The Genomic Response of Skeletal Muscle to Methylprednisolone Using Microarrays: Tailoring Data Mining to the Structure of the Pharmacogenomic Time Series

Abstract

High-throughput data collection using gene microarrays has great potential as a method for addressing the pharmacogenomics of complex biological systems. Similarly, mechanism-based pharmacokinetic/pharmacodynamic modeling provides a tool for formulating quantitative testable hypotheses concerning the responses of complex biological systems. As the response of such systems to drugs generally entails cascades of molecular events in time, a time series design provides the best approach to capturing the full scope of drug effects. A major problem in using microarrays for highthroughput data collection is sorting through the massive amount of data in order to identify probe sets and genes of interest. Due to its inherent redundancy, a rich time series containing many time points and multiple samples per time point allows for the use of less stringent criteria of expression, expression change and data quality for initial filtering of unwanted probe sets. The remaining probe sets can then become the focus of more intense scrutiny by other methods, including temporal clustering, functional clustering and pharmacokinetic/pharmacodynamic modeling, which provide additional ways of identifying the probes and genes of pharmacological interest.

Mathematical Modeling of Corticosteroid Pharmacogenomics in Rat Muscle following Acute and Chronic Methylprednisolone Dosing

“…The selection criteria for the acute study was described previously. 16 Due to fewer time points, the corresponding criteria in the chronic study were modified. A probe set had to have a call of at least 3 "P" out of the total 44 chips to be considered expressed in the tissue.…”

Microarray analysis of the temporal response of skeletal muscle to methylprednisolone: comparative analysis of two dosing regimens

“…The time points included were 6, 10, 13, 18, 24, 36, 48, 72, 96, and 168 h after pump implantation. A vehicle-treated control group of four animals was implanted with a saline-filled pump and killed at various times throughout the 7-day study period (6,18,48, and 96 h after pump implantation). Because of the fact that pump implantation requires some time to perform, an actual time zero control is not possible to obtain.…”

“…The biotinylated cRNAs were hybridized to 44 individual Affymetrix GeneChips, Rat Genome 230A (Affymetrix, Santa Clara, CA), that contained 15,967 probe sets. These gene chips contain over 7,000 more probe sets than the ones used (U-34A) in our previous acutedose MPL study in muscle (6). The high reproducibility of in situ synthesis of oligonucleotide chips allows accurate comparison of signals generated by samples hybridized to separate arrays.…”

“…The first step in the analysis involved screening the data from the 11 groups of 4 animals for variance anomalies. This was an important consideration, since the four control animals used in this study were killed at various times (6,18,48, and 96 h) within the experimental time frame. This step involved calculation of the mean, standard deviation, and coefficient of variation (CV) of the raw data for each of the 15,967 individual probe sets on the four chips at each individual time point (data from 4 drug-treated animals killed at the same time point) and for the controls (data from 4 vehicle-treated animals killed at 4 different time points).…”

“…Previously, we described the mining and analysis of microarray time series, illustrating the responses of skeletal muscle, liver, and kidney taken from the same set of animals to a single bolus dose of the corticosteroid methylprednisolone (MPL) (4,6,7). Those studies included individual chips from multiple control animals as well as multiple animals at each of 16 times over a 72-h period following acute dosing with MPL.…”