Sepsis-associated acute respiratory distress syndrome in individuals of European ancestry: a genome-wide association study

Abstract

Background: Acute respiratory distress syndrome (ARDS) is a lung inflammatory process caused mainly by sepsis. Most previous studies that identified genetic risks for ARDS focused on candidates with biological relevance. We aimed to identify novel genetic variants associated with ARDS susceptibility and to provide complementary functional evidence of their effect in gene regulation.

Methods: We did a case-control genome-wide association study (GWAS) of 1935 European individuals, using patients with sepsis-associated ARDS as cases and patients with sepsis without ARDS as controls. The discovery stage included 672 patients admitted into a network of Spanish intensive care units between January, 2002, and January, 2017. The replication stage comprised 1345 individuals from two independent datasets from the MESSI cohort study (Sep 22, 2008-Nov 30, 2017; USA) and the VISEP (April 1, 2003-June 30, 2005) and MAXSEP (Oct 1, 2007-March 31, 2010) trials of the SepNet study (Germany). Results from discovery and replication stages were meta-analysed to identify association signals. We then used RNA sequencing data from lung biopsies, in-silico analyses, and luciferase reporter assays to assess the functionallity of associated variants.

Findings: We identified a novel genome-wide significant association with sepsis-associated ARDS susceptibility (rs9508032, odds ratio [OR] 0·61, 95% CI 0·41-0·91, p=5·18 × 10^-8) located within the Fms-related tyrosine kinase 1 (FLT1) gene, which encodes vascular endothelial growth factor receptor 1 (VEGFR-1). The region containing the sentinel variant and its best proxies acted as a silencer for the FLT1 promoter, and alleles with protective effects in ARDS further reduced promoter activity (p=0·0047). A literature mining of all previously described ARDS genes validated the association of vascular endothelial growth factor A (VEGFA; OR 0·55, 95% CI 0·41-0·73; p=4·69 × 10^-5).

Interpretation: A common variant within the FLT1 gene is associated with sepsis-associated ARDS. Our findings support a role for the vascular endothelial growth factor signalling pathway in ARDS pathogenesis and identify VEGFR-1 as a potential therapeutic target.

Funding: Instituto de Salud Carlos III, European Regional Development Funds, Instituto Tecnológico y de Energías Renovables.

Figure 1.. Flow chart of quality control steps for the samples and genotyped SNPs in the discovery and replication stages.

CR, Call rate; DQC, Affymetrix dish quality control; FLD, Fisher’s Linear Discriminant; HetSO, Heterozygous Cluster Strength Offset; HWE, Hardy-Weinberg Equilibrium; MAF, minor allele frequency; mtDNA, mitochondrial DNA; Y-chr, Y chromosome.

Figure 2.. Manhattan plot of GWAS results for the discovery stage.

Axes display the -log₁₀ transformed p-values by position in each chromosome. The horizontal line indicates the threshold considered for prioritizing variants for the replication stage (p=5·0×10⁻⁵).

Figure 3.. Regional plot of association results for the genome-wide significant locus.

The -log₁₀ transformed p-values for association tests are plotted by position. The SNP rs number indicated on the plot denotes the sentinel SNP. The remaining SNPs are color coded to reflect their degree of linkage disequilibrium with the indicated SNP based on pairwise r² values from the European population from The 1000 Genomes Project. Estimated recombination rates (light blue line) are plotted on the right y-axis.

Figure 4.. Luciferase reporter assay to assess the role of intron 10 and of rs9508032 and its perfect LD proxies on FLT1 promoter activity.

A) Scheme of vector constructs. B) Experimental data showing that the intron 10 fragment harboring the reference alleles suppresses the FLT1 promoter activity in A549 and THP-1 cells. C) Experimental data showing that the intron 10 fragment harboring the alternative alleles further decreased the FLT1 promoter activity, showing a significant difference in THP-1 cells. Significance was assessed by Wilcoxon signed-rank tests (*p<0·05, #p<0·005, §p<0·0005). Ref and Alt indicate risk and protective alleles, respectively.