Abstract
BackgroundCommon genetic variants that regulate gene expression are widely suspected to contribute to the etiology and phenotypic variability of complex diseases. Although high-throughput, microarray-based assays have been developed to measure differences in mRNA expression among independent samples, these assays often lack the sensitivity to detect rare mRNAs and the reproducibility to quantify small changes in mRNA expression. By contrast, PCR-based allelic expression imbalance (AEI) assays, which use a "marker" single nucleotide polymorphism (mSNP) in the mRNA to distinguish expression from pairs of genetic alleles in individual samples, have high sensitivity and accuracy, allowing differences in mRNA expression greater than 1.2-fold to be quantified with high reproducibility. In this paper, we describe the use of an efficient PCR/next-generation DNA sequencing-based assay to analyze allele-specific differences in mRNA expression for candidate neuropsychiatric disorder genes in human brain.ResultsUsing our assay, we successfully analyzed AEI for 70 candidate neuropsychiatric disorder genes in 52 independent human brain samples. Among these genes, 62/70 (89%) showed AEI ratios greater than 1 ± 0.2 in at least one sample and 8/70 (11%) showed no AEI. Arranging log2AEI ratios in increasing order from negative-to-positive values revealed highly reproducible distributions of log2AEI ratios that are distinct for each gene/marker SNP combination. Mathematical modeling suggests that these log2AEI distributions can provide important clues concerning the number, location and contributions of cis-acting regulatory variants to mRNA expression.ConclusionsWe have developed a highly sensitive and reproducible method for quantifying AEI of mRNA expressed in human brain. Importantly, this assay allowed quantification of differential mRNA expression for many candidate disease genes entirely missed in previously published microarray-based studies of mRNA expression in human brain. Given the ability of next-generation sequencing technology to generate large numbers of independent sequencing reads, our method should be suitable for analyzing from 100- to 200-candidate genes in 100 samples in a single experiment. We believe that this is the appropriate scale for investigating variation in mRNA expression for defined sets candidate disorder genes, allowing, for example, comprehensive coverage of genes that function within biological pathways implicated in specific disorders. The combination of AEI measurements and mathematical modeling described in this study can assist in identifying SNPs that correlate with mRNA expression. Alleles of these SNPs (individually or as sets) that accurately predict high- or low-mRNA expression should be useful as markers in genetic association studies aimed at linking candidate genes to specific neuropsychiatric disorders.
Highlights
Common genetic variants that regulate gene expression are widely suspected to contribute to the etiology and phenotypic variability of complex diseases
Our results show that this assay produces allelic expression imbalance (AEI) ratios for mRNAs expressed in human brain of outstanding quality and reproducibility
In this paper we describe a PCR/next-generation DNA sequencing-based method for quantifying allele-specific differences in mRNA expression and demonstrate that it produces detailed and highly reproducible information concerning the expression of candidate neuropsychiatric candidate genes in human brain
Summary
Common genetic variants that regulate gene expression are widely suspected to contribute to the etiology and phenotypic variability of complex diseases. Unlike Mendelian disorders, which can often be traced to mutations that disrupt gene structure or coding sequences, genetic markers that associate with complex disorders often map to chromosomal sites located outside of gene coding regions [8] Such observations suggest that genetic variants that regulate gene expression, rather than disrupt gene structure, may be a major source of liability for, or protection from, complex disorders [8,9]. Unlike coding region mutations or chromosomal rearrangements, which can be identified by DNA sequencing alone, detection of regulatory variants requires experimentation, such as measurements of variation in mRNA expression or splicing To meet this challenge, hybridization-based microarray assays have been developed that are capable of measuring variation in the expression of hundreds-to-thousands of genes in multiple samples in a single experiment [12,13]
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