Pharmacogenetics at scale in real-world bioresources: CYP2C19 and clopidogrel outcomes in UK Biobank.

OBJECTIVE: The impact of CYP2C19 genotype on clopidogrel outcomes is one of the most well established pharmacogenetic interactions, supported by robust evidence and recommended by the Food and Drug Administration and clinical pharmacogenetics implementation consortium. However, there is a scarcity of large-scale real-world data on the extent of this pharmacogenetic effect, and clinical testing for the CYP2C19 genotype remains infrequent. This study utilizes the UK Biobank dataset, including 10 365 patients treated with clopidogrel, to offer the largest observational analysis of these pharmacogenetic effects to date. METHODS: Incorporating time-varying drug exposure and repeated clinical outcome, we adopted semiparametric frailty models to detect and quantify exposure-based effects of CYP2C19 (*2,*17) variants and nongenetic factors on the incidence risks of composite outcomes of death or recurrent hospitalizations due to major adverse cardiovascular events (MACE) or hemorrhage in the entire cohort of clopidogrel-treated patients. RESULTS: Out of the 10 365 clopidogrel-treated patients, 40% (4115) experienced 10 625 MACE events during an average follow-up of 9.23 years. Individuals who received clopidogrel (coverage >25%) with a CYP2C19*2 loss-of-function allele had a 9.4% higher incidence of MACE [incidence rate ratios (IRR), 1.094; 1.044-1.146], but a 15% lower incidence of hemorrhage (IRR, 0.849; 0.712-0.996). These effects were stronger with high clopidogrel exposure. Conversely, the gain-of-function CYP2C19*17 variant was associated with a 5.3% lower incidence of MACE (IRR, 0.947; 0.903-0.983). Notably, there was no evidence of *2 or *17 effects when clopidogrel exposure was low, confirming the presence of a drug-gene interaction. CONCLUSION: The impact of CYP2C19 on clinical outcomes in clopidogrel-treated patients is substantial, highlighting the importance of incorporating genotype-based prescribing into clinical practice, regardless of the reason for clopidogrel use or the duration of treatment. Moreover, the methodology introduced in this study can be applied to further real-world investigations of known drug-gene and drug-drug interactions and the discovery of novel interactions.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Effect of spatial context on perceived walking direction.

Contextual modulation occurs for many aspects of high-level vision but is relatively unexplored for the perception of walking direction. In a recent study, we observed an effect of the temporal context on perceived walking direction. Here, we examined the spatial contextual modulation by measuring the perceived direction of a target point-light walker in the presence of two flanker walkers, one on each side. Experiment 1 followed a within-subjects design. Participants (n = 30) completed a spatial context task by judging the walking direction of the target in 13 different conditions: a walker alone in the center or with two flanking walkers either intact or scrambled at a flanker deviation of ±15°, ±30°, or ±45°. For comparison, participants completed an adaptation task where they reported the walking direction of a target after adaptation to ±30° walking direction. We found the expected repulsive effects in the adaptation task but attractive effects in the spatial context task. In Experiment 2 (n = 40), we measured the tuning of spatial contextual modulation across a wide range of flanker deviation magnitudes ranging from 15° to 165° in 15° intervals. Our results showed significant attractive effects across a wide range of flanker walking directions with the peak effect at around 30°. The assimilative versus repulsive effects of spatial contextual modulation and temporal adaptation suggest dissociable neural mechanisms, but they may operate on the same population of sensory channels coding for walking direction, as evidenced by similarity in the peak tuning across the walking direction of the inducers.

Competing risks analysis for neutrophil to lymphocyte ratio as a predictor of diabetic retinopathy incidence in the Scottish population.

BACKGROUND: Diabetic retinopathy (DR) is a major sight-threatening microvascular complication in individuals with diabetes. Systemic inflammation combined with oxidative stress is thought to capture most of the complexities involved in the pathology of diabetic retinopathy. A high level of neutrophil-lymphocyte ratio (NLR) is an indicator of abnormal immune system activity. Current estimates of the association of NLR with diabetes and its complications are almost entirely derived from cross-sectional studies, suggesting that the nature of the reported association may be more diagnostic than prognostic. Therefore, in the present study, we examined the utility of NLR as a biomarker to predict the incidence of DR in the Scottish population. METHODS: The incidence of DR was defined as the time to the first diagnosis of R1 or above grade in the Scottish retinopathy grading scheme from type 2 diabetes diagnosis. The effect of NLR and its interactions were explored using a competing risks survival model adjusting for other risk factors and accounting for deaths. The Fine and Gray subdistribution hazard model (FGR) was used to predict the effect of NLR on the incidence of DR. RESULTS: We analysed data from 23,531 individuals with complete covariate information. At 10 years, 8416 (35.8%) had developed DR and 2989 (12.7%) were lost to competing events (death) without developing DR and 12,126 individuals did not have DR. The median (interquartile range) level of NLR was 2.04 (1.5 to 2.7). The optimal NLR cut-off value to predict retinopathy incidence was 3.04. After accounting for competing risks at 10 years, the cumulative incidence of DR and deaths without DR were 50.7% and 21.9%, respectively. NLR was associated with incident DR in both Cause-specific hazard (CSH = 1.63; 95% CI: 1.28-2.07) and FGR models the subdistribution hazard (sHR = 2.24; 95% CI: 1.70-2.94). Both age and HbA1c were found to modulate the association between NLR and the risk of DR. CONCLUSIONS: The current study suggests that NLR has a promising potential to predict DR incidence in the Scottish population, especially in individuals less than 65 years and in those with well-controlled glycaemic status.

Detection of Mooney faces is robust to image asymmetries produced by illumination.

Face detection relies on the visual features that are shared across different faces. An important component of the basic spatial configuration of a face is symmetry around the vertical midline. Although human faces are structurally symmetrical, they can be asymmetrical in an image due to the direction of lighting or the position of the face. In the experiments presented here, we examined how face detection from simple contrast patterns that occur across the face is affected by the image asymmetries associated with variations in the horizontal lighting direction. We presented observers with two-tone images of faces (Mooney faces) that isolated the unique pattern of contrast in the shading and shadows on a face, illuminated from a wide range of horizontal directions. In two experiments, we found that face detection is surprisingly robust to these lighting changes, with sensitivity in discriminating between face and non-face patterns reduced only at the most extreme lighting directions. This tolerance to changes in the horizontal lighting direction depended partly on the orientation of the face, vertical lighting direction, and contrast polarity. Our results provide insight into how contrast cues produced by shading and shadows occurring across the facial surface are utilized by the visual system to detect human faces.

Diabetes status modifies the long-term effect of lipoprotein-associated phospholipase A2 on major coronary events.

AIMS/HYPOTHESIS: Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity has an independent prognostic association with major coronary events (MCE). However, no study has investigated whether type 2 diabetes status modifies the effect of Lp-PLA2 activity or inhibition on the risk of MCE. We investigate the interaction between diabetes status and Lp-PLA2 activity with risk of MCE. Subsequently, we test the resulting hypothesis that diabetes status will play a role in modifying the efficacy of an Lp-PLA2 inhibitor. METHODS: A retrospective cohort study design was utilised in two study populations. Discovery analyses were performed in the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) cohort based in Scotland, UK. Participants were categorised by type 2 diabetes control status: poorly controlled (HbA1c ≥ 48 mmol/mol or ≥6.5%) and well-controlled (HbA1c < 48 mmol/mol or <6.5%) diabetes (n = 7420). In a secondary analysis of the Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy (STABILITY) trial of Lp-PLA2 inhibitor (darapladib) efficacy, 15,828 participants were stratified post hoc by type 2 diabetes diagnosis status (diabetes or no diabetes) at time of recruitment. Lp-PLA2 activity was then divided into population-specific quartiles. MCE were determined from linked medical records in GoDARTS and trial records in STABILITY. First, the interaction between diabetes control status and Lp-PLA2 activity on the outcome of MCE was explored in GoDARTS. The effect was replicated in the placebo arm of STABILITY. The effect of Lp-PLA2 on MCE was then examined in models stratified by diabetes status. This helped determine participants at higher risk. Finally, the effect of Lp-PLA2 inhibition was assessed in STABILITY in the higher risk group. Cox proportional hazards models adjusted for confounders were used to assess associations. RESULTS: In GoDARTS, a significant interaction between increased Lp-PLA2 activity (continuous and quartile divided) and diabetes control status was observed in the prediction of MCE (p < 0.0001). These effects were replicated in the placebo arm of STABILITY (p < 0.0001). In GoDARTS, stratified analyses showed that, among individuals with poorly controlled diabetes, the hazards of MCE for those with high (Q4) Lp-PLA2 activity was 1.19 compared with individuals with lower (Q1-3) Lp-PLA2 activity (95% CI 1.11, 1.38; p < 0.0001) and 1.35 (95% CI 1.16, 1.57; p < 0.0001) when compared with those with the lowest activity (Q1). Those in the higher risk group were identified as individuals with the highest Lp-PLA2 activity (Q4) and poorly controlled diabetes or diabetes. Based on these observations in untreated populations, we hypothesised that the Lp-PLA2 inhibitor would have more benefit in this higher risk group. In this risk group, Lp-PLA2 inhibitor use was associated with a 33% reduction in MCE compared with placebo (HR 0.67 [95% CI 0.50, 0.90]; p = 0.008). In contrast, Lp-PLA2 inhibitor showed no efficacy in individuals with low activity, regardless of diabetes status, or among those with no baseline diabetes and high Lp-PLA2 activity. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that diabetes status modifies the association between Lp-PLA2 activity and MCE. These results suggest that cardiovascular morbidity and mortality associated with Lp-PLA2 activity is especially important in patients with type 2 diabetes, particularly those with worse glycaemic control. Further investigation of the effects of Lp-PLA2 inhibition in diabetes appears warranted. DATA AVAILABILITY: STABILITY trial data are available from clinicaltrials.gov repository through the GlaxoSmithKline clinical study register https://clinicaltrials.gov/ct2/show/NCT00799903 . GoDARTS datasets generated during and/or analysed during the current study are available following request to the GoDARTS Access Managements Group https://godarts.org/scientific-community/ .

Young-onset diabetes in Asian Indians is associated with lower measured and genetically determined beta cell function.

AIMS/HYPOTHESIS: South Asians in general, and Asian Indians in particular, have higher risk of type 2 diabetes compared with white Europeans, and a younger age of onset. The reasons for the younger age of onset in relation to obesity, beta cell function and insulin sensitivity are under-explored. METHODS: Two cohorts of Asian Indians, the ICMR-INDIAB cohort (Indian Council of Medical Research-India Diabetes Study) and the DMDSC cohort (Dr Mohan's Diabetes Specialties Centre), and one of white Europeans, the ESDC (East Scotland Diabetes Cohort), were used. Using a cross-sectional design, we examined the comparative prevalence of healthy, overweight and obese participants with young-onset diabetes, classified according to their BMI. We explored the role of clinically measured beta cell function in diabetes onset in Asian Indians. Finally, the comparative distribution of a partitioned polygenic score (pPS) for risk of diabetes due to poor beta cell function was examined. Replication of the genetic findings was sought using data from the UK Biobank. RESULTS: The prevalence of young-onset diabetes with normal BMI was 9.3% amongst white Europeans and 24-39% amongst Asian Indians. In Asian Indians with young-onset diabetes, after adjustment for family history of type 2 diabetes, sex, insulin sensitivity and HDL-cholesterol, stimulated C-peptide was 492 pmol/ml (IQR 353-616, p<0.0001) lower in lean compared with obese individuals. Asian Indians in our study, and South Asians from the UK Biobank, had a higher number of risk alleles than white Europeans. After weighting the pPS for beta cell function, Asian Indians have lower genetically determined beta cell function than white Europeans (p<0.0001). The pPS was associated with age of diagnosis in Asian Indians but not in white Europeans. The pPS explained 2% of the variation in clinically measured beta cell function, and 1.2%, 0.97%, and 0.36% of variance in age of diabetes amongst Asian Indians with normal BMI, or classified as overweight and obese BMI, respectively. CONCLUSIONS/INTERPRETATION: The prevalence of lean BMI in young-onset diabetes is over two times higher in Asian Indians compared with white Europeans. This phenotype of lean, young-onset diabetes appears driven in part by lower beta cell function. We demonstrate that Asian Indians with diabetes also have lower genetically determined beta cell function.

Genome-wide meta-analysis and omics integration identifies novel genes associated with diabetic kidney disease.

AIMS/HYPOTHESIS: Diabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets. METHODS: We performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets. RESULTS: The meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR<60 ml/min per 1.73 m2) and DKD (microalbuminuria or worse) phenotype (p=9.8×10-9; although not withstanding correction for multiple testing, p>9.3×10-9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN-RESP18, GPR158, INIP-SNX30, LSM14A and MFF; p<2.7×10-6). Integration of GWAS with human glomerular and tubular expression data demonstrated higher tubular AKIRIN2 gene expression in individuals with vs without DKD (p=1.1×10-6). The lead SNPs within six loci significantly altered DNA methylation of a nearby CpG site in kidneys (p<1.5×10-11). Expression of lead genes in kidney tubules or glomeruli correlated with relevant pathological phenotypes (e.g. TENM2 expression correlated positively with eGFR [p=1.6×10-8] and negatively with tubulointerstitial fibrosis [p=2.0×10-9], tubular DCLK1 expression correlated positively with fibrosis [p=7.4×10-16], and SNX30 expression correlated positively with eGFR [p=5.8×10-14] and negatively with fibrosis [p<2.0×10-16]). CONCLUSIONS/INTERPRETATION: Altogether, the results point to novel genes contributing to the pathogenesis of DKD. DATA AVAILABILITY: The GWAS meta-analysis results can be accessed via the type 1 and type 2 diabetes (T1D and T2D, respectively) and Common Metabolic Diseases (CMD) Knowledge Portals, and downloaded on their respective download pages ( https://t1d.hugeamp.org/downloads.html ; https://t2d.hugeamp.org/downloads.html ; https://hugeamp.org/downloads.html ).

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans.

BACKGROUND: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans. METHODS: In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting. RESULTS: Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men. CONCLUSIONS: These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.

A genome-wide association study finds genetic variants associated with neck or shoulder pain in UK Biobank.

BACKGROUND: Common types of musculoskeletal conditions include pain in the neck and shoulder areas. This study seeks to identify the genetic variants associated with neck or shoulder pain based on a genome-wide association approach using 203 309 subjects from the UK Biobank cohort and look for replication evidence from the Generation Scotland: Scottish Family Health Study (GS:SFHS) and TwinsUK. METHODS: A genome-wide association study was performed adjusting for age, sex, BMI and nine population principal components. Significant and independent genetic variants were then sent to GS:SFHS and TwinsUK for replication. RESULTS: We identified three genetic loci that were associated with neck or shoulder pain in the UK Biobank samples. The most significant locus was in an intergenic region in chromosome 17, rs12453010, having P = 1.66 × 10-11. The second most significant locus was located in the FOXP2 gene in chromosome 7 with P = 2.38 × 10-10 for rs34291892. The third locus was located in the LINC01572 gene in chromosome 16 with P = 4.50 × 10-8 for rs62053992. In the replication stage, among four significant and independent genetic variants, rs2049604 in the FOXP2 gene and rs62053992 in the LINC01572 gene were weakly replicated in GS:SFHS (P = 0.0240 and P = 0.0202, respectively). CONCLUSIONS: We have identified three loci associated with neck or shoulder pain in the UK Biobank cohort, two of which were weakly supported in a replication cohort. Further evidence is needed to confirm their roles in neck or shoulder pain.

Spatial selectivity in adaptation to gaze direction.

A person's focus of attention is conveyed by the direction of their eyes and face, providing a simple visual cue fundamental to social interaction. A growing body of research examines the visual mechanisms that encode the direction of another person's gaze as we observe them. Here we investigate the spatial receptive field properties of these mechanisms, by testing the spatial selectivity of sensory adaptation to gaze direction. Human observers were adapted to faces with averted gaze presented in one visual hemifield, then tested in their perception of gaze direction for faces presented in the same or opposite hemifield. Adaptation caused strong, repulsive perceptual aftereffects, but only for faces presented in the same hemifield as the adapter. This occurred even though adapting and test stimuli were in the same external location across saccades. Hence, there was clear evidence for retinotopic adaptation and a relative lack of either spatiotopic or spatially invariant adaptation. These results indicate that adaptable representations of gaze direction in the human visual system have retinotopic spatial receptive fields. This strategy of coding others' direction of gaze with positional specificity relative to one's own eye position may facilitate key functions of gaze perception, such as socially cued shifts in visual attention.

The Relationship between AKI and CKD in Patients with Type 2 Diabetes: An Observational Cohort Study.

BACKGROUND: There are few observational studies evaluating the risk of AKI in people with type 2 diabetes, and even fewer simultaneously investigating AKI and CKD in this population. This limits understanding of the interplay between AKI and CKD in people with type 2 diabetes compared with the nondiabetic population. METHODS: In this retrospective, cohort study of participants with or without type 2 diabetes, we used electronic healthcare records to evaluate rates of AKI and various statistical methods to determine their relationship to CKD status and further renal function decline. RESULTS: We followed the cohort of 16,700 participants (9417 with type 2 diabetes and 7283 controls without diabetes) for a median of 8.2 years. Those with diabetes were more likely than controls to develop AKI (48.6% versus 17.2%, respectively) and have preexisting CKD or CKD that developed during follow-up (46.3% versus 17.2%, respectively). In the absence of CKD, the AKI rate among people with diabetes was nearly five times that of controls (121.5 versus 24.6 per 1000 person-years). Among participants with CKD, AKI rate in people with diabetes was more than twice that of controls (384.8 versus 180.0 per 1000 person-years after CKD diagnostic date, and 109.3 versus 47.4 per 1000 person-years before CKD onset in those developing CKD after recruitment). Decline in eGFR slope before AKI episodes was steeper in people with diabetes versus controls. After AKI episodes, decline in eGFR slope became steeper in people without diabetes, but not among those with diabetes and preexisting CKD. CONCLUSIONS: Patients with diabetes have significantly higher rates of AKI compared with patients without diabetes, and this remains true for individuals with preexisting CKD.

Asthma prescribing according to Arg16Gly beta-2 genotype: a randomised trial in adolescents.

INTRODUCTION: The A allele of rs1042713 (Arg16 amino acid) in the ß2-adrenoreceptor is associated with poor response to long-acting ß2-agonist (LABA) in young people with asthma. Our aim was to assess whether the prescribing of second-line controller with LABA or a leukotriene receptor antagonist according to Arg16Gly genotype would result in improvements in Pediatric Asthma-Related Quality of Life Questionnaire (PAQLQ). METHODS: We performed a pragmatic randomised controlled trial (RCT) via a primary care clinical research network covering England and Scotland. We enrolled participants aged 12-18 years with asthma taking inhaled corticosteroids. 241 participants (mean±sd age 14.7±1.91 years) were randomised (1:1) to receive personalised care (genotype directed prescribing) or standard guideline care. Following a 4-week run-in participants were followed for 12 months. The primary outcome measure was change in PAQLQ. Asthma control, asthma exacerbation frequency and healthcare utilisation were secondary outcomes. RESULTS: Genotype-directed prescribing resulted in an improvement in PAQLQ compared to standard care (0.16, 95% CI 0.00-0.31; p=0.049), although this improvement was below the pre-determined clinical threshold of 0.25. The AA genotype was associated with a larger improvement in PAQLQ with personalised versus standard care (0.42, 95% CI 0.02-0.81; p=0.041). CONCLUSION: This is the first RCT demonstrating that genotype-driven asthma prescribing is associated with a significant improvement in a clinical outcome compared to standard care. Adolescents with the AA homozygous genotype benefited most. The potential role of such ß2-adrenoceptor genotype directed therapy in younger and more severe childhood asthma warrants further exploration.

Genome-wide association study of asthma exacerbations despite inhaled corticosteroid use.

RATIONALE: Substantial variability in response to asthma treatment with inhaled corticosteroids (ICS) has been described among individuals and populations, suggesting the contribution of genetic factors. Nonetheless, only a few genes have been identified to date. We aimed to identify genetic variants associated with asthma exacerbations despite ICS use in European children and young adults and to validate the findings in non-Europeans. Moreover, we explored whether a gene-set enrichment analysis could suggest potential novel asthma therapies. METHODS: A genome-wide association study (GWAS) of asthma exacerbations was tested in 2681 children of European descent treated with ICS from eight studies. Suggestive association signals were followed up for replication in 538 European asthma patients. Further evaluation was performed in 1773 non-Europeans. Variants revealed by published GWAS were assessed for replication. Additionally, gene-set enrichment analysis focused on drugs was performed. RESULTS: 10 independent variants were associated with asthma exacerbations despite ICS treatment in the discovery phase (p≤5×10-6). Of those, one variant at the CACNA2D3-WNT5A locus was nominally replicated in Europeans (rs67026078; p=0.010), but this was not validated in non-European populations. Five other genes associated with ICS response in previous studies were replicated. Additionally, an enrichment of associations in genes regulated by trichostatin A treatment was found. CONCLUSIONS: The intergenic region of CACNA2D3 and WNT5A was revealed as a novel locus for asthma exacerbations despite ICS treatment in European populations. Genes associated were related to trichostatin A, suggesting that this drug could regulate the molecular mechanisms involved in treatment response.

PheGWAS: a new dimension to visualize GWAS across multiple phenotypes.

MOTIVATION: PheGWAS was developed to enhance exploration of phenome-wide pleiotropy at the genome-wide level through the efficient generation of a dynamic visualization combining Manhattan plots from GWAS with PheWAS to create a 3D 'landscape'. Pleiotropy in sub-surface GWAS significance strata can be explored in a sectional view plotted within user defined levels. Further complexity reduction is achieved by confining to a single chromosomal section. Comprehensive genomic and phenomic coordinates can be displayed. RESULTS: PheGWAS is demonstrated using summary data from Global Lipids Genetics Consortium GWAS across multiple lipid traits. For single and multiple traits PheGWAS highlighted all 88 and 69 loci, respectively. Further, the genes and SNPs reported in Global Lipids Genetics Consortium were identified using additional functions implemented within PheGWAS. Not only is PheGWAS capable of identifying independent signals but also provides insights to local genetic correlation (verified using HESS) and in identifying the potential regions that share causal variants across phenotypes (verified using colocalization tests). AVAILABILITY AND IMPLEMENTATION: The PheGWAS software and code are freely available at (https://github.com/georgeg0/PheGWAS). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ADRB2 haplotypes and asthma exacerbations in children and young adults: An individual participant data meta-analysis.

BACKGROUND: The polymorphism Arg16 in ß2 -adrenergic receptor (ADRB2) gene has been associated with an increased risk of exacerbations in asthmatic children treated with long-acting ß2 -agonists (LABA). However, it remains unclear whether this increased risk is mainly attributed to this single variant or the combined effect of the haplotypes of polymorphisms at codons 16 and 27. OBJECTIVE: We assessed whether the haplotype analysis could explain the association between the polymorphisms at codons 16 (Arg16Gly) and 27 (Gln27Glu) in ADRB2 and risk of asthma exacerbations in patients treated with inhaled corticosteroids (ICS) plus LABA. METHODS: The study was undertaken using data from 10 independent studies (n = 5903) participating in the multi-ethnic Pharmacogenomics in Childhood Asthma (PiCA) consortium. Asthma exacerbations were defined as asthma-related use of oral corticosteroids or hospitalizations/emergency department visits in the past 6 or 12 months prior to the study visit/enrolment. The association between the haplotypes and the risk of asthma exacerbations was performed per study using haplo.stats package adjusted for age and sex. Results were meta-analysed using the inverse variance weighting method assuming random-effects. RESULTS: In subjects treated with ICS and LABA (n = 832, age: 3-21 years), Arg16/Gln27 versus Gly16/Glu27 (OR: 1.40, 95% CI: 1.05-1.87, I2  = 0.0%) and Arg16/Gln27 versus Gly16/Gln27 (OR: 1.43, 95% CI: 1.05-1.94, I2  = 0.0%), but not Gly16/Gln27 versus Gly16/Glu27 (OR: 0.99, 95% CI: 0.71-1.39, I2  = 0.0%), were significantly associated with an increased risk of asthma exacerbations. The sensitivity analyses indicated no significant association between the ADRB2 haplotypes and asthma exacerbations in the other treatment categories, namely as-required short-acting ß2 -agonists (n = 973), ICS monotherapy (n = 2623), ICS plus leukotriene receptor antagonists (LTRA; n = 338), or ICS plus LABA plus LTRA (n = 686). CONCLUSION AND CLINICAL RELEVANCE: The ADRB2 Arg16 haplotype, presumably mainly driven by the Arg16, increased the risk of asthma exacerbations in patients treated with ICS plus LABA. This finding could be beneficial in ADRB2 genotype-guided treatment which might improve clinical outcomes in asthmatic patients.

Genome-wide association studies of exacerbations in children using long-acting beta2-agonists.

BACKGROUND: Some children with asthma experience exacerbations despite long-acting beta2-agonist (LABA) treatment. While this variability is partly caused by genetic variation, no genome-wide study until now has investigated which genetic factors associated with risk of exacerbations despite LABA use in children with asthma. We aimed to assess whether genetic variation was associated with exacerbations in children treated with LABA from a global consortium. METHODS: A meta-analysis of genome-wide association studies (meta-GWAS) was performed in 1,425 children and young adults with asthma (age 6-21 years) with reported regular use of LABA from six studies within the PiCA consortium using a random effects model. The primary outcome of each study was defined as any exacerbation within the past 6 or 12 months, including at least one of the following: 1) hospital admissions for asthma, 2) a course of oral corticosteroids or 3) emergency room visits because of asthma. RESULTS: Genome-wide association results for a total of 82 996 common single nucleotide polymorphisms (SNPs, MAF ≥1%) with high imputation quality were meta-analysed. Eight independent variants were suggestively (P-value threshold ≤5 × 10-6 ) associated with exacerbations despite LABA use. CONCLUSION: No strong effects of single nucleotide polymorphisms (SNPs) on exacerbations during LABA use were identified. We identified two loci (TBX3 and EPHA7) that were previously implicated in the response to short-acting beta2-agonists (SABA). These loci merit further investigation in response to LABA and SABA use.

Early experience with an opt-in research register - Scottish Health Research Register (SHARE): a multi-method evaluation of participant recruitment performance.

BACKGROUND: Recruiting participants to a clinical study is a resource-intensive process with a high failure rate. The Scottish Health Research Register (SHARE) provides recruitment support service which helps researchers recruit participants by searching patients' Electronic Health Records (EHRs). The current study aims to evaluate the performance of SHARE in participant recruitment. METHODS: Recruitment projects eligible for evaluation were those that were conducted for clinical trials or observational studies and finished before 2020. For analysis of recruitment data, projects with incomplete data were excluded. For each project we calculated, from SHARE records, 1) the fraction of the participants recruited through SHARE as a percentage of the number requested by researchers (percentage fulfilled), 2) the percentage of the potential candidates provided by SHARE to researchers that were actually recruited (percentage provided and recruited), 3) the percentage of the participants recruited through SHARE of all the potentially eligible candidates identified by searching registrants' EHRs (percentage identified and recruited). Research teams of the eligible projects were invited to participate in an anonymised online survey. Two metrics were derived from research teams' responses, including a) the fraction of the recruited over the study target number of participants (percentage fulfilled), and b) the percentage of the participants recruited through SHARE among the candidates received from SHARE (percentage provided and recruited). RESULTS: Forty-four projects were eligible for inclusion. Recruitment data for 24 projects were available (20 excluded because of missingness or incompleteness). Survey invites were sent to all the eligible research teams and received 12 responses. Analysis of recruitment data shows the overall percentage fulfilled was 34.2% (interquartile 13.3-45.1%), the percentage provided and recruited 29.3% (interquartile 20.6-52.4%) and percentage identified and recruited 4.9% (interquartile 2.6-10.2%). Based on the data reported by researchers, percentage fulfilled was 31.7% (interquartile 5.8-59.6%) and percentage provided and recruited was 20.2% (interquartile 8.2-31.0%). CONCLUSIONS: SHARE may be a valuable resource for recruiting participants for some clinical studies. Potential improvements are to expand the registrant base and to incorporate more data generated during patients' different health care encounters into the candidate-searching step.

Mitigating local over-fitting during single particle reconstruction with SIDESPLITTER.

Single particle analysis has become a key structural biology technique. Experimental images are extremely noisy, and during iterative refinement it is possible to stably incorporate noise into the reconstruction. Such "over-fitting" can lead to misinterpretation of the structure and flawed biological results. Several strategies are routinely used to prevent over-fitting, the most common being independent refinement of two sides of a split dataset. In this study, we show that over-fitting remains an issue within regions of low local signal-to-noise, despite independent refinement of half datasets. We propose a modification of the refinement process through the application of a local signal-to-noise filter: SIDESPLITTER. We show that our approach can reduce over-fitting for both idealised and experimental data while maintaining independence between the two sides of a split refinement. SIDESPLITTER refinement leads to improved density, and can also lead to improvement of the final resolution in extreme cases where datasets are prone to severe over-fitting, such as small membrane proteins.

Genetic and pharmacological relationship between P-glycoprotein and increased cardiovascular risk associated with clarithromycin prescription: An epidemiological and genomic population-based cohort study in Scotland, UK.

BACKGROUND: There are conflicting reports regarding the association of the macrolide antibiotic clarithromycin with cardiovascular (CV) events. A possible explanation may be that this risk is partly mediated through drug-drug interactions and only evident in at-risk populations. To the best of our knowledge, no studies have examined whether this association might be mediated via P-glycoprotein (P-gp), a major pathway for clarithromycin metabolism. The aim of this study was to examine CV risk following prescription of clarithromycin versus amoxicillin and in particular, the association with P-gp, a major pathway for clarithromycin metabolism. METHODS AND FINDINGS: We conducted an observational cohort study of patients prescribed clarithromycin or amoxicillin in the community in Tayside, Scotland (population approximately 400,000) between 1 January 2004 and 31 December 2014 and a genomic observational cohort study evaluating genotyped patients from the Genetics of Diabetes Audit and Research Tayside Scotland (GoDARTS) study, a longitudinal cohort study of 18,306 individuals with and without type 2 diabetes recruited between 1 December 1988 and 31 December 2015. Two single-nucleotide polymorphisms associated with P-gp activity were evaluated (rs1045642 and rs1128503 -AA genotype associated with lowest P-gp activity). The primary outcome for both analyses was CV hospitalization following prescription of clarithromycin versus amoxicillin at 0-14 days, 15-30 days, and 30 days to 1 year. In the observational cohort study, we calculated hazard ratios (HRs) adjusted for likelihood of receiving clarithromycin using inverse proportion of treatment weighting as a covariate, whereas in the pharmacogenomic study, HRs were adjusted for age, sex, history of myocardial infarction, and history of chronic obstructive pulmonary disease. The observational cohort study included 48,026 individuals with 205,227 discrete antibiotic prescribing episodes (34,074 clarithromycin, mean age 73 years, 42% male; 171,153 amoxicillin, mean age 74 years, 45% male). Clarithromycin use was significantly associated with increased risk of CV hospitalization compared with amoxicillin at both 0-14 days (HR 1.31; 95% CI 1.17-1.46, p < 0.001) and 30 days to 1 year (HR 1.13; 95% CI 1.06-1.19, p < 0.001), with the association at 0-14 days modified by use of P-gp inhibitors or substrates (interaction p-value: 0.029). In the pharmacogenomic study (13,544 individuals with 44,618 discrete prescribing episodes [37,497 amoxicillin, mean age 63 years, 56% male; 7,121 clarithromycin, mean age 66 years, 47% male]), when prescribed clarithromycin, individuals with genetically determined lower P-gp activity had a significantly increased risk of CV hospitalization at 30 days to 1 year compared with heterozygotes or those homozygous for the non-P-gp-lowering allele (rs1045642 AA: HR 1.39, 95% CI 1.20-1.60, p < 0.001, GG/GA: HR 0.99, 95% CI 0.89-1.10, p = 0.85, interaction p-value < 0.001 and rs1128503 AA 1.41, 95% CI 1.18-1.70, p < 0.001, GG/GA: HR 1.04, 95% CI 0.95-1.14, p = 0.43, interaction p-value < 0.001). The main limitation of our study is its observational nature, meaning that we are unable to definitively determine causality. CONCLUSIONS: In this study, we observed that the increased risk of CV events with clarithromycin compared with amoxicillin was associated with an interaction with P-glycoprotein.