case_images: PMC6491668_01
case_images: A novel c.584A>G/p.His195Arg variant is observed in affected family members and results in a protein where a highly conserved position critical for C2H2-type Zinc Finger domain folding is altered (H195R). (A) Sanger sequencing data showing the heterozygous c.584A>G genotype (WT/Mut) observed in the proband, his mother and maternal grandfather; his father and sister are unaffected (WT/WT). (B) Schematic representation of the IKAROS protein showing the 4 DNA binding and 2 dimerization zinc finger (ZF) domains. Approximate locations along the primary sequence for known missense variants are shown as red circles. (C) Primary protein sequence alignment of the 6 ZF domains. Conserved Cys and His residues that distinguish this class of C2H2-type ZF domains are highlighted. Known disease associated variant positions are indicated with red circles. (D) A homology model for the IKAROS ZF3 bound to DNA was constructed using SWISS-MODEL (swissmodel.expasy.org) using PDB ID 1P47 as template. The model highlights positions of disease associated variants in ZF3, which typically contact the DNA substrate (Arg184) or coordinate zinc (His191 and His195). (E) Expression of the FLAG epitope-tagged H195R IKAROS variant in NIH 3T3 fibroblasts results in a loss of the punctate pericentromeric heterochromatin staining within the nucleus (images were acquired at 600x magnification); a phenotype observed for previously reported IKAROS variants such as H167R. Wild type and mutant proteins were made the following way. A pcDNA3.1+/C-(K)DYK vector containing a wild-type version of IKZF1 transcript NM_006060 (Clone ID OHu28071) was purchased from GenScript. Using this as template, vectors containing the H167R or H195R point mutations were generated using a Q5 Site-directed mutagenesis kit (New England Biolabs) following the manufacturer's protocol. The following primer pairs were used for mutagenesis: IKZF1_H167R_F 5'-ATCAAGCTGCGTTCCGGGGAG-3' and IKZF1_H167R_R 5'- GTGCCGGAGCAGGTTGCC; IKZF1_H195R_F 5'- CTGAGGACGCGCTCCGTTGGTAAAC and IKZF1_H195R_R 5'- GTGGCCAGTGAGGGCGTC-3'. NIH3T3 cells were transfected with the IKAROS plasmids using TransIT-X2 -3T3 transfection kit (Mirus Bio). After 24 h post-transfection, the cells were seeded onto poly-L-lysine treated cover slips. The next day, the cells were washed, fixed 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated with rabbit anti-FLAG antibody (SIGMA), or normal rabbit IgG (Santa Cruz) and with Alexa Fluore 488 goat anti-rabbit IgG secondary antibody (Life Technologies). Cells were washed and counter stained with DAPI (Invitrogen). Washed cells were mounted on slides using ProLong Diamond Antifade Mountant (ThermoFisher). Images were acquired with a NiKon A1R+ confocal microscope with a 60x oil immersion objective (Nikon Instruments Inc., Melville, NY).
case_images: fped-07-00139-g0001.jpg
case_images: PMC6491668_01_fped-07-00139-g0001.jpg
case_images: F1
case_images: IKZF1 in the patient. In both the index case and his mother, genetic evaluation by Sanger sequencing verified novel heterozygous missense variant in the DNA-binding zinc finger (ZF) 3 domain of IKZF1 (c.584A>G, p.His195Arg) (Figure 1A). IKAROS is a zinc-finger protein, in which two cysteines and two histidines coordinate zinc required for protein folding and function (Figures 1B-D). Epitope-tagged mutant IKAROS proteins (Figures 1B-D) expressed in NIH3T3 cells showed an immunofluorescent staining characteristic of loss of function (Figure 1E), consistent with previous findings of other pathogenic mutations in IKZF1.