GATA1 mutant proteins with internal deletions. (A) A schema of mutant GATA1 proteins observed in patients with TAM. The amino acid sequence of GATA1-ID proteins was deduced from the sequence of GATA1 cDNA obtained from patients with TAM. Dark boxes indicate N-finger (NF) and C-finger (CF) domains. ID indicates internal deletion. (B) Somatic mutations of the GATA1 gene found in ID type 1 and type 2 patients. Missing, inserted, or substituted nucleotides are highlighted with dark color. A second translation initiation codon located in the third exon is underlined. The AGGT sequence functioning as an alternative splice donor site in mutant GATA1 genes of ID type 1 patients is circled. Note that a mutant GATA1 gene found in TAM patient 71f (ID type 2) lost a splice acceptor site in exon 3 because of the 21-nucleotide deletion. (C) Expression of GATA1 proteins in cells transfected with minigenes using anti-GATA1 antibodies recognizing the C terminus (upper) and residues between the 66th and 78th amino acids (lower) of the GATA1 protein. GATA1-ID proteins are recognized by the antibody against amino acid residues 66-78 of GATA1, whereas GATA1-S is not (lanes 6-9). Cells transfected with mock pcDNA3.1 (lane 1), pcDNA3.1-GATA1 cDNA (lane 2), original minigene (lane 3), and GATA1 minigene harboring a splicing error mutant in the 3′ boundary of intron 1¹³  (lane 4) are used as positive and negative controls for GATA1 and GATA1-S, respectively. (D) GATA1 ID type 1 protein and GATA1-S are detected in the TAM blast cells from patients 80 (lane 4) and 37 (lane 5), whereas only GATA1-S is expressed in the blast cells from patient 86 harboring a conventional type of GATA1 gene mutation in TAM cases (lane 3). Note that relatively abundant GATA1-S is recognized in patient 37 because of the intermixing of genetically distinct clone of cells expressing only GATA1-S (supplemental Table 1). Human erythroleukemia cells (HEL, lane 1) were used as a control for GATA1 and GATA1-S. DS-AMKL cells (CMK11-5, lane 2) and BHK-21 cells transfected with cDNA encoding GATA1 ID type 1 protein (lane 6) were used as controls for GATA1-S and GATA1 ID type 1, respectively.