GABRA2 Alcohol Dependence Risk Allele is Associated with Reduced Expression of Chromosome 4p12 GABAA Subunit Genes in Human Neural Cultures.
- Authors
- Lieberman, Richard; Kranzler, Henry R; Joshi, Pujan; Shin, Dong-Guk; Covault, Jonathan
- Year
- 2015
- Journal
- Alcoholism, clinical and experimental research
- PMID
- 26250693
- DOI
- 10.1111/acer.12807
- PMCID
- PMC4558268
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 polymerase chain reaction and next-generation RNA sequencing. mRNA expression in adult human brain was examined using the BrainCloud and BRAINEAC data sets. 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 3 chromosome 4p12 GABAA genes, but not other neural genes. Cluster analysis based on the relative RNA levels of the 4 chromosome 4p12 GABAA genes identified 2 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 postmortem adult cortex in either the BrainCloud or BRAINEAC data sets. 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 postmortem 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.
GABRA2 RNA Expression by rs279858 Genotype(A) qPCR data from 36 iPS-derived neural cell lines reveals a significant effect of GABRA2 rs279858 genotype on GABRA2 RNA levels. Expression was significantly lower in rs279858 C-allele carriers than in lines from T/T individuals. (B) There was no difference in RNA expression of RBFOX3 (NeuN) as a function of rs279858 genotype. (* p<0.05)
Clustering of Cell Lines by Chromosome 4p12 GABAA Expression in Differentiated Neural CellsScatterplots of qPCR RNA levels contrasting (A) GABRA2 vs. GABRG1, (B) GABRA2 vs. GABRA4, and (C) GABRA2 vs. GABRB1 RNAs for 36 iPS-derived neural cell lines. RNA levels for Cluster 1 cultures (x symbol) were lower for all 4 genes than those for Cluster 2 cultures (gray circles).
RNA Levels for Chromosome 4p12 GABAA Genes by Cluster GroupRNA levels for chr4p12 GABAA genes measured by qPCR were significantly lower in Cluster 1 cell lines (n=17) than in Cluster 2 cell lines (n=19): (A) GABRG1, (B) GABRA2, (C) GABRA4, and (D) GABRB1. There were no significant differences in RNA levels between clusters for (E) GABRD or (F) RBFOX3 (NeuN). (*** p<0.001)
RNA-Seq Confirms qPCR Results and Delineates Differences in Expression of Chromosome 4p12 GABAA GenesRNA-seq data from16 iPSC-derived neural cell lines generated from 10 donor subjects. (A) Schematic depicting the orientation of the chr4p12 GABAA cluster and adjacent genes. Arrows indicate the direction of transcription, with the approximate distance shown between each gene. (B) There were significant differences in expression between neural cell lines in Cluster 1 and Cluster 2 for the chr4p12 GABAA genes GABRG1; GABRA2; GABRA4; GABRB1, but not for the genes directly adjacent to the chr4p12 GABAA cluster GNPDA2, and COMMD8.)(C) GABAA genes located on other chromosomes did not differ in expression between neural cell lines in Cluster 1 and Cluster 2 (GABRG2, GABRB2, GABRB3, GABRD). Genes encoding cation-chloride transporter proteins also did not differ between Cluster 1 and Cluster 2 (SLC12A2 and SLC12A5). (* p<0.05; ** p<0.005)
Expression of GABRA2 RNA by rs279858 Genotype in Cluster 1 vs. Cluster 2 Neural Cell LinesCluster 1 iPSC-derived neural cell lines had lower levels of GABRA2 RNA and a greater number of C-allele carriers than Cluster 2 lines, but levels of GABRA2 RNA did not show a rs279858 allele dose effect between cell lines within each cluster.
Random Allelic Expression Bias of Chromosome 4p12 GABAA Genes in independently derived iPSC-neural cultures from a Cluster 2 SubjectAllele biased gene expression in mature neural cultures generated from two independently derived iPSC lines (727-6 and 727-8) from a Cluster 2 donor homozygous for the T-allele at GABRA2 rs279858 but heterozygous at exonic SNPs in the other three chr4p12 GABAA genes (rs976156 GABRG1 exon 3 Thr88Thr, rs7660336 GABRA4 3UTR, and rs10028945 GABRB1 3UTR). An opposite pattern of allelic bias (ΞCT between FAM and VIC probes) was observed comparing lines 727-6 and 727-8 for each of the heterozygous SNPs in (A) GABRG1, (B) GABRA4, and (C) GABRB1. Expression of opposite alleles for each gene in the two lines spanning the 4p12 GABAA gene region indicates the presence of a random process during iPSC induction that can produce bias in expression from either the maternal or paternal chromosome.
| Name | Type |
|---|---|
| 15q11 local | other |
| 4p12 local | other |
| 5-HTTLPR | variant |
| 5q34 local | other |
| AD-relevant behavioral effects local | phenotype |
| adult brain RNA expression local | phenotype |
| adult post-mortem brain local | cohort |
| AKR1C3 local | gene |
| alcohol | phenotype |
| alcohol dependence | phenotype |
| allelic bias local | phenotype |
| allelic expression imbalance local | phenotype |
| allelic imbalance local | phenotype |
| Alzheimer's disease | phenotype |
| amygdala | anatomy |
| anterior cingulate cortex | anatomy |
| anxiety | phenotype |
| Applied Biosystems 7500 instrument local | drug |
| balanced expression local | phenotype |
| Bowtie | drug |
| brain | anatomy |
| Brain Cloud local | cohort |
| BrainCloud | cohort |
| brain development | phenotype |
| Braineac | cohort |
| brain tissue | anatomy |
| cell proliferation | phenotype |
| cellular diversity of immune and neural cells local | phenotype |
| chr4p12 GABAA gene cluster local | gene |
| chr4p12 genes local | gene |
| chromosome 4 GABAA gene expression local | gene |
| chromosome 5q34 genes local | gene |
| clonal lymphoblastoid cell lines local | cohort |
| Cluster 1 | cohort |
| Cluster 1 local | phenotype |
| Cluster 2 local | cohort |
| Cluster 2 local | phenotype |
| Cluster 2 cell lines local | cohort |
| cluster membership local | cohort |
| Cluster membership local | phenotype |
| COMMD8 local | gene |
| cortex | anatomy |
| Corticogenesis local | phenotype |
| Cufflinks | drug |
| depression | phenotype |
| developing nervous system local | anatomy |
| donor 727 local | cohort |
| dorsolateral prefrontal cortex | anatomy |
| drug dependence | phenotype |
| EEG beta frequency oscillations local | phenotype |
| FAMβlabeled TaqMAN Assays local | drug |
| fast beta EEG activity local | phenotype |
| fetal brain | anatomy |
| first trimester forebrain tissue local | anatomy |
| forebrain | anatomy |
| frequency electroencephalographic activity local | phenotype |
| frontal cortex | anatomy |
| functional variant | variant |
| GABA | phenotype |
| GABAA gene region local | gene |
| GABAA genes local | gene |
| GABAA receptor | drug |
| Gabra1 | gene |
| GABRA2 | gene |
| GABRA2 expression local | phenotype |
| GABRA2 polymorphism local | variant |
| GABRA2 rs279858*C allele local | variant |
| GABRA3 | gene |
| Gabra4 | gene |
| GABRA5 | gene |
| GABRA6 | gene |
| GABRB1 | gene |
| Gabrb2 | gene |
| Gabrb3 | gene |
| Gabrd | gene |
| GABRE | gene |
| Gabrg1 | gene |
| Gabrg2 | gene |
| Gabrg3 | gene |
| GABRP | gene |
| GABRQ | gene |
| GABRR1 | gene |
| GABRR2 | gene |
| GABRR3 | gene |
| GAD2 | gene |
| genetic variants | cohort |
| GNPDA2 | gene |
| GUSB | gene |
| Heavy drinkers cohort local | cohort |
| heavy drinking | phenotype |
| hg19 | drug |
| High Capacity cDNA Reverse Transcription kit | drug |
| high-expression cluster local | cohort |
| hippocampus | anatomy |
| human | cohort |
| human clonal neural stem cells local | cohort |
| human fibroblasts | cohort |
| illicit drug dependence | phenotype |
| illicit drug use | phenotype |
| Illumina HiSeq 2000 | drug |
| Illumina TruSeq Chemistry local | drug |
| immunoglobulin genes local | gene |
| insula | anatomy |
| interleukin receptor genes local | gene |
| iPSC clones local | cohort |
| iPSC-derived forebrain neural cultures local | cohort |
| iPSC-derived neural cells local | cohort |
| iPSCβderived neural cells (one subject) local | cohort |
| iPSC-derived neural cultures local | cohort |
| iPSC lines | cohort |
| iPSC lines (36 lines from 21 donors) local | cohort |
| iPSCs | cohort |
| iPS-derived neural cell lines local | cohort |
| KCC2 local | drug |
| Klf4 | gene |
| LIN28 local | gene |
| low-expression cluster local | cohort |
| low mRNA expression of chr4p12 GABAA gene cluster local | phenotype |
| marijuana | phenotype |
| marijuana dependence | phenotype |
| mature neural cultures local | cohort |
| medial frontal cortex | anatomy |
| mice | cohort |
| monkeys | cohort |
| mouse chromosome 11 local | other |
| mouse chromosome 5 local | other |
| mouse chromosome 7 local | other |
| mouse chromosome X local | other |
| Myc | gene |
| NanoDrop 2000 spectrophotometer local | drug |
| NeuN | drug |
| neural cell lines local | cohort |
| neural culture local | cohort |
| Neural cultures (12β17 weeks postβplating) local | cohort |
| neural protocadherin genes local | gene |
| neuropsychiatric conditions local | phenotype |
| NKCC1 local | drug |
| nucleus accumbens | anatomy |
| Oct4 | gene |
| odorant receptor genes local | gene |
| placental tissue local | cohort |
| post-mortem human brain samples local | cohort |
| Post-treatment drinking local | phenotype |
| random mono-allelic expression local | phenotype |
| RBFOX3 | gene |
| relapse | phenotype |
| Retrovirus constructs local | drug |
| reward anticipation | phenotype |
| Reward pathway activation by alcohol cues local | phenotype |
| RNA | drug |
| RPKM | drug |
| rs10028945 local | variant |
| rs12529 local | variant |
| rs279856 local | variant |
| rs279858 | variant |
| rs279858*C-allele local | variant |
| rs7660336 local | variant |
| rs976156 local | variant |
| RT-qPCR local | drug |
| S-allele local | variant |
| Sendai virus constructs local | drug |
| SLC12A2 local | gene |
| SLC12A5 | gene |
| Slc17a7 | gene |
| SLC6A4 | gene |
| Social drinkers | phenotype |
| Social drinkers cohort local | cohort |
| Sox2 | gene |
| spinal cord | anatomy |
| substance-related disease local | phenotype |
| Synaptic formation local | phenotype |
| tag-SNP local | variant |
| TaqMan allele-specific probes local | drug |
| TopHat local | drug |
| Trizol | drug |
| ventral tegmental area | anatomy |
| VICβlabeled GUSB local | drug |
| Xq28 local | other |
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External
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