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Drug targets with human genetic evidence are expected to increase clinical success by at least two-fold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of early drug discovery. A key issue is that, currently, the vast majority of complex disease associations cannot be cleanly mapped to a gene. Immune disease-associated variants are enriched within regulatory elements, such as distal enhancers, found in T cell-specific open chromatin regions. To identify the genes and thus the molecular programs modulated by these regulatory elements, we developed a CRISPRi-based single-cell functional screening approach in primary human CD4+T cells. Our pipeline enables the interrogation of transcriptomic changes induced by the perturbation of regulatory elements at scale. We first optimised a highly efficient CRISPRi protocol in primary human CD4+T cells via CROPseq vectors. Subsequently, we performed a proof-of-concept screen targeting 45 non-coding regulatory elements and 35 transcription start sites and profiled approximately 250,000 CD4+T cell single-cell transcriptomes. We developed a bespoke analytical pipeline for element-to-gene (E2G) mapping and demonstrate that our method can identify both previously annotated and novel E2G links. Lastly, we integrated genetic association data for immune-related traits and demonstrate how our platform can aid in the identification of effector genes for GWAS loci.

BackgroundCertain infectious agents are recognised causes of cancer and potentially other chronic diseases. Identifying associations and understanding pathological mechanisms involving infectious agents and subsequent chronic disease risk will be possible through measuring exposure to multiple infectious agents in large-scale prospective cohorts such as UK Biobank.MethodsFollowing expert consensus we designed a Multiplex Serology platform capable of simultaneously measuring quantitative antibody responses against 45 antigens from 20 infectious agents implicated in non-communicable diseases, including human herpes, hepatitis, polyoma, papilloma, and retroviruses, as well asChlamydia trachomatis, Helicobacter pyloriandToxoplasma gondii. This panel was assayed in a random subset of UK Biobank participants (n=9,695) to test associations between infectious agents and recognised demographic and genetic risk factors and disease outcomes.FindingsSeroprevalence estimates for each infectious agent were consistent with those expected from the literature. The data confirmed epidemiological associations of infectious agent antibody responses with sociodemographic characteristics (e.g. lifetime sexual partners withC, trachomatis;P=1·8×10−149), genetic variants (e.g. rs6927022 with Epstein-Barr virus (EBV) EBNA1 antibodies,P=9·5×10−91) and disease outcomes including human papillomavirus-16 seropositivity and cervical intraepithelial neoplasia (odds ratio 2·28, 95% confidence interval 1·38-3·63), and quantitative EBV viral capsid antigen responses and multiple sclerosis through genetic correlation (MHC rG=0·30,P=0·01).InterpretationThis dataset, intended as a pilot study to demonstrate applicability of Multiplex Serology in epidemiological studies, is itself one of the largest studies to date covering diverse infectious agents in a prospective UK cohort including those traditionally under-represented in population cohorts such as human immunodeficiency virus-1 andC. trachomatis. Our results emphasise the validity of our Multiplex Serology approach in large-scale epidemiological studies opening up opportunities for improving our understanding of host-pathogen-disease relationships. These data are available to researchers interested in examining the relationship between infectious agents and human health.

Detangling gene-disease connections Many diseases are at least partially due to genetic causes that are not always understood or targetable with specific treatments. To provide insight into the biology of various human diseases as well as potential leads for therapeutic development, Pietzner et al . undertook detailed, genome-wide proteogenomic mapping. The authors analyzed thousands of connections between potential disease-associated mutations, specific proteins, and medical conditions, thereby providing a detailed map for use by future researchers. They also supplied some examples in which they applied their approach to medical contexts as varied as connective tissue disorders, gallstones, and COVID-19 infections, sometimes even identifying single genes that play roles in multiple clinical scenarios. —YN

Genetic risk factors frequently affect multiple common human diseases, providing insight into shared pathophysiological pathways and opportunities for therapeutic development. However, systematic identification of genetic profiles of disease risk is limited by the availability of both comprehensive clinical data on population-scale cohorts and the lack of suitable statistical methodology that can handle the scale of and differential power inherent in multi-phenotype data. Here, we develop a disease-agnostic approach to cluster the genetic risk profiles for 3,025 genome-wide independent loci across 19,155 disease classification codes from 320,644 participants in the UK Biobank, representing a large and heterogeneous population. We identify 339 distinct disease association profiles and use multiple approaches to link clusters to the underlying biological pathways. We show how clusters can decompose the variance and covariance in risk for disease, thereby identifying underlying biological processes and their impact. We demonstrate the use of clusters in defining disease relationships and their potential in informing therapeutic strategies.A method to cluster genetic risk profiles applied to 3,025 loci across 19,155 disease codes from over 300,000 individuals in the UK Biobank identifies 339 distinct disease association profiles and links clusters to biological pathways.

Background: Estimates of the burden of cardio-metabolic risk factors in Latin America and the Caribbean (LAC) rely on relative risks (RRs) from non-LAC countries. Whether these RRs apply to LAC remains unknown. Methods: We pooled LAC cohorts. We estimated RRs per unit of exposure to body mass index (BMI), systolic blood pressure (SBP), fasting plasma glucose (FPG), total cholesterol (TC) and non-HDL cholesterol on fatal (31 cohorts, n=168,287) and non-fatal (13 cohorts, n=27,554) cardiovascular diseases, adjusting for regression dilution bias. We used these RRs and national data on mean risk factor levels to estimate the number of cardiovascular deaths attributable to non-optimal levels of each risk factor. Results: Our RRs for SBP, FPG and TC were like those observed in cohorts conducted in high-income countries; however, for BMI, our RRs were consistently smaller in people below 75 years of age. Across risk factors, we observed smaller RRs among older ages. Non-optimal SBP was responsible for the largest number of attributable cardiovascular deaths ranging from 38 per 100,000 women and 54 men in Peru, to 261 (Dominica, women) and 282 (Guyana, men). For non-HDL cholesterol, the lowest attributable rate was for women in Peru (21) and men in Guatemala (25), and the largest in men (158) and women (142) from Guyana. Interpretation: RRs for BMI from studies conducted in high-income countries may overestimate disease burden metrics in LAC; conversely, RRs for SBP, FPG and TC from LAC cohorts are similar to those estimated from cohorts in high-income countries. Funding: Wellcome Trust (214185/Z/18/Z)

ObjectivePolymorphic variation of immune system proteins can drive variability of individual immune responses. ER aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by MHC class I molecules. Coding single nucleotide polymorphisms (SNPs) in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown to affect functional properties of the enzyme, but the interplay between combinations of these SNPs as they exist in allotypes, has not been thoroughly explored.MethodsWe used phased genotype data to estimate ERAP1 allotype frequency in 2,504 individuals across five major human populations, generated highly pure recombinant enzymes corresponding to the 10 most common ERAP1 allotypes and systematically characterized their in vitro enzymatic properties.ResultsWe find that ERAP1 allotypes possess a wide range of enzymatic activities, up to 60-fold, whose ranking is substrate-dependent. Strikingly, allotype 10, previously associated with Behçet’s disease, is consistently a low-activity outlier, suggesting that a significant percentage of individuals carry a sub-active ERAP1 gene. Enzymatic analysis revealed that ERAP1 allotypes can differ in both catalytic efficiency and substrate affinity, differences that can change intermediate accumulation in multi-step trimming reactions. Alterations in efficacy of an allosteric inhibitor that targets the regulatory site suggest that allotypic variation influences the communication between the regulatory and the active site.ConclusionOur work defines the wide landscape of ERAP1 activity in human populations and demonstrates how common allotypes can induce substrate-dependent variability in antigen processing, thus contributing, in synergy with MHC haplotypes, to immune response variability and to predisposition to chronic inflammatory conditions.

Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4’-deoxy-4’-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or ribosomal RNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy.

Translation fidelity is the limiting factor in the accuracy of gene expression. With an estimated frequency of 10−4, errors in mRNA decoding occur in a mostly stochastic manner. Little is known about the response of higher eukaryotes to chronic loss of ribosomal accuracy as per an increase in the random error rate of mRNA decoding. Here, we present a global and comprehensive picture of the cellular changes in response to translational accuracy in mammalian ribosomes impaired by genetic manipulation. In addition to affecting established protein quality control pathways, such as elevated transcript levels for cytosolic chaperones, activation of the ubiquitin-proteasome system, and translational slowdown, ribosomal mistranslation led to unexpected responses. In particular, we observed increased mitochondrial biogenesis associated with import of misfolded proteins into the mitochondria and silencing of the unfolded protein response in the endoplasmic reticulum., Communications Biology, 2, ISSN:2399-3642

OBJECTIVES: To examine the relation between breakfast frequency and incidence of diabetes in middle-aged women. METHODS: The Mexican Teacher´s Cohort is a prospective study in women. We included 71,373 participants at baseline (2006–2008). Participants were classified according to breakfast frequency 0, 1–3, 4–6, or 7 d/wk; and meal frequency 1–2, 3–4, or ≥5 meals/d. Diabetes was self-reported. We used Cox proportional hazards models to calculate hazard ratios (HR) and 95% confidence intervals (CI) to estimate the association between breakfast frequency and diabetes incidence. Models were adjusted for sociodemographic and lifestyle confounders that are associated with breakfast consumption and are risk factors for diabetes. Stratified analyses were performed for age, birth weight, indigenous background, and physical activity. RESULTS: We identified 3,613 new diabetes cases during a median of 2.2 years of follow-up. Prevalence of daily breakfast consumers was 25%. After adjustment for known risk factors for diabetes, compared to 0 d/wk, women who eat daily breakfast had 12% lower rate of diabetes (HR = 0.88; 95% CI 0.78, 0.99; p-trend = 0.0018). One day additional per week having breakfast decreased the risk of diabetes (HR = 0.98; CI 0.97, 0.99). In stratified analysis, women with indigenous background who consumed breakfast 4–6 d/wk and 7 d/wk vs. 0 d/wk shown lower risk (HR = 0.68; 95% CI 0.47, 0.98) and HR = 0.76; 95% CI 0.76 (0.51, 1.15) respectively; p-interaction = 0.197). CONCLUSIONS: Daily breakfast was associated with a lower incidence of diabetes, independently of dietary and lifestyle factors. Likely effect modifiers as ethnicity warrants more research. Daily breakfast consumption is a potential component of diabetes prevention. FUNDING SOURCES: This work is supported by the American Institute for Cancer Research (05B047) and Consejo Nacional de Ciencia y Tecnología (CONACyT) grant S0008-2009-1: 000000000115312.

Why was the cohort set up? Latin America and the Caribbean (LAC) are characterized by much diversity in terms of socio-economic status, ecology, environment, access to health care,1,2 as well as the frequency of risk factors for and prevalence or incidence of non-communicable diseases;3–7 importantly, these differences are observed both between and within countries in LAC.8,9 LAC countries share a large burden of non-communicable (e.g. diabetes and hypertension) and cardiovascular (e.g. ischaemic heart disease) diseases, with these conditions standing as the leading causes of morbidity, disability and mortality in most of LAC.10–12 These epidemiological estimates—e.g. morbidity—cannot inform about risk factors or risk prediction, which are relevant to identify prevention avenues. Cohort studies, on the other hand, could provide this evidence. Pooled analysis, using data from multiple cohort studies, have additional strengths such as increased statistical power and decreased statistical uncertainty.13 LAC cohort studies have been under-represented,14 or not included at all,15–17 in international efforts aimed at pooling data from multiple cohort studies. We therefore set out to pool data from LAC cohorts to address research questions that individual cohort studies would not be able to answer. Drawing from previous successful regional enterprises (e.g. Asia Pacific Cohort Studies Collaboration),18,19 we established the Cohorts Consortium of Latin America and the Caribbean (CC-LAC). The main aim of the CC-LAC is to start a collaborative cohort data pooling in LAC to examine the association between cardio-metabolic risk factors (e.g. blood pressure, glucose and lipids) and non-fatal and fatal cardiovascular outcomes (e.g. stroke or myocardial infarction). In so doing, we aim to provide regional risk estimates to inform disease burden metrics, as well as other ambitious projects including a cardiovascular risk score to strengthen cardiovascular prevention in LAC. Initial funding has been provided by a fellowship from the Wellcome Trust Centre for Global Health Research at Imperial College London (Strategic Award, Wellcome Trust–Imperial College Centre for Global Health Research, 100693/Z/12/Z). Additional funding is being provided by an International Training Fellowship from the Wellcome Trust (214185/Z/18/Z). At the time of writing, the daily operations and pooled database are hosted at Imperial College London, though a mid-term goal is to transfer this expertise and operations to LAC. The collaboration relies fundamentally on a strong regional network of health researchers and practitioners

Objective: Although history of preeclampsia increases a woman′s risk of developing hypertension (HTN) in adulthood, it remains unclear whether a heart-healthy post-pregnancy diet could ameliorate some of this risk. Therefore, we evaluated whether adherence to dietary patterns is related with HTN among women with prior preeclampsia Methods: At baseline, 4,781 women without HTN reported at least one pregnancy with preeclampsia in the 2008 Mexican Teachers’ Cohort follow-up cycle. From the baseline food frequency questionnaire (FFQ) we derived diet scores based on the American Heart Association (AHA) 2020 Goals and the Dietary Approaches to Stop Hypertension (DASH). The AHA score has 5 items: fruits and vegetables, fish, whole grain, sugar-sweetened beverages and sodium, whereas the DASH diet has 8: low-fat dairy, nuts/legumes, fruits, vegetables, whole grain, red and processed meats, sugar-sweetened beverages, and sodium. Incident HTN reported in 2011, was defined as high blood pressure diagnosed by a medical doctor or currently taking antihypertensive treatment. We estimated the relative risk (RR) and 95% confidence intervals (95%CI) of HTN by comparing women in the lowest score quintile to women in the highest score quintile, using log-binomial regression after adjusting for common risk factors for HTN as well as health care provider, demographic regions and indigenous heritage. Results: The mean age (SD) of women affected by preeclampsia was 41(7) years and BMI of 28(5) kg/m 2 . Over the 3-year study period, 555(12%) women developed incident HTN, which was higher that the incidence of HTN among women with normotensive pregnancies (6%). Women with the highest AHA scores had 0.82(95% CI 0.68,1.06) times the risk of developing HTN than women with lowest scores (p-trend=0.04). Similarly, highest adherence to the DASH diet had 0.74(95% CI 0.57,0.96) times the risk of developing HTN than women with the lowest score(p-trend=0.03). When individual components of the diet were separately evaluated, only sodium intake was associated with HTN. Specifically, women who consumed ≥2,500 mg/day of sodium had 1.18(0.92,1.52) times the risk of HTN compared to women who consumed Conclusion: Greater adherence to AHA and DASH diet patterns were inversely related to risk of HTN among Mexican women with a history of preeclampsia and even though lower sodium intake was also related to HTN, the FFQ underestimates absolute sodium intake. Nevertheless, post-partum recommendations on diet quality in this high-risk group may provide one strategy to prevent long-term risk of HTN.

To the Editor, As Zhou and Reveille note,1 MICA is a functionally enticing candidate gene for ankylosingspondylitis (AS). It is however challenging to study because of the technical difficulty of direct genotyping studies of the locus, its proximity and strong linkage disequilibrium with HLA-B , and the fact that it is subject to major population stratification effects. We recently performed an association study of common MICA alleles with AS susceptibility and observed no evidence of association in either HLA-B*27 positive or negative stratified analyses.2 In this study, …

The UK Biobank project is a prospective cohort study with deep genetic and phenotypic data collected on approximately 500,000 individuals from across the United Kingdom, aged between 40 and 69 at recruitment. The open resource is unique in its size and scope. A rich variety of phenotypic and health-related information is available on each participant, including biological measurements, lifestyle indicators, biomarkers in blood and urine, and imaging of the body and brain. Follow-up information is provided by linking health and medical records. Genome-wide genotype data have been collected on all participants, providing many opportunities for the discovery of new genetic associations and the genetic bases of complex traits. Here we describe the centralized analysis of the genetic data, including genotype quality, properties of population structure and relatedness of the genetic data, and efficient phasing and genotype imputation that increases the number of testable variants to around 96 million. Classical allelic variation at 11 human leukocyte antigen genes was imputed, resulting in the recovery of signals with known associations between human leukocyte antigen alleles and many diseases.

Background Among the 100 genes associated with ankylosing spondylitis (AS), RUNX3 a transcription factor (TF) involved in diverse immunological processes has a very robust (10−15) association.1 The biggest challenge following association studies remain to understand the mechanism behind this association and get insights of the disease. We have recently demonstrated that the association between AS and the single nucleotide polymorphism (SNP) rs4648889 located in a 2 kb regulatory locus upstream the promoter of RUNX3 can be explained by allele-specific effects on TF recruitment that alter gene expression, specifically in CD8 +T cells.2 In addition, another closely adjacent SNP, rs4265380 shows functional effects (i.e. TF recruitment, histone marks enrichment and cell count) on CD14 +monocytes.3 Objectives The main objectives of this work are: 1) to dissect the functional effects of the different SNPs at the RUNX3 locus, acting in different cell types (especially CD8 +T cells and monocytes); 2) to identify the different interacting partners (i.e. TFs) binding at the RUNX3 locus in the presence of the AS-associated alleles. Methods We used publicly available dataset to define the epigenetic landscape of the RUNX3 locus. In vitro functional studies were performed to characterise the effects of these specific genetic variants, providing critical functional evidence for their role in AS. Results (1) Roadmap data revealed a robust peak for open chromatin and specific histone modifications associated with regulatory elements. Hi-C data showed the interaction of RUNX3 with different genomic loci within chromosome 1, in GM12878 lymphoblastoid cell line, CD8 +T cells and monocytes; (2) ChIP-qPCR experiments on monocytes and CD8 +T cells from AS patients revealed the enrichment for several histone modifications (i.e. H3K79Me2 and H3K4Me1) at the RUNX3 locus overlapping the SNPs of interest; (3) preliminary DNA pull-down experiments, followed by Mass Spectrometry, started to identify the whole range of proteins and TFs (DNA/protein ‘interactome’) that bind at the RUNX3 locus in the presence of the AS-associated alleles, evaluated in both CD8 +T cells and monocytes; (4) initial pathway enrichment analysis highlighted the distinct contribution of proteins involved in the transcriptional machinery (AS-risk VS protective alleles). Conclusions We provide first evidence that the 2 kb region upstream the RUNX3 gene has a plausible functional role in AS. These new observations are critically important not only in identifying specific cell types that play a pathogenic role in AS, but also in defining dysregulated pathways and potential therapeutic drug targets. References [1] ] IGAS, et al. Nat Genet2013Jul;45(7):730–8. [2] Vecellio M, et al. Ann Rheum Dis2016Aug;75(8):1534–40. [3] Vecellio M, et al. RMD Open2018. Disclosure of Interest None declared

A small percentage of women with cervical HPV infection progress to cervical neoplasia, and the risk factors determining progression are incompletely understood. We sought to define the genetic loci involved in cervical neoplasia and to assess its heritability using unbiased unrelated case/control statistical approaches. We demonstrated strong association of cervical neoplasia with risk and protective HLA haplotypes that are determined by the amino-acids carried at positions 13 and 71 in pocket 4 of HLA-DRB1 and position 156 in HLA-B. Furthermore, 36% (standard error 2.4%) of liability of HPV-associated cervical pre-cancer and cancer is determined by common genetic variants. Women in the highest 10% of genetic risk scores have approximately >7.1% risk, and those in the highest 5% have approximately >21.6% risk, of developing cervical neoplasia. Future studies should examine genetic risk prediction in assessing the risk of cervical neoplasia further, in combination with other screening methods., Author summary Around 1% of women with cervical human papillomavirus (HPV) infection progress to cervical cancer. Previous studies had indicated that a person’s genetic makeup could predispose to HPV-associated cervical cancer, and that some of the genes likely to be involved include the immune-related human leukocyte antigen (HLA) genes among the major histocompatibility complex (MHC). However, it has been difficult to determine which alleles might be associated with cervical pre-cancer or cancer due to the complex and high level of co-inheritance of MHC alleles. Here, we performed a genome-wide association study that assessed the correlation of genetic variants among those with cervical cancer and healthy controls. We show that host genetics is a major determinant of HPV-associated cervical cancer, with 36% of liability due to common genetic variants in the population, and identify both risk and protective HLA alleles. Our study was also sufficiently powerful to identify particular residue variants on a number of the immune-related proteins that provide risk or protection, providing further insight into the biological basis for cervical cancer development. Our findings could lay the foundation for screening for people at increased risk of developing cancer following HPV infection, and aid in the treatment and prognosis of cervical cancer.

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 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.

The UK Biobank project is a large prospective cohort study of ~500,000 individuals from across the United Kingdom, aged between 40-69 at recruitment. A rich variety of phenotypic and health-related information is available on each participant, making the resource unprecedented in its size and scope. Here we describe the genome-wide genotype data (~805,000 markers) collected on all individuals in the cohort and its quality control procedures. Genotype data on this scale offers novel opportunities for assessing quality issues, although the wide range of ancestries of the individuals in the cohort also creates particular challenges. We also conducted a set of analyses that reveal properties of the genetic data – such as population structure and relatedness – that can be important for downstream analyses. In addition, we phased and imputed genotypes into the dataset, using computationally efficient methods combined with the Haplotype Reference Consortium (HRC) and UK10K haplotype resource. This increases the number of testable variants by over 100-fold to ~96 million variants. We also imputed classical allelic variation at 11 human leukocyte antigen (HLA) genes, and as a quality control check of this imputation, we replicate signals of known associations between HLA alleles and many common diseases. We describe tools that allow efficient genome-wide association studies (GWAS) of multiple traits and fast phenome-wide association studies (PheWAS), which work together with a new compressed file format that has been used to distribute the dataset. As a further check of the genotyped and imputed datasets, we performed a test-case genome-wide association scan on a well-studied human trait, standing height.

Free to read Objective To use high-density genotyping to investigate the genetic associations of acute anterior uveitis (AAU) in patients with and those without ankylosing spondylitis (AS). Methods We genotyped samples from 1,711 patients with AAU (either primary or combined with AS), 2,339 AS patients without AAU, and 10,000 control subjects on an Illumina Immunochip Infinium microarray. We also used data for AS patients from previous genome-wide association studies to investigate the AS risk locus ANTXR2 for its putative effect in AAU. ANTXR2 expression in mouse eyes was investigated by real-time quantitative reverse transcription–polymerase chain reaction. Results A comparison between all patients with AAU and healthy control subjects showed strong association over HLA–B, corresponding to the HLA–B27 tag single-nucleotide polymorphism rs116488202. The association of 3 non–major histocompatibility complex loci, IL23R, the intergenic region 2p15, and ERAP1, reached genome-wide significance (P < 5 × 10−8). Five loci harboring the immune-related genes IL10–IL19, IL18R1–IL1R1, IL6R, the chromosome 1q32 locus harboring KIF21B, as well as the eye-related gene EYS, were also associated, reaching a suggestive level of significance (P < 5 × 10−6). Several previously confirmed AS associations demonstrated significant differences in effect size between AS patients with AAU and AS patients without AAU. ANTXR2 expression varied across eye compartments. Conclusion These findings of both novel AAU-specific associations and associations shared with AS demonstrate overlapping but also distinct genetic susceptibility loci for AAU and AS. The associations in IL10 and IL18R1 are shared with inflammatory bowel disease, suggesting common etiologic pathways.

Ankylosing spondylitis (AS) susceptibility is strongly correlated with genetic variation within the major histocompatibility complex (MHC), and the class I HLA-B*27 allele confers the major genetic risk factor to AS. Furthermore, strong evidence for additional alleles in the MHC has been observed which affect susceptibility independently from the HLA-B*27 allele, either by direct genotyping in candidate gene studies or through large-scale imputation-driven association studies. We have previously identified variants in both class I and II which affect susceptibility to AS through imputation of classical HLA alleles...

Shared aetiopathogenic factors among immune-mediated diseases have long been suggested by their co-familiality and co-occurrence, and molecular support has been provided by analysis of human leukocyte antigen (HLA) haplotypes and genome-wide association studies. The interrelationships can now be better appreciated following the genotyping of large immune disease sample sets on a shared SNP array: the 'Immunochip'. Here, we systematically analyse loci shared among major immune-mediated diseases. This reveals that several diseases share multiple susceptibility loci, but there are many nuances. The most associated variant at a given locus frequently differs and, even when shared, the same allele often has opposite associations. Interestingly, risk alleles conferring the largest effect sizes are usually disease-specific. These factors help to explain why early evidence of extensive 'sharing' is not always reflected in epidemiological overlap.

Free to read Objective Ankylosing spondylitis (AS) is a highly heritable common inflammatory arthritis that targets the spine and sacroiliac joints of the pelvis, causing pain and stiffness and leading eventually to joint fusion. Although previous studies have shown a strong association of IL23R with AS in white Europeans, similar studies in East Asian populations have shown no association with common variants of IL23R, suggesting either that IL23R variants have no role or that rare genetic variants contribute. The present study was undertaken to screen IL23R to identify rare variants associated with AS in Han Chinese. Methods A 170-kb region containing IL23R and its flanking regions was sequenced in 50 patients with AS and 50 ethnically matched healthy control subjects from a Han Chinese population. In addition, the 30-kb region of peak association in white Europeans was sequenced in 650 patients with AS and 1,300 healthy controls. Validation genotyping was undertaken in 846 patients with AS and 1,308 healthy controls. Results We identified 1,047 variants, of which 729 were not found in the dbSNP genomic build 130. Several potentially functional rare variants in IL23R were identified, including one nonsynonomous single-nucleotide polymorphism (nsSNP), Gly149Arg (position 67421184 GA on chromosome 1). Validation genotyping showed that the Gly149Arg variant was associated with AS (odds ratio 0.61, P = 0.0054). Conclusion This is the first study to implicate rare IL23R variants in the pathogenesis of AS. The results identified a low-frequency nsSNP with predicted loss-of-function effects that was protectively associated with AS in Han Chinese, suggesting that decreased function of the interleukin-23 (IL-23) receptor protects against AS. These findings further support the notion that IL-23 signaling has an important role in the pathogenesis of AS.

Genetic discovery from the multitude of phenotypes extractable from routine healthcare data has the ability to radically transform our understanding of the human phenome, thereby accelerating progress towards precision medicine. However, a critical question when analysing high-dimensional and heterogeneous data is how to interrogate increasingly specific subphenotypes whilst retaining statistical power to detect genetic associations. Here we develop and employ a novel Bayesian analysis framework that exploits the hierarchical structure of diagnosis classifications to jointly analyse genetic variants against UK Biobank healthcare phenotypes. Our method displays a more than 20% increase in power to detect genetic effects over other approaches, such that we uncover the broader burden of genetic variation: we identify associations with over 2,000 diagnostic terms. We find novel associations with common immune-mediated diseases (IMD), we reveal the extent of genetic sharing between specific IMDs, and we expose differences in disease perception or diagnosis with potential clinical implications.

We investigated the proposal that ankylosing spondylitis (AS) is associated with unusual ERAP1 genotypes. ERAP1 haplotypes were constructed for 213 AS cases and 46 rheumatoid arthritis controls using family data. Haplotypes were generated from five common ERAP1 single nucleotide polymorphisms (SNPs)—rs2287987 (M349V), rs30187 (K528R), rs10050860 (D575N), rs17482078 (R725Q), and rs27044 (Q730E). Haplotype frequencies were compared using Q:12 Fisher’s exact test. ERAP1 haplotypes imputed from the IGAS Immunochip study were also studied. In the family study, we identified only four common ERAP1 haplotypes (“VRNQE,” “MKDRQ,” “MRDRE,” and “MKDRE”) in both AS cases and controls apart from two rare (

Thousands of genetic variants have been identified, which contribute to the development of complex diseases, but determining how to elucidate their biological consequences for translation into clinical benefit is challenging. Conflicting evidence regarding the functional impact of genetic variants in the tyrosine kinase 2 (TYK2) gene, which is differentially associated with common autoimmune diseases, currently obscures the potential of TYK2 as a therapeutic target. We aimed to resolve this conflict by performing genetic meta-analysis across disorders; subsequent molecular, cellular, in vivo, and structural functional follow-up; and epidemiological studies. Our data revealed a protective homozygous effect that defined a signaling optimum between autoimmunity and immunodeficiency and identified TYK2 as a potential drug target for certain common autoimmune disorders.

SummaryAdult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.

Spondyloarthritis (SpA) is often diagnosed late in the course of the disease and improved methods for early diagnosis are required. We have tested the ability of genetic profiling to diagnose axial SpA (axSpA) as a whole group, or ankylosing spondylitis (AS) alone, in a cohort of chronic back pain patients.282 patients were recruited from centres in the United Kingdom, Germany, Taiwan, Canada, Columbia and Turkey as part of the ASAS classification criteria for axSpA study (ASAS cohort). Subjects were classified according to the ASAS axSpA criteria, and the modified New York Criteria for AS. Patients were genotyped for ~200,000 immune-mediated disease SNPs using the Illumina Immunochip.We first established the predictive accuracy of genetic data comparing 9,638 healthy controls and 4,428 AS cases from the homogenous International Genetics of AS (IGAS) Consortium Immunochip study which showed excellent predictive power (AUC=0.91). Genetic risk scores had lower predictive power (AUC=0.83) comparing ASAS cohort axSpA cases meeting the ASAS imaging criteria with IGAS controls. Comparing genetic risk scores showed moderate discriminatory capacity between IGAS AS and ASAS imaging positive cases (AUC 0.67±0.05), indicating that significant differences in genetic makeup exist between the cohorts.In a clinical setting of referred back pain patients suspected to have axial SpA we were unable to use genetic data to construct a predictive model better than that based on existing clinical data. Potential confounding factors include significant heterogeneity in the ASAS cohort, possibly reflecting the disease heterogeneity of axSpA, or differences between centres in ascertainment or classification performance.

© 2016 Nature America, Inc. We simultaneously investigated the genetic landscape of ankylosing spondylitis, Crohn's disease, psoriasis, primary sclerosing cholangitis and ulcerative colitis to investigate pleiotropy and the relationship between these clinically related diseases. Using high-density genotype data from more than 86,000 individuals of European ancestry, we identified 244 independent multidisease signals, including 27 new genome-wide significant susceptibility loci and 3 unreported shared risk loci. Complex pleiotropy was supported when contrasting multidisease signals with expression data sets from human, rat and mouse together with epigenetic and expressed enhancer profiles. The comorbidities among the five immune diseases were best explained by biological pleiotropy rather than heterogeneity (a subgroup of cases genetically identical to those with another disease, possibly owing to diagnostic misclassification, molecular subtypes or excessive comorbidity). In particular, the strong comorbidity between primary sclerosing cholangitis and inflammatory bowel disease is likely the result of a unique disease, which is genetically distinct from classical inflammatory bowel disease phenotypes.

Inflammatory bowel disease (IBD) is a chronic gastrointestinal inflammatory disorder that affects millions worldwide. Genome-wide association studies (GWAS) have identified 200 IBD-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 IBD loci using high-density genotyping in 67,852 individuals. Of the 139 independent associations identified in these regions, 18 were pinpointed to a single causal variant with >95% certainty, and an additional 27 associations to a single variant with >50% certainty. These 45 variants are significantly enriched for protein-coding changes (n=13), direct disruption of transcription factor binding sites (n=3) and tissue specific epigenetic marks (n=10), with the latter category showing enrichment in specific immune cells among associations stronger in CD and gut mucosa among associations stronger in UC. The results of this study suggest that high-resolution, fine-mapping in large samples can convert many GWAS discoveries into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms.

The association of endoplasmic reticulum aminopeptidase 2 (ERAP2) with ankylosing spondylitis (AS) was recently described in the large International Genetics of AS Consortium Immunochip study. Variants in ERAP2 have also been associated with inflammatory bowel disease, psoriasis, acute anterior uveitis and birdshot chorioretinopathy. Subsequent investigation demonstrated an association of ERAP2 with AS which was present when one conditioned on one of the two independent haplotypes of ERAP1 associated with AS or when HLA-B27-negative patients were analysed separately. These two analyses provide analogous evidence for the association of ERAP2 with AS in HLA-B27-negative cases because of the genetic interaction between HLA-B27 and the AS-associated ERAP1 variants in AS cases. ERAP1 and ERAP2 are located on chromosome 5q15 in the opposite orientation. The locus is challenging to analyse because of the strong linkage disequilibrium (LD) across the locus and the epistasis between ERAP1 and HLA-B alleles associated with AS. We therefore sought to investigate the association of ERAP2 with AS in HLA-B27-positive patients. This is of clinical importance because functional studies have demonstrated that the strongly AS-protective variant rs2248374 causes a functional ERAP2 protein knockout, because its G allele causes a loss of ERAP2 protein expression. There is also a variant of ERAP2 which changes its enzyme catalytic activity and specificity (rs2549782, K392A). Because this is in almost complete LD with rs2248374 (1000 Genomes D′=1.00, r2=0.90), it is almost never translated in vivo. Further, the very strong LD between these markers means that analysis of rs2549782 for association would yield results almost identical to the results for rs2248374 presented below. Therefore, it is of relevance to determine whether the association of ERAP2 with HLA-B27-negative disease is also found in HLA-B27-positive cases, since ERAP inhibition may offer a novel therapeutic for AS...

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

ObjectiveTo identify genetic associations with severity of radiographic damage in ankylosing spondylitis (AS).MethodWe studied 1537 AS cases of European descent; all fulfilled the modified New York Criteria. Radiographic severity was assessed from digitised lateral radiographs of the cervical and lumbar spine using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). A two-phase genotyping design was used. In phase 1, 498 single nucleotide polymorphisms (SNPs) were genotyped in 688 cases; these were selected to capture >90% of the common haplotypic variation in the exons, exon–intron boundaries, and 5 kb flanking DNA in the 5′ and 3′ UTR of 74 genes involved in anabolic or catabolic bone pathways. In phase 2, 15 SNPs exhibiting pResultsExperiment-wise association was observed with the SNP rs8092336 (combined OR 0.32, p=1.2×10−5), which lies within RANK (receptor activator of NFκB), a gene involved in osteoclastogenesis, and in the interaction between T cells and dendritic cells. Association was also found with the SNP rs1236913 in PTGS1 (prostaglandin-endoperoxide synthase 1, cyclooxygenase 1), giving an OR of 0.53 (p=2.6×10−3). There was no observed association between radiographic severity and HLA-B*27.ConclusionsThese findings support roles for bone resorption and prostaglandins pathways in the osteoproliferative changes in AS.

A small percentage of women with cervical HPV infection progress to cervical neoplasia, and the risk factors determining progression are incompletely understood. We sought to define the genetic loci involved in cervical neoplasia and to assess its heritability using unbiased unrelated case/control statistical approaches. We demonstrated strong association of cervical neoplasia with risk and protective HLA haplotypes that are determined by the amino-acids carried at positions 13 and 71 in pocket 4 of HLA-DRB1 and position 156 in HLA-B. Furthermore, 36% (standard error 2.4%) of liability of HPV-associated cervical pre-cancer and cancer is determined by common genetic variants. Women in the highest 10% of genetic risk scores have approximately >7.1% risk, and those in the highest 5% have approximately >21.6% risk, of developing cervical neoplasia. Future studies should examine genetic risk prediction in assessing the risk of cervical neoplasia further, in combination with other screening methods.