Julian C. Knight

Systematic identification of trans eQTLs as putative drivers of known disease associations

Identifying the downstream effects of disease-associated SNPs is challenging. To help overcome this problem, we performed expression quantitative trait locus (eQTL) meta-analysis in non-transformed peripheral blood samples from 5,311 individuals with replication in 2,775 individuals. We identified and replicated trans eQTLs for 233 SNPs (reflecting 103 independent loci) that were previously associated with complex traits at genome-wide significance. Some of these SNPs affect multiple genes in trans that are known to be altered in individuals with disease: rs4917014, previously associated with systemic lupus erythematosus (SLE), altered gene expression of C1QB and five type I interferon response genes, both hallmarks of SLE. DeepSAGE RNA sequencing showed that rs4917014 strongly alters the 3′ UTR levels of IKZF1 in cis, and chromatin immunoprecipitation and sequencing analysis of the trans-regulated genes implicated IKZF1 as the causal gene. Variants associated with cholesterol metabolism and type 1 diabetes showed similar phenomena, indicating that large-scale eQTL mapping provides insight into the downstream effects of many trait-associated variants.

A ChIP-seq defined genome-wide map of vitamin D receptor binding: Associations with disease and evolution

Initially thought to play a restricted role in calcium homeostasis, the pleiotropic actions of vitamin D in biology and their clinical significance are only now becoming apparent. However, the mode of action of vitamin D, through its cognate nuclear vitamin D receptor (VDR), and its contribution to diverse disorders, remain poorly understood. We determined VDR binding throughout the human genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After calcitriol stimulation, we identified 2776 genomic positions occupied by the VDR and 229 genes with significant changes in expression in response to vitamin D. VDR binding sites were significantly enriched near autoimmune and cancer associated genes identified from genome-wide association (GWA) studies. Notable genes with VDR binding included IRF8, associated with MS, and PTPN2 associated with Crohns disease and T1D. Furthermore, a number of single nucleotide polymorphism associations from GWA were located directly within VDR binding intervals, for example, rs13385731 associated with SLE and rs947474 associated with T1D. We also observed significant enrichment of VDR intervals within regions of positive selection among individuals of Asian and European descent. ChIP-seq determination of transcription factor binding, in combination with GWA data, provides a powerful approach to further understanding the molecular bases of complex diseases.

A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria

Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF (encoding tumour necrosis factor, TNF) promoter region, we have identified a single nucleotide polymorphism (SNP) that causes the helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other known TNF polymorphisms and linked HLA alleles.

Expression of the Multiple Sclerosis-Associated MHC Class II Allele HLA-DRB1*1501 Is Regulated by Vitamin D

Multiple sclerosis (MS) is a complex trait in which allelic variation in the MHC class II region exerts the single strongest effect on genetic risk. Epidemiological data in MS provide strong evidence that environmental factors act at a population level to influence the unusual geographical distribution of this disease. Growing evidence implicates sunlight or vitamin D as a key environmental factor in aetiology. We hypothesised that this environmental candidate might interact with inherited factors and sought responsive regulatory elements in the MHC class II region. Sequence analysis localised a single MHC vitamin D response element (VDRE) to the promoter region of HLA-DRB1. Sequencing of this promoter in greater than 1,000 chromosomes from HLA-DRB1 homozygotes showed absolute conservation of this putative VDRE on HLA-DRB1*15 haplotypes. In contrast, there was striking variation among non–MS-associated haplotypes. Electrophoretic mobility shift assays showed specific recruitment of vitamin D receptor to the VDRE in the HLA-DRB1*15 promoter, confirmed by chromatin immunoprecipitation experiments using lymphoblastoid cells homozygous for HLA-DRB1*15. Transient transfection using a luciferase reporter assay showed a functional role for this VDRE. B cells transiently transfected with the HLA-DRB1*15 gene promoter showed increased expression on stimulation with 1,25-dihydroxyvitamin D3 (P = 0.002) that was lost both on deletion of the VDRE or with the homologous “VDRE” sequence found in non–MS-associated HLA-DRB1 haplotypes. Flow cytometric analysis showed a specific increase in the cell surface expression of HLA-DRB1 upon addition of vitamin D only in HLA-DRB1*15 bearing lymphoblastoid cells. This study further implicates vitamin D as a strong environmental candidate in MS by demonstrating direct functional interaction with the major locus determining genetic susceptibility. These findings support a connection between the main epidemiological and genetic features of this disease with major practical implications for studies of disease mechanism and prevention.

Innate Immune Activity Conditions the Effect of Regulatory Variants upon Monocyte Gene Expression

Introduction Many genetic variants associated with common disease susceptibility occur close to immune-related genes in noncoding DNA, suggestive of a regulatory function. The definition of functional variants and the specific genes that they regulate remains challenging and in many cases is unresolved. We hypothesized that a significant proportion of variants, including those implicated in disease, may show activity in a context-specific manner and therefore only be identifiable upon triggering of immune responses. Context-specific genetic association with differential gene expression in IFN-β signaling. (A) A local association (cis-eQTL) with IFNB1 expression for a single-nucleotide polymorphism (rs2275888) revealed after 2 hours of LPS stimulation of monocytes. (B) This genetic marker shows association with expression of 17 genes on different chromosomes (trans-eQTLs) after 24 hours of LPS stimulation, forming a gene network (C) consistent with the IFN-β signaling cascade. Methods We mapped interindividual variation in gene expression as a quantitative trait, defining expression quantitative trait loci (eQTLs). To investigate the effect of innate immune stimuli on eQTLs, we exposed primary CD14+ human monocytes from 432 European volunteers to the inflammatory proxies interferon-γ (IFN-γ) or differing durations (2 or 24 hours) of lipopolysaccharide (LPS). eQTL mapping was performed on a genome-wide basis with an additive linear model. A subset of 228 individuals with expression data available for all experimental conditions enabled cross-treatment comparisons. Results Stimulation with LPS or IFN-γ resulted in profound effects across monocyte eQTLs, with hundreds of genes and associated pathways demonstrating context-specific eQTLs dependent on the type and duration of stimulus. Context-specific eQTLs frequently intersected established canonical pathways of monocyte signaling and included key nodal genes and effector molecules. These eQTLs are typically more distal to the transcriptional start site and, in some cases, showed reversal of effect between conditions. We also found stimulation reveals novel eQTLs with simultaneous effects involving many genes (trans-eQTLs). Examples included coding polymorphisms in CYP1B1, P2RY11, and IDO2 that modulate activity and develop trans network effects upon stimulation; an LPS-specific IFN-β cytokine network response driven by a cis-eQTL for IFNB1 that was only revealed over time; an interferon regulatory factor 2 (IRF2) transcription factor modulated network up-regulated by IFN-γ involving a cis-eQTL for IRF2; and an IFN-γ–inducible trans gene network involving the transcription factor NFE2L3. We find trans associations to the major histocompatibility complex are dependent on context, paralleling the expression of class II genes. Induced eQTLs were enriched for disease-risk loci with context-specific associations to many putative causal genes, including at ATM, IRF8, and CCR3. Conditional analysis defined additional independent stimulus-specific peaks of association for a given gene. For CARD9 we observed, in addition to a constitutive eQTL informative for a genome-wide association study locus for Crohn’s disease, a stimulus-specific peak eQTL after IFN-γ, defining a further independent signal of disease association. Discussion Interindividual variation in immune responses is accompanied by diverging patterns of gene regulation dependent on underlying genotype. In human monocytes, many regulatory variants display functionality only after pathophysiologically relevant immune stimuli. By considering the cellular and environmental context relevant to disease, it is possible to more extensively resolve functional genetic variants and the specific modulated genes associated with disease. Immune Variation It is difficult to determine the mechanistic consequences of context-dependent genetic variants, some of which may be related to disease (see the Perspective by Gregersen). Two studies now report on the effects of stimulating immunological monocytes and dendritic cells with proteins that can elicit a response to bacterial or viral infection and assess the functional links between genetic variants and profiles of gene expression. M. N. Lee et al. (10.1126/science.1246980) analyzed the expression of more than 400 genes, in dendritic cells from 30 healthy subjects, which revealed how expression quantitative trait loci (eQTLs) affect gene expression within the interferon-β and the Toll-like receptor 3 and 4 pathways. Fairfax et al. (10.1126/science.1246949) performed a genome-wide analysis to show that many eQTLs affected monocyte gene expression in a stimulus- or time-specific manner. Analysis of the transcriptional responses during induced innate immune activity in primary human monocytes is explained. [Also see Perspective by Gregersen] To systematically investigate the impact of immune stimulation upon regulatory variant activity, we exposed primary monocytes from 432 healthy Europeans to interferon-γ (IFN-γ) or differing durations of lipopolysaccharide and mapped expression quantitative trait loci (eQTLs). More than half of cis-eQTLs identified, involving hundreds of genes and associated pathways, are detected specifically in stimulated monocytes. Induced innate immune activity reveals multiple master regulatory trans-eQTLs including the major histocompatibility complex (MHC), coding variants altering enzyme and receptor function, an IFN-β cytokine network showing temporal specificity, and an interferon regulatory factor 2 (IRF2) transcription factor–modulated network. Induced eQTL are significantly enriched for genome-wide association study loci, identifying context-specific associations to putative causal genes including CARD9, ATM, and IRF8. Thus, applying pathophysiologically relevant immune stimuli assists resolution of functional genetic variants.

Genetics of gene expression in primary immune cells identifies cell type–specific master regulators and roles of HLA alleles

Trans-acting genetic variants have a substantial, albeit poorly characterized, role in the heritable determination of gene expression. Using paired purified primary monocytes and B cells, we identify new predominantly cell type–specific cis and trans expression quantitative trait loci (eQTLs), including multi-locus trans associations to LYZ and KLF4 in monocytes and B cells, respectively. Additionally, we observe a B cell–specific trans association of rs11171739 at 12q13.2, a known autoimmune disease locus, with IP6K2 (P = 5.8 × 10−15), PRIC285 (P = 3.0 × 10−10) and an upstream region of CDKN1A (P = 2 × 10−52), suggesting roles for cell cycle regulation and peroxisome proliferator-activated receptor γ (PPARγ) signaling in autoimmune pathogenesis. We also find that specific human leukocyte antigen (HLA) alleles form trans associations with the expression of AOAH and ARHGAP24 in monocytes but not in B cells. In summary, we show that mapping gene expression in defined primary cell populations identifies new cell type–specific trans-regulated networks and provides insights into the genetic basis of disease susceptibility.

In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.

In vivo characterization of regulatory polymorphisms is a key requirement for next-generation human genetic analysis. Here we describe haploChIP, a method that uses chromatin immunoprecipitation (ChIP) and mass spectrometry to identify differential protein–DNA binding in vivo associated with allelic variants of a gene. We demonstrate this approach with the imprinted gene SNRPN. HaploChIP showed close correlation between the level of bound phosphorylated RNA polymerase II at the SNRPN locus and allele-specific expression. Application of the approach to the TNF/LTA locus identified functionally important haplotypes that correlate with allele-specific transcription of LTA. The haploChIP method may be useful in high-throughput screening for common DNA polymorphisms that affect gene regulation in vivo.

Regulatory polymorphisms underlying complex disease traits.

There is growing evidence that genetic variation plays an important role in the determination of individual susceptibility to complex disease traits. In contrast to coding sequence polymorphisms, where the consequences of non-synonymous variation may be resolved at the level of the protein phenotype, defining specific functional regulatory polymorphisms has proved problematic. This has arisen for a number of reasons, including difficulties with fine mapping due to linkage disequilibrium, together with a paucity of experimental tools to resolve the effects of non-coding sequence variation on gene expression. Recent studies have shown that variation in gene expression is heritable and can be mapped as a quantitative trait. Allele-specific effects on gene expression appear relatively common, typically of modest magnitude and context specific. The role of regulatory polymorphisms in determining susceptibility to a number of complex disease traits is discussed, including variation at the VNTR of INS, encoding insulin, in type 1 diabetes and polymorphism of CTLA4, encoding cytotoxic T lymphocyte antigen, in autoimmune disease. Examples where regulatory polymorphisms have been found to play a role in mongenic traits such as factor VII deficiency are discussed, and contrasted with those polymorphisms associated with ischaemic heart disease at the same gene locus. Molecular mechanisms operating in an allele-specific manner at the level of transcription are illustrated, with examples including the role of Duffy binding protein in malaria. The difficulty of resolving specific functional regulatory variants arising from linkage disequilibrium is demonstrated using a number of examples including polymorphism of CCR5, encoding CC chemokine receptor 5, and HIV-1 infection. The importance of understanding haplotypic structure to the design and interpretation of functional assays of putative regulatory variation is highlighted, together with discussion of the strategic use of experimental tools to resolve regulatory polymorphisms at a transcriptional level. A number of examples are discussed including work on the TNF locus which demonstrate biological and experimental context specificity. Regulatory variation may also operate at other levels of control of gene expression and the modulation of splicing at PTPRC, encoding protein tyrosine phosphatase receptor-type C, and of translational efficiency at F12, encoding factor XII, are discussed.

Severe Malarial Anemia and Cerebral Malaria Are Associated with Different Tumor Necrosis Factor Promoter Alleles

Experimental evidence implicates tumor necrosis factor (TNF) in the pathogenesis of malarial anemia, but there are few data relating to this hypothesis. This study found that severely anemic children with Plasmodium falciparum infection have low plasma TNF levels, in contrast to the high levels found in cerebral malaria. A previous case-control study in The Gambia found cerebral malaria, but not severe malarial anemia, was associated with the TNF-308 A allele. This study found that in the same population, severe malarial anemia was associated with the TNF-238 A allele, with an odds ratio of 2.5 (P<.001) after stratification for HLA type. These findings suggest that severe malarial anemia and cerebral malaria are influenced by separate genetic factors situated near the TNF gene.

Major Histocompatibility Complex Genomics and Human Disease

Over several decades, various forms of genomic analysis of the human major histocompatibility complex (MHC) have been extremely successful in picking up many disease associations. This is to be expected, as the MHC region is one of the most gene-dense and polymorphic stretches of human DNA. It also encodes proteins critical to immunity, including several controlling antigen processing and presentation. Single-nucleotide polymorphism genotyping and human leukocyte antigen (HLA) imputation now permit the screening of large sample sets, a technique further facilitated by high-throughput sequencing. These methods promise to yield more precise contributions of MHC variants to disease. However, interpretation of MHC-disease associations in terms of the functions of variants has been problematic. Most studies confirm the paramount importance of class I and class II molecules, which are key to resistance to infection. Infection is likely driving the extreme variation of these genes across the human population, but this has been difficult to demonstrate. In contrast, many associations with autoimmune conditions have been shown to be specific to certain class I and class II alleles. Interestingly, conditions other than infections and autoimmunity are also associated with the MHC, including some cancers and neuropathies. These associations could be indirect, owing, for example, to the infectious history of a particular individual and selective pressures operating at the population level.