GATA1 Mutants Lacking Rb-Binding Motif Observed in Transient Abnormal Myelopoiesis in Down Syndrome
Authors: Toki Tsutomu , Kobayashi Eri , Kanezaki Rika , Wang RuNan , Terui Kiminori , Kanegane Hirokazu , Maeda Miho , Endo Mikiya , Mizuochi Tatsuki , Adachi Souichi , Hayashi Yasuhide , Shimizu Ritsuko , Yamamoto Masayuki , Ito Etsuro 
Published: October 2019

Abstract 1491

Five to ten percent of neonates with Down syndrome (DS) develop transient abnormal myelopoiesis (TAM), which is characterized by a rapid growth of abnormal blast cells with the erythroid/megakaryocytic phenotype. In most cases, it resolves spontaneously within three months. However, 20 to 30% of patients develop acute megakaryocytic leukemia referred to as myeloid leukemia of DS (ML-DS) within four years. Blast cells in most of TAM and ML-DS patients have mutations in exon 2 of the gene encoding transcription factor GATA1, which is essential for the normal development of erythroid and megakaryocytic cells. These mutations lead to expression of a truncated GATA1 protein lacking the N-terminal 83 amino acids (GATA1s). However, the molecular mechanism whereby GATA1s contributes to the genesis of TAM and ML-DS remains elusive. From 2003 to 2010, we screened GATA1 mutations in clinical samples obtained from 106 patients with TAM upon request from referring hospitals. We performed direct sequencing analysis using cDNA prepared form total RNA extracted from white blood cells in peripheral blood and genomic DNA. Acquired GATA1 mutations were detected in a total of 99 (93.4%) patients among them. Most of these mutations were coding GATA1s. However, the mutations coding GATA1 mutants with internal deletion of 43 and 15 amino acids were detected in five and one patients, and we referred to them as GATA-ID type-1 and GATA1-ID type-2, respectively. Sequences of exon 3 were discrepant with that expected from the corresponding cDNA sequence. Instead, a 2-nucleotide insertion, a 2-nucleotide deletion and a 7-nucleotide deletion with a single-nucleotide substitution were detected in exon 3 of the GATA1 gene in the GATA1-ID type-1 patients, and a 21-nucleotide deletion encompassing the boundary of intron 2 and exon 3 was found in the GATA-ID type-2 patient. These findings suggest that the mutations in exon 3 lead to expression of alternative splicing forms of mRNA encoding GATA1-ID. To investigate the role of GATA1-ID on the pathogenesis of TAM and ML-DS, we next expressed GATA1-ID by retrovirus in a ML-DS cell line, KPAM1, or primary GATA1-deficient fetal megakaryocytic progenitors. Expectedly, the cell growth was markedly reduced upon transduction with full-length GATA1-expression retrovirus. However, ectopic expression of GATA1-ID type-1 and type-2 failed to restrict the proliferation of cells as similar as expression of GATAs. Interestingly, all these patients had high white blood cell counts in the peripheral blood at diagnosis. Furthermore, three patients developed liver fibrosis and two had effusion. All these factors are significantly associated with early death. Three patients were treated with low dose of Ara-C. However, early death occurred in three patients. In this study we found that GATA1-ID proteins are created by somatic mutations in DS patients and contribute to the generation of TAM phenotypes. Our newly identified GATA1-ID mutants have highlighted a much narrower set of sequences responsible for the pathogenesis of than was previously suggested. The missing region identified by the GATA1-ID proteins contains a consensus motif (LxCxE, amino acids 81–85) essential for the interaction with pRB protein and this motif is also lost in GATA1s. Our results suggested the lack of Rb-GATA1 interaction as the most likely pathogenesis for onset of TAM.

Disclosures:

No relevant conflicts of interest to declare.