Enlighten Medical Associates Inc.

Summary of the Université de Montréal Pharmacogenomics Centre Broad-Based ADME Panel

In order to address the inter-individual variability observed in the pharmacokinetic (PK) response to many medications, we have created a broad-based custom genotyping panel that interrogates variation present in ~180 key drug metabolizing genes involved in the PK pathways for many therapeutic agents. In order to make this assay as broadly applicable as possible, only genes deemed to be directly involved with Absorption, Distribution, Metabolism and Excretion (ADME) were selected for the assay. These genes can be grouped into four categories: Phase I drug metabolism enzymes, responsible for non synthetic reactions such as oxidation and reduction that render drugs more polar or water soluble; phase II drug metabolism enzymes, responsible for the conjugation with endogenous moieties such as glucuronidation or sulfation; transporters, responsible for the uptake and excretion of drugs in and out of cells; and modifiers, that can either alter the expression of other ADME genes or affect the biochemistry of ADME enzymes.

Several commercial assays are currently available that screen for the “Core List” of ~184 functional markers in drug metabolism genes that can be used for evidence based decision making. This list is only informative in a limited number of situations and a more thorough list of variants in genes involved in ADME is required if one wishes to investigate the inter-individual variability observed in a wide range of medications. Thus, the Pharmacogenomics Centre chose to develop a broad-based ADME genotyping panel, consisting of ~3,000 SNPs in ~180 genes that are not well tagged on commercial products. The panel was designed to incorporate two different types of variation to increase our ability to identify variants in clinical populations; published markers that alter gene function (~360 SNPs) and haplotype tagging markers that define blocks of linkage disequilibrium across ADME genes. Additionally, 50 SNPs were selected with known minor allele frequencies that differ greatly in different populations to be used as ancestry informative markers. This panel design can be used in two different ways, functional markers can be used to screen populations to make evidence based decisions (i.e. stratification in clinical trials) and tag markers can be used to uncover novel genotype/phenotype correlations.

The Broad-based ADME panel fills in many of the gaps in coverage, present on currently available commercial genotyping assays, which encompass many ADME genes. Table 1.; shows the percent coverage of the markers on the ADME panel (taking into account proxy SNPs that can be substituted for alternative tagging SNPs present on the commercial genotyping assays using an r-squared of 0.8 as calculated using SNAP2).

Table1: Coverage of ADME Panel Markers on Commercial Genotyping Assays.

After several phases of development and optimization, the panel has a conversion rate of close to 100% (see Table 2.) with the flexibility to add additional marker content that includes the ability to spike in PCR product in place of genomic DNA for regions that are difficult to assay. Moreover, the accuracy of the panel has been validated using samples from the International HapMap project³ as well as the testing of hundreds of previously genotyped samples. The assay has also been further validated by cross comparison with two commercial ADME products; the DMET™ Plus Premier Pack (Affymetrix)^4,5 and the VeraCode ADME Core Panel (Illumina)⁶.

Table 2: Summary Statistics and Assay Validation

To date, the assay has been part of several large research studies; it was demonstrated that the content was sufficient to be used in principal component analysis to stratify samples by geographic origin⁷. Additionally, the assay was used in a large effort to identify genetic causes of adverse drug reactions in children, to this end, novel associations were uncovered between TPMT and COMT and cisplatin induced hearing loss⁸ and SLC28A1 and anthracycline-induced cardiotoxicity⁹. Finally the assay has be utilized in two clinical trials; the first involved acute coronary syndrome patients taking part in the Rosuva-Atorva ACS trial, CENTAURUS with the aim of identifying genetic variation that can be predictive of the inter-individual variability observed in response to rosuvastatin and atorvastatin. The second involved the use of a novel 5-Lipoxygenase inhibitor in the preventions of atherosclerosis.

References:

1.        Introduction: Pharmacokinetics: Merck Manual Professional. at <http://www.merck.com/mmpe/sec20/ch303/ch303a.html#CHDHHHAD>

2.        Johnson, A.D. et al. SNAP: a web-based tool for identification and annotation of proxy SNPs using HapMap. Bioinformatics 24, 2938 -2939 (2008).

3.        A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851-861 (2007)

4.        Burmester, J.K., Sedova, M., Shapero, M.H. & Mansfield, E. DMET microarray technology for pharmacogenomics-based personalized medicine. Methods Mol. Biol 632, 99-124 (2010).

5.        Deeken, J. The Affymetrix DMET platform and pharmacogenetics in drug development. Curr. Opin. Mol. Ther 11, 260-268 (2009).

6.        VeraCode ADME Core Panel. at <http://www.illumina.com/products/veracode_adme_core_panel.ilmn>

7.        Visscher, H. et al. Application of principal component analysis to pharmacogenomic studies in Canada. Pharmacogenomics J 9, 362-372 (2009).

8.        Ross, C.J.D. et al. Genetic variants in TPMT and COMT are associated with hearing loss in children receiving cisplatin chemotherapy. Nat Genet 41, 1345-1349 (2009).

9.        Visscher, H. et al. Pharmacogenomic prediction of anthracycline-induced cardiotoxicity in children. (Article in preparation)

Broad Based Panel Content

The Université de Montréal Pharmacogenomic Centre

The Université de Montréal Pharmacogenomics Centre is a partnership between an academic institution (UdeM), and a world leading clinical institute, the Montreal Heart Institute (MHI), to develop translational medicine. One of the main objectives of the Centre is to develop and integrate innovative biomarker applications into the clinical setting using a wide-range of technical platforms. By bringing our understanding of biomarkers that influence drug efficacy and toxicity into the clinic, we hope to improve the quality of patient care through the correct selection and dosing of specific medications and therapies.

The Pharmacogenomics Centre is affiliated with the Université de Montréal faculties of Medicine and Pharmacy supporting the pharmacogenomics needs for the entire Université de Montréal Health Network. The Pharmacogenomics Centre has constructed a state of the art laboratory facility that has been built to a Good Laboratory Practices (GLP) standard.

The Pharmacogenomics Centre consists of two distinct parts: a technology development platform and a clinical operations platform. The development component is responsible for the development of genotyping panels, expression and proteomic technologies, method and technology development and the transfer of clinical grade assays to the clinical portion of the centre. The clinical component is responsible for DNA isolation, DNA and tissue banking under GLP conditions, the development and maintenance of GLP Standard Operating Procedures (SOPs) and their integration into the Montreal Heart Institute Clinical Coordinating Centre, as well as the development of necessary analytical and statistical tools.

The Centre has been designated as the NIH Heart Failure Network Genetics Core lab and is supporting the genetic testing for all the Network’s clinical trials. The lab also performs clinical sequencing for the MHI’s Cardivascular Genetic Centre screening for mutations in several cardiomyopathies (LQT, SQT, ARVS, BS, FA, HCM and DCM). The Centre has developed a genotyping panel and process for the Canadian Blood Services & HemaQuebec (the equivalent of the Red Cross in the US) to screen donors for secondary blood antigens to better match donors with patients requiring frequent transfusions. To date, we have screened >40,000 donors with >99.8% accuracy against established assays. The Centre routinely performs proficiency testing in support of several labs running ADME and GWAS technologies (i.e. DMET and Affy 6.0 for the Coriell Personalized Medicine Collaborative). Lastly, the centre has been audited on several occasions in support of clinical trial work that we have performed for large Pharmaceutical companies and CROs.

The Centre is presently not CLIA/CAP certified as this is a US standard, however, we are in the process of to submitting our CLIA/CAP application to obtain accreditation for several assays that we are performing for US collaborators and clients. We should have this accreditation within several months.