Linda C. McCarthy

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 107 000 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.

A genome-wide scan provides evidence for loci influencing a severe heritable form of common migraine

Migraine is a prevalent neurovascular disease with a significant genetic component. Linkage studies have so far identified migraine susceptibility loci on chromosomes 1, 4, 6, 11, 14, 19 and X. We performed a genome-wide scan of 92 Australian pedigrees phenotyped for migraine with and without aura and for a more heritable form of “severe” migraine. Multipoint non-parametric linkage analysis revealed suggestive linkage on chromosome 18p11 for the severe migraine phenotype (LOD*=2.32, P=0.0006) and chromosome 3q (LOD*=2.28, P=0.0006). Excess allele sharing was also observed at multiple different chromosomal regions, some of which overlap with, or are directly adjacent to, previously implicated migraine susceptibility regions. We have provided evidence for two loci involved in severe migraine susceptibility and conclude that dissection of the “migraine” phenotype may be helpful for identifying susceptibility genes that influence the more heritable clinical (symptom) profiles in affected pedigrees. Also, we concluded that the genetic aetiology of the common (International Headache Society) forms of the disease is probably comprised of a number of low to moderate effect susceptibility genes, perhaps acting synergistically, and this effect is not easily detected by traditional single-locus linkage analyses of large samples of affected pedigrees.

Genetic factors associated with response to intravitreal ranibizumab in Korean patients with neovascular age-related macular degeneration.

Purpose: To investigate the association between genetic risk variants for age-related macular degeneration (AMD) and response to intravitreal ranibizumab in Korean patients with neovascular AMD. Methods: This prospective study included 273 treatment-naive patients (273 eyes) who underwent 5 monthly injections (Months 0, 1, 2, 3, and 4) of intravitreal ranibizumab for neovascular AMD. Patients were genotyped for 23 single-nucleotide polymorphisms within 12 AMD-relevant genes. For each polymorphism, genotypic association with good response at Month 5, predetermined as visual improvement of ≥8 Early Treatment Diabetic Retinopathy Study letters from baseline, was investigated with logistic regression analysis adjusted for age, gender, smoking, baseline Early Treatment Diabetic Retinopathy Study letter, central retinal thickness, lesion area, and type of choroidal neovascularization. Results: At Month 5, visual acuity improved by 9.1 ± 17.6 letters from baseline, and 136 patients (49.8%) were classified as good responders. In logistic regression, no tested polymorphism showed statistically significant association with favorable visual outcome at Month 5. When unadjusted for multiple tests, AA genotype for VEGF rs699947 had an increased chance of good response compared with other genotypes (odds ratio, 3.61; 95% confidence interval, 1.42–9.18; P = 0.0071). Conclusion: In this Korean neovascular AMD cohort, there was no statistically significant effect of genotype on early visual outcome after ranibizumab treatment.

Replication study of the insulin receptor gene in migraine with aura.

We performed the first replication study for the reported association of the insulin receptor gene (INSR) with migraine with aura (MA). Two of 35 SNPs (rs1052371 and rs2860174) reached borderline significance (best uncorrected allelic p value of 0.052 for rs2860174) in stage 1 of our study (270 MA patients, 280 controls). As rs2860174 was 1 of the 5 SNPs with prior evidence of association, we also genotyped this SNP in our stage 2 sample (679 MA patients, 368 controls), and it was nonsignificant (allelic p value 0.478). The combined analysis of our samples showed just a nonsignificant trend for rs2860174 (p=0.1). However, the joint analysis of our study and the initial study reporting an association-including 1278 Caucasian MA patients and 1337 Caucasian controls altogether-displayed a significant allelic p value of 0.005. In conclusion, further association studies for rs2860174 with even larger numbers of individuals are required to exclude or confirm definitely a small effect of this SNP on migraine susceptibility.

Pharmacogenetics and obesity: common gene variants influence weight loss response of the norepinephrine/dopamine transporter inhibitor Gw320659 in obese subjects

Background GW320659, a highly selective neuronal norepinephrine and dopamine re-uptake inhibitor, has been evaluated for the treatment of obesity. Scrutiny of the weight loss data from a phase II study (GlaxoSmithKline study OBS20001) showed a wide variation in weight loss response following GW320659 treatment and the possibility that the study population might include subgroups with enhanced weight loss response. Methods Pharmacogenetic analysis was performed in 191 subjects prospectively ascertained from a Phase II dose ranging study to evaluate the influence of genotype on weight loss efficacy and safety of GW320659 in obese subjects. Results Common genetic polymorphisms in the drug target (norepinephrine transporter protein 1, SLC6A2) and mechanism pathway (NMDA receptor channel NR1 subunit, GRIN1) were associated with increased weight loss following GW320659 treatment in a proportion (36%) of the study population. In the patient subgroup selected for these genotypes, GW320659 (15 mg/day) produced a significant difference in mean weight loss of 7.84 kg (SD 5.23, n=14), compared to 2.53 kg (SD 5.17, n=24) in the subgroup that did not possess the genotypes (P=0.006). This subgroup also showed a highly significant weight loss response for GW320659 compared to placebo (+0.31 kg, SD 3.32, n=16) with the same genotypes (P<0.0001). In addition, there was no difference in placebo response between either subgroup. Conclusions Polymorphisms in SLC6A2 and GRIN1 could be used to maximize effective obesity pharmacotherapy by norepinephrine/dopamine transporter inhibitors by identifying patients that may be predisposed to particularly good treatment weight loss response.

Pharmacogenetics in diverse ethnic populations - implications for drug discovery and development

It is widely acknowledged that the vast quantities of data now publicly available as a result of the human genome initiative have the potential to revolutionize the pharmaceutical industry. More tangibly to the drug development business, the dawn of the pharmacogenetics era has the potential to impact not only the discovery of new medicines but also the safety and efficacy of pharmaceutical agents. Coincident with these scientific advances is the emergence of new markets for pharmaceutical agents. Japan, which represents the worlds second biggest market, is a good example. With the ICH E5 agreement in 1998 and a rapid change in the drug registration process in Japan, there are increasing opportunities to improve access to more medicines in all parts of the world. However, it is increasingly clear that significant genetic variation still exists between populations, with a host of data on interethnic variation in drug metabolizing enzyme and drug transporter activity. Evidence suggesting that this genetic variation may play an important role in defining some of the interethnic variation in drug response to currently marketed compounds is reviewed here, and future possibilities of using such information to better streamline the drug development process are discussed.

Genetic variants in the epithelial sodium channel associate with oedema in type 2 diabetic patients receiving the peroxisome proliferator-activated receptor gamma agonist farglitazar

Peroxisome proliferator-activated receptor gamma (PPAR&ggr;) agonists are highly effective in the treatment of type 2 diabetes. In some patients, PPAR&ggr; ligands are associated with fluid retention/oedema, for which the mechanism is not fully understood. A pharmacogenetic study was undertaken to investigate effects of variations in 21 candidate genes related to epithelial sodium channel (ENaC) pathways on oedema. This study used DNA samples collected from type 2 diabetes phase III clinical trials of the PPAR&ggr; agonist farglitazar (administered alone or in combination with insulin or glyburide) and investigated oedema reported as an adverse event as phenotype. Initial case–control analysis of oedema identified candidate gene single nucleotide polymorphisms with significant associations. These included three polymorphisms in ENaC&bgr; subunit (SCNN1B) that showed significant associations (P<0.05) with the two combination treatments in discrete regions of the gene, but not farglitazar treatment alone. Sequencing of SCNN1B in 207 Caucasian participants receiving farglitazar plus insulin or glyburide combination therapies, identified additional polymorphisms that were also significantly associated with oedema (P<0.0005) and maintained the treatment-regional associations. Further covariate analysis accounting for clinical factors influencing oedema supported these observations. One of the SCNN1B polymorphisms, at position −405 of the 5′ flanking region (rs34241435), was predicted to modify transcriptional interactions and in a transfected COS cell luciferase reporter gene assay exhibited higher promoter activity. These exploratory studies provide clinical pharmacogenetic and functional genomic evidence to support a pivotal role for ENaC regulation in PPAR&ggr;-induced oedema and provide insight into mechanisms and possible management of this side effect.

Predictive models of choroidal neovascularization and geographic atrophy incidence applied to clinical trial design.

PURPOSE To develop comprehensive predictive models for choroidal neovascularization (CNV) and geographic atrophy (GA) incidence within 3 years that can be applied realistically to clinical practice. DESIGN Retrospective evaluation of data from a longitudinal study to develop and validate predictive models of CNV and GA. METHODS The predictive performance of clinical, environmental, demographic, and genetic risk factors was explored in regression models, using data from both eyes of 2011 subjects from the Age-Related Eye Disease Study (AREDS). The performance of predictive models was compared using 10-fold cross-validated receiver operating characteristic curves in the training data, followed by comparisons in an independent validation dataset (1410 AREDS subjects). Bayesian trial simulations were used to compare the usefulness of predictive models to screen patients for inclusion in prevention clinical trials. RESULTS Logistic regression models that included clinical, demographic, and environmental factors had better predictive performance for 3-year CNV and GA incidence (area under the receiver operating characteristic curve of 0.87 and 0.89, respectively), compared with simple clinical criteria (AREDS simplified severity scale). Although genetic markers were associated significantly with 3-year CNV (CFH: Y402H; ARMS2: A69S) and GA incidence (CFH: Y402H), the inclusion of genetic factors in the models provided only marginal improvements in predictive performance. CONCLUSIONS The logistic regression models combine good predictive performance with greater flexibility to optimize clinical trial design compared with simple clinical models (AREDS simplified severity scale). The benefit of including genetic factors to screen patients for recruitment to CNV prevention studies is marginal and is dependent on individual clinical trial economics.

Phenome-wide association study using research participants’ self-reported data provides insight into the Th17 and IL-17 pathway

A phenome-wide association study of variants in genes in the Th17 and IL-17 pathway was performed using self-reported phenotypes and genetic data from 521,000 research participants of 23andMe. Results replicated known associations with similar effect sizes for autoimmune traits illustrating self-reported traits can be a surrogate for clinically assessed conditions. Novel associations controlling for a false discovery rate of 5% included the association of the variant encoding p.Ile684Ser in TYK2 with increased risk of tonsillectomy, strep throat occurrences and teen acne, the variant encoding p.Arg381Gln in IL23R with a decrease in dandruff frequency, the variant encoding p.Asp10Asn in TRAF3IP2 with risk of male-pattern balding, and the RORC regulatory variant (rs4845604) with protection from allergies. This approach enabled rapid assessment of association with a wide variety of traits and investigation of traits with limited reported associations to overlay meaningful phenotypic context on the range of conditions being considered for drugs targeting this pathway.

Identification of new therapeutic targets for osteoarthritis through genome-wide analyses of UK Biobank

Osteoarthritis is the most common musculoskeletal disease and the leading cause of disability globally. Here, we perform the largest genome-wide association study for osteoarthritis to date (77,052 cases and 378,169 controls), analysing 4 phenotypes: knee osteoarthritis, hip osteoarthritis, knee and/or hip osteoarthritis, and any osteoarthritis. We discover 64 signals, 52 of them novel, more than doubling the number of established disease loci. Six signals fine map to a single variant. We identify putative effector genes by integrating eQTL colocalization, fine-mapping, human rare disease, animal model, and osteoarthritis tissue expression data. We find enrichment for genes underlying monogenic forms of bone development diseases, and for the collagen formation and extracellular matrix organisation biological pathways. Ten of the likely effector genes, including TGFB1, FGF18, CTSK and IL11 have therapeutics approved or in clinical trials, with mechanisms of action supportive of evaluation for efficacy in osteoarthritis.