As early as the 1918 flu pandemic, clinicians have documented the hyper-inflammatory response and its contribution to unexpected mortality in young, previously healthy patients. Trauma surgeons today continue to encounter hyper-inflammatory mediated syndromes: Adult Respiratory Distress Syndrome2
, Abdominal Compartment Syndrome6
and Multi-Organ Failure7
. It is well documented that C2 polymorphisms and deficiencies have been linked to several chronic inflammatory conditions: age-related macular degeneration (AMD)8, 9
and systemic lupus erythematosus (SLE),10
respectively. Although functional differences between the C2 variants have not been well elucidated, it is possible that each influences the level of complement activation differently. In the rare cases of C2 deficiency, the effect is clearer. Individuals lacking C2 have an impaired ability to activate complement through the lectin and classical pathways. It has been shown that C2 deficient patients have higher levels of circulating immune complexes. The impaired ability of these individuals to clear circulating immune complexes may predispose them to autoimmune disease.4
Consequently, we chose to look at the Complement System in general, and C2 specifically, because we believe an understanding of complement activation may lead to understanding inflammation and identify new diagnoses, new ways to stratify patient risk, and new therapy.
In this manuscript we describe preliminary results of an association study which suggest an association between C2 E318D and increased mortality and infection. In the univariate model, the presence of E318D appears to double the mortality rate. In the multivariate model, which controlled for confounders (age, injury severity, gender and lactate), the effect is equally pronounced. Patients with the C2 E318D polymorphism have increased mortality (OR: 2.65; p = .02) and increased probability of VAP (OR: 2.00; p = .04)
The genetics of the inflammatory response is complex. Complement activation is one key component of the acute inflammatory response. Complement protects against infection, kills diseased cells, introduces inflammation, eliminates damaged tissue and promotes wound healing.4
There are three major pathways of complement activation: The Classical pathway is
activated by antigen bound antibodies. The Lectin pathway
is activated by polysaccharides, particularly those containing mannose. The Alternative pathway
is activated by many microbial substances and other foreign surfaces.4
The three pathways for complement activation converge with the proteolytic activation of Complement Component 3 (C3) through the formation of enzymatic complexes termed C3 convertases. There are two types of C3 convertase. The Alternative pathway C3 convertase consists of activated forms of C3 and Factor B. The Classical and Lectin pathways share the Classical pathway C3 convertase which consists of activated forms of C2 and C4.4
Both types of C3 convertase proteolytically activate C3 into two peptides, C3a and C3b. C3b possesses a reactive thioester that allows it to covalently attach to a variety of surfaces. Factor B then associates with C3b to form the precursor C3 convertase (C3bB). This complex is subsequently acted upon by another complement protease, Factor D, to form the active C3 convertase (C3Bb). All pathways are thus amplified by the alternative pathway through C3 activation. In the shared terminal pathway, C5 is proteolytically activated next, to form C5a and C5b, Assembly of the membrane attack complex
follows, by sequential addition of components C6 through C9 to C5b.4
In addition to triggering target cell lysis, complement activation also induces inflammation. The activation of C3 and C5 produces two small peptides, the anaphylatoxins C3a and C5a which are potent inflammatory mediators. These peptides have partially overlapping effects, including immune cell activation, increased vascular permeability and histamine release.4
It is these conflicting effects which make the pathway so interesting in trauma patients.
Because the complement system has both positive and negative effects, it is highly regulated. The cascade possesses at least ten negative regulators. Once the C2 zymogen is activated, it forms a proteolytic subunit of the Classical Pathway C3 convertase. This convertase is shared by the Lectin and Classical pathways of complement activation.4
There is evidence that circulating self-reactive antibodies can activate complement in the model of ischemia/reperfusion injury.11
Individuals genetically lacking C2 are prone to autoimmune disease and possess circulating auto-antibodies.10
Rapid complement activation has been shown to occur following trauma, both in animal models and in man. The level of complement activation in the hours immediately after injury correlates well with the risk of mortality. It is therefore conceivable that variants in complement proteins may influence the level of complement activation following injury, particularly for the proteolytic subunit of the Classical pathway C3 convertase enzyme complex.
Strengths and Limitations
This is a genetics association study. Such association studies have two potential limitations that we attempted to avoid: selection bias and clinical variability. To minimize selection bias, we employed a population-based, prospectively collected, cohort of consecutive patients to a single trauma ICU from a large catchment area.
Clinical variability is minimized by aggregating all trauma patients in the hands of a small number of physicians practicing under standardized evidence-based protocols. This structure was designed to minimize variation in clinical care and maximize the strength of the genetic signal. Additionally, the exclusion of patients with an ISS of > 45 was designed to magnify the genetics signal by excluding patients with a low probability of survival. We believe that in the highest decile of acuity, the genetic signal contributing to mortality risk is overwhelmed by the magnitude of injury.
Finally, the C2 polymorphisms were selected because of prior epidemiological evidence suggesting associations with specific phenotypes.10, 11
This lends biological plausibility to their potential involvement. It remains possible the C2 E318D polymorphism is in linkage disequilibrium with the causative polymorphism. Additional future research into the functional status of this well-known polymorphism is warranted.
Clinical studies and the ICU environment are difficult. Genetic studies are especially difficult as both the phenotype and the environment are difficult control, the regulatory environment is rigorous and the polymorphisms are highly complex and involve many potential confounding variables. Additionally, there are many polymorphisms worthy of investigation.
Consequently, interpretation of this study requires perspective. This is an association study. We have only demonstrated an association between the specific C2 polymorphism E318D and the outcome criteria: death and infection. This work requires scientific validation in an entirely new population, in thousands of patients, prospectively collected under similar conditions present in this study. This project is currently underway.
The emerging era of personalized medicine mandates defining the relationship between individual genetic variation and outcome in critical care patients. To understand the interaction between the genome, the ICU environment and inflammation will require: thousands of patients, powerful informatics tools and DNA sequencing.
In the future, advancements will be made in chip technology and the cost will decline. This will allow us to move from pathway analysis to genome wide association studies. Genome wide association studies now can analyze one million single nucleotide polymorphisms (SNP) that begin to cover a significant proportion of an individual's genetic variation. These studies probe a wide variety of biochemical pathways and can generate new hypotheses based on unanticipated associations.
The simultaneous analysis of millions of genes will challenge our computational and analytical skills. We will need to develop new methods of computing, such as parallel processing and quantum computing. We are preparing for the day when trauma patients routinely have DNA genotyping upon admission. Ideally select populations, such as the military, will be genotyped prior to injury. We envision the use of microarray chips containing numerous polymorphisms, all validated, which stratify patients by risk of disease or complication and predict response to therapy. We expect regulatory proteins associated with complement activation to be part of that chip.
In summary, trauma patients generate a robust but variable acute inflammatory response, which may protect against infection or may be pathologic. We have demonstrated an association between the C2 polymorphism E318D, a regulator of complement activation, and mortality and VAP in a population of trauma patients. Of the 702 trauma patients in this study group, 8.3% had the high risk genetic variation C2 E318D which was associated with increased mortality (OR: 2.65) and infection risk (OR: 2.00). The C2 E318D variation:
- Identifies a previously unknown high risk group for infection and mortality;
- Can potentially be determined on admission;
- May provide opportunity for early therapeutic intervention; and
- Requires validation in a new, larger and distinct cohort of patients.