On the trail of anxiety

With the advances in genome wide screening arrays and sequencing techniques scientists were enabled to examine genetic variations and their effects on behavioral phenotypes. While single nucleotide polymorphisms (SNPs) are the most widely studied form of genomic variations to date, another type of variants has become increasingly important in recent research, the copy number variants (CNVs). These large segments of DNA that can comprise up to several megabasepairs and differ in copy number with respect to a reference genome have been associated with several disorders and behavioral phenotypes before. This study investigated the influence of CNVs on anxiety related behavior. The detection of these variants turned out to be a major challenge since all methods available are biased by limitations of the design of the approach and the subsequent computational analyses. Therefore, three different techniques (next generation sequencing and two distinct whole genome genotyping arrays) were employed to identify CNVs in a CD 1 derived mouse model consisting of two mouse strains showing high (HAB) and low (LAB) anxiety related behavior, respectively. By comparing CNVs in HAB vs. LAB mice with expression data of four distinct brain regions of high relevance to the limbic system (central and basolateral amygdala, cingulate cortex and the hypothalamic paraventricular nucleus), it was shown that CNVs can influence the expression of protein coding genes by the alteration of the genes’ copy number per se. Therefore, the genes mapping into regions where CNVs were detected in HAB vs. LAB mice (by all three detection methods) were suggested to be possible effectors of anxiety related behavior. Amongst these candidate genes those were considered to be the most interesting ones that were additionally found to map into regions of CNVs associated with anxiety related behavior in CD 1 mice. CNVs in these mice were detected by means of a whole genome genotyping array and subsequent processing of the raw data with a novel computational approach that was adapted from existing analysis methods. Furthermore, to test the effect of a specific CNV on anxiety related behavior in vivo, a breeding approach was used to generate animals with a full genetic background of HAB mice except for one LAB derived locus harboring a CNV that included the Glo1 gene. No direct effect on the phenotype could be observed, however, the respective CNV might be involved in the manipulation of anxiety related behavior taken into account the interaction with other factors. Taken together, this study provides not only a comprehensive catalogue of CNVs in HAB/LAB mice but also the evidence that these variants can influence anxiety related behavior. Furthermore, it gives a first insight into the functionality of CNVs with respect to anxiety related behavior. Therefore, this thesis provides a profound basis for multiple advanced studies

Connecting Anxiety and Genomic Copy Number Variation: A Genome-Wide Analysis in CD-1 Mice.

Genomic copy number variants (CNVs) have been implicated in multiple psychiatric disorders, but not much is known about their influence on anxiety disorders specifically. Using next-generation sequencing (NGS) and two additional array-based genotyping approaches, we detected CNVs in a mouse model consisting of two inbred mouse lines showing high (HAB) and low (LAB) anxiety-related behavior, respectively. An influence of CNVs on gene expression in the central (CeA) and basolateral (BLA) amygdala, paraventricular nucleus (PVN), and cingulate cortex (Cg) was shown by a two-proportion Z-test (p = 1.6 x 10-31), with a positive correlation in the CeA (p = 0.0062), PVN (p = 0.0046) and Cg (p = 0.0114), indicating a contribution of CNVs to the genetic predisposition to trait anxiety in the specific context of HAB/LAB mice. In order to confirm anxiety-relevant CNVs and corresponding genes in a second mouse model, we further examined CD-1 outbred mice. We revealed the distribution of CNVs by genotyping 64 CD 1 individuals using a high-density genotyping array (Jackson Laboratory). 78 genes within those CNVs were identified to show nominally significant association (48 genes), or a statistical trend in their association (30 genes) with the time animals spent on the open arms of the elevated plus-maze (EPM). Fifteen of them were considered promising candidate genes of anxiety-related behavior as we could show a significant overlap (permutation test, p = 0.0051) with genes within HAB/LAB CNVs. Thus, here we provide what is to our knowledge the first extensive catalogue of CNVs in CD-1 mice and potential corresponding candidate genes linked to anxiety-related behavior in mice

Distribution of CNVs in CD-1 mice.

<p>Chromosomes are indicated by grey horizontal lines. Start points of CNVs are marked by dots and lines are drawn to the end points. Due to limitations in resolution, a small CNV might appear as dot only. CNVs highlighted in blue or red were associated with anxiety-related behavior (time on the open arm of the EPM) with a nominal <i>p</i>-value less than 0.1 or 0.05, respectively.</p

Protein coding genes in genomic regions of CNVs detected in HAB/LAB and CD-1 mice.

<p>All genes listed overlap both CNVs in HAB/LAB mice detected with aCGH, JaxMDGA and NGS, and CNVs in CD-1 mice which were best associated with the time the animals spent on the open arm of the EPM (nominal <i>p</i>-value < 0.1).</p><p>Protein coding genes in genomic regions of CNVs detected in HAB/LAB and CD-1 mice.</p

Association of copy number with anxiety-related behavior in CD-1 mice.

<p>Exemplarily, data of three associations resulting in nominal <i>p</i>-values reaching significance (<i>p</i> < 0.05), a trend (<i>p</i> < 0.1), and not reaching significance (<i>p</i> > 0.05), respectively, are shown. Each dot represents data of a single animal (N = 64). The relative copy number is represented by the mean normalized intensities of JaxMDGA probes within the respective CNV. <b>(A)</b> CNV no. 498; <i>P</i>_nom = 0.0009; regression line: y = 0.0091x + 9.4389. <b>(B)</b> CNV no. 164; <i>P</i>_nom = 0.0554; regression line: y = 0.0061x + 10.201. <b>(C)</b> CNV no. 453; <i>P</i>_nom = 0.9791; regression line: y = 0.0008x + 9.6225.</p

Genomic positions of CNVs on chromosome 3.

<p>The chromosome is indicated by a thick horizontal line (grey). Depending on the detection method, CNVs in HAB/LAB mice are depicted in orange (aCGH), dark red (JaxMDGA) and red (NGS), respectively. Data displayed above the grey line represent a copy number gain in HAB vs. LAB animals, data below a copy number loss. Data printed on the grey line show CNVs in 64 CD-1 mice, with those highlighted in color that could be associated with anxiety-related behavior (time on the open arm of EPM) with a nominal <i>p</i>-value less than 0.1 (light blue) or less than 0.05 (blue). Start points of CNVs are marked by dots and lines are drawn to the end points.</p