Genetic Variation in MAP3K1 Associates with Ventilator-Free Days in Acute Respiratory Distress Syndrome

Abstract

Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) regulates numerous intracellular signaling pathways involved in inflammation and apoptosis. We hypothesized that genetic variation in MAP3K1 might be associated with outcomes in patients with acute respiratory distress syndrome (ARDS), and that these variants would alter MAP3K1-mediated changes in inflammation and transcriptional regulation. To test this hypothesis, we genotyped single-nucleotide polymorphisms covering linkage disequilibrium bins in MAP3K1 in 306 subjects with ARDS from the ARDSNet FACTT (Fluid and Catheter Treatment Trial) study, and tested for associations between MAP3K1 single-nucleotide polymorphisms and ventilator-free days (VFDs) and mortality. We then validated these associations in a separate cohort of 241 patients with ARDS from Harborview Medical Center (Seattle, WA). We found the variant allele of rs832582 (MAP3K1_906Val) was significantly associated with decreased VFDs using multivariate linear regression (-6.1 d, false discovery rate = 0.06) in the FACTT cohort. In the Harborview Medical Center cohort, subjects homozygous for MAP3K1_906Val also had decreased VFDs (-15.1 d, false discovery rate < 0.01), and increased 28-day mortality (all subjects homozygous for the rare allele died). In whole blood stimulated with various innate immune agonists ex vivo, MAP3K1_906Val was associated with increased IL-1β, IL-6, IL-8, monocyte chemoattractant protein 1, and TNF-α production. Transcriptome analysis of whole blood stimulated with Toll-like receptor 4 agonist ex vivo demonstrated enrichment of inflammatory gene sets in subjects homozygous for MAP3K1_906Val. Our findings show a robust association between the variant allele of rs832582 (MAP3K1_906Val) and decreased VFDs in patients with ARDS and suggest that this variant may predispose individuals to a greater inflammatory response.

Keywords: acute respiratory distress syndrome; mitogen-activated protein kinase kinase kinase 1.

Figure 1.

Location of the nonsynonymous coding single-nucleotide polymorphism (SNP) rs832582 in mitogen-activated protein kinase kinase kinase 1 (MAP3K1). Left: linkage disequilibrium (LD) plot showing r² values for pairwise LD analysis for the FACTT (Fluid and Catheter Treatment Trial) white cohort (plot created using SVS, Golden Helix, Bozeman, MT). Center: diagram of the MAP3K1 gene with exons and the locations of the nine LD bin tagging SNP (tagSNPs) that we identified. Right: cartoon of the MAP3K1 protein with the coding SNP identified along the noncatalytic regulatory segment of the protein. MAP3K1 contains a Pleckstrin homology (PH) domain as well as a kinase domain.

Figure 2.

Whole-blood cytokine levels after ex vivo stimulation with Toll-like receptor (TLR) agonists by rs832582 genotype. Whole blood was stimulated for 6 hours on 96-well plates coated with stimulating agents or media alone. Cytokine levels were measured by cytometric bead–based immunoassays, adjusted for monocyte counts, and log₂ transformed. Summary plots showing mean (±SD) cytokine levels in: (A) whole blood after stimulation with normal media based on rs832582 genotype (AG or AA: n = 328 versus GG: n = 18); (B) whole blood after stimulation with Pam3CysSerLys4 (Pam3CSK4) (TLR1/2 agonist) based on rs832582 genotype (AG or AA: n = 328 versus GG: n = 18); (C) whole blood after stimulation with Yersinia pestis LPS (TLR4 agonist) based on rs832582 genotype (AG or AA: n = 169 versus GG: n = 10); and (D) whole blood after stimulation with R848 (TLR7/8) based on rs832582 genotype (AG or AA: n = 169 versus GG: n = 10). P values for all comparisons were generated using the unpaired Student’s t test, and significant comparisons are designated with an asterisk. MCP-1 = monocyte chemoattractant protein-1.

Figure 3.

Luciferase reporter activity for p53 (n = 3) (A), NF-κB (n = 3) (B), and activator protein 1 (AP-1) (n = 4) (C) in human embryonic kidney cells 293 (HEK-293) are not influenced by coding variants in MAP3K1 SNP rs832582. All samples were processed in triplicate. The experimental output was relative luminescence units, which were derived by dividing firefly luciferase luminescence for each reporter construct by Renilla (control) luminescence at 48 hours after transfection with a MAP3K1 construct. Data are shown with each construct normalized to the empty vector value. P values for all pair-wise comparisons were generated using the Mann–Whitney test on normalized data.