Validating Discovered Cis-Acting Regulatory Genetic Variants: Application of an Allele Specific Expression Approach to HapMap Populations

Susana Campino,Srilakshmi Raj,Kirk Rockett,Anna Richardson,Dominic P Kwiatkowski,Valentina D Mangano,Bert Mohr,Andrew Fry,Taane G Clark,Claire Vandiedonck,Julian Forton,Sarah Auburn,Geraldine Butler

doi:10.1371/journal.pone.0004105

Abstract

BackgroundLocalising regulatory variants that control gene expression is a challenge for genome research. Several studies have recently identified non-coding polymorphisms associated with inter-individual differences in gene expression. These approaches rely on the identification of signals of association against a background of variation due to other genetic and environmental factors. A complementary approach is to use an Allele-Specific Expression (ASE) assay, which is more robust to the effects of environmental variation and trans-acting genetic factors.Methodology/Principal FindingsHere we apply an ASE method which utilises heterozygosity within an individual to compare expression of the two alleles of a gene in a single cell. We used individuals from three HapMap population groups and analysed the allelic expression of genes with cis-regulatory regions previously identified using total gene expression studies. We were able to replicate the results in five of the six genes tested, and refined the cis- associated regions to a small number of variants. We also showed that by using multi-populations it is possible to refine the associated cis-effect DNA regions.Conclusions/SignificanceWe discuss the efficacy and drawbacks of both total gene expression and ASE approaches in the discovery of cis-acting variants. We show that the ASE approach has significant advantages as it is a cleaner representation of cis-acting effects. We also discuss the implication of using different populations to map cis-acting regions and the importance of finding regulatory variants which contribute to human phenotypic variation.

Highlights

Understanding the cellular mechanisms that modulate gene expression is fundamental to defining the genetic contribution to human phenotypic variation and disease susceptibility
Gene selection was based on: (1) the presence of a transcribed single nucleotide polymorphisms (SNPs), as this is required to measure relative transcript abundance (2) modest/high minor allele frequencies (MAF) of transcribed SNPs, to maximise the number of heterozygous individuals available for Allele-Specific Expression (ASE) analysis 3) PCR-primer design that can be applied to both cDNA and DNA samples, so that relative abundance of genomic DNA can be ascertained and used as a control 4) PCR-Primers that do not include other SNPs to avoid differential primer hybridization that could confound results
Using the ASE approach we observed allelic expression imbalance for five of six candidate genes described by Cheung et al [5]

Summary

Introduction

Understanding the cellular mechanisms that modulate gene expression is fundamental to defining the genetic contribution to human phenotypic variation and disease susceptibility. Identifying non-coding regions that alter gene expression by affecting transcriptional efficiency or by modulating mRNA splicing is difficult. Cis-regulatory elements reside on the same chromosome as the gene they regulate, and act only on the copy of the gene on the same chromosome. Trans-regulatory elements may be located on a different chromosome and regulate both copies of the gene. Several studies have recently identified non-coding polymorphisms associated with inter-individual differences in gene expression. These approaches rely on the identification of signals of association against a background of variation due to other genetic and environmental factors. A complementary approach is to use an Allele-Specific Expression (ASE) assay, which is more robust to the effects of environmental variation and trans-acting genetic factors

Methods

Results

Conclusion