Discussion

Our current results suggest that it is

possible to identify susceptibility regions

using this methodology.

The presented method takes advantage

of the large amount of phenotype data

generated by high-content imaging and the

detailed genotype information available for

inbred mouse strains. This allows us to

perform a high-throughput scan for QTLs

affecting cytotoxic responses.

This information, combined with the

increasing amount of protein interaction and

functional data available, can potentially

help the discovery of genes involved in drug

adverse reactions.

Results

We currently have 35 drugs screened that

are in different stages of the image

acquisition and analysis. We have not

advanced to the point of QTL mapping yet.

Partial results show some differences

between strains in drug response (Figures 1

and 2).

Methods

We have screened embryonic fibroblasts from

32 inbred mouse strains with 35

toxicants/drugs in a high-content imaging

screen that determines changes in specific

cell-health status phenotypes (nuclear

changes, membrane permeability,

mitochondrial membrane potential and

apoptosis).

The phenotype data generated will be used

in haplotype-based genome-wide association

scans. The experimental design is outlined in

the following sequence.

Introduction

Pharmacogenetic studies have successfully

identified genetic variants that contribute to

variation in susceptibility to drug responses,

but it is still a complex and challenging task

to evaluate broadly across the human

genome to identify the genetic components

of response to drugs. Despite limitations

there is a pressing need to identify genetic

components that contribute to the efficacy

and toxicity of drugs, across a wide spectrum

of agents. We have proposed that using an

alternative model population approach will

provide many underlying mechanisms and

pathways that are implicated in drug activity

and responses. We have developed a

platform from genetically well defined mouse

strains which will enable us to assess effects

of toxicity and efficacy of current and novel

agents in drug therapies.

The use of inbred mouse strains has

potential advantages in screening a large

number of compounds for response

variations. It is a genetically stable population

that allows us to quickly perform association

scans using available genotype information

and the large amounts of phenotypic data

generated by this project.

Here, we present a new strategy to

identify genes and gene pathways that

underlie susceptibility to cellular-level

adverse drug reaction. No targets have been

validated yet, but we demonstrate the ability

to multiplex cell-based assays for high-

throughput QTL discovery.

The cells are treated using 9 different drug concentrations, ranging from 15 nM to 100 M.

Genome-wide association scans are used to discover QTLs involved in cytotoxic response.

Candidate gene selection based on functional and expression information and validation.

-logP

Cumulative position

SNPster (haplotype association mapping)

Cell fixation and multiplex staining after 24 h and 72 h for high-content imaging analysis

Hoechst Permeability dyeMitochondria

membrane potential Cytochrome c

Control

Cycloheximide 100M

Pharmacogenetics: In-vitro drug toxicity screeningO. Suzuki1, N. Butz1, R. Singh2, B. Steffy1, D. Scoville1,

B. Parks2, R. Thomas2 , T. Wiltshire1 1) School of Pharmacy, University of North Carolina, Chapel Hill, NC;

2) The Hamner Institute for Health Sciences, Research Triangle Park, NC.

NOR/LtJWSB/EiJ

Some strains display markedly different responses to drug treatment

Figure 2: Cell permeability dose-response curves (A) after 24h of treatment with Cycloheximide, showing differences between two inbred mouse strains. WSB/EiJ cells have increased cell permeability (B) after treatment with 100M Cycloheximide for 24 h (green fluorescence).

A)

B)

DNA content Cell membrane permeability

Mitochondrial membrane potential changes

Cell loss

Nuclear morphology

Cytochrome c localization and release from

mitochondria

Segmentation of acquired images and calculation of different parameters relevant to cytotoxic response:

Mouse embryonic fibroblasts from 32 different inbred mouse strains are simultaneously grown and seeded onto single 384-well plates.

Nonlinear regression and extraction of quantitative phenotypic data.

Figure 1: Strains display different sensitivities to Cycloheximide after 24 h of treatment.

Dose: log[Cycloheximide] (nM)

Cell permeability

Differences in dose-response curves between strains