An integrated transcriptome and expressed variant analysis of sepsis survival and death

Abstract

Background: Sepsis, a leading cause of morbidity and mortality, is not a homogeneous disease but rather a syndrome encompassing many heterogeneous pathophysiologies. Patient factors including genetics predispose to poor outcomes, though current clinical characterizations fail to identify those at greatest risk of progression and mortality.

Methods: The Community Acquired Pneumonia and Sepsis Outcome Diagnostic study enrolled 1,152 subjects with suspected sepsis. We sequenced peripheral blood RNA of 129 representative subjects with systemic inflammatory response syndrome (SIRS) or sepsis (SIRS due to infection), including 78 sepsis survivors and 28 sepsis non-survivors who had previously undergone plasma proteomic and metabolomic profiling. Gene expression differences were identified between sepsis survivors, sepsis non-survivors, and SIRS followed by gene enrichment pathway analysis. Expressed sequence variants were identified followed by testing for association with sepsis outcomes.

Results: The expression of 338 genes differed between subjects with SIRS and those with sepsis, primarily reflecting immune activation in sepsis. Expression of 1,238 genes differed with sepsis outcome: non-survivors had lower expression of many immune function-related genes. Functional genetic variants associated with sepsis mortality were sought based on a common disease-rare variant hypothesis. VPS9D1, whose expression was increased in sepsis survivors, had a higher burden of missense variants in sepsis survivors. The presence of variants was associated with altered expression of 3,799 genes, primarily reflecting Golgi and endosome biology.

Conclusions: The activation of immune response-related genes seen in sepsis survivors was muted in sepsis non-survivors. The association of sepsis survival with a robust immune response and the presence of missense variants in VPS9D1 warrants replication and further functional studies.

Trial registration: ClinicalTrials.gov NCT00258869. Registered on 23 November 2005.

Figure 1

A systems survey of sepsis survival. (A) Schematic representing the different trajectories enrolled subjects might take. X-axis represents time (not to scale), emphasizing the illness progresses from local to systemic infection prior to clinical presentation (t₀). The green line is flat only to distinguish subjects without infection, although these individuals could also have the full spectrum of clinical illness severity. Blue lines represent subjects with sepsis of different severities, all of whom survive at 28 days. This is in contrast to subjects with sepsis who die within 28 days, independent of initial sepsis severity. (B) Analytical plan for the CAPSOD cohort including previously published metabolome and proteome [11]. Metabolomic and proteomic analyses were performed on samples obtained at t₀ and 24 h later. Transcriptomic analysis was performed on samples obtained at t₀.

Figure 2

CONSORT flow chart of patient enrollment and selection. The planned study design was to analyze 30 subjects each with uncomplicated sepsis, severe sepsis (sepsis with organ dysfunction), septic shock, sepsis deaths, and SIRS (no infection present). However, limited sample quality or quantity in some cases decreased the number available per group. The analysis population includes 78 sepsis survivors, 28 sepsis non-survivors, and 23 SIRS survivors. Three SIRS non-survivors represented too few subjects to define their own analysis subgroup and were therefore removed prior to analysis.

Figure 3

Differentially expressed genes and pathways. (A) Number and overlap among the differentially expressed, annotated genes in each pairwise comparison. (B) Hierarchical clustering of 2,140 differentially expressed gene (including 314 unannotated loci) using Pearson’s moment correlations applied to subjects with SIRS, Sepsis Non-survivors, and Sepsis Survivors. ANOVA with 7.5% FDR correction; −log10 P value = 2.21. (C) Highly represented ToppGene pathways and processes among the annotated genes differentially expressed between SIRS and Sepsis Survivors as well as Sepsis Survivors and Sepsis Non-survivors.

Figure 4

Protein structure of VPS9D1 showing approximate location of variants associated with sepsis survival.

Figure 5

Expression of VPS9D1 . VPS9D1 is represented by two different genetic loci: XLOC_011354 (Cufflinks Transcript ID TCONS_00032132; RefSeq ID NM_004913) and XLOC_010886 (Cufflinks Transcript ID TCONS_00030416; RefSeq ID NM_004913). The former demonstrated greater sequencing coverage and is presented here. Results for XLOC_010886 were similar (data not shown). (A) Level of VPS9D1 expression in sepsis survivors (n = 74) and sepsis non-survivors (n = 26). (B) Level of VPS9D1 expression as a function of the VPS9D1 reference (n = 64) or variant sequence (n = 36) among subjects with adequate coverage. (C) Volcano plot depicting differentially expressed genes as a function of the VPS9D1 reference or variant allele.