Coding and noncoding variants in EBF3 are involved in HADDS and simplex autism

Abstract

Background: Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737.

Results: We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10^-3), and combined dataset (p = 1.1 × 10^-4). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10^-35, loss-of-function p = 2.26 × 10^-13) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10^-6, OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia.

Conclusions: In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.

Keywords: Autism; De novo; EBF3; Enhancer; Gene regulatory network; Genome; Neurodevelopmental disorder; Variant; hs737.

Fig. 1

Characterization of DNVs in hs737. A Pedigrees of families with de novo variants in hs737. Lightning symbols indicate de novo variants with red = regulatory, purple = missense, and blue = deletion. Family identifiers are shown above the pedigree and the full-scale IQ is shown below each proband. B Sequence analysis of each of the three hs737 de novo mutations, identified in individuals with autism, including transcription factor binding site analysis results. C Results of luciferase assays in neuroblastoma (Neuro2a) cell lines with rs2435357 (RET+3) as a positive control for enhancer activity, promoter only (Basal), the wild type sequence of hs737 (hs737wt), and each of the three DNVs identified in individuals with autism. Error bars represent standard error (SE). D log2 normalized expression of genes from the transcription factor binding site analysis in the brain throughout development and adulthood. E Correlogram of candidate genes and EBF3 after performing regression, with positive control MECP2 and negative control CFTR

Fig. 2

Copy number variation over hs737. Displays the counts for both deletions and duplications over hs737 in individuals with neurodevelopmental disorders and controls

Fig. 3

hs737 is a prenatal, brain-specific enhancer. A Genome browser view (chr7:136,079,964–136,087,591; mm10) of chromatin states in mouse from [43] called by chromHMM [44] based on eight histone modifications: H3K4me1, H3K4me2, H3K4me3, H3K27ac, H3K27me3, H3K36me3, H3K9me3, and H3K9ac. B Genome browser view (chr7:136,079,964–136,087,591; mm10) of ATAC-seq and H3K27ac ChIP-seq signal in midbrain, hindbrain, and forebrain at multiple developmental mouse stages from E11.5 to the day of birth (P0)

Fig. 4

EBF3 is the gene target of hs737. A Schematic of hs737 and target genes. Gray boxes represent promoters and colored boxes represent gene bodies and red box represents hs737. Hi-C contact map generated using data from Won et al. [49] visualized with Juicebox [48] at 25 kbp. Heatmaps are symmetrical across the diagonal, except that HiCCUPS loop calls are shown as black boxes in the upper right half of each heatmap. B Hi-C contact maps from Bonev et al. [47] visualized with Juicebox [48] at 5-kbp resolution. C EBF3 protein diagram (plotted using the DOG protein plotter [67]) with DNVs identified in NDDs. Shown in blue are the missense variants and in red are the loss-of-function variants. D 3D model (plotted using the MuPIT program [68, 69]) of the EBF3 protein with DNVs identified in individuals with NDDs shown in green. E Genes with promoters bound by EBF3 based on ChIP sequencing in SK-N-SH cells. Enrichment is seen for the promoters of known NDD genesets

Fig. 5

EBF3 gene network analysis. A Correlation matrix of EBF3 with other high scoring SFARI genes (score < 3) after performing regression and hierarchical clustering and having an absolute correlation greater than 0.6. There is a significant enrichment of genes involved in chromatin binding. B Network analysis of genes from cluster 2 from Genemania where each gene is a node and different forms of supporting evidence for an interaction are edges

Fig. 6

Consequence of coding and noncoding variation in EBF3. A GTEx expression data of EBF3 with color corresponding to the organ system. B Human Protein Atlas highlighting where EBF3 expression is detected in the human body. C LacZ staining assay for reporter activity driven by the hs737 enhancer at mouse E11.5. D Phenotypic analysis comparing the frequency of ataxia, hypotonia, ID and GDD, autism, and having 7 or more symptoms between all patients, individuals with EBF3 mutations, individuals with mutations specifically in the EBF3 DNA binding domain, and in the hs737 enhancer. E Gene regulatory network encompassing EBF3 built using current molecular biological knowledge