Molecular dissection of germline chromothripsis in a developmental context using patient-derived iPS cells.

Background: Germline chromothripsis causes complex genomic rearrangements that are likely to affect multiple genes and their regulatory contexts. The contribution of individual rearrangements and affected genes to the phenotypes of patients with complex germline genomic rearrangements is generally unknown.

Methods: To dissect the impact of germline chromothripsis in a relevant developmental context, we performed trio-based RNA expression analysis on blood cells, induced pluripotent stem cells (iPSCs), and iPSC-derived neuronal cells from a patient with de novo germline chromothripsis and both healthy parents. In addition, Hi-C and 4C-seq experiments were performed to determine the effects of the genomic rearrangements on transcription regulation of genes in the proximity of the breakpoint junctions.

Results: Sixty-seven genes are located within 1 Mb of the complex chromothripsis rearrangements involving 17 breakpoints on four chromosomes. We find that three of these genes (FOXP1, DPYD, and TWIST1) are both associated with developmental disorders and differentially expressed in the patient. Interestingly, the effect on TWIST1 expression was exclusively detectable in the patient's iPSC-derived neuronal cells, stressing the need for studying developmental disorders in the biologically relevant context. Chromosome conformation capture analyses show that TWIST1 lost genomic interactions with several enhancers due to the chromothripsis event, which likely led to deregulation of TWIST1 expression and contributed to the patient's craniosynostosis phenotype.

Conclusions: We demonstrate that a combination of patient-derived iPSC differentiation and trio-based molecular profiling is a powerful approach to improve the interpretation of pathogenic complex genomic rearrangements. Here we have applied this approach to identify misexpression of TWIST1, FOXP1, and DPYD as key contributors to the complex congenital phenotype resulting from germline chromothripsis rearrangements.