An Enrichment Strategy Yields Seven Novel Single Nucleotide Polymorphisms Associated With Mortality and Altered Th17 Responses Following Blunt Trauma

Abstract

Trauma is the leading cause of death worldwide for individuals under the age of 55. Interpatient genomic differences, in the form of candidate single-nucleotide polymorphisms (SNPs), have been associated previously with adverse outcomes after trauma. However, the utility of these SNPs to predict outcomes based on a meaningful endpoint such as survival is as yet undefined. We hypothesized that specific SNP haplotypes could segregate trauma survivors from non-survivors. Genomic DNA samples were obtained from 453 blunt trauma patients, for whom complete daily clinical and biomarker data were available for 397. Of these, 13 patients were non-survivors and the remaining 384 were survivors. All 397 DNA samples were amplified, fragmented, and examined for 551,839 SNPs using the Illumina Infinium CoreExome-24 v1.1 BeadChip (Illumina). To enrich for likely important SNPs, we initially compared SNPs of the 13 non-survivors versus 13 matched survivors, who were matched algorithmically for injury severity score (ISS), age, and gender ratio. This initial enrichment yielded 126 SNPs; a further comparison to the haplotypes of the remaining 371 survivors yielded a final total of 7 SNPs that distinguished survivors from non-survivors. Furthermore, severely injured survivors with the same seven SNPs as non-survivor exhibited distinct inflammatory responses from similarly injured survivors without those SNPs, and specifically had evidence of altered Th17 cell phenotypes based on computational modeling. These studies suggest an interaction among genetic polymorphism, injury severity, and initial inflammatory responses in driving trauma outcomes.

Figure 1. Study schematic and enrichment strategy

A) Patient cohort derivation: 493 blunt trauma patients were enrolled in this study, 453 from whom DNA samples were obtained. 397 of these patients had complete daily clinical and biomarker records. B) Enrichment strategy: Allele frequencies for each genotype of all 551,839 SNPs were compared between the 13 non-survivors vs the 13 matched survivors. Based on this initial step, 126 SNPs were identified. In a second step, the frequencies of the discriminative 126 SNPs were compared between the non-survivors and the remaining 371 survivors, resulting in a total of 7 SNPs that were present at 100% in the 13 non-survivors and less than 50% in the 384 survivors.

Figure 2. Inflammatory properties of trauma non-survivors and matched survivors

A) IL-17A response of non-survivors and matched survivors: trauma non-survivors (n=13) exhibit significantly higher IL-17A responses compared to algorithmically matched survivors (n=13; see Materials and Methods) over a time course of seven days after admission; p= 0.0086 by two-way ANOVA. B) Trauma non-survivors (n=13) showed rising dynamic inflammatory network complexity as compared to matched survivors (n=13) over a time course of six days.

Figure 3. Circulating levels of inflammatory mediators over a time course of seven days after admission of S-7 SNP vs matched S-0 SNP patients

Survivors with the same 7 SNPs as non-survivors (S-7 SNP patients; n = 10) expressed significantly different levels of multiple circulating inflammatory mediators as compared to survivors with none of the same SNPs as non-survivors (matched S-0 SNP; n = 10).

Figure 4. Principal component analysis suggests a role for type 17 immunity in the circulating inflammatory response to traumatic injury

Principal component analysis was carried out using the data on all four patient sub-groups (non-survivors, matched survivors, S-7 SNP, and matched S-0 SNP) as described in the Materials and Methods. In S-7 SNP patients, IL-33, IL-22, IL-17E/25, IL-17A, and IL-9 were the most relevant inflammatory mediators (A). In the matched S-0 SNP patients, IL-17A. IL-1β, IL-2, IL-10, and IL-17E/25 were the most relevant inflammatory mediators (B). In the non-survivos, IL-22, IL-33, IL-23, IL-17E/25, and IL-1β appeared as the principal characteristics of the inflammatory response (C). In the matched survivors, sIL-2Rα, IL-4, IFN-γ, IL-7 and IL-17 appeared as the principal characteristics (D).

Figure 5. Spearman correlations of IL-17A vs. GM-CSF or IL-10 in non-survivors, matched survivors, S-7 SNP and matched S-0 SNP patients suggest differential Th17 responses

Non-survivors showed a significant, positive correlation between IL-17A and GM-CSF (r= 0.51, p<0.0001) (A), and a significant, negative correlation between IL-17A and IL-10 (r= −0.34; p=0.007) (B). In contrast, matched survivors showed no correlations between either IL-17A and GM-CSF (r= −0.06; p= 0.63) (C) or between IL-17A and IL-10 (r= −0.05; p= 0.66) (D). S-7 SNP patients exhibited no significant correlation between IL-17A and GM-CSF (r= −0.17; p= 0.21; Fig. 4E), but showed a significant, positive correlation between IL-17A and IL-10 (r= 0.51; p<0.0001) (F). In contrast, matched S-0 SNP patients showed a significant, positive correlation between IL-17A and GM-CSF (r= 0.52; p< 0.0001) (G), but no significant correlation between IL-17A and IL-10 (r= 0.21; p= 0.08) (H).