Ian Mongrain

Pharmacogenomic Determinants of the Cardiovascular Effects of Dalcetrapib

Background—Dalcetrapib did not improve clinical outcomes, despite increasing high-density lipoprotein cholesterol by 30%. These results differ from other evidence supporting high-density lipoprotein as a therapeutic target. Responses to dalcetrapib may vary according to patients’ genetic profile. Methods and Results—We conducted a pharmacogenomic evaluation using a genome-wide approach in the dal-OUTCOMES study (discovery cohort, n=5749) and a targeted genotyping panel in the dal-PLAQUE-2 imaging trial (support cohort, n=386). The primary endpoint for the discovery cohort was a composite of cardiovascular events. The change from baseline in carotid intima-media thickness on ultrasonography at 6 and 12 months was evaluated as supporting evidence. A single-nucleotide polymorphism was found to be associated with cardiovascular events in the dalcetrapib arm, identifying the ADCY9 gene on chromosome 16 (rs1967309; P=2.41×10–8), with 8 polymorphisms providing P<10–6 in this gene. Considering patients with genotype AA at rs1967309, there was a 39% reduction in the composite cardiovascular endpoint with dalcetrapib compared with placebo (hazard ratio, 0.61; 95% confidence interval, 0.41–0.92). In patients with genotype GG, there was a 27% increase in events with dalcetrapib versus placebo. Ten single-nucleotide polymorphism in the ADCY9 gene, the majority in linkage disequilibrium with rs1967309, were associated with the effect of dalcetrapib on intima-media thickness (P<0.05). Marker rs2238448 in ADCY9, in linkage disequilibrium with rs1967309 (r2=0.8), was associated with both the effects of dalcetrapib on intima-media thickness in dal-PLAQUE-2 (P=0.009) and events in dal-OUTCOMES (P=8.88×10–8; hazard ratio, 0.67; 95% confidence interval, 0.58–0.78). Conclusions—The effects of dalcetrapib on atherosclerotic outcomes are determined by correlated polymorphisms in the ADCY9 gene. Clinical Trial Information—URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00658515 and NCT01059682

Genotype-Dependent Effects of Dalcetrapib on Cholesterol Efflux and Inflammation: Concordance with Clinical Outcomes

Background—Dalcetrapib effects on cardiovascular outcomes are determined by adenylate cyclase 9 gene polymorphisms. Our aim was to determine whether these clinical end point results are also associated with changes in reverse cholesterol transport and inflammation. Methods and Results—Participants of the dal-OUTCOMES and dal-PLAQUE-2 trials were randomly assigned to receive dalcetrapib or placebo in addition to standard care. High-sensitivity C-reactive protein was measured at baseline and at end of study in 5243 patients from dal-OUTCOMES also genotyped for the rs1967309 polymorphism in adenylate cyclase 9. Cholesterol efflux capacity of high-density lipoproteins from J774 macrophages after cAMP stimulation was determined at baseline and 12 months in 171 genotyped patients from dal-PLAQUE-2. Treatment with dalcetrapib resulted in placebo-adjusted geometric mean percent increases in high-sensitivity C-reactive protein from baseline to end of trial of 18.1% (P=0.0009) and 18.7% (P=0.00001) in participants with the GG and AG genotypes, respectively, but the change was −1.0% (P=0.89) in those with the protective AA genotype. There was an interaction between the treatment arm and the genotype groups (P=0.02). Although the mean change in cholesterol efflux was similar among study arms in patients with GG genotype (mean: 7.8% and 7.4%), increases were 22.3% and 3.5% with dalcetrapib and placebo for those with AA genotype (P=0.005). There was a significant genetic effect for change in efflux for dalcetrapib (P=0.02), but not with placebo. Conclusions—Genotype-dependent effects on C-reactive protein and cholesterol efflux are supportive of dalcetrapib benefits on atherosclerotic cardiovascular outcomes in patients with the AA genotype at polymorphism rs1967309. Clinical Trials Registration—ClinicalTrials.gov; Unique Identifiers: NCT00658515 and NCT01059682.

CKM and LILRB5 Are Associated With Serum Levels of Creatine Kinase

Background—Statins (HMG-CoA reductase inhibitors) are the most prescribed class of lipid-lowering drugs for the treatment and prevention of cardiovascular disease. Creatine kinase (CK) is a commonly used biomarker to assist in the diagnosis of statin-induced myotoxicity but the normal range of CK concentrations is wide, which limits its use as a diagnostic biomarker. Methods and Results—We conducted a genome-wide association study of serum CK levels in 3412 statin users. Patients were recruited in Quebec, Canada, and genotyped on Illumina Human610-Quad and an iSelect panel enriched for lipid homeostasis, hypertension, and drug metabolism genes. We found a strong association signal between serum levels of CK and the muscle CK (CKM) gene (rs11559024: P=3.69×10−16; R2=0.02) and with the leukocyte immunoglobulin-like receptor subfamily B member 5 (LILRB5) gene (rs2361797: P=1.96×10−10; R2=0.01). Genetic variants in those 2 genes were independently associated with CK levels in statin users. Results were successfully replicated in 5330 participants from the Montreal Heart Institute Biobank in statin users for CKM (rs11559024: P=4.32×10−16; R2=0.02) and LILRB5 (rs12975366 P=4.45×10−10; R2=0.01) and statin nonusers (P=4.08×10−7, R2=0.01; P=3.17×10−9, R2=0.02, respectively). Conclusions—This is the first genome-wide study to report on the underlying genetic determinants of CK variation in a population of statin users. We found statistically significant association for variants in the CKM and LILRB5 genes.

PRKCB is associated with calcineurin inhibitor-induced renal dysfunction in heart transplant recipients.

Objectives Single nucleotide polymorphisms (SNPs) in the transforming growth factor-&bgr;1 gene (TGFB1) have been inconsistently associated with calcineurin inhibitor (CNI)-induced renal dysfunction following cardiac transplantation. The impact of genetic variants related to the renin-angiotensin-aldosterone system (RAAS) and natriuretic peptides, which are implicated in CNI nephrotoxicity, is unknown. The primary objective of this study was to validate the association between two common variants in TGFB1 (rs1800470, rs1800471) and postcardiac transplant renal function. The secondary objective was to investigate the effect of candidate genes related to the RAAS, natriuretic peptides, and other elements involved in the intracellular signaling of these pathways. Methods We conducted a retrospective cohort study of 158 heart transplant recipients treated with CNIs, and evaluated the association between select SNPs and the estimated glomerular filtration rate as calculated by the Modification of Diet in Renal Disease simplified formula. A total of 273 SNPs distributed in 44 genes were tested. Results No association was observed between TGFB1 variants and renal function. One polymorphism in the protein kinase C-&bgr; gene (PRKCB; rs11074606), which is implicated in the RAAS intracellular signaling, was significantly associated with post-transplant estimated glomerular filtration rate after adjusting for possible confounders (P=0.00049). This marker is in linkage disequilibrium with two variants located in putative regulatory regions of the gene (rs2283541, rs1013316). Conclusion Our results suggest that PRKCB may be a potential predictor of CNI-induced nephrotoxicity in heart transplant recipients, and could therefore be a promising candidate to identify patients who are most susceptible to this adverse drug reaction.

Testing the role of predicted gene knockouts in human anthropometric trait variation

Although the role of complete gene inactivation by two loss-of-function mutations inherited in trans is well-established in recessive Mendelian diseases, we have not yet explored how such gene knockouts (KOs) could influence complex human phenotypes. Here, we developed a statistical framework to test the association between gene KOs and quantitative human traits. Our method is flexible, publicly available, and compatible with common genotype format files (e.g. PLINK and vcf). We characterized gene KOs in 4498 participants from the NHLBI Exome Sequence Project (ESP) sequenced at high coverage (>100×), 1976 French Canadians from the Montreal Heart Institute Biobank sequenced at low coverage (5.7×), and >100 000 participants from the Genetic Investigation of ANthropometric Traits (GIANT) Consortium genotyped on an exome array. We tested associations between gene KOs and three anthropometric traits: body mass index (BMI), height and BMI-adjusted waist-to-hip ratio (WHR). Despite our large sample size and multiple datasets available, we could not detect robust associations between specific gene KOs and quantitative anthropometric traits. Our results highlight several limitations and challenges for future gene KO studies in humans, in particular when there is no prior knowledge on the phenotypes that might be affected by the tested gene KOs. They also suggest that gene KOs identified with current DNA sequencing methodologies probably do not strongly influence normal variation in BMI, height, and WHR in the general human population.

A pharmacogenetic investigation of intravenous furosemide in decompensated heart failure: a meta-analysis of three clinical trials.

We conducted a meta-analysis of pharmacogenomic substudies of three randomized trials conducted in patients with decompensated heart failure (HF) that were led by National Heart Lung and Blood Institute (NHLBI)-funded HF Network to test the hypothesis that candidate genes modulate net fluid loss and weight change in patients with decompensated HF treated with a furosemide-based diuretic regimen. Although none of the genetic variants previously shown to modulate the effects of loop diuretics in healthy individuals were associated with net fluid loss after 72 h of treatment, a set of rare variants in the APOL1 gene, which codes for apolipoprotein L1 (P=0.0005 in the random effects model), was associated with this end point. Moreover, a common variant in the multidrug resistance protein-4 coding gene (ABCC4, rs17268282) was associated with weight loss with furosemide use (P=0.0001). Our results suggest that both common and rare genetic variants modulate the response to a furosemide-based diuretic regimen in patients with decompensated HF.

Development of a broad-based ADME panel for use in pharmacogenomic studies

AIM To optimally address the interindividual variability observed in pharmacokinetic drug response, we have created a custom genotyping panel that interrogates most of the key genetic variations present in a set of 181 prioritized genes responsible for the absorption, distribution, metabolism and excretion (ADME) of many therapeutic agents. This consensus list of genes and variants was based on the ADME core and extended gene lists compiled by a group of pharmaceutical companies as having relevance. Although these pharmacokinetic genes and pathways are well known, tools that can interrogate a large number of these genes simultaneously within a single experiment are not currently available. METHODS Using novel design strategies, we have developed an optimized and validated ADME genotyping panel, encompassing approximately 3000 variants, that has broad applicability to any study or clinical trial that would benefit from the evaluation of an extensive list of ADME genes. RESULTS & CONCLUSION Over the course of three design iterations, overall assay conversion rates were improved from 83 to 97% resulting in a panel that fills in many of the gaps in coverage present on currently available commercial genotyping assays. The utility of the assay has been demonstrated by the screening of more than 1000 samples resulting in the discovery of novel pharmacogenomic associations. The assay, and the underlying methods, will continue to be a valuable tool for use in future pharmacogenomic studies.

Rationale, design, and preliminary results of the Quebec Warfarin Cohort Study

Over‐ and undercoagulation with warfarin are associated with hemorrhagic and thromboembolic events, respectively. Genetic and clinical factors affect warfarin response, and the causes of this variability remain unclear. We present descriptive statistics and test for predictors of poor anticoagulation control. The Quebec Warfarin Cohort (QWC) comprises 1059 new warfarin users, with prospective follow‐up using telephone questionnaires every 3 months for 1 year, and using healthcare administrative databases (RAMQ and Med‐Echo) for 5 years prior to cohort entry and up to 10 years following active patient participation. Genetic material was collected, and genotyping of CYP2C9 and VKORC1 genes was conducted. Measured outcomes included the percentage of time patients spent within therapeutic range, anticoagulation control, warfarin dose, bleeding, and thromboembolic events. We report baseline characteristics and outcomes after 1 year of follow‐up. Poor anticoagulation control was defined as time in therapeutic range <60% in the 3‐ to 12‐month interval. Participants had a mean age of 71 years, and 62% were men. The most common indication for warfarin was atrial fibrillation (87%). Mean time in therapeutic range was 56% (±25%) in the 3 months following warfarin initiation, and 70% (±21%) in the 3‐ to 12‐month interval. During follow‐up, the rate of stroke or systemic embolism was 1.8 events per 100 person‐years; for major bleeding events, 3.3 events per 100 person‐years. Independent predictors of poor anticoagulation control were chronic kidney disease, heart failure, dyslipidemia, and age. The QWC represents a good research cohort to investigate clinical and genetic factors in a warfarin‐anticoagulated population.

Nuclear receptor gene polymorphisms and warfarin dose requirements in the Quebec Warfarin Cohort

Warfarin is primarily metabolized by cytochrome 2C9, encoded by gene CYP2C9. Here, we investigated whether variants in nuclear receptor genes which regulate the expression of CYP2C9 are associated with warfarin response. We used data from 906 warfarin users from the Quebec Warfarin Cohort (QWC) and tested the association of warfarin dose requirement at 3 months following the initiation of therapy in nine nuclear receptor genes: NR1I3, NR1I2, NR3C1, ESR1, GATA4, RXRA, VDR, CEBPA, and HNF4A. Three correlated SNPs in the VDR gene (rs4760658, rs11168292, and rs11168293) were associated with dose requirements of warfarin (P = 2.68 × 10−5, P = 5.81 × 10−4, and P = 5.94 × 10−4, respectively). Required doses of warfarin were the highest for homozygotes of the minor allele at the VDR variants (P < 0.0026). Variants in the VDR gene were associated with the variability in response to warfarin, emphasizing the possible clinical relevance of nuclear receptor gene variants on the inter-individual variability in drug metabolism.

Pharmacogenetic content of commercial genome-wide genotyping arrays

AIM We have evaluated the pharmacogenetic content of commercial human genome-wide genotyping arrays, as it is a critical determinant to enabling pharmacogenomic discoveries. METHODS Using bioinformatics approaches, we assessed 27,811 genetic variants in 3146 genes for their presence in 18 Illumina and 15 Affymetrix genome-wide arrays. RESULTS The pharmacogenetic content of the arrays varied greatly. The combination of the Affymetrix precision medicine array and PharmacoScan arrays (Affymetrix) had the highest coverage for a set of clinically actionable absorption, distribution, metabolism and excretion (ADME) variants, single nucleotide ADME variants and ADME insertions/deletions, with a physical coverage of 125/130 (96.2%), 9924/24,138 (41.1%) and 2252/3994 (56.4%), respectively. CONCLUSION The combination of the Affymetrix precision medicine array and PharmacoScan arrays provided both genome-wide and pharmacogene coverage, which is crucial in the discovering of new variants responsible for drug adverse effects. These results will help in the design of pharmacogenomic studies and will enable a critical review of results from past studies.