibrium (p ⁇ 0.01) were regenotyped. Reproducibihty of genotyping was validated by scoring a panel of 6 polymo ⁇ hisms in 100 randomly selected patients using direct sequencing on an ABI3700 capillary sequencer (Applied Biosystems, Foster City, CA).
• Angiotensin converting enzyme (ACE) deletion and insertion alleles were identified on the basis of polymerase chain reaction amplification of the respective fragments from the intron 16 of the ACE gene, size fractionation and electrophoretic visualization, as described (Lindpaintner et al, N. Engl. J. Med. 332:706-711 (1995)). The results were scored by two independent investigators, blinded to the clinical phenotype.
• ACE Angiotensin converting enzyme
• a second predictive model was developed using clinical, operative, and demographic variables to predict CTD (the "clinical” model). Finally, all of the predictors from the genetic and clinical models were combined into one model, to address the question of whether genetic information would add predictive ability to the clinical model. Potential bias from population admixture was investigated in the following ways. First, self-reported race (African American or Caucasian; no other race was sufficiently represented) was tested directly as a predictor of CTD. Secondly, each polymo ⁇ hism was tested individually in interaction with race as a predictor of CTD. Finally, after the development of the final combined genetic and clinical model, race was included as a covariate in the multivariable model and tested again. All statistical analysis was performed using SAS/Genetics system version 8.02 (SAS Inc, Cary, NC). Continuous variables will be described as mean +/- standard deviation, categorical variables will be described as percentages.
• Results Total enrollment in the perioperative genomic project was 3300. The final size of the sample for this endpoint was 740. Self-reported black or white race was not associated with bleeding either as an independent predictor or as an interaction term with any of the candidate polymo ⁇ hisms. A similar analysis using structure genes to assess population admixture also did not correlate with postoperative bleeding. Therefore, the sample was assessed as a single population for all further analyses. Characteristics of the 19 prospectively identified candidate polymo ⁇ hisms studied are defined for this population in Table 1.
• Prothrombotic physiology is associated with minor alleles except for ⁇ 2 /3; integrin -52C/T and ACE intron 16 del/ins polymo ⁇ hisms, where the wild- type major alleles are prothrombotic.
• prothrombotic minor alleles are relatively common in the study population (frequency > 0.1 ; see Table 1), however, a significant subgroup are present at lower frequency (0.02-0.09).
• Study population demographics and clinical co- variates considered related to postoperative bleeding are detailed in Table 2. Seven polymo ⁇ hisms demonstrate significant associations with postoperative bleeding, either independently or in interaction with each other (Table 3).
• Figure 1 models the predicted chest tube drainage for all possible genotype combinations of the seven polymo ⁇ hisms listed in Table 3, depicting the observed and hypothetical genotype combinations associated with the highest and lowest values of chest tube drainage. This Figure illustrates the major effect that a combination of multi-locus, genetic polymo ⁇ hisms may have on the variability in bleeding seen after primary CABG surgery. Genetic factors predict bleeding.
• the final genetic multivariate linear regression model including these seven polymo ⁇ hisms had an R 2 value of 5 0.09, predicting 9% of the variability in CTD. Remarkably, this was more predictive than the model encompassing both preoperative co-morbidities and intraoperative risk factors (Table 3). Combining these clinical and genetic predictors produced a powerful model, predicting 14% of CTD variability (R value of 0.14). This indicates that genetic effects are primarily independent of,0 and carry more predictive power than, clinical effects in the study. Combining the genetic and clinical factors improves prediction of postoperative bleeding. Su ⁇ risingly, three of the four two-way genetic interactions identified in the multivariable model identify less bleeding with wild type genotypes than with any combination of the pro-thrombotic alleles.
• prothrombotic polymo ⁇ hisms appear to be associated with more rather than less bleeding after coronary bypass surgery. Linkage disequilibrium was present between some of the candidate o polymo ⁇ hisms (Table 4).
• thrombotic elements may be modulated by the polymo ⁇ hisms described, and contribute to more bleeding in this setting.
• the functional effects of the alleles evaluated in this study include modulation of platelet activation and thrombin generation.
• the 807C and -52T minor alleles in the ⁇ 2 gene of the ⁇ ⁇ integrin downregulate the surface expression of this primary collagen receptor (Jacquelin et al, Blood 97:1721-1726 (2001)), Kunicki et al, Blood 89:1939-1943 (1997)), reducing platelet affinity for collagen and prothrombotic outomes (Kunicki, Arteriosclerosis, Thrombosis & Vascular Biology 22:14-20 (2002), Santoso et al, Blood 93:2449-2453 (1999)).
• the 524C allele of GPIb ⁇ the platelet adhesion receptor binding to exposed collagen via von Willebrand factor
• GPIb ⁇ the platelet adhesion receptor binding to exposed collagen via von Willebrand factor
• prothrombotic alleles are associated with increased bleeding, probably by promoting consumptive platelet activation and thrombin generation during cardiopulmonary bypass.
• Genetic association studies have limitations. Many prothrombotic o polymo ⁇ hisms that broadly involve the same effect in the same physiologic pathway were studied — while some alleles decrease function and others increase, they all affect thrombosis in a similar way. By the nature of examining numerous multi-locus interactions, multiple comparisons are necessary, and prospective studies, including mechanistic aspects, will be5 required to confirm the present results.
• Platelet glycoprotein Ialla otjft receptor integrin
• Tissue factor (TF) -1208del/ins -1208ins 5 t level 034 bSS -603A G -603G 5 t level 0.49 0.57
• Table 3 Results of three multivanate linear regression models testing the association of polymo ⁇ hisms and clinical co-variates with chest tube drainage.
• the minor allele is tested as absent or present (heterozygote or homozygote)
• the R 2 values for the clinical, genetics and combined models are 0.07, 0.09 and 0.14 respectively.
• a statistically significant p value ⁇ 0.05 is denoted by *. oi T3 ⁇ U C IS ⁇ ⁇ ⁇ t
• Shear mediated platelet thrombus deposition is significantly dependent upon the P LA 1/2 polymo ⁇ hism of the Ilia chai ', as is resposiveness to epinephrine 2 increased thrombin generation in response lo microvascular injury 3 and a prothrombotic phenotype 4 .
• the 5T/C substitution (Kozak polymorphism) and the 524 C T substitution encoding Metl45Thr on the GPlb_ subunit of the GPlb complex (GPlb_, GPlb_, GP IX and GPV), are associated with increased transcription rate and surface expression of the GPlb complex 5 and a prothrombotic phenotype respectively. * .
• the 807C/T substitution on the 2 gene regulates the surface density of GPIalTa (integrin _2_1) and the T allele has prothrombotic associations.
• c-52T also regulates the surface density of GPIaJIa 9 but has not clinical associations as yet
• G 1006A also related to post heparin TFPI levels
• Factor V (F5) 16 1 A (Factor V Leiden) is associated with reduced bleeding after CABG [Donahue, 2003 #3614] and there is a strong association with venous and arterial thrombosis via activated Protein C resistance Prothrombin (F2) 20210A of 20210 G/A is associated with venous and arterial thrombosis l ⁇ Fibrinogen _ (FGB) All three polymorphisms are associated with fibrinogen levels but they are in linkage disequilibrium such that -455 A is always associated with increased fibrinogen levels (in Caucasians) and the 4 common haplotypes are shown below ". However -148T is associated with higher fibriogen in UK blacks and not 100% in linkage
• Thrombomodulin Increased MI risk in blacks only ", functional/mechanistic basis for this unclear.
• Plasminogen activator Inhibitor - 1 PAI-1, SERPI E 1
• CTSG Cathepsin
• Homozygoles (freq 0.10) are associated with arterial and venous thrombosis by mcreasmg plasma homocysteine levels " a .
• Angiotensin converting enzyme ACE
• Indel polymorphism increases risk of venous thrombosis u u 24 ".
• Afshar-Khargban V Li CQ, Khoshnevis-Asl M, Lopez JA Kozak sequence polymorphism of the glycoprotein (GP) Ibalpha gene is a major dete ⁇ ninant of the plasma membrane levels of the platelet GP Ib-IX-V complex. Blood. 1999;94:186-91. 6.
• Gonzalez-Conejeio R Lozano ML, Rivera J, Corral J, Iniesia JA, Moraleda JM, Vicente V. Polymorphisms of platelet membrane glycoprotein lb associated with arterial thrombolic disease. Blood 1998,92 2771-6 7.
• Novel platelet membrane glycoprotein VI dimo ⁇ hism is a ⁇ sk factor for myocardial infarction.[comment]. Circulation. 2001;104: 1459-63. 13. Amaud E, Barbalat V, Nicaud V, CambJen F, Evans A, Morrison C, Arveiler D. Luc G, Ruidaveis JB, Emmerich J, Fiessinger JN, Aiach M. Porymo ⁇ hisms in the 5' iegulatory region of the tissue factor gene and the risk of myocardial infarction and venous thromboembolisnr the ECTIM and PATHROS studies.
• Doggen CJ Cats VM, Bertina RM, Rosendaal FR. Interaction of coagulation defects and cardiovascular risk factors: increased nsk of myocaidial infarction associated with factor V Leiden or prothrombin 20210A [comment ⁇ Circulation. 1998,97: 1037-41. 17 Green FR Fibrinogen polymo ⁇ hisms and atherothrombolic disease. Annals of the New York Academy of Sciences. 2001;936 549-59 18. Cook DG, Cappuccio FP, Atkinson RW, Wicks PD, Chitolie A, Nakandakare ER, Sagnella GA, Humphries SE.
• Allele-spe ific increase in basal transcription of the plasminogen-activalor inhibitor 1 gene is associated with myocardial infarction. Proceedings of the National Academy of Sciences of the United Slates of America. 1 95;92:1851-5. 21. Herrmann SM, Funke-Kaiser H, Schmidt-Petersen K, Nicaud V, Gautier-Bertrand M, Evans A, Kee F, ArveiJer D, Morrison C, OrzechowsJ ⁇ HD, Elbaz A, A arenco P, Cambien F, Paul M Characterization of pohmo ⁇ hic structure of cathepsin G gene role in cardiovascular and cerebrovascular diseases.
• EXPERIMENTAL DETAILS Study Population This analysis is a sub-study of the Perioperative Genetics and Safety Outcomes Study (PEGASUS), an ongoing LRB approved, prospective, longitudinal study at Duke University Medical Center, where 3,149 patients have been prospectively enrolled and consented to have clinical and genetic data analyzed in relation to perioperative outcomes.
• 1,464 were Caucasian
• 207 were African American
• 97 were of another race; due to small numbers in each of the "other" race categories, the analysis was limited to Caucasians and African Americans.
• the primary outcome variable selected was peak fractional change in postoperative serum creatinine (% ⁇ Cr), defined as percent difference between preoperative serum creatinine (CrPre) and highest of the daily in-hospital postoperative values (Cr max Post); this is a commonly used, validated continuous variable reflecting relative reduction in renal filtration function (Andersson et al, Thorac. Cardiovasc. Surg. 41(4):237-241 (1993)). Serum creatinine was determined using a dry slide enzymatic reflectance technique (Vitros 950, Johnson and Johnson, New Brunswick, NJ) with a normal range of 44-133 ⁇ mol/L (0.5-1.4 mg/dl). Preoperative clinical covariate data includes demographic variables and pre-existing comorbidities (Table 7).
• Intra- and postoperative variables include duration of CPB, duration of aortic crossclamp, number of aortacoronary bypass grafts, blood product usage, requirement for inotropic drugs and/or intra-aortic balloon pump (IABP) counterpulsation (Table 7).
• Use of agents with potential renal effects e.g., intravenous dopamine, furosemide and mannitol was recorded and taken into account during analysis, but not regulated, since these agents have not demonstrated significant beneficial effects in the setting of cardiac surgery (Conger, Am. J. Kidney Dis. 26(4):565-576 (1995), Vijayan and Miller, Semin. Nephrol.
• Table 7 Patient, Procedural, and Renal Function Characteristics
• Demographic Variables Age (years) 64+/-11 63+/-11 Body mass index (kg/m 2 ) 29 7+/-14 1 31 4+/-18 5 Female (%) 26 50 Weight (kg) 86 2+/-1 1 842+/-18 2 Preoperative Comorbidities Carotid Son (%) 6 5 Chronic Steroid Therapy (%) 1 3 Hannan Mortality Risk Score* 0 02 5 +/-0029 0 028+/-0036 History of congestive heart failure (%) 16 21 History of diabetes (%) 32 42 History of hypertension (%) 6 5 82 Preoperative inotropic drug mfus ⁇ on(s) (%) 1 1 Preoperative lntraaortic balloon counterpulsation (%) 1 2 History of myocardial infarction (%) 27 28 History of obstructive lung disease (%) 12 8 History of peripheral vascular disease (%) 13 18 History of stroke (%) 6 10 Preoperative ejection fraction (%) 52+/-14 49+/-14 Preoperative hematoc ⁇ t (%) 39+/-7 37+/-8
• Transfusion - a marker of perioperative transfusion (>2 units packed red cells and at least one other blood product within 24 hours of surgery) Peak percentage change in postoperative creatinine (% ⁇ Cr) defined as the difference between preoperative and peak postoperative values represented as a percentage of the preoperative value.
• the Hannan Score is a risk factor score for in-hospital mortality following CABG surgery, identified by Hannan et al., in the New York State population (Hannan et al, JAMA 264(21):2768-2774 (1990)).
• Inotropic drug use defined as postoperative infusion of either dopamine >5 ⁇ g/kg/min and/or dobutamine >5 ⁇ g/kg/min, or epinephrine >0.03 ⁇ g/kg/min.
• Table 8 Association of Genetic Polymorphisms with Peak Postoperative Rise in Serum Creatinine (% ⁇ Cr) Caucasian African American % ⁇ Cr Gene Polymorphism Genotype Freq (%) Freq (%) % ⁇ Cr (+/-SD) (+/-SD)
• Vasomotor regulation genes Angiotensin Converting Enzyme Intron 16 DD 32 27 (39) 41 32(44) Delet ⁇ on(D)/ DI 46 27 (60) 46 36(51) Insert ⁇ on(I) II 22 29 (40) 13 33 (27) Angiotensinogen T842C TT 3 5 26 (45) 7 23 (46) TC 47 28 (40) 30 31(48) CC 18 36 (82) 63 37 (43) Angiotensin Receptor 1 A1166C AA 50 27 (43) 83 31 (43) CA 40 29 (45) 14 35 (43) CC 10 36 (96) 3 29 (29) Endothelial Constitutive G894T GG 42 29 (50) 74 34 (46) Nitric Oxide Synthase TG 44 28 (55) 23 31 (36) TT 13 33 (48) 3 52 (68)
• % ⁇ Cr - Peak percentage change in postoperative creatinine is defined as the difference between the preoperative and peak postoperative values and represented as a percentage of the preoperative value. SD - standard deviation
• MALDI-TOF Matrix Assisted Laser Desorption/Ionization, Time-Of- Flight
• Angiotensin converting enzyme (ACE) deletion and insertion (D/I) alleles were identified on the basis of polymerase chain reaction amplification of the respective fragments from the intron 16 of the ACE gene, followed by size-fractionation via electrophoresis as previously described (Rigat et al, J. Clin. Invest. 86(4):1343-1346 (1990). Results were scored by 2 independent investigators blinded to the clinical phenotype. After completion of genotype analysis, genetic samples were linked to covariate and phenotypic variables in a relational database with extensive quality control features.
• Genotyping Assays for the 12 Candidate Gene Polymorphisms PCR primers (top-forward, bottom-reverse) and extension primers for MALDI-TOF mass spectrometry assays
• Table 12 Genetic, Clinical, and Combined Genetic and Clinical Multivariable Risk Factor Models Predicting Peak Postoperative Serum Creatinine Rise in 1464 Caucasians after Aortocoronary Surgery.
• Gaudino and colleagues noted increased postoperative renal dysfunction in carriers of the -I74C polymo ⁇ hism in 111 coronary bypass patients (Gaudino et al, J. Thorac. Cardiovasc. Surg. 126(4):1107-1112 (2003)). While in the present study, the IL6 -572C and not the -174C was related to acute renal injury, the findings of Gaudino and colleagues support the present findings since significant linkage disequilibrium was noted between these 2 alleles in Caucasians, indicating they are included in a haplotype block (Tables 9-11).
• both of these IL6 polymo ⁇ hisms have been associated with greater rises in IL 6 protein levels after cardiac surgery (Kelberman et al, Biochim. Biophys. Acta 1688(2):160-167 (2004), Burzotta et al, Am. J. Cardiol. 88(10):1125-1128
• Ischemic stroke was the primary outcome variable. During the study period, all patients with suspected stroke (defined as any new focal abnormality on neurologic examination occurring within one week of surgery) l o underwent evaluation by an independent neurologist and confirmatory brain imaging (magnetic resonance [MRI] or computerized tomography [CT]). Patients with cerebral hemorrhage or generalized encephalopathy were specifically excluded. Fulfillment of these criteria were confirmed after independent review of patient hospital records. Preoperative climcal covariate
• TIA transient ischemic attack
• Beta blocker beta-adrenergic antagonist therapy
• COPD chronic obstructive pulmonary disease
• CPB cardiopulmonary bypass
• Stroke ischemic stroke. *P-values are from Wilcoxon Rank-Sum test for continuous measures, and from exact Chi-Squared test for categorical (%) characteristics. + Note: Preoperative beta blocker became non-significant when added to the genetic model, whereas age remained significant.
• the 26 SNPs represented 13 genes, which were classified on the basis of biological function into categories of coagulation, inflammation, and lipid metabolism (Table 15).
• the coagulation and inflammatory categories were chosen based on extensive literature supporting a role of thrombosis and inflammation in stroke whereas the lipid metabolism category (specifically, apolipoprotein E) was chosen because of its previous demonstration of a relationship to other neurologic disorders, including post-cardiac surgery cognitive dysfunction (Tardiff et al, Ann. Thorac. Surg. 64:715-720 (1997)).
• Genotyping assays for SNPs were conducted at Agencourt Bioscience Co ⁇ oration (Beverly, MA) by Matrix Assisted Laser Deso ⁇ tion/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry, using the SequenomTM MassARRAYTM system (Sequenom, San Diego, CA) (Sun et al, Nucleic Acids Res. 28(12):E68 (2000)). Primers used and polymo ⁇ hism details can be found at anesthesia.duhs.duke.edu/pegasus/stroke/1/.
• Genotyping accuracy of the SequenomTM MassARRAYTM system was estimated at 99.6% (Gabriel et al, Science 296(5576):2225-2229 (2002)).
• genotyping reproducibihty in this study was validated to be >99% by scoring a panel of six polymo ⁇ hisms in 100 randomly selected patients. After completion of genotype analysis, genetic results were linked to covariate and phenotypic variables in a relational database.
• F2 Prothrombin 11 p1 1 -q12 RS1799963 2021 OG/A ACGTTGGATGAGAGAGCTGCCCATGAATAG CCAATAAAAGTGACTCTCAGC ACG ⁇ GGATGTCTTCTACCCCACGAAGGTC RS1361600 -603A/G ACGTTGGATGA ⁇ CCCACCGCCTrTCTCCTG CTACCCCACGAAGGTCAAGAATAC
• F3 (Tissue factor) 1 p22-p21 ACG ⁇ GGATGTTTrGCACAGTTTTA ⁇ CTG P021" ⁇ 1208indel(18bp) ACGTTGGATGAGTCAGTCnGCATTTTAAT CTG ⁇ AAAACAAGTGG ⁇ CAGTA ACG ⁇ GGATGCTGAAAGG ⁇ ACTTCAAGGAC
• F5 (Factor V) 1q23 RS6025 1691G/A ACG ⁇ GGATGTGGGCTAATAGGACTACTTC AGGACAAAATACCTGTA ⁇ CCT ACGTTGGATGCAT ⁇ AAGCAACATC ⁇ CCC RS1800787 •148C T ACG ⁇ GGATGAAC ⁇ CCCATCATTTTGTCC AAGCAACATC ⁇ CCCAGCAAA ACGTTGGATGGCTTATGTTTTCTGACAATG
• association analyses for each candidate polymo ⁇ hism were based on two genotypic classes, distinguished by the presence of at least one copy of the minor allele. Pearson Chi square tests were used to test for independence between stroke and race or gender. Association between SNP genotype and self- declared race was investigated using Chi square tests. In addition, the presence of cryptic population structure was investigated using 58 unlinked, non-candidate SNPs to fit a hierarchical model of population structure (Humphries and Morgan, Lancet Neurol. 3(4):227-235 (2004)). Chi square tests for independence between stroke and SNP genotype were computed for each of the 26 candidate SNPs.
• Tests for genetic effects of a pair of SNPs were investigated separately within each functional category by distinguishing genotypes bearing at least one minor allele at both SNPs. This particular partition of genotypes yielded a 2x2 table of genotype by stroke thereby allowing a test for independence with individual SNPs. There exist three other partitions of genotypes based on joint presence or absence of the minor allele in pairs of SNPs and tests were carried out for independence on these genotype partitions as well. After these genetic association tests, a logistic regression model was used to test the joint independent effects of significant SNPs and age, a factor repeatedly shown to be of importance to post-cardiac surgery stroke (Roach et al, N. Engl. J. Med.
• Random permutation analysis was used to adjust p- values (Chasman et al, JAMA 291(23):2821-2827 (2004)). 5000 copies of the data set were generated, randomly reassigning stroke events to study subjects, thereby disassociating genotype from stroke events in the data. For each permutation, p-values were calculated for all four genotype partitions of each pair of SNPs. For each genotype partition, the smallest p-value was retained to estimate the distribution of 5000 "smallest" p-values under the null hypothesis of no association. An adjusted p-value was computed as the fraction of permutation p-values that were smaller than the observed p-value.
• *P-values represent significance level of race or gender with stroke in final multivariable model controlling for both age and genotype; percentage represented by parentheses ( ).
• Age for example, is a robust risk factor for perioperative stroke and was reconfirmed in this present study (Newman et al, Circulation 94(9 Suppl):II74-II80 (1996)).
• the present findings reveal that the concurrent presence of at least one minor allele at each of two loci (CRP: 3'UTR 1846C/T; IL6: -174G/C) is a risk factor for stroke, increasing risk more than three-fold.
• CRP 3'UTR 1846C/T
• IL6 -174G/C
• the IL6 polymo ⁇ hism is thought to influence plasma levels and functional activity of the IL6 protein and has been associated with other perioperative inflammatory complications (Gaudino et al, J. Thorac. Cardiovasc. Surg. 126(4).T 107-1112 (2003), Gaudino et al, Circulation 108(Suppl l):II195- ⁇ i99 (2003), Burzotta et al, Am. J. Cardiol. 88(10):1125- 1128 (2001)).
• association between IL6 polymo ⁇ hism and perioperative neurologic outcome has not been previously described.
• the C/T polymo ⁇ hism in the V UTR of the CRP gene has been associated with increased basal plasma levels of CRP and is part of a haplotype associated with elevated peak CRP levels after CABG (Russell et al, Hum. Mol. Genet. 13(1):137-147 (2004), Brull et al,
• genetic variants modulating IL6 levels and the expression of CRP may contribute to the perioperative pro-inflammatory phenotype seen in cardiac surgical patients.

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