Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is a devastating
form of respiratory failure characterized by intense inflammation and increased permeability
in the lungs that usually develops in response to a major insult such as sepsis, trauma,
pneumonia, burns, or multiple transfusions [1]. Despite the common occurrence of these
risk factors, only a minority of patients who have these injuries develops ALI [2],
[3]. ALI/ARDS is now recognized as being more prevalent than initially thought, with
an age-adjusted incidence of 86.2/100,000 person-years, with a mortality of 38.5%,
and with significant morbidity among the survivors [4], [5].
Because ALI has such high mortality and morbidity, any intervention that could prevent
or treat ALI would have a significant impact on critical care medicine and on public
health. Epidemiologic studies can contribute to prevention and treatment by determining
the risk factors associated with variable susceptibility and outcomes that could be
modified to decrease the risk of developing the disease or of having a poor outcome.
The current understanding of why some patients develop and die from ALI and others
do not is incomplete. Recently, discoveries about the genetic control and regulation
of innate immunity and inflammatory response have raised the question of whether the
multiple polymorphic alleles of genes that encode for cytokines and other mediators
of inflammation may result in phenotypic differences in host inflammatory response.
These differences may account for some of the heterogeneity in individual susceptibility
to and prognosis in ARDS.
Since the initial description of ALI, there has been much research on the role of
complement, endotoxin, and pro- and anti-inflammatory cytokine response in the pathogenesis
and course of ALI/ARDS [6]. Protein biomarkers, such as tumor necrosis factor-α (TNF-α),
interleukin-6 (IL-6), plasminogen activator inhibitor-1, surfactant protein B (SFTPB),
and von Willebrand's factor antigen, may be useful in predicting either development
of or outcomes in ALI [7], [8], [9], [10], [11]. Although research on protein biomarkers
in ALI/ARDS has contributed greatly to the understanding of the pathogenesis of ALI,
it has not yet led to novel interventions.
Genetic epidemiology is a relatively new discipline that seeks to determine the role
of genetic factors and their interactions with the environment in the occurrence of
the disease or its outcome within a population [12]. Genetic epidemiology has been
applied to the study of ALI. only recently. Genes hold several advantages over protein
markers of lung injury, especially for possible prevention. Unlike cytokines, which
can vary with the precipitant factor for ALI and with the time course of critical
illness, a person's genotype is constant throughout the individual's life, regardless
of health status. Thus, there is inherently less variability to the determination
of genotypes than protein markers. The variation of many of the protein markers before
and during critical illness means that the window of opportunity for assessment must
be consistent and is likely to be narrow. Such a window for assessment may be especially
impractical in the prevention of ALI/ARDS, because lung injury tends to develop rapidly,
within hours to days of the predisposing injury. In addition, in ALI regional differences
in the expression and concentrations of some cytokines, such as TNF-α, means that
biomarkers may be best measured from alveolar fluid [13]. Measurements from the lungs
are invasive, are vulnerable to technical variation, and are not always appropriate
for severely hypoxic patients who have ARDS or for the nonintubated patients at risk.
DNA for genotype assessment can be obtained easily from peripheral blood samples,
and thus genotype assessment can be performed safely for any patient. Another advantage
of genes is that any true genetic association with the disease is unlikely to be an
epiphenomenon related to lung injury. Any variation in a protein marker may be a product
rather than the cause of developing lung injury. The individual's genotype, however,
precedes the lung injury and the precipitant to lung injury. Thus, any true genetic
association supports the biologic causality of the gene or its product in the development
of ALI/ARDS and the targeting of the gene in future preventions. Last, the invariant
nature of the genome means that an individual's genetic predisposition to developing
lung injury could be determined in advance and noted in the individual's medical records
or, conceivably, on an encrypted microchip worn by the individual. This precaution
would be especially useful in interventions to prevent ALI/ARDS, because the injury
leading to ALI/ARDS is almost always unanticipated, and the window for intervention
to prevent lung injury after the insult is narrow.
In the last few years, there has been a sudden explosion of studies of the genetic
susceptibility of ALI/ARDS. The following sections review the recently published studies
in the genetic epidemiology of ALI/ARDS and discuss the relative strengths and limitations
of the current approach with a focus on the implications for future prevention and
treatment. The possible applications and potential limitations to the translation
of genomics and genetic epidemiology to future prevention and treatment of ALI/ARDS
are discussed also.
Current approach and recent studies in the genetic epidemiology of acute lung injury/acute
respiratory distress syndrome
Candidate-gene approach
Traditionally, the term “pharmacogenomics” referred to the application of whole-genome
scanning for the discovery of new drug targets [14]. Genome-wide studies examining
anonymous markers spaced throughout the entire genome are not yet practical in ALI/ARDS.
Rather, all studies thus far have used the candidate-gene approach, which focuses
on specific genes whose products have been well characterized as biologically important
in the pathogenesis, progression, or manifestation of ALI/ARDS [15]. The candidate-gene
approach is hypothesis driven and founded on current knowledge of the disease process.
The validity of the candidate gene rests on the evidence supporting its selection
as a candidate in ALI/ARDS.
Table 1 details the candidate genes that have been studied in ALI/ARDS and the evidence
supporting their selection.
Table 1
Candidate genes and polymorphisms examined in acute lung injury/acute respiratory
distress syndrome and the evidence supporting their biological plausibility
Polymorphisms studied in ALI/ARDS
Evidence supporting importance in ALI/ARDS
Candidate gene
Polymorphism
Functional significance
ACE
[16]
Insertion/deletion polymorphism in intron 16
Yes
D allele associated with severity of and mortality in meningococcal disease [17]
ACE levels or activity is variable in ARDS patients [18], [19]
More recently, ACE linked to ALI in ACE knockout mice [20]
CC16
[21]
−226GA promoter SNP
Yes
−226GA polymorphism associated with asthma but not critical illnesses
Lower Clara cell protein levels correlate with severity of bacterial pneumonia but
no reports in ALI/ARDS [22]
IL-6
[23]
−174GC promoter SNP
Yes
Plasma IL-6 correlate with ARDS mortality [10]
IL-6 Haplotypes associated with mortality in systemic inflammatory response [24]
Functional genomics indicate altered IL-6 gene expression in ALI [25]
IL-10
[26]
−1082GA promoter SNP
Yes
In pneumonia,−1082GG genotype is associated with increased mortality [27]
−1082GG genotype occurs less frequently in critical illness compared to healthy controls
and is associated with lower severity of illness, organ failure, and mortality [28],
[29], [30]
Low bronchoalveolar lavage IL-10 correlate with ARDS and mortality in ARDS but high
plasma IL-10 correlate with ARDS and sepsis mortality [31], [32]
MBL-2
codon −221 promoter SNP
codon 52 polymorphism
codon 54 polymorphism
codon 57 polymorphism
Yes
Variant X, D, B, and C alleles of codon −221, 52, 54, and 57 are associated with low
serum MBL deficiency, greater risk of sepsis, greater severity of sepsis, and/or increased
mortality in sepsis [33], [34]
MLCK
Haplotypes examined
No
Functional genomics indicate altered MLCK gene expression in ALI [35]
MLCK involved in ventilator and sepsis associated lung injury in animals [35], [36]
PBEF
[37]
T-1001G promoter SNP
C-1543T promoter SNP
No
Yes
Functional genomics indicate altered PBEF gene expression in ALI [35]
Increased PBEF protein in animal models of ALI and in humans with ALI [37]
SFTPB
[38], [39], [40]
Insertion/deletion polymorphism in intron 4
+1580CT SNP in codon 131
No
Suspected but not known
SFTP-B limits lung injury in animals and correlate with respiratory failure in humans
[41], [42]
Insertion/deletion polymorphism in intron 4 is associated with neonatal respiratory
distress syndrome [43]
TNF-α and TNF-β [44]
−308GA SNP in TNF-α
TNF-β1/2 Ncol SNP in TNFB
Yes in some but not all studies
Increased plasma or bronchoalveolar TNF-α correlate with development of or mortality
in ARDS in some but not all studies [9], [10], [32], [45]
−308A allele and TNF-β2 homozygotes associated with sepsis in some studies [46], [47],
[48]
VGEF
[49]
+936CT SNP
Yes
Plasma VGEF increases and pulmonary VGEF decreased with ARDS and then normalizes with
recovery in ARDS [50]
No known association between +936CT polymorphism and critical illnesses
Abbreviations: ACE, angiotensin-converting enzyme; ALI, acute lung injury; ARDS, acute
respiratory distress syndrome; CC16, Clara cell protein 16; IL-6, interleukin-6; IL-10,
interleukin-10; MBL-2, mannose-binding lectin-2; MLCK, myosin light-chain kinase;
PBEF, pre-B-cell colony-enhancing factor; SFTPB, surfactant protein B; SNP, single
nucleotide polymorphism; TNF-α, tumor necrosis factor-α; TNF-β, tumor necrosis factor-β;
VEGF, vascular endothelial growth factor.
The strongest candidates for investigation are the genes that have been linked to
ALI in previous linkage studies, in association studies, or in animal models of the
disease (
Fig. 1) [51]. Investigations into the genetic determinants of ALI/ARDS have been undertaken
only recently. The selections of many of the candidate genes in recently published
studies were supported by previously published reports in other, similar conditions,
such as neonatal respiratory distress syndrome for the SFTPB gene and sepsis for the
TNF-α, IL-10, mannose binding lectin-2 (MBL-2), and IL-6 genes. Conversely, several
candidate genes found to be associated with ARDS (ie, the +1580CT polymorphisms in
the SFTPB gene, the T-1001G and C-1543T polymorphisms in the pre–B-cell colony-enhancing
factor [PBEF] gene, and the codon 54 polymorphism in the MBL-2 gene) were also found
to be associated with increased risk for sepsis or septic shock in the same population
[37], [38], [52]. Overall this finding suggests that genes and polymorphisms that
have been implicated in sepsis would serve as strong candidate genes in ALI/ARDS.
Fig. 1
Criteria for strong candidate genes in ALI. The strongest candidates for investigation
in ALI/ARDS are genes in which specific alleles have been linked with ALI/ARDS or
related diseases such as sepsis, neonatal respiratory distress syndrome, or other
critical illnesses. Alternately, in the absence of such data, there should be evidence
supporting the importance of the gene product or function in ALI/ARDS. If a direct
candidate-gene approach is used, additional evidence for the functional significance
of the allele of interest should exist. (Adapted from Gong MN, Christiani DC. Genetic
epidemiology of acute lung injury. In: Mathay MA, editor. Acute respiratory distress
syndrome. New York: Marcel Dekker, Inc.; 2003. p. 392; with permission.)
In the absence of studies of ALI or related conditions, the biologic plausibility
of the candidate gene in the pathogenesis of lung injury is important (see Fig. 1).
There should be evidence supporting the importance of the gene product or function
specifically in ALI.
More recently, novel candidate genes in ALI have come from functional genomic studies
that established their biologic plausibility in lung injury. PBEF is a cytokine and
adipokine with a variety of functions including the maturation of B-cell precursors,
inhibition of neutrophil apoptosis in sepsis, and stimulation of glucose uptake with
action similar to insulin [5], [53]. Its role in ALI had not been reported until expression
of the PBEF gene was found to be increased in a series of animal models of stretch
and liposaccharide-induced lung injury and in vivo studies of patients who had ALI
[25]. Other potential candidates in ALI/ARDS that had increased expression included
genes previously suspected to be important, such as IL-6, plasminogen activator inhibitor-1,
and Myosin light-chain kinase (MLCK). PBEF protein expression also was increased in
the lungs, bronchoalveolar lavage fluid, and serum of patients who had ALI. After
identifying two common-promoter single-nucleotide polymorphisms (SNPs) in the PBEF
gene, Ye and colleagues [37] found that the variant of the T-1001G polymorphism was
associated with increased risk of sepsis-induced ALI compared with healthy controls,
whereas the variant C-1543T polymorphism was associated with a protective effect in
sepsis-induced ALI compared with healthy adults.
After the selection of the candidate genes, there are two approaches to investigation.
The direct approach focuses on the association between ALI/ARDS and specific polymorphisms,
often SNPs, in the candidate gene that are thought to be functional, either because
of linkage with other disease processes or because of the known effect on the levels,
function, or effectiveness of the gene. Such an approach is effective for hypothesis
testing but is limited to previously studied polymorphisms of a gene. This approach
is the one most commonly used in ALI/ARDS.
Alternatively, the indirect approach examines all common SNPs in the gene (> 1% in
a sample population), regardless of whether the SNPs have any functional significance.
Often these SNPs are examined individually and in combination with other SNPs on the
same gene. The term “haplotype” refers to two or more SNPs that are linked and tend
to be inherited together en bloc. Multilocus haplotypes can be viewed as signature
patterns of allelic variation on a gene that capture and characterize all polymorphisms
within the haplotype block. The functional or disease polymorphism may be one of the
loci genotyped, or it may reside within the haplotype block and be captured by the
haplotype. Thus, the haplotype would serve as a surrogate marker for the functional
polymorphism that is truly linked to the disease state. As such, some argue that haplotype
analyses could identify functional or disease loci better than a single polymorphism,
especially if the penetrance is low, as would be expected in complex diseases like
ARDS [54], [55]. Haplotype analyses also can be more efficient in large epidemiology
studies, because genotyping can be confined to the minimum number of SNPs that define
that haplotype block (haplotype-tagging SNPs) [56]. Haplotype analyses also can capture
cis interaction between SNPs. If one polymorphism increases the risk of disease only
in the presence of another polymorphism in the same gene, haplotype analysis will
be able to discern this relationship, whereas separate analyses of the polymorphisms
will not. Last, haplotype analysis can help localize the disease locus to within the
haplotype block in the gene and thus may help focus the search for functional variants
in subsequent studies. The haplotype approach has become increasing popular in the
genetic epidemiology of complex diseases, and this approach was used in the investigation
of the PBEF and MLCK genes in ALI.
Together, these studies have validated the candidate-gene approach in the search for
genetic determinants of ALI/ARDS. Although this approach is hypothesis driven and
is well validated in the genetic epidemiology of complex diseases, it is only as strong
as the hypothesis supporting the choice of candidates. Thus, the possibility that
any candidate gene in ALI/ARDS can serve as a potential target for future preventive
and therapeutic measures will rest on the strength of the evidence supporting its
role as a candidate gene in ALI/ARDS. This evidence will not depend on any one genetic
epidemiology study. Rather, it must be grounded in a series of genetic, molecular,
bioinformatics, and clinical studies and confirmatory studies that support the biologic
plausibility of the gene in ALI/ARDS.
Case-control study design
Given the high mortality in ARDS and the generally late age of onset, traditional
family-based approaches in genetic epidemiology are either not feasible or impractical.
Rather, studies in ALI/ARDS have established the unrelated case-control study as an
effective and well-validated design in the investigation of the genetic determinants
of ALI/ARDS. Case-control studies require the delineation of a control group and focus
on whether the gene of interest occurs at a significantly greater frequency among
the patients who have the disease than among the controls. One of the most important
advantages of case-control studies in complex disorders such as ALI is the power of
the design. Association studies are the most sensitive and powerful of all of the
study designs described thus far in detecting common, low-penetrant susceptibility
genes in complex disease [12]. In addition, the case-control design is well suited
to the study of genetic markers of disease. Genes are stable indicators of disease
susceptibility, because they do not change with time or circumstances. The use of
genetic markers as the exposure eliminates recall bias that often plagues case-control
studies. Case-control studies also are amendable to multivariate modeling, which allows
adjustment for important nongenetic factors and interactions.
Because of the power and versatility of association studies, many believe that the
future deciphering of the genetics of complex diseases will involve case-control studies
[51], [57]. With increasing use of this design, however, comes some misuse as well.
The most common and troubling criticisms of association studies are inconsistency
and lack of reproducibility. This heterogeneity is caused by a number of factors.
The epidemiologic quality of published genetic studies is quite variable [58]. Other
factors include the lack of power in some studies (type II errors) and the lack of
control for confounders such as population differences or gene–environment interaction.
As is true in any case-control design in epidemiology, the strength of the study depends
entirely on the proper selection of cases and controls and on the appropriate accounting
of the potential confounders, power and type I error [59]. The following section focuses
on the features of genetic case-control design as illustrated by studies in ALI.
Table 2 details some of these features and the results of recent genetic epidemiology
studies in ALI/ARDS.
Table 2
Summary of published genetic epidemiology studies in acute lung injury/acute respiratory
distress syndrome
Patient population
Major findings
Candidate gene
Genotype studied
Study
Case
Controls
Susceptibility to ALI/ARDS
Outcomes in ALI/ARDS
ACE
Insertion/deletion polymorphism in intron 16
Marshall et al [16]
96 whites with AECC defined ARDS
88 whites with non-ARDS respiratory failure
174 whites after heart surgery
1906 healthy white males
D allele and DD genotype associated with increased susceptibility to ARDS compared
to all control groups
Increasing mortality in ARDS associated with increasing number of D alleles carried
Chan et al [60]
17 Chinese patients with AECC defined ARDS from SARS
123 Chinese patients with SARS326 healthy Chinese individuals
No association found
Not examined
CC16
−226GA promoter SNP
Frerking et al [21]
117 German with AECC-defined ARDS
373 healthy German newborns
No association found
Not examined
IL-6
−174GC promoter SNP
Marshall et al [23]
96 whites with AECC defined ARDS
88 whites with non-ARDS respiratory failure
174 whites after heart surgery
1906 healthy whites males
No association found
−174C allele and −174CC genotype correlated with serum IL-6 levels, and was associated
with survival in ARDS and in non-ARDS with respiratory failure
IL-10
−1082GA promoter SNP
Gong et al [26]
211 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma,
aspiration, and massive transfusion
429 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration,
and massive who did not develop ARDS
−1082GG genotype was associated with ARDS but only in presence of significant interaction
between genotype and age
−1082GG genotype associated with less organ failure and lower mortality in ARDS
MBL-2
codon −221 promoter SNP
codon 52 SNP
codon 54 SNP
codon 57 SNP
Gong et al [52]
212 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma,
aspiration, and massive transfusion
442 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration,
and massive transfusions who did not develop ARDS.
Homozygotes for variant codon 54B allele was associated with greater severity of illness
and increased susceptibility to ARDS
Homozygotes for variant codon 54B allele was associated with greater daily organ failures
and increased ARDS mortality
MLCK
28 SNPs in whites
25 SNPs in African Americans
Gao et al [61]
92 whites with sepsis related AECC defined ALI
114 whites with sepsis
85 healthy whites
51 AA with sepsis
61 healthy African Americans
One SNP and one haplotype associated with ALI in whites compared with septic controls
Not examined
46 African Americans sepsis-related AECC defined ALI
2 haplotypes associated with ALI in African Americans compared to septic controls
PBEF
T-1001G promoter SNP
C-1543T promoter SNP
Ye et al [37]
87 whites with sepsis-related AECC-defined ALI
100 whites with sepsis
84 healthy whites
Compared to healthy controls, variant G1001 allele and 1001G:1543C haplotype were
associated with increased susceptibility to ALI while the variant T1543 allele was
associated with decreased susceptibility to ALI
No association between variant G1001 allele and ARDS mortality
No association seen in comparison with septic controls
Bajwa et al [62]
375 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma,
aspiration, and massive transfusion
787 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration,
and massive transfusions who did not develop ARDS
Variant G1001 allele and 1001G:1543C haplotype associated with increased susceptibility
to ALI in septic and noninfectious risks for ARDS
No association between either polymorphism and ARDS mortality
Variant T1543 allele not associated with ARDS
SFTPB
Insertion/deletion polymorphism in intron 4
Max et al [63]
15 Germans with AECC-defined ARDS
21 healthy Americans
Variant allele associated with increased susceptibility to ARDS
Not examined
Gong et al [40]
72 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma,
aspiration, and massive transfusion
117 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration,
and massive who did not develop ARDS
Variant allele associated with increased susceptibility to ARDS and increased susceptibility
to severe direct pulmonary injury like pneumonia in women
Not examined
+1580CT SNP in codon 131
Lin et al [39]
52 German patients with AECC-defined ARDS
46 healthy German adults
25 whites with trauma, pneumonia, and heart failure
+1580C allele and + 1580CC genotype were associated with increased susceptibility
to ARDS compared to both control groups
Not examined
Quasney et al [38]
12 whites and African Americans with ARDS caused by pneumonia
390 whites and African Americans with pneumonia
+1580CC genotype were associated with increased susceptibility to respiratory failure,
septic shock, and ARDS
No association with mortality in pneumonia ARDS mortality not specifically examined
TNF-α and TNF-β
−308GA SNP in TNF-α TNFβ1/2 Ncol SNP in TNFB
Gong et al [44]
237 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma,
aspiration, and massive transfusion
476 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration,
and massive who did not develop ARDS
−308A allele and −308A:TNF-β1 haplotype was associated with increased susceptibility
to ARDS in direct pulmonary injury
Increasing ARDS mortality with increasing number of −308A alleles with greatest mortality
found in younger patients carrying the −308A allele
No association with ARDS found for TNF-β1/2
VGEF
+936CT SNP
Medford et al [49]
117 whites with AECC-defined ARDS
137 healthy whites
103 EA who had respiratory failure
+936CT and +936TT genotype associated with more susceptibility to ARDS compared with
both control groups
+936CT and +936TT genotype associated with greater severity of illness in ARDS but
no association with ARDS mortality was found
Abbreviations: ACE, angiotensin-converting enzyme; EA, European-Americans; IL-6, interleukin-6;
IL-10, interleukin-10; PBEF, pre-B-cell colony-enhancing factor; SARS, severe acute
respiratory syndrome; SFTPB, surfactant protein B; SNP, single-nucleotide polymorphism;
TNF-α, tumor necrosis factor-α; TNF-β, tumor necrosis factor-β; VEGF, vascular endothelial
growth factor.
Case definition
As with any case-control study, the choice and phenotype of cases and controls is
pivotal to the design, strength, validity, and generalizability of the study. The
case definition will differ, depending on the whether the focus is on prevention or
treatment. Studies of susceptibility to developing ARDS are more relevant for future
prevention, whereas studies on outcomes in ALI/ARDS are more relevant for treatment.
In molecular epidemiology studies, factors important in susceptibility studies may
not be important in prognostication of outcomes, and vice versa. For example, mutations
in the BRCA1 gene, now known to be important in DNA repair, are associated with increased
susceptibility to developing early-onset breast or ovarian cancer. However, the BRCA1
gene is not associated with differences in breast cancer recurrence or disease-free
survival after therapy, even though BRCA1-associated breast cancer tends to present
at a more advanced stage [64].
When the focus is on prevention and susceptibility rather than on treatment and outcomes
in ALI/ARDS, there are inherently more challenges. Genetic epidemiology studies examining
outcomes in ALI/ARDS usually use mortality or ventilator-free days as end points.
The outcome of ALI/ARDS in genetic susceptibility studies is more heterogeneous and
is prone to misclassification, because there is no definitive diagnostic test. The
American-European Consensus criteria serve as a uniformly accepted guideline for defining
lung injury, but certain criteria, specifically the radiologic criteria, are not always
clear and are subject to interobserver variability [65]. In addition, the ratio of
partial pressure of arterial carbon dioxide to fraction of inspired oxygen represents
a continuum of hypoxemic respiratory failure. The use of a cutoff of 300 mm Hg in
the criteria for ALI will result in inevitable random misclassification of cases and
controls that tends to bias results toward the null hypothesis. In addition, autopsy
studies indicate that the American-European Consensus criteria for ALI/ARDS are sensitive
but are not very specific [66]. Nevertheless, the American-European Consensus definition
is used uniformly in the studies of ALI/ARDS. Care must be taken to assess carefully
the rigor with which the cases adhere to the ALI/ARDS criteria. Similar attention
also must be taken to ensure that controls do not actually have ARDS. Reliance on
chart review and clinical diagnosis is inadequate, given recent evidence for the underdiagnosis
of ALI/ARDS [67]. Thus, the same screening procedures used to determine the cases
of ALI/ARDS should be applied to the controls to ensure that they do not also have
the condition. Even so, misclassification will occur, and large, well-phenotyped sample
sizes will be needed to detect an association.
Choice of controls
The choice of controls in case-control studies is equally important, although often
neglected. In case-controls studies, controls are not simply people who do not have
the disease. Rather, they should represent the population that is at risk for the
disease. In other words, they should not have the disease at the time of selection
but, under the study design, they would have been included as a case if they did develop
the disease [68]. A poor choice of control may result in hidden confounding. In one
review, 30% of the genetic epidemiology studies did not delineate adequately the criteria
by which the controls were selected, and the controls were improperly chosen in 13.5%
of the studies [58].
The most common problem is the selection of controls who are not at risk for the disease,
making comparisons with the cases difficult. The controls in many of the studies of
ALI/ARDS were healthy individuals or hospitalized patients who did not have a clear
prior injury placing them at risk for ALI [21], [39], [49], [63]. As discussed previously,
genes associated with sepsis are strong candidate genes for ALI, but because sepsis
also is the leading precipitant for ALI, one must be careful to avoid confounding
from a genetic association with the predisposing injury. When the controls are healthy
or have conditions that differ from the precipitating injuries in the ARDS cases,
any association found between a candidate gene and ALI/ARDS may actually be caused
by an association between the polymorphism and the risk condition for ALI/ARDS, such
as sepsis. It is important to use at-risk individuals who have similar conditions
as the cases to avoid this confounder. In the investigation of the PBEF gene, both
healthy individuals and patients who had sepsis were used as controls. The variant
T-1001G and C-1543T alleles were found to be associated with the development of sepsis-associated
ALI when compared with healthy controls, but no association was found between patients
who had sepsis-associated ALI compared with sepsis patients who did not have ALI [37].
Thus, it is not clear whether the PBEF polymorphisms are associated with ALI or with
the severe sepsis that placed the patients at risk for ALI. In a subsequent study
in a different cohort of patients who had sepsis, trauma, aspiration, or multiple
transfusions, the variant T-1001G, but not the C-1543T allele, was confirmed to be
associated with ARDS compared with at-risk individuals [62]. This association was
present even among patients who had ARDS of noninfectious origin, extending the generalizability
of the genetic association.
One potential issue with using at-risk controls is that the patients are not drawn
randomly from the general population. Rather they are selected as controls on the
basis of their critical illness. If the genotype of interest is associated with critical
illness, then the genotype frequency may deviate from that predicted by random mating
(Hardy Weinberg equilibrium) [69]. Indeed, such was the case with the -1082GA IL-10
and MBL-2 polymorphisms. In such cases, extra effort is needed to ensure that the
deviation from Hardy Weinberg equilibrium is not from genotype or clerical error.
Such efforts include repeat genotyping, blinding of personnel, or validation of genotyping
in a different population.
Race and genetic epidemiology of acute lung injury/acute respiratory distress syndrome
Recently the role of race in critical illnesses has been explored. In a retrospective
study of decedents, nonwhite race was associated with increased mortality in ARDS,
with African Americans, especially young African Americans, having the higher mortality
from ARDS than whites and other minorities [70]. It is not clear why African Americans
have higher mortality in ARDS than whites. Precipitants for lung injury and other
predictors of ARDS were not available for comparison. Because the study focused on
deaths from ARDS, it is not clear whether minorities have a higher risk of developing
ARDS in the first place.
Many have postulated that genetic variability may contribute to the racial disparities
in ARDS. Many of the polymorphisms found to be associated with ALI/ARDS susceptibility
or outcome, such as the insertion/deletion polymorphism in intron 4 of the SFTP-B
gene [71], the -308GA polymorphism in the TNF-α gene [72], and the codon 54 polymorphism
in the MBL-2 gene [73], are known to vary in frequency among major racial groups.
This variation may be especially important when the haplotype approach is used. The
extent of linkage disequilibrium and, hence, of haplotype blocks and frequencies differs
between African Americans and non-Africans [74]. Thus, any genotype analysis must
be restricted to one racial group or be stratified by race to avoid confounding from
differences in ethnic groups (population stratification). In one study, haplotypes
in the MLCK gene were found to be associated with variable susceptibility to developing
sepsis-induced ALI in both American whites of European heritage and in African Americans
[61]. Whites and African Americans differed in the linkage disequilibrium between
SNPs and in haplotype block definition, however. Although significant associations
between the gene and ALI were found, the disease-associated haplotypes differed between
racial groups. The similar location of the race-specific at-risk haplotypes in whites
and African Americans suggests that the true disease-associated variant may be located
within the 5′ region of the gene.
After stratifying by major racial groups, additional methods to adjust for population
stratification may not be necessary, especially for studies conducted in the United
States. Wacholder and colleagues [75] demonstrated that, among whites, bias from population
stratification is small and decreases as the number of ethnic subgroups within the
white population increases. This finding may be especially pertinent for whites in
the United States, a classification that tends to be composed of many different ethnic
subgroups. Similar results were found in the classification of African Americans that
contained large numbers of ethnic subgroups [76]. Consistent with these stimulation
studies, Gao and colleagues [61] found evidence for ethnic differences within their
African Americans subjects, but adjusting for these differences did not significantly
change the associations between haplotypes and SNPs in the MLCK gene and ALI except
for one SNP, in which the association was actually strengthened.
Currently, most, although not all, studies have restricted their analyses to whites
or have stratified their analyses by race. Thus, the findings in genetic epidemiology
studies of ALI/ARDS cannot be generalized to nonwhites. Large cohorts of nonwhites
will be necessary to confirm previously detected genetic associations in other racial
groups.
Gene–environment interaction
The role of the environment is particularly critical in determining the genetic determinants
in a complex disorder in which the gene may have no influence on the risk of disease
unless there is concomitant exposure to a particular environmental insult. Such interaction
is important in understanding and interpreting the genetic contribution to complex
disease such as ALI. Failure to examine the role of environmental exposure can lead
to decreased sensitivity in detecting an association between the gene of interest
and the disease [77]. Neglect of the gene–environment interaction contributes to the
inconsistent findings from genetic association studies of complex disease [78].
Recently, there is growing evidence to suggest potential gene–environment interaction,
from whether the initial precipitant for ARDS is a direct pulmonary injury such as
pneumonia or aspiration or an indirect pulmonary injury such as extrapulmonary sepsis
or massive transfusion.
Two SNPs in the SFTPB gene have been found to be associated with ARDS. SFTPB is essential
for the surface tension–lowering properties of pulmonary surfactant, which is known
to be dysfunctional in ALI/ARDS. In two small studies, the variant allele in the insertion/deletion
polymorphism in intron 4 of the SFTPB gene was found to be associated with susceptibility
to ARDS or severe direct pulmonary injury such as pneumonia, especially among women
[40], [63]. In another polymorphism in the SFTPB gene, the C allele of +1580CT SNP
was found to be associated with ARDS, but this association was confined to patients
who had idiopathic insults, mostly direct pulmonary injuries such as pneumonia [39].
No associations were found in the group of patients who had exogenic ARDS, mostly
extrapulmonary causes of ARDS [38]. Although healthy controls were used, a subsequent
study using ARDS cases and controls who had community-acquired pneumonia confirmed
this association between the C allele and ARDS, suggesting that the +1580C allele
is associated with ALI/ARDS and not with severe pneumonia. Together, these studies
suggest that the SFTPB gene may be important in ARDS susceptibility in direct pulmonary
injuries such as pneumonia. This gene also may influence susceptibility to direct
pulmonary injury, such as severe pneumonia, that places these patients at risk for
ALI/ARDS. The role of the SFTPB gene in lung injury resulting from other causes is
not yet clear.
A similar gene–environment interaction was found with the −308GA polymorphisms in
the TNF-α gene [44]. No association was found between the variant −308A allele and
ARDS compared with other critically ill non-ARDS controls who had sepsis, aspiration,
massive transfusion, or trauma. After stratification by the site of injury, however,
the −308A allele was associated with decreased likelihood of developing ARDS among
patients who had direct pulmonary injury (adjusted odds ration [OR], 0.52; 95% confidence
interval [CI], 0.30–0.91) but with a nonsignificant increased likelihood of ARDS in
indirect pulmonary injury (adjusted OR, 1.7; 95% CI, 0.93–3.2) with evidence for significant
effect modification (P = .01).
The reasons for this interaction are unclear. The risk of ARDS is different in direct
pulmonary injuries and indirect pulmonary injuries [79]. The cytokine profile and
inflammatory markers differ in patients who have ARDS and at-risk patients who do
not have ARDS, depending on whether the predisposing injury was sepsis, trauma, acute
pancreatitis, or massive transfusion [80]. Certainly, the inflammatory response and
the radiologic, histologic, and mechanical properties of the lung differ depending
on whether the site of infection or the etiology of ARDS is pulmonary or extrapulmonary
[81], [82]. Although these results need to be confirmed in larger studies, these findings
overall indicate important gene–environment interactions in the genetic susceptibility
to developing ALI/ARDS that depend on the risk factor that predisposes the individual
to lung injury.
Another source of potential gene–environment interaction is age. Older patients have
a higher risk than younger individuals of developing and dying from ARDS [4], [83].
In complex diseases, the genetic contribution may be greatest in diseases with an
early age of onset rather than in a disease with a late age of onset, in which environmental
factors such as comorbidities may figure more prominently. Potential interactions
with age have been found in genetic epidemiology studies of ALI/ARDS. Among 212 patients
who had ARDS, the −308A allele was associated with more daily organ dysfunction and
increased 60-day mortality in ARDS (adjusted OR, 3.5; 95% CI, 1.4–8.6) after adjusting
for age, severity of illness, septic shock, transfusion and other potential predictors
[44]. Age seemed to be important, with the strongest association found among the 117
ARDS patients younger than median age of 67 years (adjusted OR, 14.9; 95% CI, 3.0–74;
P < .001). In the same cohort of critically ill at-risk patients, the −1082GG genotype
was associated with increased susceptibility to ARDS in critically ill patients (P
< .001), but only in the presence of a statistically significant interaction between
age and the −1082GG genotype [26]. When the nature of this interaction was explored
further by stratifying the analyses by the median age of 67 years, the −1082GG genotype
was protective against ARDS among the older (adjusted OR, 0.63; 95% CI, 0.34–1.2)
but not among the younger patients (OR, 1.7; 95% CI, 0.89–3.2), with significant effect
modification by age of the association between −1082GG and ARDS (P < .001). Further
study with a larger sample size is needed to confirm and define better the age effect
on the genetic susceptibility to developing and dying from ARDS. If such interaction
does exist, future interventions aimed at preventing and treating ARDS may have variable
efficacy, depending on the age of the individual.
Other potential factors that will be worthwhile examining for gene–environment interaction
in the future are diabetes and chronic alcohol abuse. A history of diabetes has been
found to be protective in ARDS [79], [84]. A growing body of literature is suggesting
a role for chronic alcohol abuse in increased susceptibility and poorer outcomes in
ALI/ARDS [85]. It is likely that there may be genotypes that are important in ALI/ARDS
only in the context of diabetes or chronic alcohol abuse.
Defining these gene–environment interactions is important. Many of the polymorphisms
identified in ARDS are common, with frequency greater than 1%. Given their persistence
in the genome and the lethality of ARDS, it is unlikely that these polymorphisms are
universally detrimental. Rather, it is likely that these variants may be detrimental
in some situations and benign or even beneficial in others. Otherwise, there would
be selection pressure against their persistence in the population. Thus, any intervention
that targets the same causal pathway as the implicated at-risk genes may be beneficial
in some circumstances but less helpful in others. Understanding the gene–drug–environment
interaction will be important in identifying the patient population that has the most
favorable risk/benefit ratio for any particular therapy.
Type I errors and power
Type I and type II errors also are important in genetic case-control studies. Type
I error is the likelihood of a false-positive finding. Although a p-value or a type
I error rate of 5% is generally considered acceptable, one may be more likely to find
an association by chance alone if multiple comparisons of different genetic loci to
the development of disease are performed. Although adjustment for multiple comparisons
is ideal, it is not entirely clear what the best strategy is, and current studies
of ALI/ARDS may still too small to accommodate statistical correction for multiple
comparisons. Ultimately, the likelihood of a cause-and-effect relationship underlying
any genetic association will depend on the reproducibility of well-designed studies
in different populations and in the strength of the biologic rationale behind the
selection of that gene for analysis. Although troublesome to classical geneticists,
the need to confirm studies is common in epidemiology. Any population study needs
to be validated for different populations and in larger studies.
Type II errors involve the statistical power of the study. Power to detect an association
depends upon the size of the effect, the frequency of the genotype in the population,
and the sensitivity of the analysis deployed. Some of the negative studies in genetic
epidemiology in ALI are probably caused by the lack of adequate power [21]. The power
of the study is especially important when there may be phenotype misclassification
and gene–gene or gene–environment interaction. Currently, most ALI/ARDS studies are
relatively small for genetic epidemiology studies, and their small size makes examination
of interactions difficult. Only the Boston cohort has sufficient sample size to explore
for gene–environment interaction. Additional large cohorts will be necessary to confirm
previously found associations and interactions [26], [44].
Genetic epidemiology and its potential application in the prevention and treatment
of acute lung injury/acute respiratory distress syndrome
With the completion of the HapMap and Human Genome Project, there has been much interest
in how genetics may lead to future prevention and treatment of complex diseases. This
interest must be tempered to avoid raising false hope. Genetic epidemiology has been
applied to the study of ALI/ARDS only recently. Technical and methodological issues
in approach and study design are still evolving, and the large cohorts needed for
effective genetic epidemiology studies and for the required confirmatory studies in
different populations are still being developed. Because the translation of research
findings into clinical practice usually takes years, it is likely that genetic epidemiology
studies will not lead to any change in clinical practice for years or decades to come.
Nevertheless, genetic epidemiology may contribute to future prevention and therapeutic
strategies in ALI/ARDS by (1) identifying targets for intervention, (2) enabling risk
assessment, and (3) identifying the appropriate patient groups or conditions for interventions
(genetic pharmacoepidemiology).
Identification of novel targets for intervention
Unlike diseases with simple Mendelian inheritance, ALI/ARDS is unlikely to be caused
by discrete mutations in a particular gene. Rather, multiple genes with incomplete
penetrance and much gene–gene and gene–environment interaction will be important in
ALI/ARDS. As such, expecting gene therapy to correct a specific disease-causing mutation
or locus is unrealistic. More likely, genetic epidemiology studies may help identify
important pathways in the pathogenesis and evolution of lung injury and new therapeutic
targets within these pathways for intervention. Hence, the potential of any gene or
its product in the future prevention and treatment of ALI/ARDS will depend greatly
on the strength of the evidence supporting the biologic role for the candidate gene
in ALI/ARDS. In such cases, a multidisciplinary translational approach involving genetic
epidemiology, functional genomics, animal models, and bioinformatics will be important.
The translational approach may be bidirectional [86]. The “benchside” work may occur
before the association study to lend support to its selection as a candidate gene,
as was the case with the PBEF and MLCK genes [37], [61]. Alternately, such investigation
may occur after the association study to explain better the nature of the genetic
association. For example, after an association between the D allele in the angeiotensin-converting
enzyme (ACE) gene and ARDS was reported, greater support for the role of ACE in lung
injury was established when the loss of ACE activity in ACE-knockout mice was found
to protect against lung injury [20]. In contrast, mice deficient in ACE 2, a homologue
of ACE, were more susceptible to sepsis and endotoxin-induced lung injury. Inactivation
of the ACE gene reduced the injury seen in these ACE 2-knockout mice. These results
lend greater strength to the biologic plausibility of ACE in the development of ARDS
and, consequently, its potential as a target for intervention.
The lack of functional significance of a specific SNP or haplotype found to be associated
with disease does not negate the importance of the candidate gene and its pathway
in pathogenesis and development of ALI/ARDS. The functional consequence may depend
on the stimulus or on activation of other genes. The SNP or haplotype may result in
changes that are not easily measured, such as the posttranslational modification or
alternative splicing of the gene product. In addition, the disease-associated SNP
may not be functional itself but, rather, may be linked to the actual functional susceptibility
locus. Nevertheless, if the candidate gene was chosen with sound scientific rationale,
a positive association between the candidate gene and the disease supports its importance
in ALI, even if the polymorphism studied is not the direct cause of the disease. In
such cases, functional studies help support the role of the gene or its product in
ALI/ARDS.
Risk assessment
Another way that genetic epidemiology studies can contribute to future prevention
and treatment is in risk assessment. In the past, clinical trials of surfactant replacement
in ARDS and anti-TNF therapy in sepsis have proved disappointing. It is possible that
these therapies may not be beneficial in all patients. For example, anti-TNF therapy
may be beneficial only for patients who are genetically predisposed to be exuberant
TNF secretors. Anti-TNF therapy may be useless or even detrimental in patients who
have the low TNF-secreting genotypes. Such genotype-dependent responses to therapy
were demonstrated with recombinant interleukin-1 receptor antagonist (IL1-ra) and
the rare IL-1ra +3954 allele in rheumatoid arthritis and with salmeterol therapy and
a β-adrenergic receptor genotype in asthma [87], [88]. Better risk assessment of the
patient, based on the patient's genetic profile and likelihood of response or adverse
reaction to an intervention, will allow better design of future clinical trials. Future
trials can target specific patient populations that have genotypes that are more likely
to respond. Alternatively, patients can be stratified on the basis of their genotype
to allow analyses of drug response by genotype. Given the acuity of the condition,
the targeting of individuals who have a certain genotype or the stratification of
subjects by genotype before randomization will require rapid and accurate genotyping
assays that are not yet available.
Understanding genetic risk factors can help with risk assessment in health policy
decisions, as well. Young, healthy patients often are considered to have a low risk
of serious or complicated influenza infection or pneumonia and are not recommended
for routine vaccination or close observation in the hospital [89]. Gene–age interaction
for the TNF-α and IL-10 genes in ARDS, however, suggests that certain young individuals
have a particularly high risk of developing and dying from ARDS. Knowledge of the
genetic predisposition to developing ALI/ARDS might help identify young, healthy patients
who would benefit either from early vaccination while still healthy or from closer
observation in the event of any insult such as a community-acquired pneumonia.
Identification of appropriate patient populations or conditions for intervention
In clinical practice, outside the strict inclusion and exclusion criteria and methodology
of a randomized, control trial, the patient population is more heterogeneous, and
there is a larger variability in the response and the complication rate associated
with the intervention [90]. Given potential gene–environment interaction with the
site of injury, it is possible that interventions based on surfactant or TNF-α may
have varying efficacy depending on whether the initial injury predisposing to ARDS
is pulmonary or extrapulmonary. Defining this heterogeneity in response in the context
of both environmental and genetic factors in a population falls within the emerging
field of genetic pharmacoepidemiology [91]. Obviously, the genes that are directly
targeted by the intervention are important. Genes governing drug metabolism, receptor
binding to the drug, and other genes in the causal pathways of the disease process
probably are important, as well.
In essence, this consideration is a special example of gene–environment interaction,
in which one of the key environmental factors is the drug or intervention. Identifying
these interactions is important in understanding and interpreting the genetic contribution
to ALI and in identifying which patient populations and what conditions have the most
appropriate risk/benefit ratios warranting a particular intervention. This understanding
will be important especially in interventions to prevent ALI/ARDS because of the many
different causes that can lead to lung injury.
Limitations and barriers to future prevention and treatment in acute lung injury/acute
respiratory distress syndrome
There is great excitement about how the rapid advances in genomics and genetic epidemiology
may lead to individualized medicine in complex diseases such as ALI. This excitement
should be tempered to avoid unrealistic expectations. Significant limitations and
barriers may limit the application of genomics and genetic epidemiology to future
preventive and therapeutic interventions in ALI/ARDS.
One possible limitation is that any novel intervention based on genetic variation
will not be universally beneficial. Rather, its applicability and benefit will be
limited to those who have the disease genotype. Thus, the population prevalence of
the disease-associated genotypes will determine the size of the patient population
that may benefit from this intervention and the generalizability of the intervention.
The efficacy of the intervention will be further limited by gene–gene and gene–environment
interactions. The pathogenesis of ALI/ARDS consists of interactions and balances between
multiple pathways involved in inflammation, coagulation, fibrogenesis, fluid transport,
and apoptosis [6]. With such a complex, interdependent process, it is likely that
multiple genes are important, and any intervention based on one gene is unlikely to
be uniformly and universally beneficial. The presence of gene–environment interactions
would further limit the context in which novel therapy will be appropriate.
Another limitation and barrier to the application of genetic epidemiology in ALI/ARDS
is the need for large, well-phenotyped cohorts that are sufficiently powered to account
adequately for gene–gene and gene–environment interactions. This need is especially
pronounced in genetic pharmacoepidemiology. With the need for large DNA databases
comes the need for more research on issues surrounding genetic research in the critical
care setting, where mortality is high, and consent is obtained from family members
or surrogates. There is a need for a better understanding of the concerns of the patients
and their surrogates and how best to protect those interests. In addition, the racial
difference in ALI means that large cohorts of minority groups will be vital to determine
the efficacy of an intervention in different racial groups.
Last, the narrow window of opportunity for intervention presents another barrier for
any interventions in ALI/ARDS. For example, ALI/ARDS develops rapidly after the initial
injury with a median of 1 day after admission to the ICU (interquartile range, 0–5
days) [2], [79]. Such a narrow window for intervention requires rapid identification
of appropriate patients for intervention and initiation of intervention as early as
possible, possibly in the emergency room. Therapy based on a specific genotype would
require either rapid DNA testing or prior genotyping of all patients and storage of
this information, either in the medical record or in a secure device on the persons
themselves. Neither of these measures is available currently.
Summary
The application of genetic epidemiology and genomics to the study of ALI/ARDS is in
its infancy. Optimal study designs and approaches are still being discussed, and the
large, prospective cohorts that will be necessary to examine gene–environment interaction
and to confirm prior findings are being developed. There will be technological and
analytic challenges to the proper study of genetic determinants of ALI/ARDS that will
benefit from a multifaceted approach. There will be significant barriers to the translation
of genetic epidemiology studies and genomics to preventive and therapeutic interventions,
and any intervention is unlikely to occur in the near future. In oncology, where there
is a longer history of genetic and molecular epidemiology studies, commercially available
genetic tests now allow individualized risk assessment and tailored therapy for breast
cancer. Although significant challenges lie ahead, there is a similar potential for
such individualized risk assessment and therapy in critical care medicine. Large,
well-phenotyped studies will be crucial to this goal.