Network Effects of the 15q13.3 Microdeletion on the Transcriptome and Epigenome in Human-Induced Neurons

Abstract

Background: The 15q13.3 microdeletion is associated with several neuropsychiatric disorders, including autism and schizophrenia. Previous association and functional studies have investigated the potential role of several genes within the deletion in neuronal dysfunction, but the molecular effects of the deletion as a whole remain largely unknown.

Methods: Induced pluripotent stem cells, from 3 patients with the 15q13.3 microdeletion and 3 control subjects, were generated and converted into induced neurons. We analyzed the effects of the 15q13.3 microdeletion on genome-wide gene expression, DNA methylation, chromatin accessibility, and sensitivity to cisplatin-induced DNA damage. Furthermore, we measured gene expression changes in induced neurons with CRISPR (clustered regularly interspaced short palindromic repeats) knockouts of individual 15q13.3 microdeletion genes.

Results: In both induced pluripotent stem cells and induced neurons, gene copy number change within the 15q13.3 microdeletion was accompanied by significantly decreased gene expression and no compensatory changes in DNA methylation or chromatin accessibility, supporting the model that haploinsufficiency of genes within the deleted region drives the disorder. Furthermore, we observed global effects of the microdeletion on the transcriptome and epigenome, with disruptions in several neuropsychiatric disorder-associated pathways and gene families, including Wnt signaling, ribosome function, DNA binding, and clustered protocadherins. Individual gene knockouts mirrored many of the observed changes in an overlapping fashion between knockouts.

Conclusions: Our multiomics analysis of the 15q13.3 microdeletion revealed downstream effects in pathways previously associated with neuropsychiatric disorders and indications of interactions between genes within the deletion. This molecular systems analysis can be applied to other chromosomal aberrations to further our etiological understanding of neuropsychiatric disorders.

Keywords: 15q13.3; CRISPR–Cas9; Copy number variants; Genomics; Induced pluripotent stem cells; Neurons.

Figure 1.

Generation and characterization of CNV lines. (A) Information on donors of fibroblasts, including age, sex, and genotype. (B) Induced neuron generation timeline. (C) Sequencing read depth of fibroblast samples. (D) Immunocytochemistry for the pluripotency markers Nanog, TRA-1–60, and SSEA-4 on iPSCs (scale bar = 200 μm). (E) Immunocytochemistry for the neuronal markers MAP2, VGLUT1, and TUJ1 on day 6–induced neurons (scale bar = 200 μm). (F) Top 10 GO biological process terms from gene set enrichment analysis of genes differentially expressed between iPSCs and iNs, ordered by NES. Chr, chromosome; CNV, copy number variant; GO, Gene Ontology; iN, induced neuron; iPSC, induced pluripotent stem cell; NES, normalized enrichment score.

Figure 2.

Gene expression changes within the 15q13.3 microdeletion and genome wide. (A, B) Log2 FC of protein-coding 15q13.3 genes in deletion samples compared with control subjects in iPSCs (A) and iNs (B) (95% confidence intervals shown). (C, D) Manhattan plots of RNA-Seq genes in iPSCs (C) and iNs (D), with the red threshold line indicating .05 FDR significance. (E) Significant GO terms from gene set enrichment analysis of 15q13.3 iN RNA-Seq dataset. The top 10 terms in each category based on FDR are listed, ordered by NES. (F) Log2 FC of EIF2 signaling genes differentially expressed in deletion iNs compared with control iNs (95% confidence intervals shown). Chr, chromosome; DEG, differentially expressed gene; FC, fold change; FDR, false discovery rate; GO, Gene Ontology; iN, induced neuron; iPSC, induced pluripotent stem cell; NES, normalized enrichment score; rRNA, ribosomal RNA; RNA-Seq, RNA sequencing.

Figure 3.

DMRs in 15q13.3 microdeletion lines. (A, B) Genome-wide distribution of DMRs in iPSCs (A) and iNs (B). DMRs above red threshold line have ≥0.1 mean methylation difference and adjusted p ≤ .05. Arrows indicate PCDH gene clusters. (C, D) DMRs in iPSCs and iNs, split by DMR location, direction of mean methylation difference, and direction of expression change in nearest gene. (E, F) Significant terms from gene set enrichment analysis of 15q13.3 iN and iPSC Methyl-Seq datasets, sorted by NES. (G–I) DMRs located near the alpha (G), beta (H), and gamma (I) PCDH families. DMRs are colored red for higher mean methylation, and blue for lower mean methylation, in deletion samples. Chr, chromosome; DMR, differentially methylated region; GABA, gammaaminobutyric acid; iN, induced neuron; iPSC, induced pluripotent stem cell; Methyl-Seq, DNA methylation sequencing; NES, normalized enrichment score; PCDH, protocadherin.

Figure 4.

ATAC-Seq shows altered chromatin accessibility in 15q13.3 microdeletion lines. (A, B) Genome-wide distribution of peaks identified from ATAC-Seq in iPSCs (A) and iNs (B). Peaks above red threshold line have adjusted p # .05. (C, D) Differentially accessible ATAC-Seq peaks in iPSCs and iNs, split by peak location, direction of chromatin accessibility change, and direction of expression change in nearest gene. (E, F) Enriched motifs in iPSCs and iNs from de novo Homer analysis with p value ≤ 10⁻¹², best match TF expressed in the same tissue, and best match score ≥ 0.6. *Gene has known association with Wnt signaling pathway. ATAC-Seq, assay for transposase-accessible chromatin sequencing; iN, induced neuron; iPSC, induced pluripotent stem cell; TF, transcription factor.

Figure 5.

Multiomics correlation analysis. Correlations between gene expression log2 FC and chromatin accessibility log2 FC (A, D), between gene expression log2 FC and mean DNA methylation difference (B, E), and between mean DNA methylation difference and chromatin accessibility log2 FC (C, F) are shown. FC, fold change; iN, induced neuron; iPSC, induced pluripotent stem cell;

Figure 6.

CRISPR–Cas9 knockout RNA-Seq analysis. Significant GO terms from gene set enrichment analysis of RNA-Seq datasets for knockouts of CHRNA7 (A), KLF13 (B), FAN1 (C), MTMR10 (D), and OTUD7A (E) are shown. The top 10 terms in each category based on FDR are listed, ordered by NES. *GO term also enriched in 15q13.3 microdeletion induced neurons. CRISPR, clustered regularly interspaced short palindromic repeats; FDR, false discovery rate; GO, Gene Ontology; ncRNA, noncoding RNA; NES, normalized enrichment score; NF, nuclear factor; RNA-Seq, RNA sequencing; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA.

Figure 7.

Cell survival after cisplatin exposure as a measure of altered DNA damage response in 15q13.3 microdeletion iNs. Mean percentages (with 95% confidence intervals) of surviving cisplatin-treated cells normalized to untreated cells in 15q13.3 microdeletion iNs and control iNs are shown. *p <.005 by nested t test. iN, induced neuron.