Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in GATA1

Abstract

Haematopoietic development is regulated by nuclear protein complexes that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis in embryonic stem cells and mice has revealed roles for the X-linked gene Gata1 in erythrocyte and megakaryocyte differentiation. GATA-1 is the founding member of a family of DNA-binding proteins that recognize the motif WGATAR through a conserved multifunctional domain consisting of two C4-type zinc fingers. Here we describe a family with X-linked dyserythropoietic anaemia and thrombocytopenia due to a substitution of methionine for valine at amino acid 205 of GATA-1. This highly conserved valine is necessary for interaction of the amino-terminal zinc finger of GATA-1 with its essential cofactor, FOG-1 (for friend of GATA-1; refs 9-12). We show that the V205M mutation abrogates the interaction between Gata-1 and Fog-1, inhibiting the ability of Gata-1 to rescue erythroid differentiation in an erythroid cell line deficient for Gata-1 (G1E). Our findings underscore the importance of FOG-1:Gata-1 associations in both megakaryocyte and erythroid development, and suggest that other X-linked anaemias or thrombocytopenias may be caused by defects in GATA1.

Fig. 1

Peripheral blood and bone marrow abnormalities in patients II-1 and II-2. a, Peripheral blood smear. Note the severe deficiency of platelets and heterogeneity in red blood cell size (poikilocytosis) and shape (anisocytosis). An abnormal red blood cell with a bilobed nucleus is indicated (arrow). Original magnification ×50. b, Bone marrow core biopsy. Megakaryocytes, distinguished by their pale pink cytoplasm, are abundant and abnormally small. Representative megakaryocytes are indicated (arrows). Original magnification ×10. c, Bone marrow aspirate showing a cluster of developing erythroid precursors. Dyserythropoiesis is illustrated by the large, multinuclear erythroblast (centre). Original magnification ×100. d, Bone marrow core biopsy. Megakaryocytes (m) are dysplastic with small ecentric nuclei. Original magnification ×50.

Fig. 2

Ultrastructural abnormalities in megakaryocytes and platelets revealed by electron microscopy. a, Low-power micrograph of a normal megakaryocyte. Note the centrally located nucleus (Nu) and the well-developed demarcation membrane system (DM). Original magnification ×2,880. b, Megakaryocyte from affected patient showing showing abnormal features that include an abundance of smooth endoplasmic reticulum (Ser), ecentric nucleus and a relative paucity of granules. Original magnification ×2,880. c, High-power micrograph of a normal megakaryocyte showing well-organized membrane demarcation system. Original magnification ×20,000. d, High-power micrograph of mutant megakaryocyte showing abnormal abundance of smooth endoplasmic reticulum (Ser). The membrane demarcation system is poorly developed. Original magnification ×20,000. e, Platelet from normal individual. Note the abundance of a-granules (G). Original magnification ×20,000. f,g,h, Circulating platelets from affected patients. Note the paucity of granules (G), abundance of smooth endoplasmic reticulum (Ser) and abnormal membranous complexes (MC). Original magnification ×20,000.

Fig. 3

Mutational analysis of GATA1 in the affected pedigree. a, Partial pedigree of the affected kindred and DNA sequence analysis of GATA1. Patients with severe manifestations are represented by filled symbols, and patients with milder symptoms by half-filled symbols. Genotypes are shown below the pedigree members (other family members were not tested). b, Amino acid alignment of the N-terminal zinc finger of GATA-1 protein from various species and the Drosophila GATA-A protein. Amino acids known to interact with FOG-1 are labelled with black dots. The V205M mutation is indicated. c, Three-dimensional structure of the N-terminal zinc finger. Amino acids presumed to interact with DNA are shown in red. Valine 205 is indicated in yellow. Other FOG-1–interacting residues are shown in green.

Fig. 4

Biochemical characterization of mutant V205M Gata-1. a, The V205M mutation impairs binding of Gata-1 to Fog-1. Wild-type or V205M Gata-1 cDNAs were transfected into COS cells along with a FLAG-tagged version of Fog-1 consisting of zinc fingers 5 and 6. Gata-1 bound to Fog-1 was detected by immunoprecipitation with anti-FLAG antibody (α-FLAG IP) followed by western-blot analysis with anti-Gata-1 antibody (top). Gata-1 is indicated (arrow). The upper band (asterisk) corresponds to the IgG heavy chain of the anti-FLAG antibody. Total Gata-1 was determined by western-blot analysis of nuclear extracts from the transfected cells (NE, bottom). Equivalent amounts of FLAG-tagged Fog-1 were immunoprecipitated in Zfpm1 cDNA-transfected samples as determined by western blot with anti-FLAG antibody (data not shown). b, V205M Gata-1 binds DNA normally. The stability of Gata-1–DNA interactions was measured by a dissociation gel shift assay. Labelled probe bound to Gata-1 (arrow) migrates more slowly than free probe seen in the lower portion of the panel. V205M and wild-type (WT) Gata-1 dissociate from the labelled probe at similar rates. In contrast, the C204R mutation, which disrupts the N-terminal zinc finger, increases the dissociation rate between Gata-1 and bound DNA probe.

Fig. 5

Impaired function of V205M mutant Gata-1 in erythroid cells. a, Plasmids encoding oestrogen-inducible forms of wild-type (WT) or V205M Gata-1 were introduced into the Gata-1–null erythroid line G1E. Nuclear extracts from stably expressing clones were analysed by western blot for Gata-1 expression (arrow). Cells were treated with β-oestradiol for 72 h and erythroid maturation was determined by benzidine staining, which detects haemoglobin. Note that V205M/Gata-1 fails to induce differentiation in eight independent clones. b, Representative data from clones V205M-8 and WT-4. May Grunwald-Giemsa (MGG) and benzidine staining after 72 h of β-oestradiol treatment. c, Northern-blot analysis of clones V205M-8 and WT-4 after treatment with β-oestradiol for the indicated times. WT-4 exhibits mRNA changes that are characteristic of erythroid maturation, including induction of α-globin and band 3, and concommitant downregulation of Gata-2. No changes in gene expression are seen in V205M-8 cells after β-oestradiol treatment. Each lane represents 15 μg of total RNA.