Identification of a patient with CDA III who has compound heterozygous variants in RACGAP1. (A) Bone marrow aspirate from the proband showing a characteristic multinucleated erythroblast (top) and gigantoblast (bottom; Wright-Giemsa stain; scale bar, 10 μm). (B) Electropherograms of genomic DNA for the mother (top), father (middle), and proband (bottom) for variants c.1187T>A (left) and c.1294C>T (right). (C) Domain structure of CYK4/MgcRacGAP encoded by RACGAP1 showing the coiled-coil (CC), C1, and RhoGAP (GTPase-activating protein [GAP]) domains as well as the MKLP1-interacting region. Sequence alignment of CYK4 orthologs shows strong conservation of the indicated residues affected by the variants. The GAP domain fragment (amino acids 343-547) was used for the biochemical assays in Figure 2B. (D) Primary human CD34⁺ erythroid cells were treated with short hairpin RNA (shRNA)-targeted luciferase (i) or RACGAP1 (ii) and cultured for 15 days. Arrows and arrowheads indicate enucleated and multinucleated cells, respectively (scale bar, 10 μm). (E-F) Immortalized cord blood–derived erythroid (ImCBE) cells control depleted (luciferase) (i), RACGAP1 depleted (ii), or RACGAP1 depleted and rescued with an RNAi-resistant RACGAP1 transgene (iii-v) (scale bar, 20 μm). (F) The frequency of multinucleate cells (arrowheads) in total >500 cells in biologic triplicates is shown with the 95% confidence interval. The P values of the statistical test for the difference of the cells rescued with the P432S or L396Q variants from those rescued with the wild-type (WT) RACGAP1 were corrected for multiple comparisons by Dunnett’s method. ***P < .001.