Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death.

Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.

Toll-like receptor 1 polymorphisms and associated outcomes in sepsis after traumatic injury: a candidate gene association study.

OBJECTIVE: To determine whether single nucleotide polymorphisms (SNPs) in TLR1 are associated with mortality, specifically sepsis-associated mortality, in a traumatically injured population. BACKGROUND: Innate immune responses mediated by toll-like receptors (TLRs) induce early inflammatory responses to pathogen and damage-associated molecular patterns. Genetic variation in TLRs has been associated with susceptibility and outcomes in a number of infectious and noninfectious disease states. METHODS: Patients admitted to the trauma intensive care unit at a level 1 trauma center serving 4 states were enrolled and followed for development of infection, sepsis, and death. Genomic DNA was genotyped and logistic regression analysis was performed to determine associations between TLR1 SNPs and mortality. We further examined for associations between TLR1 SNPs and mortality in subgroups on the basis of the presence of sepsis and the type of sepsis-associated organism. RESULTS: We enrolled 1961 patients. TLR1-7202G (rs5743551) was associated with increased mortality after traumatic injury and this association was primarily observed in the subset of patients who developed sepsis [adjusted odds ratio (OR): 3.16; 95% confidence interval (CI): 1.43-6.97, P=0.004]. This association persisted after further restriction to gram-positive sepsis. TLR1(742A/G(Asn248Ser)) (rs4833095), a coding SNP in LD with TLR1-7202G, was also associated with mortality in gram-positive sepsis (adjusted OR: 4.16; 95% CI: 1.22-14.19, P=0.023). CONCLUSIONS: Genetic variation in TLR1 is associated with increased mortality in patients with sepsis after traumatic injury and may represent a novel marker of risk for death in critically injured patients.

A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease.

Recent progress in cataloguing common genetic variation has made possible genome-wide studies that are beginning to elucidate the causes and consequences of our genetic differences. Approaches that provide a mechanistic understanding of how genetic variants function to alter disease susceptibility and why they were substrates of natural selection would complement other approaches to human-genome analysis. Here we use a novel cell-based screen of bacterial infection to identify human variation in Salmonella-induced cell death. A loss-of-function allele of CARD8, a reported inhibitor of the proinflammatory protease caspase-1, was associated with increased cell death in vitro (p = 0.013). The validity of this association was demonstrated through overexpression of alternative alleles and RNA interference in cells of varying genotype. Comparison of mammalian CARD8 orthologs and examination of variation among different human populations suggest that the increase in infectious-disease burden associated with larger animal groups (i.e., herds and colonies), and possibly human population expansion, may have naturally selected for loss of CARD8. We also find that the loss-of-function CARD8 allele shows a modest association with an increased risk of systemic inflammatory response syndrome in a small study (p = 0.05). Therefore, a by-product of the selected benefit of loss of CARD8 could be increased inflammatory diseases. These results demonstrate the utility of genome-wide cell-based association screens with microbes in the identification of naturally selected variants that can impact human health.

Genetic variation in the FAS gene and associations with acute lung injury.

RATIONALE: Fas (CD95) modulates apoptosis and inflammation and is believed to play an important role in lung injury. OBJECTIVES: To determine if common genetic variation in FAS is associated with acute lung injury (ALI) susceptibility, risk of death, and FAS gene expression. METHODS: We genotyped 14 single nucleotide polymorphisms (tagSNPS) in FAS in samples from healthy white volunteers (control subjects, n = 294) and patients with ALI (cases, n = 324) from the ARDSnet Fluid and Catheter Treatment Trial (FACTT). FAS genotypes associated with ALI in the discovery study were confirmed in a nested case-control validation study of critically ill patients at risk for ALI (n = 657). We also tested for associations between selected tagSNPS and FAS mRNA levels in whole blood from healthy control subjects exposed to media alone or LPS ex vivo. MEASUREMENTS AND MAIN RESULTS: We identified associations between four tagSNPs in FAS (FAS(-11341A>T) [rs17447140], FAS(9325G>A) [rs2147420], FAS(21541C>T) [rs2234978], and FAS(24484A>T) [rs1051070]) and ALI case status. Haplotype-based analyses suggested that three of the tagSNPs (FAS(9325G>A), FAS(21541C>T), and FAS(24484A>T)) function as a unit. The association with this haplotype and ALI was validated in a nested case-control study of at-risk subjects (P = 0.05). This haplotype was also associated with increased FAS mRNA levels in response to LPS stimulation. There was no association between FAS polymorphisms and risk of death among ALI cases. CONCLUSIONS: Common genetic variants in FAS are associated with ALI susceptibility. This is the first genetic evidence supporting a role for FAS in ALI.

Strong toll-like receptor responses in cystic fibrosis patients are associated with higher lung function.

BACKGROUND: Cystic fibrosis (CF) airways disease varies widely among patients with identical cystic fibrosis transmembrane conductance regulator (CFTR) genotypes. Robust airway inflammation is thought to be deleterious in CF; inter-individual variation in Toll-like receptor (TLR)-mediated innate immune inflammatory responses (TMIIR) might account for a portion of the phenotypic variation. We tested if TMIIR in people with CF are different than those of healthy controls, and whether higher TMIIR in people with CF are associated with reduced lung function. METHODS: We cultured whole blood from clinically stable subjects with CF (n = 76) and healthy controls (n = 45) with TLR agonists, and measured cytokine production and expression of TLR-associated genes. We tested for differences in TLR-stimulated cytokine levels between subjects with CF and healthy subjects, and for associations between cytokine and gene expression levels with baseline lung function (forced expiratory volume in one second percent predicted (FEV1%)) and decline in FEV1% over time. RESULTS: TMIIR in blood from subjects with CF were lower than in healthy controls. Expression of TLR regulators SARM1, TOLLIP, and AKT1 were downregulated in CF. In subjects with CF we found that lower TLR4-agonist-induced IL-8 was associated with lower FEV1% at enrollment (p<0.001) and with greater five year FEV1% decline (p<0.001). CONCLUSIONS: TMIIR were lower in people with CF relative to healthy controls; however, unexpectedly, greater whole blood TMIIR were positively associated with lung function in people with CF. These findings suggest a complex interaction between inflammation and disease in people with CF.

Toll-like receptor 1 polymorphisms affect innate immune responses and outcomes in sepsis.

RATIONALE: Polymorphisms affecting Toll-like receptor (TLR)-mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis. OBJECTIVES: To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis. METHODS: We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case-control study in a cohort of intensive care unit patients with sepsis, and a case-control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects. MEASUREMENTS AND MAIN RESULTS: The SNP demonstrating the most hypermorphic effect was the G allele of TLR1(-7202A/G) (rs5743551), which associated with elevated TLR1-mediated cytokine production (P < 2 x 10(-20)). TLR1(-7202G) marked a coding SNP that causes higher TLR1-induced NF-kappaB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1(-7202G) predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07-3.09). In the case-control study TLR1(-7202G) was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59-7.27). TLR1(-7202G) also associated with a higher prevalence of gram-positive cultures in both clinical studies. CONCLUSIONS: Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis.

Inflammation and immune-related candidate gene associations with acute lung injury susceptibility and severity: a validation study.

INTRODUCTION: Common variants in genes related to inflammation, innate immunity, epithelial cell function, and angiogenesis have been reported to be associated with risks for Acute Lung Injury (ALI) and related outcomes. We tested whether previously-reported associations can be validated in an independent cohort at risk for ALI. METHODS: We identified 37 genetic variants in 27 genes previously associated with ALI and related outcomes. We prepared allelic discrimination assays for 12 SNPs from 11 genes with MAF>0.05 and genotyped these SNPs in Caucasian subjects from a cohort of critically ill patients meeting criteria for the systemic inflammatory response syndrome (SIRS) followed for development of ALI, duration of mechanical ventilation, and in-hospital death. We tested for associations using additive and recessive genetic models. RESULTS: Among Caucasian subjects with SIRS (n = 750), we identified a nominal association between rs2069832 in IL6 and ALI susceptibility (OR(adj) 1.61; 95% confidence interval [CI], 1.04-2.48, P = 0.03). In a sensitivity analysis limiting ALI cases to those who qualified for the Acute Respiratory Distress Syndrome (ARDS), rs61330082 in NAMPT was nominally associated with risk for ARDS. In terms of ALI outcomes, SNPs in MBL2 (rs1800450) and IL8 (rs4073) were nominally associated with fewer ventilator-free days (VFDs), and SNPs in NFE2L2 (rs6721961) and NAMPT (rs61330082) were nominally associated with 28-day mortality. The directions of effect for these nominal associations were in the same direction as previously reported but none of the associations survived correction for multiple hypothesis testing. CONCLUSION: Although our primary analyses failed to statistically validate prior associations, our results provide some support for associations between SNPs in IL6 and NAMPT and risk for development of lung injury and for SNPs in IL8, MBL2, NFE2L2 and NAMPT with severity in ALI outcomes. These associations provide further evidence that genetic factors in genes related to immunity and inflammation contribute to ALI pathogenesis.