Loss of B Cells in Patients with Heterozygous Mutations in IKAROS

Abstract

Background: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells.

Methods: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates.

Results: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients.

Conclusions: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Figure 1. Mutation in IKAROS (IKZF1) in Families with Common Variable Immunodeficiency

Panel A shows the pedigrees of the six families. Circles and squares denote female and male family members, respectively. Black symbols represent family members with a heterozygous mutation in IKZF1, and slashes indicate deceased family members. In Family F, the diamond with the number in it indicates the number of family members (male or female) who were not screened. Arrows indicate the probands. The specific complementary DNA (IKZF1 transcript variant NM_006060) and protein mutation are indicated above each pedigree. Mut denotes mutation, and WT wild type. Panel B, top, shows the exon structure of full-length IKAROS isoform 1 in light-gray boxes (IKZF1 transcript variant NM_006060), with the zinc fingers (ZFs) indicated in dark-gray boxes. The sites of the mutations in Families A, B, C, and D and the deletion of c.161-8388_589+2308del in IKZF1 in Family E are shown. The lower part of the panel shows a schematic representation of the 4.7-Mb deletion on the short arm of chromosome 7 (7p12.3-p12.1) in Family F, with the deletion indicated in red.

Figure 2. Low Serum IgG Levels and Progressive Loss of Peripheral-Blood B-Cell Counts in Patients with IKZF1 Mutations

The symbols represent individual family members, with black symbols indicating those who were asymptomatic; gray shading represents mean values ±1 SD in healthy controls. Only patients who were not being treated with gamma globulin are included in the data shown for IgG levels.

Figure 3. Immunohistochemical Analysis of Bone Marrow–Biopsy Specimens and Flow-Cytometric Analysis of Bone Marrow Aspirates

Panel A shows immunohistochemical staining for CD34+ cells (hematopoietic stem cells) and CD138+ cells (plasma cells) in bone marrow–biopsy specimens from a healthy control, Patients C1 and C2, and a patient with typical common variable immunodeficiency (CVID). CD34+ and CD138+ cells are present in the control and in Patients C1 and C2 (red staining); CD34+ cells are present but CD138+ cells are absent in the patient with typical CVID. Panel B shows the results of flow-cytometric analysis of bone marrow aspirates from a healthy control and Patient C2. A total of 500,000 events were analyzed for both persons. A gate was used that collected all cells that were positive for CD34, CD19, or both (top and middle graphs) and cells that were negative for CD34 but positive for CD19 (bottom graph). The graph at the top shows a marked decrease in CD34+CD19+ pro-B cells in Patient C2 as compared with the control. The middle graph shows that precursors of pro-B cells that express CD34+ and cTdT+ (cytoplasmic terminal deoxynucleotidyl transferase) but lack CD19 are also decreased. The bottom graph shows that the relative numbers of pre-B cells (CD34−CD19+sIg− [surface immunoglobulin]) to immature B cells (CD34−CD19+sIg+), as documented by the expression of surface kappa or surface lambda, in the patient is similar to that in the control, although the total number of cells in both populations is decreased in the patient.