A Novel Splice Site Mutation in the 5'UTR of GATA1 resulting in Abnormal Hematopoiesis

Diamond-Blackfan anemia (DBA) is a rare disorder of impaired erythropoiesis belonging to a broader family of inherited and acquired anemias known as pure red cell aplasias (PRCA). The cardinal feature of DBA is aregenerative anemia accompanied by macrocytosis, reticulocytopenia and decreased or absent erythroid precursors in an otherwise normocellular marrow. Classic DBA is associated with mutations of genes encoding ribosome subunits. Advancements in molecular techniques have prompted discussion regarding the genetic basis of the disease and its prognostic implications as non-ribosomal genes are beginning to be implicated in DBA.

Acquired mutations within the X-linked transcription factor GATA1 have been described in Down syndrome-related transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AKML), while germline GATA1 mutations affecting both isoforms are associated with congenital thrombocytopenia. Normal alternative splicing of GATA1 produces a full-length GATA1 isoform (flGATA1) as well as a shortened GATA1 isoform (GATA1s), which lacks the N-terminal domain that activates GATA1-driven erythropoiesis program and recruits flGATA1 to a subset of megakaryocyte and erythroblast genes. Rare germline defects known as "GATA1s mutations" are characterized by an unbalanced production of the GATA1s isoform at the expense of a flGATA1 transcript due to disrupting exon 2 splice sites or the ATG initiation codon. GATA1s mutations had been reported to phenocopy Diamond-Blackfan anemia by causing isolated red blood cell aplasia in some patients, although thrombocytopenia with structural platelet abnormalities and dyserythropoiesis as well as DBA-like picture progressing into MDS had been described in other GATA1s families. Therefore, while GATA1s mutations uniformly disrupt erythropoiesis, the impact of defective flGATA production on megakaryopoiesis in non-trisomy 21 individuals is less clear.

Here we describe a child with dyserthropoietic anemia, marked megakaryocyte dysplasia, peripheral thrombocytosis and platelet dysfunction due to a novel disease-causing mutation within the 5' UTR of GATA1. DBA was initially suspected due to progressive macrocytic anemia beginning in infancy. Bone marrow analysis revealed not only a paucity of erythroid precursor cells and dyserythropoiesis, but also prominent megakaryocytosis and megakaryocyte dysplasia, not typically associated with classic DBA. Ribosome mutational analysis was unremarkable, as were cytogenetics and MDS-FISH analysis. Given aregenerative anemia mimicking DBA as well as megakaryocyte dysplasia, the question was raised whether the findings could be associated with a germline mutation in GATA1. Indeed, sequencing revealed a novel, pathogenic mutation in the 5'UTR of GATA1 (c.-21 A>G). We explored the impact of this novel mutation in silico, found it to disrupt a consensus splice site, and further demonstrated that this mutation conferred a striking predominance of the GATA1s isoform at the transcript and protein level. Two-color immunohistochemistry confirmed loss of full-length GATA1 protein in the patient's bone marrow and showed that full-length GATA1 expression is restricted to megakaryocytes and erythroblasts in healthy bone marrow, consistent with dysmegakaryopoiesis and decreased erythropoiesis in our patient.

Our findings support a role for the N-terminus of GATA1 in both erythroblast and megakaryocyte maturation and function in vivo. Furthermore, the findings of prominent megakaryocytes in the bone marrow and resultant functional platelet abnormalities may provide subtle clinical clues to differentiate Diamond-Blackfan anemia due to ribosomopathy from dyserythropoiesis secondary to GATA1s mutations. We conclude that GATA1 sequencing, including non-coding GATA1 regions, should be considered in males with congenital multi-lineage dysplasia and/or DBA-like clinical presentation.