Congenital pancytopenia and absence of B lymphocytes in a neonate with a mutation in the Ikaros gene

Abstract

Background: Congenital pancytopenia is a rare and often lethal condition. Current knowledge of lymphoid and hematopoietic development in mice, as well as understanding regulators of human hematopoiesis, have led to the recent discovery of genetic causes of bone marrow failure disorders. However, in the absence of mutations of specific genes or a distinct clinical phenotype, many cases of aplastic anemia are labeled as idiopathic, while congenital immune deficiencies are described as combined immune deficiency.

Procedure: We describe the case of a 33-week gestation age male with severe polyhydramnios, hydrops, and ascites who was noted to be pancytopenic at birth. Bone marrow examination revealed a hypocellular marrow with absent myelopoiesis. An immune workup demonstrated profound B lymphopenia, near absent NK cells, and normal T cell number. Due to the similarity of the patient's phenotype with the IKAROS knockout mouse, studies were performed on bone marrow and peripheral blood to assess a potential pathogenic role of Ikaros.

Results: DNA studies revealed a point mutation in one allele of the IKAROS gene, resulting in an amino acid substitution in the DNA-binding zinc finger domain. Functional studies demonstrated that the observed mutation decreased Ikaros DNA-binding affinity, and immunofluorescence microscopy revealed aberrant Ikaros pericentromeric localization.

Conclusions: Our report describes a novel case of congenital pancytopenia associated with mutation of the IKAROS gene. Furthermore, these data suggest a critical role of IKAROS in human hematopoiesis and immune development.

Fig. 1

Abnormal subcellular localization of Ikaros. Immunofluorescence microscopy was used to detect and compare Ikaros' subcellular localization in lymphocytes of the patient (top panel); with those from his unaffected sister (middle panel) and Jurkat T cells—as a control (bottom panel). DNA was labeled with propidium iodine (red)—left panel. Subcellular localization of Ikaros was detected by anti-IK-CTS antibodies (green)—middle vertical panel. The right panel shows the combined images with yellow indicating co-localization of Ikaros and DNA.

Fig. 2

Sequencing of Ikaros. Bidirectional sequencing of Ikaros exon 5 was performed from genomic DNA isolated from the peripheral blood of the patient (A). Arrow indicates an ambiguous sequence in exon 5 at position 629. Bidirectional sequencing of cloned Ikaros' alleles reveals the presence of an Ikaros' mutated allele, with the point mutation indicated by an arrow at position 629 (B), and the wild-type allele (C).

Fig. 3

Mutated Ikaros protein has altered DNA binding affinity. 293T cells were transfected to express wild-type or mutated Ikaros and analyzed by EMSA. Loading was verified by Western blotting with IK-CTS antibody as shown above each EMSA. EMSA was performed with three probes derived from human PC-HC (g satellite 8, satellite 3 and CENP-B, lines 1–9) and with a probe derived from the upstream regulatory region of Ikaros target gene VPAC-1 (lines 10–12). Ikaros containing complexes are indicated by arrows.