Verification and rectification of cell type-specific splicing of a Seckel syndrome-associated ATR mutation using iPS cell model

Seckel syndrome (SS) is a rare spectrum of congenital severe microcephaly and dwarfism. One SS-causative gene is Ataxia Telangiectasia and Rad3-Related Protein (ATR), and ATR (c.2101 A>G) mutation causes skipping of exon 9, resulting in a hypomorphic ATR defect in patients. Because ATR governs DNA repair response, the mutation has been considered the cause of an impaired response to DNA replication stress in neuronal progenitor cells (NPCs), which is associated with the pathogenesis of microcephaly. However, the precise mechanism through which the mutation causes SS remains unclear. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts carrying the ATR mutation and an isogenic ATR-corrected counterpart iPSC clone by genome editing. Interestingly, SS-patient-derived iPSCs (SS-iPSCs) exhibited cell type-specific splicing; exon 9 was dominantly skipped in fibroblasts and iPSC-derived NPCs, but it was included in undifferentiated iPSCs and definitive endodermal cells. SS-iPSC-derived NPCs (SS-NPCs) showed distinct expression profiles from ATR non-mutated NPCs. In SS-NPCs, abnormal mitotic spindles were observed more frequently than in gene-corrected counterparts, and the alignment of NPCs in the surface of the neurospheres was perturbed. Finally, we tested several splicing-modifying compounds and found that a CLK1 inhibitor, TG003, could pharmacologically rescue the exon 9 skipping in SS-NPCs. Furthermore, treatment with TG003 restored the function of ATR in SS-NPCs and decreased the frequency of abnormal mitotic events. In conclusion, our iPSC model of SS revealed a novel function of the ATR mutation in NPCs and NPC-specific missplicing, proving its usefulness for dissecting the pathophysiology of ATR-SS.