Plasma angiopoietin-2 as a potential causal marker in sepsis-associated ARDS development: evidence from Mendelian randomization and mediation analysis

Abstract

Purpose: A causal biomarker for acute respiratory distress syndrome (ARDS) could fuel precision therapy options. Plasma angiopoietin-2 (ANG2), a vascular permeability marker, is a strong candidate on the basis of experimental and observational evidence. We used genetic causal inference methods-Mendelian randomization and mediation-to infer potential effects of plasma ANG2.

Methods: We genotyped 703 septic subjects, measured ICU admission plasma ANG2, and performed a quantitative trait loci (QTL) analysis to determine variants in the ANGPT2 gene associated with plasma ANG2 (p < 0.005). We then used linear regression and post-estimation analysis to genetically predict plasma ANG2 and tested genetically predicted ANG2 for ARDS association using logistic regression. We estimated the proportion of the genetic effect explained by plasma ANG2 using mediation analysis.

Results: Plasma ANG2 was strongly associated with ARDS (OR 1.59 (95% CI 1.35, 1.88) per log). Five ANGPT2 variants were associated with ANG2 in European ancestry subjects (n = 404). Rs2442608C, the most extreme cis QTL (coefficient 0.22, 95% CI 0.09-0.36, p = 0.001), was associated with higher ARDS risk: adjusted OR 1.38 (95% CI 1.01, 1.87), p = 0.042. No significant QTL were identified in African ancestry subjects. Genetically predicted plasma ANG2 was associated with ARDS risk: adjusted OR 2.25 (95% CI 1.06-4.78), p = 0.035. Plasma ANG2 mediated 34% of the rs2442608C-related ARDS risk.

Conclusions: In septic European ancestry subjects, the strongest ANG2-determining ANGPT2 genetic variant is associated with higher ARDS risk. Plasma ANG2 may be a causal factor in ARDS development. Strategies to reduce plasma ANG2 warrant testing to prevent or treat sepsis-associated ARDS.

Keywords: Angiopoietin-2; Mediation analysis; Mendelian randomization analysis; Respiratory distress syndrome, adult.

Figure 1:. Regional association plots demonstrate a consistent region of association between the ANGPT2 gene and plasma ANG2 (panel A) and previously reported trauma-associated ARDS (panel B).

Panel A depicts the regional association plot between loci on the ANGPT2 gene (chromosome 8) on the x-axis, and the strength of association with plasma ANG2 in early sepsis among EA shown as -log(p-value for ANG2 association) on the y-axis. Single nucleotide polymorphisms (SNPs) with association more extreme than p=0.005 are labeled, with rs2442608 being the most extreme SNP from the QTL analysis. Color-coding depicts the strength of linkage disequilibrium (LD) between rs2442608 and other loci, with increasing LD represented by increasing red. Navy represents minimal LD with rs2442608. A schematic of the ANGPT2 gene is depicted as a red bar with blue exons and an arrow to indicate the direction of transcription. Panel B depicts the regional association plot between the same region of chromosome 8 with trauma-associated ARDS as reported in our prior study [22]. The most extreme SNP from that association, rs7825407, is in moderate LD with rs2442608 (r²=0.37) in EA populations [42], replicating the importance of this ANGPT2 intron for ARDS and providing functional relevance for this locus. Plots were created using LocusZoom [43].

Figure 2:. Mendelian Randomization conceptual model to infer whether plasma ANG2 has a causal effect on ARDS risk.

We first used linear regression to identify 5 SNPs near ANGPT2 that strongly associated with plasma ANG2 and jointly explained 8% of the variance in measured ANG2 among EA subjects. Each EA individual was then assigned a genetically predicted ANG2 value using post-estimation prediction. The predicted values should be less affected by unmeasured confounders beyond population stratification, since they derive from each individual’s genetic assortment of parental alleles. Genetically predicted plasma ANG2 values are then tested for an association with ARDS risk by multivariable logistic regression adjusting for genetic ancestry, severity of illness (APACHE III score), and pulmonary versus non-pulmonary source of sepsis. The statistically significant association between the genetically-determined component of plasma ANG2 and ARDS risk is evidence for a potential causal effect of plasma ANG2 towards ARDS development. SNPs: single nucleotide polymorphisms; ANGPT2: angiopoietin-2 gene; ANG2: angiopoietin-2 protein; EA: European ancestry.