University of Groningen Gene expression profiling in C 57 BL / 6 J and A / J mouse inbred strains reveals gene networks specific for brain regions independent of genetic background

Background: We performed gene expression profiling of the amygdala and hippocampus taken from inbred mouse strains C57BL/6J and A/J. The selected brain areas are implicated in neurobehavioral traits while these mouse strains are known to differ widely in behavior. Consequently, we hypothesized that comparing gene expression profiles for specific brain regions in these strains might provide insight into the molecular mechanisms of human neuropsychiatric traits. We performed a whole-genome gene expression experiment and applied a systems biology approach using weighted gene co-expression network analysis. Results: We were able to identify modules of co-expressed genes that distinguish a strain or brain region. Analysis of the networks that are most informative for hippocampus and amygdala revealed enrichment in neurologically, genetically and psychologically related pathways. Close examination of the strain-specific gene expression profiles, however, revealed no functional relevance but a significant enrichment of single nucleotide polymorphisms in the probe sequences used for array hybridization. This artifact was not observed for the modules of co-expressed genes that distinguish amygdala and hippocampus. Conclusions: The brain-region specific modules were found to be independent of genetic background and are therefore likely to represent biologically relevant molecular networks that can be studied to complement our knowledge about pathways in neuropsychiatric disease. Background Genome-wide gene expression profiling has been used to aid in the discovery of genes involved in human diseases and to discriminate between disease subtypes. This approach has already been successfully applied in cancer and obesity research [1,2], but similar approaches have not yet been widely applied to human neuropsychiatric traits. An important issue concerning expression profiling in these disorders in man is the lack of the most relevant tissue, i.e. the brain, for study. Except for limited numbers of post-mortem samples, there is no easy access to human neuronal tissue for expression studies [3]. Mouse models, however, have long been used to study neuropsychiatric traits. There is consensus that symptoms of disorders such as major depression and anxiety can be studied by observing behavior in inbred strains [4,5]. Moreover, mouse brain tissue is accessible and most brain regions are highly conserved between mouse and human [6,7]. Regional gene expression in the brain has been shown to be conserved between non-human primates, rodents and man, making it possible to study mice in relation to human neurobiology [8,9]. This study focuses on two brain regions, the hippocampus and amygdala, because of their importance in neural behavior and psychiatric disease. These areas are both part of the limbic system and known to be heavily * Correspondence: r.a.ophoff@umcutrecht.nl Department of Medical Genetics and Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands de Jong et al. BMC Genomics 2010, 11:20 http://www.biomedcentral.com/1471-2164/11/20 © 2010 de Jong S et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. interconnected. Hippocampal function has been extensively studied and it has been shown to be a key structure in learning and memory processes [10]. The amygdala is important in processing fear and anxiety and is also thought to be involved in associative learning and memory. Because of its small size and complicated anatomy the amygdala has been less well studied in man. The structure consists of about 10 different subnuclei with distinct roles for the input and output of information [11]. Both brain regions have been implicated in neuropsychiatric disorders such as schizophrenia and depression, as well as epilepsy, but there is increasing evidence that they might play differential roles as well [12,13]. Using the mouse as an animal model is advantageous for the study of neurobehavioral traits because the genetic make-up of these inbred strains is known and they have been extensively phenotyped for more than two decades. This makes it possible to select strains most divergent for the (behavioral) trait of interest and to search for underlying genetic factors [14]. A recent study estimated that there are ~8 million single nucleotide polymorphisms (SNPs) between classical mouse inbred strains, which results in an estimated strain diversity of 1 SNP every 250-300 base pairs [15]. Although gene expression carries information about biological state and environment, a number of studies have shown that up to 85% of the measured transcripts in human lymphocytes are significantly heritable [16,17]. This suggests that gene expression in general is under strong genetic control. An association analysis of expression levels with SNPs and genomic copy number variations (CNVs) in human lymphoblastoid cell lines was performed and showed that SNPs and CNVs capture 83.6% and 17.7% of the total detected genetic variation in gene expression, respectively [18]. Expression levels are shown to be more variable among individuals than among the populations they belong to, as studied in fundulus fish and man [17,19,20]. Differences in allele frequencies seem to explain most of the variation in transcript levels between human populations [21]. To explore gene expression differences specific to a strain and/or brain region, we studied the amygdala and hippocampus brain tissues of mouse inbred strains A/J and C57BL/6J. These strains have been consistently shown to differ in behavioral, physiological and developmental processes. For example, studies have reported that A/J show more anxiety-like behaviors and are less social towards other mice [22]. A/J is also known to have lower motor activity levels [23] and be more resistant to developing epilepsy [24] than C57BL/6J. Although they are heavily interconnected, the amygdala and hippocampus perform separate functions in memory and emotion (see earlier). The amygdala functions mostly through GABA-ergic transmission, while the hippocampus uses excitatory connections [6]. Here we explore whether behavioral differences between the mouse strains and functional specificity of the two brain regions are reflected in differential gene expression profiles. It is plausible that differences in expression profiles represent a combination of genetic and functional variation. Considering the involvement of the hippocampus and amygdala in neurobehavioral symptoms in both mouse and man, the results from our mouse analysis may shed new light on pathways underlying neuropsychiatric traits in humans as well. Since we were interested in finding co-expression modules that relate to amygdala and hippocampus in the different mouse strains, we used gene co-expression network analysis. Gene co-expression network methods have been successfully applied in a variety of different settings [8,25-29]. This systems biology analysis method starts out by defining clusters of co-expressed genes (called ‘modules’) which may represent molecular networks involved in a common biological pathway. Genes that are highly connected within these groups are thought to drive the modules and are considered to be ‘hub genes’. As described below, we identified large coexpression modules that are significantly associated with differences between mouse strains and brain regions. We found that the observed strain-specific modules in fact reflect hybridization artifacts but, strikingly, two large modules distinguish brain regions irrespective of genetic background. Detailed functional enrichment analyses revealed an overrepresentation of genes involved in neurological and psychological disorders, in addition to nervous system function and development.