CREATE Abstract, 2005

Functional Polymorphism Analysis in Drug Pathways

Overview

Most pharmacogenetic strategies to date have focused on the role of single genes in the regulation of drug activity and have made important steps towards the goal of optimizing therapy for individual patients. 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 through the coordinated efforts of investigators from Washington University and University of California, San Francisco, with expertise in the fields of Genomics, Pharmacogenetics, Clinical Pharmacology, Pathology, Bioinformatics/Computational Biology, Molecular Genetics, Statistical Genetics, Population Genetics, Clinical Trials, and Translational Research. Together they are evaluating the following general aims to develop validation strategies for applied pharmacogenetics.

General Aims

• Identification of polymorphisms in members of pathways regulating drug activity
• Novel approaches to pathway dissection
• Validation and functional assessment of drug pathway variants

Based on our discoveries and current experience in the NIH PGRN since 2001, we have designed a program in which three collaborating Projects are supported by three core platforms to answer critical questions in the field of pharmacogenetics, make major contributions to PharmGKB, and provide the infrastructure for collaborative research across the PGRN. We believe that this approach, in which we pursue Gene Variant Discovery (Project 1), use state-of-the-art and novel approaches for Pathway Dissection (Project 2), and subject our findings to rigorous Assessment and Validation (Project 3), will allow us to continue to progress the understanding of the role of genes in pharmacologic response.

Project 1: Identification of polymorphisms in members of pathways regulating drug activity

Much has changed in the field of genomics since the inception of CREATE in 2001. The sequencing of the human genome has been completed; the genomes of a multitude of other organisms are either completely sequenced or will be sequenced over the next five years; almost 10 million single nucleotide polymorphisms (SNPs) have been deposited into public databases such as dbSNP (www.ncbi.nlm.nih.gov/SNP); and the International HapMap Consortium has obtained genotypes for almost 1,500,000 SNPs and is on track to genotype close to 3 million SNPs by the end of 2005. During this exciting period of genome research, it becomes abundantly clear that in addition to the coding sequences (which accounts for 1.5% of the human genome), there is another 3 to 8% of the human genome that is highly conserved when compared to other mammalian genomes. Initial examination of these non-coding conserved sequences shows that they harbor gene regulatory elements such as enhancers. Of equal importance is the realization that only a fraction of the variants found in gene loci are in the public databases and that the SNPs used to construct linkage disequilibrium (LD) maps (or haplotype maps) will not capture the genetic diversity in the population, even when the HapMap project is completed at the end of 2005. The data produced in this project thus far have convinced us that for a credible genetic study of genes involved in the pathway regulating drug activity, we need to continue to take a comprehensive approach and expand our SNP discovery effort to include non-coding conserved regions in these gene loci.

In this project, we fully survey the 124 drug pathway genes and new genes from Project 2, concentrating on the multi-species conserved sequences. As we are trying to identify common polymorphisms useful in association studies by the pharmacogenetics community, we continue to use the most cost effective and efficient approach, namely, comparative pooled DNA sequencing, in our SNP discovery process. In addition, we continue to use computational biology approaches to evaluate the identified variants and predict which are most likely to be of functional importance. We also determine the frequency of identified variants in common ethnic/racial groups. With this proven strategy, we not only identify a comprehensive panel of gene-associated variants for the pharmacogenetic community, but also provide a framework to select the most promising variants (as predicted by a computational approach) for analysis in the other projects of this network. Specifically, we propose the following:

• Specific Sub-aim 1: Design and validate PCR assays for the multi-species conserved sequences in candidate genes
• Specific Sub-aim 2: Sequence and analyze all PCR fragments from pooled DNA samples
• Specific Sub-aim 3: Characterize genotype and allele frequency of drug pathway variants in European, Hispanic, Asian and African populations

The data produced will be of great interest to the pharmacogenetics community. Researchers in the field will have a validated and characterized set of polymorphisms in gene loci important in the pathways regulating drug activity together with their predicted functional consequences.

Project 2: Novel approaches to pathway dissection

An important challenge to the field of pharmacogenetics is the selection of the "right" genes for validation and assessment. The construction of drug pathways has been an important step forward and allows for a level of prioritization for candidate evaluation. However, most of the over 33,000 genes in the human genome have not been evaluated in the context of drug response and have the potential to have unanticipated impact on pharmacology. Tools are desperately needed to generate more reliable and comprehensive gene lists for in vitro, ex vivo, and clinical evaluation. Therefore, we have chosen to focus project 2 on the following specific subaims with the common theme of "let the biology identify the genes for pharmacogenetics analysis":

• Specific Sub-aim 1: Evaluate genome-wide pharmacogenetics discovery strategies using linkage analysis
• Specific Sub-aim 2: Assess statistical tools for association analysis of familial and nonfamilial pharmacogenetic data
• Specific Sub-aim 3: Define gene networks association with drug targets

The results of these studies will identify new candidate genes for analysis in Projects 1 and 3 and stimulate the development of additional strategies for the discovery and prioritization of candidate genes for pharmacogenetics studies.

Project 3: Validation and functional assessment of drug pathway variants

A major goal of the PGRN is to develop the evidence for applied pharmacogenetics. The CREATE application continues to push toward that goal, with more comprehensive approaches to candidate gene identification (Project 2) and targeted resequencing (Project 1). An important bottleneck to progress in this context is the development of meaningful methods of assessing the relative impact of any particular candidate gene from drug pathways. Project 3 provides a wide-ranging approach to the prioritization of candidate genes, including assessment in clinically relevant tissue, in vitro credentialing using small inhibitory RNAs, and association studies in large prospective randomized trials. The project also takes bold steps into the clinical arena, with both genotype-guided therapy studies and questionnaire assays of patient and clinician fears/expectations about pharmacogenetics to define the landscape on which the field will develop. This project will not only generate large amounts of interesting data for deposition in PharmGKB, but will also advance our quest to move pharmacogenetics from an observational discipline to one that facilitates intervention (e.g., choice of drug, starting dose, etc) through the following specific aims:

• Specific Sub-aim 1: Determine the genotype-gene expression relationship in malignant tissue to validate gene variants
• Specific Sub-aim 2: Define the relative contribution of individual drug pathway members to aid prioritization of candidate genes
• Specific Sub-aim 3: Establish molecular marker, study design, and patient/clinician evidence to expedite the development of pharmacogenetically-guided studies