Fig. 2

KLHL13 NDD-causing variants affect cell division and cycle regulation at the M/G1 phase in heterologous cells. A. Confocal image of COS-7 cells overexpressing GFP-tagged KLHL13WT or NDD-associated variants synchronized at the G0/G1M phase of the cell cycle. Immunolabeling with anti-GFP (green) and anti-β-tubulin (red) antibodies revealed chromosomal misalignment at metaphase (green arrows) in p.His60Arg and p.Val594Glyfs∗6, whereas all four variants caused abnormal segregation and lagging of sister chromatids (white arrows) at anaphase. Chromosomal bridging (yellow arrows) at telophase was also observed in cells overexpressing KLHL13 with p.Ala318Val, p.His60Arg, and p.Val594Glyfs∗6 variants. Moreover, all four variants affected the completion of cytokinesis via the formation of giant multinucleated cells (red arrows). Nuclear contents were labeled with DAPI (blue). Scale bar: 10 μm. Note: Figure 2A shows merged images, whereas Supplemental Figures 3-7 display individual channels for each stage of mitosis. B. Graphical representation of mitotic abnormalities observed in cells overexpressing KLHL13 constructs. All four NDD-associated variants showed incomplete cytokinesis, with significantly high (∗P < .02, ∗∗P < .001, ∗∗∗P < .0001) formation of bi/multinucleated cells when compared with KLHL13WT-expressing cells. At least 120 GFP-positive cells were imaged for each condition, with 40 cells quantified per experiment over three independent experiments (n = 3). C. Bar graph representing the percentage of KLHL13WT and variants overexpressing cells at metaphase, containing aligned vs misaligned chromosomes. There was a significantly higher number of cells affecting chromosomal alignment during metaphase in variants-expressing cells compared with KLHL13WT (∗∗P < .001, ∗∗∗P < .0001). A total of 90 GFP-positive cells were imaged at metaphase, with 30 cells quantified per experiment across three independent experiments (n = 3). D. The bar graph represents the percentage of cells observed with lagging and bridging chromosomes showing an incomplete segregation of the sister chromatids. The variants overexpressing cells were found to have a significantly higher number of cells observed with the lagging and bridging phenotypes during anaphase compared with the KLHL13WT overexpressing cells (∗P < .01, ∗∗P < .001). A total of 60 GFP-positive cells were imaged at anaphase, with 20 cells quantified per experiment over 3 independent experiments (n = 3). E. Graphical representation of the percentages of KLHL13WT and NDD variants overexpressing HEK293 cells at different phases of the cell cycle. In contrast to WT-expressing cells, KLHL13 NDD-associated variants-expressing cells had significantly higher (∗P < .01, ∗∗P < .001) cell-cycle abnormalities, leading to genomic instability. A total of 2 × 105 events were collected for cell-cycle analysis per condition, and each experiment was performed in triplicate (n = 3). G. Visual motor startle behavior analysis showed a significant improvement in the overall locomotor activity in the human KLHL13WT mRNA and ATG_MO coinjected larvae in the light/dark transition period compared with the ATG_MO injected larvae (∗∗P <.001, ∗∗∗P <.0001, ∗∗∗∗P <.00001). H. The graph represents the number of ATG_MO injected morphants, as well as human KLHL13WT and NDD-associated variants mRNA coinjected morphants in various morphological categories. Microinjections of human KLHL13WT encoding mRNA, along with ATG_MOs, significantly rescued the number of larvae in the normal developmental class compared with ATG_MO alone (∗∗∗P <.0002). However, NDD-associated KLHL13 hemizygous variants (c.179A>G, c.557G>A, c.953C>T, and c.1781_1782del) encoding mRNAs did not rescue the phenotype when compared with the normal class of KLHL13WT encoding mRNA (∗∗∗P <.0001), supporting their pathogenic nature. Moreover, significantly more larvae in the severe class were found when ATG_MO was coinjected with human KLHL13 mRNAs harboring c.179A>G (∗∗P <.0012) and c.1781_1782del variants (∗∗P <.0052).