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1.  Toll-like Receptor 1 Polymorphisms and Associated Outcomes in Sepsis Following Traumatic Injury, A Candidate Gene Association Study 
Annals of surgery  2014;259(1):10.1097/SLA.0b013e31828538e8.
To determine if SNPs in TLR1 are associated with mortality, specifically sepsis-associated mortality, in a traumatically injured population.
Summary Background Data
Innate immune responses mediated by toll-like receptors (TLRs), induce early inflammatory responses to pathogen and damage-associated molecular patterns. Genetic variation in TLRs has been associated with susceptibility and outcomes in a number of infectious and non-infectious disease states.
Patients admitted to the trauma intensive care unit at a level one trauma center serving 4 states were enrolled and followed for development of infection, sepsis, and death. Genomic DNA was genotyped and logistic regression analysis performed to determine associations between TLR1 SNPs and mortality. We further examined for associations between TLR1 SNPs and mortality in subgroups based on the presence of sepsis and the type of sepsis-associated organism.
We enrolled 1,961 patients.TLR1-7202G (rs5743551) was associated with increased mortality after traumatic injury and this association was primarily observed in the subset of patients who developed sepsis (adjusted OR 3.16; 95% CI 1.43–6.97, P=0.004). This association persisted after further restriction to gram-positive sepsis. TLR1742A/G(Asn248Ser) (rs4833095), a coding SNP in LD with TLR1-7202Gwas also associated with mortality in gram-positive sepsis (adjusted OR 4.16; 95% CI 1.22–14.19, P=0.023).
Genetic variation in TLR1 is associated with increased mortality in patients with sepsis following traumatic injury and may represent a novel marker of risk for death in critically injured patients.
PMCID: PMC3686843  PMID: 23478521
2.  A variational Bayes discrete mixture test for rare variant association 
Genetic epidemiology  2014;38(1):21-30.
Recently, many statistical methods have been proposed to test for associations between rare genetic variants and complex traits. Most of these methods test for association by aggregating genetic variations within a predefined region, such as a gene. Although there is evidence that “aggregate” tests are more powerful than the single marker test, these tests generally ignore neutral variants and therefore are unable to identify specific variants driving the association with phenotype. We propose a novel aggregate rare-variant test that explicitly models a fraction of variants as neutral, tests associations at the gene-level, and infers the rare-variants driving the association. Simulations show that in the practical scenario where there are many variants within a given region of the genome with only a fraction causal our approach has greater power compared to other popular tests such as the Sequence Kernel Association Test (SKAT), the Weighted Sum Statistic (WSS), and the collapsing method of Morris and Zeggini (MZ). Our algorithm leverages a fast variational Bayes approximate inference methodology to scale to exome-wide analyses, a significant computational advantage over exact inference model selection methodologies. To demonstrate the efficacy of our methodology we test for associations between von Willebrand Factor (VWF) levels and VWF missense rare-variants imputed from the National Heart, Lung, and Blood Institute’s Exome Sequencing project into 2,487 African Americans within the VWF gene. Our method suggests that a relatively small fraction (~10%) of the imputed rare missense variants within VWF are strongly associated with lower VWF levels in African Americans.
PMCID: PMC4030763  PMID: 24482836
Exome sequencing study; approximate inference; von Willebrand Factor genetics
4.  Differential Regulation of Membrane CD14 Expression and Endotoxin-Tolerance in Alveolar Macrophages 
CD14 is important in the clearance of bacterial pathogens from lungs. However, the mechanisms that regulate the expression of membrane CD14 (mCD14) on alveolar macrophages (AM) have not been studied in detail. This study examines the regulation of mCD14 on AM exposed to Escherichia coli in vivo and in vitro and explores the consequences of changes in mCD14 expression. The expression of mCD14 was decreased on AM exposed to E. coli in vivo and AM incubated with lipopolysaccharide (LPS) or E. coli in vitro. Polymyxin B abolished LPS effects but only partially blocked the effects of E. coli. Blockade of extracellular signal-regulated kinase pathways attenuated LPS and E. coli-induced decrease in mCD14 expression. Inhibition of proteases abrogated the LPS-induced decrease in mCD14 expression on AM and the release of sCD14 into the supernatants, but did not affect the response to E. coli. The production of TNF-α in response to a second challenge with Staphylococcus aureus or zymosan was decreased in AM following incubation with E. coli but not LPS. These studies show that distinct mechanisms regulate the expression of mCD14 and the induction of endotoxin-tolerance in AM and suggest that AM function is impaired at sites of bacterial infection.
PMCID: PMC4096031  PMID: 15059784
alveolar macrophages; CD14; lungs; lipopolysaccharide; rabbit
5.  IL1RN Coding Variant Is Associated with Lower Risk of Acute Respiratory Distress Syndrome and Increased Plasma IL-1 Receptor Antagonist 
Rationale: Acute respiratory distress syndrome (ARDS) behaves as a complex genetic trait, yet knowledge of genetic susceptibility factors remains incomplete.
Objectives: To identify genetic risk variants for ARDS using large scale genotyping.
Methods: A multistage genetic association study was conducted of three critically ill populations phenotyped for ARDS. Stage I, a trauma cohort study (n = 224), was genotyped with a 50K gene-centric single-nucleotide polymorphism (SNP) array. We tested SNPs associated with ARDS at P < 5 × 10−4 for replication in stage II, a trauma case–control population (n = 778). SNPs replicating their association in stage II (P < 0.005) were tested in a stage III nested case–control population of mixed subjects in the intensive care unit (n = 2,063). Logistic regression was used to adjust for potential clinical confounders. We performed ELISA to test for an association between ARDS-associated genotype and plasma protein levels.
Measurements and Main Results: A total of 12 SNPs met the stage I threshold for an association with ARDS. rs315952 in the IL1RN gene encoding IL-1 receptor antagonist (IL1RA) replicated its association with reduced ARDS risk in stages II (P < 0.004) and III (P < 0.02), and was robust to clinical adjustment (combined odds ratio = 0.81; P = 4.2 × 10−5). Plasma IL1RA level was associated with rs315952C in a subset of critically ill subjects. The effect of rs315952 was independent from the tandem repeat variant in IL1RN.
Conclusions: The IL1RN SNP rs315952C is associated with decreased risk of ARDS in three populations with heterogeneous ARDS risk factors, and with increased plasma IL1RA response. IL1RA may attenuate ARDS risk.
PMCID: PMC3707367  PMID: 23449693
functional genetic polymorphism; acute lung injury; acute respiratory distress syndrome; IL-1 receptor antagonist; replication
6.  Survey of Innate Immune Responses to Burkholderia pseudomallei in Human Blood Identifies a Central Role for Lipopolysaccharide 
PLoS ONE  2013;8(11):e81617.
B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.
PMCID: PMC3841221  PMID: 24303060
7.  On-time clinical phenotype prediction based on narrative reports 
In this paper we describe a natural language processing system which is able to predict whether or not a patient exhibits a specific phenotype using the information extracted from the narrative reports associated with the patient. Furthermore, the phenotypic annotations from our report dataset were performed at the report level which allows us to perform the prediction of the clinical phenotype at any point in time during the patient hospitalization period. Our experiments indicate that an important factor in achieving better results for this problem is to determine how much information to extract from the patient reports in the time interval between the patient admission time and the current prediction time.
PMCID: PMC3900127  PMID: 24551325
8.  Pneumonia identification using statistical feature selection 
This paper describes a natural language processing system for the task of pneumonia identification. Based on the information extracted from the narrative reports associated with a patient, the task is to identify whether or not the patient is positive for pneumonia.
A binary classifier was employed to identify pneumonia from a dataset of multiple types of clinical notes created for 426 patients during their stay in the intensive care unit. For this purpose, three types of features were considered: (1) word n-grams, (2) Unified Medical Language System (UMLS) concepts, and (3) assertion values associated with pneumonia expressions. System performance was greatly increased by a feature selection approach which uses statistical significance testing to rank features based on their association with the two categories of pneumonia identification.
Besides testing our system on the entire cohort of 426 patients (unrestricted dataset), we also used a smaller subset of 236 patients (restricted dataset). The performance of the system was compared with the results of a baseline previously proposed for these two datasets. The best results achieved by the system (85.71 and 81.67 F1-measure) are significantly better than the baseline results (50.70 and 49.10 F1-measure) on the restricted and unrestricted datasets, respectively.
Using a statistical feature selection approach that allows the feature extractor to consider only the most informative features from the feature space significantly improves the performance over a baseline that uses all the features from the same feature space. Extracting the assertion value for pneumonia expressions further improves the system performance.
PMCID: PMC3422830  PMID: 22539080
9.  Variation in the TLR10/TLR1/TLR6 Locus is the Major Genetic Determinant of Inter-Individual Difference in TLR1/2-Mediated Responses 
Genes and immunity  2012;14(1):52-57.
Toll-like receptor (TLR)-mediated innate immune responses are important in early host defense. Using a candidate gene approach, we previously identified genetic variation within TLR1 that is associated with hyper-responsiveness to a TLR1/2 agonist in vitro and with death and organ dysfunction in patients with sepsis. Here we report a genome-wide association study designed to identify genetic loci controlling whole blood cytokine responses to the TLR1/2 lipopeptide agonist, Pam3CSK4 ex vivo. We identified a very strong association (p<1×10−27) between genetic variation within the TLR10/1/6 locus on chromosome 4, and Pam3CSK4-induced cytokine responses. This was the predominant association explaining over 35% of the population variance for this phenotype. Notably, strong associations were observed within TLR10 suggesting genetic variation in TLR10 may influence bacterial lipoprotein-induced responses. These findings establish the TLR10/1/6 locus as the dominant common genetic factor controlling inter-individual variability in Pam3CSK4-induced whole blood responses in the healthy population.
PMCID: PMC3554851  PMID: 23151486
TLR; polymorphism; genomics; innate immunity
10.  Automated Tools for Phenotype Extraction from Medical Records  
Clinical research studying critical illness phenotypes relies on the identification of clinical syndromes defined by consensus definitions. Historically, identifying phenotypes has required manual chart review, a time and resource intensive process. The overall research goal of C ritical I llness PH enotype E xt R action (deCIPHER) project is to develop automated approaches based on natural language processing and machine learning that accurately identify phenotypes from EMR. We chose pneumonia as our first critical illness phenotype and conducted preliminary experiments to explore the problem space. In this abstract, we outline the tools we built for processing clinical records, present our preliminary findings for pneumonia extraction, and describe future steps.
PMCID: PMC3845784  PMID: 24303281
11.  Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis 
Rationale: Polymorphisms affecting Toll-like receptor (TLR)–mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis.
Objectives: To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis.
Methods: We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case–control study in a cohort of intensive care unit patients with sepsis, and a case–control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects.
Measurements and Main Results: The SNP demonstrating the most hypermorphic effect was the G allele of TLR1−7202A/G (rs5743551), which associated with elevated TLR1-mediated cytokine production (P < 2 × 10−20). TLR1−7202G marked a coding SNP that causes higher TLR1-induced NF-κB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1−7202G predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07–3.09). In the case-control study TLR1−7202G was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59–7.27). TLR1−7202G also associated with a higher prevalence of gram-positive cultures in both clinical studies.
Conclusions: Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis.
PMCID: PMC2556453  PMID: 18635889
innate immunity; genetic variation; genetic predisposition
12.  Impaired TLR5 Functionality Is Associated with Survival in Melioidosis 
Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR51174C>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney–293 cells transfected with TLR5 and found that B. pseudomallei induced TLR51174C- but not TLR51174T-dependent activation of NF-κB. We tested the association of TLR51174C>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR51174C>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08–0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19–0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR51174C>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR51174T compared with carriers of TLR51174C. B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR51174T. We conclude that the hypofunctional genetic variant TLR51174C>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.
PMCID: PMC3607401  PMID: 23447684
13.  Inflammation and Immune-Related Candidate Gene Associations with Acute Lung Injury Susceptibility and Severity: A Validation Study 
PLoS ONE  2012;7(12):e51104.
Common variants in genes related to inflammation, innate immunity, epithelial cell function, and angiogenesis have been reported to be associated with risks for Acute Lung Injury (ALI) and related outcomes. We tested whether previously-reported associations can be validated in an independent cohort at risk for ALI.
We identified 37 genetic variants in 27 genes previously associated with ALI and related outcomes. We prepared allelic discrimination assays for 12 SNPs from 11 genes with MAF>0.05 and genotyped these SNPs in Caucasian subjects from a cohort of critically ill patients meeting criteria for the systemic inflammatory response syndrome (SIRS) followed for development of ALI, duration of mechanical ventilation, and in-hospital death. We tested for associations using additive and recessive genetic models.
Among Caucasian subjects with SIRS (n = 750), we identified a nominal association between rs2069832 in IL6 and ALI susceptibility (ORadj 1.61; 95% confidence interval [CI], 1.04–2.48, P = 0.03). In a sensitivity analysis limiting ALI cases to those who qualified for the Acute Respiratory Distress Syndrome (ARDS), rs61330082 in NAMPT was nominally associated with risk for ARDS. In terms of ALI outcomes, SNPs in MBL2 (rs1800450) and IL8 (rs4073) were nominally associated with fewer ventilator-free days (VFDs), and SNPs in NFE2L2 (rs6721961) and NAMPT (rs61330082) were nominally associated with 28-day mortality. The directions of effect for these nominal associations were in the same direction as previously reported but none of the associations survived correction for multiple hypothesis testing.
Although our primary analyses failed to statistically validate prior associations, our results provide some support for associations between SNPs in IL6 and NAMPT and risk for development of lung injury and for SNPs in IL8, MBL2, NFE2L2 and NAMPT with severity in ALI outcomes. These associations provide further evidence that genetic factors in genes related to immunity and inflammation contribute to ALI pathogenesis.
PMCID: PMC3522667  PMID: 23251429
14.  Assessing Pneumonia Identification from Time-Ordered Narrative Reports 
AMIA Annual Symposium Proceedings  2012;2012:1119-1128.
In this paper, we present a natural language processing system that can be used in hospital surveillance applications with the purpose of identifying patients with pneumonia. For this purpose, we built a sequence of supervised classifiers, where the dataset corresponding to each classifier consists of a restricted set of time-ordered narrative reports. In this way the pneumonia surveillance application will be able to invoke the most suitable classifier for each patient based on the period of time that has elapsed since the patient was admitted into the hospital. Our system achieves significantly better results when compared with a baseline previously proposed for pneumonia identification.
PMCID: PMC3540463  PMID: 23304388
15.  Microarray-based Analysis of Ventilator-induced Lung Injury 
Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are a frequent cause of intensive care unit admission, affecting over 200,000 patients in the United States each year. Mechanical ventilation is a life-saving intervention in the setting of ARDS and ALI, but clinical trials have demonstrated that mechanical ventilation with excessive tidal volumes plays a role in promoting and perpetuating lung injury and leads to excess mortality. This process has been labeled ventilator-induced lung injury (VILI), but the molecular mechanisms driving this process and its interactions with predisposing risk factors such as sepsis and chemical injury remain incompletely understood. Genome-wide measurements of gene expression using microarray technology represent a powerful tool to examine the pathophysiology of VILI. Several recent studies have used this approach to study VILI in isolation and associated with endotoxin instillation or saline lavage. These studies and others examining gene expression profiles in epithelial cells subjected to cyclic stretch have provided novel insights on the molecular mechanisms underlying VILI. This review will summarize these findings and discuss implications for future studies.
PMCID: PMC2647617  PMID: 17202295
microarray analysis; lung injury; mechanical ventilation; gene expression; genomics
16.  A Nonsynonymous Polymorphism of IRAK4 Associated with Increased Prevalence of Gram-Positive Infection and Decreased Response to Toll-Like Receptor Ligands 
Journal of Innate Immunity  2011;3(5):447-458.
Mutations in IRAK4 have been associated with recurrent Gram-positive infections in children. Given the central role of IRAK4 in innate immunity signaling, we hypothesized that common genetic variants of IRAK4 may be associated with prevalence of Gram-positive infection in critically ill adults. Haplotype clade tag single nucleotide polymorphisms (SNPs) of the IRAK4 gene were selected and genotyped in a cohort of 1,029 critically ill patients with systemic inflammatory response syndrome (SIRS). We found that a haplotype clade tagged by the A allele of the htSNP G29429A (Ala428Thr) was associated with increased relative risk of Gram-positive infection at admission to ICU (RR = 1.2, p < 0.05). Furthermore, the 29429A allele was associated with decreased lymphoblastoid cell response to CpG (as measured by IL-6 production) (raw values ± 95% CI 40.3 ± 32.3 vs. 85.8 ± 29.4 pg/ml; log-transformed values ± 95% CI 1.13 ± 0.37 vs. 1.55 ± 0.18, p < 0.04). We also found that IRAK4-deficient fibroblasts transfected with an IRAK4 expression plasmid containing the 29429A allele produced less IL-6 in response to lipopolysaccharide (p = 0.07). Our data suggest that the IRAK4 haplotype clade marked by 29429A (428Thr) alters susceptibility to Gram-positive bacteria, by decreasing cellular response to TLR ligands.
Copyright © 2011 S. Karger AG, Basel
PMCID: PMC3186712  PMID: 21576904
Bacterial infections; Protein kinase; Inflammation
17.  A Phase II Randomized Placebo-Controlled Trial of Omega-3 Fatty Acids for the Treatment of Acute Lung Injury 
Critical care medicine  2011;39(7):1655-1662.
Administration of eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA), omega-3 fatty acids in fish oil, has been associated with improved patient outcomes in acute lung injury (ALI) when studied in a commercial enteral formula. However, fish oil has not been tested independently in ALI. We therefore sought to determine if enteral fish oil alone would reduce pulmonary and systemic inflammation in patients with ALI.
Phase II randomized controlled trial.
Four North American medical centers.
Mechanically ventilated patients with ALI ≥ 18 years of age.
Subjects were randomized to receive enteral fish oil (9.75g EPA and 6.75g DHA daily) or saline placebo for up to 14 days.
Measurements and Main Results
Bronchoalveolar lavage fluid (BALF) and blood were collected at baseline (day 0), day 4±1, and day 8±1. The primary endpoint was BALF interleukin (IL)-8 levels. Forty-one participants received fish oil and 49 received placebo. Enteral fish oil administration was associated with increased serum EPA concentration (p<0.0001). However, there was no significant difference in the change in BALF IL-8 from baseline to day 4 (p=0.37) or day 8 (p=0.55) between treatment arms. There were no appreciable improvements in other BALF or plasma biomarkers in the fish oil group compared to the control group. Similarly, organ failure score, ventilator-free days, ICU-free days, and 60-day mortality did not differ between the groups.
Fish oil did not reduce biomarkers of pulmonary or systemic inflammation in patients with ALI, and the results do not support the conduct of a larger clinical trial in this population with this agent. This experimental approach is feasible for proof of concept studies evaluating new treatments for ALI.
PMCID: PMC3125670  PMID: 21423000
Acute lung injury; randomized controlled trial; eicosapentaenoic acid; docosahexaenoic acid; acute respiratory distress syndrome; fish oils
18.  ANGPT2 Genetic Variant Is Associated with Trauma-associated Acute Lung Injury and Altered Plasma Angiopoietin-2 Isoform Ratio 
Rationale: Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI.
Objectives: To identify ALI risk variants after major trauma using a large-scale candidate gene approach.
Methods: We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease–centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10−4) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot.
Measurements and Main Results: Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06–1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038).
Conclusions: An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.
PMCID: PMC3114062  PMID: 21257790
acute lung injury; acute respiratory distress syndrome; functional genetic polymorphism; genetic association study
19.  Genetic Variation in the FAS Gene and Associations with Acute Lung Injury 
Rationale: Fas (CD95) modulates apoptosis and inflammation and is believed to play an important role in lung injury.
Objectives: To determine if common genetic variation in FAS is associated with acute lung injury (ALI) susceptibility, risk of death, and FAS gene expression.
Methods: We genotyped 14 single nucleotide polymorphisms (tagSNPS) in FAS in samples from healthy white volunteers (control subjects, n = 294) and patients with ALI (cases, n = 324) from the ARDSnet Fluid and Catheter Treatment Trial (FACTT). FAS genotypes associated with ALI in the discovery study were confirmed in a nested case-control validation study of critically ill patients at risk for ALI (n = 657). We also tested for associations between selected tagSNPS and FAS mRNA levels in whole blood from healthy control subjects exposed to media alone or LPS ex vivo.
Measurements and Main Results: We identified associations between four tagSNPs in FAS (FAS−11341A>T [rs17447140], FAS9325G>A [rs2147420], FAS21541C>T [rs2234978], and FAS24484A>T [rs1051070]) and ALI case status. Haplotype-based analyses suggested that three of the tagSNPs (FAS9325G>A, FAS21541C>T, and FAS24484A>T) function as a unit. The association with this haplotype and ALI was validated in a nested case-control study of at-risk subjects (P = 0.05). This haplotype was also associated with increased FAS mRNA levels in response to LPS stimulation. There was no association between FAS polymorphisms and risk of death among ALI cases.
Conclusions: Common genetic variants in FAS are associated with ALI susceptibility. This is the first genetic evidence supporting a role for FAS in ALI.
PMCID: PMC3056231  PMID: 20813889
adult respiratory distress syndrome; FAS receptor; genetic predisposition; apoptosis
20.  Genome Wide Association Identifies PPFIA1 as a Candidate Gene for Acute Lung Injury Risk Following Major Trauma 
PLoS ONE  2012;7(1):e28268.
Acute Lung Injury (ALI) is a syndrome with high associated mortality characterized by severe hypoxemia and pulmonary infiltrates in patients with critical illness. We conducted the first investigation to use the genome wide association (GWA) approach to identify putative risk variants for ALI. Genome wide genotyping was performed using the Illumina Human Quad 610 BeadChip. We performed a two-stage GWA study followed by a third stage of functional characterization. In the discovery phase (Phase 1), we compared 600 European American trauma-associated ALI cases with 2266 European American population-based controls. We carried forward the top 1% of single nucleotide polymorphisms (SNPs) at p<0.01 to a replication phase (Phase 2) comprised of a nested case-control design sample of 212 trauma-associated ALI cases and 283 at-risk trauma non-ALI controls from ongoing cohort studies. SNPs that replicated at the 0.05 level in Phase 2 were subject to functional validation (Phase 3) using expression quantitative trait loci (eQTL) analyses in stimulated B-lymphoblastoid cell lines (B-LCL) in family trios. 159 SNPs from the discovery phase replicated in Phase 2, including loci with prior evidence for a role in ALI pathogenesis. Functional evaluation of these replicated SNPs revealed rs471931 on 11q13.3 to exert a cis-regulatory effect on mRNA expression in the PPFIA1 gene (p = 0.0021). PPFIA1 encodes liprin alpha, a protein involved in cell adhesion, integrin expression, and cell-matrix interactions. This study supports the feasibility of future multi-center GWA investigations of ALI risk, and identifies PPFIA1 as a potential functional candidate ALI risk gene for future research.
PMCID: PMC3266233  PMID: 22295056
21.  Identification and Characterization of a Loss-of-Function Human MPYS Variant 
Genes and immunity  2011;12(4):263-269.
MPYS, also known as STING and MITA, is an IFNβ stimulator essential for host defense against RNA, DNA viruses and intracellular bacteria. MPYS also facilitates the adjuvant activity of DNA vaccines. Here we report identification of a distinct human MPYS haplotype that contains three non-synonymous SNPs, R71H-G230A-R293Q (thus named the HAQ haplotype). We estimate, in two cohorts (1074 individuals), that ~3% of Americans are homozygous for this HAQ haplotype. HAQ MPYS exhibits a >90% loss in the ability to stimulate IFNβ production. Furthermore, fibroblasts and macrophage cells expressing HAQ are defective in Listeria monocytogenes infection-induced IFNβ production. Lastly, we find that the loss of IFNβ activity is due primarily to the R71H and R293Q SNPs in HAQ. We hypothesize that individuals carrying HAQ may exhibit heightened susceptibility to viral infection and respond poorly to DNA vaccines.
PMCID: PMC3107388  PMID: 21248775
SNP; IFNβ; anti-viral signaling; MPYS/STING/MITA
22.  Automated Classification of Radiology Reports for Acute Lung Injury: Comparison of Keyword and Machine Learning Based Natural Language Processing Approaches 
This paper compares the performance of keyword and machine learning-based chest x-ray report classification for Acute Lung Injury (ALI). ALI mortality is approximately 30 percent. High mortality is, in part, a consequence of delayed manual chest x-ray classification. An automated system could reduce the time to recognize ALI and lead to reductions in mortality. For our study, 96 and 857 chest x-ray reports in two corpora were labeled by domain experts for ALI. We developed a keyword and a Maximum Entropy-based classification system. Word unigram and character n-grams provided the features for the machine learning system. The Maximum Entropy algorithm with character 6-gram achieved the highest performance (Recall=0.91, Precision=0.90 and F-measure=0.91) on the 857-report corpus. This study has shown that for the classification of ALI chest x-ray reports, the machine learning approach is superior to the keyword based system and achieves comparable results to highest performing physician annotators.
PMCID: PMC2998031  PMID: 21152268
23.  Transglutaminase 2, a Novel Regulator of Eicosanoid Production in Asthma Revealed by Genome-Wide Expression Profiling of Distinct Asthma Phenotypes 
PLoS ONE  2010;5(1):e8583.
A frequent manifestation of asthma, exercise-induced bronchoconstriction (EIB), occurs in 30–50% of asthmatics and is characterized by increased release of inflammatory eicosanoids. The objective of this study was to identify genes differentially expressed in EIB and to understand the function of these genes in the biology of asthma.
Methodology/Principal Findings
Genome-wide expression profiling of airway leukocytes and epithelial cells obtained by induced sputum was conducted in two groups of subjects with asthma with and without EIB (n = 7 per group), at baseline and following exercise challenge. Based on the results of the gene expression study, additional comparisons were made with a normal control group (n = 10). Localization studies were conducted on epithelial brushings and biopsies from an additional group of asthmatics with EIB (n = 3). Genes related to epithelial repair and mast cell infiltration including β-tryptase and carboxypeptidase A3 were upregulated by exercise challenge in the asthma group with EIB. A gene novel to asthma pathogenesis, transglutaminase 2 (TGM2), was the most differentially expressed at baseline between the groups. In vivo studies confirmed the increased expression of TGM2 in airway cells and airway lining fluid, and demonstrate that TGM2 is avidly expressed in the asthmatic airway epithelium. In vitro studies using recombinant human enzymes reveal that TGM2 augments the enzymatic activity of secreted phospholipase A2 (PLA2) group X (sPLA2-X), an enzyme recently implicated in asthma pathogenesis.
This study found that TGM2, a mediator that is novel to asthma pathogenesis, is overexpressed in asthmatic airways and functions to increase sPLA2-X enzymatic activity. Since PLA2 serves as the first rate-limiting step leading to eicosanoid formation, these results suggest that TGM2 may be a key initiator of the airway inflammatory cascade in asthma.
PMCID: PMC2797392  PMID: 20052409
24.  Proteomic and Computational Analysis of Bronchoalveolar Proteins during the Course of the Acute Respiratory Distress Syndrome 
Rationale: Acute lung injury causes complex changes in protein expression in the lungs. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of pathogenesis and new targets for treatment.
Objectives: The purpose of this study was to examine the changes in protein expression in the bronchoalveolar lavage fluid (BALF) of patients during the course of the acute respiratory distress syndrome (ARDS).
Methods: Using two-dimensional difference gel electrophoresis (DIGE), the expression of proteins in the BALF from patients on Days 1 (n = 7), 3 (n = 8), and 7 (n = 5) of ARDS were compared with findings in normal volunteers (n = 9). The patterns of protein expression were analyzed using principal component analysis (PCA). Biological processes that were enriched in the BALF proteins of patients with ARDS were identified using Gene Ontology (GO) analysis. Protein networks that model the protein interactions in the BALF were generated using Ingenuity Pathway Analysis.
Measurements and Main Results: An average of 991 protein spots were detected using DIGE. Of these, 80 protein spots, representing 37 unique proteins in all of the fluids, were identified using mass spectrometry. PCA confirmed important differences between the proteins in the ARDS and normal samples. GO analysis showed that these differences are due to the enrichment of proteins involved in inflammation, infection, and injury. The protein network analysis showed that the protein interactions in ARDS are complex and redundant, and revealed unexpected central components in the protein networks.
Conclusions: Proteomics and protein network analysis reveals the complex nature of lung protein interactions in ARDS. The results provide new insights about protein networks in injured lungs, and identify novel mediators that are likely to be involved in the pathogenesis and progression of acute lung injury.
PMCID: PMC2556452  PMID: 18658106
acute respiratory distress syndrome; acute lung injury; proteomic analysis; bronchoalveolar lavage; 2D gel electrophoresis
25.  Polymorphisms of the IL1-receptor antagonist gene (IL1RN) are associated with multiple markers of systemic inflammation 
Circulating levels of acute phase reactant proteins such as plasma C-reactive protein (CRP) are likely influenced by multiple genes regulating the innate immune response.
We screened a set of 16 inflammation-related genes for association with CRP in a large, population-based study of healthy young adults (n=1,627). Results were validated in two independent studies (n=1,208 and n=4,310), including a pooled analysis of all 3 studies.
In the pooled analysis, the minor allele of IL1RN 1018 (rs4251961) within the gene encoding interleukin-1 receptor antagonist (IL-1RA) was significantly associated with higher mean plasma log(CRP) level (p < 1 × 10−4). The same IL1RN 1018 allele was associated with higher mean plasma log(IL-6) levels (p=0.004). In the pooled analysis, the minor allele of IL1RN 13888 (rs2232354) was associated with higher fibrinogen, (p = 0.001). The IL1RN 1018 and 13888 variant alleles tag a clade of IL1RN haplotypes linked to allele 1 of a 86 bp VNTR polymorphism. We confirmed that the IL1RN 1018 variant (rs4251961) was associated with decreased cellular IL-1RA production ex vivo.
Common functional polymorphisms of the IL1RN gene are associated with several markers of systemic inflammation.
PMCID: PMC2748384  PMID: 18451331
IL-1receptor antagonist; C-reactive protein; inflammation; fibrinogen

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