Xanthine oxidoreductase gene polymorphisms are associated with high risk of sepsis and organ failure

This is the first study reporting an association between XDH polymorphisms and sepsis and sepsis-associated ARDS. The major findings of our study are: (i) XDH variants may increase the risk of sepsis and renal dysfunction as well as risk of death in sepsis-associated ARDS; (ii) XOR activity may serve as a biomarker for evaluating disease prognosis, and (iii) knowledge of XDH SNPs could potentially identify sub-groups that might benefit from development of targeted therapies to improve clinical outcomes.

Sepsis is a major cause of death worldwide [3, 37, 38] and is characterized by a dysregulated acute inflammatory response, frequently leading to multisystem organ failure. Oxidant injury related to formation of ROS (generated by XOR and other pro-oxidant enzymes) has been postulated in the pathogenesis of sepsis and organ dysfunction [39]. We found that XOR mediated damage leads to enhanced lung injury whereas inhibition of XOR with allopurinol is protective [16, 40, 41]. We also reported that XOR prevents restoration of the endothelial barrier (key to resolution of lung injury), and XOR inhibition improved survival in an endotoxemia murine model [42]. Despite a role of XOR in sepsis and ARDS, their utility as genetic markers for identifying patients at high probability of fatal outcome remains unclear.

The gene XDH encodes XOR, spans 80.42 kb on chromosome 2p23.1 and contains 36 exons. This gene has been tested for association with diseases including purine-pyrimidine metabolism and xanthinuria (type I), and may participate in pathways and processes including free radical induced apoptosis, purine metabolism, electron transport, lactation, and epithelial cell differentiation. XDH variants have been associated with xanthinuria [43], and recently with systemic hypertension and oxidative stress [44]. Activation of XOR may contribute to ARDS [41], and variation in XOR activity could be used to predict outcomes, i.e., XOR activity was increased in non-survivor septic patients [45].

Ethnic disparities in sepsis prevalence and outcome have been reported [6]. In the CELEG cohort, AA sepsis patients with ARDS had a higher mortality than in the ARDS Network trials [46]. These findings highlight a genetic predisposition among AA patients and warrant further study to explore a potential mechanistic basis for targeted therapy [19]. In our study, we observed association with risk of sepsis and renal dysfunction among AA patients. The most compelling signals derived from a cluster of three markers (rs185925, rs561525, rs2163059) from a region spanning introns 8 to 12. Indirect functional assessment provided further supporting evidence for potential functional consequences of these SNPs. First, we assessed patterns of LD, which is important in mapping genes associated with complex diseases. That region was in relatively strong LD among AA control subjects compared to the EAs (Supplementary Fig. 1, Additional File 1). Second, this region corresponds to amino acid position 245 to 404 (NP_000370), which aligns with “FAD_binding_4 super family (cl10516)”, a family with various enzymes that are similar to XOR and use FAD as a co-factor (Fig. 1, panel C). Third, this region was also enriched with regulatory elements identified by ENCODE regulatory tracks (Supplementary Fig. 2, Additional File 1). Finally, multiple association signals discovered for susceptibility of ARDS from the replication cohort of Spanish sepsis patients, highlighted the potential importance of this region influencing sepsis outcome. However, we cannot rule out the potential functional relevance of any of the markers studied in this region, as they maybe in LD with some causal variant(s) rather than being causal themselves. Additionally, two common SNPs in tight LD (including a coding-synonymous SNP rs1884725 in exon 27) provided the strongest evidence for association with risk of renal dysfunction among AA sepsis patients. Carriers had an increase of serum creatinine concentration compared to non-carriers. Interestingly, the SNP rs1884725 aligns with “Ald_Xan_dh_C2 superfamily (cl10595)” (Fig. 1, panel C), the largest functional domain for XOR. Given the potential importance of these regions in influencing susceptibility to and outcome of sepsis, further studies are warranted, such as extensive sequencing of this genomic region to unravel the likely causal variants (i.e., unknown low-frequency variants with potential biological roles), and mechanistic studies to understand the causal role of the risk variants identified. Nevertheless, our findings may help to understand health disparities in terms of higher morbidity and mortality observed in this vulnerable population.

Of note, a coding non-synonymous SNP (rs17011368, Ile703Val), which predicts a replacement of isoleucine by valine at amino acid position 703, was associated with 60-day mortality among EA sepsis patients with ARDS. This finding, coupled with a recent observation showing markedly enhanced activity of the Ile703Val variant [43], provides a compelling link between enhanced XOR activity and mortality in ARDS. Oxidative stress was identified as a key lung injury pathway controlling ARDS severity in animal models [47]. Recently reported associations between several oxidant/antioxidant pathway genes and ARDS highlight the importance of regulation of oxidant-mediated response in ARDS. It is likely that multiple genes in the same or interacting biological pathways contribute to sepsis/ARDS severity and outcomes. Thus, exploring the possible gene-gene interactions between XDH and other oxidative stress pathway genes (e.g., NRF2), or innate immunity genes (e.g., CD14 and TLRs), will be important for future research.

For prioritization of sepsis-associated causal variants, especially those noncoding XDH variants, we applied multiple tools integrating various genomic and epigenomic annotations to elucidate multiple aspects of biological functionality of XDH variants. Multiple lines of evidence were discovered supporting our hypothesis that prioritized variants identified in our study could affect XDH expression and/or function, therefore influencing the severity and outcome of sepsis (Table 5). Further investigation to validate prioritized XDH variants on their functional impact in experimental studies is warranted.

We acknowledge some limitations of our study. Although our findings suggest that XDH variants are risk factors for sepsis, it deserves further validation in non-white cohorts with a larger sample size of sepsis cases. We found higher serum XOR activity in the combined group of sepsis patients, and sepsis patients with ARDS had the highest level of XOR. Further evidence was found for the lead variant rs185925, which was associated with XOR activity in a small subset of AA sepsis patients with ARDS (P < 0.005). These findings suggest that XOR activity is a genetically determined biomarker for sepsis and sepsis associated ARDS but clearly warrants further investigation. The high mortality rate in sepsis patients is likely due to environmental and genetic factors, which influence the host response to infection. However, we were unable to explore the genotype by environment (GxE) interactions in our study to confirm the possible modifying effects of local tissue/cell type specific environment (e.g., systemic inflammation) on XDH genetic risk factors in sepsis. With its capacity to generate ROS, XOR has been implicated in the development of tissue oxidative damage in a wide variety of respiratory and cardiovascular disorders such as ARDS, ischemia reperfusion injury, atherosclerosis, heart failure, and arterial hypertension [14, 48]. Other conditions where XOR has also been incriminated include COPD, obstructive sleep apnea, pulmonary manifestations of sickle cell disease, interstitial pneumonitis, as well as rejection of heart-lung transplant [14, 49]. Thus, our study may have a broader implication in assessing genetic influence of XDH on disease susceptibility and outcome wherever oxidative damage is involved.