Interpreting the impact of noncoding structural variation in neurodevelopmental disorders

The emergence of novel sequencing technologies has greatly improved the identification of structural variation, revealing that a human genome harbors tens of thousands of structural variants (SVs). Since these SVs primarily impact noncoding DNA sequences, the next challenge is one of interpretation, not least to improve our understanding of human disease etiology. However, this task is severely complicated by the intricacy of the gene regulatory landscapes embedded within these noncoding regions, their incomplete annotation, as well as their dependence on the three-dimensional (3D) conformation of the genome. Also in the context of neurodevelopmental disorders (NDDs), reports of putatively causal, noncoding SVs are accumulating and understanding their impact on transcriptional regulation is presenting itself as the next step toward improved genetic diagnosis

Identification of long non-coding RNAs involved in neuronal development and intellectual disability

Recently, exome sequencing led to the identification of causal mutations in 16–31% of patients with intellectual disability (ID), leaving the underlying cause for many patients unidentified. In this context, the noncoding part of the human genome remains largely unexplored. For many long non-coding RNAs (lncRNAs) a crucial role in neurodevelopment and hence the human brain is anticipated. Here we aimed at identifying lncRNAs associated with neuronal development and ID. Therefore, we applied an integrated genomics approach, harnessing several public epigenetic datasets. We found that the presence of neuron-specific H3K4me3 confers the highest specificity for genes involved in neurodevelopment and ID. Based on the presence of this feature and GWAS hits for CNS disorders, we identified 53 candidate lncRNA genes. Extensive expression profiling on human brain samples and other tissues, followed by Gene Set Enrichment Analysis indicates that at least 24 of these lncRNAs are indeed implicated in processes such as synaptic transmission, nervous system development and neurogenesis. The bidirectional or antisense overlapping orientation relative to multiple coding genes involved in neuronal processes supports these results. In conclusion, we identified several lncRNA genes putatively involved in neurodevelopment and CNS disorders, providing a resource for functional studies

CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells

The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naive state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naive stem cells. Editing efficiencies of respectively 1.3-8.4% and 3.819% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naive hESCs were successfully differentiated to neural progenitor cells. As a proofof- principle of our workflow, two monoclonal genome-edited naive hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naive hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning

A neuronal enhancer network upstream of MEF2C is compromised in patients with Rett-like characteristics

Mutations in myocyte enhancer factor 2C (MEF2C), an important transcription factor in neurodevelopment, are associated with a Rett-like syndrome. Structural variants (SVs) upstream of MEF2C, which do not disrupt the gene itself, have also been found in patients with a similar phenotype, suggesting that disruption of MEF2C regulatory elements can also cause a Rett-like phenotype. To characterize those elements that regulate MEF2C during neural development and that are affected by these SVs, we used genomic tools coupled with both in vitro and in vivo functional assays. Through circularized chromosome conformation capture sequencing (4C-seq) and the assay for transposase-accessible chromatin using sequencing (ATAC-seq), we revealed a complex interaction network in which the MEF2C promoter physically contacts several distal enhancers that are deleted or translocated by disease-associated SVs. A total of 16 selected candidate regulatory sequences were tested for enhancer activity in vitro, with 14 found to be functional enhancers. Further analyses of their in vivo activity in zebrafish showed that each of these enhancers has a distinct activity pattern during development, with eight enhancers displaying neuronal activity. In summary, our results disentangle a complex regulatory network governing neuronal MEF2C expression that involves multiple distal enhancers. In addition, the characterized neuronal enhancers pose as novel candidates to screen for mutations in neurodevelopmental disorders, such as Rett-like syndrome

Identification of long non-coding RNAs in neuronal development and intellectual disability

Recent studies have assigned important functions to lncRNAs in gene regulation and protein interactions. Since many of these lncRNAs emerged recently during vertebrate and primate evolution, a crucial role in the human brain is anticipated. Here, we aimed at identifying candidate lncRNAs associated with neuronal development and intellectual disability (ID). To do this, we combined the latest genomic annotations of lncRNAs (i.e. LNCipedia database) with functional data (neuron-specific H3K4 trimethylation, REST binding & DNaseI hypersensitivity). These three datasets were applied as filters, both to RefSeq protein-coding genes and LNCipedia lncRNA transcripts. To assess the specificity of these potential filters, we performed an enrichment analysis of ID genes and genome wide association study (GWAS) hits for central nervous system (CNS) disorders, on the resulting sets of protein-coding genes (ID genes & GWAS hits) and lncRNA transcripts (GWAS hits only). We found the neuron-specific H3K4me3 mark to confer the highest specificity for genes involved in ID and neurodevelopment. Applying this mark as a filter for all LNCipedia transcripts resulted in a set of 4188 lncRNAs, of which 53 harbour a GWAS hit for CNS disorders. As the presence of such a SNP directly implicates these lncRNA loci in neuropathogenesis, we focused during subsequent analyses on this set of 53 lncRNAs. This approach was complemented by extensive expression profiling of all protein-coding genes and ca. 23,000 lncRNA transcripts in 15 human tissues, among which 8 different brain samples. This allowed us to construct coexpression profiles for 30 of the lncRNAs that were identified by our filtering strategy (no unique probes could be designed for the other 23 transcripts). Using Gene Set Enrichment Analysis (GSEA) we evaluated the involvement of the selected lncRNAs in neuronal processes. For 19 out of the 30 selected lncRNAs, gene sets linked to synaptic transmission, nervous system development or neurogenesis were highly enriched among the top positively correlated genes. Five lncRNAs were negatively correlated to genes involved in these neuronal processes, suggesting that these lncRNAs may be involved in suppressive regulation. In conclusion, we set up a strategy to identify lncRNAs involved in neuronal development and 30 interesting lncRNA transcripts remained. The relevance of our strategy was underscored by the fact that at least 24 of these lncRNAs are implicated in neuronal processes through correlated expression profiles. In our further research we will validate these top candidates by targeted functional analysis

Identification of lncRNAs involved in neuronal differentiation and intellectual disability

Recent studies have assigned important functions to lncRNAs in gene regulation and protein interactions. Since many of these lncRNAs emerged recently during vertebrate and primate evolution, a crucial role in the human brain is anticipated. Here, we aimed at identifying candidate lncRNAs associated with neuronal development and intellectual disability (ID). To do this, we combined genomic annotations (i.e. LNCipedia database) with functional genomics data (neuron-specific H3K4me3, REST binding & DNaseI hypersensitivity). For each of these three potential filters, we performed an enrichment analysis of ID genes and genome wide association studies (GWAS) hits for central nervous system (CNS) disorders, both on RefSeq protein-coding genes (ID genes & GWAS hits) and LNCipedia lncRNA transcripts (GWAS hits only). We found the neuron-specific H3K4me3 mark to confer the highest specificity for genes involved in ID and neurodevelopment. Applying this mark as a filter resulted in a set of 4188 lncRNA transcripts, of which 53 harbour a GWAS hit for CNS disorders. As the presence of such a SNP directly implicates these lncRNA loci in neuropathogenesis, we focused during subsequent analyses on this set of 53 lncRNAs. This approach was complemented by extensive expression profiling of all protein-coding genes and ca. 23,000 lncRNA transcripts in 15 human tissues, among which 8 brain samples. This allowed us to construct coexpression profiles for the lncRNAs identified by our filtering strategy (30 out of 53 were covered on the expression array). Using Gene Set Enrichment Analysis (GSEA) we assigned putative functions to the selected lncRNAs. For 13 candidates, highly correlated protein-coding genes were enriched in gene sets linked to neuronal processes and development, underscoring the relevance of the strategy used here. Subsequently, these lncRNAs will be functionally validated