The online version of this article (https://doi.org/10.1186/s13229-018-0191-y) contains supplementary material, which is available to authorized users.
The inability to analyze gene expression in living neurons from Angelman (AS) and Duplication 15q (Dup15q) syndrome subjects has limited our understanding of these disorders at the molecular level.
Here, we use dental pulp stem cells (DPSC) from AS deletion, 15q Duplication, and neurotypical control subjects for whole transcriptome analysis. We identified 20 genes unique to AS neurons, 120 genes unique to 15q duplication, and 3 shared transcripts that were differentially expressed in DPSC neurons vs controls.
Copy number correlated with gene expression for most genes across the 15q11.2-q13.1 critical region. Two thirds of the genes differentially expressed in 15q duplication neurons were downregulated compared to controls including several transcription factors, while in AS differential expression was restricted primarily to the 15q region. Here, we show significant downregulation of the transcription factors FOXO1 and HAND2 in neurons from 15q duplication, but not AS deletion subjects suggesting that disruptions in transcriptional regulation may be a driving factor in the autism phenotype in Dup15q syndrome. Downstream analysis revealed downregulation of the ASD associated genes EHPB2 and RORA, both genes with FOXO1 binding sites. Genes upregulated in either Dup15q cortex or idiopathic ASD cortex both overlapped significantly with the most upregulated genes in Dup15q DPSC-derived neurons.
Finding a significant increase in both HERC2 and UBE3A in Dup15q neurons and significant decrease in these two genes in AS deletion neurons may explain differences between AS deletion class and UBE3A specific classes of AS mutation where HERC2 is expressed at normal levels. Also, we identified an enrichment for FOXO1-regulated transcripts in Dup15q neurons including ASD-associated genes EHPB2 and RORA indicating a possible connection between this syndromic form of ASD and idiopathic cases.
Additional file 1: Tables S1–S9. Complete GSEA analysis for all samples and FOXO1 and functional enrichment analysis of ASD brain vs Dup15q DPSC neurons. S9 is a table of primers used for qRT-PCR. (XLSX 68 kb)13229_2018_191_MOESM1_ESM.xlsx
Additional file 2: Figure S1. Copy number and breakpoint analysis by FISH. Examples of each cell line are shown. All cell lines were analyzed with NIPA1 + SNRPN or TRPM1 + SNRPN. At least 15 cell preparations were analyzed for each line to determine the breakpoints and copy numbers. Note, DPSC line TP-041 is an interstitial duplication line with three copies of the BP2-BP3 region (green signals) but only one copy of the BP1-BP3 or BP4-BP5 regions (red signals). Fig. S2 Boxplots for specific genes of interest mentioned in the text. These are the RPKM averages across all three biological replicates for DPSC and DPSC neuronal cultures and for control (red), Dup15q (blue), and AS (green) for each gene of interest. Error bars are standard deviation. Fig. S3 PSD95 staining of DPSC and DPSC-derived neurons. A band correlating to PSD95 is seen in the DPSC-neuronal cultures. Unfortunately, a cross-reactive band also present in the α-goat secondary alone blot interfered with the signal for PSD95 in DPSC alone making quantification impossible (top versus middle lane). GAPDH was used as a loading control. (PDF 3128 kb)13229_2018_191_MOESM2_ESM.pdf
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- Significant transcriptional changes in 15q duplication but not Angelman syndrome deletion stem cell-derived neurons
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T. Grant Belgard
Quynh T. Tran
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