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1.  Genome-wide association study of survival from sepsis due to pneumonia: an observational cohort study 
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.
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.
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.
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.
European Commission and the Wellcome Trust.
PMCID: PMC4314768  PMID: 25533491
2.  Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach 
Genome Biology  2014;15(10):494.
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.
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 and infectious disease with novel loci including major histone gene clusters. We resolve 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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0494-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4243378  PMID: 25366989
3.  Transcriptomic profiling facilitates classification of response to influenza challenge 
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-014-1212-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4281383  PMID: 25345603
Influenza; Challenge; Transcriptome; Expression; RNA; Microarray
4.  Innate Immune Activity Conditions the Effect of Regulatory Variants upon Monocyte Gene Expression 
Science (New York, N.Y.)  2014;343(6175):1246949.
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.
PMCID: PMC4064786  PMID: 24604202
5.  Systematic identification of trans-eQTLs as putative drivers of known disease associations 
Nature genetics  2013;45(10):1238-1243.
Identifying the downstream effects of disease-associated single nucleotide polymorphisms (SNPs) is challenging: the causal gene is often unknown or it is unclear how the SNP affects the causal gene, making it difficult to design experiments that reveal functional consequences. To help overcome this problem, we performed the largest expression quantitative trait locus (eQTL) meta-analysis so far reported in non-transformed peripheral blood samples of 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. Although we did not study specific patient cohorts, we identified trait-associated SNPs that affect multiple trans-genes that are known to be markedly altered in patients: for example, systemic lupus erythematosus (SLE) SNP rs49170141 altered C1QB and five type 1 interferon response genes, both hallmarks of SLE2-4. Subsequent ChIP-seq data analysis on these trans-genes implicated transcription factor IKZF1 as the causal gene at this locus, with DeepSAGE RNA-sequencing revealing that rs4917014 strongly alters 3’ UTR levels of IKZF1. 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.
PMCID: PMC3991562  PMID: 24013639
6.  Fine mapping genetic determinants of the highly variably expressed MHC gene ZFP57 
ZFP57 is an important transcriptional regulator involved in DNA methylation and genomic imprinting during development. Here we demonstrate that gene expression also occurs at a low level in adult peripheral blood cells and other tissues including the kidney and thymus, but is critically dependent on underlying local genetic variation within the MHC. We resolve a highly significant expression quantitative trait locus for ZFP57 involving single-nucleotide polymorphisms (SNPs) in the first intron of the gene co-localizing with a DNase I hypersensitive site and evidence of CTCF recruitment. These data identify ZFP57 as a candidate gene underlying reported MHC disease associations, notably for putative regulatory variants associated with cancer and HIV-1. The work highlights the role that ZFP57 may play in DNA methylation and epigenetic regulation beyond early development into adult life dependent on genetic background, with important potential implications for disease.
PMCID: PMC3953924  PMID: 24193346
ZFP57; expression quantitative trait; genetic variation; MHC; disease susceptibility
7.  Allele-specific transcription of the asthma-associated PHD finger protein 11 gene (PHF11) modulated by octamer-binding transcription factor 1 (Oct-1) 
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.
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.
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.
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.
These results suggest a mechanism by which rs1046295 may act as a regulatory variant modulating transcription at this locus and altering asthma susceptibility.
PMCID: PMC3955022  PMID: 21320718
Asthma genetics; PHF11; IgE; rs1046295; electrophoretic mobility shift assay; Oct-1; gene expression
8.  Vitamin D receptor gene methylation is associated with ethnicity, tuberculosis and TaqI polymorphism 
Human immunology  2010;72(3):262-268.
The Vitamin D Receptor (VDR) gene encodes a transcription factor which, on activation by vitamin D, modulates diverse biological 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 characterise TB predisposition. We use bisulphite conversion and/or pyrosequencing to analyse the methylation status of 17 CpGs of VDR and to genotype 7 SNPs in the 3′ 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 (MVPs) 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.
PMCID: PMC3955023  PMID: 21168462
VDR (vitamin D (1,25- dihydroxyvitamin D3) receptor); gene polymorphism; CpG methylation; TB (tuberculosis); ethnic differences
9.  An Integrated Approach to Defining Genetic and Environmental Determinants for Major Clinical Outcomes Involving Vitamin D 
Molecular Diagnosis & Therapy  2014;18(3):261-272.
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.
PMCID: PMC4031425  PMID: 24557774
10.  Associations of HLA alleles with specific language impairment 
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.
We perform quantitative association analyses of three linguistic measures and case-control association analyses using both SNP data and imputed HLA types.
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).
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.
PMCID: PMC3906746  PMID: 24433325
Specific language impairment (SLI); HLA; Neurodevelopmental disorders; Genetic association
11.  Genomic modulators of the immune response 
Trends in Genetics  2013;29(2):74-83.
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.
PMCID: PMC3605582  PMID: 23122694
immunity; major histocompatibility complex; gene regulation; autoimmunity; immunodeficiency; leukocyte
Nature genetics  2012;44(5):502-510.
Trans-acting genetic variants play a substantial, albeit poorly characterized, role in the heritable determination of gene expression. Using paired purified primary monocytes and B-cells we identify novel, predominantly cell-specific, cis- and trans-eQTL (expression quantitative trait loci). These include multi-locus trans-associations to LYZ in monocytes and to KLF4 in B-cells. Additionally, we observe B-cell specific trans-association of rs11171739 at 12q13.2, a known autoimmune disease locus, to IP6K2 (pB-cell=5.8×10−15), PRIC285 (pB-cell=3.0×10−10) and an upstream region of CDKN1A (pB-cell=2×10−52; pmonocyte=1.8×10−4), suggesting roles for cell cycle regulation and PPARγ signaling in disease pathogenesis. We also find specific HLA alleles forming trans-association with the expression of AOAH and ARHGAP24 in monocytes but not in B-cells. In summary, we demonstrate that mapping gene expression in defined primary cell populations identifies new cell-specific trans-regulated networks and provides insights into the genetic basis of disease susceptibility.
PMCID: PMC3437404  PMID: 22446964
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 inter-individual variation of endotoxin tolerance induction in man remain poorly characterized. Here we describe a genetic association study of variation in endotoxin tolerance amongst 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.
PMCID: PMC3211060  PMID: 21282507
14.  Regulatory polymorphisms underlying complex disease traits 
There is growing evidence that genetic variation plays an important role in the determination of individual susceptibility to complex disease traits. In contrast to coding sequence polymorphisms, where the consequences of non-synonymous variation may be resolved at the level of the protein phenotype, defining specific functional regulatory polymorphisms has proved problematic. This has arisen for a number of reasons, including difficulties with fine mapping due to linkage disequilibrium, together with a paucity of experimental tools to resolve the effects of non-coding sequence variation on gene expression. Recent studies have shown that variation in gene expression is heritable and can be mapped as a quantitative trait. Allele-specific effects on gene expression appear relatively common, typically of modest magnitude and context specific. The role of regulatory polymorphisms in determining susceptibility to a number of complex disease traits is discussed, including variation at the VNTR of INS, encoding insulin, in type 1 diabetes and polymorphism of CTLA4, encoding cytotoxic T lymphocyte antigen, in autoimmune disease. Examples where regulatory polymorphisms have been found to play a role in mongenic traits such as factor VII deficiency are discussed, and contrasted with those polymorphisms associated with ischaemic heart disease at the same gene locus. Molecular mechanisms operating in an allele-specific manner at the level of transcription are illustrated, with examples including the role of Duffy binding protein in malaria. The difficulty of resolving specific functional regulatory variants arising from linkage disequilibrium is demonstrated using a number of examples including polymorphism of CCR5, encoding CC chemokine receptor 5, and HIV-1 infection. The importance of understanding haplotypic structure to the design and interpretation of functional assays of putative regulatory variation is highlighted, together with discussion of the strategic use of experimental tools to resolve regulatory polymorphisms at a transcriptional level. A number of examples are discussed including work on the TNF locus which demonstrate biological and experimental context specificity. Regulatory variation may also operate at other levels of control of gene expression and the modulation of splicing at PTPRC, encoding protein tyrosine phosphatase receptor-type C, and of translational efficiency at F12, encoding factor XII, are discussed.
PMCID: PMC3132451  PMID: 15592805
Gene expression; Genetics; Gene polymorphism; Promoter; Transcription
15.  Origins of magic: review of genetic and epigenetic effects 
BMJ : British Medical Journal  2007;335(7633):1299-1301.
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.
PMCID: PMC2151141  PMID: 18156238
16.  Regulation of MHC class II gene expression, genetic variation and disease 
Genes and immunity  2009;11(2):99-112.
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 explore 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 explore 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.
PMCID: PMC2987717  PMID: 19890353
MHC; HLA; transcription; gene regulation; polymorphism; autoimmune
17.  An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations 
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 traits1.
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)2,3. 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 markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 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.
PMCID: PMC3157858  PMID: 21085102
18.  Genetic determinants of HSP70 gene expression following heat shock 
Human Molecular Genetics  2010;19(24):4939-4947.
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.
PMCID: PMC2989893  PMID: 20876613
19.  Identification of a Novel β-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  2009;58(8):1929-1935.
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 β-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands.
The β-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.
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 (≥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.
A novel β-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 β-cell GCK promoter should be included.
PMCID: PMC2712784  PMID: 19411616
20.  Leprosy and the Adaptation of Human Toll-Like Receptor 1 
PLoS Pathogens  2010;6(7):e1000979.
Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae and remains endemic in many parts of the world. Despite several major studies on susceptibility to leprosy, few genomic loci have been replicated independently. We have conducted an association analysis of more than 1,500 individuals from different case-control and family studies, and observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to leprosy (TLR1 I602S, case-control P = 5.7×10−8, OR = 0.31, 95% CI = 0.20–0.48, and HLA-DQA1 rs1071630, case-control P = 4.9×10−14, OR = 0.43, 95% CI = 0.35–0.54). The effect sizes of these associations suggest that TLR1 and HLA-DRB1/DQA1 are major susceptibility genes in susceptibility to leprosy. Further population differentiation analysis shows that the TLR1 locus is extremely differentiated. The protective dysfunctional 602S allele is rare in Africa but expands to become the dominant allele among individuals of European descent. This supports the hypothesis that this locus may be under selection from mycobacteria or other pathogens that are recognized by TLR1 and its co-receptors. These observations provide insight into the long standing host-pathogen relationship between human and mycobacteria and highlight the key role of the TLR pathway in infectious diseases.
Author Summary
Mycobacterium leprae is an obligate intracellular pathogen that causes leprosy, a disease that shares a long history with the human population but which remains endemic in many parts of the world. Despite the fact that the genome of M. leprae has been sequenced, our understanding of its pathogenesis and interaction with the human host is limited, in part due to the inability to culture the bacterium in vitro. In this gene-centric microarray study, we have genotyped SNPs in over 2,000 genes and identified TLR1 and HLA-DRB1/DQA1 as major leprosy susceptibility genes. Studying the geographical distribution of this hypo-functional TLR1 variant demonstrated extreme population differentiation at this locus. These results suggest that leprosy may have contributed to the evolution of this genomic region, and provide insight into the long history of the host-pathogen relationship between humans and M. leprae.
PMCID: PMC2895660  PMID: 20617178
21.  An integrated expression phenotype mapping approach defines common variants in LEP, ALOX15 and CAPNS1 associated with induction of IL-6 
Human Molecular Genetics  2009;19(4):720-730.
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 ∼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.
PMCID: PMC2807371  PMID: 19942621
22.  A Common Variant Associated with Dyslexia Reduces Expression of the KIAA0319 Gene 
PLoS Genetics  2009;5(3):e1000436.
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.
Author Summary
Dyslexia, or reading disability, is a common disorder caused by both genetic and environmental factors. Genetic studies have implicated a number of genes as candidates for playing a role in dyslexia. We functionally characterized one such gene (KIAA0319) to identify variant(s) that might affect gene expression and contribute to the disorder. We discovered a variant residing outside of the protein-coding region of KIAA0319 that reduces expression of the gene. This variant creates a binding site for the transcription factor OCT-1. Previous studies have shown that OCT-1 binding to a specific DNA sequence upstream of a gene can reduce the expression of that gene. In this case, reduced KIAA0319 expression could lead to improper development of regions of the brain involved in reading ability. This is the first study to identify a functional variant implicated in dyslexia. More broadly, our study illustrates the steps that can be utilized for identifying mutations causing other complex genetic disorders.
PMCID: PMC2653637  PMID: 19325871
23.  Expression of the Multiple Sclerosis-Associated MHC Class II Allele HLA-DRB1*1501 Is Regulated by Vitamin D 
PLoS Genetics  2009;5(2):e1000369.
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.
Author Summary
Multiple Sclerosis (MS) is a complex neurological disease with a strong genetic component. The Major Histocompatibility Complex (MHC) on chromosome 6 exerts the strongest genetic effect on disease risk. A region at or near the HLA-DRB1 locus in the MHC influences the risk of MS. HLA-DRB1 has over 400 different alleles. The dominant haplotype of Northern Europe, marked by the presence of DRB1*1501, increases risk of MS by 3-fold. The environment also plays a key role in MS. The most striking illustration of this is the geographical distribution of the disease in populations matched for ethnicity. This has led to the proposal that sunshine, and in particular, vitamin D, is an environmental factor influencing the risk of MS. Circumstantial evidence supporting this comes from studies showing the involvement of vitamin D in immune and nervous system function. The current investigation sought to uncover any relationship between vitamin D and HLA-DRB1. It was found that vitamin D specifically interacts with HLA-DRB1*1501 to influence its expression. This study therefore provides more direct support for the already strong epidemiological evidence implicating sunlight and vitamin D in the determination of MS risk, and implies that vitamin D supplementation at critical time periods may be key to disease prevention.
PMCID: PMC2627899  PMID: 19197344
24.  Chromatin profiling across the human tumour necrosis factor gene locus reveals a complex, cell type-specific landscape with novel regulatory elements 
Nucleic Acids Research  2008;36(15):4845-4862.
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.
PMCID: PMC2528168  PMID: 18653526

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