Shwu-Fan Ma

Association Between the MUC5B Promoter Polymorphism and Survival in Patients With Idiopathic Pulmonary Fibrosis

IMPORTANCE Current prediction models of mortality in idiopathic pulmonary fibrosis (IPF), which are based on clinical and physiological parameters, have modest value in predicting which patients will progress. In addition to the potential for improving prognostic models, identifying genetic and molecular features that are associated with IPF mortality may provide insight into the underlying mechanisms of disease and inform clinical trials. OBJECTIVE To determine whether the MUC5B promoter polymorphism (rs35705950), previously reported to be associated with the development of pulmonary fibrosis, is associated with survival in IPF. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of survival in 2 independent cohorts of patients with IPF: the INSPIRE cohort, consisting of patients enrolled in the interferon-γ1b trial (n = 438; December 15, 2003-May 2, 2009; 81 centers in 7 European countries, the United States, and Canada), and the Chicago cohort, consisting of IPF participants recruited from the Interstitial Lung Disease Clinic at the University of Chicago (n = 148; 2007-2010). The INSPIRE cohort was used to model the association of the MUC5B genotype with survival, accounting for the effect of matrix metalloproteinase 7 (MMP-7) blood concentration and other demographic and clinical covariates. The Chicago cohort was used for replication of findings. MAIN OUTCOMES AND MEASURES The primary end point was all-cause mortality. RESULTS The numbers of patients in the GG, GT, and TT genotype groups were 148 (34%), 259 (59%), and 31 (7%), respectively, in the INSPIRE cohort and 41 (28%), 98 (66%), and 9 (6%), respectively, in the Chicago cohort. The median follow-up period was 1.6 years for INSPIRE and 2.1 years for Chicago. During follow-up, there were 73 deaths (36 GG, 35 GT, and 2 TT) among INSPIRE patients and 64 deaths (26 GG, 36 GT, and 2 TT) among Chicago patients. The unadjusted 2-year cumulative incidence of death was lower among patients carrying 1 or more copies of the IPF risk allele (T) in both the INSPIRE cohort (0.25 [95% CI, 0.17-0.32] for GG, 0.17 [95% CI, 0.11-0.23] for GT, and 0.03 [95% CI, 0.00-0.09] for TT) and the Chicago cohort (0.50 [95% CI, 0.31-0.63] for GG, 0.22 [95% CI, 0.13-0.31] for GT, and 0.11 [95% CI, 0.00-0.28] for TT). In the INSPIRE cohort, the TT and GT genotypes (risk for IPF) were associated with improved survival compared with GG (hazard ratios, 0.23 [95% CI, 0.10-0.52] and 0.48 [95% CI, 0.31-0.72], respectively; P < .001). This finding was replicated in the Chicago cohort (hazard ratios, 0.15 [95% CI, 0.05-0.49] and 0.39 [95% CI, 0.21-0.70], respectively; P < .002). The observed association of MUC5B with survival was independent of age, sex, forced vital capacity, diffusing capacity of carbon monoxide, MMP-7, and treatment status. The addition of the MUC5B genotype to the survival models significantly improved the predictive accuracy of the model in both the INSPIRE cohort (C = 0.71 [95% CI, 0.64-0.75] vs C = 0.68 [95% CI, 0.61-0.73]; P < .001) and the Chicago cohort (C = 0.73 [95% CI, 0.62-0.78] vs C = 0.69 [95% CI, 0.59-0.75]; P = .01). CONCLUSIONS AND RELEVANCE Among patients with IPF, a common risk polymorphism in MUC5B was significantly associated with improved survival. Further research is necessary to refine the risk estimates and to determine the clinical implications of these findings.

MicroRNAs implicated in dysregulation of gene expression following human lung transplantation

BackgroundLung transplantation remains the only viable treatment option for the majority of patients with advanced lung diseases. However, 5-year post-transplant survival rates remain low primarily secondary to chronic rejection. Novel insights from global gene expression profiles may provide molecular phenotypes and therapeutic targets to improve outcomes after lung transplantation.MethodsWhole-genome gene expression profiling was performed in a cohort of patients that underwent lung transplantation as well as healthy controls using the Affymetrix Human Exon 1.0ST Array. To explore the potential roles of microRNAs (miRNAs) in regulating lung transplantation-associated gene dysregulation, miRNA expression levels were also profiled in the same samples using the Exiqon miRCURY LNA Array.ResultsIn a cohort of 18 lung transplant patients, 364 dysregulated genes were identified in Caucasian patients relative to normal individuals without pulmonary disorders. Pathway enrichment analysis of the dysregulated genes pointed to Gene Ontology biological processes such as “defense response”, “immune response” and “response to wounding”. We then compared the expression profiles of potential regulating miRNAs, suggesting that dysregulation of a number of lung transplantation-associated genes (e.g., ATR, FUT8, LRRC8B, NFKBIA) may be attributed to the dysregulation of their respective regulating miRNAs.ConclusionsFollowing human lung transplantation, a substantial proportion of genes, particularly those genes involved in certain biological processes like immune response, were dysregulated in patients relative to their healthy counterparts. This exploratory analysis of the relationships between miRNAs and their gene targets in the context of lung transplantation warrants further investigation and may serve as novel therapeutic targets in lung transplant complications.

Expression Profiling Elucidates a Molecular Gene Signature for Pulmonary Hypertension in Sarcoidosis

Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.

Correction: Lung Transcriptomics during Protective Ventilatory Support in Sepsis-Induced Acute Lung Injury

The accession number for the complete microarray data set deposited in ArrayExpress appears incorrectly throughout the article as E-MEXP-12345. The correct accession number should be E-MTAB-2673.

Additional file 3: Figure S2. of A functional genomic model for predicting prognosis in idiopathic pulmonary fibrosis

Receiver-Operating-Characteristic (ROC) analysis of genomic model for diagnosis prediction. ROC curves of UCV cohort consisting of IPF patients and healthy individuals were plotted based on the Prognostic Index (PI) derived from IPF genomic model. AUC (Area-Under-Curve) is displayed in the graph. The red line denotes 10 % false alarm (1-Specificity). (PPTX 79 kb)

50 A NOVEL REGION OF THE MYOSIN LIGHT CHAIN KINASE GENE IS ASSOCIATED WITH SUSCEPTIBILITY TO ACUTE LUNG INJURY IN A SPANISH POPULATION.

Rationale Acute lung injury (ALI) is a life-threatening syndrome with both susceptibility and outcome known to be influenced by genetic factors. Nonmuscle myosin light chain kinase (nmMLCK) encoded by the MYLK gene is a cytoskeleton protein involved in the endothelial cell barrier regulation and in the inflammatory response. We have reported the association of MYLK variants with susceptibility to sepsis-induced ALI in African and European Americans (AJRCMB 2006;34:487). Here we tested the association of MYLK variants in ALI patients from Spain. Methods DNA samples of 96 controls and 80 patients with severe septics were obtained from a Spanish ICU Network. Using TaqMan assays, we first genotyped 16 SNPs selected from our previous report and assessed individual SNPs and haplotype association by Armitage tests and a sliding-window approach, respectively. A new set of nine tagging SNPs (tSNPs) were further selected from the strongest associated region based on the HapMap and data were similarly analyzed. Results Significant association was observed with an SNP (p = .027) previously associated with sepsis and ALI in European Americans with exploratory haplotype analysis of the first 16 SNPs identifying the strongest association (p = .01) in the specific region encoding the nmMLCK N-terminus. The additional nine tSNPs selected from this region and the 4 kb region upstream reinforced the association of the 5′ end of the gene (N-terminus) with susceptibility to ALI (permuted p = .027). Conclusions Our haplotype analyses confirm that MYLK gene variants are associated with ALI in a well-phenotyped replicate population. A fine map genotyping with tSNPs has allowed us to identify a novel region of MYLK associated with ALI, which may be useful to delineate a smaller region for further functional studies of these susceptibility variants. Funded by Specialized Centers of Clinically Oriented Research P50 HL-073994.

37 AN ACUTE LUNG INJURY-ASSOCIATED CORTACTIN POLYMORPHISM ALTERS PULMONARY ENDOTHELIAL CELL BARRIER FUNCTION.

Introduction Acute lung injury (ALI) syndromes are highly morbid consequences of systemic inflammatory conditions such as sepsis. Inflammation-induced disruption during ALI of the endothelial cell (EC) barrier that lines the pulmonary vasculature results in leakage of fluid, protein, and cells into the airspaces of the lung, resulting in respiratory failure. We have previously described (Dudek et al. J Biol Chem 2004;279:24692-700) a critical role for the actin-binding protein cortactin in mediating EC cytoskeletal rearrangements that regulate in vitro barrier function. Methods/Results We now report immunofluorescence and coimmunoprecipitation data demonstrating increased association of cortactin with the junctional proteins β-catenin, focal adhesion kinase, and vinculin during the recovery phase following thrombin-induced permeability. Thus, cortactin interacts at critical cell-cell and cell-matrix junctional sites as the EC monolayer recovers after inflammation-induced disruption. As a translational approach building upon these in vitro observations, single-nucleotide polymorphism (SNP) discovery of the cortactin gene was performed using direct fluorescence-based resequencing. Cortactin SNP discovery in 36 patients, subdivided into those with sepsis-induced ALI, sepsis alone and healthy controls, identified 26 SNPs within the human cortactin gene, including a single novel coding variant. This SNP at amino acid position 484 results in a serine to asparagine change (Ser484Asn), which was enriched in patients with ALI and sepsis compared with controls. Since this S484N site is in close proximity to a critical p60src-targeted tyrosine residue (Y486), a major regulatory site of cortactin function, we generated wild-type and S484N mutant constructs for overexpression in human pulmonary ECs. These studies revealed greatly increased phosphorylation of cortactin at Y486 in the S484N mutant under baseline and EC barrier-altering conditions. In addition, overexpression of S484N in ECs inhibited the potent barrier-promoting effects of sphingosine 1-phosphate as well as delayed barrier recovery after the barrier disruption by thrombin. Summary Cortactin plays a critical role in mediating cytoskeletal and junctional protein rearrangements that regulate EC barrier function. Given that phosphorylation of Y486 modulates cortactin function, the S484N cortactin SNP may increase ALI susceptibility via this mechanism.

76 ATTENUATION OF A RODENT MODEL OF PULMONARY HYPERTENSION: A NOVEL ROLE FOR SORAFENIB, A MULTIKINASE INHIBITOR.

Drawing from new drug discovery studies is the observation that severe pulmonary hypertension (PH) and cancer pathophysiology share common signal transduction pathways leading to abnormal smooth muscle and endothelial cell (EC) interactions and angioproliferative vasculopathy. Sorafenib (Sor), a chemotherapeutic agent in clinical trials for the treatment of renal cell cancer, is an inhibitor of multiple kinases, including Raf-1 kinase, MAPK, VEGFR-2, and VEGFR-3, genes implicated in angiogenesis, proliferation, and the inhibition of apoptosis. We therefore tested the hypothesis that Sor will attenuate the development of PH using an established rodent model of the disease. We performed two 3-week hypoxia (FiO2 10%) and SU5416 (a selective VEGFR-2 inhibitor known to dramatically augment hypoxia-induced PH) studies to induce PH in Dahl salt-sensitive rats (SS). Rat groups were normoxia/vehicle (Norm), hypoxia/vehicle (H), H-Su, hypoxia/sorafenib (H-Sor), and hypoxia/sorafenib/ SU5416 (H-Su-Sor). Except for Norm, all rats were kept in hypoxia, whereas the H-Su group received SU5416 at day 1 (20 mg/kg, sc) and Sor was gavaged daily (2.5 mg/kg). Echocardiography, pulmonary artery pressures (PAPs), right ventricular pressures (RVPs), and lung gene microarray analyses were assessed at 3 weeks. Our results showed that H-Su rats developed severe PH compared with Norm, rats in the H alone group had mildly elevated pressures compared with Norm, and no changes were seen in pressures, weights, or remodeling in the H-Sor or H-SU-Sor groups compared with Norm. The H-Su-Sor rats showed significant reductions in PAP (56%), RVP (55%), and RV hypertrophy (52%). Gene expression profiling data were compared with Norm using GCRMA normalization in R and SAM (> .639, MFC > 1.7). With false discovery rates (FDRs) of 5.1% and 0.7%, respectively, 356 and 293 genes were up- or down-regulated. Forty-seven of the 356 H genes were recapitulated from previous H studies in the rodent model. In addition, 45 genes were differentially expressed between H-Su and H-Su-Sor (FDR 12%), with ECM, cytoskeleton, and angiogenesis gene ontologies, and 81 genes were changed in the H and H-Su groups but not in the H-Su-Sor group. These studies suggest Sor as a powerful novel treatment in PH.

59 A TWO-STEP TAGGING SINGLE NUCLEOTIDE POLYMORPHISM APPROACH FOR THE IDENTIFICATION OF GENETIC VARIANTS OF INTERLEUKIN-6 ASSOCIATED WITH ACUTE LUNG INJURY.

Rationale Interleukin-6 (IL-6) is a key proinflammatory cytokine in the inflammatory diseases including acute lung injury (ALI) and sepsis. Genetic associations of IL-6 variants with disease have focused on the -174 G>C single nucleotide polymorphism (SNP) within the IL-6 promoter. However, it is not clear whether these associations are due to linkage disequilibrium to flanking regions or to other SNPs of the gene. Methods Eighty-four healthy controls (H), 91 septic (S), and 64 ALI patients of European American descent (ED) were analyzed for association. A set of seven evenly distributed intergenic SNPs covering ≈74 kb plus the -174 SNP were chosen to evaluate the associations with the flanking regions of IL-6 gene. TagIT software (http://www.genome.duke.edu/resourses/computation/software>) was used to select IL-6 cosmopolitan tagging SNPs (tSNPs) from 23 ED and 24 African American descent (AD) individuals. SNP dropping with resampling method was used to predict the properties of tSNPs covering variation. Results Only the -174 G>C SNP was significantly associated with ALI (ALI vs H: OR = 0.28, 95% CI 0.08-0.96, p = .039; ALI vs S: OR = 0.26, 95% CI 0.08-0.87, p = .038). Additionally, a set of 14 cosmopolitan tSNPs were identified covering more than 85% of known and unknown ED or AD common variations in the IL-6 gene. Conclusion Our data demonstrate that variants in the IL-6 gene, but not flanking regions, are involved in susceptibility to ALI. The analysis of haplotypes defined by tSNPs within the IL-6 gene may confirm whether genetic variants other than -174 G>C SNP may be associated with ALI. This approach will allow us to delimitate the search for causal variants in the IL-6 gene. Funding Specialized Centers of Clinically Oriented Research P50 HL-073994 and Fundacion Canaria Dr. Manuel Morales.

23 IDENTIFICATION OF ORGAN-SHARED AND STRAIN-INDEPENDENT CANDIDATE GENES DURING COMBINED VENTILATOR-ASSOCIATED LUNG AND KIDNEY INJURY.

Combined ventilator-induced lung injury (VILI) and acute renal failure (ARF) have 80% mortality in the ICU. We hypothesized that analysis of global genetic changes in lung and kidney tissues during developing of ALI will identify similar genomic responses to injury that could underlie injurious effects in both organs. We performed gene expression profiling of lung and kidney tissues in three different mouse strains in a model of VILI, speculating that key transcriptional changes would be tissue and strain independent. Three inbred mouse strains C57BL/6J (B), C3H/HeJ (H), and DBA/2J (D) were selected and young 4-week-old mice were mechanically ventilated (Vt = 17 mL/kg, 110 breaths/min) for 2 hours (n = 6-8) and compared to spontaneous ventilated (n = 6) mice. Analysis of bronchoalveolar lavage proteins demonstrated a significant (p < .05) increase in B, H, and D strains by 133 ± 47SE μg/mL, 125 ± 28SE μg/mL, and 108 ± 53SE μg/mL, respectively, with no significant differences between groups at this age. Gene expression profiles of VILI-affected lung and kidney tissues were generated using MG-U74A (12,488 genes) GeneChips (n = 12) and simultaneously analyzed using GC-Robust Multichip Average and Significance Analysis of Microarrays softwares. Genes with the lowest false discovery rate (q = 0.092%) and ± 50% fold change (FC) were considered affected by VILI in both tissues. Gene Ontology (GO) analysis of identified genes was conducted by GenMAPP and MAPPFinder tools and biological processes with z score > 1.96 were considered significantly affected by VILI. This cross-tissue and cross-strain analysis identified 58 genes that behaved similarly in lung and kidney tissues during VILI. Gene ontology analysis of these genes revealed significant up-regulation of genes involved in response to stress (z = 2.51), including Mig-6 (5.97FCLung (L), 3.02FCKidney (K)) and Gadd-45{g (3.68FCL, 6.06FCK); and transcription activity (z = 4.04), including Ceb/pd} (3.97FCL, 3.74FCK) and Ceb/pb (2.87FCL, 4.58FCK). These data demonstrate that VILI activates specific transcription factors and stress responsive genes in both lung and kidney in all tested strains. Detailed studies of these pathways can reveal organ-shared VILI targets for the development of new therapeutic strategies for this devastating disorder.