GABRA2 Alcohol Dependence Risk Allele is Associated with Reduced Expression of Chromosome 4p12 GABA_A Subunit Genes in Human Neural Cultures

Abstract

Background Genetic variation in a region of chromosome 4p12 that includes the GABAA-subunit gene GABRA2 has been reproducibly associated with alcohol dependence (AD). However, the molecular mechanisms underlying the association are unknown. This study examined correlates of in vitro gene expression of the AD-associated GABRA2 rs279858*C-allele in human neural cells using an induced pluripotent stem cell (iPSC) model system. Methods We examined mRNA expression of chromosome 4p12 GABAA subunit genes (GABRG1, GABRA2, GABRA4, and GABRB1 in 36 human neural cell lines differentiated from iPSCs using quantitative PCR and Next Generation RNA Sequencing. mRNA expression in adult human brain was examined using the BrainCloud and Braineac datasets. Results We found significantly lower levels of GABRA2 mRNA in neural cell cultures derived from rs279858*C-allele carriers. Levels of GABRA2 RNA were correlated with those of the other three chromosome 4p12 GABAA genes, but not other neural genes. Cluster analysis based on the relative RNA levels of the four chromosome 4p12 GABAA genes identified two distinct clusters of cell lines, a low-expression cluster associated with rs279858*C-allele carriers and a high-expression cluster enriched for the rs279858*T/T genotype. In contrast, there was no association of genotype with chromosome 4p12 GABAA gene expression in post-mortem adult cortex in either the BrainCloud or Braineac datasets. Conclusions AD-associated variation in GABRA2 is associated with differential expression of the entire cluster of GABAA subunit genes on chromosome 4p12 in human iPSC-derived neural cell cultures. The absence of a parallel effect in post-mortem human adult brain samples suggests that AD-associated genotype effects on GABAA expression, although not present in mature cortex, could have effects on regulation of the chromosome 4p12 GABAA cluster during neural development.

Examining FKBP5 mRNA expression in human iPSC-derived neural cells

“…Expression of these genes was normalized to the VIC-labeled housekeeping gene GUSB (beta-glucuronidase, 4326320E, Life Technologies). GUSB was selected as a control due to robust, detectable expression using qPCR in our neural cells and fibroblasts, and our prior demonstration of its reliability in studies of alcohol and genetic variation in iPSC-derived neural cultures (Lieberman et al, 2012; Lieberman et al, 2015). cDNA synthesized from RNA extracted from each culture well was assayed in triplicate 20 μL reactions using Gene Expression Master Mix (Life Technologies) per the manufacturer’s protocol, with the addition of 0.1% bovine serum albumin (BSA, Life Technologies).…”

“…While the whole-transcriptome profiles of iPSC clones derived from the same donor subject are similar (Schuster et al, 2015), we previously identified differences in targeted gene expression between clones (Lieberman et al, 2015). Therefore, we assumed for the statistical analysis that iPSC clones were separate lines even when derived from the same donor subject.…”

GABAA receptor subtype involvement in addictive behaviour

“…How these genetic variants may influence GABA A receptor function at selective neuronal loci remains an open question though, as highlighted earlier, a recent study indicates a specific SNP, rs279858 to associate with reduced α2mRNA in an iPSC culture model (Lieberman et al . ). In this respect, the observation that in humans high levels of impulsivity in adolescents associate with a selective reduction in the GABA signal in the anterior cingulate cortex of adult subjects (Silveri et al .…”

“…Changes in lateral inhibition, due to decreased α2 GABA A R in rodents (as a result of early life stress; Lambert ) or humans (dependent on specific GABRA2 variants, e.g. SNP rs279858; Lieberman et al ) might be the basis for the increased beta power measured in EEGs (Costa & Bauer ; Edenberg et al ; Lydall et al ; Rangaswamy et al ).…”

GRIK1 and GABRA2 Variants Have Distinct Effects on the Dose‐Related Subjective Response to Intravenous Alcohol in Healthy Social Drinkers

“…“Extrasynaptic” subtypes of GABA A receptors are direct targets for alcohol, while synaptic GABA A receptors have low sensitivity to alcohol but respond to GABA that may be released during alcohol administration. Rs279858 is a coding SNP in exon 5 that produces a synonymous substitution, but a gene expression study in cultured human neural cells showed that the risk allele “C” was associated with significantly reduced mRNA levels for the α 2 subunit of the GABA A receptor ( GABRA2 ), and altered expression of the cluster of four GABA A receptor subunit genes on chromosome 4 (Lieberman et al, 2015). The rs279858*C allele conveys risk for AUD (cf.…”