Loss of GATA-1 Full Length as a Cause of Diamond–Blackfan Anemia Phenotype

INTRODUCTION

In mammals, hematopoietic homeostasis is supported by the balance of the expression of multiple genes specific to each process. The GATA-1 gene is located on the X-chromosome (Xp11.23) and encodes a transcription factor that regulates the development of erythrocytes, megakaryocytic cells, eosinophils, mast cells, and dendritic cells [1]. GATA-1 protein has three functional domains (Fig. 1A): an N-terminal transactivation domain (TD), essential for transcriptional activation activity, a N-terminal zinc finger (NF), and a C-terminal zinc finger (CF) responsible for the binding to DNA [1,2]. Accumulating evidence supports the notion that defects in GATA-1 are linked to hematopoietic disorders. Exon 4 mutations have been identified in families with dyserythropoietic anemia, thrombocytopenia, thalassemia, and erythropoietic porphyria [3]. Furthermore, acquired somatic mutations in exon 2, that cause the loss of the N-terminal TD of GATA-1, have been found in individuals with Down Syndrome (DS) who developed either transient myeloproliferative disorder (TMD) or acute megakaryoblastic leukemia (AMKL) [4]. These mutations prevent the synthesis of full-length GATA-1 (GATA-1 FL), but allow for the synthesis of a short isoform of GATA-1 (GATA-1s), that lacks the N-terminal TD [4]. Related germline mutations have also been described. Hollanda et al. [5] reported a c.220G > C mutation in seven-male members of a family all with anemia and trilineage dysplasia. Sankaran et al. [6] identified a mutation in two siblings affected by DBA and another mutation in a third male from a cohort of 62 additional DBA patients (Table I). Both mutations occur at the donor splice site of exon 2 in the GATA-1 gene resulting in exon skipping. Thus only GATA-1s is produced.

DBA is a normochromic–macrocytic anemia characterized by impaired proliferation/maturation of the erythroid precursors in the bone marrow. Approximately, 30% of the patients have a heterogeneous mix of congenital malformations [7]. Sixty percent of patients carry mutations in genes encoding ribosomal proteins (RP). Systematic sequencing of all RP genes in patients with DBA has allowed the identification of mutations in 11 RP genes [8–10]. The remaining 40% of patients have mutations in unknown genes.

MATERIALS AND METHODS

RESULTS

One of the 23 patients studied showed a c.2T > C hemizygous mutation in the initiation codon used to synthetize the GATA-1 FL. This mutation was inherited from his mother (Fig. 1B). As a consequence of this change, GATA-1s is the only isoform expressed. Support for this view comes from a study of DS patients with TMD [4] where mutations in the initiation codon for GATA-1 FL, that cause the loss of the first methionine, including a c.2T > C mutation, were shown to lead to the exclusive synthesis of GATA-1s.

Our is the first study that reports a germline mutation in the first ATG codon of GATA-1 (Fig. 1B).

The proband reported here (Supplementary Table I, patient number 10) was born at term after an uneventful pregnancy. At the age of 9 months he developed a severe hyporegenerative anemia (Hb 5.5 g/dl, MCV 93 fL, reticulocyte count 40,000/μl) and was referred to us based on a suspected diagnosis of DBA. No somatic malformations were evident. His erythrocyte adenosine deaminase activity was slightly increased (1.9 U/g Hb, normal values 1 ± 0.2 U/g Hb). Leukocytes and platelets were in the normal range. The bone marrow aspirate showed a selective deficiency in erythroid precursors without abnormalities of the other hematopoietic lineages. He was treated with a standard dose of prednisone (2 mg/kg/day) for 4 weeks which was gradually reduced. Only a partial response was observed and his hemoglobin level reached 8 g/dl. Treatment was discontinued after 3 months and his hemoglobin level persisted between 8 and 9 g/dl without the need of further treatment including red blood cell transfusion.

At the age of 4 years he developed a progressive three linear cytopenia. A bone marrow biopsy showed a severe hypocellular marrow. The karyotype showed mosaicism with a 45XY, −7 clone (65%) and a further 50XXY, +3, +8, +21 clone. On this basis, he was diagnosed with Myelodysplastic Syndrome (MDS) after which he underwent Hematopoietic Stem Cell Transplantation (HSCT) from an unrelated donor. Ten years posttransplant, the patient is transfusion independent and in good health.

DISCUSSION

Our study reports a patient with a hyporegenerative macrocytic anemia with a mutation in GATA-1 that interferes with the expression of GATA-1 FL, leaving GATA-1s as the sole isoform expressed. Mutations that abolish the expression of GATA-1 FL have been previously identified in 10 DBA patients from three independent families (Table I) [5,6]. These patients showed various degrees of anemia, with or without an involvement of other hematopoietic lineages (Table I). Our patient displayed a progressive trilinear cytopenia evolving to MDS requiring HSCT. This evolution occurs infrequently in DBA but at an incidence higher than the general population [12]. Progression to MDS is not restricted to GATA-1 genotypes since it has also been observed in patients with mutations in RP genes.

DBA is considered the prototype of ribosomopathies, since the discovery that a mutation in a RP can lead to the impairment of the biogenesis and function of the ribosomes. GATA-1 is the first and so far the only non-ribosomal protein gene that when mutated leads to DBA. In contrast to ribosomal proteins, whose essential function in all cells make it difficult to explain such a selective phenotype, a role for GATA-1 as a causative gene in DBA is much more intuitive. Given its role in the transcription of a number of genes required for erythroid development, loss of its transcription activation domain as occurs in patients with DBA is consistent with the erythroid phenotype.

Unresolved is the relationship between DBA caused by mutations in GATA-1 and RPs. It may be possible that RP haploinsufficiency interferes with the expression of GATA-1 FL. Alternatively, the pathways involving GATA-1 and ribosome function may be independent but result in similar phenotypes. The association of somatic GATA-1 mutations with TMD in patients with DS, raises concerns regarding risks to patients with DBA harboring similar mutations in GATA-1. While the sample size is far too small to calculate relative risks for such progression, the trilineage cytopenia and outgrowth of myelodysplastic clones observed in our patient indicate a need for further studies in this area and the careful surveillance of affected individuals.

Supplementary Material