Diagnostic and therapeutic potential of RNASET2 in Crohn's disease: Disease-risk polymorphism modulates allelic-imbalance in expression and circulating protein levels and recombinant-RNASET2 attenuates pro-inflammatory cytokine secretion.

Ribonuclease T2 gene (RNASET2) variants are associated in genome wide association studies (GWAS) with risk for several autoimmune diseases, including Crohn's disease (CD). In T cells, a functional and biological relationship exists between TNFSF15-mediated enhancement of IFN-γ production, mucosal inflammation and RNASET2. Disease risk variants are associated with decreased mRNA expression and clinical characteristics of severe CD; however, functional classifications of variants and underlying molecular mechanisms contributing to pathogenesis remain largely unknown. In this study we demonstrate that allelic imbalance of RNASET2 disease risk variant rs2149092 is associated with transcriptional and post-transcriptional mechanisms regulating transcription factor binding, promoter-transactivation and allele-specific expression. RNASET2 mRNA expression decreases in response to multiple modes of T cell activation and recovers following elimination of activator. In CD patients with severe disease necessitating surgical intervention, preoperative circulating RNASET2 protein levels were decreased compared to non-IBD subjects and rebounded post-operatively following removal of the inflamed region, with levels associated with allelic carriage. Furthermore, overexpression or treatment with recombinant RNASET2 significantly reduced IFN-γ secretion. These findings reveal that RNASET2 cis- and trans-acting variation contributed regulatory complexity and determined expression and provide a basis for linking genetic variation with CD pathobiology. These data may ultimately identify RNASET2 as an effective therapeutic target in a subset of CD patients with severe disease.

Identification of ATP2B4 Regulatory Element Containing Functional Genetic Variants Associated with Severe Malaria.

Genome-wide association studies for severe malaria (SM) have identified 30 genetic variants mostly located in non-coding regions. Here, we aimed to identify potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium (LD) with the malaria-associated genetic variants. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing five ATP2B4 SNPs in LD with rs10900585. We found significant associations between SM and rs10900585 and our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we demonstrated that both individual SNPs and the combination of SNPs had regulatory effects. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Our data demonstrate that severe malaria-associated genetic variants alter the expression of ATP2B4 encoding a plasma membrane calcium-transporting ATPase 4 (PMCA4) expressed on red blood cells. Altering the activity of this regulatory element affects the risk of SM, likely through calcium concentration effect on parasitaemia.

Transcriptional Response in a Sepsis Mouse Model Reflects Transcriptional Response in Sepsis Patients.

Mortality due to sepsis remains unacceptably high, especially for septic shock patients. Murine models have been used to better understand pathophysiology mechanisms. However, the mouse model is still under debate. Herein we investigated the transcriptional response of mice injected with lipopolysaccharide (LPS) and compared it to either human cells stimulated in vitro with LPS or to the blood cells of septic patients. We identified a molecular signature composed of 2331 genes with an FDR median of 0%. This molecular signature is highly enriched in regulated genes in peritoneal macrophages stimulated with LPS. There is significant enrichment in several inflammatory signaling pathways, and in disease terms, such as pneumonia, sepsis, systemic inflammatory response syndrome, severe sepsis, an inflammatory disorder, immune suppression, and septic shock. A significant overlap between the genes upregulated in mouse and human cells stimulated with LPS has been demonstrated. Finally, genes upregulated in mouse cells stimulated with LPS are enriched in genes upregulated in human cells stimulated in vitro and in septic patients, who are at high risk of death. Our results support the hypothesis of common molecular and cellular mechanisms between mouse and human sepsis.

Genetic Predisposition to the Mortality in Septic Shock Patients: From GWAS to the Identification of a Regulatory Variant Modulating the Activity of a CISH Enhancer.

The high mortality rate in septic shock patients is likely due to environmental and genetic factors, which influence the host response to infection. Two genome-wide association studies (GWAS) on 832 septic shock patients were performed. We used integrative bioinformatic approaches to annotate and prioritize the sepsis-associated single nucleotide polymorphisms (SNPs). An association of 139 SNPs with death based on a false discovery rate of 5% was detected. The most significant SNPs were within the CISH gene involved in cytokine regulation. Among the 139 SNPs associated with death and the 1311 SNPs in strong linkage disequilibrium with them, we investigated 1439 SNPs within non-coding regions to identify regulatory variants. The highest integrative weighted score (IW-score) was obtained for rs143356980, indicating that this SNP is a robust regulatory candidate. The rs143356980 region is located in a non-coding region close to the CISH gene. A CRISPR-Cas9-mediated deletion of this region and specific luciferase assays in K562 cells showed that rs143356980 modulates the enhancer activity in K562 cells. These analyses allowed us to identify several genes associated with death in patients with septic shock. They suggest that genetic variations in key genes, such as CISH, perturb relevant pathways, increasing the risk of death in sepsis patients.