Identification of a heterozygous variant in IKZF2 and effects on Helios function and transcription. (A) Family pedigree of the patient under study. The 16-year-old girl (A1) is the only affected member of the family. (B) Illustration of the Helios transcription factor, with 4 N-terminal zinc fingers responsible for DNA binding to the consensus sequence and 2 C-terminal zinc fingers that form the homo/heterodimerization domain. Location of the variant (transcript ID ENST00000434687.5; c.871C>T, p.Arg291Ter, rs146574423) is shown (red arrow). (C) Nuclear extracts from 293T cells transfected with wild-type (WT) or the mutant were prepared, and electrophoretic mobility shift assay was performed using the IK-BS1 probe, an Ikaros consensus-binding sequence. Representative image of 7 independent experiments. In the empty vector (EV) and WT lanes, 2 faint bands are visible, the top being DNA complexes, the bottom free probe. The lower band (red asterisk) for Helios R291X, the size of which coincides with that of the DNA complex band seen in the EV and WT lanes, is the monomer binding to the DNA probe, indicating that this variant cannot bind DNA as a homodimer. (D) Immunofluorescence staining of NIH3T3 cells transfected with Helios WT or Helios R291X using an anti-Helios antibody, showing the formation of foci at pericentromeric heterochromatin regions. Graph on the bottom shows the quantification of number of foci per cell. Results represent 3 independent experiments. For statistical analysis, an unpaired t test was performed. (E) Coimmunoprecipitation experiments after cotransfection of HEK293T cells with Streptavidin (Strep)-HA-Helios WT, Strep-HA-Helios R291X, FLAG-Helios WT, or an EV. Immunoprecipitations (IPs) were performed with Strep beads, and western blot analysis was performed by running both the (IP) and whole-cell lysate (input) on a gel and blotting with HA, FLAG, Helios, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Results are representative of 3 to 5 independent experiments. The expected size of untagged Helios was 60 kDa; the observed size increase is explained by addition of amino acids by the protein tag, with the Strep-HA tag being substantially longer than the FLAG tag. The sizes of the truncated variant R291X tagged with either FLAG or with Strep-HA were 45 and 55 KDa, respectively. (F) The top panel shows a low-dimensional projection (uniform manifold approximation and projection [UMAP] plot) of the combined scRNA-seq data set comprising 23 946 cells from the patient (P) and 4 controls. Numbers indicate clusters (graph-based clustering), and colors correspond to cell type (curation based on supplemental Figure 3A). The bottom panel shows the same projection, with colors indicating the distribution of patient cells (blue) within the clusters compared with controls (gray). (G) Bar plot showing the distribution of cells from each individual across the clusters defined in panel F. (H) Heatmap showing differentially expressed genes in clusters 2a and 2b, corresponding to T cells and T and NK cells, respectively. cDC, conventional dendritic cell; DAPI, 4′,6-diamidino-2-phenylindole; pDC, plasmacytoid dendritic cell. Scale bar: 25 µm.