Evaluating the pathogenic significance of unique chromosomal variants in craniosynostosis using patient-derived...

Unraveling causal links between unique structural/copy-number variants (SV/CNV) and associated phenotypes is essential for correct genetic counseling. We investigated 2 families in which patients with craniosynostosis had SV/CNV that potentially dysregulated a fibroblast growth factor (FGF)-encoding gene: a 730 kb duplication on chromosome 4q21.21 (dup(4)) including FGF5 and a complex 568 kb interspersed 13q12.11 duplication, located 841 kb from FGF9.

We combined bioinformatic predictions of altered topologically associating domain (TAD) structure with experimental analysis (RNA- and assay for transposase-accessible chromatin-sequencing) of patient-induced pluripotent stem cell lines differentiated to neural crest cell and osteoprogenitor cell identities. For the dup(4), we generated a mouse bearing an equivalent rearrangement using Clustered Regularly Interspaced Short Palindromic Repeats-Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 targeting.

TAD analysis suggested the potential dysregulation of the FGF5/FGF9 gene by bringing it into a novel genomic milieu. The RNA and assay for transposase-accessible chromatin sequencing assays demonstrated FGF5/FGF9 upregulation (2.7- to 18-fold) and local opening of chromatin in 3/4 cell lines. For the dup(4), a causal role was supported by the mouse model, whereas interpretation of the 13q12.11 SV was confounded by a coexisting FOXP2 pathogenic variant.

Patient-derived induced pluripotent stem cell (iPSC)-differentiated neural crest cell and osteoprogenitor cell lines, combined with TAD-based modeling to generate testable functional hypotheses, provide valuable functional evidence when evaluating causation of unique SV/CNV in craniosynostosis.