|
Figure 2
(A) Sanger sequencing chromatogram showing the cDNA sequences from a healthy control and a cataract patient. The heterozygous c.5828G>A missense variant in the patient is indicated by the red arrow. (B) A schematic diagram showing the human PIKFYVE domains. The p.G1943E variant in the PIPK domain is indicated by the red arrow. (C) Protein sequence alignment of PIKFYVE orthologs in vertebrates. The black triangle denotes the conserved glycine at position 1943. (D) Western blot analysis of PIKFYVEWT and PIKFYVEG1943E expression in HEK293T cells that were transiently transfected with either pCS2(+)-CMV-PIKFYVEWT or pCS2(+)-CMV-PIKFYVEG1943E. The protein levels were normalized by GAPDH expression. Experiments were repeated three times. (E) Predicted structure model of the p.G1943E variant form of PIKFYVE PIPK domain generated by the PHYPRE2 server (http://www.sbg.bio.ic.ac.uk/~phyre2/html/). N-lobe, C-lobe, and the hinge linker are shown in gold, cyan, and red, respectively. The variant residue E1943 is shown in sticks and labeled with green. The negatively charged residue D1872 close to E1943 side chain is also shown in sticks. (F) A schematic demonstrating the organization of PIKFYVE PIPK domain. N-lobe, C-lobe, and the hinge linker are shown in gold, cyan, and red, respectively. The position of the p.G1943E variant is labeled with a yellow star. (G) Surface electrostatic potential comparison of the PIPK domain of PIKFYVE between wild-type (WT) and p.G1943E variant. The electrostatic potentials are presented as heatmaps from red to blue, and the electrostatic potential scales are shown in the lower panel. See Figure 2—source data 1 for details.The PIKFYVE variant identified from the congenital cataract family.