Astrid Yeo

Detection of genotyping errors by Hardy–Weinberg equilibrium testing

Genotyping data sets may contain errors that, in some instances, lead to false conclusions. Deviation from Hardy–Weinberg equilibrium (HWE) in random samples may be indicative of problematic assays. This study has analysed 107u2009000 genotypes generated by TaqMan, RFLP, sequencing or mass spectrometric methods from 443 single-nucleotide polymorphisms (SNPs). These SNPs are distributed both within genes and in intergenic regions. Genotype distributions for 36 out of 313 assays (11.5%) whose minor allele frequencies were >0.05 deviated from HWE (P<0.05). Some of the possible reasons for this deviation were explored: assays for five SNPs proved nonspecific, and genotyping errors were identified in 21 SNPs. For the remaining 10 SNPs, no reasons for deviation from HWE were identified. We demonstrate the successful identification of a proportion of nonspecific assays, and assays harbouring genotyping error. Consequently, our current high-throughput genotyping system incorporates tests for both assay specificity and deviation from HWE, to minimise the genotype error rate and therefore improve data quality.

GLCCI1 rs37973 does not influence treatment response to inhaled corticosteroids in white subjects with asthma

To The Editor: n nInhaled corticosteroids (ICS) are the primary anti-inflammatory therapy for the control and management of asthma, but their effects are characterised by some inter-individual variability that may have a genetic basis1–4. Identification of genetic markers that predict ICS treatment response will facilitate individualised treatment for asthma patients in the future, particularly in those with more severe disease. An association between genetic variation in GLCCI1 and response to ICS therapy in non-Hispanic white asthma subjects was observed by Tantisira et al.5 Genome wide association analysis of 118 trios (one asthmatic child and two parents) from the NHLBI Childhood Asthma Management Program (CAMP) identified 13 single nucleotide polymorphisms (SNPs) with evidence for association with the level of ICS treatment response. These 13 SNPs which included the GLCCI1 promoter polymorphism rs37972, were genotyped and evaluated in four additional independent collections: adult studies SOCS (Salmeterol Or CorticosteroidS) and SLIC (SalmeteroL ± Inhaled CorticosteroidS) (n=264); a second adult study (n=385); LOCCS (Leukotriene modifier Or Corticosteroid or Corticosteroid-Salmeterol) (n=185) and CARE (Childhood Asthma Research and Education) (n=101). In three of the four replicate populations GLCCI1 rs37973, a functional promoter polymorphism5 in complete linkage disequilibrium with rs37972 in white populations, was associated (P<0.05) with change in FEV1 (forced expiratory volume in one second) after 4–8 weeks of ICS treatment. The combined P value measuring association between rs37973 and ICS response over the four collections (n=935) was 0.0007. Tantisira et al. concluded that this functional GLCCI1 polymorphism, rs37973, was associated with response to ICS in asthma patients. n nUsing data from a recently completed genome wide association study of response to steroid therapy, we sought to confirm this observation in a homogeneous, well-characterised population of n=1,924 non-Hispanic white subjects by testing for association between rs37973 and measures of corticosteroid response in subjects treated with either fluticasone furoate (FF) or fluticasone propionate (FP) in seven GSK-sponsored clinical trials (NCT01165138; NCT01134042; NCT01086384; NCT01159912; NCT00603382; NCT00603278; NCT00603746). All seven studies were randomised, double blind, placebo controlled, parallel group multicentre studies in adolescent and adult subjects and employed change from baseline in FEV1 as their primary end-point, apart from HZA106837 which also investigated asthma exacerbations. HZA106837 (N=616) required each subject to have an asthma exacerbation within the 12 months prior to enrolment, whereas the other studies excluded subjects with previous asthma exacerbations. FEV1 was assessed at week 8 for all studies except HZA106837, which used week 12 data. Overall, except for FF and FP dose, baseline demographics and study characteristics were similar across all seven studies (Table 1). n n n nTABLE 1 n nBaseline, demographic and steroid response characteristics in seven GSK-sponsored clinical studies n n n nGermline DNA was extracted from peripheral blood collected from all 1,924 subjects all of whom provided consent for genetic analysis. Genotyping used either the KBiosciences Competitive Allele Specific PCR SNP genotype System (KASPar) (Hoddesdon, Herts, UK) or the Illumina Omni1-Quad panel (Expression Analysis, Durham, NC, USA). Analysis was undertaken in 1,916 subjects, including four with imputed data from rs37972. Eight subjects had missing covariate data. n nGenetic association between rs37973 and ICS response was evaluated, with ICS treatment response defined as change from baseline in trough FEV1 using the last observation carried forward (in any subject who did not complete the specific trial), to week 8 or week 12 of FF or FP treatment. Change in trough FEV1 was regressed against covariates identified in this asthma population: age, percent of predicted baseline FEV1, study, height, asthma duration and drug (FF versus FP). We also evaluated the influence of rs37973 on subject placement within the highest and lowest response quartiles. Subjects who fell into the lowest (n=479) and highest (n=479) response quartiles were identified. Logistic regression was used to fit a model with quartile of response as the dependent variable, and genotype and relevant covariates as the independent variables. The P value measuring heterogeneity among the seven studies was 0.98, allowing pooling of data at the subject level. n nThe minor allele frequency of rs37973 was 0.44 and its genotype frequencies were consistent with Hardy-Weinberg equilibrium (P=0.48). Covariate-adjusted FEV1 change was regressed on rs37973 genotype (Figure 1). Rs37973 did not influence change from baseline in FEV1 in this sample of 1,916 non-Hispanic white subjects treated with either FF or FP (P=0.15). However, this regression analysis suggested a trend toward a slightly lower ICS response for each additional copy of the rs37973 G allele. This direction of effect was consistent with that observed by Tantisira et al. In addition, the percentage change from baseline FEV1 (unadjusted for covariates, results not shown) was 10.4±0.8% in AA homozygotes and 8.8±1.0% in GG homozygotes, while the overall mean response was 9.8±0.4%. n n n nFIGURE 1 n nGLCCI1 rs37973 genotype does not significantly influence steroid response in 1,916 asthma patients n n n nThis genetic marker did not influence subject membership within response quartiles (P=0.08, Odds Ratio (OR) =1.39, 95% CI 0.96–2.00). The ORs in each clinical study ranged from 0.61 (HZA106827) (N=616) to 4.42 (FFA112059), which is one of two smallest clinical trials, N=143 (Table 1). Meta-analysis of the influence of rs37973 on subject placement within response quartile across all seven clinical studies, revealed similar results to the subject level pooled data, suggesting a non-significant trend towards lower ICS response in GLCCI1 rs37973 GG homozygotes, compared to AA homozygotes (P=0.11, OR 1.32, 95% CI 0.94–1.87). In order to closely mimic the analyses of Tantisira et al 5, change in trough FEV1 was also regressed against age, sex and height. All analyses in our sample were repeated using these covariates; there were no qualitative differences between the two sets of results. n nAsthma is currently estimated to affect ~315 million people worldwide6. A robust genetic predictor of ICS response in asthma patients would provide clinical value7 as inter-individual variability in ICS treatment response is commonly observed. Tantisira et al 5 reported an association (P=0.0007) in a pooled analysis between GLCCI1 rs37973 and ICS treatment response as measured over 4–8 weeks in 935 white non-Hispanic adults and children, and an OR of 2.36 in a subject level pooled analysis evaluating subject placement with response quartiles. In this larger sample set, n=1,916, drawn from seven clinical studies, we did not confirm GLCCI1 rs37973 as a predictor of ICS response. However, the discrepant outcomes might have been due to various factors: the GSK studies were clinical trials specifically designed with FEV1 change as the primary endpoint for all studies except one, whereas those of Tantisira were designed around a range of other primary endpoints, including lung growth, time to treatment failure and the percentage of asthma control days; paediatric study participants were only included in the initial Tantisira5 cohort; and the duration of ICS treatment at the time of FEV1 assessment varied between the two evaluations. Further genetic studies will be required to fully elucidate the potential role of GLCCI1 in ICS treatment response in asthma patients.

Modulation of β-amyloid by a single dose of GSK933776 in patients with mild Alzheimer’s disease: a phase I study

IntroductionIn this study, we evaluated the safety and pharmacodynamic effects of the Fc-inactivated anti-β-amyloid (anti-Aβ) monoclonal antibody GSK933776 in patients with mild Alzheimer’s disease and mild cognitive impairment. Aβ and tau levels were investigated in cerebrospinal fluid (CSF), and the relationship between Aβ levels and Aβ modulation in plasma was explored. The feasibility of a continuous sampling method using a lumbar catheter was assessed.MethodsThis trial was a phase I, open-label, uncontrolled, single-dose, exploratory experimental medicine study of intravenous GSK933776 at doses of 1 mg/kg, 3 mg/kg or 6 mg/kg (nu2009=u20096/group). The time course of plasma and CSF concentrations of GSK933776 and Aβ was assessed. Sample size was based on feasibility, and no formal statistical analyses were performed.ResultsFollowing administration of GSK933776 at doses of 1 mg/kg, 3 mg/kg and 6 mg/kg, there were decreases from baseline in CSF Aβ1–42 (from 0 to 12 hours) by 22.8 pg/ml (6.2%), 43.5 pg/ml (9.2%) and 60.5 pg/ml (12.5%), respectively. Plasma concentrations of total Aβ18–35 and Aβ4228–42 increased immediately after infusion and CSF tau concentration increased slightly, but did not significantly change, following administration of all doses of GSK933776. Pharmacokinetics confirmed the presence of GSK933776 in the CSF, which exhibited a dose–response relationship. One patient underwent minor surgery without sequelae following a ruptured lumbar catheter.ConclusionGSK933776 demonstrated pharmacological activity and target engagement in CSF and plasma, and the continuous sampling method via a catheter successfully assessed the Aβ changes following single-dose administration of GSK933776.Trial registrationClinicalTrials.gov Identifier: NCT01424436. Registered 4 August 2011

First Administration of the Fc-Attenuated Anti-β Amyloid Antibody GSK933776 to Patients with Mild Alzheimer's Disease: A Randomized, Placebo-Controlled Study

Objective To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the Fc-inactivated anti-β amyloid (Aβ) monoclonal antibody (mAb) GSK933776 in patients with mild Alzheimer’s disease (AD) or mild cognitive impairment (MCI). Methods This was a two-part, single blind, placebo-controlled, first-time-in-human (FTIH) study of single (n = 18) and repeat dose (n = 32) intravenous GSK933776 0.001–6 mg/kg (ClinicalTrials.gov: NCT00459550). Additional safety data from an open-label, uncontrolled, single dose study of intravenous GSK933776 1–6 mg/kg (n = 18) are included (ClinicalTrials.gov: NCT01424436). Results There were no cases of amyloid-related imaging abnormalities-edema (ARIA-E) or –hemorrhage (ARIA-H) after GSK933776 administration in both studies. Three patients across the two studies developed anti-GSK933776 antibodies. Plasma GSK933776 half-life (t1/2) was 10–15 days after repeat dosing. After each of three administrations of GSK933776, plasma levels of total Aβ42 and Aβ increased whereas plasma levels of free Aβ decreased dose dependently; no changes were observed for placebo. For total Aβ42 the peak:trough ratio was ≤2 at doses ≥3 mg/kg; for total Aβ the ratio was ≤2 at 6 mg/kg. CSF concentrations of Aβ showed increases from baseline to week 12 for Aβ X–38 (week 12:baseline ratio: 1.65; 95%CI: 1.38, 1.93) and Aβ X–42 (week 12:baseline ratio: 1.18; 95%CI: 1.06, 1.30) for values pooled across doses. Conclusion In this FTIH study the Fc-inactivated anti-Aβ mAb GSK933776 engaged its target in plasma and CSF without causing brain ARIA-E/H in patients with mild AD or MCI. Trial Registration ClinicalTrials.gov NCT00459550

Lipoprotein-Associated Phospholipase A2 Loss-of-Function Variant and Risk of Vascular Diseases in 90,000 Chinese Adults

Lipoprotein-associated phospholipase A2 (Lp-PLA2) produces the proinflammatory mediators lysophosphatidylcholine and oxidized free fatty acids through hydrolysis of oxidized phospholipids carried on low-density lipoproteins in atherosclerotic plaques. Although increased Lp-PLA2 activity has been associated with higher risks of occlusive vascular diseases, recent phase III trials of the Lp-PLA2 inhibitor darapladib, which reduces Lp-PLA2 activity by ∼60%, failed to establish a protective role of darapladib for prevention of major vascular events in patients with stable coronary heart disease (CHD) or acute coronary syndrome 1, 2.

Pharmacogenetic meta-analysis of baseline risk factors, pharmacodynamic, efficacy and tolerability endpoints from two large global cardiovascular outcomes trials for darapladib

Darapladib, a lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor, failed to demonstrate efficacy for the primary endpoints in two large phase III cardiovascular outcomes trials, one in stable coronary heart disease patients (STABILITY) and one in acute coronary syndrome (SOLID-TIMI 52). No major safety signals were observed but tolerability issues of diarrhea and odor were common (up to 13%). We hypothesized that genetic variants associated with Lp-PLA2 activity may influence efficacy and tolerability and therefore performed a comprehensive pharmacogenetic analysis of both trials. We genotyped patients within the STABILITY and SOLID-TIMI 52 trials who provided a DNA sample and consent (n = 13,577 and 10,404 respectively, representing 86% and 82% of the trial participants) using genome-wide arrays with exome content and performed imputation using a 1000 Genomes reference panel. We investigated baseline and change from baseline in Lp-PLA2 activity, two efficacy endpoints (major coronary events and myocardial infarction) as well as tolerability parameters at genome-wide and candidate gene level using a meta-analytic approach. We replicated associations of published loci on baseline Lp-PLA2 activity (APOE, CELSR2, LPA, PLA2G7, LDLR and SCARB1) and identified three novel loci (TOMM5, FRMD5 and LPL) using the GWAS-significance threshold P≤5E-08. Review of the PLA2G7 gene (encoding Lp-PLA2) within these datasets identified V279F null allele carriers as well as three other rare exonic null alleles within various ethnic groups, however none of these variants nor any other loci associated with Lp-PLA2 activity at baseline were associated with any of the drug response endpoints. The analysis of darapladib efficacy endpoints, despite low power, identified six low frequency loci with main genotype effect (though with borderline imputation scores) and one common locus (minor allele frequency 0.24) with genotype by treatment interaction effect passing the GWAS-significance threshold. This locus conferred risk in placebo subjects, hazard ratio (HR) 1.22 with 95% confidence interval (CI) 1.11–1.33, but was protective in darapladib subjects, HR 0.79 (95% CI 0.71–0.88). No major loci for tolerability were found. Thus, genetic analysis confirmed and extended the influence of lipoprotein loci on Lp-PLA2 levels, identified some novel null alleles in the PLA2G7 gene, and only identified one potentially efficacious subgroup within these two large clinical trials.