1. Introduction  Acute respiratory distress syndrome (ARDS) is characterized by impaired gas exchange with a systemic release of inflammatory mediators, hypoxemia, multiple organ failure and high mortality rates (30-50%).  The etiology and pathogenesis of ARDS are still not fully understood and there are few effective therapeutic options.  It is increasingly recognized that genetic predisposition significantly contributes to the pathogenesis of ARDS. Our objective of this study was to identify novel genetic markers for susceptibility, severity and mortality associated with ARDS. Methods  The outcomes of interest in ARDS cases are described in Table 2.  These three novel SNPs that are associated with susceptibility (rs78142040, rs9605146, and rs3848719), severity (rs3848719) and outcome (rs78142040) of ARDS(Table 3).  Profile of the 3 potential SNP markers are described in Table 4 rs78142040 (C>T) is suspected to occur within a histone mark in intron 6 of the Arylsulfatase D gene rs9605146 (G>A) causes a coding change from proline to leucine with a deleterious effect in the XK, Kell blood group complex subunit-related family, member 3 gene. rs3848719 (G>A) is a synonymous SNP in the 5th exon of the Zinc-Finger/Leucine-Zipper Co- Transducer NIF1 gene. Summary  In our study cohort, 49,834 SNPs were common across the ARDS subgroups (all ARDS, Caucasian ARDS, African American ARDS, all sepsis, and all pneumonia) and the 1000 Genomes Project. Of these, the significance level for association with susceptibility was p<0.01 for 156 SNPs, and p<2.95x10 -7 for 102 SNPs 53 of the 102 SNPs were located in coding regions - 24 caused nonsynonymous changes in amino acid coding; 10 of these changes had a negative predicted functional effect. Of the 27 SNPs analyzed (24 nonsynonymous, 2 synonymous, 1 splice variant), the following SNPs emerged as potential markers to be associated with: Susceptibility: rs78142040, rs9605146, rs3848719 Severity (APACHEII): rs3848719 Outcome (60-day Mortality): rs7814204 Conclusion  In our cohort of ARDS patients, we identified three novel genetic biomarkers of susceptibility, severity, and mortality to ARDS.  Further studies are warranted to validate these findings in larger populations and to identify the underlying molecular mechanisms. Table 4. Profile of the 3 SNPs ARDS PatientsParticipants (N) ARDS Total213 Caucasian145 African American43 Other ancestry25 ARDS with Sepsis total107 Caucasian71 African American20 Other ancestry16 ARDS with Pneumonia total106 Caucasian74 African American23 Other ancestry9 Secondary Controls from the 1000 Genomes Project Participants(N) 1000 genomes Total Control1092 1000 genomes European Ancestry Control379 1000 genomes African Ancestry Control246 ARDS cases APACHE II Score mean ±SD Ventilator-free days/28 days mean ±SD 60-day mortality (%) 213(Total)101.6± 31.912.3± 10.330.2 96(by Exome-Seq) 97.2±30.412.6±10.229.4 117(by TaqMan) 105.1±32.712.0±10.531 Table 1. Distribution of Race/Ethnicity Table 2. Outcomes among ARDS cases NK: Not known, HWE: Hardy-Weinberg equilibrium, AA: alternate genotype or homozygous minor genotype, Ar: heterozygous genotype, rr: reference genotype or homozygous major genotype 1:http://provean.jcvi.org/genome_submit.php, 2:http://genetics.bwh.harvard.edu/pph2/ Data collection SNP Data of 96 patients were obtained using exome-sequencing (Illumina HiScanSQ) Selected SNPs were validated in additional 117 patients using the TaqMan Assay Study sample Test association of SNPs with ARDS using SNP & Variation Suite 7 software package (Golden Helix, Bozeman, MT) for: Susceptibility: Presence of disease Severity: APACHE II score, Ventilator-free days/28 days Outcome: Mortality at 60 days Data analysis Cases: 213 ARDS patients in total Controls: 1092 participants from the 1000 Genomes Project rs3848719rs9605146rs78142040 Location20:4459654522:17265194X:2832771 Gene IDZNF335XKR3ARSD Call Rate (Cases + Controls)0.9810.9980.995 Call Rate (Cases)0.8830.9860.972 HWE P-value (Cases)2.86E-044.22E-011.18E-03 HWE P-value (Controls)6.69E-107.80E-191.81E-239 HWE P-value (Cases + Controls)6.14E-152.06E-307.47E-02 Reference: Alternate AlleleG>A C>T Alt. Allele Freq. (Cases)39.4%38.6%22.0% Alt. Allele Freq. (Controls)22.0%3.4%0.2% Number AA(%) (Cases)41(21.8%)34(16.2%)2(1.0%) Number AA(%) (Controls)88(8.1%)11(1.0%)2(0.2%) Number Ar(%) (Cases)66(35.1%)94(44.8%)87(42.0%) Number Ar(%) (Controls)305(27.9%)53(4.9%)0(0.0%) Number rr(%) (Cases)81(45.1%)82(39.1%)118(57.0%) Number rr(%) (Controls)699(64.0%)1028(94.1%)1090(99.8%) SNP classificationCoding Coding classificationSynonymousNonsynonymousIntronic Reference amino acidSPNK Alternate amino acidSLNK Provean prediction (cutoff=-25) 1 NeutralDeleteriousNK Sift prediction (cutoff=0.05) 1 Tolerated NK Pph2 prediction 2 NKbenignNK rs3848719rs9605146rs78142040 Susceptibility(cases vs. controls) Chi-squared p-value5.47E-132.44E-1164.38E-104 Odds Ratio (95% CI) 1 2.30(1.83-2.89)17.66(13.04-23.90)153.54(56.03-420.77) Severity(ventilator-free days/28 days) p-valueNS Odds Ratio(95% CI) 2 NS Severity(APACHE II score) p-value0.032NS0.061 Odds Ratio(95% CI) 2 0.55(0.31-0.96)NS Outcome(60-day mortality) p-valueNS*NS0.017 Odds Ratio(95% CI) 2 NS 2.04(1.13-3.68) Table 3. Overall Associations of 3 SNPs NS: Not significant; * Significantly associated in genotyped Caucasian, pneumonia, and Caucasian pneumonia subgroups; 1:Odds ratio for alternate allele (allelic test); 2:additive genotypic model Identification of Three Novel SNPs Associated With ARDS by Exome-Seq K. Shortt 1,2, S. Chaudhary 2, D. Grigoryev 1,2, D.P. Heruth 2, L.Venkitachalam 1, L.Q. Zhang 2, and S.Q. Ye 1,2. Department of Biomedical and Health Informatics 1 and Department of Pediatrics 2, Children's Mercy Hospitals and Clinics, University of Missouri, Kansas City, MO. Contact: Katherine Shortt( kasb84@umkc.edu) IRB Approval: This study protocol was determined to be IRB exempt. Funded By: NHLBI/NIH Grant (HL080042 & HL080042- S1, Ye, SQ), start-up fund of Children's Mercy Hospitals and Clinics, UMKC (Ye, SQ), and a Sarah Morrison Student Research Award of UMKC (Shortt, K). Results  The distribution of race/ethnicity in the study sample is given in Table 1. Among ARDS cases, 68% are Caucasian, 20% are African American, and 12% are other races. Among controls, 35% are of European Ancestry, 23% are of African ancestry, and 43% are of other ancestries.