To the editor:
We read with interest the description by Hoeller et al of an exon 2 GATA-1 mutation leading to severe transient myeloproliferative disease (TMD),1 in which the authors speculated that the severe phenotype might reflect loss of both full-length (FL) GATA-1 and the shorter isoform, GATA-1s, due to the position of the mutation in codon 2. Although almost all GATA-1 mutations in children with Down syndrome are in exon 22-4 and lead to loss of GATA-1FL, exon 2 mutations are also predicted to leave GATA-1s protein production unaffected,4 by translation of an alternatively spliced mRNA comprising exons 1/3/4/5/6.2 In humans, alternative splicing producing an exon 1/2/3/4/5/6 mRNA for GATA-1FL and exon 1/3/4/5/6 splice variant for GATA-1s has been demonstrated in adult bone marrow CD34+ cells.2 Using exon 1 and 3 primers, we have also found that both variants are consistently expressed in all normal cord blood and second-trimester fetal liver and bone marrow cells we have tested (n = 12; Figure 1). It seems likely, therefore, that exon 2 mutations would still allow expression of GATA-1s mRNA, which may be difficult to detect at the protein level due to the relative insensitivity of commercial GATA-1 antibodies in immunohistochemical reactions. Interestingly, and by contrast, although mice produce the 2 Gata-1 isoforms by alternative translation of a single mRNA,5 Gata-1s transcripts have not been reported in murine tissues and, in our experience, exon 1 and 3 primers consistently amplify only the FL transcript in mice (Figure 1B) despite evidence of Gata-1s protein production (Figure 1C). Further investigation of the functional consequences of different GATA-1 mutations may shed further light on the enigmatic role of GATA-1s in early hematopoiesis and differences between alternative splicing in human and murine GATA-1.
GATA-1 mRNA alternative splicing in human and murine tissues. (A) Reverse transcription–polymerase chain reaction (RT-PCR) of cDNA from human bone marrow, cord blood, and second-trimester (15 weeks) fetal blood, liver, and bone marrow, the expected size of the exon 1/2/3 splice variant (348 bp) and an exon 1/3 splice variant (109 bp) are marked. (B) RT-PCR of cDNA from murine hematopoietic tissues and the erythroleukemia cell line MEL. The expected size of an exon 1/2/3 splice variant (356 bp) and an exon 1/3 splice variant (117 bp) are marked. (C) Western blot of a MEL nuclear extract using M20 GATA-1 antibody (Santa Cruz Biotechnology), the predicted size of GATA-1FL is 47 kDa and GATA-1s 40 kDa.
GATA-1 mRNA alternative splicing in human and murine tissues. (A) Reverse transcription–polymerase chain reaction (RT-PCR) of cDNA from human bone marrow, cord blood, and second-trimester (15 weeks) fetal blood, liver, and bone marrow, the expected size of the exon 1/2/3 splice variant (348 bp) and an exon 1/3 splice variant (109 bp) are marked. (B) RT-PCR of cDNA from murine hematopoietic tissues and the erythroleukemia cell line MEL. The expected size of an exon 1/2/3 splice variant (356 bp) and an exon 1/3 splice variant (117 bp) are marked. (C) Western blot of a MEL nuclear extract using M20 GATA-1 antibody (Santa Cruz Biotechnology), the predicted size of GATA-1FL is 47 kDa and GATA-1s 40 kDa.
Authorship
Acknowledgments: This work was supported by a Leukaemia Research Clinical Training Fellowship (C.H.) and a Kay Kendall Leukaemia Fund Junior Fellowship (O.T.). Approval was obtained from the Hammersmith and Queen Charlotte’s Hospital Research Ethics Committee for these studies. Informed consent was provided according to the Declaration of Helsinki.
Contribution: C.H. performed the research and wrote the paper; O.T. performed the research and analyzed the data; and B.G., I.R., and G.G. designed the research and analyzed the data.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Christina Halsey, Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow Biomedical Research Centre, 120 University Pl, Glasgow, G12 8TA, United Kingdom; e-mail: c.halsey@clinmed.gla.ac.uk.