Role of Transporters and Enzymes in Metabolism and Distribution of 4-Chlorokynurenine (AV-101).

4-Chlorokynurenine (4-Cl-KYN, AV-101) is a prodrug of a NMDA receptor antagonist and is in clinical development for potential CNS indications. We sought to further understand the distribution and metabolism of 4-Cl-KYN, as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays, and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and its metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite has the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.

Characterization of Drug-Specific CD4+ T-Cells Reveals Possible Roles of HLA Class II in the Pathogenesis of Carbamazepine Hypersensitivity Reactions.

Carbamazepine (CBZ) is an aromatic anticonvulsant known to cause drug hypersensitivity reactions, which range in severity from relatively mild maculopapular exanthema to potentially fatal Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS-TEN). These reactions are known to be associated with human leukocyte antigen (HLA) class I alleles, and CBZ interacts preferentially with the related HLA proteins to activate CD8+ T-cells. This study aimed to evaluate the contribution of HLA class II in the effector mechanism(s) of CBZ hypersensitivity. CBZ-specific T-cells clones were generated from two healthy donors and two hypersensitive patients with high-risk HLA class I markers. Phenotype, function, HLA allele restriction, response pathways, and cross-reactivity of CBZ-specific T-cells were assessed using flow cytometry, proliferation analysis, enzyme-linked immunosorbent spot, and enzyme-linked immunosorbent assay. The association between HLA class II allele restriction and CBZ hypersensitivity was reviewed using Allele Frequency Net Database. Forty-four polyclonal CD4+ CBZ-specific T-cell clones were generated and found to be restricted to HLA-DR, particularly HLA-DRB1*07:01. This CD4+-mediated response proceeded through a direct pharmacological interaction between CBZ and HLA-DR molecules. Similar to the CD8+ response, CBZ-stimulated CD4+ clones secreted granulysin, a key mediator of SJS-TEN. Our database review revealed an association between HLA-DRB1*07:01 and CBZ-induced SJS-TEN. These findings implicate HLA class II antigen presentation as an additional pathogenic factor for CBZ hypersensitivity reactions. Both HLA class II molecules and drug-responsive CD4+ T-cells should be evaluated further to gain better insights into the pathogenesis of drug hypersensitivity reactions.

Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.

Platelet response to aspirin in UK and Irish pregnancy cohorts: a genome-wide approach.

A multi-center prospective cross-sectional and genome-wide association study (GWAS) recruited pregnant women taking low dose aspirin. Objectives were to (i) develop pregnancy-specific 95% reference intervals for a range of laboratory based platelet function tests (PFTs); (ii) select an optimal and acceptable PFT that reflected aspirin's COX-1 inhibition in women with confirmed aspirin adherence in pregnancy; and (iii) identify genomic variants that may influence pregnant women's platelet response to aspirin.The study included two independent cohorts of pregnant women. A range of PFTs and matched phenotyping with urinary 11-dehydrothromboxane B2 (11DTXB2) and nuclear magnetic resonance (NMR) spectroscopy detection of urinary salicyluric acid as a measure of aspirin adherence were performed. Genome-wide data was acquired from the UK Biobank Axiom® (Thermo Fisher Scientific). 11DTXB2 in combination with adherence testing with NMR salicyluric acid was an accurate and acceptable testing strategy for detecting biochemical aspirin responsiveness in pregnant women, with the provision of relevant reference ranges. GWAS meta-analysis found no significant single nucleotide polymorphisms in association with response to aspirin in pregnancy. Further evaluation in relation to effective dosing of aspirin in pregnancy and optimizing the benefits to specific subgroups should now be a priority for future research.

Allele frequency net database (AFND) 2020 update: gold-standard data classification, open access genotype data and new query tools.

The Allele Frequency Net Database (AFND, www.allelefrequencies.net) provides the scientific community with a freely available repository for the storage of frequency data (alleles, genes, haplotypes and genotypes) related to human leukocyte antigens (HLA), killer-cell immunoglobulin-like receptors (KIR), major histocompatibility complex Class I chain related genes (MIC) and a number of cytokine gene polymorphisms in worldwide populations. In the last five years, AFND has become more popular in terms of clinical and scientific usage, with a recent increase in genotyping data as a necessary component of Short Population Report article submissions to another scientific journal. In addition, we have developed a user-friendly desktop application for HLA and KIR genotype/population data submissions. We have also focused on classification of existing and new data into 'gold-silver-bronze' criteria, allowing users to filter and query depending on their needs. Moreover, we have also continued to expand other features, for example focussed on HLA associations with adverse drug reactions. At present, AFND contains >1600 populations from >10 million healthy individuals, making AFND a valuable resource for the analysis of some of the most polymorphic regions in the human genome.

Probenecid Increases the Concentration of 7-Chlorokynurenic Acid Derived from the Prodrug 4-Chlorokynurenine within the Prefrontal Cortex.

Recent advances in the understanding of depression have led to increasing interest in ketamine and the role that N-methyl-d-aspartate (NMDA) receptor inhibition plays in depression. l-4-Chlorokynurenine (4-Cl-KYN, AV-101), a prodrug, has shown promise as an antidepressant in preclinical studies, but this promise has not been realized in recent clinical trials. We sought to determine if transporters in the CNS could be playing a role in this clinical response. We used radiolabeled uptake assays and microdialysis studies to determine how 4-Cl-KYN and its active metabolite, 7-chlorokynurenic acid (7-Cl-KYNA), cross the blood-brain barrier (BBB) to access the brain and its extracellular fluid compartment. Our data indicates that 4-Cl-KYN crosses the blood-brain barrier via the amino acid transporter LAT1 (SLC7A5) after which the 7-Cl-KYNA metabolite leaves the brain extracellular fluid via probenecid-sensitive organic anion transporters OAT1/3 (SLC22A6 and SLC22A8) and MRP4 (ABCC4). Microdialysis studies further validated our in vitro data, indicating that probenecid may be used to boost the bioavailability of 7-Cl-KYNA. Indeed, we found that coadministration of 4-Cl-KYN with probenecid caused a dose-dependent increase by as much as an 885-fold increase in 7-Cl-KYNA concentration in the prefrontal cortex. In summary, our data show that 4-Cl-KYN crosses the BBB using LAT1, while its active metabolite, 7-Cl-KYNA, is rapidly transported out of the brain via OAT1/3 and MRP4. We also identify a hitherto unreported mechanism by which the brain extracellular concentration of 7-Cl-KYNA may be increased to produce significant boosting of the drug concentration at its site of action that could potentially lead to an increased therapeutic effect.

Plasma long-chain omega-3 fatty acid status and risk of recurrent early spontaneous preterm birth: a prospective observational study.

INTRODUCTION: A 2018 Cochrane review found that omega-3 supplementation in pregnancy was associated with a risk reduction of early preterm birth of 0.58; prompting calls for universal supplementation. Recent analysis suggests the benefit may be confined to women with a low baseline omega-3 fatty acid status. However, the contemporary omega-3 fatty acid status of pregnant women in the UK is largely unknown. This is particularly pertinent for women with a previous preterm birth, in whom a small relative risk reduction would have a larger reduction of absolute risk. This study aimed to assess the omega-3 fatty acid status of a UK pregnant population and determine the association between the long-chain omega-3 fatty acids and recurrent spontaneous early preterm birth. MATERIAL AND METHODS: A total of 283 high-risk women with previous early preterm birth were recruited to the prospective observational study in Liverpool, UK. Additionally, 96 pregnant women with previous term births and birth ≥39+0  weeks in the index pregnancy provided a low-risk population sample. Within the high-risk group we assessed the odds ratio of recurrent early preterm birth compared with birth at ≥37+0  weeks of gestation according to plasma eicosapentaenoic acid plus docosahexaenoic acid (EPA+DHA) at 15-22 weeks of gestation. RESULTS: Our participants had low EPA+DHA; 62% (143/229) of women with previous preterm birth and 69% (68/96) of the population sample had levels within the lowest two quintiles of a previously published pregnancy cohort. We found no association between long-chain omega-3 status and recurrent early preterm birth (n = 51). The crude odds ratio of a recurrent event was 0.91 (95% CI 0.38-2.15, p = 0.83) for women in the lowest, compared with the highest three quintiles of EPA+DHA. CONCLUSIONS: In the majority of our participants, levels of long-chain omega-3 were low; within the range that may benefit from supplementation. However, levels showed no association with risk of recurrent early spontaneous preterm birth. This could be because our population levels were too low to show benefit in being omega-3 "replete"; or else omega-3 levels may be of lesser importance in recurrent early preterm birth.

Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers.

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10-250 µM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

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.

The longitudinal NIHR ARC North West Coast Household Health Survey: exploring health inequalities in disadvantaged communities.

BACKGROUND: The Household Health Survey (HHS) was developed to understand the socioeconomic determinants of mental and physical health, and health inequalities in health and social care. This paper aims to provide a detailed rationale of the development and implementation of the survey and explore socio-economic variations in physical and mental health and health care. METHODS: This comprehensive longitudinal public health survey was designed and piloted in a disadvantaged area of England, comprising questions on housing, physical health, mental health, lifestyle, social issues, environment, work, and finances. After piloting, the HHS was implemented across 28 neighbourhoods - 10 disadvantaged neighbourhoods for learning (NfLs), 10 disadvantaged comparator sites, and eight relatively advantaged areas, in 2015 and 2018. Participants were recruited via random sampling of households in pre-selected neighbourhoods based on their areas of deprivation. RESULTS: 7731 residents participated in Wave 1 (N = 4319) and 2 (n = 3412) of the survey, with 871 residents having participated in both. Mental health, physical health, employment, and housing quality were poorer in disadvantaged neighbourhoods than in relatively advantaged areas. CONCLUSIONS: This survey provides important insights into socio-economic variations in physical and mental health, with findings having implications for improved care provision to enable residents from any geographical or socio-economic background to access suitable care.

Implementation of genotype-guided dosing of warfarin with point-of-care genetic testing in three UK clinics: a matched cohort study.

BACKGROUND: Warfarin is a widely used oral anticoagulant. Determining the correct dose required to maintain the international normalised ratio (INR) within a therapeutic range can be challenging. In a previous trial, we showed that a dosing algorithm incorporating point-of-care genotyping information ('POCT-GGD' approach) led to improved anticoagulation control. To determine whether this approach could translate into clinical practice, we undertook an implementation project using a matched cohort design. METHODS: At three clinics (implementation group; n = 119), initial doses were calculated using the POCT-GGD approach; at another three matched clinics (control group; n = 93), patients were dosed according to the clinic's routine practice. We also utilised data on 640 patients obtained from routinely collected data at comparable clinics. Primary outcome was percentage time in target INR range. Patients and staff from the implementation group also provided questionnaire feedback on POCT-GGD. RESULTS: Mean percentage time in INR target range was 55.25% in the control group and 62.74% in the implementation group; therefore, 7.49% (95% CI 3.41-11.57%) higher in the implementation group (p = 0.0004). Overall, patients and staff viewed POCT-GGD positively, suggesting minor adjustments to allow smooth implementation into practice. CONCLUSIONS: In the first demonstration of the implementation of genotype-guided dosing, we show that warfarin dosing determined using an algorithm incorporating genetic and clinical factors can be implemented smoothly into clinic, to ensure target INR range is reached sooner and maintained. The findings are like our previous randomised controlled trial, providing an alternative method for improving the risk-benefit of warfarin use in daily practice.

The Effect of Inhibitory Signals on the Priming of Drug Hapten-Specific T Cells That Express Distinct Vß Receptors.

Drug hypersensitivity involves the activation of T cells in an HLA allele-restricted manner. Because the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T cell response. Thus, we have used a T cell-priming assay and nitroso sulfamethoxazole (SMX-NO) as a model Ag to investigate the activation of specific TCR Vß subtypes, the impact of programmed death -1 (PD-1), CTL-associated protein 4 (CTLA4), and T cell Ig and mucin domain protein-3 (TIM-3) coinhibitory signaling on activation of naive and memory T cells, and the ability of regulatory T cells (Tregs) to prevent responses. An expansion of the TCR repertoire was observed for nine Vß subtypes, whereas spectratyping revealed that SMX-NO-specific T cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vß-restricted Ag-specific T cell responses is governed by regulatory signals. Blockade of PD-L1/CTLA4 signaling dampened activation of SMX-NO-specific naive and memory T cells, whereas blockade of TIM-3 produced no effect. Programmed death-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T cell activation, and expression of each receptor was enhanced on dividing T cells. Because these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T cell activation was investigated. Tregs significantly dampened the priming of T cells. In conclusion, our findings demonstrate that distinct TCR Vß subtypes, dysregulation of coinhibitory signaling pathways, and dysfunctional Tregs may influence predisposition to hypersensitivity.

Genetic testing for prevention of severe drug-induced skin rash.

BACKGROUND: Drug-induced skin reactions present with a range of clinical symptoms, from mild maculopapular skin rashes to potentially fatal blistering skin rashes - such as Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) - which may result in death. Milder reactions may be troublesome and lead to low drug compliance. The pathogenesis of these drug reactions is not yet fully understood; however, there is evidence that pretreatment genetic testing may help to predict and prevent these reactions in some cases. OBJECTIVES: To assess the effects of prospective pharmacogenetic screening to reduce drug-associated skin reactions in a patient population. SEARCH METHODS: We searched the following databases up to July 2018: the Cochrane Skin Specialised Register, CENTRAL, MEDLINE, Embase and LILACS. We also searched five trials registers, and checked the reference lists of included studies and relevant reviews for further references to relevant randomised controlled trials (RCTs). SELECTION CRITERIA: We included RCTs of participants who had prospective pharmacogenetic screening to determine genetic variants associated with hypersensitivity reactions, compared with those who did not have prospective pharmacogenetic screening. We included participants in any setting, who were of any age, gender, and ethnicity, who had been prescribed drugs known to cause delayed type hypersensitivity reactions. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. To assess studies for inclusion, two review authors independently screened all of the titles and abstracts of publications identified by the searches. Because there was only one included study, many of the planned data analyses were not applicable to the review. We used GRADE to assess the quality of the included study.The review's primary outcomes were the incidence of severe skin rashes with systemic symptoms (such as fever and multiple organ involvement), and long-term effects (such as scarring of eyelids or lung tissue). Secondary outcomes were hospitalisation for drug-induced skin reactions, blistering skin reactions (such as SJS, hypersensitivity (HSS) syndrome), and death. MAIN RESULTS: One study, which was a randomised, double-blind, controlled, multicentre trial, fulfilled our inclusion criteria. The trial included 1956 adult participants (74% men, with a mean age of 42 years) across 265 centres (medical centres, hospitals, outpatient clinics) in 19 countries around the world who were infected with HIV-type 1 and who had not received abacavir previously. The participants, who had a clinical need for treatment with an antiretroviral-drug regimen containing abacavir, were randomly assigned to undergo prospective human leukocyte antigen (HLA) Class I, locus B, allele 57:01 (HLA-B*57:01) screening (prospective-screening group) before this treatment, or to undergo a standard-care approach of abacavir use without prospective HLA-B*57:01 screening (control group). Participants who tested positive for HLA-B*57:01 were not given abacavir; instead, they received antiretroviral therapy that did not include abacavir. The control group did have retrospective HLA-B*57:01 pharmacogenetic testing. The trial duration was six months. Each participant was observed for six weeks. Assessments were performed at the time of study entry, at baseline (day one of abacavir treatment), and at weeks one, two and six. This study was funded by the manufacturer of abacavir, GlaxoSmithKline.The study did not assess any of our primary outcomes, and it measured none of our secondary outcomes in isolation. However, it did assess an outcome of (characteristically severe) hypersensitivity reaction which included (but was not limited to) our secondary outcomes of HSS and SJS/TEN.The study demonstrated that prospective HLA-B*57:01 screening probably reduces the incidence of hypersensitivity reaction to abacavir. The incidence of clinically diagnosed HSS reaction to abacavir was lower in the screening arm (risk ratio (RR) 0.43, 95% confidence interval (CI) 0.28 to 0.67; 1650 participants; moderate-quality evidence), as was immunologically confirmed HSS reaction (RR 0.02, 95% 0.00 to 0.37; 1644 participants; moderate-quality evidence). A positive result from an epicutaneous patch test performed six to ten weeks after clinical diagnosis provided immunological confirmation.Overall, the study demonstrates a low risk of bias across five out of seven domains. There was a high risk of detection bias because hypersensitivity reactions were diagnosed by the principal investigator at the recruitment site without the use of predefined clinical criteria. Although there was also high risk of attrition bias due to excluding participants with incomplete follow-up from analyses, the authors did undertake a series of sensitivity analyses based on the intention-to-treat population, which demonstrated consistent results with the primary analysis. We rated the study quality as moderate-quality using GRADE criteria. AUTHORS' CONCLUSIONS: Prospective screening for HLA-B*57:01 probably reduces severe hypersensitivity skin reactions to abacavir in patients positive for HIV-type 1. However, these results are only based on one study, which was at high risk of attrition and detection bias.Our primary outcomes (incidence of severe skin rashes with systemic symptoms, and long-term effects) were not assessed by the trial, and only one of the review's secondary outcomes was measured (hypersensitivity reaction); thus, we found no evidence relating to hospitalisation, death, or long-term conditions resulting from drug injury.We found no eligible evidence on genetic testing for severe drug-induced skin rash in relation to different drugs and classes of drugs. Further clinical trials based on other drugs, and in different patient populations, would be useful for advising policy changes for improving the prevention of adverse skin reactions to drug treatments.

Public involvement in the dissemination of the North West Coast Household Health Survey: Experiences and lessons of co-producing research together.

BACKGROUND: Engaging with the public is a key element of health research; however, little work has examined experiences of public involvement in research dissemination. The aim of this paper was to assess the extent of public involvement, experiences of public advisers and resulting changes in the dissemination of the North West Coast Household Health Survey (HHS). METHODS: Three writing groups allowed public advisers to contribute to the dissemination of the HHS. A public workshop was set up to aid the co-production of the research evidence and discuss the experiences of public advisers involved with the survey in March 2018. A focus group with public advisers was conducted in August 2018 to understand their experiences of involvement. Data were analysed using thematic analysis and coded by two researchers. Writing groups are still on-going. RESULTS: Fourteen public advisers contributed via three face-to-face writing groups, by actively interpreting findings and helping in the write-up of research articles and by presenting talks at the public workshop. At the workshop, seven public advisors contributed to setting priorities for data analysis from the HHS. Five public advisers took part in the focus group, which highlighted that whilst public advisers were generally satisfied with their involvement, they would like to be involved in more activities. CONCLUSIONS: Members of the public shaped the dissemination of evidence and provided guidance for future steps. Public advisers were mostly positive about their involvement in the dissemination of the HHS, but highlighted the need for more transparency and support from researchers.

Application of in Vitro T Cell Assay Using Human Leukocyte Antigen-Typed Healthy Donors for the Assessment of Drug Immunogenicity.

It is unclear whether priming of naïve T cells to drugs is detectable in healthy human donors expressing different human leukocyte antigen (HLA) alleles. Thus, we examined T cell priming with drugs associated with HLA risk alleles and control compounds in 14 HLA-typed donors. Nitroso sulfamethoxazole and piperacillin activated T cells from all donors, whereas responses to carbamazepine and oxypurinol were only seen in donors expressing HLA-B*15:02 and HLA-B*58:01, respectively. Weak flucloxacillin-specific T cell responses were detected in donors expressing HLA-B*57:01 and HLA-B*58:01. These data show that the priming of T cells with certain drugs is skewed toward donors expressing specific HLA alleles.

A common missense variant of LILRB5 is associated with statin intolerance and myalgia.

Aims: A genetic variant in LILRB5 (leukocyte immunoglobulin-like receptor subfamily-B) (rs12975366: T > C: Asp247Gly) has been reported to be associated with lower creatine phosphokinase (CK) and lactate dehydrogenase (LDH) levels. Both biomarkers are released from injured muscle tissue, making this variant a potential candidate for susceptibility to muscle-related symptoms. We examined the association of this variant with statin intolerance ascertained from electronic medical records in the GoDARTS study. Methods and results: In the GoDARTS cohort, the LILRB5 Asp247 variant was associated with statin intolerance (SI) phenotypes; one defined as having raised CK and being non-adherent to therapy [odds ratio (OR) 1.81; 95% confidence interval (CI): 1.34-2.45] and the other as being intolerant to the lowest approved dose of a statin before being switched to two or more other statins (OR 1.36; 95% CI: 1.07-1.73). Those homozygous for Asp247 had increased odds of developing both definitions of intolerance. Importantly the second definition did not rely on CK elevations. These results were replicated in adjudicated cases of statin-induced myopathy in the PREDICTION-ADR consortium (OR1.48; 95% CI: 1.05-2.10) and for the development of myalgia in the JUPITER randomized clinical trial of rosuvastatin (OR1.35, 95% CI: 1.10-1.68). A meta-analysis across the studies showed a consistent association between Asp247Gly and outcomes associated with SI (OR1.34; 95% CI: 1.16-1.54). Conclusion: This study presents a novel immunogenetic factor associated with statin intolerance, an important risk factor for cardiovascular outcomes. The results suggest that true statin-induced myalgia and non-specific myalgia are distinct, with a potential role for the immune system in their development. We identify a genetic group that is more likely to be intolerant to their statins.

Genome-wide association study of nevirapine hypersensitivity in a sub-Saharan African HIV-infected population.

Background: The antiretroviral nevirapine is associated with hypersensitivity reactions in 6%-10% of patients, including hepatotoxicity, maculopapular exanthema, Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Objectives: To undertake a genome-wide association study (GWAS) to identify genetic predisposing factors for the different clinical phenotypes associated with nevirapine hypersensitivity. Methods: A GWAS was undertaken in a discovery cohort of 151 nevirapine-hypersensitive and 182 tolerant, HIV-infected Malawian adults. Replication of signals was determined in a cohort of 116 cases and 68 controls obtained from Malawi, Uganda and Mozambique. Interaction with ERAP genes was determined in patients positive for HLA-C*04:01 . In silico docking studies were also performed for HLA-C*04:01 . Results: Fifteen SNPs demonstrated nominal significance ( P < 1 × 10 -5 ) with one or more of the hypersensitivity phenotypes. The most promising signal was seen in SJS/TEN, where rs5010528 ( HLA-C locus) approached genome-wide significance ( P < 8.5 × 10 -8 ) and was below HLA -wide significance ( P < 2.5 × 10 -4 ) in the meta-analysis of discovery and replication cohorts [OR 4.84 (95% CI 2.71-8.61)]. rs5010528 is a strong proxy for HLA-C*04:01 carriage: in silico docking showed that two residues (33 and 123) in the B pocket were the most likely nevirapine interactors. There was no interaction between HLA-C*04:01 and ERAP1 , but there is a potential protective effect with ERAP2 [ P = 0.019, OR 0.43 (95% CI 0.21-0.87)]. Conclusions: HLA-C*04:01 predisposes to nevirapine-induced SJS/TEN in sub-Saharan Africans, but not to other hypersensitivity phenotypes. This is likely to be mediated via binding to the B pocket of the HLA-C peptide. Whether this risk is modulated by ERAP2 variants requires further study.

Cost effectiveness analysis of HLA-B*58:01 genotyping prior to initiation of allopurinol for gout.

Objective: To determine whether prospective testing for HLA-B*58:01, as a strategy to prevent serious adverse reactions to allopurinol in patients with gout, is cost-effective from the perspective of the National Health Service in the UK. Methods: A systematic review and meta-analysis for the association of HLA-B*58:01 with cutaneous and hypersensitivity adverse drug reactions informed a decision analytic and Markov model to estimate lifetime costs and outcomes associated with testing vs standard care (with febuxostat prescribed for patients who test positive). Scenario analyses assessed alternative treatment assumptions and patient populations. Results: The number of patients needed to test to prevent one case of adverse drug reaction was 11 286 (95% central range (CR): 2573, 53 594). Cost and quality-adjusted life-year (QALY) gains were small, £103 (95% CR: £98, £106) and 0.0023 (95% CR: -0.0006, 0.0055), respectively, resulting in an incremental cost-effectiveness ratio (ICER) of £44 954 per QALY gained. The probability of testing being cost-effective at a threshold of £30 000 per QALY was 0.25. Reduced costs of testing or febuxostat resulted in an ICER below £30 000 per QALY gained. The ICER for patients with chronic renal insufficiency was £38 478 per QALY gained. Conclusion: Routine testing for HLA-B*58:01 in order to reduce the incidence of adverse drug reactions in patients being prescribed allopurinol for gout is unlikely to be cost-effective in the UK; however testing is expected to become cost-effective with reductions in the cost of genotyping, and with the future availability of cheaper, generic febuxostat.

Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations.

It has been 12 years since the Allele Frequency Net Database (AFND; http://www.allelefrequencies.net) was first launched, providing the scientific community with an online repository for the storage of immune gene frequencies in different populations across the world. There have been a significant number of improvements from the first version, making AFND a primary resource for many clinical and scientific areas including histocompatibility, immunogenetics, pharmacogenetics and anthropology studies, among many others. The most widely used part of AFND stores population frequency data (alleles, genes or haplotypes) related to human leukocyte antigens (HLA), killer-cell immunoglobulin-like receptors (KIR), major histocompatibility complex class I chain-related genes (MIC) and a number of cytokine gene polymorphisms. AFND now contains >1400 populations from more than 10 million healthy individuals. Here, we report how the main features of AFND have been updated to include a new section on 'HLA epitope' frequencies in populations, a new section capturing the results of studies identifying HLA associations with adverse drug reactions (ADRs) and one for the examination of infectious and autoimmune diseases associated with KIR polymorphisms-thus extending AFND to serve a new user base in these growing areas of research. New criteria on data quality have also been included.