Assessing the Functional Relevance of Variants in the IKAROS Family Zinc Finger Protein 1 (IKZF1) in a Cohort of Patients With Primary Immunodeficiency

Abstract

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency. Patients with CVID are prone to recurrent bacterial infection due to the failure of adequate immunoglobulin production. Monogenetic defects have been identified in ~25% of CVID patients. Recently, mutations in IKZF1, encoding the zinc-finger transcription factor IKAROS which is broadly expressed in hematopoietic cells, have been associated with a CVID-like phenotype. Herein we describe 11 patients with heterozygous IKZF1 variants from eight different families with autosomal dominant CVID and two siblings with an IKZF1 variant presenting with inflammatory bowel disease (IBD). This study shows that mutations affecting the DNA binding domain of IKAROS can impair the interaction with the target DNA sequence thereby preventing heterochromatin and pericentromeric localization (HC-PC) of the protein. Our results also indicate an impairment of pericentromeric localization of IKAROS by overexpression of a truncated variant, caused by an immature stop codon in IKZF1. We also describe an additional variant in TNFSF10, encoding Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL), additionally presented in individuals of Family A. Our results indicate that this variant may impair the TRAIL-induced apoptosis in target cell lines and prohibit the NFκB activation by TRAIL and may act as a modifier in Family A.

Keywords: CVID; IKAROS; TRAIL DNA binding; monogenic defects; nuclear localization.

Figure 1

Autosomal dominant inheritance of IKAROS heterozygous mutations in CVID families of a cohort of 650 individuals. (A) Pedigrees of families A–I. Circles, female; squares, male; black filled symbols, affected individual; gray filled symbols; individual with hypogammaglobulinemia without CVID-related symptoms, open symbol; unaffected; slash, diseased. (B) Schematic presentation of the structure of human IKAROS. The N-terminal DNA binding domain composed of four zinc finger motifs (yellow boxes, ZF1–ZF4), the central activation/repression domain (green) and the C-terminal dimerization domain (ZF5–ZF6). Exon borders and amino acid positions are indicated. Dotted lines in the upper part indicate heterozygous variants identified in this study. Lower parts shows previously published IKZF1 mutations (, –38).

Figure 2

(A) The sequence, used for the EMSA experiments is shown. Motifs that resemble the IKAROS consensus are shown in red in the rectangle. (B) Mutations affecting the DNA binding domain of IKAROS, preventing its binding to its target sequence. A specific gel-shift band, indicating binding of IKAROS multimers to the IKBS-4 DNA probe, was observed in the nuclear extract of HEK293T cells expressing the wildtype IKAROS. This band was absent in cells expressing the IKAROS variants Arg143Trp, Cys150Arg, and Arg162Gln and was weaker in the nuclear extracts of cells co-transfected with equal amounts of wildtype and one of the three mutant forms. A smaller band was also detected in the co-transfected samples, compared to the wildtype ones which can indicate the DNA-bound dimers of IKAROS. The DNA binding activity remained unaffected when the mutant variants Lys286*, Gly337Ser, Ala448Arg, and Met494Val were expressed.

Figure 3

IKAROS subnuclear and pericentromeric-heterochromatin localization. (A) NIH3T3 cells, transiently transfected with the pCR3 vector expressing wildtype or mutant IKAROS were incubated with rabbit-anti-human-IKAROS antibody and N-terminus anti-human IKAROS antibody and stained with goat anti-rabbit Alexa fluor 488. Cells overexpressing the wildtype IKAROS, showed the punctate staining pattern, observed in green. In contrast, a diffuse nuclear staining was observed in NIH3T3 cells overexpressing the Arg143Trp, Cys150Arg, Arg162Gln, and Lys286* mutant forms. (B) The DNA binding ability of wildtype IKAROS was not inhibited by the presence of a mutant variant. NIH3T3 cells were co-transfected with equal amounts of wild-type and each mutant pCR3 expression vectors and incubated and stained with the above-mentioned antibodies. In NIH3T3 cells co-transfected with equal amounts of wild-type and each mutant forms, the punctate nuclear staining pattern were obtained in all samples.

Figure 4

The results of confocal microscopy with epitope-tagged constructs were consistent with the results of Immunofluorescent microscopy. (A) NIH3T3 cells, transiently overexpressing epitope-myc/FLAG-tagged wildtype IKAROS and myc-tagged mutant forms were incubated with monoclonal rabbit anti myc-tag-antibody and mouse monoclonal anti-FLAG M2 antibody and stained with goat anti-rabbit Alexa fluor 488 (green) and goat anti-mouse Alexa 546 (red). In NIH3T3 cells transfected with epitope-myc/FLAG-tagged wildtype IKAROS the speckled nuclear localization was observed in contrast to a diffuse nuclear staining, observed in NIH3T3 cells expressing the myc-tagged Arg143Trp, Cys150Arg, Arg162Gln, and Lys286* mutant forms. (B) Wildtype components were dominant to form IKAROS complexes in PC-HC sites. NIH3T3 cells were co-transfected with 50% of myc/FLAG-tagged wildtype IKAROS and 50% of myc-tagged, expressing each mutant form. The diffuse nuclear localization of the mutant forms was significantly reduced in NIH3T3 cells co-transfected with equal amounts of the FLAG-tagged wildtype IKAROS together with the myc-tagged Arg143Trp, Cys150Arg, and Arg162Gln. Accumulation of FLAG-tagged wildtype proteins, observed as a punctuated nuclear staining pattern in the co-transfected samples, showed the dominance of wildtype components to form IKAROS complexes in PC-HC sites.