Julian Knight group

Research overview

Our research aims to understand how genetic variation impacts genes critical to mounting an appropriate immune response and may contribute to susceptibility to infectious, inflammatory and autoimmune diseases. There is a wide spectrum of genetic variation modulating inter-individual differences in immune response with functional consequences ranging from severe primary immunodeficiency disorders to risk of multifactorial traits involving inflammation and immunity. Our recent discovery that non-coding regulatory variants are major drivers of diversity in the immune response transcriptome identifies an important mechanism for this.

The disease relevance of regulatory variants is highlighted by genome-wide association studies (GWAS) in which the majority of reported associations have been found to involve non-coding variants. To take forward the results of GWAS and translate into potential clinical benefit, we now need to define causal regulatory variants, resolve their mode of action and identify the specific modulated genes and pathways which may be therapeutic targets.

Local and distant associations with gene expression in human monocytes (Science 2014)

Our goal is to leverage recent advances in human genetics to improve understanding of biological process in immune disease pathogenesis, validate drug targets and advance opportunities for precision medicine.

Our work combines bioinformatics with functional genomic approaches studying genetic variants in primary cells in disease relevant contexts and establishing mechanism. This includes analysis of allele-specific gene expression, expression quantitative trait mapping and detailed characterisation of how sequence diversity modulates the epigenetic and genetic control of gene expression. 

We have established translational programmes in the genomics of sepsis and ankylosing spondylitis, together with a programme on genetics-led drug target prioritisation as part of an Innovative Medicines Initiative.  

Resolving heterogeneity in sepsis (Lancet Respir Med 2016)

We aim to promote use of genomics for drug target identification and validation, public engagement with genomics and implementation of genomic medicine in the clinic through education, training and a multidisciplinary team approach.


Podcast 'Genetic Variation in Inflammation and Immunity'

Funding sources

Wellcome Trust, Arthritis Research UK, European Commission, Psoriasis Society, Oxford Biomedical Research Centre

Research area

Genomics of immunity and inflammation


Keywords: Gene expression, regulatory variants, genome editing, MHC, immunity, inflammation, sepsis, transcription, single nucleotide polymorphism, ankylosing spondylitis, eQTL, functional genomics, epigenomics, drug targets


Antcliffe DB, Burnham KL, Al-Beidh F, Santhakumaran S, Brett SJ, Hinds CJ, Ashby D, Knight JC, Gordon AC. 2018. Transcriptomic Signatures in Sepsis and a Differential Response to Steroids: From the VANISH Randomized Trial. Am J Respir Crit Care Med, | Show Abstract | Read more

RATIONALE: There remains uncertainty about the role of corticosteroids in sepsis with clear beneficial effects on shock duration but conflicting survival effects. Two transcriptomic sepsis response signatures (SRS) have been identified. SRS1 is relatively immunosuppressed whilst SRS2 is relatively immunocompetent. OBJECTIVES: We aimed to categorized patients based on SRS endotypes to determine if these profiles influenced response to either norepinephrine or vasopressin, or to corticosteroids in septic shock. METHODS: A post-hoc analysis was performed of a double-blind randomized clinical trial in septic shock (VANISH). Patients were included within 6 hours of onset of shock and were randomized to receive norepinephrine or vasopressin followed by hydrocortisone or placebo. Genome-wide gene expression profiling was performed and SRS endotype was determined using a previously established model using seven discriminant genes. MEASUREMENTS AND MAIN RESULTS: Samples were available from 176 patients, 83 SRS1 and 93 SRS2. There was no significant interaction between SRS group and vasopressor assignment (p=0·50). However, there was an interaction between assignment to hydrocortisone or placebo, and SRS endotype (p=0·02). Hydrocortisone use was associated with increased mortality in those with an SRS2 phenotype (OR 7·9, 95%CI 1·6-39·9). CONCLUSIONS: Transcriptomic profile at onset of septic shock was associated with response to corticosteroids. Those with the immuno-competent SRS2 endotype had significantly higher mortality when given corticosteroids compared to placebo. Clinical trial registration available at www.isrctn.com, ID ISRCTN20769191.

Binks S, Varley J, Lee W, Makuch M, Elliott K, Gelfand JM, Jacob S, Leite MI, Maddison P, Chen M et al. 2018. Distinct HLA associations of LGI1 and CASPR2-antibody diseases. Brain, 141 (8), pp. 2263-2271. | Citations: 3 (Scopus) | Show Abstract | Read more

The recent biochemical distinction between antibodies against leucine-rich, glioma-inactivated-1 (LGI1), contactin-associated protein-2 (CASPR2) and intracellular epitopes of voltage-gated potassium-channels (VGKCs) demands aetiological explanations. Given established associations between human leucocyte antigen (HLA) alleles and adverse drug reactions, and our clinical observation of frequent adverse drugs reactions in patients with LGI1 antibodies, we compared HLA alleles between healthy controls (n = 5553) and 111 Caucasian patients with VGKC-complex autoantibodies. In patients with LGI1 antibodies (n = 68), HLA-DRB1*07:01 was strongly represented [odds ratio = 27.6 (95% confidence interval 12.9-72.2), P = 4.1 × 10-26]. In contrast, patients with CASPR2 antibodies (n = 31) showed over-representation of HLA-DRB1*11:01 [odds ratio = 9.4 (95% confidence interval 4.6-19.3), P = 5.7 × 10-6]. Other allelic associations for patients with LGI1 antibodies reflected linkage, and significant haplotypic associations included HLA-DRB1*07:01-DQA1*02:01-DQB1*02:02, by comparison to DRB1*11:01-DQA1*05:01-DQB1*03:01 in CASPR2-antibody patients. Conditional analysis in LGI1-antibody patients resolved further independent class I and II associations. By comparison, patients with both LGI1 and CASPR2 antibodies (n = 3) carried yet another complement of HLA variants, and patients with intracellular VGKC antibodies (n = 9) lacked significant HLA associations. Within LGI1- or CASPR2-antibody patients, HLA associations did not correlate with clinical features. In silico predictions identified unique CASPR2- and LGI1-derived peptides potentially presented by the respective over-represented HLA molecules. These highly significant HLA associations dichotomize the underlying immunology in patients with LGI1 or CASPR2 antibodies, and inform T cell specificities and cellular interactions at disease initiation.

Osgood JA, Knight JC. 2018. Translating GWAS in rheumatic disease: approaches to establishing mechanism and function for genetic associations with ankylosing spondylitis. Brief Funct Genomics, 17 (5), pp. 308-318. | Show Abstract | Read more

Ankylosing spondylitis (AS) is a highly heritable chronic inflammatory arthritis characterized by osteoproliferation, fusion of affected joints and systemic manifestations. Many disease associations for AS have been reported through genome-wide association studies; however, identifying modulated genes and functional mechanism remains challenging. This review summarizes current genetic associations involving AS and describes strategic approaches for functional follow-up of disease-associated variants. Fine mapping using methods leveraging Bayesian approaches are outlined. Evidence highlighting the importance of context specificity for regulatory variants is reviewed, noting current evidence in AS for the relevant cell and tissue type to conduct such analyses. Technological advances for understanding the regulatory landscape within which functional variants may act are discussed using exemplars. Approaches include defining regulatory elements based on chromatin accessibility, effects of variants on genes at a distance through evidence of physical interactions (chromatin conformation capture), expression quantitative trait loci mapping and single-cell methodologies. Opportunities for mechanistic studies to investigate the function of specific variants, regulatory elements and genes enabled by genome editing using clustered regularly interspaced short palindromic repeats/Cas9 are also described. Further progress in our understanding of the genetics of AS through functional genomic and epigenomic approaches offers new opportunities to understand mechanism and develop innovative treatments.

Berlanga-Taylor AJ, Plant K, Dahl A, Lau E, Hill M, Sims D, Heger A, Emberson J, Armitage J, Clarke R, Knight JC. 2018. Genomic Response to Vitamin D Supplementation in the Setting of a Randomized, Placebo-Controlled Trial. EBioMedicine, 31 pp. 133-142. | Citations: 1 (Web of Science Lite) | Show Abstract | Read more

BACKGROUND: Vitamin D deficiency has been associated with multiple diseases, but the causal relevance and underlying processes are not fully understood. Elucidating the mechanisms of action of drug treatments in humans is challenging, but application of functional genomic approaches in randomized trials may afford an opportunity to systematically assess molecular responses. METHODS: In the Biochemical Efficacy and Safety Trial of Vitamin D (BEST-D), a double-blind, placebo-controlled, dose-finding, randomized clinical trial, 305 community-dwelling individuals aged over 65 years were randomly allocated to treatment with vitamin D3 4000 IU, 2000 IU or placebo daily for 12 months. Genome-wide genotypes at baseline, and transcriptome and plasma levels of cytokines (IFN-γ, IL-10, IL-8, IL-6 and TNF-α) at baseline and after 12 months, were measured. The trial had >90% power to detect 1.2-fold changes in gene expression. FINDINGS: Allocation to vitamin D for 12-months was associated with 2-fold higher plasma levels of 25-hydroxy-vitamin D (25[OH]D, 4000 IU regimen), but had no significant effect on whole-blood gene expression (FDR < 5%) or on plasma levels of cytokines compared with placebo. In pre-specified analysis, rs7041 (intron variant, GC) had a significant effect on circulating levels of 25(OH)D in the low dose, but not in the placebo or high dose vitamin D regimen. A gene expression quantitative trait locus analysis (eQTL) demonstrated evidence of 31,568 cis-eQTLs (unique SNP-probe pairs) among individuals at baseline and 34,254 after supplementation for 12 months (any dose). No significant associations involving vitamin D supplementation response eQTLs were found. INTERPRETATION: We performed a comprehensive functional genomics and molecular analysis of vitamin D supplementation in a randomized, placebo-controlled trial. Although this study was limited to mostly Caucasian individuals aged over 65 years, the results differ from many previous studies and do not support a strong effect of vitamin D on long-term transcriptomic changes in blood or on plasma cytokine levels. The trial demonstrates the feasibility of applying functional genomic and genetic approaches in randomized trials to assess molecular and individual level responses. KEY RESULT: Supplementation with high-dose vitamin D in older people for 12 months in a randomized, placebo-controlled trial had no significant effect on gene expression or on plasma concentrations of selected cytokines. TRIAL REGISTRATION: SRCTN registry (Number 07034656) and the European Clinical Trials Database (EudraCT Number 2011-005763-24).

Gilchrist JJ, Rautanen A, Fairfax BP, Mills TC, Naranbhai V, Trochet H, Pirinen M, Muthumbi E, Mwarumba S, Njuguna P et al. 2018. Risk of nontyphoidal Salmonella bacteraemia in African children is modified by STAT4. Nat Commun, 9 (1), pp. 1014. | Citations: 1 (Scopus) | Show Abstract | Read more

Nontyphoidal Salmonella (NTS) is a major cause of bacteraemia in Africa. The disease typically affects HIV-infected individuals and young children, causing substantial morbidity and mortality. Here we present a genome-wide association study (180 cases, 2677 controls) and replication analysis of NTS bacteraemia in Kenyan and Malawian children. We identify a locus in STAT4, rs13390936, associated with NTS bacteraemia. rs13390936 is a context-specific expression quantitative trait locus for STAT4 RNA expression, and individuals carrying the NTS-risk genotype demonstrate decreased interferon-γ (IFNγ) production in stimulated natural killer cells, and decreased circulating IFNγ concentrations during acute NTS bacteraemia. The NTS-risk allele at rs13390936 is associated with protection against a range of autoimmune diseases. These data implicate interleukin-12-dependent IFNγ-mediated immunity as a determinant of invasive NTS disease in African children, and highlight the shared genetic architecture of infectious and autoimmune disease.

Lee W, Plant K, Humburg P, Knight JC. 2018. AltHapAlignR: improved accuracy of RNA-seq analyses through the use of alternative haplotypes. Bioinformatics, 34 (14), pp. 2401-2408. | Show Abstract | Read more

Motivation: Reliance on mapping to a single reference haplotype currently limits accurate estimation of allele or haplotype-specific expression using RNA-sequencing, notably in highly polymorphic regions such as the major histocompatibility complex. Results: We present AltHapAlignR, a method incorporating alternate reference haplotypes to generate gene- and haplotype-level estimates of transcript abundance for any genomic region where such information is available. We validate using simulated and experimental data to quantify input allelic ratios for major histocompatibility complex haplotypes, demonstrating significantly improved correlation with ground truth estimates of gene counts compared to standard single reference mapping. We apply AltHapAlignR to RNA-seq data from 462 individuals, showing how significant underestimation of expression of the majority of classical human leukocyte antigen genes using conventional mapping can be corrected using AltHapAlignR to allow more accurate quantification of gene expression for individual alleles and haplotypes. Availability and implementation: Source code freely available at https://github.com/jknightlab/AltHapAlignR. Supplementary information: Supplementary data are available at Bioinformatics online.

Sweeney TE, Perumal TM, Henao R, Nichols M, Howrylak JA, Choi AM, Bermejo-Martin JF, Almansa R, Tamayo E, Davenport EE et al. 2018. A community approach to mortality prediction in sepsis via gene expression analysis. Nat Commun, 9 (1), pp. 694. | Citations: 6 (Web of Science Lite) | Show Abstract | Read more

Improved risk stratification and prognosis prediction in sepsis is a critical unmet need. Clinical severity scores and available assays such as blood lactate reflect global illness severity with suboptimal performance, and do not specifically reveal the underlying dysregulation of sepsis. Here, we present prognostic models for 30-day mortality generated independently by three scientific groups by using 12 discovery cohorts containing transcriptomic data collected from primarily community-onset sepsis patients. Predictive performance is validated in five cohorts of community-onset sepsis patients in which the models show summary AUROCs ranging from 0.765-0.89. Similar performance is observed in four cohorts of hospital-acquired sepsis. Combining the new gene-expression-based prognostic models with prior clinical severity scores leads to significant improvement in prediction of 30-day mortality as measured via AUROC and net reclassification improvement index These models provide an opportunity to develop molecular bedside tests that may improve risk stratification and mortality prediction in patients with sepsis.

Vecellio M, Cortes A, Roberts AR, Ellis J, Cohen CJ, Knight JC, Brown MA, Bowness P, Wordsworth BP. 2018. Evidence for a second ankylosing spondylitis-associated RUNX3 regulatory polymorphism. RMD Open, 4 (1), pp. e000628. | Citations: 2 (Scopus) | Show Abstract | Read more

Objectives: To explore the functions of RUNX3 single nucleotide polymorphisms (SNPs) associated with ankylosing spondylitis (AS). Methods: Individual SNP associations were evaluated in 4230 UK cases. Their effects on transcription factor (TF) binding, transcription regulation, chromatin modifications, gene expression and gene interactions were tested by database interrogation, luciferase reporter assays, electrophoretic mobility gel shifts, chromatin immunoprecipitation and real-time PCR. Results: We confirmed the independent association of AS with rs4265380, which was robust (P=4.7×10-6) to conditioning on another nearby AS-associated RUNX3 SNP (rs4648889). A RUNX3 haplotype incorporating both SNPs was strongly associated with AS (OR 6.2; 95% CI 3.1 to 13.2, P=1.4×10-8). In a large UK cohort, rs4265380 is associated with leucocyte counts (including monocytes). RUNX3 expression is lower in AS peripheral blood mononuclear cells than healthy controls (P<0.002), independent of rs4265380 genotype. Enhancer function for this RUNX3 region was suggested by increased luciferase activity (approximately tenfold; P=0.005) for reporter constructs containing rs4265380. In monocytes, there was differential allelic binding of nuclear protein extracts to a 50 bp DNA probe containing rs4265380 that was strongly augmented by lipopolysaccharide activation. TF binding also included the histone modifier p300. There was enrichment for histone modifications associated with active enhancer elements (H3K27Ac and H3K79Me2) that may be allele dependent. Hi-C database interrogation showed chromosome interactions of RUNX3 bait with the nearby RP4-799D16.1 lincRNA. Conclusions: The association of AS with this RUNX3 regulatory region involves at least two SNPs apparently operating in different cell types. Monocytes may be potential therapeutic targets in AS.

Al-Mossawi MH, Chen L, Fang H, Ridley A, de Wit J, Yager N, Hammitzsch A, Pulyakhina I, Fairfax BP, Simone D et al. 2017. Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis. Nat Commun, 8 (1), pp. 1510. | Citations: 8 (Web of Science Lite) | Show Abstract | Read more

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.

Ormondroyd E, Mackley MP, Blair E, Craft J, Knight JC, Taylor JC, Taylor J, Watkins H. 2018. "Not pathogenic until proven otherwise": perspectives of UK clinical genomics professionals toward secondary findings in context of a Genomic Medicine Multidisciplinary Team and the 100,000 Genomes Project. Genet Med, 20 (3), pp. 320-328. | Citations: 3 (Web of Science Lite) | Show Abstract | Read more

PurposeApproaches to secondary findings in genome sequencing (GS) are unresolved. In the United Kingdom, GS is now routinely available through the 100,000 Genomes Project, which offers participants feedback of limited secondary findings.MethodsIn Oxford, a Genomic Medicine Multidisciplinary Team (GM-MDT) governs local access to GS, and reviews findings. Semistructured interviews were conducted with 19 GM-MDT members to explore perspectives on secondary findings.ResultsWhile enthusiastic about GS for diagnosing rare disease, members question the rationale for genome screening largely because of lack of evidence for clinical utility and limited justification for use of resources. Members' views are drawn from diverse experiences; they feel a strong sense of responsibility to act in participants' best interests. The capacity to return limited secondary findings should be enabled, but members favor a cautious approach that is responsive to accumulating evidence. Informed participant choice is considered critical, yet challenging. Discrimination of variants is considered essential, and requiring of specialist input and consensus. Multiple areas requiring enhanced engagement and education are identified, i.e., for patients, the public, and health-care professionals; at present, mainstreaming of genomics may be premature.ConclusionUK experts believe that evidence to inform policy toward secondary findings is lacking, arguing for caution.

Scicluna BP, van Vught LA, Zwinderman AH, Wiewel MA, Davenport EE, Burnham KL, Nürnberg P, Schultz MJ, Horn J, Cremer OL et al. 2017. Classification of patients with sepsis according to blood genomic endotype: a prospective cohort study. Lancet Respir Med, 5 (10), pp. 816-826. | Citations: 19 (Scopus) | Show Abstract | Read more

BACKGROUND: Host responses during sepsis are highly heterogeneous, which hampers the identification of patients at high risk of mortality and their selection for targeted therapies. In this study, we aimed to identify biologically relevant molecular endotypes in patients with sepsis. METHODS: This was a prospective observational cohort study that included consecutive patients admitted for sepsis to two intensive care units (ICUs) in the Netherlands between Jan 1, 2011, and July 20, 2012 (discovery and first validation cohorts) and patients admitted with sepsis due to community-acquired pneumonia to 29 ICUs in the UK (second validation cohort). We generated genome-wide blood gene expression profiles from admission samples and analysed them by unsupervised consensus clustering and machine learning. The primary objective of this study was to establish endotypes for patients with sepsis, and assess the association of these endotypes with clinical traits and survival outcomes. We also established candidate biomarkers for the endotypes to allow identification of patient endotypes in clinical practice. FINDINGS: The discovery cohort had 306 patients, the first validation cohort had 216, and the second validation cohort had 265 patients. Four molecular endotypes for sepsis, designated Mars1-4, were identified in the discovery cohort, and were associated with 28-day mortality (log-rank p=0·022). In the discovery cohort, the worst outcome was found for patients classified as having a Mars1 endotype, and at 28 days, 35 (39%) of 90 people with a Mars1 endotype had died (hazard ratio [HR] vs all other endotypes 1·86 [95% CI 1·21-2·86]; p=0·0045), compared with 23 (22%) of 105 people with a Mars2 endotype (HR 0·64 [0·40-1·04]; p=0·061), 16 (23%) of 71 people with a Mars3 endotype (HR 0·71 [0·41-1·22]; p=0·19), and 13 (33%) of 40 patients with a Mars4 endotype (HR 1·13 [0·63-2·04]; p=0·69). Analysis of the net reclassification improvement using a combined clinical and endotype model significantly improved risk prediction to 0·33 (0·09-0·58; p=0·008). A 140-gene expression signature reliably stratified patients with sepsis to the four endotypes in both the first and second validation cohorts. Only Mars1 was consistently significantly associated with 28-day mortality across the cohorts. To facilitate possible clinical use, a biomarker was derived for each endotype; BPGM and TAP2 reliably identified patients with a Mars1 endotype. INTERPRETATION: This study provides a method for the molecular classification of patients with sepsis to four different endotypes upon ICU admission. Detection of sepsis endotypes might assist in providing personalised patient management and in selection for trials. FUNDING: Center for Translational Molecular Medicine, Netherlands.

Lashin HMS, Nadkarni S, Oggero S, Jones HR, Knight JC, Hinds CJ, Perretti M. 2018. Microvesicle Subsets in Sepsis Due to Community Acquired Pneumonia Compared to Faecal Peritonitis. Shock, 49 (4), pp. 393-401. | Citations: 4 (Scopus) | Show Abstract | Read more

RATIONALE: Microvesicles (MV) act as a nonsoluble means of intercellular communication, with effector roles in disease pathogenesis and potentially as biomarkers. Previously, we reported that neutrophil MV expressing alpha-2-macroglobulin (A2MG) are protective in experimental sepsis and associate with survival in a small cohort of patients with sepsis due to community acquired pneumonia (CAP). OBJECTIVES: To characterize MV profiles in sepsis due to CAP or fecal peritonitis (FP) and determine their relation to outcome. To investigate the effects of novel sepsis treatments (granulocyte-macrophage colony stimulating factor (GM-CSF) and interferon-υ (IFN-γ)) on MV production and functions in vitro. METHODS: Flow cytometry analysis of MV identified the cell of origin and the proportion of A2MG expression in the plasma of patients with sepsis secondary to CAP (n = 60) or FP (n = 40) and compared with healthy volunteers (HV, n = 10). The association between MV subsets and outcome was examined. The ability of GM-CSF and IFN-γ on A2MG MV production from whole blood was examined together with the assessment of their effect on neutrophil and endothelial functions. RESULTS: Circulating cell-derived and A2MG MV were higher in CAP compared with FP and HV. A2MG MV were higher in survivors of CAP, but not in FP. GM-CSF and IFN-γ enhanced A2MG MV production, with these MV eliciting pathogen clearance in vitro. CONCLUSIONS: Plasma MV profiles vary according to the source of infection. A2MG MV are associated with survival in CAP but not FP. We propose specific MV subsets as novel biomarkers in sepsis and potential effector for some of the actions of experimental therapeutic interventions.

Burnham KL, Davenport EE, Radhakrishnan J, Humburg P, Gordon AC, Hutton P, Svoren-Jabalera E, Garrard C, Hill AVS, Hinds CJ, Knight JC. 2017. Shared and Distinct Aspects of the Sepsis Transcriptomic Response to Fecal Peritonitis and Pneumonia. Am J Respir Crit Care Med, 196 (3), pp. 328-339. | Citations: 14 (Web of Science Lite) | Show Abstract | Read more

RATIONALE: Heterogeneity in the septic response has hindered efforts to understand pathophysiology and develop targeted therapies. Source of infection, with different causative organisms and temporal changes, might influence this heterogeneity. OBJECTIVES: To investigate individual and temporal variations in the transcriptomic response to sepsis due to fecal peritonitis, and to compare these with the same parameters in community-acquired pneumonia. METHODS: We performed genome-wide gene expression profiling in peripheral blood leukocytes of adult patients admitted to intensive care with sepsis due to fecal peritonitis (n = 117) or community-acquired pneumonia (n = 126), and of control subjects without sepsis (n = 10). MEASUREMENTS AND MAIN RESULTS: A substantial portion of the transcribed genome (18%) was differentially expressed compared with that of control subjects, independent of source of infection, with eukaryotic initiation factor 2 signaling being the most enriched canonical pathway. We identified two sepsis response signature (SRS) subgroups in fecal peritonitis associated with early mortality (P = 0.01; hazard ratio, 4.78). We defined gene sets predictive of SRS group, and serial sampling demonstrated that subgroup membership is dynamic during intensive care unit admission. We found that SRS is the major predictor of transcriptomic variation; a small number of genes (n = 263) were differentially regulated according to the source of infection, enriched for IFN signaling and antigen presentation. We define temporal changes in gene expression from disease onset involving phagosome formation as well as natural killer cell and IL-3 signaling. CONCLUSIONS: The majority of the sepsis transcriptomic response is independent of the source of infection and includes signatures reflecting immune response state and prognosis. A modest number of genes show evidence of specificity. Our findings highlight opportunities for patient stratification and precision medicine in sepsis.

Roberts AR, Vecellio M, Cortes A, Knight JC, Cohen CJ, Wordsworth BP. 2017. Investigation of a possible extended risk haplotype in the IL23R region associated with ankylosing spondylitis. Genes Immun, 18 (2), pp. 105-108. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

The IL23R region on chromosome 1 exhibits complex associations with ankylosing spondylitis (AS). We used publicly available epigenomic information and historical genetic association data to identify a putative regulatory element (PRE) in the intergenic region between IL23R and IL12RB2, which includes two single-nucleotide polymorphisms (SNPs) independently associated with AS-rs924080 (P=2 × 10-3) and rs11578380 (P=2 × 10-4). In luciferase reporter assays, this PRE showed silencer activity (P<0.001). Haplotype and conditional analysis of 4230 historical AS cases and 9700 controls revealed a possible AS-associated extended haplotype, including the PRE and risk variants at three SNPs (rs11209026, rs11209032 and rs924080), but excluding the rs11578380 risk variant. However, the rs924080 association was absent after conditioning on the primary association with rs11209032, which, in contrast, was robust to conditioning on all other AS-associated SNPs in this region (P<2 × 10-8). The role of this putative silencer on some IL23R extended haplotypes therefore remains unclear.

Ormondroyd E, Mackley MP, Blair E, Craft J, Knight JC, Taylor J, Taylor JC, Wilkie AO, Watkins H. 2017. Insights from early experience of a Rare Disease Genomic Medicine Multidisciplinary Team: a qualitative study. Eur J Hum Genet, 25 (6), pp. 680-686. | Citations: 8 (Web of Science Lite) | Show Abstract | Read more

Whole-exome/whole-genome sequencing (WES/WGS) has the potential to enhance genetic diagnosis of rare disease, and is increasingly becoming part of routine clinical care in mainstream medicine. Effective translation will require ongoing efforts in a number of areas including: selection of appropriate patients, provision of effective consent, pre- and post-test genetic counselling, improving variant interpretation algorithms and practices, and management of secondary findings including those found incidentally and those actively sought. Allied to this is the need for an effective education programme for all members of clinical teams involved in care of patients with rare disease, as well as to maintain public confidence in the use of these technologies. We established a Genomic Medicine Multidisciplinary Team (GM-MDT) in 2014 to build on the experiences of earlier successful research-based WES/WGS studies, to address these needs and to review results including pertinent and secondary findings. Here we report on a qualitative study of decision-making in the GM-MDT combined with analysis of semi-structured interviews with GM-MDT members. Study findings show that members appreciate the clinical and scientific diversity of the GM-MDT and value it for education and oversight. To date, discussions have focussed on case selection including the extent and interpretation of clinical and family history information required to establish likely monogenic aetiology and inheritance model. Achieving a balance between effective use of WES/WGS - prioritising cases in a diverse and highly complex patient population where WES/WGS will be tractable - and meeting the recruitment targets of a large project is considered challenging.

Neville MJ, Lee W, Humburg P, Wong D, Barnardo M, Karpe F, Knight JC. 2017. High resolution HLA haplotyping by imputation for a British population bioresource. Hum Immunol, 78 (3), pp. 242-251. | Citations: 4 (Web of Science Lite) | Show Abstract | Read more

This study aimed to establish the occurrence and frequency of HLA alleles and haplotypes for a healthy British Caucasian population bioresource from Oxfordshire. We present the results of imputation from HLA SNP genotyping data using SNP2HLA for 5553 individuals from Oxford Biobank, defining one- and two-field alleles together with amino acid polymorphisms. We show that this achieves a high level of accuracy with validation using sequence-specific primer amplification PCR. We define six- and eight-locus HLA haplotypes for this population by Bayesian methods implemented using PHASE. We determine patterns of linkage disequilibrium and recombination for these individuals involving classical HLA loci and show how analysis within a haplotype block structure may be more tractable for imputed data. Our findings contribute to knowledge of HLA diversity in healthy populations and further validate future large-scale use of HLA imputation as an informative approach in population bioresources.

Kasela S, Kisand K, Tserel L, Kaleviste E, Remm A, Fischer K, Esko T, Westra H-J, Fairfax BP, Makino S et al. 2017. Pathogenic implications for autoimmune mechanisms derived by comparative eQTL analysis of CD4+ versus CD8+ T cells. PLoS Genet, 13 (3), pp. e1006643. | Citations: 13 (Web of Science Lite) | Show Abstract | Read more

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.

Goh C, Knight JC. 2017. Enhanced understanding of the host-pathogen interaction in sepsis: new opportunities for omic approaches. Lancet Respir Med, 5 (3), pp. 212-223. | Citations: 5 (Scopus) | Show Abstract | Read more

Progress in sepsis research has been severely hampered by a heterogeneous disease phenotype, limiting the interpretation of clinical trials and the development of effective therapeutic interventions. Application of omics-based methodologies is advancing understanding of the dysregulated host immune response to infection in sepsis. However, the frequently elusive nature of the infecting organism in sepsis has limited efforts to understand the effect of disease heterogeneity involving the pathogen. Recent advances in nucleic acid sequencing-based pathogen analysis provide the opportunity for more accurate and comprehensive microbiological diagnosis. In this Review, we explore how better understanding of the host-pathogen interaction can substantially enhance, and in turn benefit from, current and future application of omics-based approaches to understand the host response in sepsis. We illustrate this using recent work accounting for heterogeneity involving the pathogen. We propose that there is a timely opportunity to further resolve sepsis heterogeneity by considering host-pathogen interactions, enabling progress towards a precision medicine approach.

Kumar D, Puan KJ, Andiappan AK, Lee B, Westerlaken GHA, Haase D, Melchiotti R, Li Z, Yusof N, Lum J et al. 2017. A functional SNP associated with atopic dermatitis controls cell type-specific methylation of the VSTM1 gene locus. Genome Med, 9 (1), pp. 18. | Citations: 2 (Web of Science Lite) | Show Abstract | Read more

BACKGROUND: Expression quantitative trait loci (eQTL) databases represent a valuable resource to link disease-associated SNPs to specific candidate genes whose gene expression is significantly modulated by the SNP under investigation. We previously identified signal inhibitory receptor on leukocytes-1 (SIRL-1) as a powerful regulator of human innate immune cell function. While it is constitutively high expressed on neutrophils, on monocytes the SIRL-1 surface expression varies strongly between individuals. The underlying mechanism of regulation, its genetic control as well as potential clinical implications had not been explored yet. METHODS: Whole blood eQTL data of a Chinese cohort was used to identify SNPs regulating the expression of VSTM1, the gene encoding SIRL-1. The genotype effect was validated by flow cytometry (cell surface expression), correlated with electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and bisulfite sequencing (C-methylation) and its functional impact studied the inhibition of reactive oxygen species (ROS). RESULTS: We found a significant association of a single CpG-SNP, rs612529T/C, located in the promoter of VSTM1. Through flow cytometry analysis we confirmed that primarily in the monocytes the protein level of SIRL-1 is strongly associated with genotype of this SNP. In monocytes, the T allele of this SNP facilitates binding of the transcription factors YY1 and PU.1, of which the latter has been recently shown to act as docking site for modifiers of DNA methylation. In line with this notion rs612529T associates with a complete demethylation of the VSTM1 promoter correlating with the allele-specific upregulation of SIRL-1 expression. In monocytes, this upregulation strongly impacts the IgA-induced production of ROS by these cells. Through targeted association analysis we found a significant Meta P value of 1.14 × 10-6 for rs612529 for association to atopic dermatitis (AD). CONCLUSION: Low expression of SIRL-1 on monocytes is associated with an increased risk for the manifestation of an inflammatory skin disease. It thus underlines the role of both the cell subset and this inhibitory immune receptor in maintaining immune homeostasis in the skin. Notably, the genetic regulation is achieved by a single CpG-SNP, which controls the overall methylation state of the promoter gene segment.

Chen L, Al-Mossawi MH, Ridley A, Sekine T, Hammitzsch A, de Wit J, Simone D, Shi H, Penkava F, Kurowska-Stolarska M et al. 2017. miR-10b-5p is a novel Th17 regulator present in Th17 cells from ankylosing spondylitis. Ann Rheum Dis, 76 (3), pp. 620-625. | Citations: 9 (Scopus) | Show Abstract | Read more

OBJECTIVE: To determine the microRNA (miR) signature in ankylosing spondylitis (AS) T helper (Th)17 cells. METHODS: Interleukin (IL)-17A-producing CD4+ T cells from patients with AS and healthy controls were FACS-sorted for miR sequencing and qPCR validation. miR-10b function was determined by miR mimic expression followed by cytokine measurement, transcriptome analysis, qPCR and luciferase assays. RESULTS: AS Th17 cells exhibited a miR signature characterised by upregulation of miR-155-5p, miR-210-3p and miR-10b. miR-10b has not been described previously in Th17 cells and was selected for further characterisation. miR-10b is transiently induced in in vitro differentiated Th17 cells. Transcriptome, qPCR and luciferase assays suggest that MAP3K7 is targeted by miR-10b. Both miR-10b overexpression and MAP3K7 silencing inhibited production of IL-17A by both total CD4 and differentiating Th17 cells. CONCLUSIONS: AS Th17 cells have a specific miR signature and upregulate miR-10b in vitro. Our data suggest that miR-10b is upregulated by proinflammatory cytokines and may act as a feedback loop to suppress IL-17A by targeting MAP3K7. miR-10b is a potential therapeutic candidate to suppress pathogenic Th17 cell function in patients with AS.

Fang H, Knezevic B, Burnham KL, Knight JC. 2016. XGR software for enhanced interpretation of genomic summary data, illustrated by application to immunological traits. Genome Med, 8 (1), pp. 129. | Citations: 6 (European Pubmed Central) | Show Abstract | Read more

BACKGROUND: Biological interpretation of genomic summary data such as those resulting from genome-wide association studies (GWAS) and expression quantitative trait loci (eQTL) studies is one of the major bottlenecks in medical genomics research, calling for efficient and integrative tools to resolve this problem. RESULTS: We introduce eXploring Genomic Relations (XGR), an open source tool designed for enhanced interpretation of genomic summary data enabling downstream knowledge discovery. Targeting users of varying computational skills, XGR utilises prior biological knowledge and relationships in a highly integrated but easily accessible way to make user-input genomic summary datasets more interpretable. We show how by incorporating ontology, annotation, and systems biology network-driven approaches, XGR generates more informative results than conventional analyses. We apply XGR to GWAS and eQTL summary data to explore the genomic landscape of the activated innate immune response and common immunological diseases. We provide genomic evidence for a disease taxonomy supporting the concept of a disease spectrum from autoimmune to autoinflammatory disorders. We also show how XGR can define SNP-modulated gene networks and pathways that are shared and distinct between diseases, how it achieves functional, phenotypic and epigenomic annotations of genes and variants, and how it enables exploring annotation-based relationships between genetic variants. CONCLUSIONS: XGR provides a single integrated solution to enhance interpretation of genomic summary data for downstream biological discovery. XGR is released as both an R package and a web-app, freely available at http://galahad.well.ox.ac.uk/XGR .

Vince N, Li H, Ramsuran V, Naranbhai V, Duh F-M, Fairfax BP, Saleh B, Knight JC, Anderson SK, Carrington M. 2016. HLA-C Level Is Regulated by a Polymorphic Oct1 Binding Site in the HLA-C Promoter Region. Am J Hum Genet, 99 (6), pp. 1353-1358. | Citations: 11 (Web of Science Lite) | Show Abstract | Read more

Differential HLA-C levels influence several human diseases, but the mechanisms responsible are incompletely characterized. Using a validated prediction algorithm, we imputed HLA-C cell surface levels in 228 individuals from the 1000 Genomes dataset. We tested 68,726 SNPs within the MHC for association with HLA-C level. The HLA-C promoter region variant, rs2395471, 800 bp upstream of the transcription start site, gave the most significant association with HLA-C levels (p = 4.2 × 10-66). This imputed expression quantitative trait locus, termed impeQTL, was also shown to associate with HLA-C expression in a genome-wide association study of 273 donors in which HLA-C mRNA expression levels were determined by quantitative PCR (qPCR) (p = 1.8 × 10-20) and in two cohorts where HLA-C cell surface levels were determined directly by flow cytometry (n = 369 combined, p < 10-15). rs2395471 is located in an Oct1 transcription factor consensus binding site motif where the A allele is predicted to have higher affinity for Oct1 than the G allele. Mobility shift electrophoresis demonstrated that Oct1 binds to both alleles in vitro, but decreased HLA-C promoter activity was observed in a luciferase reporter assay for rs2395471_G relative to rs2395471_A on a fixed promoter background. The rs2395471 variant accounts for up to 36% of the explained variation of HLA-C level. These data strengthen our understanding of HLA-C transcriptional regulation and provide a basis for understanding the potential consequences of manipulating HLA-C levels therapeutically.

Hore V, Viñuela A, Buil A, Knight J, McCarthy MI, Small K, Marchini J. 2016. Tensor decomposition for multiple-tissue gene expression experiments. Nat Genet, 48 (9), pp. 1094-1100. | Citations: 22 (Scopus) | Show Abstract | Read more

Genome-wide association studies of gene expression traits and other cellular phenotypes have successfully identified links between genetic variation and biological processes. The majority of discoveries have uncovered cis-expression quantitative trait locus (eQTL) effects via mass univariate testing of SNPs against gene expression in single tissues. Here we present a Bayesian method for multiple-tissue experiments focusing on uncovering gene networks linked to genetic variation. Our method decomposes the 3D array (or tensor) of gene expression measurements into a set of latent components. We identify sparse gene networks that can then be tested for association against genetic variation across the genome. We apply our method to a data set of 845 individuals from the TwinsUK cohort with gene expression measured via RNA-seq analysis in adipose, lymphoblastoid cell lines (LCLs) and skin. We uncover several gene networks with a genetic basis and clear biological and statistical significance. Extensions of this approach will allow integration of different omics, environmental and phenotypic data sets.

Humburg P, Maugeri N, Lee W, Mohr B, Knight JC. 2016. Characterisation of the global transcriptional response to heat shock and the impact of individual genetic variation. Genome Med, 8 (1), pp. 87. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

BACKGROUND: The heat shock transcriptional response is essential to effective cellular function under stress. This is a highly heritable trait but the nature and extent of inter-individual variation in heat shock response remains unresolved. METHODS: We determined global transcription profiles of the heat shock response for a panel of lymphoblastoid cell lines established from 60 founder individuals in the Yoruba HapMap population. We explore the observed differentially expressed gene sets following heat shock, establishing functional annotations, underlying networks and nodal genes involving heat shock factor 1 recruitment. We define a multivariate phenotype for the global transcriptional response to heat shock using partial least squares regression and map this quantitative trait to associated genetic variation in search of the major genomic modulators. RESULTS: A comprehensive dataset of differentially expressed genes following heat shock in humans is presented. We identify nodal genes downstream of heat shock factor 1 in this gene set, notably involving ubiquitin C and small ubiquitin-like modifiers together with transcription factors. We dissect a multivariate phenotype for the global heat shock response which reveals distinct clustering of individuals in terms of variance of the heat shock response and involves differential expression of genes involved in DNA replication and cell division in some individuals. We find evidence of genetic associations for this multivariate response phenotype that involves trans effects modulating expression of genes following heat shock, including HSF1 and UBQLN1. CONCLUSION: This study defines gene expression following heat shock for a cohort of individuals, establishing insights into the biology of the heat shock response and hypotheses for how variation in this may be modulated by underlying genetic diversity.

Vecellio M, Roberts AR, Cohen CJ, Cortes A, Knight JC, Bowness P, Wordsworth BP. 2016. The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression. Ann Rheum Dis, 75 (8), pp. 1534-1540. | Citations: 10 (Web of Science Lite) | Show Abstract | Read more

OBJECTIVES: To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). METHODS: We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. RESULTS: The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10(-14)) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 'G' allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk 'A' allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). CONCLUSION: We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS.

Dhalla F, Fox H, Davenport EE, Sadler R, Anzilotti C, van Schouwenburg PA, Ferry B, Chapel H, Knight JC, Patel SY. 2016. Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility. Clin Exp Immunol, 184 (2), pp. 216-227. | Citations: 14 (Web of Science Lite) | Show Abstract | Read more

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)-17 and IL-22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex-vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in-vitro techniques, such as intracellular cytokine staining and enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6⁺ CXCR3⁻ CCR4⁺ CD161⁺ T helper cells generates results that correlate with intracellular cytokine staining for IL-17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6⁺ CXCR3⁻ CD4⁺ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

Davenport EE, Burnham KL, Radhakrishnan J, Humburg P, Hutton P, Mills TC, Rautanen A, Gordon AC, Garrard C, Hill AVS et al. 2016. Genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study. Lancet Respir Med, 4 (4), pp. 259-271. | Citations: 97 (Scopus) | Show Abstract | Read more

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.

Roberts AR, Vecellio M, Chen L, Ridley A, Cortes A, Knight JC, Bowness P, Cohen CJ, Wordsworth BP. 2016. An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation. Ann Rheum Dis, 75 (12), pp. 2150-2156. | Citations: 14 (Scopus) | Show Abstract | Read more

OBJECTIVES: To explore the functional basis for the association between ankylosing spondylitis (AS) and single-nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region. METHODS: We performed conditional analysis on genetic association data and used epigenetic data on chromatin remodelling and transcription factor (TF) binding to identify the primary AS-associated IL23R-IL12RB2 intergenic SNP. Functional effects were tested in luciferase reporter assays in HEK293T cells and allele-specific TF binding was investigated by electrophoretic mobility gel shift assays. IL23R and IL12RB2 mRNA levels in CD4+ T cells were compared between cases homozygous for the AS-risk 'A' allele and the protective 'G' allele. The proportions of interleukin (IL)-17A+ and interferon (IFN)-γ+ CD4+ T-cells were measured by fluorescence-activated cell sorting and compared between these AS-risk and protective genotypes. RESULTS: Conditional analysis identified rs11209032 as the probable causal SNP within a 1.14 kb putative enhancer between IL23R and IL12RB2. Reduced luciferase activity was seen for the risk allele (p<0.001) and reduced H3K4me1 methylation observed in CD4+ T-cells from 'A/A' homozygotes (p=0.02). The binding of nuclear extract to the risk allele was decreased ∼3.5-fold compared with the protective allele (p<0.001). The proportion of IFN-γ+ CD4+ T-cells was increased in 'A/A' homozygotes (p=0.004), but neither IL23R nor IL12RB2 mRNA was affected. CONCLUSIONS: The rs11209032 SNP downstream of IL23R forms part of an enhancer, allelic variation of which may influence Th1-cell numbers. Homozygosity for the risk 'A' allele is associated with more IFN-γ-secreting (Th1) cells. Further work is necessary to explain the mechanisms for these important observations.

Bentham J, Morris DL, Graham DSC, Pinder CL, Tombleson P, Behrens TW, Martín J, Fairfax BP, Knight JC, Chen L et al. 2015. Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus. Nat Genet, 47 (12), pp. 1457-1464. | Citations: 138 (Scopus) | Show Abstract | Read more

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.

Owen HC, Torrance H, Barnes MR, Brohi K, Knight JC, Hinds CJ, O'Dwyer MJ. 2015. The Role of Micrornas in The Development of Hospital Acquired Infection in Polytrauma Patients. Intensive Care Med Exp, 3 (Suppl 1), pp. A35. | Read more

Naranbhai V, Fletcher HA, Tanner R, O'Shea MK, McShane H, Fairfax BP, Knight JC, Hill AVS. 2015. Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood Monocytes Dependent on the Ratio of Monocytes: Lymphocytes. EBioMedicine, 2 (11), pp. 1619-1626. | Citations: 11 (European Pubmed Central) | Show Abstract | Read more

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 R2=11%, p=0.02) dominated over in vitro ratios (R2=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.

Holt RJ, Vandiedonck C, Willis-Owen SA, Knight JC, Cookson WO, Moffatt MF, Zhang Y. 2015. A functional AT/G polymorphism in the 5′-untranslated region of SETDB2 in the IgE locus on human chromosome 13q14 Genes and Immunity, 16 (7), pp. 488-494. | Citations: 2 (Scopus) | Show Abstract | Read more

© 2015 Macmillan Publishers Limited All rights reserved. The immunoglobulin E (IgE)-associated locus on human chromosome 13q14 influencing asthma-related traits contains the genes PHF11 and SETDB2. SETDB2 is located in the same linkage disequilibrium region as PHF11 and polymorphisms within SETDB2 have been shown to associate with total serum IgE levels. In this report, we sequenced the 15 exons of SETDB2 and identified a single previously ungenotyped mutation (AT/G, rs386770867) in the 5′-untranslated region of the gene. The polymorphism was found to be significantly associated with serum IgE levels in our asthma cohort (P=0.0012). Electrophoretic mobility shift assays revealed that the transcription factor Ying Yang 1 binds to the AT allele, whereas SRY (Sex determining Region Y) binds to the G allele. Allele-specific transcription analysis (allelotyping) was performed in 35 individuals heterozygous for rs386770867 from a panel of 200 British families ascertained through probands with severe stage 3 asthma. The AT allele was found to be significantly overexpressed in these individuals (P=1.26 × 10-21). A dual-luciferase assay with the pGL3 luciferase reporter gene showed that the AT allele significantly affects transcriptional activities. Our results indicate that the IgE-associated AT/G polymorphism (rs386770867) regulates transcription of SETDB2.

Holt RJ, Vandiedonck C, Willis-Owen SA, Knight JC, Cookson WO, Moffatt MF, Zhang Y. 2015. A functional AT/G polymorphism in the 5'-untranslated region of SETDB2 in the IgE locus on human chromosome 13q14. Genes Immun, 16 (7), pp. 488-494. | Citations: 5 (Web of Science Lite) | Show Abstract | Read more

The immunoglobulin E (IgE)-associated locus on human chromosome 13q14 influencing asthma-related traits contains the genes PHF11 and SETDB2. SETDB2 is located in the same linkage disequilibrium region as PHF11 and polymorphisms within SETDB2 have been shown to associate with total serum IgE levels. In this report, we sequenced the 15 exons of SETDB2 and identified a single previously ungenotyped mutation (AT/G, rs386770867) in the 5'-untranslated region of the gene. The polymorphism was found to be significantly associated with serum IgE levels in our asthma cohort (P=0.0012). Electrophoretic mobility shift assays revealed that the transcription factor Ying Yang 1 binds to the AT allele, whereas SRY (Sex determining Region Y) binds to the G allele. Allele-specific transcription analysis (allelotyping) was performed in 35 individuals heterozygous for rs386770867 from a panel of 200 British families ascertained through probands with severe stage 3 asthma. The AT allele was found to be significantly overexpressed in these individuals (P=1.26×10(-21)). A dual-luciferase assay with the pGL3 luciferase reporter gene showed that the AT allele significantly affects transcriptional activities. Our results indicate that the IgE-associated AT/G polymorphism (rs386770867) regulates transcription of SETDB2.

Naranbhai V, Fairfax BP, Makino S, Humburg P, Wong D, Ng E, Hill AVS, Knight JC. 2015. Genomic modulators of gene expression in human neutrophils. Nat Commun, 6 (1), pp. 7545. | Citations: 33 (Scopus) | Show Abstract | Read more

Neutrophils form the most abundant leukocyte subset and are central to many disease processes. Technical challenges in transcriptomic profiling have prohibited genomic approaches to date. Here we map expression quantitative trait loci (eQTL) in peripheral blood CD16+ neutrophils from 101 healthy European adults. We identify cis-eQTL for 3281 neutrophil-expressed genes including many implicated in neutrophil function, with 450 of these not previously observed in myeloid or lymphoid cells. Paired comparison with monocyte eQTL demonstrates nuanced conditioning of genetic regulation of gene expression by cellular context, which relates to cell-type-specific DNA methylation and histone modifications. Neutrophil eQTL are markedly enriched for trait-associated variants particularly autoimmune, allergy and infectious disease. We further demonstrate how eQTL in PADI4 and NOD2 delineate risk variant function in rheumatoid arthritis, leprosy and Crohn's disease. Taken together, these data help advance understanding of the genetics of gene expression, neutrophil biology and immune-related diseases.

van Schouwenburg PA, Davenport EE, Kienzler A-K, Marwah I, Wright B, Lucas M, Malinauskas T, Martin HC, WGS500 Consortium, Lockstone HE et al. 2015. Application of whole genome and RNA sequencing to investigate the genomic landscape of common variable immunodeficiency disorders. Clin Immunol, 160 (2), pp. 301-314. | Citations: 26 (Web of Science Lite) | Show Abstract | Read more

Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways.

Naranbhai V, Fletcher HA, Tanner R, O'Shea MK, McShane H, Fairfax BP, Knight JC, Hill AVS. 2015. Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood Monocytes Dependent on the Ratio of Monocytes: Lymphocytes. EBioMedicine, 2 (11), pp. 1619-1626. | Citations: 16 (Scopus) | Show Abstract | Read more

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.

Taylor JC, Martin HC, Lise S, Broxholme J, Cazier J-B, Rimmer A, Kanapin A, Lunter G, Fiddy S, Allan C et al. 2015. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nat Genet, 47 (7), pp. 717-726. | Citations: 101 (Scopus) | Show Abstract | Read more

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

Westra H-J, Arends D, Esko T, Peters MJ, Schurmann C, Schramm K, Kettunen J, Yaghootkar H, Fairfax BP, Andiappan AK et al. 2015. Cell Specific eQTL Analysis without Sorting Cells. PLoS Genet, 11 (5), pp. e1005223. | Citations: 21 (Web of Science Lite) | Show Abstract | Read more

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.

Edwards AM, Arrowsmith CH, Bountra C, Bunnage ME, Feldmann M, Knight JC, Patel DD, Prinos P, Taylor MD, Sundström M, SGC Open Source Target-Discovery Partnership. 2015. Preclinical target validation using patient-derived cells. Nat Rev Drug Discov, 14 (3), pp. 149-150. | Citations: 25 (Scopus) | Show Abstract | Read more

The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.

Gilchrist JJ, Mills TC, Naranbhai V, Chapman SJ, Fairfax BP, Knight JC, Williams TN, Scott JAG, MacLennan CA, Rautanen A et al. 2015. Genetic variants associated with non-typhoidal Salmonella bacteraemia in African children. Lancet, 385 Suppl 1 pp. S13. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

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.

Simpson NH, Ceroni F, Reader RH, Covill LE, Knight JC, SLI Consortium, Hennessy ER, Bolton PF, Conti-Ramsden G, O'Hare A et al. 2015. Genome-wide analysis identifies a role for common copy number variants in specific language impairment. Eur J Hum Genet, 23 (10), pp. 1370-1377. | Citations: 16 (Scopus) | Show Abstract | Read more

An exploratory genome-wide copy number variant (CNV) study was performed in 127 independent cases with specific language impairment (SLI), their first-degree relatives (385 individuals) and 269 population controls. Language-impaired cases showed an increased CNV burden in terms of the average number of events (11.28 vs 10.01, empirical P=0.003), the total length of CNVs (717 vs 513 Kb, empirical P=0.0001), the average CNV size (63.75 vs 51.6 Kb, empirical P=0.0005) and the number of genes spanned (14.29 vs 10.34, empirical P=0.0007) when compared with population controls, suggesting that CNVs may contribute to SLI risk. A similar trend was observed in first-degree relatives regardless of affection status. The increased burden found in our study was not driven by large or de novo events, which have been described as causative in other neurodevelopmental disorders. Nevertheless, de novo CNVs might be important on a case-by-case basis, as indicated by identification of events affecting relevant genes, such as ACTR2 and CSNK1A1, and small events within known micro-deletion/-duplication syndrome regions, such as chr8p23.1. Pathway analysis of the genes present within the CNVs of the independent cases identified significant overrepresentation of acetylcholine binding, cyclic-nucleotide phosphodiesterase activity and MHC proteins as compared with controls. Taken together, our data suggest that the majority of the risk conferred by CNVs in SLI is via common, inherited events within a 'common disorder-common variant' model. Therefore the risk conferred by CNVs will depend upon the combination of events inherited (both CNVs and SNPs), the genetic background of the individual and the environmental factors.

Rautanen A, Mills TC, Gordon AC, Hutton P, Steffens M, Nuamah R, Chiche J-D, Parks T, Chapman SJ, Davenport EE et al. 2015. Genome-wide association study of survival from sepsis due to pneumonia: an observational cohort study. Lancet Respir Med, 3 (1), pp. 53-60. | Citations: 52 (Web of Science Lite) | Show Abstract | Read more

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.

Davenport EE, Antrobus RD, Lillie PJ, Gilbert S, Knight JC. 2015. Transcriptomic profiling facilitates classification of response to influenza challenge. J Mol Med (Berl), 93 (1), pp. 105-114. | Citations: 13 (Web of Science Lite) | Show Abstract | Read more

UNLABELLED: Despite increases in vaccination coverage, reductions in influenza-related mortality have not been observed. Better vaccines are therefore required and influenza challenge studies can be used to test the efficacy of new vaccines. However, this requires the accurate post-challenge classification of subjects by outcome, which is limited in current methods that use artificial thresholds to assign 'symptomatic' and 'asymptomatic' phenotypes. We present data from an influenza challenge study in which 22 healthy adults (11 vaccinated) were inoculated with H3N2 influenza (A/Wisconsin/67/2005). We generated genome-wide gene expression data from peripheral blood taken immediately before the challenge and at 12, 24 and 48 h post-challenge. Variation in symptomatic scoring was found amongst those with laboratory confirmed influenza. By combining the dynamic transcriptomic data with the clinical parameters this variability can be reduced. We identified four subjects with severe laboratory confirmed influenza that show differential gene expression in 1103 probes 48 h post-challenge compared to the remaining subjects. We have further reduced this profile to six genes (CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3) that can be used to define these subjects. We have used this gene set to predict symptomatic infection from an independent study. This analysis gives further insight into host-pathogen interactions during influenza infection. However, the major potential value is in the clinical trial setting by providing a more quantitative method to better classify symptomatic individuals post influenza challenge. KEY MESSAGE: Differential gene expression signatures are seen following influenza challenge. Expression of six predictive genes can classify response to influenza challenge. The genomic influenza response classification replicates in an independent dataset.

Knight JC. 2014. Approaches for establishing the function of regulatory genetic variants involved in disease. Genome Med, 6 (10), pp. 92. | Citations: 21 (Scopus) | Show Abstract | Read more

The diversity of regulatory genetic variants and their mechanisms of action reflect the complexity and context-specificity of gene regulation. Regulatory variants are important in human disease and defining such variants and establishing mechanism is crucial to the interpretation of disease-association studies. This review describes approaches for identifying and functionally characterizing regulatory variants, illustrated using examples from common diseases. Insights from recent advances in resolving the functional epigenomic regulatory landscape in which variants act are highlighted, showing how this has enabled functional annotation of variants and the generation of hypotheses about mechanism of action. The utility of quantitative trait mapping at the transcript, protein and metabolite level to define association of specific genes with particular variants and further inform disease associations are reviewed. Establishing mechanism of action is an essential step in resolving functional regulatory variants, and this review describes how this is being facilitated by new methods for analyzing allele-specific expression, mapping chromatin interactions and advances in genome editing. Finally, integrative approaches are discussed together with examples highlighting how defining the mechanism of action of regulatory variants and identifying specific modulated genes can maximize the translational utility of genome-wide association studies to understand the pathogenesis of diseases and discover new drug targets or opportunities to repurpose existing drugs to treat them.

Wong D, Lee W, Humburg P, Makino S, Lau E, Naranbhai V, Fairfax BP, Chan K, Plant K, Knight JC. 2014. Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach. Genome Biol, 15 (10), pp. 494. | Citations: 12 (Scopus) | Show Abstract | Read more

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.

Fairfax BP, Knight JC. 2014. Genetics of gene expression in immunity to infection. Curr Opin Immunol, 30 (1), pp. 63-71. | Citations: 26 (Scopus) | Show Abstract | Read more

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.

Petousi N, Copley RR, Lappin TRJ, Haggan SE, Bento CM, Cario H, Percy MJ, WGS Consortium, Ratcliffe PJ, Robbins PA, McMullin MF. 2014. Erythrocytosis associated with a novel missense mutation in the BPGM gene. Haematologica, 99 (10), pp. e201-e204. | Citations: 6 (Scopus) | Read more

Berlanga-Taylor AJ, Knight JC. 2014. An integrated approach to defining genetic and environmental determinants for major clinical outcomes involving vitamin D Molecular Diagnosis and Therapy, 18 (3), pp. 261-272. | Citations: 8 (Scopus) | Show Abstract | Read more

There is substantial genetic and epidemiological evidence implicating vitamin D in the pathogenesis of many common diseases. A number of studies have sought to define an association for disease with sequence variation in the VDR gene, encoding the ligand-activated nuclear hormone receptor for vitamin D. The results of such studies have been difficult to replicate and are likely to need to account for specific environmental exposures. Here, we review recent work that has begun to study the interactions between VDR gene polymorphisms, vitamin D blood levels, and complex disease susceptibility, notably in the context of major clinical outcomes. We highlight the challenges moving forward in this area and its importance for effective clinical translation of current research. © 2014 The Author(s).

Cazier J-B, Rao SR, McLean CM, Walker AK, Wright BJ, Jaeger EEM, Kartsonaki C, Marsden L, Yau C, Camps C et al. 2014. Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden. Nat Commun, 5 (1), pp. 3756. | Citations: 49 (Web of Science Lite) | Show Abstract | Read more

Bladder cancers are a leading cause of death from malignancy. Molecular markers might predict disease progression and behaviour more accurately than the available prognostic factors. Here we use whole-genome sequencing to identify somatic mutations and chromosomal changes in 14 bladder cancers of different grades and stages. As well as detecting the known bladder cancer driver mutations, we report the identification of recurrent protein-inactivating mutations in CDKN1A and FAT1. The former are not mutually exclusive with TP53 mutations or MDM2 amplification, showing that CDKN1A dysfunction is not simply an alternative mechanism for p53 pathway inactivation. We find strong positive associations between higher tumour stage/grade and greater clonal diversity, the number of somatic mutations and the burden of copy number changes. In principle, the identification of sub-clones with greater diversity and/or mutation burden within early-stage or low-grade tumours could identify lesions with a high risk of invasive progression.

Fairfax BP, Humburg P, Makino S, Naranbhai V, Wong D, Lau E, Jostins L, Plant K, Andrews R, McGee C, Knight JC. 2014. Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression. Science, 343 (6175), pp. 1246949. | Citations: 260 (Scopus) | Show Abstract | Read more

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.

Nudel R, Simpson NH, Baird G, O'Hare A, Conti-Ramsden G, Bolton PF, Hennessy ER, SLI Consortium, Monaco AP, Knight JC et al. 2014. Associations of HLA alleles with specific language impairment. J Neurodev Disord, 6 (1), pp. 1. | Citations: 15 (European Pubmed Central) | Show Abstract | Read more

BACKGROUND: Human leukocyte antigen (HLA) loci have been implicated in several neurodevelopmental disorders in which language is affected. However, to date, no studies have investigated the possible involvement of HLA loci in specific language impairment (SLI), a disorder that is defined primarily upon unexpected language impairment. We report association analyses of single-nucleotide polymorphisms (SNPs) and HLA types in a cohort of individuals affected by language impairment. METHODS: We perform quantitative association analyses of three linguistic measures and case-control association analyses using both SNP data and imputed HLA types. RESULTS: Quantitative association analyses of imputed HLA types suggested a role for the HLA-A locus in susceptibility to SLI. HLA-A A1 was associated with a measure of short-term memory (P = 0.004) and A3 with expressive language ability (P = 0.006). Parent-of-origin effects were found between HLA-B B8 and HLA-DQA1*0501 and receptive language. These alleles have a negative correlation with receptive language ability when inherited from the mother (P = 0.021, P = 0.034, respectively) but are positively correlated with the same trait when paternally inherited (P = 0.013, P = 0.029, respectively). Finally, case control analyses using imputed HLA types indicated that the DR10 allele of HLA-DRB1 was more frequent in individuals with SLI than population controls (P = 0.004, relative risk = 2.575), as has been reported for individuals with attention deficit hyperactivity disorder (ADHD). CONCLUSION: These preliminary data provide an intriguing link to those described by previous studies of other neurodevelopmental disorders and suggest a possible role for HLA loci in language disorders.

Plant K, Fairfax BP, Makino S, Vandiedonck C, Radhakrishnan J, Knight JC. 2014. Fine mapping genetic determinants of the highly variably expressed MHC gene ZFP57. Eur J Hum Genet, 22 (4), pp. 568-571. | Citations: 6 (European Pubmed Central) | Show Abstract | Read more

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.

Simpson NH, Addis L, Brandler WM, Slonims V, Clark A, Watson J, Scerri TS, Hennessy ER, Bolton PF, Conti-Ramsden G et al. 2014. Increased prevalence of sex chromosome aneuploidies in specific language impairment and dyslexia. Dev Med Child Neurol, 56 (4), pp. 346-353. | Citations: 13 (Scopus) | Show Abstract | Read more

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.

Westra H-J, Peters MJ, Esko T, Yaghootkar H, Schurmann C, Kettunen J, Christiansen MW, Fairfax BP, Schramm K, Powell JE et al. 2013. Systematic identification of trans eQTLs as putative drivers of known disease associations. Nat Genet, 45 (10), pp. 1238-1243. | Citations: 678 (Web of Science Lite) | Show Abstract | Read more

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.

Trowsdale J, Knight JC. 2013. Major histocompatibility complex genomics and human disease. Annu Rev Genomics Hum Genet, 14 (1), pp. 301-323. | Citations: 159 (Scopus) | Show Abstract | Read more

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.

Bønnelykke K, Matheson MC, Pers TH, Granell R, Strachan DP, Alves AC, Linneberg A, Curtin JA, Warrington NM, Standl M et al. 2013. Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization. Nat Genet, 45 (8), pp. 902-906. | Citations: 122 (Web of Science Lite) | Show Abstract | Read more

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.

Guo Y, Lanktree MB, Taylor KC, Hakonarson H, Lange LA, Keating BJ, IBC 50K SNP array BMI Consortium. 2013. Gene-centric meta-analyses of 108 912 individuals confirm known body mass index loci and reveal three novel signals. Hum Mol Genet, 22 (1), pp. 184-201. | Citations: 52 (Scopus) | Show Abstract | Read more

Recent genetic association studies have made progress in uncovering components of the genetic architecture of the body mass index (BMI). We used the ITMAT-Broad-Candidate Gene Association Resource (CARe) (IBC) array comprising up to 49 320 single nucleotide polymorphisms (SNPs) across ~2100 metabolic and cardiovascular-related loci to genotype up to 108 912 individuals of European ancestry (EA), African-Americans, Hispanics and East Asians, from 46 studies, to provide additional insight into SNPs underpinning BMI. We used a five-phase study design: Phase I focused on meta-analysis of EA studies providing individual level genotype data; Phase II performed a replication of cohorts providing summary level EA data; Phase III meta-analyzed results from the first two phases; associated SNPs from Phase III were used for replication in Phase IV; finally in Phase V, a multi-ethnic meta-analysis of all samples from four ethnicities was performed. At an array-wide significance (P < 2.40E-06), we identify novel BMI associations in loci translocase of outer mitochondrial membrane 40 homolog (yeast) - apolipoprotein E - apolipoprotein C-I (TOMM40-APOE-APOC1) (rs2075650, P = 2.95E-10), sterol regulatory element binding transcription factor 2 (SREBF2, rs5996074, P = 9.43E-07) and neurotrophic tyrosine kinase, receptor, type 2 [NTRK2, a brain-derived neurotrophic factor (BDNF) receptor gene, rs1211166, P = 1.04E-06] in the Phase IV meta-analysis. Of 10 loci with previous evidence for BMI association represented on the IBC array, eight were replicated, with the remaining two showing nominal significance. Conditional analyses revealed two independent BMI-associated signals in BDNF and melanocortin 4 receptor (MC4R) regions. Of the 11 array-wide significant SNPs, three are associated with gene expression levels in both primary B-cells and monocytes; with rs4788099 in SH2B adaptor protein 1 (SH2B1) notably being associated with the expression of multiple genes in cis. These multi-ethnic meta-analyses expand our knowledge of BMI genetics.

Palles C, Cazier J-B, Howarth KM, Domingo E, Jones AM, Broderick P, Kemp Z, Spain SL, Guarino E, Salguero I et al. 2013. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet, 45 (2), pp. 136-144. | Citations: 403 (Scopus) | Show Abstract | Read more

Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.

Knight JC. 2013. Genomic modulators of the immune response. Trends Genet, 29 (2), pp. 74-83. | Citations: 23 (Web of Science Lite) | Show Abstract | Read more

Our understanding of immunity has historically been informed by studying heritable mutations in both the adaptive and innate immune responses, including primary immunodeficiency and autoimmune diseases. Recent advances achieved through the application of genomic and epigenomic approaches are reshaping the study of immune dysfunction and opening up new avenues for therapeutic interventions. Moreover, applying genomic techniques to resolve functionally important genetic variation between individuals is providing new insights into immune function in health. This review describes progress in the study of rare variants and primary immunodeficiency diseases arising from whole-exome sequencing (WES), and discusses the application, success, and challenges of applying genome-wide association studies (GWAS) to disorders of immune function and how they may inform more rational use of therapeutics. In addition, the application of expression quantitative-trait mapping to immune phenotypes, progress in understanding MHC disease associations, and insights into epigenetic mechanisms at the interface of immunity and the environment are reviewed.

Simpson PD, Moysi E, Wicks K, Sudan K, Rowland-Jones SL, McMichael AJ, Knight J, Gillespie GM. 2012. Functional differences exist between TNFα promoters encoding the common -237G SNP and the rarer HLA-B*5701-linked A variant. PLoS One, 7 (7), pp. e40100. | Citations: 7 (Scopus) | Show Abstract | Read more

A large body of functional and epidemiological evidence have previously illustrated the impact of specific MHC class I subtypes on clinical outcome during HIV-1 infection, and these observations have recently been re-iterated in genome wide association studies (GWAS). Yet because of the complexities surrounding GWAS-based approaches and the lack of knowledge relating to the identity of rarer single nucleotide polymorphism (SNP) variants, it has proved difficult to discover independent causal variants associated with favourable immune control. This is especially true of the candidate variants within the HLA region where many of the recently proposed disease influencing SNPs appear to reflect linkage with 'protective' MHC class I alleles. Yet causal MHC-linked SNPs may exist but remain overlooked owing to the complexities associated with their identification. Here we focus on the ancestral TNFα promoter -237A variant (rs361525), shown historically to be in complete linkage disequilibrium with the 'protective' HLA-B*5701 allele. Many of the ancestral SNPs within the extended TNFα promoter have been associated with both autoimmune conditions and disease outcomes, however, the direct role of these variants on TNFα expression remains controversial. Yet, because of the important role played by TNFα in HIV-1 infection, and given the proximity of the -237 SNP to the core promoter, its location within a putative repressor region previously characterized in mice, and its disruption of a methylation-susceptible CpG dinucleotide motif, we chose to carefully evaluate its impact on TNFα production. Using a variety of approaches we now demonstrate that carriage of the A SNP is associated with lower TNFα production, via a mechanism not readily explained by promoter methylation nor the binding of transcription factors or repressors. We propose that the -237A variant could represent a minor causal SNP that additionally contributes to the HLA-B*5701-mediated 'protective' effect during HIV-1 infection.

Knight JC. 2012. Resolving the variable genome and epigenome in human disease. J Intern Med, 271 (4), pp. 379-391. | Citations: 13 (Scopus) | Show Abstract | Read more

The individual human genome and epigenome are being defined at unprecedented resolution by current advances in sequencing technologies with important implications for human disease. This review uses examples relevant to clinical practice to illustrate the functional consequences of genetic and epigenetic variation. The insights gained from genome-wide association studies are described together with current efforts to understand the role of rare variants in common disease, set in the context of recent successes in Mendelian traits through the application of whole exome sequencing. The application of functional genomics to interrogate the genome and epigenome, build up an integrated picture of the regulatory genomic landscape and inform disease association studies is discussed, together with the role of expression quantitative trait mapping and analysis of allele-specific gene expression.

Fairfax BP, Makino S, Radhakrishnan J, Plant K, Leslie S, Dilthey A, Ellis P, Langford C, Vannberg FO, Knight JC. 2012. Genetics of gene expression in primary immune cells identifies cell type-specific master regulators and roles of HLA alleles. Nat Genet, 44 (5), pp. 502-510. | Citations: 250 (Web of Science Lite) | Show Abstract | Read more

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.

Knight J. 2012. Resolving the Variable Genome and Epigenome in Human Disease. J Intern Med, pp. no-no. | Show Abstract | Read more

The individual human genome and epigenome are being defined at unprecedented resolution by current advances in sequencing technologies with important implications for human disease. This review uses examples relevant to clinical practice to illustrate the functional consequences of genetic and epigenetic variation. The insights gained from genome-wide association studies are described together with current efforts to understand the role of rare variants in common disease, set in the context of recent successes in Mendelian traits through application of whole exome sequencing. The application of functional genomics to interrogate the genome and epigenome, build up an integrated picture of the regulatory genomic landscape and inform disease association studies is discussed, together with the role of expression quantitative trait mapping and analysis of allele-specific gene expression.



Vandiedonck C, Taylor MS, Lockstone HE, Plant K, Taylor JM, Durrant C, Broxholme J, Fairfax BP, Knight JC. 2011. Pervasive haplotypic variation in the spliceo-transcriptome of the human major histocompatibility complex. Genome Res, 21 (7), pp. 1042-1054. | Citations: 44 (Scopus) | Show Abstract | Read more

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.

Holt RJ, Zhang Y, Binia A, Dixon AL, Vandiedonck C, Cookson WO, Knight JC, Moffatt MF. 2011. Allele-specific transcription of the asthma-associated PHD finger protein 11 gene (PHF11) modulated by octamer-binding transcription factor 1 (Oct-1) Journal of Allergy and Clinical Immunology, 127 (4), | Citations: 9 (Scopus) | Show Abstract | Read more

Background: Asthma is a common, chronic inflammatory airway disease of major public health importance with multiple genetic determinants. Previously, we found by positional cloning that PHD finger protein 11 (PHF11) on chromosome 13q14 modifies serum immunoglobulin E (IgE) concentrations and asthma susceptibility. No coding variants in PHF11 were identified. Objective: Here we investigate the 3 single nucleotide polymorphisms (SNPs) in this gene most significantly associated with total serum IgE levels - rs3765526, rs9526569, and rs1046295 - for a role in transcription factor binding. Methods: We used electrophoretic mobility shift assays to examine the effect of the 3 SNPs on transcription factor binding in 3 cell lines relevant to asthma pathogenesis. Relative preferential expression of alleles was investigated by using the allelotyping method. Results: Electrophoretic mobility shift assays show that rs1046295 modulates allele-specific binding by the octamer-binding transcription factor 1 (Oct-1). Analysis of the relative expression levels of the 2 alleles of this SNP in heterozygous individuals showed a modest, but highly significant (P = 6.5 × 10-16), preferential expression of the A allele consistent with a functional role for rs1046295. Conclusion: These results suggest a mechanism by which rs1046295 may act as a regulatory variant modulating transcription at this locus and altering asthma susceptibility. © 2010 American Academy of Allergy, Asthma & Immunology.

Andraos C, Koorsen G, Knight JC, Bornman L. 2011. Vitamin D receptor gene methylation is associated with ethnicity, tuberculosis, and TaqI polymorphism Human Immunology, 72 (3), pp. 262-268. | Citations: 41 (Scopus) | Show Abstract | Read more

The Vitamin D receptor (VDR) gene encodes a transcription factor which, on activation by vitamin D, modulates diverse biologic processes, including calcium homeostasis and immune function. Genetic variation involving VDR shows striking differences in allele frequency between populations and has been associated with disease susceptibility, including tuberculosis and autoimmunity, although results have often been conflicting. We hypothesized that methylation of VDR may be population specific and that the combination of differential methylation and genetic variation may characterize tuberculosis (TB) predisposition. We use bisulfite conversion and/or pyrosequencing to analyze the methylation status of 17 CpGs of VDR and to genotype 7 SNPs in the 3' CpG Island (CpG island [CGI] 1060), including the commonly studied SNPs ApaI (rs7975232) and TaqI (rs731236). We show that, for lymphoblastoid cell lines from two ethnically diverse populations (Yoruba from HapMap, n = 30 and Caucasians, n = 30) together with TB cases (n = 32) and controls (n = 29) from the Venda population of South Africa, there are methylation variable positions in the 3' end that significantly distinguish ethnicity (9/17 CpGs) and TB status (3/17 CpGs). Moreover, methylation status shows complex association with TaqI genotype highlighting the need to consider both genetic and epigenetic variants in genetic studies of VDR association with disease. © 2011 American Society for Histocompatibility and Immunogenetics.

Wicks K, Knight JC. 2011. Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-Β gene Genes and Immunity, 12 (2), pp. 126-135. | Citations: 9 (Scopus) | Show Abstract | Read more

Transcriptional regulation has a critical role in the coordinate and context-specific expression of a cluster of genes encoding members of the tumour necrosis factor (TNF) superfamily found at chromosome 6p21, comprising TNF, LTA (encoding lymphotoxin-α) and LTB (encoding lymphotoxin-Β). This is important, as dysregulated expression of these genes is implicated in susceptibility to many autoimmune, inflammatory and infectious diseases. We describe here a novel regulatory element in the fourth exon of LTB, which is highly conserved, localises to the only CpG island in the locus, and is associated with a DNase I hypersensitive site and specific histone modifications. We find evidence of binding by Yin Yang 1 (YY1), cyclic AMP response element (CRE)-binding protein (CREB) and CCCTC-binding factor (CTCF) to this region in Jurkat T cells, which is associated with transcriptional repression on reporter gene analysis. Chromatin conformation capture experiments show evidence of DNA looping, involving interaction of this element with the LTB promoter, LTA promoter and TNF 3′ untranslated region (UTR). Small interfering RNA (siRNA) experiments demonstrate a functional role for YY1 and CREB in LTB expression. Our findings provide evidence of additional complexity in the transcriptional regulation of LTB with implications for coordinate expression of genes in this important genomic locus. © 2011 Macmillan Publishers Limited All rights reserved.

Fairfax BP, Davenport EE, Makino S, Hill AVS, Vannberg FO, Knight JC. 2011. A common haplotype of the TNF receptor 2 gene modulates endotoxin tolerance. J Immunol, 186 (5), pp. 3058-3065. | Citations: 9 (Web of Science Lite) | Show Abstract | Read more

Endotoxin tolerance is characterized by the suppression of further TNF release upon recurrent exposure to LPS. This phenomenon is proposed to act as a homeostatic mechanism preventing uncontrolled cytokine release such as that observed in bacterial sepsis. The regulatory mechanisms and interindividual variation of endotoxin tolerance induction in man remain poorly characterized. In this paper, we describe a genetic association study of variation in endotoxin tolerance among healthy individuals. We identify a common promoter haplotype in TNFRSF1B (encoding TNFR2) to be strongly associated with reduced tolerance to LPS (p = 5.82 × 10(-6)). This identified haplotype is associated with increased expression of TNFR2 (p = 4.9 × 10(-5)), and we find basal expression of TNFR2, irrespective of genotype and unlike TNFR1, is associated with secondary TNF release (p < 0.0001). Functional studies demonstrate a positive-feedback loop via TNFR2 of LPS-induced TNF release, confirming this previously unrecognized role for TNFR2 in the modulation of LPS response.

Maugeri N, Radhakrishnan J, Knight JC. 2010. Genetic determinants of HSP70 gene expression following heat shock. Hum Mol Genet, 19 (24), pp. 4939-4947. | Citations: 14 (Scopus) | Show Abstract | Read more

The regulation of heat shock protein expression is of significant physiological and pathophysiological significance. Here we show that genetic diversity is an important determinant of heat shock protein 70 expression involving local, likely cis-acting, polymorphisms. We define DNA sequence variation for the highly homologous HSPA1A and HSPA1B genes in the major histocompatibility complex on chromosome 6p21 and establish quantitative and specific assays for determining transcript abundance. We show for lymphoblastoid cell lines established from individuals of African ancestry that following heat shock, expression of HSPA1B is associated with rs400547 (P 3.88 × 10(-8)) and linked single nucleotide polymorphisms (SNPs) located 62-93 kb telomeric to HSPA1B. This association was found to explain 31 and 29% of the variance in HSPA1B expression following heat shock or in resting cells, respectively. The associated SNPs show marked variation in minor allele frequency among populations, being more common in individuals of African ancestry, and are located in a region showing population-specific haplotypic block structure. The work illustrates how analysis of a heritable induced expression phenotype can be highly informative in defining functionally important genetic variation.

Ramagopalan SV, Heger A, Berlanga AJ, Maugeri NJ, Lincoln MR, Burrell A, Handunnetthi L, Handel AE, Disanto G, Orton S-M et al. 2010. A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution. Genome Res, 20 (10), pp. 1352-1360. | Citations: 490 (Scopus) | Show Abstract | Read more

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 Crohn's 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.

Knight JC. 2010. Understanding human genetic variation in the era of high-throughput sequencing. EMBO Rep, 11 (9), pp. 650-652. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

The EMBO/EMBL symposium 'Human Variation: Cause and Consequence' highlighted advances in understanding the molecular basis of human genetic variation and its myriad implications for biology, human origins and disease. As high-throughput sequencing allows us to define genetic variation and its functional consequences at genome-wide resolution for a large number of people, important questions need to be asked about how to use new technologies to maximize the translational relevance of genetic research for society and the individual patient.

Wong SH, Gochhait S, Malhotra D, Pettersson FH, Teo YY, Khor CC, Rautanen A, Chapman SJ, Mills TC, Srivastava A et al. 2010. Leprosy and the adaptation of human toll-like receptor 1. PLoS Pathog, 6 (7), pp. e1000979. | Citations: 91 (Scopus) | Show Abstract | Read more

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.

Paracchini S, Monaco AP, Knight JC. 2010. An allele-specific gene expression assay to test the functional basis of genetic associations Journal of Visualized Experiments, (45), | Citations: 2 (Scopus) | Show Abstract | Read more

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits. This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants). Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells. DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers. The primer extension assay is carried out using the MassARRAY (Sequenom) platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results. © 2010 Journal of Visualized Experiments.

Fairfax BP, Vannberg FO, Radhakrishnan J, Hakonarson H, Keating BJ, Hill AVS, Knight JC. 2010. An integrated expression phenotype mapping approach defines common variants in LEP, ALOX15 and CAPNS1 associated with induction of IL-6. Hum Mol Genet, 19 (4), pp. 720-730. | Citations: 18 (Scopus) | Show Abstract | Read more

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.

Handunnetthi L, Ramagopalan SV, Ebers GC, Knight JC. 2010. Regulation of major histocompatibility complex class II gene expression, genetic variation and disease. Genes Immun, 11 (2), pp. 99-112. | Citations: 63 (Scopus) | Show Abstract | Read more

Major histocompatibility complex (MHC) class II molecules are central to adaptive immune responses and maintenance of self-tolerance. Since the early 1970s, the MHC class II region at chromosome 6p21 has been shown to be associated with a remarkable number of autoimmune, inflammatory and infectious diseases. Given that a full explanation for most MHC class II disease associations has not been reached through analysis of structural variation alone, in this review we examine the role of genetic variation in modulating gene expression. We describe the intricate architecture of the MHC class II regulatory system, indicating how its unique characteristics may relate to observed associations with disease. There is evidence that haplotype-specific variation involving proximal promoter sequences can alter the level of gene expression, potentially modifying the emergence and expression of key phenotypic traits. Although much emphasis has been placed on cis-regulatory elements, we also examine the role of more distant enhancer elements together with the evidence of dynamic inter- and intra-chromosomal interactions and epigenetic processes. The role of genetic variation in such mechanisms may hold profound implications for susceptibility to common disease.

Knight JC. 2009. Genetics and the general physician: insights, applications and future challenges. QJM, 102 (11), pp. 757-772. | Citations: 10 (Scopus) | Show Abstract | Read more

Scientific and technological advances in our understanding of the nature and consequences of human genetic variation are now allowing genetic determinants of susceptibility to common multifactorial diseases to be defined, as well as our individual response to therapy. I review how genome-wide association studies are robustly identifying new disease susceptibility loci, providing insights into disease pathogenesis and potential targets for drug therapy. Some of the remarkable advances being made using current genetic approaches in Crohn's disease, coronary artery disease and atrial fibrillation are described, together with examples from malaria, HIV/AIDS, asthma, prostate cancer and venous thrombosis which illustrate important principles underpinning this field of research. The limitations of current approaches are also noted, highlighting how much of the genetic risk remains unexplained and resolving specific functional variants difficult. There is a need to more clearly understand the significance of rare variants and structural genomic variation in common disease, as well as epigenetic mechanisms. Specific examples from pharmacogenomics are described including warfarin dosage and prediction of abacavir hypersensitivity that illustrate how in some cases such knowledge is already impacting on clinical practice, while in others prospective evaluation of clinical utility and cost-effectiveness is required to define opportunities for personalized medicine. There is also a need for a broader debate about the ethical implications of current advances in genetics for medicine and society.

Vandiedonck C, Knight JC. 2009. The human Major Histocompatibility Complex as a paradigm in genomics research. Brief Funct Genomic Proteomic, 8 (5), pp. 379-394. | Citations: 51 (Scopus) | Show Abstract | Read more

Since its discovery more than 50 years ago, the human Major Histocompatibility Complex (MHC) on chromosome 6p21.3 has been at the forefront of human genetic research. Here, we review from a historical perspective the major advances in our understanding of the nature and consequences of genetic variation which have involved the MHC, as well as highlighting likely future directions. As a consequence of its particular genomic structure, its remarkable polymorphism and its early implication in numerous diseases, the MHC has been considered as a model region for genomics, being the first substantial region to be sequenced and establishing fundamental concepts of linkage disequilibrium, haplotypic structure and meiotic recombination. Recently, the MHC became the first genomic region to be entirely re-sequenced for common haplotypes, while studies mapping gene expression phenotypes across the genome have strongly implicated variation in the MHC. This review shows how the MHC continues to provide new insights and remains in the vanguard of contemporary research in human genomics.

Gasperíková D, Tribble ND, Staník J, Hucková M, Misovicová N, van de Bunt M, Valentínová L, Barrow BA, Barák L, Dobránsky R et al. 2009. Identification of a novel beta-cell glucokinase (GCK) promoter mutation (-71G>C) that modulates GCK gene expression through loss of allele-specific Sp1 binding causing mild fasting hyperglycemia in humans. Diabetes, 58 (8), pp. 1929-1935. | Citations: 20 (Scopus) | Show Abstract | Read more

OBJECTIVE: Inactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK beta-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands. RESEARCH DESIGN AND METHODS: The beta-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding. RESULTS: A novel -71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (> or = 5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele. CONCLUSIONS: A novel beta-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the beta-cell GCK promoter should be included.

Ramagopalan SV, Knight JC, Ebers GC. 2009. Multiple sclerosis and the major histocompatibility complex. Curr Opin Neurol, 22 (3), pp. 219-225. | Citations: 66 (Scopus) | Show Abstract | Read more

PURPOSE OF REVIEW: Multiple sclerosis (MS) is the most common neurological disease affecting young adults. The cause is unknown, but detailed epidemiological and genetic studies have shown a clear inherited component. We review here some of the recent findings of MS genetics with a particular focus on genes of the major histocompatibility complex (MHC). RECENT FINDINGS: Recent studies add further complexity to the role of the MHC in MS. Reported MHC associations are complex, involving haplotypes rather than single alleles and may involve epigenetic mechanisms and other modulators of gene expression. MHC class II haplotypes display a hierarchy of risks, including protective effects and epistatic interactions, which together dwarf any non-MHC genetic effect. Genes in the MHC region have been shown to influence disease severity, display parent-of-origin effects and interact with a major environmental candidate for MS, vitamin D. SUMMARY: The MHC class II association with MS is not as straightforward as previously thought. A complete understanding of the epistatic interactions and epigenetic features of this region will be important to understand disease pathogenesis and likely aid the discovery of new therapeutics.

Dennis MY, Paracchini S, Scerri TS, Prokunina-Olsson L, Knight JC, Wade-Martins R, Coggill P, Beck S, Green ED, Monaco AP. 2009. A common variant associated with dyslexia reduces expression of the KIAA0319 gene. PLoS Genet, 5 (3), pp. e1000436. | Citations: 66 (Web of Science Lite) | Show Abstract | Read more

Numerous genetic association studies have implicated the KIAA0319 gene on human chromosome 6p22 in dyslexia susceptibility. The causative variant(s) remains unknown but may modulate gene expression, given that (1) a dyslexia-associated haplotype has been implicated in the reduced expression of KIAA0319, and (2) the strongest association has been found for the region spanning exon 1 of KIAA0319. Here, we test the hypothesis that variant(s) responsible for reduced KIAA0319 expression resides on the risk haplotype close to the gene's transcription start site. We identified seven single-nucleotide polymorphisms on the risk haplotype immediately upstream of KIAA0319 and determined that three of these are strongly associated with multiple reading-related traits. Using luciferase-expressing constructs containing the KIAA0319 upstream region, we characterized the minimal promoter and additional putative transcriptional regulator regions. This revealed that the minor allele of rs9461045, which shows the strongest association with dyslexia in our sample (max p-value = 0.0001), confers reduced luciferase expression in both neuronal and non-neuronal cell lines. Additionally, we found that the presence of this rs9461045 dyslexia-associated allele creates a nuclear protein-binding site, likely for the transcriptional silencer OCT-1. Knocking down OCT-1 expression in the neuronal cell line SHSY5Y using an siRNA restores KIAA0319 expression from the risk haplotype to nearly that seen from the non-risk haplotype. Our study thus pinpoints a common variant as altering the function of a dyslexia candidate gene and provides an illustrative example of the strategic approach needed to dissect the molecular basis of complex genetic traits.

Ramagopalan SV, Maugeri NJ, Handunnetthi L, Lincoln MR, Orton S-M, Dyment DA, Deluca GC, Herrera BM, Chao MJ, Sadovnick AD et al. 2009. Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1*1501 is regulated by vitamin D. PLoS Genet, 5 (2), pp. e1000369. | Citations: 345 (Scopus) | Show Abstract | Read more

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.

Knight JC. 2009. Insights into the nature and consequences of our variable genome. Brief Funct Genomic Proteomic, 8 (5), pp. 343-344. | Read more

Chao MJ, Barnardo MCNM, Lincoln MR, Ramagopalan SV, Herrera BM, Dyment DA, Montpetit A, Sadovnick AD, Knight JC, Ebers GC. 2008. HLA class I alleles tag HLA-DRB1*1501 haplotypes for differential risk in multiple sclerosis susceptibility. Proc Natl Acad Sci U S A, 105 (35), pp. 13069-13074. | Citations: 70 (Scopus) | Show Abstract | Read more

The major locus for multiple sclerosis (MS) susceptibility is located within the class II region of the Major Histocompatibility Complex (MHC). HLA-DRB1 alleles, constituting the strongest MS susceptibility factors, have been widely exploited in research including construction of transgenic animal models of MS. Many studies have concluded that HLA-DRB1*15 allele itself determines MS-associated susceptibility. If this were true, haplotypes bearing this allele would confer equal risk. If HLA-DRB1*15 bearing haplotypes differed for risk, roles for other loci in this region would be implied and further study of the fine structure of this locus would be compelling. We have tested the hypothesis comparing haplotypes stratified by HLA class I tagging. We show here that HLA-DRB1*15-bearing-haplotypes in 1970 individuals from 494 MS families are indeed heterogeneous. Some HLA-DRB1*15 haplotypes determine susceptibility while others do not. Three groups of class I tagged HLA-DRB1*15 haplotypes were not over-transmitted: (i) HLA-DRB1*15-HLA-B*08 (TR = 25, NT = 23, Odds Ratio = 1.09), (ii) -HLA-B*27 (TR = 18, NT = 17, Odds Ratio = 1.06), and (iii) rare HLA-DRB1*15 haplotypes (frequency <0.02). Rare haplotypes were significantly different from common haplotypes, and transmissions were remarkably similar to those for class-I-matched non-HLA-DRB1*15 haplotypes. These results unambiguously indicate that HLA-DRB1*15 is part of a susceptibility haplotype but cannot be the susceptibility allele itself, requiring either epistatic interactions, epigenetic modifications on some haplotypes, or nearby structural variation. These findings strongly imply that differences among HLA-DRB1*15 haplotypes will furnish the basis for MHC-associated susceptibility in MS and raise the possibility that the MHC haplotype is the fundamental unit of genetic control of immune response.

Taylor JM, Wicks K, Vandiedonck C, Knight JC. 2008. Chromatin profiling across the human tumour necrosis factor gene locus reveals a complex, cell type-specific landscape with novel regulatory elements. Nucleic Acids Res, 36 (15), pp. 4845-4862. | Citations: 20 (Scopus) | Show Abstract | Read more

The TNF locus on chromosome 6p21 encodes a family of proteins with key roles in the immune response whose dysregulation leads to severe disease. Transcriptional regulation is important, with cell type and stimulus-specific enhancer complexes involving the proximal TNF promoter. We show how quantitative chromatin profiling across a 34 kb region spanning the TNF locus has allowed us to identify a number of novel DNase hypersensitive sites and characterize more distant regulatory elements. We demonstrate DNase hypersensitive sites corresponding to the lymphotoxin alpha (LTA) and tumour necrosis factor (TNF) promoter regions, a CpG island in exon 4 of lymphotoxin beta (LTB), the 3' end of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1 (NFKBIL1) and 3.4 kb upstream of LTA. These sites co-localize to highly conserved DNA sequences and show evidence of cell type specificity when lymphoblastoid, Jurkat, U937, HeLa and HEK293T cell lines are analysed using Southern blotting. For Jurkat T cells, we define histone modifications across the locus. Peaks of acetylated histone H3 and H4, together with tri-methyl K4 of histone H3, correspond to hypersensitive sites, notably in exon 4 of LTB. We provide evidence of a functional role for an intergenic DNase I hypersensitive site distal to LTA in Jurkat cells based on reporter gene analysis, with evidence of recruitment of upstream stimulatory factors (USF) transcription factors.

Gordon A, Knight JC, Hinds CJ. 2008. Genes and sepsis: how tight is the fit? Crit Care Med, 36 (5), pp. 1652-1654. | Citations: 2 (European Pubmed Central) | Read more

Ramagopalan SV, Knight M, Ebers GC, Knight JC. 2007. Origins of magic: review of genetic and epigenetic effects. BMJ, 335 (7633), pp. 1299-1301. | Show Abstract | Read more

OBJECTIVE: To assess the evidence for a genetic basis to magic. DESIGN: Literature review. SETTING: Harry Potter novels of J K Rowling. PARTICIPANTS: Muggles, witches, wizards, and squibs. INTERVENTIONS: Limited. MAIN OUTCOME MEASURES: Family and twin studies, magical ability, and specific magical skills. RESULTS: Magic shows strong evidence of heritability, with familial aggregation and concordance in twins. Evidence suggests magical ability to be a quantitative trait. Specific magical skills, notably being able to speak to snakes, predict the future, and change hair colour, all seem heritable. CONCLUSIONS: A multilocus model with a dominant gene for magic might exist, controlled epistatically by one or more loci, possibly recessive in nature. Magical enhancers regulating gene expressionmay be involved, combined with mutations at specific genes implicated in speech and hair colour such as FOXP2 and MCR1.

Knight J. 2007. Polymorphisms in tumor necrosis factor and other cytokines as risks for infectious diseases and the septic syndrome. Curr Infect Dis Rep, 3 (5), pp. 427-439. | Show Abstract | Read more

An increasing number of genetic association studies have implicated polymorphisms of cytokine genes as host genetic factors influencing susceptibility to infectious disease, primarily using a candidate gene approach based on knowledge of disease pathogenesis. The application and limitations of association studies are reviewed together with the impact of recent advances in single nucleotide polymorphism mapping on strategic approaches to defining genetic susceptibility loci. It often remains unclear whether associated genetic polymorphisms are themselves functionally relevant or acting only as markers within an extended haplotype, and experimental approaches to investigating the functional impact of polymorphisms in noncoding regulatory DNA sequences are discussed. An overview of genetic associations of cytokine genes with infectious disease is presented, together with discussion of recent studies in a number of infectious diseases including hepatitis, HIV, malaria, and sepsis.

Hull J, Campino S, Rowlands K, Chan M-S, Copley RR, Taylor MS, Rockett K, Elvidge G, Keating B, Knight J, Kwiatkowski D. 2007. Identification of common genetic variation that modulates alternative splicing. PLoS Genet, 3 (6), pp. e99. | Citations: 106 (Web of Science Lite) | Show Abstract | Read more

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

Hull J, Campino S, Rowlands K, Chan MS, Copley RR, Taylor MS, Rockett K, Elvidge G, Keating B, Knight J, Kwiatkowski D. 2007. Identification of common genetic variation that modulates alternative splicing. PLoS genetics, 3 (6), | Citations: 36 (Scopus) | Show Abstract | Read more

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

Paracchini S, Thomas A, Castro S, Lai C, Paramasivam M, Wang Y, Keating BJ, Taylor JM, Hacking DF, Scerri T et al. 2006. The chromosome 6p22 haplotype associated with dyslexia reduces the expression of KIAA0319, a novel gene involved in neuronal migration. Hum Mol Genet, 15 (10), pp. 1659-1666. | Citations: 186 (Scopus) | Show Abstract | Read more

Dyslexia is one of the most prevalent childhood cognitive disorders, affecting approximately 5% of school-age children. We have recently identified a risk haplotype associated with dyslexia on chromosome 6p22.2 which spans the TTRAP gene and portions of THEM2 and KIAA0319. Here we show that in the presence of the risk haplotype, the expression of the KIAA0319 gene is reduced but the expression of the other two genes remains unaffected. Using in situ hybridization, we detect a very distinct expression pattern of the KIAA0319 gene in the developing cerebral neocortex of mouse and human fetuses. Moreover, interference with rat Kiaa0319 expression in utero leads to impaired neuronal migration in the developing cerebral neocortex. These data suggest a direct link between a specific genetic background and a biological mechanism leading to the development of dyslexia: the risk haplotype on chromosome 6p22.2 down-regulates the KIAA0319 gene which is required for neuronal migration during the formation of the cerebral neocortex.

Knight JC. 2006. Analysis of allele-specific gene expression. Methods Mol Biol, 338 pp. 153-165. | Citations: 2 (European Pubmed Central) | Show Abstract | Read more

The analysis of allele-specific gene expression has been of long-standing interest in the study of genomic imprinting, but there is growing awareness that differences in allelic expression are widespread among autosomal nonimprinted genes. Recent research into cis-acting regulatory polymorphisms has utilized the analysis of allele-specific gene expression to identify functionally important regulatory haplotypes and specific genetic polymorphisms. Allele-specific effects are typically of modest magnitude, requiring techniques for analysis of high sensitivity and specificity. Here, strategic approaches to the analysis of allele-specific gene expression are reviewed with protocols for in vivo analysis. These include analysis of the relative allelic abundance of transcribed RNA and of transcription factor recruitment and Pol II loading by chromatin immunoprecipitation.

Charles Knight J. 2005. HaploChIP: an in vivo assay. Methods Mol Biol, 311 pp. 49-60. | Citations: 1 (European Pubmed Central) | Show Abstract | Read more

The characterization of protein-deoxyribonucleic acid (DNA) interactions occurring at an allele-specific level is important to resolving the functional consequences of genetic variation in non-coding DNA for gene expression and regulation. The approach of haplotype-specific chromatin immunoprecipitation (i.e., haploChIP) resolves in living cells relative protein-DNA binding to a particular allele through immunoprecipitation of proteins crosslinked to DNA. Single-nucleotide polymorphisms present in a heterozygous form are used as markers to differentiate allelic origin. This in turn allows resolution of specific haplotypes showing differences in relative protein occupancy. The haploChIP approach allows testing of in vitro hypotheses that a transcription factor protein shows haplotype specific occupancy. In addition, the haploChIP approach allows screening of haplotypes for differences in relative gene expression by immunoprecipitation using antibodies to phosphorylated Pol II.

Knight JC. 2005. Regulatory polymorphisms underlying complex disease traits. J Mol Med (Berl), 83 (2), pp. 97-109. | Citations: 167 (Scopus) | Show Abstract | Read more

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.

Hacking D, Knight JC, Rockett K, Brown H, Frampton J, Kwiatkowski DP, Hull J, Udalova IA. 2004. Increased in vivo transcription of an IL-8 haplotype associated with respiratory syncytial virus disease-susceptibility. Genes Immun, 5 (4), pp. 274-282. | Citations: 106 (Scopus) | Show Abstract | Read more

Interleukin-8 (IL-8) has been implicated in the pathogenesis of RSV-induced bronchiolitis. Previously, we have described an association between bronchiolitis disease severity and a specific IL-8 haplotype comprising six single-nucleotide polymorphisms (SNPs) (-251A/+396G/+781T/+1238delA/+1633T/+2767T, haplotype 2). Here we investigated the functional basis for this association by measuring haplotype-specific transcription in vivo in human primary cells. We found a significant increase in transcript level derived from the IL-8 haplotype 2 relative to the mirror haplotype 1 (-251T/+396T/+781C/+1238insA/+1633C/+2767A) in respiratory epithelial cells but not in lymphocytes. A promoter polymorphism, -251A, present on the high producer haplotype, had no significant affect on the allele-specific level of transcription when analyzed in reporter gene experiments in human respiratory epithelial A549 cells. We proceeded to systematically screen for allele-specific protein-DNA binding in this functional haplotype, which revealed significant differential binding at the +781T/C polymorphism. C/EBP beta was identified as being part of a transcription factor binding complex that preferentially bound in the presence of the +781 T allele. These results suggest that the mechanism for disease susceptibility to RSV-induced bronchiolitis may occur through a haplotype-specific increase in IL-8 transcription, which may be mediated by functional polymorphisms within that haplotype.

Knight JC, Keating BJ, Kwiatkowski DP. 2004. Allele-specific repression of lymphotoxin-alpha by activated B cell factor-1. Nat Genet, 36 (4), pp. 394-399. | Citations: 92 (Scopus) | Show Abstract | Read more

Genetic variation at the human LTA locus, encoding lymphotoxin-alpha, is associated with susceptibility to myocardial infarction, asthma and other diseases. By detailed haplotypic analysis of the locus, we identified a single-nucleotide polymorphism (SNP) at LTA+80 as a main predictor of LTA protein production by human B cells. We found that activated B-cell factor-1 (ABF-1) binds to this site in vitro and suppresses reporter gene expression, but only in the presence of the LTA+80A allele. Using haplotype-specific chromatin immunoprecipitation, we confirmed that ABF-1 is preferentially recruited to the low-producer allele in vivo. These findings provide a molecular model of how LTA expression may be genetically regulated by allele-specific recruitment of the transcriptional repressor ABF-1.

Knight JC. 2004. Allele-specific gene expression uncovered. Trends Genet, 20 (3), pp. 113-116. | Citations: 133 (Scopus) | Show Abstract | Read more

Genetic variation in populations can result in variation in levels of gene expression but the extent to which this occurs has been unclear. In this article, recent studies of allele-specific expression among autosomal non-imprinted genes are reviewed. These new data provide evidence that differential expression is relatively common and that allelic differences are heritable and can be highly context specific.

Knight JC. 2003. Functional implications of genetic variation in non-coding DNA for disease susceptibility and gene regulation. Clin Sci (Lond), 104 (5), pp. 493-501. | Citations: 52 (Web of Science Lite) | Show Abstract | Read more

The role of host genetic variation in determining susceptibility to complex disease traits is the subject of much research effort, but it often remains unclear whether disease-associated genetic polymorphisms are themselves functionally relevant or acting only as markers within an extended haplotype. Experimental approaches to investigate the functional impact of polymorphisms in non-coding regulatory DNA sequences for gene expression are discussed, including the role of gel-shift assays, DNA footprinting and reporter gene analysis. The limitations of different experimental approaches are presented together with future prospects for in vivo analysis. The strategic application of these functional approaches is discussed and illustrated by analysis of the role of genetic variation in the tumour necrosis factor promoter region in determining susceptibility to severe malaria.

Knight JC, Keating BJ, Rockett KA, Kwiatkowski DP. 2003. In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading. Nat Genet, 33 (4), pp. 469-475. | Citations: 206 (Scopus) | Show Abstract | Read more

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.

Knight J. 2001. Polymorphisms in Tumor Necrosis Factor and Other Cytokines As Risks for Infectious Diseases and the Septic Syndrome. Curr Infect Dis Rep, 3 (5), pp. 427-439. | Citations: 15 (European Pubmed Central) | Show Abstract | Read more

An increasing number of genetic association studies have implicated polymorphisms of cytokine genes as host genetic factors influencing susceptibility to infectious disease, primarily using a candidate gene approach based on knowledge of disease pathogenesis. The application and limitations of association studies are reviewed together with the impact of recent advances in single nucleotide polymorphism mapping on strategic approaches to defining genetic susceptibility loci. It often remains unclear whether associated genetic polymorphisms are themselves functionally relevant or acting only as markers within an extended haplotype, and experimental approaches to investigating the functional impact of polymorphisms in noncoding regulatory DNA sequences are discussed. An overview of genetic associations of cytokine genes with infectious disease is presented, together with discussion of recent studies in a number of infectious diseases including hepatitis, HIV, malaria, and sepsis.

Knight JC, Knight M, Smith MJ. 2000. Two cases of pulmonary complications associated with a recently recognised Salmonella enteritidis phage type, 21b, affecting immunocompetent adults. Eur J Clin Microbiol Infect Dis, 19 (9), pp. 725-726. | Citations: 3 (European Pubmed Central) | Read more

Knight JC, McGuire W, Kortok MM, Kwiatkowski D. 1999. Accuracy of genotyping of single-nucleotide polymorphisms by PCR-ELISA allele-specific oligonucleotide hybridization typing and by amplification refractory mutation system. Clin Chem, 45 (10), pp. 1860-1863. | Citations: 18 (Web of Science Lite)

Knight JC, Kwiatkowski D. 1999. Inherited variability of tumor necrosis factor production and susceptibility to infectious disease. Proc Assoc Am Physicians, 111 (4), pp. 290-298. | Citations: 107 (Scopus) | Show Abstract | Read more

Tumor necrosis factor (TNF) is a critical mediator of host defense against infection but may cause severe pathology when produced in excess. Individuals vary in the amount of TNF produced when their peripheral blood mononuclear cells are stimulated in vitro, and family studies indicate that much of this variability is genetically determined. Since the TNF response to infection is partly regulated at the transcriptional level, TNF promoter polymorphisms have been the subject of intense interest as potential determinants of disease susceptibility. A single nucleotide polymorphism at nucleotide -308 relative to the transcriptional start site has been associated with susceptibility to severe malaria, leishmaniasis, scarring trachoma, and lepromatous leprosy. Some experimental data indicate that this polymorphism acts to upregulate TNF transcription, but this remains controversial. Detailed analysis of multiple genetic markers at this locus and more sophisticated investigations of TNF transcriptional regulation, in different cell types and with a wide range of stimuli, are required to understand the molecular basis of these disease associations.

Knight JC, Udalova I, Hill AV, Greenwood BM, Peshu N, Marsh K, Kwiatkowski D. 1999. A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria. Nat Genet, 22 (2), pp. 145-150. | Citations: 395 (Scopus) | Show Abstract | Read more

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.

McGuire W, Knight JC, Hill AV, Allsopp CE, Greenwood BM, Kwiatkowski D. 1999. Severe malarial anemia and cerebral malaria are associated with different tumor necrosis factor promoter alleles. J Infect Dis, 179 (1), pp. 287-290. | Citations: 198 (Scopus) | Show Abstract | Read more

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.

Udalova IA, Knight JC, Vidal V, Nedospasov SA, Kwiatkowski D. 1998. Complex NF-kappaB interactions at the distal tumor necrosis factor promoter region in human monocytes. J Biol Chem, 273 (33), pp. 21178-21186. | Citations: 111 (Web of Science Lite) | Show Abstract | Read more

We describe a dense cluster of DNA-protein interactions located 600 nucleotides upstream of the transcriptional start site of the human tumor necrosis factor (TNF) gene. This area was identified as being of potential importance for lipopolysaccharide-inducible TNF expression in the human monocyte cell line Mono Mac 6, based on reporter gene analysis of point mutations at a number of nuclear factor kappaB (NF-kappaB)-like motifs within the human TNF promoter region. The area contains two NF-kappaB sites, which are here shown by DNase I and methylation interference footprinting to flank a novel binding site. UV cross-linking studies reveal that the novel site can also bind NF-kappaB as well as an unknown protein(s) of approximately 40 kDa. We show that these three adjacent kappaB-binding sites differ markedly in their relative affinities for p50/p50, p65/p65, and p65/p50, yet this 39-nucleotide segment of DNA appears capable of binding up to three NF-kappaB heterodimers simultaneously. Reporter gene studies indicate that each element of the cluster contributes to lipopolysaccharide-induced transcriptional activation in Mono Mac 6 cells. These findings suggest that NF-kappaB acts in a complex manner to activate TNF transcription in human monocytes.

Kwiatkowski D, Bate CA, Scragg IG, Beattie P, Udalova I, Knight JC. 1997. The malarial fever response--pathogenesis, polymorphism and prospects for intervention. Ann Trop Med Parasitol, 91 (5), pp. 533-542. | Citations: 32 (Scopus) | Show Abstract | Read more

It is estimated that over 200 million people each year suffer debilitating attacks of malarial fever, and roughly 2 million of these episodes are fatal. The fever is caused by tumour necrosis factor (TNF) and other pyrogenic cytokines that are released by the host immune system response to products of schizont rupture. TNF has anti-parasitic properties but excessive TNF production is thought to play an important role in the pathogenesis of cerebral malaria. This review summarizes recent attempts to achieve molecular characterization of the parasite components that stimulate the host TNF response, and to define the host and parasite factors that affect the level of TNF production. Of particular interest are host polymorphisms that may regulate TNF gene expression, and naturally acquired antibodies that prevent the parasite from inducing TNF, both of which correlate with the clinical severity of infection. Our understanding of these processes, which are potentially of considerable therapeutic relevance, remains very limited at both the molecular and the epidemiological level.

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