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Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach.
BACKGROUND: The master transactivator CIITA is essential to the regulation of Major Histocompatibility Complex (MHC)class II genes and an effective immune response. CIITA is known to modulate a small number of non-MHC genes involved in antigen presentation such as CD74 and B2M but its broader genome-wide function and relationship with underlying genetic diversity has not been resolved. RESULTS: We report the first genome-wide ChIP-seq map for CIITA and complement this by mapping inter-individual variation in CIITA expression as a quantitative trait. We analyse CIITA recruitment for pathophysiologically relevant primary human B cells and monocytes, resting and treated with interferon-gamma, in the context of the epigenomic regulatory landscape and DNA-binding proteins associated with the CIITA enhanceosome including RFX, CREB1/ATF1 and NFY. We confirm recruitment to proximal promoter sequences in MHC class II genes and more distally involving the canonical CIITA enhanceosome. Overall, we map 843 CIITA binding intervals involving 442 genes and find 95% of intervals are located outside the MHC and 60% not associated with RFX5 binding. Binding intervals are enriched for genes involved in immune function n and infectious disease with novel loci including major histone gene clusters. Were solve differentially expressed genes associated in trans with a CIITA intronic sequence variant, integrate with CIITA recruitment and show how this is mediated by allele-specific recruitment of NF-kB. CONCLUSIONS: Our results indicate a broader role for CIITA beyond the MHC involving immune-related genes.We provide new insights into allele-specific regulation of CIITA informative for understanding gene function and disease.
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 x 10(-8), OR = 0.31, 95% CI = 0.20-0.48, and HLA-DQA1 rs1071630, case-control P = 4.9 x 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.
Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE.
Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood Monocytes Dependent on the Ratio of Monocytes: Lymphocytes.
The ratio of monocytes and lymphocytes (ML ratio) in peripheral blood is associated with tuberculosis and malaria disease risk and cancer and cardiovascular disease outcomes. We studied anti-mycobacterial function and the transcriptome of monocytes in relation to the ML ratio. Mycobacterial growth inhibition assays of whole or sorted blood were performed and mycobacteria were enumerated by liquid culture. Transcriptomes of unstimulated CD14 + monocytes isolated by magnetic bead sorting were characterised by microarray. Transcript expression was tested for association with ML ratio calculated from leucocyte differential counts by linear regression. The ML ratio was associated with mycobacterial growth in vitro (β = 2.23, SE 0.91, p = 0.02). Using sorted monocytes and lymphocytes, in vivo ML ratio (% variance explained R(2) = 11%, p = 0.02) dominated over in vitro ratios (R(2) = 5%, p = 0.10) in explaining mycobacterial growth. Expression of 906 genes was associated with the ML ratio and 53 with monocyte count alone. ML-ratio associated genes were enriched for type-I and -II interferon signalling (p = 1.2 × 10(− 8)), and for genes under transcriptional control of IRF1, IRF2, RUNX1, RELA and ESRRB. The ML-ratio-associated gene set was enriched in TB disease (3.11-fold, 95% CI: 2.28-4.19, p = 5.7 × 10(− 12)) and other inflammatory diseases including atopy, HIV, IBD and SLE. The ML ratio is associated with distinct transcriptional and anti-mycobacterial profiles of monocytes that may explain the disease associations of the ML ratio.
Genetics of gene expression in immunity to infection.
Mapping gene expression as a quantitative trait (eQTL mapping) can reveal local and distant associations with functionally important genetic variation informative for disease. Recent studies are reviewed which have demonstrated that this approach is particularly informative when applied to diverse immune cell populations and situations relevant to infection and immunity. Context-specific eQTL have now been characterised following endotoxin activation, induction with interferons, mycobacteria, and influenza, together with genetic determinants of response to vaccination. The application of genetical genomic approaches offers new opportunities to advance our understanding of gene-environment interactions and fundamental processes in innate and adaptive immunity.
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 common haplotype lowers PU.1 expression in myeloid cells and delays onset of Alzheimer's disease.
A genome-wide survival analysis of 14,406 Alzheimer's disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and 14 novel loci associated with age at onset. Linkage disequilibrium score regression of 220 cell types implicated the regulation of myeloid gene expression in AD risk. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability was enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affected the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.
Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization.
Allergen-specific immunoglobulin E (present in allergic sensitization) has a central role in the pathogenesis of allergic disease. We performed the first large-scale genome-wide association study (GWAS) of allergic sensitization in 5,789 affected individuals and 10,056 controls and followed up the top SNP at each of 26 loci in 6,114 affected individuals and 9,920 controls. We increased the number of susceptibility loci with genome-wide significant association with allergic sensitization from three to ten, including SNPs in or near TLR6, C11orf30, STAT6, SLC25A46, HLA-DQB1, IL1RL1, LPP, MYC, IL2 and HLA-B. All the top SNPs were associated with allergic symptoms in an independent study. Risk-associated variants at these ten loci were estimated to account for at least 25% of allergic sensitization and allergic rhinitis. Understanding the molecular mechanisms underlying these associations may provide new insights into the etiology of allergic disease.
Pervasive haplotypic variation in the spliceo-transcriptome of the human major histocompatibility complex.
The human major histocompatibility complex (MHC) on chromosome 6p21 is a paradigm for genomics, showing remarkable polymorphism and striking association with immune and non-immune diseases. The complex genomic landscape of the MHC, notably strong linkage disequilibrium, has made resolving causal variants very challenging. A promising approach is to investigate gene expression levels considered as tractable intermediate phenotypes in mapping complex diseases. However, how transcription varies across the MHC, notably relative to specific haplotypes, remains unknown. Here, using an original hybrid tiling and splice junction microarray that includes alternate allele probes, we draw the first high-resolution strand-specific transcription map for three common MHC haplotypes (HLA-A1-B8-Cw7-DR3, HLA-A3-B7-Cw7-DR15, and HLA-A26-B18-Cw5-DR3-DQ2) strongly associated with autoimmune diseases including type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis. We find that haplotype-specific differences in gene expression are common across the MHC, affecting 96 genes (46.4%), most significantly the zing finger protein gene ZFP57. Differentially expressed probes are correlated with polymorphisms between haplotypes, consistent with cis effects that we directly demonstrate for ZFP57 in a cohort of healthy volunteers (P = 1.2 × 10(-14)). We establish that alternative splicing is significantly more frequent in the MHC than genome-wide (72.5% vs. 62.1% of genes, P ≤ 1 × 10(-4)) and shows marked haplotypic differences. We also unmask novel and abundant intergenic transcription involving 31% of transcribed blocks identified. Our study reveals that the renowned MHC polymorphism also manifests as transcript diversity, and our novel haplotype-based approach marks a new step toward identification of regulatory variants involved in the control of MHC-associated phenotypes and diseases.
Cell Specific eQTL Analysis without Sorting Cells.
The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.
Increased prevalence of sex chromosome aneuploidies in specific language impairment and dyslexia.
AIM: Sex chromosome aneuploidies increase the risk of spoken or written language disorders but individuals with specific language impairment (SLI) or dyslexia do not routinely undergo cytogenetic analysis. We assess the frequency of sex chromosome aneuploidies in individuals with language impairment or dyslexia. METHOD: Genome-wide single nucleotide polymorphism genotyping was performed in three sample sets: a clinical cohort of individuals with speech and language deficits (87 probands: 61 males, 26 females; age range 4 to 23 years), a replication cohort of individuals with SLI, from both clinical and epidemiological samples (209 probands: 139 males, 70 females; age range 4 to 17 years), and a set of individuals with dyslexia (314 probands: 224 males, 90 females; age range 7 to 18 years). RESULTS: In the clinical language-impaired cohort, three abnormal karyotypic results were identified in probands (proband yield 3.4%). In the SLI replication cohort, six abnormalities were identified providing a consistent proband yield (2.9%). In the sample of individuals with dyslexia, two sex chromosome aneuploidies were found giving a lower proband yield of 0.6%. In total, two XYY, four XXY (Klinefelter syndrome), three XXX, one XO (Turner syndrome), and one unresolved karyotype were identified. INTERPRETATION: The frequency of sex chromosome aneuploidies within each of the three cohorts was increased over the expected population frequency (approximately 0.25%) suggesting that genetic testing may prove worthwhile for individuals with language and literacy problems and normal non-verbal IQ. Early detection of these aneuploidies can provide information and direct the appropriate management for individuals.
Fine mapping genetic determinants of the highly variably expressed MHC gene ZFP57.
ZFP57 is an important transcriptional regulator involved in DNA methylation and genomic imprinting during development. Here we demonstrate that gene expression also occurs at a low level in adult peripheral blood cells and other tissues including the kidney and thymus, but is critically dependent on underlying local genetic variation within the MHC. We resolve a highly significant expression quantitative trait locus for ZFP57 involving single-nucleotide polymorphisms (SNPs) in the first intron of the gene co-localizing with a DNase I hypersensitive site and evidence of CTCF recruitment. These data identify ZFP57 as a candidate gene underlying reported MHC disease associations, notably for putative regulatory variants associated with cancer and HIV-1. The work highlights the role that ZFP57 may play in DNA methylation and epigenetic regulation beyond early development into adult life dependent on genetic background, with important potential implications for disease.
Pathogenic implications for autoimmune mechanisms derived by comparative eQTL analysis of CD4+ versus CD8+ T cells.
Inappropriate activation or inadequate regulation of CD4+ and CD8+ T cells may contribute to the initiation and progression of multiple autoimmune and inflammatory diseases. Studies on disease-associated genetic polymorphisms have highlighted the importance of biological context for many regulatory variants, which is particularly relevant in understanding the genetic regulation of the immune system and its cellular phenotypes. Here we show cell type-specific regulation of transcript levels of genes associated with several autoimmune diseases in CD4+ and CD8+ T cells including a trans-acting regulatory locus at chr12q13.2 containing the rs1131017 SNP in the RPS26 gene. Most remarkably, we identify a common missense variant in IL27, associated with type 1 diabetes that results in decreased functional activity of the protein and reduced expression levels of downstream IRF1 and STAT1 in CD4+ T cells only. Altogether, our results indicate that eQTL mapping in purified T cells provides novel functional insights into polymorphisms and pathways associated with autoimmune diseases.
Genetic variants associated with non-typhoidal Salmonella bacteraemia in African children.
BACKGROUND: Non-typhoidal Salmonella (NTS) causes invasive and frequently fatal disease in African children. Existing strategies to prevent, diagnose, and treat NTS disease are inadequate. An improved understanding of the biology of invasive Salmonella infection will facilitate the development of novel NTS control measures. Despite evidence in mice and man showing a clear role for host genetics in NTS susceptibility, there are no published studies investigating host genetic susceptibility to NTS in African populations. METHODS: We conducted a genome-wide association study (SNP Array 6.0, Affymetrix, CA, USA) of NTS bacteraemia in Kenyan children, with replication in Malawian children. We assessed the function of NTS-associated variants in an expression quantitative trait locus (eQTL) dataset of interferon γ (IFNγ) and lipopolysaccharide-stimulated monocytes from 432 healthy European adults. Serum IFNγ (Bio-Plex immunoassay, Bio-Rad Laboratories, CA, USA) in Malawian NTS cases (n=106) during acute disease was correlated with genotype by linear regression. FINDINGS: After whole-genome imputation and quality control, 180 Kenyan cases and 2677 controls were included in an association analysis at 7 951 614 (additive model) and 4 669 537 (genotypic model) loci. After quality control, 143 Malawian cases and 336 controls were included in the replication analysis. An intronic variant in STAT4 was associated (recessive model) with NTS in both Kenyan and Malawian children (Kenya p=5·6 × 10(-9), Malawi p=0·02, combined p=1·4 × 10(-9); odds ratio 7·2, 95% CI 3·8-13·5). The NTS-associated variant was an eQTL for STAT4 expression in IFNγ-stimulated monocytes (p=9·59 × 10(-6)), the NTS risk allele being associated with lower STAT4 expression. In Malawian children with NTS bacteraemia, the same NTS risk allele was associated with lower serum concentrations of IFNγ (p=0·02) at presentation. INTERPRETATION: STAT4 is highly plausible as a susceptibility locus for invasive NTS disease. STAT4 mediates IFNγ release in T cells and natural killer cells in response to interleukin 12 (IL12). Individuals with rare mutations elsewhere in the IL12-IFNγ axis are at risk of disseminated NTS infection. We provide the first evidence, to our knowledge, of a host genetic determinant of NTS disease in African children, and of a STAT4 variant conferring susceptibility to an infectious disease in man. FUNDING: Wellcome Trust.
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.
Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis.
Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A+GM-CSF+ double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.
The GABA(B2) subunit is critical for the trafficking and function of native GABA(B) receptors.
Studies in heterologous systems have demonstrated that heterodimerisation of the two GABA(B) receptor subunits appears to be crucial for the trafficking and signalling of the receptor. Gene targeting of the GABA(B1) gene has demonstrated that the expression of GABA(B1) is essential for GABA(B) receptor function in the central nervous system (CNS). However, the contribution of the GABA(B2) subunit in the formation of native GABA(B) receptors is still unclear, in particular whether other proteins can substitute for this subunit. We have created a transgenic mouse in which the endogenous GABA(B2) gene has been mutated in order to express a C-terminally truncated version of the protein. As a result, the GABA(B1) subunit does not reach the cell surface and concomitantly both pre- and post-synaptic GABA(B) receptor functions are abolished. Taken together with previous gene deletion studies for the GABA(B1) subunit, this suggests that classical GABA(B) function in the brain is exclusively mediated by GABA(B1/2) heteromers.
An integrated expression phenotype mapping approach defines common variants in LEP, ALOX15 and CAPNS1 associated with induction of IL-6.
Interleukin-6 (IL-6) is an important modulator of inflammation and immunity whose dysregulation is associated with a number of disease states. There is evidence of significant heritability in inter-individual variation in IL6 gene expression but the genetic variants responsible for this remain to be defined. We adopted a combined approach of mapping protein and expression quantitative trait loci in peripheral blood mononuclear cells using high-density single-nucleotide polymorphism (SNP) typing for approximately 2000 loci implicated in cardiovascular, metabolic and inflammatory syndromes to show that common SNP markers and haplotypes of LEP (encoding leptin) associate with a 1.7- to 2-fold higher level of lipopolysaccharide (LPS)-induced IL-6 expression. We subsequently demonstrate that basal leptin expression significantly correlates with LPS-induced IL-6 expression and that the same variants at LEP which associate with IL-6 expression are also major determinants of leptin expression in these cells. We find that variation involving two other genomic regions, CAPNS1 (encoding calpain small subunit 1) and ALOX15 (encoding arachidonate 15-lipoxygenase), show significant association with IL-6 expression. Although this may be a subset of all such trans-acting effects, we find that the same ALOX15 variants are associated with induced expression of tumour necrosis factor and IL-1beta consistent with a broader role in acute inflammation for ALOX15. This study provides evidence of novel genetic determinants of IL-6 production with implications for understanding susceptibility to inflammatory disease processes and insight into cross talk between metabolic and inflammatory pathways. It also provides proof of concept for use of an integrated expression phenotype mapping approach.
Fatal case of sorafenib-associated idiosyncratic hepatotoxicity in the adjuvant treatment of a patient with renal cell carcinoma.
BACKGROUND: Sorafenib is an orally available kinase inhibitor with activity at Raf, PDGFβ and VEGF receptors that is licensed for the treatment of advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Current evidence-based post-nephrectomy management of individuals with localized RCC consists of surveillance-based follow up. The SORCE trial is designed to investigate whether treatment with adjuvant sorafenib can reduce recurrence rates in this cohort. CASE PRESENTATION: Here we report an idiosyncratic reaction to sorafenib resulting in fatal hepatotoxicity and associated renal failure in a 62 year-old man treated with sorafenib within the SORCE trial. CONCLUSION: This is the first reported case of sorafenib exposure associated fatal toxicity in the adjuvant setting and highlights the unpredictable adverse effects of novel adjuvant therapies.