Fig 2: ACTL6B p.(Val421_Cys425del) abolishes conserved amino acid residues in the COOH terminus. (a) Conservation of Val421_Cys425 residues across actin-related proteins in humans. The five amino acids deleted in EIEE-76 are marked in red in ACTL6B (above). The conserved domain found in ACTB, ACTG1, POTEKP, ACTBL2, AFAP1, and ACTL7A represented as a red block in the schematic (below). The UniProt entry number for each gene is given next to the gene name. (b) ACTL6B alterations (red) modeled onto the solved structure for ACTB visualized with Swiss-PdbViewer (https://spdbv.vital-it.ch/, RRID:SCR_013295) (left). Enlarged view showing eight intramolecular hydrogen bonds associated with residues 370–374 (orange dotted lines). Enlarged view showing p.(Val370_ 374Cysdel) disrupts the eight inferred hydrogen bonds

Fig 3: Generation of induced pluripotent stem cells (iPSCs) derived from EIEE-76 patients with ACTL6B p.(Val421_Cys425del) and family-matched controls. (a) Brightfield images of patient-derived iPSC clones. Expression of stem cell markers NANOG and SOX2 was detected in all clonal iPSC lines (Unaffected: CW067 U5 and CW067 U9; Affected: CW066 A4 and CW066 A5). Representative images for each clone, shown with DAPI (4',6-diamidino-2-phenylindole) counterstain. Scale bar, 100 µm. (b) RT-PCR for stem cell identity markers (OCT4 and PODXL) in iPSC clones derived from a healthy parent (Unaffected, CW067) and an EIEE-76 patient (Affected, CW066) primary fibroblast cell lines show effective reprogramming. GAPDH, technical control. (c) RT-PCR for endoderm (AFP) and mesoderm (MSX1) lineage markers from unaffected (CW067) and affected (CW066) spontaneously differentiated day (d)7 embryoid bodies demonstrate iPSC pluripotency. GAPDH, technical control. (d) Karyostat karyotyping analysis of iPSC clones derived from a healthy parent (Unaffected, CW067) and an EIEE-76 patient (Affected, CW066) depicting genome-wide chromosomal copy number at a resolution of 1–5 Mb

Fig 4: Neural differentiation of patient-derived induced pluripotent stem cells (iPSCs) to neural progenitor cells and neurons. (a) Schematic of neural differentiation from EIEE-76 patient-specific iPSCs. (b) Neural progenitor cells (NPCs) differentiated from control (Unaffected: CW067 U5; CW067 U9) and an EIEE-76 patient (Affected: CW066 A4; CW066 A5) iPSC clones are indistinguishable. The iPSCs clones form embryoid bodies (EBs) and neural rosettes (yellow, dashed outline) similarly upon neural induction. Isolated NPCs express neural progenitor cell markers PAX6 and NESTIN. Representative images shown with DAPI (4',6-diamidino-2-phenylindole) counterstain. Scale bar, 100 µm. (c) Characterization of NPC lines derived from unaffected familial control (CW067) and an EIEE-76 affected patient (CW066) by RT-PCR for expression of neural crest cell (SOX10 and FOXD3), stem cell (OCT4), and neural progenitor cell (NESTIN) identity genes show the NPC cultures are not contaminated by neural crest derivatives or undifferentiated stem cells. GAPDH, loading control. (d) Co-immunocytochemistry for neuronal markers MAP2 and TUJ1 at day (d)25 of differentiation show positive expression in familial unaffected controls and EIEE-76 patient-specific affected neurons. Representative images shown with DAPI counterstain. Scale bar, 50 µm. (e) Dual color infrared western blot for HA (green) and ACTL6B (red) in HEK293T cell lysates transfected with a 2xHA-epitope tagged ACTL6B expression construct showing strong colocalization at the expected size of 49 kDa. (f) Temporal analysis of ACTL6B expression during neural differentiation found ACTL6B was detected at increasing levels after day d3, while TUJ1 expression was first visible at d10. GAPDH, loading control. Western blot from neurons differentiated from iPSC clone CW067 U9 is displayed