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PgmNr 163: Mechanistic dissection of chromatin topology disruption as an indirect, strong effect driver of neurodevelopmental disorders.

K. Mohajeri 1,2,3; E. D'haene 4,5; R. Yadav 1,3; H. Gu 6,7; B. Menten 4,5; A. Presser Aiden 6,7; C. Lowther 1,3; S. Erdin 1,3; M. Moyses Oliveria 1,3; P. Boone 1,3; E. Lieberman-Aiden 6,7; J. Gusella 1,3; S. Vergult 4,5; M. Talkowski 1,3

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1) Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA; 2) Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA; 3) Program in Medical and Population Genetics, Broad Institute, Cambridge, MA; 4) Center for Medical Genetics, Ghent University, Ghent, Belgium; 5) Dept. of Biomolecular Medicine, Ghent University, Ghent, Belgium; 6) The Center for Genome Architecture, Baylor College of Medicine, Houston, TX; 7) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX

Structural variants have the potential to create long-range positional effects, uncouple genes from regulatory elements, and facilitate aberrant 3D chromatin folding. In an independent study, we analyzed the breakpoints of balanced chromosomal abnormalities (BCAs) from 387 congenital anomaly cases and 247 BCA-harboring controls, revealing genome-wide significant enrichments of intergenic BCA breakpoints predicted to disrupt topologically associating domains (TADs) at multiple loci, with the most significant enrichment at chromosome 5q14.3. Among the 11 5q14.3 BCA carriers, all were cases with neurodevelopmental disorders (NDD) with breakpoints localized to a single TAD housing a known NDD driver, MEF2C. Our previous targeted expression studies revealed BCA breakpoints disrupting a distal loop boundary but not MEF2C directly, resulted in decreased MEF2C expression, while HiC analyses of 6 5q14.3 BCA cases found alterations to canonical regulatory contacts. Given these results, we performed a mechanistic dissection of the regulatory network associated with the 5q14.3 locus and its constituent 3D functional elements using Cas9-based genome editing. We generated >180 cell lines in an isogenic background, representing deletions of MEF2C alongside 4 TAD and loop boundary targets within the 5q14.3 region in iPS-derived neural stem cells (NSCs) and cortical induced neurons (iNs). Using Nanostring-based targeted expression profiling, we surveyed expression of all 9 protein-coding genes within a 6Mbp window of 5q14.3 in each line. In NSCs, deletion of the case-disrupted distal loop boundary resulted in a statistically significant 20% increase in MEF2C expression, with MEF2C as the only differentially expressed gene within the TAD. In contrast, MEF2C was not differentially expressed in matched iNs. Probing the underlying contact patterns revealed evidence of loop maintenance via CTCF buffering demonstrated by UMI-4C in both iNs and NSCs when we deleted the distal loop boundary, while deletion of the proximal boundary partner displayed increased contacts with predicted enhancers in the adjacent TAD. Our findings highlight compensatory mechanisms against 3D chromatin disruption while underscoring their associated complexity on a cell type and variant class basis. These results suggest potentially novel regulatory mechanisms driving phenotypic outcomes for this genomic disorder region, with significant implications for interpretation of pathogenic structural variation.