Integrative omics provide biological and clinical insights into acute respiratory distress syndrome.

PURPOSE: Acute respiratory distress syndrome (ARDS) is accompanied by a dysfunctional immune-inflammatory response following lung injury, including during coronavirus disease 2019 (COVID-19). Limited causal biomarkers exist for ARDS development. We sought to identify novel genetic susceptibility targets for ARDS to focus further investigation on their biological mechanism and therapeutic potential. METHODS: Meta-analyses of ARDS genome-wide association studies were performed with 1250 cases and 1583 controls in Europeans, and 387 cases and 387 controls in African Americans. The functionality of novel loci was determined in silico using multiple omics approaches. The causality of 114 factors potentially involved in ARDS development was assessed using Mendelian Randomization analysis. RESULTS: There was distinct genetic heterogeneity in ARDS between Europeans and African Americans. rs7967111 at 12p13.2 was functionally associated with ARDS susceptibility in Europeans (odds ratio = 1.38; P = 2.15 × 10-8). Expression of two genes annotated at this locus, BORCS5 and DUSP16, was dynamic but ultimately decreased during ARDS development, as well as downregulated in immune cells alongside COVID-19 severity. Causal inference implied that comorbidity of inflammatory bowel disease and elevated levels of C-reactive protein and interleukin-10 causally increased ARDS risk, while vitamin D supplementation and vasodilator use ameliorated risk. CONCLUSION: Our findings suggest a novel susceptibility locus in ARDS pathophysiology that implicates BORCS5 and DUSP16 as potentially acting in immune-inflammatory processes. This locus warrants further investigation to inform the development of therapeutic targets and clinical care strategies for ARDS, including those induced by COVID-19.

Plasma Insulin-like Growth Factor Binding Protein 7 Contributes Causally to ARDS 28-Day Mortality: Evidence From Multistage Mendelian Randomization.

BACKGROUND: ARDS is a devastating syndrome with heterogeneous subtypes, but few causal biomarkers have been identified. RESEARCH QUESTION: Would multistage Mendelian randomization identify new causal protein biomarkers for ARDS 28-day mortality? STUDY DESIGN AND METHODS: Three hundred moderate to severe ARDS patients were selected randomly from the Molecular Epidemiology of ARDS cohort for proteomics analysis. Orthogonal projections to latent structures discriminant analysis was applied to detect the association between proteins and ARDS 28-day mortality. Candidate proteins were analyzed using generalized summary data-based Mendelian randomization (GSMR). Protein quantitative trait summary statistics were retrieved from the Efficiency and safety of varying the frequency of whole blood donation (INTERVAL) study (n = 2,504), and a genome-wide association study for ARDS was conducted from the Identification of SNPs Predisposing to Altered Acute Lung Injury Risk (iSPAAR) consortium study (n = 534). Causal mediation analysis detected the role of platelet count in mediating the effect of protein on ARDS prognosis. RESULTS: Plasma insulin-like growth factor binding protein 7 (IGFBP7) moderately increased ARDS 28-day mortality (OR, 1.11; 95% CI, 1.04-1.19; P = .002) per log2 increase. GSMR analysis coupled with four other Mendelian randomization methods revealed IGFBP7 as a causal biomarker for ARDS 28-day mortality (OR, 2.61; 95% CI, 1.33-5.13; P = .005). Causal mediation analysis indicated that the association between IGFBP7 and ARDS 28-day mortality is mediated by platelet count (OR, 1.03; 95% CI, 1.02-1.04; P = .01). INTERPRETATION: We identified plasma IGFBP7 as a novel causal protein involved in the pathogenesis of ARDS 28-day mortality and platelet function in ARDS, a topic for further experimental and clinical investigation.

Plasma Levels of Proresolving and Prophlogistic Lipid Mediators: Association With Severity of Respiratory Failure and Mortality in Acute Respiratory Distress Syndrome.

OBJECTIVES: Acute respiratory distress syndrome is characterized by an overly exuberant inflammatory state in the lung. Specialized proresolving mediators are endogenous agonists for the resolution of lung inflammation and injury in health, yet their association with acute respiratory distress syndrome severity and outcomes remains to be defined. In the current study, we investigate associations between plasma levels of specialized proresolving mediators and acute respiratory distress syndrome severity and mortality. DESIGN: Translational pilot study nested within a large prospective cohort of patients with risk factors for acute respiratory distress syndrome. SETTING: ICU from a large medical center. PATIENTS: Twenty-six Caucasian patients with acute respiratory distress syndrome and available plasma from early in critical illness. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Here, in samples from 26 acute respiratory distress syndrome patients, we examined plasma levels of select specialized proresolving mediators that promote lung injury resolution in preclinical systems, namely lipoxin A4 and maresin 1, and select prophlogistic lipid mediators linked to acute respiratory distress syndrome pathogenesis, namely cysteinyl leukotrienes and thromboxane B2. These mediators were detected by sensitive enzyme-linked immunosorbent assay: lipoxin A4 (assay range) (8.2-5,000 pg/mL), maresin 1 (7.8-1,000 pg/mL), cysteinyl leukotrienes (7.8-1,000 pg/mL), and thromboxane B2 (15.6-2,000 pg/mL). Lower plasma levels of specialized proresolving mediators were associated with increased duration of ventilatory support and ICU length of stay. Even in this small sample size, trends were evident for increased cysteinyl leukotrienes to specialized proresolving mediator ratios (cysteinyl leukotrienes/maresin 1 and cysteinyl leukotrienes/lipoxin A4) in acute respiratory distress syndrome nonsurvivors. CONCLUSIONS: Lower specialized proresolving mediator levels in acute respiratory distress syndrome patients may disrupt timely resolution of lung inflammation and/or injury and contribute to clinical severity and mortality.

Deconstructing ARDS Variability: Platelet Count, an ARDS Intermediate Phenotype and Novel Mediator of Genetic Effects in ARDS.

Genome-wide association studies (GWASs) in acute respiratory distress syndrome (ARDS) have been hampered by the heterogeneity of the clinical phenotypes and the large sample size requirement. As the limitations of these studies to uncover the complex genetic architecture of ARDS are evident, new approaches intended to reduce data complexity need to be applied. Intermediate phenotypes are mechanism-related manifestations of the disease, located closer to the genetic substrate than to disease phenotype, and therefore able to reflect more directly and more strongly the effect of causal genes. The dissection of complex phenotypes into less complex intermediate phenotypes is a valuable strategy to facilitate the discovery of those genetic variants whose effect is not strong enough to be detected as markers of disease in traditional GWASs. Genetic causal inference methodologies can be then applied to estimate the implication of the intermediate trait in the causal circuit between genes and disease. By following this strategy, platelet count, a relevant intermediate quantitative trait in ARDS, has been recently identified as a novel mediator in the genetic contribution to ARDS risk and mortality. The use of intermediate phenotypes and causal inference are emerging methodological and statistical strategies that can help to overcome the limitations of traditional GWASs in ARDS. Moreover, these approaches can provide evidence for the mechanisms linking genes to ARDS and help to prioritize therapeutic targets for the treatment of this devastating syndrome.

Whole blood microRNA markers are associated with acute respiratory distress syndrome.

BACKGROUND: MicroRNAs (miRNAs) can play important roles in inflammation and infection, which are common manifestations of acute respiratory distress syndrome (ARDS). We assessed if whole blood miRNAs were potential diagnostic biomarkers for human ARDS. METHODS: This nested case-control study (N = 530) examined a cohort of ARDS patients and critically ill at-risk controls. Whole blood miRNA profiles and logistic regression analyses identified miRNAs correlated with ARDS. Stratification analysis also assessed selected miRNA markers for their role in sepsis and pneumonia associated with ARDS. Receiver operating characteristic (ROC) analysis evaluated miRNA diagnostic performance, along with Lung Injury Prediction Score (LIPS). RESULTS: Statistical analyses were performed on 294 miRNAs, selected from 754 miRNAs after quality control screening. Logistic regression identified 22 miRNAs from a 156-patient discovery cohort as potential risk or protective markers of ARDS. Three miRNAs-miR-181a, miR-92a, and miR-424-from the discovery cohort remained significantly associated with ARDS in a 373-patient independent validation cohort (FDR q < 0.05) and meta-analysis (p < 0.001). ROC analyses demonstrated a LIPS baseline area-under-the-curve (AUC) value of ARDS of 0.708 (95% CI 0.651-0.766). Addition of miR-181a, miR-92a, and miR-424 to LIPS increased baseline AUC to 0.723 (95% CI 0.667-0.778), with a relative integrated discrimination improvement of 2.40 (p = 0.005) and a category-free net reclassification index of 27.21% (p = 0.01). CONCLUSIONS: miR-181a and miR-92a are risk biomarkers for ARDS, whereas miR-424 is a protective biomarker. Addition of these miRNAs to LIPS can improve the risk estimate for ARDS.

A Missense Genetic Variant in LRRC16A/CARMIL1 Improves Acute Respiratory Distress Syndrome Survival by Attenuating Platelet Count Decline.

RATIONALE: Platelets are believed to contribute to acute respiratory distress syndrome (ARDS) pathogenesis through inflammatory coagulation pathways. We recently reported that leucine-rich repeat-containing 16A (LRRC16A) modulates baseline platelet counts to mediate ARDS risk. OBJECTIVES: To examine the role of LRRC16A in ARDS survival and its mediating effect through platelets. METHODS: A total of 414 cases with ARDS from intensive care units (ICUs) were recruited who had exome-wide genotyping data, detailed platelet counts, and follow-up data during ICU hospitalization. Association of LRRC16A single-nucleotide polymorphisms (SNPs) and ARDS prognosis, and the mediating effect of SNPs through platelet counts were analyzed. LRRC16A mRNA expression levels for 39 cases with ARDS were also evaluated. MEASUREMENTS AND MAIN RESULTS: Missense SNP rs9358856G>A within LRRC16A was associated with favorable survival within 28 days (hazard ratio [HR], 0.57; 95% confidence interval [CI], 0.38-0.87; P = 0.0084) and 60 days (P = 0.0021) after ICU admission. Patients with ARDS who carried the variant genotype versus the wild-type genotype showed an attenuated platelet count decline (∆PLT) within 28 days (difference of ∆PLT, -27.8; P = 0.025) after ICU admission. Patients with ∆PLT were associated with favorable ARDS outcomes. Mediation analysis indicated that the SNP prognostic effect was mediated through ∆PLT within 28 days (28-day survival: HRIndirect, 0.937; 95% CI, 0.918-0.957; P = 0.0009, 11.53% effects mediated; 60-day survival: HRIndirect, 0.919; 95% CI, 0.901-0.936; P = 0.0001, 14.35% effects mediated). Functional exploration suggested that this SNP reduced LRRC16A expression at ICU admission, which was associated with a lesser ∆PLT during ICU hospitalization. CONCLUSIONS: LRRC16A appears to mediate ∆PLT after ICU admission to affect the prognosis in patients with ARDS.

Late-onset moderate to severe acute respiratory distress syndrome is associated with shorter survival and higher mortality: a two-stage association study.

PURPOSE: To evaluate the association between acute respiratory distress syndrome (ARDS) onset time and prognosis. METHODS: Patients with moderate to severe ARDS (N = 876) were randomly assigned into derivation (N = 520) and validation (N = 356) datasets. Both 28-day and 60-day survival times after ARDS onset were analyzed. A data-driven cutoff point between early- and late-onset ARDS was determined on the basis of mortality risk effects of onset times. We estimated the hazard ratio (HR) and odds ratio (OR) of late-onset ARDS using a multivariate Cox proportional hazards model of survival time and a multivariate logistic regression model of mortality rate, respectively. RESULTS: Late-onset ARDS, defined as onset over 48 h after intensive care unit (ICU) admission (N = 273, 31%), was associated with shorter 28-day survival time: HR = 2.24, 95% CI 1.48-3.39, P = 1.24 × 10-4 (derivation); HR = 2.16, 95% CI 1.33-3.51, P = 1.95 × 10-3 (validation); and HR = 2.00, 95% CI 1.47-2.72, P = 1.10 × 10-5 (combined dataset). Late-onset ARDS was also associated with shorter 60-day survival time: HR = 1.70, 95% CI 1.16-2.48, P = 6.62 × 10-3 (derivation); HR = 1.78, 95% CI 1.15-2.75, P = 9.80 × 10-3 (validation); and HR = 1.59, 95% CI 1.20-2.10, P = 1.22 × 10-3 (combined dataset). Meanwhile, late-onset ARDS was associated with higher 28-day mortality rate (OR = 1.46, 95% CI 1.04-2.06, P = 0.0305) and 60-day mortality rate (OR = 1.44, 95% CI 1.03-2.02, P = 0.0313). CONCLUSIONS: Late-onset moderate to severe ARDS patients had both shorter survival time and higher mortality rate in 28-day and 60-day observations.

Predictors of Circulating Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-Binding Protein-3 in Critical Illness.

OBJECTIVE: To characterize predictors of insulin-like growth factor-1 and insulin-like growth factor-binding protein-3 in acute critical illness with the hypothesis that acute factors associated with critical illness will more strongly predict circulating insulin-like growth factor-1 and insulin-like growth factor-binding protein-3 than chronic clinical or genetic factors. DESIGN: Observational study nested within a large prospective study using multivariable linear regression to model circulating insulin-like growth factor-1 and insulin-like growth factor-binding protein-3 with acute and chronic clinical variables, and genotype from five polymorphisms in insulin-like growth factor pathway genes. SETTING: ICUs from two large academic medical centers. PATIENTS: Five hundred forty-three Caucasian patients with risk factors for acute respiratory distress syndrome and available plasma from early in critical illness. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Total insulin-like growth factor-1 and insulin-like growth factor-binding protein-3 were measured in plasma using IMMULITE assays (Siemens, Malvern, PA). We examined age, gender, body mass index, cirrhosis, and diabetes, as well as Acute Physiology, Age, and Chronic Health Evaluation III score, acute hepatic dysfunction, pneumonia and aspiration, sepsis/septic shock, acute respiratory distress syndrome, and receipt of corticosteroids. Body mass index, cirrhosis, and acute respiratory distress syndrome were strongly associated with insulin-like growth factor-1 and insulin-like growth factor-binding protein-3 levels; Acute Physiology, Age, and Chronic Health Evaluation III was strongly associated with insulin-like growth factor-1 levels; and age was strongly associated with insulin-like growth factor-binding protein-3. Five polymorphisms (IGF1: rs1520220, rs35767, rs2946834; IGFBP1: rs4619; IGFBP3: rs2854746) were analyzed for associations with plasma levels. When genotypes were added to models, rs2854746 was significantly associated with plasma insulin-like growth factor-binding protein-3. Genotype explained an additional 2% of variability with an overall adjusted R-square of 0.18. CONCLUSIONS: Despite the acute derangements of critical illness, both acute and chronic health factors significantly influence circulating levels of insulin-like growth factor-1 and insulin-like growth factor-binding protein-3 early in critical illness. rs2854746 is also significantly associated with insulin-like growth factor-binding protein-3 levels in this ICU cohort. Overall, phenotypic and genotypic factors explained only a modest amount of variability in insulin-like growth factor-1 and insulin-like growth factor-binding protein-3. Further research is needed to understand how to apply these findings to patient care.

B Cell-Activating Factor. An Orchestrator of Lymphoid Follicles in Severe Chronic Obstructive Pulmonary Disease.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) have increased pulmonary lymphoid follicle (LF) counts. B cell-activating factor of tumor necrosis factor family (BAFF) regulates B cells in health, but its role in COPD pathogenesis is unclear. OBJECTIVES: To determine whether BAFF expression in pulmonary LFs correlates with COPD severity, LF size or number, and/or readouts of B-cell function in LFs. METHODS: We correlated BAFF immunostaining in LFs in lung explants or biopsies from nonsmoking control subjects (NSC), smokers without COPD (SC), and patients with COPD with the number and size of LFs, and LF B-cell apoptosis, activation, and proliferation. We analyzed serum BAFF levels and BAFF expression in B cells in blood and bronchoalveolar lavage samples from the same subject groups. We assessed whether: (1) cigarette smoke extract (CSE) increases B-cell BAFF expression and (2) recombinant BAFF (rBAFF) rescues B cells from CSE-induced apoptosis by inhibiting activation of nuclear factor-κB (NF-κB). MEASUREMENTS AND MAIN RESULTS: Patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD had increased numbers and larger pulmonary LFs than patients with GOLD stages I-II COPD and SC. We identified two main types of pulmonary LFs: (1) type A, the predominant type in GOLD stages I-II COPD and SC, characterized by abundant apoptotic but few BAFF-positive cells (mostly B cells); and (2) type B, the main type in GOLD stage IV COPD, characterized by abundant BAFF-positive cells but few apoptotic cells (mostly B cells). BAFF levels were also higher in blood and bronchoalveolar lavage B cells in patients with COPD versus NSC and SC. Surprisingly, rBAFF blocked CSE-induced B-cell apoptosis by inhibiting CSE-induced NF-κB activation. CONCLUSIONS: Our data support the hypothesis that B-cell BAFF expression creates a self-perpetuating loop contributing to COPD progression by promoting pulmonary B-cell survival and LF expansion.

Platelet count mediates the contribution of a genetic variant in LRRC16A to ARDS risk.

BACKGROUND: Platelets are believed to be critical in pulmonary-origin ARDS as mediators of endothelial damage through their interactions with fibrinogen and multiple signal transduction pathways. A prior meta-analysis identified five loci for platelet count (PLT): BAD, LRRC16A, CD36, JMJD1C, and SLMO2. This study aims to validate the quantitative trait loci (QTLs) of PLT within BAD, LRRC16A, CD36, JMJD1C, and SLMO2 among critically ill patients and to investigate the associations of these QTLs with ARDS risk that may be mediated through PLT. METHODS: ARDS cases and at-risk control subjects were recruited from the intensive care unit of the Massachusetts General Hospital. Exome-wide genotyping data of 629 ARDS cases and 1,026 at-risk control subjects and genome-wide gene expression profiles of 18 at-risk control subjects were generated for analysis. RESULTS: Single-nucleotide polymorphism (SNP) rs7766874 within LRRC16A was a significant locus for PLT among at-risk control subjects (ß = -13.00; 95% CI, -23.22 to -2.77; P = .013). This association was validated using LRRC16A gene expression data from at-risk control subjects (ß = 77.03 per 1 SD increase of log2-transformed expression; 95% CI, 27.26-126.80; P = .005). Further, rs7766874 was associated with ARDS risk conditioned on PLT (OR = 0.68; 95% CI, 0.51-0.90; P = .007), interacting with PLT (OR = 1.15 per effect allele per 100 × 103/µL of PLT; 95% CI, 1.03-1.30; P = .015), and mediated through PLT (indirect OR = 1.045; 95% CI, 1.007-1.085; P = .021). CONCLUSIONS: Our findings support the role of LRRC16A in platelet formation and suggest the importance of LRRC16A in ARDS pathophysiology by interacting with, and being mediated through, platelets.

Functional characterization of polymorphisms in the peptidase inhibitor 3 (elafin) gene and validation of their contribution to risk of acute respiratory distress syndrome.

Elafin (peptidase inhibitor 3 [PI3]) and its biologically active precursor, pre-elafin, are neutrophil serine proteinase inhibitors with an important role in preventing excessive tissue injury during inflammatory events. Recently, we reported an association between single-nucleotide polymorphism (SNP) rs2664581 in the PI3 gene, increased risk of acute respiratory distress syndrome (ARDS) and pre-elafin circulating levels. This study aims to validate the legitimacy of this association by using a cohort of patients who met the criteria for systemic inflammatory response syndrome and were at risk of developing ARDS (n = 840). A comprehensive functional study of SNPs in PI3 gene was also performed. Luciferase assays and electrophoretic mobility shift assays were conducted to determine the functional relevance of promoter region variants. The effect of the coding SNP rs2664581 on the neutrophil elastase inhibitory activity and transglutaminase binding properties of pre-elafin was also investigated. The variant allele of rs2664581 (C) was significantly associated with increased ARDS risk, mainly among subjects with sepsis (odds ratio = 1.44; 95% confidence interval = 1.04-1.99; P = 0.0276, adjusted by age, sex, and Acute Physiology and Chronic Health Evaluation III). Pre-elafin recombinant protein carrying the amino acid change associated with rs2664581 (Thr34Pro, mutant protein [MT]) had greater capacity to undergo transglutaminase-mediated cross-linking to immobilized fibronectin than wild-type protein in vitro (P < 0.003). No differences were observed in the neutrophil elastase inhibitory activities of wild-type versus MT proteins. In addition, the risk allele-promoter construct had significantly lower cytokine-induced transcriptional activity. Electrophoretic mobility shift assay results indicated a differential binding of nuclear proteins to the G and A alleles of SNP -338G > A. Our results confirm the association between SNP rs2664581 and enhanced risk of ARDS, further supporting the role of PI3 in ARDS development. SNPs in the PI3 locus may act synergistically by regulating PI3 gene expression and pre-elafin biological functions.

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.

Inflammation and immune-related candidate gene associations with acute lung injury susceptibility and severity: a validation study.

INTRODUCTION: 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. METHODS: 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. RESULTS: Among Caucasian subjects with SIRS (n = 750), we identified a nominal association between rs2069832 in IL6 and ALI susceptibility (OR(adj) 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. CONCLUSION: 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.

Distinct and replicable genetic risk factors for acute respiratory distress syndrome of pulmonary or extrapulmonary origin.

BACKGROUND: The role of genetics in the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) from direct or indirect lung injury has not been specifically investigated. The aim of this study was to identify genetic variants contributing to ALI/ARDS from pulmonary or extrapulmonary causes. METHODS: We conducted a multistage genetic association study. We first performed a large-scale genotyping (50K ITMAT-Broad_CARe Chip) in 1717 critically ill Caucasian patients with either pulmonary or extrapulmonary injury, to identify single nucleotide polymorphisms (SNPs) associated with the development of ARDS from direct or indirect insults to the lung. Identified SNPs (p≤0.0005) were validated in two separated populations (Stage II), with trauma (Population I; n=765) and pneumonia/pulmonary sepsis (Population II; n=838), as causes for ALI/ARDS. Genetic variants replicating their association with trauma related-ALI in Stage II were validated in a second trauma-associated ALI population (n=224, Stage III). RESULTS: In Stage I, non-overlapping SNPs were significantly associated with ARDS from direct/indirect lung injury, respectively. The association between rs1190286 (POPDC3) and reduced risk of ARDS from pulmonary injury was validated in Stage II (p<0.003). SNP rs324420 (FAAH) was consistently associated with increased risk of ARDS from extrapulmonary causes in two independent ALI-trauma populations (p<0.006, Stage II; p<0.05, Stage III). Meta-analysis confirmed these associations. CONCLUSIONS: Different genetic variants may influence ARDS susceptibility depending on direct versus indirect insults. Functional SNPs in POPDC3 and FAAH genes may be driving the association with direct and indirect ALI/ARDS, respectively.

Interleukin-1 receptor-associated kinase 3 gene associates with susceptibility to acute lung injury.

Sepsis is the most common cause of acute lung injury (ALI), leading to organ dysfunction and death in critically ill patients. Previous studies associated variants of interleukin-1 receptor-associated kinase genes (IRAKs) with differential immune responses to pathogens and with outcomes during sepsis, and revealed that increased expression levels of the IRAK3 gene were correlated with poor outcomes during sepsis. Here we explored whether common variants of the IRAK3 gene were associated with susceptibility to, and outcomes of, severe sepsis. After our discovery of polymorphism, we genotyped a subset of seven single-nucleotide polymorphisms (SNPs) in 336 population-based control subjects and 214 patients with severe sepsis, collected as part of a prospective study of adults from a Spanish network of intensive care units. Whereas IRAK3 SNPs were not associated with susceptibility to severe sepsis, rs10506481 showed a significant association with the development of ALI among patients with sepsis (P = 0.007). The association remained significant after adjusting for multiple comparisons, population stratification, and clinical variables (odds ratio, 2.50; 95% confidence interval, 1.15-5.47; P = 0.021). By imputation, we revealed three additional SNPs independently associated with ALI (P < 0.01). One of these (rs1732887) predicted the disruption of a putative human-mouse conserved transcription factor binding site, and demonstrated functional effects in vitro (P = 0.017). Despite the need for replication in independent studies, our data suggest that common SNPs in the IRAK3 gene may be determinants of sepsis-induced ALI.

Ure2 is involved in nitrogen catabolite repression and salt tolerance via Ca2+ homeostasis and calcineurin activation in the yeast Hansenula polymorpha.

Disruption of HpURE2 resulted in a low expression of genes encoding nitrate-assimilatory proteins; sensitivity to Li(+), Na(+), and Cd(2+); no induction of ENA1; low levels of the GATA-type transcription factor Gat1; and low intracellular Ca(2+) levels. Gat1 levels were also very low in a Δcnb1 mutant lacking the regulatory subunit of calcineurin. The strain Δure2 was very sensitive to the calcineurin inhibitor FK506 and displayed several phenotypes reminiscent of Δcnb1. The reporter 4xCDRE-lacZ, containing calcineurin-dependent response elements in its promoter, revealed that calcineurin activation was reduced in HpΔure2. Expression of ScURE2 in Δure2 rescued nitrogen catabolite repression and Cd(2+) tolerance but not those phenotypes depending on calcineurin activation, such as salt tolerance and nitrate assimilation gene derepression. HpΔure2 showed an increased expression of the gene PMR1 encoding the Golgi Ca(2+)-ATPase, whereas that of PMC1 encoding the vacuolar Ca(2+)-ATPase remained unaltered. PMR1 up-regulation was abolished by deletion of the GATA-type transcription factor GAT2 in a HpΔure2 genetic background, and normal Ca(2+) levels were recovered. Moreover, overexpression of GAT2 or PMR1 yielded strains mimicking the phenotype of the HpΔure2. This suggests that the low Ca(2+) levels in the HpΔure2 mutant are due to the high levels of Pmr1 that replenish the Golgi Ca(2+) content, thus acting as a negative signal for Ca(2+) entry into the cell. We conclude that HpUre2 is involved in salt tolerance and also in nitrate assimilation gene derepression via Ca(2+) homeostasis regulation and calcineurin activation, which control the levels of Gat1.

Heme oxygenase-1 microsatellite polymorphism and haplotypes are associated with the development of acute respiratory distress syndrome.

OBJECTIVE: Heme oxygenase-1 (HO-1) acts in cytoprotection against acute lung injury. The polymorphic (GT)n repeat in the HO-1 gene (HMOX1) promoter regulates HMOX1 expression. We investigated the associations of HMOX1 polymorphisms with acute respiratory distress syndrome (ARDS) risk and plasma HO-1 levels. DESIGN: Unmatched, nested case-control study. SETTING: Academic medical center. PATIENTS: Consecutive patients with ARDS risk factors upon ICU admission were prospectively enrolled. Cases were 437 Caucasians who developed ARDS and controls were 1,014 Caucasians who did not. MEASUREMENTS AND RESULTS: We genotyped the (GT)n polymorphism and three tagging single nucleotide polymorphisms (tSNPs) in 1,451 patients, and measured the plasma HO-1 levels in 106 ARDS patients. We clustered the (GT)n repeats into: S-allele (<24 repeats), M-allele (24-30 repeats) and L-allele (> or = 31 repeats). We found that longer (GT)n repeats were associated with reduced ARDS risk (Ptrend = 0.004 for both alleles and genotypes), but no individual tSNP was associated with ARDS risk. HMOX1 haplotypes were significantly associated with ARDS risk (global test, P = 0.016), and the haplotype S-TAG was associated with increased ARDS risk (OR, 1.75; 95% CI, 1.15-2.68; P = 0.010). Intermediate-phenotype analysis showed longer (GT)n repeats were associated with higher plasma HO-1 levels (Ptrend = 0.019 for alleles and 0.027 for genotypes). CONCLUSIONS: Longer (GT)n repeats in the HMOX1 promoter are associated with higher plasma HO-1 levels and reduced ARDS risk. The common haplotype S-TAG is associated with increased ARDS risk. Our results suggest that HMOX1 variation may modulate ARDS risk through the promoter microsatellite polymorphism.

Genetic variants in the angiopoietin-2 gene are associated with increased risk of ARDS.

OBJECTIVE: Angiopoietin-2 (Ang-2) is a potent regulator of vascular permeability and inflammation in acute lung injury and acute respiratory distress syndrome (ARDS). Genetic variants in the Ang-2 gene may lead to altered activities of Ang-2 (or ANGPT2) gene. The aim of this study was to assess if genetic variants of Ang-2 are associated with the risk of ARDS. DESIGN: Unmatched, case-control study nested within a prospectively enrolled cohort. SETTING: Intensive care units (ICU) of an academic medical center. PATIENTS: About 1,529 critically ill patients with risk factors for ARDS consecutively admitted to the ICUs from 1999 to 2006. Cases were 449 patients who developed ARDS and controls were 1,080 subjects who did not developed ARDS. INTERVENTION: None. MEASUREMENTS AND RESULTS: Nine tagging SNPs (tSNPs) spanning the entire Ang-2 gene were genotyped in all patients. The results were analyzed using logistic regression models, adjusting for covariates. The variant T allele of one tSNP (rs2515475) was significantly associated with increased risk of ARDS (OR(adjusted) = 1.28; P = 0.042). This association was stronger in subjects with extrapulmonary injuries (OR(adjusted) = 1.79; P = 0.004). Haplotype TT in block 2 containing the T allele of the rs2515475 was also significantly associated with higher risk of ARDS (OR(adjusted) = 1.42; P = 0.009), particularly in subjects with extrapulmonary injuries (OR(adjusted) = 1.90; P = 0.004). CONCLUSION: Common genetic variation in the Ang-2 gene may be associated with increased risk of ARDS, especially among patients with extrapulmonary injuries.