High-throughput transcriptome sequencing identifies candidate genetic modifiers of vulnerability to fetal alcohol spectrum disorders

Abstract

Background: Fetal alcohol spectrum disorders (FASD) is a leading cause of neurodevelopmental disability. Genetic factors can modify vulnerability to FASD, but these elements are poorly characterized.

Methods: We performed high-throughput transcriptional profiling to identify gene candidates that could potentially modify vulnerability to ethanol's (EtOH's) neurotoxicity. We interrogated a unique genetic resource, neuroprogenitor cells from 2 closely related Gallus gallus lines having well-characterized robust or attenuated EtOH responses with respect to intracellular calcium mobilization and CaMKII/β-catenin-dependent apoptosis. Samples were not exposed to EtOH prior to analysis.

Results: We identified 363 differentially expressed genes in neuroprogenitors from these 2 lines. Kyoto Encyclopedia of Genes and Genomes analysis revealed several gene clusters having significantly differential enrichment in gene expression. The largest and most significant cluster comprised ribosomal proteins (38 genes, p = 1.85 × 10(-47) ). Other significantly enriched gene clusters included metabolism (25 genes, p = 0.0098), oxidative phosphorylation (18 genes, p = 1.10 × 10(-11) ), spliceosome (13 genes, p = 7.02 × 10(-8) ), and protein processing in the endoplasmic reticulum (9 genes, p = 0.0011). Inspection of gene ontogeny (GO) terms identified 24 genes involved in the calcium/β-catenin signals that mediate EtOH's neurotoxicity in this model, including β-catenin itself and both calmodulin isoforms.

Conclusions: Four of the identified pathways with altered transcript abundance mediate the flow of cellular information from RNA to protein. Importantly, ribosome biogenesis also senses nucleolar stress and regulates p53-mediated apoptosis in neural crest. Human ribosomopathies produce craniofacial malformations and 11 known ribosomopathy genes were differentially expressed in this model of neural crest apoptosis. Rapid changes in ribosome expression are consistently observed in EtOH-treated mouse embryo neural folds, a model that is developmentally similar to ours. The recurring identification of ribosome biogenesis suggests it is a candidate modifier of EtOH vulnerability. These results highlight this approach's efficacy to formulate new, mechanistic hypotheses regarding EtOH's developmental damage.

Keywords: Apoptosis; Diamond-Blackfan Anemia; Fetal Alcohol Spectrum Disorders; Gallus gallus; Neural Crest; Oxidative Phosphorylation; Ribosome Biogenesis; Transcriptome Profiling.

Figure 1. Differential ethanol sensitivity of W98S and W98D neural progenitors

For both panels (●) is W98S and (○) is W98D. (A) Elevation of intracellular calcium within neural progenitors following ethanol challenge at the indicated concentrations as quantified using ratiometric imaging for Fura-2.Values are mean ± SD for 3–8 embryos per treatment. Data are plotted using non-linear regression analysis and global curve fitting to evaluate each line's response maxima and relative binding affinity for the dose-response. (B) The numbers of acridine orange-labeled cells within rhombomere 4 were enumerated within HH12/13 hindbrain following treatment at HH8 with the indicated ethanol doses. * indicates values significantly different from 0 mmol ethanol within that line at p< 0.001 using one-way ANOVA. †indicates values significantly different between lines at the same ethanol exposure at p< 0.001 using t-test. N = 9–15 embryos per treatment group.

Figure 2. Distribution plots for differentially expressed transcripts in neural folds of ethanol-sensitive (W98S) and ethanol-resistant (W98D) lines in the absence of ethanol treatment

(A) Normalized dispersion plot showing variance in Read Counts across the W98S and W98D RNA-Seq libraries. Black dots indicate the empirical dispersion. As expected, dispersion decreases as read count increases, as indicated by the fitted line (red). (B). Comparison of transcriptome abundance between W98S and W98D neural progenitors including neural crest. Fold-changes (mean expression W98D – mean expression W98S) are presented as log2. Genes with significantly different expression are shown in red.

Figure 3. Gene ontogeny modules showing significant differential enrichment in ethanol-sensitive versus ethanol-resistant neural crest and neural progenitors at p≤0.05

The top enrichments of GOTERMS involved Molecular Function (A), Biological Processes (B), and Cellular Components (C). For each category, the number of transcripts represented out of the 363 DE genes is indicated. For each column the percentage of transcripts within each GOTERM is indicated.