(Chest. 2003;124:1103-1115.)
© 2003
American College of Chest Physicians
Genetic Polymorphisms in Sepsis and Septic Shock*
Role in Prognosis and Potential for Therapy
Cheryl L. Holmes, MD;
James A. Russell, MD and
Keith R. Walley, MD
* From the University of British Columbia, McDonald Research Laboratories/The iCAPTURE Centre, Vancouver, BC, Canada.
Correspondence to: Keith R. Walley, MD, McDonald Research Laboratories/The iCAPTURE Centre, Providence Health Care/University of British Columbia, 1081 Burrard St, Vancouver, BC Canada V6Z 1Y6; e-mail: kwalley{at}mrl.ubc.ca
 |
Abstract
|
|---|
Genetic epidemiologic studies suggest a strong genetic influence on the outcome from sepsis, and genetics may explain the wide variation in the individual response to infection that has long puzzled clinicians. Several candidate genes have been identified as important in the inflammatory response and investigated in case-controlled studies, including the tumor necrosis factor (TNF)-
and TNF-ß genes, positioned next to each other within the cluster of human leukocyte antigen class III genes on chromosome 6. Other candidate genes for sepsis and septic shock include the interleukin (IL)-1 receptor antagonist gene, the heat shock protein gene, the IL-6 gene, the IL-10 gene, the CD-14 gene, the Toll-like receptor (TLR)-4 gene, and the TLR-2 gene, to name a few. In this review, we summarize the evidence for a genetic susceptibility to development of sepsis and death from sepsis, discuss design of clinical genetics studies relevant to the study of complex disorders, consider the candidate genes likely to be involved in the pathogenesis of sepsis, and discuss the potential for targeted therapy of sepsis and septic shock based on genetic variability.
Key Words: heat-shock proteins • interleukins • polymorphism • septicemia • sepsis syndrome • tumor necrosis factor
|
Introduction
|
|---|
The significant problems we face cannot be solved at the same level of thinking we were at when we created them. Albert Einstein
|
Theoretical Case Presentation – Year 2010
|
|---|
A 35-year-old man is brought to the emergency department complaining of back pain and fever of 3 days in duration. His medical and family history are unremarkable. Examination reveals a heart rate of 120 beats/min, a temperature of 40°C, a respiratory rate of 35 breaths/min, and a BP of 80/30 mm Hg. He is confused, and his extremities are cool and mottled. Percussion of his left flank reveals marked tenderness. Preliminary investigations reveal a WBC count of 30,000/µL, an increased alveolar-arterial oxygen gradient, lactic acidosis, and diffuse infiltrates on the chest radiograph. He receives resuscitation including intubation, ventilation, fluid and vasopressor therapy, and broad-spectrum antibiotics. An ultrasound is performed in the emergency department that reveals hydronephrosis of the left kidney associated with ureteral obstruction, and a percutaneous stent is placed. He is carrying a subcutaneous silicon chip, which identifies his genotypic profile. The chip is scanned in the emergency department, and his allelic variants are identified. He is homozygous for the tumor necrosis factor (TNF)-ß2 allele, which in a multicenter study completed in 2009 was associated with improved outcome in septic shock when combined with anti-TNF therapy. He is also homozygous for the allelic variant in the promoter region of the protein C gene, associated with low protein C levels, disseminated intravascular coagulation, and a higher mortality in sepsis in a clinical trial in 2008. Based on genetic profile, he is administered monoclonal antibody to TNF- and activated protein C. On arrival to the ICU, he is entered in a matched cohort study designed to compare the outcome of patients who receive therapy based on their genetic profile vs patients in whom this information is not available.
|
Introduction
|
|---|
Although technologic, pharmacologic, and surgical advances have improved the outcomes of many diseases, the mortality of septic shock continues to be distressingly high. It is ironic that the mortality from septic shock, the most common cause of death in the critical care unit,1
has not decreased dramatically in the past decade.2
Furthermore, despite efforts expended to refine prognostication tools such as scoring systems to predict the outcome of groups of critically ill patients, intensivists continue to be frustrated in their inability to predict the outcome of any one patient. Even more frustrating is that despite an explosion of knowledge of the inflammatory response to sepsis and despite the enormous financial resources invested in randomized controlled trials of anti-inflammatory therapies, intensivists still lack effective therapies targeting the inflammatory response to sepsis.3
4
Recently, attention has shifted to derangements in the coagulation system in the pathogenesis of sepsis. Activated protein C (APC), an endogenous protein that inhibits thrombosis and inflammation while promoting fibrinolysis, plays an important role in the pathogenesis of sepsis. Recombinant human APC, drotrecogin alfa (activated), decreased the relative risk of death at 28 days by 19.4% (95% confidence interval [CI], 6.6 to 30.5%; p = 0.005) in a randomized, double-blind study of 1,690 patients with severe sepsis (the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis trial5
). This is the first study of new treatment for severe sepsis that showed a positive result, yet many questions remain; importantly, which group of patients with sepsis will benefit most from this very costly therapy?
Will the study of genetic variability in susceptibility to sepsis and response to therapy revolutionize the treatment of sepsis and septic shock? Francis Collins has stated, "Gene isolation provides the best hope for understanding human disease at its most fundamental level. Knowledge about genetic control of cellular functions will underpin future strategies to prevent or treat disease phenotypes."6
This promise is being fulfilled for many single-gene diseases, but teasing out the genetic components of complex disorders such as sepsis remains a formidable task. In this review, we summarize the evidence for a genetic susceptibility to development of sepsis and death from sepsis, discuss design of clinical genetics studies relevant to the study of complex disorders, consider the candidate genes likely to be involved in the pathogenesis of sepsis, and discuss the potential for targeted therapy of sepsis and septic shock based on genetic variability.
|
Genetic Epidemiology of Sepsis
|
|---|
Genetic epidemiologic studies suggest a strong genetic influence on the outcome from sepsis. The septic response is a classic example of genetic influence modifying response to an environmental stimulus: the infectious insult. Genetic epidemiology is the study of the interaction between genes and the environment. The traditional approach analyzes family history data to estimate the relative risk of a disease in relatives of affected vs nonaffected individuals. Adoption and twin studies have been extensively employed in research designed to disentangle the genetic and environmental contribution to disease.7
The Danish Adoption Register has been an important source of information in genetic epidemiology. Sorensen et al8
conducted a study of adoptees born between 1924 and 1926, focusing on death from all causes; death from natural causes; death involving infections, cardiovascular, or cerebrovascular conditions; and death from cancer. The adoptees and their biological and adoptive parents were followed up through population registers to 1982. The influence of a biological parent dying before the age of 50 years and before the age of 70 years was assessed, using a proportional hazards regression model, by comparing the adoptee mortality to that seen if both biological parents were alive at these ages. A similar analysis was conducted for the adoptive parents. The results were compelling. If a biological parent died of infection before the age of 50 years, the child had a 5.81 relative risk of also dying of infection (95% CI, 2.47 to 13.7). If a biological parent died of a cardiovascular or cerebrovascular event, the child had a 4.52 times greater chance (95% CI, 1.32 to 15.4) of also dying of a cardiovascular or cerebrovascular event. A biological parent dying of cancer did not confer a greater risk in the child of dying of cancer. Thus, susceptibility and responses to infection have a surprisingly important genetic influence. In contrast, the death of an adoptive parent of infection or vascular causes did not confer a greater risk of the child dying of these causes, whereas an adoptive parent dying of cancer resulted in a 5.16 times greater risk (95% CI, 1.32 to 15.4) of the adoptive child dying of cancer. Thus, development of cancer has a marked environmental influence, whereas development and response to infection does not. A similar but weaker pattern of effects was observed when the parents died before the age of 70 years.8
Based on this study, the authors conclude that genetic influences are responsible for the major part of the familial influence on overall mortality. The findings of this study emphasize the role of genotype in the susceptibility to infection.
Twin studies show, by a factor > 2, greater concordance in monozygotic twins than dizygotic twins for tuberculosis, leprosy, poliomyelitis, and hepatitis B.9
Since infection itself is not a genetic disease, this means that genetic variation in inflammatory responses leads to significant differences in outcome.
|
Nomenclature and Examples of Genetic Variants
|
|---|
A genetic polymorphism is an allelic variant that exists stably in a population in a frequency that cannot be accounted for by new mutations (generally > 1%). The most frequent type of polymorphism is the single-nucleotide polymorphism (SNP), which can be a substitution, a deletion, or an insertion of a single nucleotide (for nomenclature, see Table 1
). Approximately 1 in every 300 to 500 bases of human DNA may be a SNP. The mutation can be in coding (exon) or noncoding (intron) regions of the gene or in the promoter region. When the SNP is within a protein-coding region of a gene, the variant allele may lead to an amino acid substitution that renders the resulting protein functionally altered. An example is sickle-cell anemia. The mutated gene consists of a substitution of thymine for adenine (GTG for GAG) in a glutamic acid codon. The codon leads to a substitution of valine for glutamic acid at the ß-6 position of the ß-globin chain and produces hemoglobin S.
Much more commonly, SNPs occur in nontranslated regions; however, SNPs in these regions may still have biological effects. For example, SNPs in the promoter region may alter binding affinity of transcription factors, such as nuclear factor (NF)-
B, and therefore alter the rate of gene transcription and thus translation resulting in higher protein levels. SNPs within the 5'upstream region and 3'downstream region may alter stability of the transcribed messenger RNA (mRNA) or may alter enhancer activity, and therefore alter the efficiency of gene transcription and mRNA translation. SNPs within introns are less likely to have biological effects yet have still been observed to be associated with biological effect, although this association may frequently be due to linkage with biologically active SNPs within other parts of the gene. It is interesting to note that amino acid-altering SNPs occur with the lowest frequency, SNPs within coding regions that do not alter amino acid sequences occur somewhat more frequently, SNPs within promoter regions even more frequently, and SNPs within introns most frequently.10
Natural selection against SNPs with adverse biological effects may account for these differences.
|
Genetic Polymorphisms Influencing the Susceptibility and Resistance to Infectious Diseases
|
|---|
Many generations of physicians have observed and pondered the individual variability in the susceptibility to infectious disease. Why do some infants contract respiratory syncytial virus and acquire respiratory failure while others experience little more than a runny nose?11
Why do some individuals develop a Gram-negative urinary tract infection that responds to one dose of antibiotic and others acquire septic shock and multiple system organ failure despite adequate antimicrobial treatment? The evidence for genetic susceptibility to common infectious diseases is growing. There is compelling evidence for a genetic component, including twin studies of diseases such as tuberculosis, leprosy, malaria, and Helicobacter pylori.12
There are > 100 examples of single-gene mutations giving rise to severe immunodeficiency disorders.12
Malaria was the first infectious disease to be studied extensively for genetic variability and susceptibility to disease. Many polymorphisms conferring susceptibility and resistance have been identified.13
Examples of genetic polymorphisms conferring resistance to malaria include and ß thalassemias, hemoglobins S, E, and C, South East Asian ovalocytosis, glucose-6-phosphate dehydrogenase deficiency, and the Duffy blood group.14
The highest frequency of these polymorphisms is usually found in highly malarious areas and explains why many individuals in these areas remain in good health despite being continuously infected with this parasite.
More complex determinants of susceptibility to infection and possibly the sepsis syndrome are found in the genes located on chromosome 6 in the major histocompatibility complex (MHC). Polymorphisms in human leukocyte antigen (HLA) genes correlate with susceptibility to infections including malaria, tuberculosis, HIV and hepatitis B.12
The areas flanking the HLA-B locus are up to 20 times more polymorphic than the reported average degree of human neutral polymorphism.15
Since the functional role of the HLA is to present antigens to the immune system, it has been postulated that the extraordinary genetic variability of the HLA arose in response to antigenic diversity in infectious organisms.12
A large number of genes that are known or predicted to have immunologic function reside alongside the HLA genes including heat shock protein (HSP); complement factor B; complement components 2, 4A, and 4B; TNF-; and TNF-ß (lymphotoxin).16
These genes, along with others influencing the inflammatory response, are strong candidates as genetic determinants of susceptibility to sepsis and septic shock. Before we discuss these and other candidate genes in detail, it is important to review the strengths and weaknesses of genetic study design.
|
The Challenge of Study Design for Studies of Polygenic Diseases
|
|---|
The observed variation in the traits that Mendel studied was due to a simple difference at a single gene. Most traits and diseases, however, are polygenic and exhibit nonmendelian inheritance (for nomenclature, see Table 1
). The identification of susceptibility genes for traits and diseases caused by single gene defects (eg, Huntington chorea, glucose-6-phosphate dehydrogenase deficiency, sickle-cell anemia) has become both rapid and efficient through linkage analysis and positional mapping. Linkage analysis compares the inheritance of the disease with the inheritance of genetic markers in families with multiple affected members. Markers closest to the disease gene show the strongest correlation with disease patterns in families, and tracking of recombination events can narrow the region of the disease gene to between 100 and several thousand kilobase pairs. Using linkage analysis and positional cloning, success has also been achieved in identifying several genes that account for uncommon subsets of relatively common disorders. Examples include genes for breast cancer (BRCA-1 and BRCA-2), colon cancer (familial adenomatous polyposis, and hereditary nonpolyposis colorectal cancer), Alzheimer disease (ß-amyloid precursor protein, and presenilin-1 and presenelin-2) and diabetes (maturity-onset diabetes of youth [MODY]-1, MODY-2, and MODY-3).17
In contrast, most phenotypic traits and common diseases are determined by many genes collaborating at different loci rather than by single-gene effects. These are referred to as polygenic traits and diseases. Height and intelligence are examples of polygenic traits, exhibiting "nonmendelian" inheritance, in which the extremes of the distribution are not necessarily considered abnormal. Diseases that are examples of polygenic inheritance include diabetes, atherosclerosis, and susceptibility to sepsis and septic shock.
The identification of the genetic basis of nonmendelian polygenic disorders (such as susceptibility to sepsis and septic shock) is exceedingly more difficult than that of simple (one-gene) traits. Linkage analysis has proven to be much less reliable for the study of nonmendelian diseases because of the high rate of false-positive results. The study of polygenic traits is complicated by the lack of familial segregation patterns and the environmental variability, as might occur with infectious exposure. Identification of weakly penetrant alleles that contribute to common disorders requires new and more powerful approaches. One approach to studying the candidate genes of these disorders is high-density genome scans that depend on linkage disequilibrium,18
which looks for the association of disease with marker alleles—usually evenly spaced SNPs. SNPs can be used as markers in whole-genome linkage analysis of families with affected members, as well as in association studies of individuals in a population. This type of study views large human populations as evolutionary families and does not rely on studies of nuclear families for gene mapping.6
A second method that has been used to identify polymorphisms involved in disease pathogenesis is to select candidate genes that are known or hypothesized to have a biological effect important in the pathogenesis of disease and apply association analysis to test for the association of the polymorphisms with the disease phenotype. An example is the study of the association of the TNF-ß gene with susceptibility to septic shock. Monocytes from individuals homozygous for the variant TNF-ß allele have been shown to develop higher circulating levels of TNF- in response to stimulation by endotoxin.19
An association study of trauma patients homozygous for the variant allele found higher TNF- levels and greater risk of severe sepsis (odds ratio, 3.07; 95% CI, 1.42 to 6.63) relative to other genotypes.20
Association studies using candidate genes and contrasting allele frequencies in cases vs control subjects are useful for genes with modest displacement (small effect) and do not depend on family studies but are subject to confounding factors. For genetic studies, the most serious confounder is ethnicity. If cases and control subjects are not ethnically comparable, then differences in allele frequency will emerge at all loci that differentiate these groups whether the alleles are related to disease or not. Therefore, false-positive results arise due to this stratification artifact. Investigators have tried to prevent this by restricting study subjects to a particular ethnic group; however, ethnic background is often difficult to define precisely. A solution is to use a matched case-control design using homogeneous and randomly mating populations17
; however, nonrandom mating patterns in heterogeneous populations such as North Americans do not ensure ethnic comparability. Another solution is to use relatives as control subjects for the cases. A statistical test is then applied termed the transmission-disequilibrium test, which evaluates the frequency of transmission of specific alleles at a single locus from heterozygous parents to their affected children. Each allele should be transmitted 50% of the time. If a marker allele is transmitted significantly more often than 50% of the time, this implies that the allele must be linked to and in linkage disequilibrium with the disease- causing allele, or is the disease-causing allele. The advantage of this strategy is that stratification artifact is eliminated. The problem is that a significant result does not imply a causal relationship between the tested allele and the disease, and again spurious associations can arise. There are many other suggestions for optimal genome strategies that are beyond the scope of this review.17
It should be emphasized that epidemiologic and genetic case-control studies are at best observational and do not prove causation. Associations with complex diseases are hypothesis generating but do not prove the role or the function of the gene in disease pathogenesis.21
In vitro and transgenic studies are required to prove that an allelic variant of the gene is nonneutral.22
Association studies do, however, provide the needed first step toward understanding genetic factors in polygenic disease, and statistical and design strategies that will optimize these studies continue to be refined.
|
Candidate Genes Influencing the Intensity of the Inflammatory Response
|
|---|
The systemic inflammatory response to infection or injury, termed systemic inflammatory response syndrome, is likely a complex trait. Most of the candidate genes hypothesized to influence the intensity of the inflammatory response are located on the highly polymorphic region of chromosome 6 known as the MHC. The ideal candidate gene for a given phenotype should have biological plausibility, and the common variant should be phenotypically neutral. A review of the innate immune response to infection and tissue injury involving the inflammatory cytokines and the coagulation cascade is first necessary (Fig 1
).
View larger version (132K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 1. A simplified diagram of the innate immune response to infection and tissue injury involving the inflammatory cytokines and the coagulation cascade. –ve = negative; +ve = positive; PAI1 = platelet-activation inhibitor-1; ICAM = intracellular adhesion molecule.
|
|
|
The Innate Immune Response
|
|---|
The stimulus for inflammation and coagulation can result from microbial invasion (exogenous injury) or direct tissue injury (endogenous injury). Cellular receptors that recognize danger are called pattern recognition receptors (PRRs) and nucleotide-binding oligomerization domain (NOD) receptors.23
PRRs are expressed on extracellular membranes—both luminal and nonluminal surfaces. Examples of PRRs are Toll-like receptor (TLR)-4 and CD-14 that are expressed on the surface of monocytes and macrophages and are important for lipopolysaccharide (LPS) recognition.24
Other PRRs include TLR-2, a transducer for various bacterial products; TLR-5; TLR-6; and TLR-9.24
New families of receptors, NOD, appear to respond to both injury/pathogen-related signals and normal physiologic signals involved with apoptosis.23
PRRs recognize pathogen-associated microbial patterns (PAMPs),25
which are essential products of microbial physiology. Examples of PAMPs include LPS (Gram-negative bacteria), lipotechoic acid and peptidoglycan (Gram-positive bacteria), and zymosan (yeast).25
During tissue injury, HSP-70 is an example of an endogenous ligand produced by cellular injury that functions as a PAMP.25
PAMP binding to PRRs results in release of NF-B from its inactive complex in I-B. NF-B is an important intracellular protein that then translocates to the nucleus causing transcription of cytokines, both proinflammatory (eg, TNF, interleukin [IL]-1, IL-6) and anti-inflammatory (eg, IL-10). Proinflammatory cytokines activate neutrophils, causing expression of -integrin (CD-11) and ß2-integrin (CD-18), which are cofactors in cell adhesion. Proinflammatory cytokines also up-regulate expression of cognate receptors for integrins on endothelial cells such as intracellular adhesion molecule and vascular adhesion molecule, causing neutrophil adhesion to the endothelium. Activation of endothelial cells by proinflammatory cytokines also results in increased nitric oxide (NO) production via inducible NO synthase. Neutrophils cause further tissue injury by production of reactive oxygen intermediates. Reactive oxygen intermediates plus NO result in the formation of peroxynitrite, which contributes to further cell damage.
Tissue factor (TF) can be produced by direct cellular injury and neutrophil adhesion. TF activates the coagulation cascade, resulting in thrombin production and fibrin deposition that up-regulates of the inflammatory cascade.5
Additionally, inflammatory cytokines stimulate release of TF from monocytes and endothelium. Counterregulatory molecules such as APC inhibit this process at several levels. For example, APC inhibits thrombin generation, and inhibits the rolling of monocytes and neutrophils on injured endothelium, thereby inhibiting further production of inflammatory cytokines. APC also increases the fibrinolytic response by inhibiting platelet-activation inhibitor 1.
Thus, inflammation and coagulation are intimately related and costimulatory. This simplified description of the innate immune response to infection highlights many targets for study of genetic polymorphisms that could be key to understanding the genetic susceptibility to sepsis. We now review the genetic polymorphisms currently thought to influence the severity of the inflammatory response and summarize the nine clinical studies to date testing these hypotheses.
|
TNF-
|
|---|
TNF- plays a central role in the pathogenesis of the acute inflammatory response, and in some studies high levels of TNF- correlate with severity of disease. Genetic variability at the TNF loci within the MHC on chromosome 6 (Fig 2
) have been well characterized.26
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 2. Structure of the TNF genes. The locations of MHC loci are shown. Hatched boxes indicate the relative positions of the indicated loci. Open boxes represent the untranslated portions of the TNF exons, and closed boxes represent translated portions. Lines indicate introns (areas of the gene that are spliced out of the mature RNA product). Arrows indicate the transcriptional orientation of the genes. Reprinted with permission by Webb et al.26
kb = kilobase. C2 = complement component 2; Bf = complement factor B; C4A = complement component 4A; C4B = complement component C4B; 21-OH = 21-hydroxylase.
|
|
A TNF- polymorphism at position -308 in the promoter region of the TNF- gene, consisting of a G (TNF--308G) in the common allele and an A (TNF--308A) in the uncommon (wild-type) allele, modifies gene expression.27
The TNF--308A allele has a prevalence of approximately 30% in the general white population.28
In vitro studies show that TNF--308A variant displays increased gene transcription as compared with the wild-type allele,27
and is associated with increased secretion of TNF- from macrophages in vitro and elevated TNF- blood concentration in vivo. There is evidence that the TNF--308A allele is associated with adverse outcome in a variety of infectious and inflammatory diseases including cerebral malaria,29
meningococcal disease,30
and celiac disease.31
The TNF- promoter gene is in linkage disequilibrium with several HLA alleles that may be involved with the control of TNF- secretion, or that may be independent risk factors for the development of meningococcal disease or other forms of sepsis.
There have been four studies that examined the association between the TNF--308A polymorphism and outcome from sepsis (Table 2
). Nadel and coworkers30
reported an association study of 98 consecutive children admitted with meningococcal disease in the United Kingdom and found a significantly higher variant allele (TNF--308A) prevalence in nonsurvivors compared to survivors. Possession of at least one copy of the variant allele was associated with a significantly increased risk of more fulminant meningococcal infection and death. The relative risk of death in heterozygotes was 2.5 compared with children homozygous for the wild-type allele. All three children who were homozygous for the variant allele died. Ethnicity was not reported, and TNF- levels were not measured; therefore, the possibility of a spurious association exists. Stuber and coworkers32
reported an association study of the TNF--308 allele and outcome of 80 postoperative patients with severe sepsis. They found no difference in allele frequency between healthy German blood donors and German patients admitted to the surgical ICU for severe postoperative sepsis. They also found no difference in allele frequency between survivors and nonsurvivors of severe sepsis. The strength of this study was that the cases and control subjects were well matched in terms of disease severity and were ethnically homogeneous; all were German whites and from the same area. An important finding was that patients grouped according to their genotypes did not differ in their serum levels of TNF- in vivo or after in vitro stimulation of monocytes by endotoxin. These investigators also performed transfection studies to assess allelic variation in the TNF--308 promoter region. G to A variation did not change the endotoxin-inducible promoter activity.32
Mira and coworkers28
reported an association study of the TNF--308 promoter region SNP and septic shock. In contrast to Stuber et al,32
they found an increased prevalence of the variant allele in the patients admitted to the ICU with severe septic shock compared to healthy, French, white blood donors. The important finding was that among the patients with septic shock, the variant allele frequency was significantly greater in nonsurvivors compared to survivors (Table 2)
. After controlling for age and severity of illness, multiple logistic regression analysis showed that patients possessing at least one TNF--308A allele had a 3.7-fold risk of death (95% CI, 1.37 to 10.24). The authors concluded that the variant allele is strongly associated with susceptibility to septic shock and with death due to septic shock. One weakness of this study is that these investigators did not stratify or match for ethnicity. Additionally, supporting the findings of Stuber et al,32
there were no differences in serum TNF- levels between groups stratified by genotype.
The most recent association study of the TNF--308 promoter region and septic shock is by Tang and co-workers33
in Taiwan. They genotyped consecutive patients admitted to a postoperative ICU. They found no difference in allele frequency between survivors and nonsurvivors, and no increase in variant allele frequency in patients with septic shock. In the subgroup of patients with septic shock, they found that a significantly greater proportion of nonsurvivors possessed at least one copy of the variant allele compared with survivors of septic shock (Table 2)
. The mortality in the patients with septic shock possessing at least one variant allele was 92% compared to 62% mortality (p < 0.05) in those who did not. They also report higher serum TNF- levels in patients with septic shock who did not survive compared with those who survived (p < 0.05). They do not comment whether the subgroup of patients with septic shock was well matched for other confounders. Ethnicity was not stated, although most of the patients were elderly, male, Taiwanese veterans.
In summary, the relevance of the TNF--308 promoter region SNP and the susceptibility to sepsis and outcome from sepsis has not been supported by association studies32
33
except in meningococcal disease.30
In contrast, the outcome from septic shock has been associated with the G to A polymorphism in the TNF--308 promoter region in at least two studies to date.28
33
The TNF--308A allele for exaggerated TNF- gene expression is not uniformly supported by in vitro and ex-vivo studies.32
35
Stuber et al32
hypothesized that the findings of association studies emphasizing the importance of the TNF--308A allele are due to linkage between the TNF--308 locus and another MHC locus.
|
TNF-ß
|
|---|
In contrast to TNF-, which is expressed by macrophages, TNF-ß (lymphotoxin A) is expressed and released by lymphocytes. A TNF-ß polymorphism exists at position 252 within the first intron of the TNF-ß gene, consisting of a G (TNF-ß-252G) on one allele and an A (TNF-ß-252A) on the alternate allele.36
The TNF- and TNF-ß genes are positioned next to each other within the cluster of HLA class III genes on chromosome 6 (Fig 2)
.16
Evolutionary studies suggest a common ancestor for both genes that duplicated during evolution.37
Individuals homozygous for TNF-ß-252A (known as "TNF-ß2" in most studies) have been shown to secrete increased levels of TNF- in response to sepsis.34
In vitro studies confirm the association of this polymorphism with increased secretion of TNF- in response to endotoxin.38
There have been three clinical studies to date testing the association between the TNF-ß-252 G to A polymorphism and outcome from severe sepsis (Table 2)
. Stuber and co-workers34
compared nonsurvivors of postoperative severe sepsis with survivors and found that 65% of nonsurvivors were homozygous for the variant (TNF-ß252A) allele compared to 12% of survivors (p < 0.005). In another larger study,32
they confirmed these findings. Additionally, the homozygotes for the variant allele had significantly higher serum TNF- levels than did other genotypes.34
The patients were well matched for age and severity of illness, and were from the same ethnic background.
Another study20
examined the association between outcome from blunt trauma and the TNF-ß-252A polymorphism. Genotype frequency in patients with an uncomplicated clinical course differed significantly from that of patients with severe posttraumatic sepsis (Table 2)
. When matched for age and injury, patients with the homozygous TNF-ß-252A genotype had an odds ratio for death of 3.45 (95% CI, 1.54 to 7.77; p = 0.0026) compared with heterozygotes. They also found that patients with this genotype had significantly higher serum TNF- levels compared with the heterozygotes. This study suggests that detection of homozygosity for the TNF-ß-252A genotype may be a useful tool for evaluating patients at high risk for sepsis after blunt trauma. Furthermore, this strategy may theoretically identify patients who might benefit from anti-TNF treatment. The authors stress that the polymorphism may not be directly linked to sepsis susceptibility but may be a marker for another gene in the MHC region.
Waterer et al39
reported a prospective cohort study of 280 consecutive patients admitted with community-acquired pneumonia and found an association between the TNF-ß- 252AA genotype (high secretors of TNF) and development of septic shock. This did not translate to a higher mortality in the TNF-ß-252AA group. They did not find any significant association between TNF--308A allele and susceptibility to sepsis, septic shock, or death. They also analyzed the TNF--308A:TNF-ß-252A haplotype (high secretors of TNF) and found no association between the risk of septic shock and this haplotype.
Why do the aforementioned clinical studies seem to have such contradictory results? A major limitation of association studies is that they are frequently confounded by a type I (false-positive) error. This highlights the need for much larger sample sizes to detect true association with 80% power when 
10% of the variance in the trait is explained by the locus.
|
Other Candidate Genes
|
|---|
Other candidate genes for sepsis and septic shock located include the IL-1 receptor antagonist (IL-1ra) gene,40
the HSP gene,41
the IL-6 gene,42
the IL-10 gene,43
the CD-14 gene,44
the TLR-4 gene,45
and the TLR-2 gene (Fig 1)
.46
|
IL-1
|
|---|
IL-1 is an important proinflammatory cytokine. IL-1 increases plasma concentrations of platelet activating factor, prostaglandins, and NO.47
These are potent vasodilators and induce shock in animal models. IL-1ra is a naturally occurring inhibitor of IL-1 activity that competes with IL-1 for occupancy of cell-surface receptors but possesses no agonist activity. After experimental endotoxemia, the concentration of IL-1ra increases in parallel with the decrease in IL-1B concentration in the plasma of human volunteers.48
A polymorphic region within intron 2 of the IL-1ra gene contains a variable number of 86 base-pair tandem repeats.40
Six alleles at this polymorphic site have been identified. The allele frequency in healthy control subjects is 54% for IL-lraA1 (four repeats) and 34% for IL-1raA2 (two repeats), with the other alleles being much less common.49
The IL-raA2 allele is associated with increased IL-1ra protein production and reduced IL-1 production by monocytes.50
The IL-1raA2 allele occurs with increased frequency in patients with severe sepsis compared to normal individuals48
; therefore, it has been suggested as a genetic risk factor for sepsis.49
50
However, IL-1raA2 was not associated with a worse outcome.49
While no association with outcome and this polymorphism were detected, coincidence of homozygous TNF-ß2 allele and IL-1raA2 genotypes identified a group with a 100% mortality rate from sepsis,49
suggesting a potentially important interaction of TNF- and IL-1ra.
|
HSPs
|
|---|
HSPs are expressed in response to heat shock and a variety of other stimuli, including endotoxin and other mediators of severe sepsis.51
Three genes encoding members of the HSP family (HSP70) lie in the class III region of the MHC.16
Individuals carrying the polymorphism HSP70–2G exhibit lower levels of mRNA ex-vivo.41
Schroeder and coworkers41
hypothesized that individuals homozygous for the HSP70–2G allele should have greater susceptibility to and/or higher mortality from sepsis compared to other genotypes. They tested this hypothesis in 87 patients admitted to a surgical ICU with severe sepsis and found no association.41
They did find a linkage between HSP70–2A (the "protective" allele) and TNF-ß2, an allelic variant previously shown to be associated with higher TNF- levels and worse outcome from sepsis.32
The overall mortality in the group of HSP-2G homozygotes was not increased possibly because of linkage with the non-TNF-ß2 haplotype. This finding highlights the importance of knowing all the polymorphisms relevant to the inflammatory response in individuals rather than interpreting a polymorphism in isolation.
|
IL-6
|
|---|
IL-6 is a prominent proinflammatory cytokine52
associated with an increased occurrence of shock and death in septic patients.53
Because IL-6 is expression is triggered by many early proinflammatory pathways, IL-6 may represent an integrated index of the acute inflammatory response. Recently, a G to C polymorphism at -174 in the promoter region of the IL-6 gene, found on chromosome 7, was reported.42
The frequency of the IL-6–174C allele is 40% in the general population and is decreased in patients with the inflammatory disease, juvenile rheumatoid arthritis.42
Transfection of the IL-6–174C allele into HeLa cells in vitro resulted in reduced production of IL-6 compared to the IL-6–174G allele. In healthy subjects, those with the IL-6–174C allele had significantly lower plasma concentrations of IL-6.42
However, IL-6 expression may be cell-line specific.54
Additionally, other polymorphisms in the promoter region of the IL-6 gene may influence IL-6 transcription with complex interaction determined by the haplotype.54
To date, there have been no studies of this SNP in sepsis.
|
IL-10
|
|---|
TOP
Abstract
Introduction
