Abstract
Autism Spectrum Disorder (ASD) has a complex genetic basis, but the role of non-coding variation remains largely unexplored. This project aims to identify rare de novo variants in non-coding regulatory elements contributing to ASD risk, focusing on human-mouse syntenic Long Range Interactions (hmsLRI) identified through RNApolIII-ChIA-PET in neural stem cells. We will analyze whole genome sequencing data from ASD cohorts to identify variants overlapping conserved regulatory regions and assess their target genes. We will prioritize enhancers hosting multiple rare de novo variants and/or connected to target genes representing known or novel candidates for ASD. To assess their function, we will modulate selected enhancers in mouse neural stem cells using CRISPR-dCas9 and test effects on gene expression and cellular phenotypes. This work will advance understanding of non-coding variation in ASD and uncover novel regulatory elements and genes involved in neurodevelopment.
Results achieved
The project investigated how rare genetic variants located outside protein-coding regions may contribute to Autism Spectrum Disorder (ASD). Specifically, it focused on the identification and analysis of variants found in individuals with ASD that affect enhancers, DNA elements that regulate when, where and to what extent genes are expressed and may therefore influence brain development even when the coding sequences of the genes themselves remain intact. Whole-genome sequencing data from four ASD cohorts were analysed, including a cohort of 105 ASD families recruited by Unit 1 (HOUSE) and three large ASD family collections (MSSNG, Simons Simplex Collection (SSC) and Korean cohorts), for a total of more than 9,000 individuals. Rare de novo single-nucleotide variants and copy-number variants were identified and overlapped with human–mouse syntenic long-range interaction regions (hmsLRI regions) previously mapped in neural stem cells. Overall, more than 400 de novo variants were found within these candidate regulatory regions. The association between ASD and conserved de novo variants within hmsLRI regions was tested in the SSC collection, which allows a direct comparison between 1902 ASD cases and their 1902 unaffected siblings as controls. Evolutionarily conserved de novo variants whin hmsLRI regions were significantly enriched in individuals with ASD compared with their unaffected siblings, supporting the hypothesis that alterations in non-coding regulatory DNA contribute to ASD susceptibility. The project then linked the candidate enhancers to their potential target genes. This analysis identified hundreds of connected genes, including genes already implicated in ASD or other neurodevelopmental disorders and genes with important functions in the brain. Twelve enhancers carrying at least two de novo variants were prioritized for functional studies, together with additional regulatory elements connected to relevant genes such as FOXG1, SYNGAP1, QKI, EID1, TPM1, OLIG1 and OLIG2. Functional experiments demonstrated that selected enhancers have a direct role in controlling gene expression. Specifically, in mouse neural stem cells, CRISPR-dCas9-mediated repression of a distal FOXG1 enhancer reduced FOXG1 expression by approximately 50%. Two additional enhancers located closer to FOXG1 were also shown to regulate the gene, and simultaneous repression of two enhancers produced an additive effect. These findings provide direct evidence that multiple non-coding regulatory elements contribute to the control of FOXG1, a gene strongly associated with neurodevelopmental disorders. The project also investigated an enhancer connected to OLIG1 and OLIG2, genes involved in the generation of GABAergic interneurons and in maintaining the balance between excitatory and inhibitory signals in the brain. Repression of this enhancer reduced the expression of both genes and revealed broader regulatory interactions with neighboring genes, highlighting the complexity of long-range gene regulation. To extend the analysis to a larger number of candidate elements, selected human enhancers were tested in transgenic zebrafish embryos. Several enhancers connected to FOXG1, SYNGAP1 and OLIG1/2 drove reporter-gene expression in the developing forebrain, confirming their activity in vivo and indicating the brain regions in which alterations of these regulatory elements may have an impact. Overall, the project established an integrated strategy combining whole-genome sequencing, enhancer–promoter interaction maps and functional assays in neural stem cells and zebrafish. The results provide new evidence that non-coding variants can alter gene-regulatory networks relevant to brain development and ASD, and they identify candidate enhancers and target genes for future genetic, functional and diagnostic studies. The project was supported by the European Union – NextGenerationEU through the PRIN 2022 PNRR programme.Dettagli del progetto
Responsabile scientifico: Elena Maestrini
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Contributo totale di progetto: Euro (EUR) 228.361,00
Contributo totale Unibo: Euro (EUR) 114.882,00
Durata del progetto in mesi: 24
Data di inizio
30/11/2023
Data di fine:
28/02/2026
