Generalised Anxiety Disorder--A Twin Study of Genetic Architecture, Genome-Wide Association and Differential Gene Expression

Abstract

Generalised Anxiety Disorder (GAD) is a common anxiety-related diagnosis, affecting approximately 5% of the adult population. One characteristic of GAD is a high degree of anxiety sensitivity (AS), a personality trait which describes the fear of arousal-related sensations. Here we present a genome-wide association study of AS using a cohort of 730 MZ and DZ female twins. The GWAS showed a significant association for a variant within the RBFOX1 gene. A heritability analysis of the same cohort also confirmed a significant genetic component with h2 of 0.42. Additionally, a subset of the cohort (25 MZ twins discordant for AS) was studied for evidence of differential expression using RNA-seq data. Significant differential expression of two exons with the ITM2B gene within the discordant MZ subset was observed, a finding that was replicated in an independent cohort. While previous research has shown that anxiety has a high comorbidity with a variety of psychiatric and neurodegenerative disorders, our analysis suggests a novel etiology specific to AS.

Fig 1. a:b: Manhattan and qq plot of ASI GWAS with RBFOX1 gene region highlighted.

(a) The rs13334105 SNP (p values 4.39 x 10–8) highlighted in green is the only variant to show genomewide significant association with ASI. (b) The qq plot of ASI GWAS.

Fig 2. a-b Association plots of the genomewide significant variant rs13334105, showing its location with the RBFOX1 gene region.

Fig 3. a-b. Otowa et al. identified SNPs within the RBFOX1 intronic region with unadjusted p-values of significance, (a) rs17664315 (p = 0.00037) for case-control and (b) rs7203983 (p = 0.00250) for factor scores.

Fig 4. Relative expression of the six ITM2B exon between case and control ASI discordant twins.

Significantly higher levels of expression are shown in the twins with higher levels of ASI in the case of exon 5 and exon 6.

Fig 5. Probe location of exon 4, 5 and 6 of ITM2B in the U219 Affy gene expression array.

Fig 6. Residualised gene expression showing higher expression levels of the ITM2B gene in the affected twin in comparison to the unaffected sibling in the independent sample.

Fig 7. Neuronal depolarisation induces the movement of Fox1 protein from cytoplasm to the nucleus where it is coded by RBFOX1 and increases its capacity to induce splice sites (1).

Gabrg2 is regulated by RBFOX1 through the binding of Fox1 protein 1 and Nova near the 5′ and 3′ splice sites of the downstream intron through the targeting of the RNA hexanucleotide UGCAUG (2). Binding to the conserved UGCAUG elements cause an inclusion of 8 amino acids (LLRMFSFK) on the intracellular loop between the M3 and M4 transmembrane domain site. This creates a putative for protein kinase C (PKC) which is a point for serine phosphorylation (3). Phosphorylation of serine alters the γ2 subunit structure of the GABA (A) receptor which controls the regulation of GABAergic neuronal transmission (4).