A Child With Dyserythropoietic Anemia and Megakaryocyte Dysplasia Due to a Novel 5'UTR GATA1s Splice Mutation

Abstract

We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5' untranslated region (5'UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5'UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N-terminus; and (iii) expression of the GATA1 N-terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patient's abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N-terminal domain of GATA1 in human hematopoiesis.

Keywords: GATA1; dyserythropoietic anemia; megakaryocyte dysplasia.

Figure 1. Hypoplastic anemia, megakaryocyte dysplasia and thrombocytosis due to mutation within the 5′UTR of GATA1

(A) Chronic anemia and thrombocytosis over the course of 5 years. (B) Decreased erythropoiesis and megakaryocyte dysplasia seen on bone marrow aspirate. (C) Immunohistochemistry with CD71 (erythroblast marker) and CD61 (megakaryocyte marker) reveals decreased erythropoiesis and accumulation of megakaryocytes in the patient’s bone marrow compared to a healthy individual. Right panel shows dysplastic megakaryocytes (black arrows) in the patient’s marrow (Wright-Giemsa stain). (D) GATA1 sequencing reveals a novel mutation in the affected child. RT-PCR and Western blotting demonstrate decreased full-length GATA1 transcript (E) and protein (F) in the patient. (G) Schematic representation of GATA1 alternative splicing (only first three exons are shown for simplicity). The GATA1^c-.21A>G mutation produces GATA1s phenotype by disrupting full-length GATA1 splicing.

Figure 2. Full-length GATA1 expression is restricted to erythrocyte and megakaryocyte precursors during hematopoiesis

(A) Antibodies used for immunohistochemistry. Antibody against the N-terminus of GATA1 recognizes only full-length GATA1, while the C-terminal antibody recognizes both GATA1 isoforms (flGATA1 and GATA1s). (B) Loss of flGATA1 expression in the GATA1^c.-21A>G patient’s bone marrow. Note that (i) flGATA1 is not expressed in all hematopoietic cells of a healthy individual, and (ii) GATA1s production is not affected by the GATA1^c.-21A>G mutation. (C) Expression of N-terminal GATA1 domain (flGATA1; blue) is restricted to erythroblasts and megakaryocytes during human hematopoiesis. Appropriate hematopoietic lineage markers (brown) were co-stained as shown. (D) GATA1s mutations cause a range of phenotypes from Diamond-Blackfan anemia (red) to multilineage hematopoietic dysplasia (blue). Novel mutation described in this work is marked with asterisk. The arrow indicates that the long-term risk of MDS in GATA1s patients presenting with pure RBC aplasia remains to be determined as one GATA1s patient initially diagnosed with DBA developed MDS later in childhood [12].