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

Characterisation of the global transcriptional response to heat shock and the impact of individual genetic variation

Abstract 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

A common variant associated with dyslexia reduces expression of the KIAA0319 gene

This work was supported by the Wellcome Trust (MYD, SP, TSS, JCK, RWM, PC, SB, and APM), the Intramural Research Programs of the National Human Genome Research Institute (MYD and EDG) and National Cancer Institute (MPO), and the NIH/Ox-Cam Graduate Partnership Program (MYD).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.PostprintPeer reviewe

Distinct Transcriptional and Anti-Mycobacterial Profiles of Peripheral Blood Monocytes Dependent on the Ratio of Monocytes: Lymphocytes.

The ratio of monocytes and lymphocytes (ML ratio) in peripheral blood is associated with tuberculosis and malaria disease risk and cancer and cardiovascular disease outcomes. We studied anti-mycobacterial function and the transcriptome of monocytes in relation to the ML ratio. Mycobacterial growth inhibition assays of whole or sorted blood were performed and mycobacteria were enumerated by liquid culture. Transcriptomes of unstimulated CD14 + monocytes isolated by magnetic bead sorting were characterised by microarray. Transcript expression was tested for association with ML ratio calculated from leucocyte differential counts by linear regression. The ML ratio was associated with mycobacterial growth in vitro (β = 2.23, SE 0.91, p = 0.02). Using sorted monocytes and lymphocytes, in vivo ML ratio (% variance explained R(2) = 11%, p = 0.02) dominated over in vitro ratios (R(2) = 5%, p = 0.10) in explaining mycobacterial growth. Expression of 906 genes was associated with the ML ratio and 53 with monocyte count alone. ML-ratio associated genes were enriched for type-I and -II interferon signalling (p = 1.2 × 10(− 8)), and for genes under transcriptional control of IRF1, IRF2, RUNX1, RELA and ESRRB. The ML-ratio-associated gene set was enriched in TB disease (3.11-fold, 95% CI: 2.28-4.19, p = 5.7 × 10(− 12)) and other inflammatory diseases including atopy, HIV, IBD and SLE. The ML ratio is associated with distinct transcriptional and anti-mycobacterial profiles of monocytes that may explain the disease associations of the ML ratio

Circularly polarized luminescence from helically chiral N,N,O,O-boron-chelated dipyrromethenes

Helically chiral N,N,O,O-boron chelated dipyrromethenes showed solution-phase circularly polarized luminescence (CPL) in the red region of the visible spectrum (λem(max) from 621 to 663 nm). The parent dipyrromethene is desymmetrised through O chelation of boron by the 3,5-ortho-phenolic substituents, inducing a helical chirality in the fluorophore. The combination of high luminescence dissymmetry factors (|glum| up to 4.7 ×10−3) and fluorescence quantum yields (ΦF up to 0.73) gave exceptionally efficient circularly polarized red emission from these simple small organic fluorophores, enabling future application in CPL-based bioimaging

Chromosome conformation capture approaches to investigate 3D genome architecture in Ankylosing Spondylitis

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies

Controversies Surrounding Renal Denervation:Lessons Learned From Real-World Experience in Two United Kingdom Centers

Renal denervation (RDN) is a therapy that targets treatment‐resistant hypertension (TRH). The Renal Denervation in Patients With Uncontrolled Hypertension (Symplicity) HTN‐1 and Symplicity HTN‐2 trials reported response rates of >80%; however, sham‐controlled Symplicity HTN‐3 failed to reach its primary blood pressure (BP) outcome. The authors address the current controversies surrounding RDN, illustrated with real‐world data from two centers in the United Kingdom. In this cohort, 52% of patients responded to RDN, with a 13±32 mm Hg reduction in office systolic BP (SBP) at 6 months (n=29, P=.03). Baseline office SBP and number of ablations correlated with office SBP reduction (R=−0.47, P=.01; R=−0.56, P=.002). RDN appears to be an effective treatment for some patients with TRH; however, individual responses are highly variable. Selecting patients for RDN is challenging, with only 10% (33 of 321) of the screened patients eligible for the study. Medication alterations and nonadherence confound outcomes. Adequate ablation is critical and should impact future catheter design/training. Markers of procedural success and improved patient selection parameters remain key research aims

Transcriptomic Signatures in Sepsis and a Differential Response to Steroids. From the VANISH Randomized Trial.

Non-commercial use onlyRATIONALE: 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 (SRSs) have been identified. SRS1 is relatively immunosuppressed, whereas SRS2 is relatively immunocompetent. OBJECTIVES: We aimed to categorize 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 [Vasopressin vs. Norepinephrine as Initial Therapy in Septic Shock]). 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 by 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 (odds ratio = 7.9; 95% confidence interval = 1.6-39.9). CONCLUSIONS: Transcriptomic profile at onset of septic shock was associated with response to corticosteroids. Those with the immunocompetent SRS2 endotype had significantly higher mortality when given corticosteroids compared with placebo. Clinical trial registered with www.clinicaltrials.gov (ISRCTN 20769191).Supported by the UK National Institute for Health Research (NIHR) under Research for Patient Benefit program grant PB-PG-0610-22350, NIHR Clinician Scientist Award NIHR/CS/009/007, and NIHR Research Professor award RP-2015-06-018 (A.C.G.); also supported by the NIHR Imperial Biomedical Research Centre, the UK Intensive Care Foundation, Wellcome Trust grant 090532/Z/09/Z to core facilities at the Wellcome Centre for Human Genetics, Wellcome Trust Investigator Award 204969/Z/16/Z (J.C.K.), and by the NIHR Oxford Biomedical Research Centre

Risk of nontyphoidal Salmonella bacteraemia in African children is modified by STAT4

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-gamma (IFN gamma) production in stimulated natural killer cells, and decreased circulating IFN gamma 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 IFN gamma-mediated immunity as a determinant of invasive NTS disease in African children, and highlight the shared genetic architecture of infectious and autoimmune disease.Peer reviewe

Genetic determinants of HSP70 gene expression following heat shock

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