CREATE Abstract, 2003

Functional Polymorphism Analysis in Drug Pathways

Goals

Most pharmacogenetic strategies to date have focused on the role of single genes in the regulation of drug activity. However, there is clear evidence that medications, like most common diseases, are under the control of a network of genes, each contributing to the patient's phenotype. The CREATE Pharmacogenetic Research Network (Comprehensive Research on Expressed Alleles in Therapeutic Evaluation) brings together infrastructure for the evaluation of pathways regulating drug activity. This is being achieved by investigators with expertise in the fields of Genomics, Pharmacogenetics, Clinical Pharmacology, Bioinformatics, Computational Biology, Medical Genetics, Statistical and Population Genetics, and Translational Research to evaluate the following general projects:

• Identify polymorphisms in genes that are components of pathways regulating drug activity
• Establish the population characteristics of drug pathway polymorphisms in Caucasian, Hispanic, Asian, and African American subjects
• Determine the genotype-expression relationship in therapeutically relevant tissue

These aims are initially being achieved using human gastrointestinal cancer as a model system. The CREATE Network has identified 124 genes in drug pathways for 5-fluorouracil, irinotecan, and oxaliplatin (complete list available on CREATE website). With the support of administrative, molecular analysis, gene expression, tissue bank, and bioinformatics cores, we are now performing a comprehensive evaluation approach to the discovery of genetic variants, their frequency in the most common USA ethnic/racial groups, and functional significance in ex vivo colorectal cancer and adjacent normal tissue.

Progress

• Project 1: In Project 1, we have designed PCR assays for 103 pathway genes, totaling 855 exons and regulatory regions (1,000 bases before the first exon and the 1000 bases after the last exon). This includes > 40 genes shared with other PGRN groups (PAAR, PMT, COBRA, PAT). A bioinformatics program for characterization of regulatory SNPs is also used for additional focused resequencing (http://polly.wustl.edu/promolign/main.html). We have performed over 16,000 sequencing reactions to date and generated over 6 Mb of raw data. Two hundred seventy six SNPs have been identified (1 SNP per 500 bp scanned) in the 270 exons analyzed. These data provide the primary reagents for Projects 2 and 3, in order to validate the functional importance of drug pathway gene variants on drug therapy.
• Project 2: There are three main sources of human genetic polymorphisms for the CREATE network: published data, publicly available databases, and resequencing data from Project 1. Our initial validation efforts have focused on the African and European populations, as they contain the most dramatic ethnic/racial differences in allele frequency. A total of 34,813 genotypes from 55 of the pathway genes have been submitted to PharmGKB, giving us a clear view of the degree of ethnic/racial difference in target genes. This has also identified the most informative sources of SNP information, with Project 1 resequencing data and literature genetic variants being validated at a much higher rate (90-100%) compared to publicly available databases (40-70%). Prof Templeton is now developing novel methods for nested cladistic analysis to define gene flow for variants within the drug pathway genes.
• Project 3: The goal of this project is to use ex vivo human tissue to establish the relationship between DNA variants and both RNA and protein expression. Using an initial panel of 52 colorectal tumors and the adjacent normal mucosa (104 samples total), genotyping for variants of interest has been conducted in both germline and somatic DNA using Pyrosequencing. We have generated RNA analysis probes for 124 drug pathway genes and have completed analysis for 74 genes (7696 expression profiles submitted to PharmGKB). Antibodies are available for 84% of our 124 candidate genes and are being generated for the remaining genes. Using tissue arrays constructed by the Tissue Bank Core, we are able to evaluate protein expression in the same tumor/adjacent tissue pairs used for genotype and RNA expression analysis. Genotype-RNA/protein analysis is identifying genetic variants with an impact on RNA/protein concentration. The mechanistic basis for these relationships is now under investigation, in collaboration with PMT, COBRA, PAT, and PAAR. These data are now facilitating the selection of genetic variants for application in clinical studies. Initial studies of patients (n=87) receiving camptothecins have observed an impact of ABCB1 and ABCG2 on pharmacokinetics. More importantly we have integrated pharmacogenetic sampling into large, NIH cooperative group studies. A randomized, controlled trial of 588 patients with advanced colorectal cancer, receiving 5-fluorouracil, irinotecan, and/or oxaliplatin, has identified predictive markers for each of the three agents and a "therapy selection panel" is being constructed. Further NCI cooperative group studies are underway, in collaboration with members of the PAAR, PMT, PAT, and COBRA, to develop validation strategies for applied pharmacogenetics.

Phenotyping Strategies and Criteria

Pharmacodynamic phenotypes. We have chosen to emphasize pharmacologic phenotypes that are in part dictated by the particular disease states, as well as by an initial phenotyping strategy that emphasizes regional (rather than systemic) vascular responses, so as to remove pharmacokinetic and baroreflex variables from their confounding influence, and thereby to isolate and focus upon pharmacodynamic (receptor and post-receptor) determinants of drug responses. The human regional responses are characterized in the pulmonary (Phenotyping project 1), renal (Phenotyping project 2), forearm (Phenotyping project 3), and hand (Phenotyping project 4) circulatory beds.