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.

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.

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.

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.

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.

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.

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.

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.

Genome-wide association study of angioedema induced by angiotensin-converting enzyme inhibitor and angiotensin receptor blocker treatment.

Angioedema in the mouth or upper airways is a feared adverse reaction to angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blocker (ARB) treatment, which is used for hypertension, heart failure and diabetes complications. This candidate gene and genome-wide association study aimed to identify genetic variants predisposing to angioedema induced by these drugs. The discovery cohort consisted of 173 cases and 4890 controls recruited in Sweden. In the candidate gene analysis, ETV6, BDKRB2, MME, and PRKCQ were nominally associated with angioedema (p < 0.05), but did not pass Bonferroni correction for multiple testing (p < 2.89 × 10-5). In the genome-wide analysis, intronic variants in the calcium-activated potassium channel subunit alpha-1 (KCNMA1) gene on chromosome 10 were significantly associated with angioedema (p < 5 × 10-8). Whilst the top KCNMA1 hit was not significant in the replication cohort (413 cases and 599 ACEi-exposed controls from the US and Northern Europe), a meta-analysis of the replication and discovery cohorts (in total 586 cases and 1944 ACEi-exposed controls) revealed that each variant allele increased the odds of experiencing angioedema 1.62 times (95% confidence interval 1.05-2.50, p = 0.030). Associated KCNMA1 variants are not known to be functional, but are in linkage disequilibrium with variants in transcription factor binding sites active in relevant tissues. In summary, our data suggest that common variation in KCNMA1 is associated with risk of angioedema induced by ACEi or ARB treatment. Future whole exome or genome sequencing studies will show whether rare variants in KCNMA1 or other genes contribute to the risk of ACEi- and ARB-induced angioedema.

Novel Genetic Locus Influencing Retinal Venular Tortuosity Is Also Associated With Risk of Coronary Artery Disease.

OBJECTIVE: The retina may provide readily accessible imaging biomarkers of global cardiovascular health. Increasing evidence suggests variation in retinal vascular traits is highly heritable. This study aimed to identify the genetic determinants of retinal vascular traits. Approach and Results: We conducted a meta-analysis of genome-wide association studies for quantitative retinal vascular traits derived using semi-automatic image analysis of digital retinal photographs from the GoDARTS (Genetics of Diabetes Audit and Research in Tayside; N=1736) and ORCADES (Orkney Complex Disease Study; N=1358) cohorts. We identified a novel genome-wide significant locus at 19q13 (ACTN4/CAPN12) for retinal venular tortuosity (TortV), and one at 13q34 (COL4A2) for retinal arteriolar tortuosity (TortA); these 2 loci were subsequently confirmed in 3 independent cohorts (Ntotal=1413). In the combined analysis of discovery and replication cohorts, the lead single-nucleotide polymorphism in ACTN4/CAPN12 was rs1808382 (ßs.d.=-0.109; SE=0.015; P=2.39×10-13) and in COL4A2 was rs7991229 (ßs.d.=0.103; SE=0.015; P=4.66×10-12). Notably, the ACTN4/CAPN12 locus associated with TortV is also associated with coronary artery disease, heart rate, and atrial fibrillation. CONCLUSIONS: Genetic determinants of retinal vascular tortuosity are also linked to cardiovascular health. These findings provide a molecular pathophysiological foundation for the use of retinal vascular traits as biomarkers for cardiovascular diseases.

Serum kidney injury molecule 1 and ß2-microglobulin perform as well as larger biomarker panels for prediction of rapid decline in renal function in type 2 diabetes.

AIMS/HYPOTHESIS: As part of the Surrogate Markers for Micro- and Macrovascular Hard Endpoints for Innovative Diabetes Tools (SUMMIT) programme we previously reported that large panels of biomarkers derived from three analytical platforms maximised prediction of progression of renal decline in type 2 diabetes. Here, we hypothesised that smaller (n ≤ 5), platform-specific combinations of biomarkers selected from these larger panels might achieve similar prediction performance when tested in three additional type 2 diabetes cohorts. METHODS: We used 657 serum samples, held under differing storage conditions, from the Scania Diabetes Registry (SDR) and Genetics of Diabetes Audit and Research Tayside (GoDARTS), and a further 183 nested case-control sample set from the Collaborative Atorvastatin in Diabetes Study (CARDS). We analysed 42 biomarkers measured on the SDR and GoDARTS samples by a variety of methods including standard ELISA, multiplexed ELISA (Luminex) and mass spectrometry. The subset of 21 Luminex biomarkers was also measured on the CARDS samples. We used the event definition of loss of >20% of baseline eGFR during follow-up from a baseline eGFR of 30-75 ml min-1 [1.73 m]-2. A total of 403 individuals experienced an event during a median follow-up of 7 years. We used discrete-time logistic regression models with tenfold cross-validation to assess association of biomarker panels with loss of kidney function. RESULTS: Twelve biomarkers showed significant association with eGFR decline adjusted for covariates in one or more of the sample sets when evaluated singly. Kidney injury molecule 1 (KIM-1) and ß2-microglobulin (B2M) showed the most consistent effects, with standardised odds ratios for progression of at least 1.4 (p < 0.0003) in all cohorts. A combination of B2M and KIM-1 added to clinical covariates, including baseline eGFR and albuminuria, modestly improved prediction, increasing the area under the curve in the SDR, Go-DARTS and CARDS by 0.079, 0.073 and 0.239, respectively. Neither the inclusion of additional Luminex biomarkers on top of B2M and KIM-1 nor a sparse mass spectrometry panel, nor the larger multiplatform panels previously identified, consistently improved prediction further across all validation sets. CONCLUSIONS/INTERPRETATION: Serum KIM-1 and B2M independently improve prediction of renal decline from an eGFR of 30-75 ml min-1 [1.73 m]-2 in type 2 diabetes beyond clinical factors and prior eGFR and are robust to varying sample storage conditions. Larger panels of biomarkers did not improve prediction beyond these two biomarkers.

Polymorphism in a lincRNA Associates with a Doubled Risk of Pneumococcal Bacteremia in Kenyan Children.

Bacteremia (bacterial bloodstream infection) is a major cause of illness and death in sub-Saharan Africa but little is known about the role of human genetics in susceptibility. We conducted a genome-wide association study of bacteremia susceptibility in more than 5,000 Kenyan children as part of the Wellcome Trust Case Control Consortium 2 (WTCCC2). Both the blood-culture-proven bacteremia case subjects and healthy infants as controls were recruited from Kilifi, on the east coast of Kenya. Streptococcus pneumoniae is the most common cause of bacteremia in Kilifi and was thus the focus of this study. We identified an association between polymorphisms in a long intergenic non-coding RNA (lincRNA) gene (AC011288.2) and pneumococcal bacteremia and replicated the results in the same population (p combined = 1.69 × 10(-9); OR = 2.47, 95% CI = 1.84-3.31). The susceptibility allele is African specific, derived rather than ancestral, and occurs at low frequency (2.7% in control subjects and 6.4% in case subjects). Our further studies showed AC011288.2 expression only in neutrophils, a cell type that is known to play a major role in pneumococcal clearance. Identification of this novel association will further focus research on the role of lincRNAs in human infectious disease.

Genome-wide association study of inhaled corticosteroid response in admixed children with asthma.

BACKGROUND: Inhaled corticosteroids (ICS) are the most widely prescribed and effective medication to control asthma symptoms and exacerbations. However, many children still have asthma exacerbations despite treatment, particularly in admixed populations, such as Puerto Ricans and African Americans. A few genome-wide association studies (GWAS) have been performed in European and Asian populations, and they have demonstrated the importance of the genetic component in ICS response. OBJECTIVE: We aimed to identify genetic variants associated with asthma exacerbations in admixed children treated with ICS and to validate previous GWAS findings. METHODS: A meta-analysis of two GWAS of asthma exacerbations was performed in 1347 admixed children treated with ICS (Hispanics/Latinos and African Americans), analysing 8.7 million genetic variants. Those with P ≤ 5 × 10-6 were followed up for replication in 1697 asthmatic patients from six European studies. Associations of ICS response described in published GWAS were followed up for replication in the admixed populations. RESULTS: A total of 15 independent variants were suggestively associated with asthma exacerbations in admixed populations (P ≤ 5 × 10-6 ). One of them, located in the intergenic region of APOBEC3B and APOBEC3C, showed evidence of replication in Europeans (rs5995653, P = 7.52 × 10-3 ) and was also associated with change in lung function after treatment with ICS (P = 4.91 × 10-3 ). Additionally, the reported association of the L3MBTL4-ARHGAP28 genomic region was confirmed in admixed populations, although a different variant was identified. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed the novel association of APOBEC3B and APOBEC3C with asthma exacerbations in children treated with ICS and replicated previously identified genomic regions. This contributes to the current knowledge about the multiple genetic markers determining responsiveness to ICS which could lead in the future the clinical identification of those asthma patients who are not able to respond to such treatment.

Neutrophil extracellular traps are associated with disease severity and microbiota diversity in patients with chronic obstructive pulmonary disease.

BACKGROUND: Neutrophil extracellular traps (NETs) have been observed in the airway in patients with chronic obstructive pulmonary disease (COPD), but their clinical and pathophysiologic implications have not been defined. OBJECTIVE: We sought to determine whether NETs are associated with disease severity in patients with COPD and how they are associated with microbiota composition and airway neutrophil function. METHODS: NET protein complexes (DNA-elastase and histone-elastase complexes), cell-free DNA, and neutrophil biomarkers were quantified in soluble sputum and serum from patients with COPD during periods of disease stability and during exacerbations and compared with clinical measures of disease severity and the sputum microbiome. Peripheral blood and airway neutrophil function were evaluated by means of flow cytometry ex vivo and experimentally after stimulation of NET formation. RESULTS: Sputum NET complexes were associated with the severity of COPD evaluated by using the composite Global Initiative for Obstructive Lung Disease scale (P < .0001). This relationship was due to modest correlations between NET complexes and FEV1, symptoms evaluated by using the COPD assessment test, and higher levels of NET complexes in patients with frequent exacerbations (P = .002). Microbiota composition was heterogeneous, but there was a correlation between NET complexes and both microbiota diversity (P = .009) and dominance of Haemophilus species operational taxonomic units (P = .01). Ex vivo airway neutrophil phagocytosis of bacteria was reduced in patients with increased sputum NET complexes. Consistent results were observed regardless of the method of quantifying sputum NETs. Failure of phagocytosis could be induced experimentally by incubating healthy control neutrophils with soluble sputum from patients with COPD. CONCLUSION: NET formation is increased in patients with severe COPD and associated with more frequent exacerbations and a loss of microbiota diversity.

Lp-PLA2 activity is associated with increased risk of diabetic retinopathy: a longitudinal disease progression study.

AIMS/HYPOTHESIS: The aim of the study was to examine the association between lipoprotein-associated phospholipase A2 (Lp-PLA2) activity levels and incident diabetic retinopathy and change in retinopathy grade. METHODS: This was a cohort study of diabetic participants with serum collected at baseline and routinely collected diabetic retinal screening data. Participants with type 2 diabetes from the GoDARTS (Genetics of Diabetes Audit and Research in Tayside Scotland) cohort were used. This cohort is composed of individuals of white Scottish ancestry from the Tayside region of Scotland. Survival analysis accounting for informative censoring by modelling death as a competing risk was performed for the development of incident diabetic retinopathy from a disease-free state in a 3 year follow-up period (n = 1364) by stratified Lp-PLA2 activity levels (in quartiles). The same analysis was performed for transitions to more severe grades. RESULTS: The hazard of developing incident diabetic retinopathy was 2.08 times higher (95% CI 1.64, 2.63) for the highest quartile of Lp-PLA2 activity compared with the lowest. Higher Lp-PLA2 activity levels were associated with a significantly increased risk for transitions to all grades. The hazards of developing observable (or more severe) and referable (or more severe) retinopathy were 2.82 (95% CI 1.71, 4.65) and 1.87 (95% CI 1.26, 2.77) times higher for the highest quartile of Lp-PLA2 activity compared with the lowest, respectively. CONCLUSIONS/INTERPRETATION: Higher Lp-PLA2 levels are associated with increased risk of death and the development of incident diabetic retinopathy, as well as transitions to more severe grades of diabetic retinopathy. These associations are independent of calculated LDL-cholesterol and other traditional risk factors. Further, this biomarker study shows that the association is temporally sensitive to the proximity of the event to measurement of Lp-PLA2.

Loss of Cardioprotective Effects at the ADAMTS7 Locus as a Result of Gene-Smoking Interactions.

BACKGROUND: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk. METHODS: We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of <1.0×10-3 (Bonferroni correction for 50 tests). RESULTS: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers (P=1.3×10-16) in comparison with 5% in ever-smokers (P=2.5×10-4), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value=8.7×10-5). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. CONCLUSIONS: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.

Analysis with the exome array identifies multiple new independent variants in lipid loci.

It has been hypothesized that low frequency (1-5% minor allele frequency (MAF)) and rare (<1% MAF) variants with large effect sizes may contribute to the missing heritability in complex traits. Here, we report an association analysis of lipid traits (total cholesterol, LDL-cholesterol, HDL-cholesterol triglycerides) in up to 27 312 individuals with a comprehensive set of low frequency coding variants (ExomeChip), combined with conditional analysis in the known lipid loci. No new locus reached genome-wide significance. However, we found a new lead variant in 26 known lipid association regions of which 16 were >1000-fold more significant than the previous sentinel variant and not in close LD (six had MAF <5%). Furthermore, conditional analysis revealed multiple independent signals (ranging from 1 to 5) in a third of the 98 lipid loci tested, including rare variants. Addition of our novel associations resulted in between 1.5- and 2.5-fold increase in the proportion of heritability explained for the different lipid traits. Our findings suggest that rare coding variants contribute to the genetic architecture of lipid traits.

Genetically determined height and coronary artery disease.

BACKGROUND: The nature and underlying mechanisms of an inverse association between adult height and the risk of coronary artery disease (CAD) are unclear. METHODS: We used a genetic approach to investigate the association between height and CAD, using 180 height-associated genetic variants. We tested the association between a change in genetically determined height of 1 SD (6.5 cm) with the risk of CAD in 65,066 cases and 128,383 controls. Using individual-level genotype data from 18,249 persons, we also examined the risk of CAD associated with the presence of various numbers of height-associated alleles. To identify putative mechanisms, we analyzed whether genetically determined height was associated with known cardiovascular risk factors and performed a pathway analysis of the height-associated genes. RESULTS: We observed a relative increase of 13.5% (95% confidence interval [CI], 5.4 to 22.1; P<0.001) in the risk of CAD per 1-SD decrease in genetically determined height. There was a graded relationship between the presence of an increased number of height-raising variants and a reduced risk of CAD (odds ratio for height quartile 4 versus quartile 1, 0.74; 95% CI, 0.68 to 0.84; P<0.001). Of the 12 risk factors that we studied, we observed significant associations only with levels of low-density lipoprotein cholesterol and triglycerides (accounting for approximately 30% of the association). We identified several overlapping pathways involving genes associated with both development and atherosclerosis. CONCLUSIONS: There is a primary association between a genetically determined shorter height and an increased risk of CAD, a link that is partly explained by the association between shorter height and an adverse lipid profile. Shared biologic processes that determine achieved height and the development of atherosclerosis may explain some of the association. (Funded by the British Heart Foundation and others.).