PT  - JOURNAL ARTICLE
AU  - Andrew McEwan
AU  - Elizabeth Hay
AU  - Connor Davidson
AU  - Yvonne Turnbull
AU  - Pietro Marini
AU  - Dana Wilson
AU  - Andrew M. McIntosh
AU  - Mark J. Adams
AU  - Chris Murgatroyd
AU  - Perry Barrett
AU  - Mirela Delibegovic
AU  - Toni-Kim Clarke
AU  - Alasdair MacKenzie
TI  - CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests its role in anxiety and male alcohol intake
AID  - 10.1101/572065
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 572065
4099  - http://biorxiv.org/content/early/2019/03/09/572065.short
4100  - http://biorxiv.org/content/early/2019/03/09/572065.full
AB  - Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake against only 4% in women. Previous studies identified a significant interaction between haplotypes of the GAL gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n=115,865; p=0.0008) between allelic variation (GG or CA haplotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men that was consistent with these previous studies. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly and specifically reduced GAL expression in amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found reduced anxiety in male animals lacking GAL5.1 demonstrating sexual dimorphism mirroring that seen in humans. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co expressed with GAL in amygdala and hypothalamus, as being important in the PKC supported activity of the GG haplotype of GAL5.1 but less so in the CA haplotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to anxiety induced alcohol abuse in men.