Tara C. Mills

Leprosy and the adaptation of human toll-like receptor 1.

Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae and remains endemic in many parts of the world. Despite several major studies on susceptibility to leprosy, few genomic loci have been replicated independently. We have conducted an association analysis of more than 1,500 individuals from different case-control and family studies, and observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to leprosy (TLR1 I602S, case-control P = 5.7×10−8, OR = 0.31, 95% CI = 0.20–0.48, and HLA-DQA1 rs1071630, case-control P = 4.9×10−14, OR = 0.43, 95% CI = 0.35–0.54). The effect sizes of these associations suggest that TLR1 and HLA-DRB1/DQA1 are major susceptibility genes in susceptibility to leprosy. Further population differentiation analysis shows that the TLR1 locus is extremely differentiated. The protective dysfunctional 602S allele is rare in Africa but expands to become the dominant allele among individuals of European descent. This supports the hypothesis that this locus may be under selection from mycobacteria or other pathogens that are recognized by TLR1 and its co-receptors. These observations provide insight into the long standing host-pathogen relationship between human and mycobacteria and highlight the key role of the TLR pathway in infectious diseases.

CISH and Susceptibility to Infectious Diseases

BACKGROUND The interleukin-2-mediated immune response is critical for host defense against infectious pathogens. Cytokine-inducible SRC homology 2 (SH2) domain protein (CISH), a suppressor of cytokine signaling, controls interleukin-2 signaling. METHODS Using a case-control design, we tested for an association between CISH polymorphisms and susceptibility to major infectious diseases (bacteremia, tuberculosis, and severe malaria) in blood samples from 8402 persons in Gambia, Hong Kong, Kenya, Malawi, and Vietnam. We had previously tested 20 other immune-related genes in one or more of these sample collections. RESULTS We observed associations between variant alleles of multiple CISH polymorphisms and increased susceptibility to each infectious disease in each of the study populations. When all five single-nucleotide polymorphisms (SNPs) (at positions -639, -292, -163, +1320, and +3415 [all relative to CISH]) within the CISH-associated locus were considered together in a multiple-SNP score, we found an association between CISH genetic variants and susceptibility to bacteremia, malaria, and tuberculosis (P=3.8x10(-11) for all comparisons), with -292 accounting for most of the association signal (P=4.58x10(-7)). Peripheral-blood mononuclear cells obtained from adult subjects carrying the -292 variant, as compared with wild-type cells, showed a muted response to the stimulation of interleukin-2 production--that is, 25 to 40% less CISH expression. CONCLUSIONS Variants of CISH are associated with susceptibility to diseases caused by diverse infectious pathogens, suggesting that negative regulators of cytokine signaling have a role in immunity against various infectious diseases. The overall risk of one of these infectious diseases was increased by at least 18% among persons carrying the variant CISH alleles.

Genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study

Summary Background Effective targeted therapy for sepsis requires an understanding of the heterogeneity in the individual host response to infection. We investigated this heterogeneity by defining interindividual variation in the transcriptome of patients with sepsis and related this to outcome and genetic diversity. Methods We assayed peripheral blood leucocyte global gene expression for a prospective discovery cohort of 265 adult patients admitted to UK intensive care units with sepsis due to community-acquired pneumonia and evidence of organ dysfunction. We then validated our findings in a replication cohort consisting of a further 106 patients. We mapped genomic determinants of variation in gene transcription between patients as expression quantitative trait loci (eQTL). Findings We discovered that following admission to intensive care, transcriptomic analysis of peripheral blood leucocytes defines two distinct sepsis response signatures (SRS1 and SRS2). The presence of SRS1 (detected in 108 [41%] patients in discovery cohort) identifies individuals with an immunosuppressed phenotype that included features of endotoxin tolerance, T-cell exhaustion, and downregulation of human leucocyte antigen (HLA) class II. SRS1 was associated with higher 14 day mortality than was SRS2 (discovery cohort hazard ratio (HR) 2·4, 95% CI 1·3–4·5, p=0·005; validation cohort HR 2·8, 95% CI 1·5–5·1, p=0·0007). We found that a predictive set of seven genes enabled the classification of patients as SRS1 or SRS2. We identified cis-acting and trans-acting eQTL for key immune and metabolic response genes and sepsis response networks. Sepsis eQTL were enriched in endotoxin-induced epigenetic marks and modulated the individual host response to sepsis, including effects specific to SRS group. We identified regulatory genetic variants involving key mediators of gene networks implicated in the hypoxic response and the switch to glycolysis that occurs in sepsis, including HIF1α and mTOR, and mediators of endotoxin tolerance, T-cell activation, and viral defence. Interpretation Our integrated genomics approach advances understanding of heterogeneity in sepsis by defining subgroups of patients with different immune response states and prognoses, as well as revealing the role of underlying genetic variation. Our findings provide new insights into the pathogenesis of sepsis and create opportunities for a precision medicine approach to enable targeted therapeutic intervention to improve sepsis outcomes. Funding European Commission, Medical Research Council (UK), and the Wellcome Trust.

Genome-wide association study of survival from sepsis due to pneumonia: an observational cohort study.

Summary Background Sepsis continues to be a major cause of death, disability, and health-care expenditure worldwide. Despite evidence suggesting that host genetics can influence sepsis outcomes, no specific loci have yet been convincingly replicated. The aim of this study was to identify genetic variants that influence sepsis survival. Methods We did a genome-wide association study in three independent cohorts of white adult patients admitted to intensive care units with sepsis, severe sepsis, or septic shock (as defined by the International Consensus Criteria) due to pneumonia or intra-abdominal infection (cohorts 1–3, n=2534 patients). The primary outcome was 28 day survival. Results for the cohort of patients with sepsis due to pneumonia were combined in a meta-analysis of 1553 patients from all three cohorts, of whom 359 died within 28 days of admission to the intensive-care unit. The most significantly associated single nucleotide polymorphisms (SNPs) were genotyped in a further 538 white patients with sepsis due to pneumonia (cohort 4), of whom 106 died. Findings In the genome-wide meta-analysis of three independent pneumonia cohorts (cohorts 1–3), common variants in the FER gene were strongly associated with survival (p=9·7 × 10−8). Further genotyping of the top associated SNP (rs4957796) in the additional cohort (cohort 4) resulted in a combined p value of 5·6 × 10−8 (odds ratio 0·56, 95% CI 0·45–0·69). In a time-to-event analysis, each allele reduced the mortality over 28 days by 44% (hazard ratio for death 0·56, 95% CI 0·45–0·69; likelihood ratio test p=3·4 × 10−9, after adjustment for age and stratification by cohort). Mortality was 9·5% in patients carrying the CC genotype, 15·2% in those carrying the TC genotype, and 25·3% in those carrying the TT genotype. No significant genetic associations were identified when patients with sepsis due to pneumonia and intra-abdominal infection were combined. Interpretation We have identified common variants in the FER gene that associate with a reduced risk of death from sepsis due to pneumonia. The FER gene and associated molecular pathways are potential novel targets for therapy or prevention and candidates for the development of biomarkers for risk stratification. Funding European Commission and the Wellcome Trust.

Two Human MYD88 Variants, S34Y and R98C, Interfere with MyD88-IRAK4-Myddosome Assembly

Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.

IFITM3 and susceptibility to respiratory viral infections in the community

Abstract Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM 1,2, and 3) are viral restriction factors that mediate cellular resistance to several viruses. We have genotyped a possible splice-site altering single-nucleotide polymorphism (rs12252) in the IFITM3 gene in 34 patients with H1N1 influenza and severe pneumonia, and >5000 individuals comprising patients with community-acquired mild lower respiratory tract infection and matched controls of Caucasian ancestry. We found evidence of an association between rs12252 rare allele homozygotes and susceptibility to mild influenza (in patients attending primary care) but could not confirm a previously reported association between this single-nucleotide polymorphism and susceptibility to severe H1N1 infection.

A Naturally Occurring Variant in Human TLR9, P99L, Is Associated with Loss of CpG Oligonucleotide Responsiveness

The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.

MyD88 Adaptor-Like D96N Is a Naturally Occurring Loss-of-Function Variant of TIRAP

Signals elicited by TLRs following the detection of microbes are integrated and diversified by a group of four cytoplasmic adaptor molecules featuring an evolutionarily conserved Toll/IL-1R signaling domain. Single nucleotide polymorphisms (SNPs) in TLRs and their adaptor molecules have been shown to influence susceptibility to a range of infectious and other diseases. The adaptor MyD88 adaptor-like (Mal)/Toll/IL-1R–containing adaptor protein is involved in TLR2 and 4 signal transduction by recruiting another adaptor molecule, MyD88, to the plasma membrane. In this study, we used naturally occurring variants of Mal as tools to study the molecular biology of Mal in more detail in cellular model systems and to thereby identify functionally interesting variants whose corresponding nonsynonymous SNPs might be of further epidemiological interest. Of seven reported variants for Mal, we found Mal D96N associated with reduced NF-κB signaling and cytokine production after overexpression in HEK293 and Huh-7 cells. The D96N mutation prevented Mal from recruiting its signaling partner MyD88 to the plasma membrane and altered posttranslational modification of Mal. These findings led us to investigate the frequency of heterozygosity for the corresponding SNP rs8177400 in a Caucasian case-control study on the etiology of lymphoma, a disease in which TLRs have been implicated. Although rs8177400 did not modify lymphoma risk in general, its frequency of heterozygosity was accurately determined to 0.97%. Our data add rs8177400 (D96N) to the list of functionally important variants of Mal and warrant further research into its immunological, epidemiological, and diagnostic relevance.

Variants in the mannose-binding lectin gene MBL2 do not associate with sepsis susceptibility or survival in a large European cohort

We use a large cohort of immune competent adults to analyze the influence of MBL2 genetic variants on sepsis susceptibility and survival. We find no significant associations with the 4 main functional single nucleotide polymorphisms in MBL2, or any combination of genotypes.

Polymorphism in a lincRNA Associates with a Doubled Risk of Pneumococcal Bacteremia in Kenyan Children

Bacteremia (bacterial bloodstream infection) is a major cause of illness and death in sub-Saharan Africa but little is known about the role of human genetics in susceptibility. We conducted a genome-wide association study of bacteremia susceptibility in more than 5,000 Kenyan children as part of the Wellcome Trust Case Control Consortium 2 (WTCCC2). Both the blood-culture-proven bacteremia case subjects and healthy infants as controls were recruited from Kilifi, on the east coast of Kenya. Streptococcus pneumoniae is the most common cause of bacteremia in Kilifi and was thus the focus of this study. We identified an association between polymorphisms in a long intergenic non-coding RNA (lincRNA) gene (AC011288.2) and pneumococcal bacteremia and replicated the results in the same population (p combined = 1.69 × 10−9; OR = 2.47, 95% CI = 1.84–3.31). The susceptibility allele is African specific, derived rather than ancestral, and occurs at low frequency (2.7% in control subjects and 6.4% in case subjects). Our further studies showed AC011288.2 expression only in neutrophils, a cell type that is known to play a major role in pneumococcal clearance. Identification of this novel association will further focus research on the role of lincRNAs in human infectious disease.