Functional Annotation of Genetic Loci Associated With Sepsis Prioritizes Immune and Endothelial Cell Pathways

Figure 1. Expression QTL mapping and differential expression analyses prioritized potential genes. (A) Among 45eQTL genes, there are 12 genes that are differentially expressed in at least one condition in stimulated PBMCs. (B) Expression levels of cis genes that have not eQTL effect in blood, but differentially expressed upon stimulation in PBMC. Heatmap was plotted based on log2 (Fold-change) of RNA expression levels in P.aeruginosa, S.pneumoniae, and C.albicans-stimulated PBMCs. RNA expression levels were measured after 4 or 24 h of stimulation. Colors represent the RNA expression levels, red, significantly induced genes; blue, significantly suppressed genes; gray, not significantly different between stimulated and non-stimulated PBMCs.

Figure 2. Subsets of prioritized genes are also associated with severity of sepsis. Among 55 genes, there are seven genes that are DE in patients (FC > 1.5 and FDR ≤ 0.05). Heat map shows RNA expression levels of seven genes in both discovery and validation cohort. Colors represent expression levels by fold-change between two groups: severe patients SR1 vs. mild patients SR2. Blue, significantly lowly expressed in the severe group; red, significantly highly expressed in the severe group; white, non-significantly different between the severe and mild groups (13).

Figure 3. Suggestive GWAS loci could influence the production of cytokines from PBMC in response to infection. Heat map shows cytokine- QTL (cQTL) effect of the suggestive SNPs (P-value ≤0.05), based on 500 FG cytokine QTL data (10). Empty boxes indicate no cQTL relationship between the SNPs and cytokine production. Color darkness was scaled base on –log10 (P-value).

Figure 4. Pascal pathway enrichment for 39 independent suggestive-GWAS loci (21). Y axis: pathways enriched by Reactome and KEGG database. X axis: –log(10) of q-value.

Figure 5. Validation of rs4957796 SNP. (A) EMSA (electrophoresis molecular shift assay) of oligos resembling the sequence of 30 nts surrounding the top SNP: rs4957796, containing either T or C allele. The shift in the position of the probe carrying T or C allele indicated the effect of nucleotide alteration at rs4957796 in changing the binding affinity of transcription factor. (B) Effect of FER or MAN2A1 deficiency in HUVEC on the expression of adhesion molecules and cytokines. RNA expression levels of E-selectin, VCAM-1, ICAM-1, and IL-8 in HUVEC after 4 h of stimulation were measured by RT-qPCR. Each dot represents one sample. Data represent for three replications.