PAT: Pharmacogenomics of Arrhythmia TherapyVanderbilt University, Nashville, TN

ABSTRACTWe submit here a proposal for renewal of the Pharmacogenomics of Arrhythmia Therapy (PAT) node of the PGRN. The studies we propose combine candidate, genome-wide, and targeting resequencing approaches to identify the genomic basis for arrhythmia drug response phenotypes of major public health importance. In addition, each Specific Aim includes a major focus on multi-institutional accumulation of large numbers of patients with well-defined drug response phenotypes. Studies in Specific Aim 1 further define the genomic basis underlying susceptibility to drug-induced long QT-related arrhythmias, a continuing challenge in clinical drug use and in drug development. In Specific Aim 2, we build on previous work to prospectively assess the relationships among drug response, genotypes, and clinical endophenotypes in patients with atrial fibrillation (AF). While AF therapy often includes warfarin anticoagulation whose major complication is bleeding, pharmacogenomic studies of warfarin response to date have focused on determinants of dose. In Specific Aim 3, we will identify a large set of cases of major warfarin-related bleeding in BioVU, our DNA repository linked to de-identified electronic medical records, and undertake a case-control study of the genomic determinants of this complication. We include here two proposals for network resources: (1) PGPop (PharmacoGenomic discovery and replication in very large patient POPulations) that brings together multiple healthcare system-based nodes, including BioVU, that each include extensive drug response phenotypes and extant or planned large DNA collections. PGPop will serve as a “real-world” platform for pharmacogenomic discovery; one initial project will be generation of a replication set for warfarin-related bleeding. (2) P-STAR (PGRN Statistical Analysis Resource), that will provide statistical support to PGRN sites and develop advanced methods in the field. These studies build on progress within our own site, across PGRN, and reach out beyond the network to develop expanded populations and new tools to enable application of pharmacogenetic knowledge to clinical care.

We have a long-standing interest in the mechanisms and genomics of the drug-induced long QT syndrome (diLQTS) and the associated arrhythmia (torsades de pointes). As a result, we have established collaborations across arrhythmia centers world-wide to accrue a large set of patients with diLQTS. In the previous cycle, we used this resource to execute an intensive candidate gene/common haplotype analysis and a GWAS in collaboration with RIKEN. Multiple candidate genes for mediating diLQTS risk are now identified, by GWAS, human genetics, and molecular electrophysiology. In the present cycle, we will•follow-up GWAS results•test the hypothesis that rare variants contribute to diLQTS risk•work with the Severe Adverse Events Consortium to establish an expanded international diLQTS study group.

Aim 1: Genomics of drug-induced torsades de pointes Aim 3: BioVU: linking DNA variants to human phenotypesOne area of focus in the previous cycle was evaluation of the genomic determinants of the dose of warfarin, an anticoagulant widely used in AF. However, a major unaddressed issue is whether these predictors of steady state dosage requirement also predict the “hard” endpoint of bleeding. Our aims here are to •use our large DNA databank, BioVU, to accrue a very large cohort of patients with bleeding complications during warfarin therapy and controls. Further data on BioVU are presented in the PGPop poster.•test candidate genetic variants (in CYP2C9 and VKORC1) as predictors of bleeding•conduct genome-wide analysis to identify common variants mediating bleeding risk•replicate our findings in datasets accrued in other large populations•extend this approach to analyze other variable drug actions

Aim 2: Genomics of atrial fibrillation – toward subtype-specific therapy

The Team

ECG findings in a patient with lone AF and a loss of function mutation resulting in R28W in a sodium channel modulatory protein, SCN2B. The red arrows indicate the J-point which is >1 mm elevated above the baseline. This ECG phenotype is present in ~10% of patients with lone AF and is also seen with other loss-of-function sodium channel variants. We propose to challenge AF patients with a sodium channel blocker, and determine the genomic basis for this phenotype and its implications for therapy.

Studies in the Vanderbilt atrial fibrillation (AF) Registry, which now contains >1000 probands with AF, have identified both common and rare variants contributing to AF susceptibility and variability in response to drug therapy. In mid-late 2009, the registry set underwent genome-wide genotyping as part of the RIKEN-PGRN collaboration. The experiments we propose will •determine the genomic predictors of variable drug response in AF•build on previous findings to test the hypothesis that challenge with a sodium channel blocking drug identifies specific clinical and genetic subsets of patients with AF•create and initiate analyses in a DNA databank for CABANA, an upcoming 3,000-patient NHLBI-sponsored study of AF therapies.

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Rare non-synonymous variants in the cardiac sodium channel gene SCN5A were identified in 22/375 probands with AF. The panel below shows the locations of previously unreported variants. The bottom panel shows 8 pedigrees in which more than one individual had AF (solid symbol: documented AF; shaded: AF by history; gray unknown; white: unaffected).

Results of an Illumina GoldenGate assay of 1536 SNPs in 19 high priority candidate genes for the phenotype of drug-induced torsades de pointes; this analysis included 157 cases. The control groups were a cohort of subjects exposed to QT-prolonging antiarrhythmics without exaggerated QT prolongation (now >600), and population controls. One SNP, rs1805128 in KCNE1 (arrow), had the lowest P value in comparisons of cases versus drug-exposed controls (top), and cases versus population controls (bottom). Expanding the case definition to TdP + exaggerated QT prolongation (n=182) yielded very similar results.

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157 cases of drug-induced TdP vs controls

An analysis in 1536 common SNPs across 19 candidate genes

vs 353 drug-exposed controls

vs 837 population controls TdP cases (183) vs drug-exposed controls (519)

age and sex adjusted

Initial result from a genome-wide analysis of 183 cases of drug-induced TdP and 519 drug-exposed controls. None of the top SNPs are near genes known to modulate cardiac ion channel function.

Nancy Brown*

Dan and Thomas RodenPI and gene-sharer

Christie Ingram

Dawood Darbar

Prince Kannankeril

Al George

Tao Yang

Russ Wilke

Mike Stein

Andrea Havens Ramirez

Jessica Delaney

Marylyn Ritchie(P*star)

Dana Crawford

Hua Xu(PGPop)

Josh Denny

(PGPop)

Dan Masys*

Shannon Carter, Kris Norris, Gayle Kucera,

Tanya Stubblefield

Host Oct. 2010 PGRN

Electrophysiology and genomicsProgram management Clinical pharmacologyResearch fellows

Jonathan Haines*

Statistical genetics Biomedical informatics Ascertainment and nursing informatics

*Internal Advisory Panel

Studies in the Vanderbilt AF registry, as well as in other large US and European collections shown above, replicated the association between rs220733 on chr4q25 and AF. We and others have also shown that 4q25 variants predict post-cardiac surgery AF, as well as response to antiarrhythmic drug therapy and to ablation therapy.

Screen-shot (above) of the results of a search in April 2009 for “warfarin” in the medication list of patients in BioVU. This tool provides a crude estimate of possible cases; extensive natural language processing and hand curation is then required to validate cases and controls for epidemiologic and genomic research. A preliminary analysis suggested that of these 4842 subjects, 932 were admitted for possible warfarin-related bleeding and hand curation of a subset suggested that about a third were actual cases. The search below was conducted July 2, 2010, showing that the number of subjects has doubled in the last year.

4842total

1275potential

932admitted

~372true cases

Time to first therapeutic INR:

Time to therapeutic INR during initiation of warfarin therapy in 297 patients at Vanderbilt. There was a significant impact of VKORC1 haplotype, but no effect of CYP2C9 genotype. Variants in both genes contributed to the steady state dose requirement in this and other studies.

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CYP2C9 and VKORC1 genotypes vary by ethnicity

IWPC, NEJM, 2009

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Genome-wide association to analyze warfarin response

Cooper et al., Blood 2008

Genome-wide association study conducted by Rieder and colleagues at the University of Washington examining warfarin dose requirement . The Vanderbilt samples were used in the replication phase of the study. Only SNPs at the VKORC1 and CYP2C9 sites survived replication. These data support the design of prospective clinical trials examining the utility of genotype-guided warfarin therapy.

Comparing dosing algorithms

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Data from the International Warfarin Pharmacogenomics Consortium. The upper panel shows the ancestry-dependence of common VKORC1 and CYP2C9 variants; these contribute to ancestry-dependent variability in dose requirement. The bottom panel shows the ability of three dosing approaches (fixed dose, dosing based on clinical characteristics, dosing based on clinical characteristics+ genotypes) to predict steady state warfarin dose requirement. The ordinate shows the success of the algorithm. For subjects whose dose requirement is “average” any of the three approaches is satisfactory (middle set of bars). However, for those with high or low doses, the genetic algorithm is superior to the other two.