Haematologica 2002; 87:(07)ELT30
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Transcription factor GATA-4 is not involved in Diamond-Blackfan anemia
Jana Cmejlova,* Dagmar Pospisilova,° Radek Cmejla*
*Institute of Hematology and Blood Transfusion, Prague, Czech Republic, °Department of Pediatrics, Faculty of Medicine, Palacky University and Faculty Hospital, Olomouc, Czech Republic

Correspondence: Radek Cmejla, Institute of Hematology and Blood Transfusion, P.O. Box 74, U Nemocnice 1, 128 20, Prague, Czech Republic. Tel: +420-2-21977276. Fax: +420-2-21977370. E-mail address: racm@centrum.cz or cmejla@uhkt.cz
Diamond-Blackfan anaemia is a congenital disorder characterised by severe hypoplastic anaemia accompanied in 40% of patients by various physical anomalies. Ribosomal protein S19 (19q13 region) was identified to be the DBA1 gene, and the transcription factor GATA-4 (8p23 region) was hypothesised to be the DBA2 gene. Here we report that in 15 patients no mutations in GATA-4 gene have been identified. GATA-4 mRNA was not detected either in mononuclear cells or in CD34+ cells isolated from cord blood. We conclude that GATA-4 is not the gene responsible for DBA. The importance of 8p23 locus in DBA pathogenesis is discussed.

Diamond-Blackfan anaemia (DBA; OMIM: 205900) is a congenital disorder affecting 5-7 children per million live births (for DBA review see1). The DBA diagnosis is based on the following criteria: 1) normochromic macrocytic anaemia presenting usually in the first year of life, 2) profound reticulocytopenia, 3) normocellular bone marrow with a selective deficiency in red blood cell precursors, 4) normal or slightly increased platelet count and normal or slightly decreased white blood cell count. In about 40% of patients various congenital anomalies are present, including craniofacial dysmorphism, thumb and neck anomalies, congenital heart defects, urogenital malformations, prenatal or postnatal growth retardation independent of steroid therapy.2
As for the genetic background, the locus on 19q13.2 was the first one recognised to be associated with DBA, and later ribosomal protein S19 (RPS19) residing in this region was found mutated in 25% of DBA patients.3 Recently, Gazda and co-workers presented evidence that the second DBA gene might be located within an 8.1-cM interval on the 8p23.3-8p22 chromosome (LOD score +3.55).4 From tens of genes in this region already assigned to contigs (Human Genome Sequencing, www.ncbi.nlm.nih.gov/genome/seq/HsHome.shtml), the transcription factor GATA-4 was considered a candidate to be the DBA2 gene.4 GATA-4 is a member of GATA transcription factor family of proteins with two zinc fingers that direct DNA binding.5 The GATA family can be divided into two groups - a subfamily containing GATA-1, 2, 3 which are predominantly expressed in haematopoietic cells, and GATA-4, 5, 6 subfamily with the expression detected in various mesoderm and endoderm derived tissues (heart, liver, lung, gonads, small intestine).5
GATA-4 null mice embryos die between embryonic days 7 and 9.5 because of severe developmental abnormalities - embryos lack a primitive heart tube and foregut and develop partially outside the yolk sac.6 GATA-4 was concluded to be required for the ventral morphogenesis and the heart tube formation, but not by the regulation of cardiac cell lineage differentiation per se, but rather by its involvement in the lateral to ventral folding throughout the embryo, the process important for the heart tube formation.6 Moreover, it was shown that the ectopic expression of GATA-4 can compensate the erythropoietic defect in GATA-1 deficient embryonic stem cells induced to form embryoid bodies, and that it can activate globin genes transcription as well.7
To test the possibility of GATA-4 involvement in DBA we have sequenced GATA-4 gene in 15 DBA patients (13 with normal karyotypes, in 2 patients karyotype not done). No mutations in RPS19 gene were identified in all patients. Thirteen patients were from the Czech DBA Registry, two patients were from Croatia. Four of them presented with congenital heart defects - one patient with tetralogy of Fallot, three patients with secundum atrial septal defects. DNA was isolated from peripheral blood mononuclear cells (MNC), and all of six GATA-4 exons were PCR amplified and sequenced (for primers sequences see Table 1). No mutations in exons and splice sites were identified at the genomic level.
We have also focused on GATA-4 mRNA expression in haematopoietic tissues. RT-PCR was performed from child bone marrow MNC, adult peripheral blood and cord blood MNC, as well as from cord blood CD34+ cells to find, whether GATA-4 is expressed. However, GATA-4 mRNA was not detected (Figure 1).
We therefore conclude that although GATA-4 can compensate for GATA-1 deficiency in erythroid differentiation (at least in embryoid bodies),7 the spatial pattern of its expression and our sequencing results do not support its role in normal erythropoiesis or in DBA development. It is also of interest to note that mice heterozygous for GATA-4 exhibit normal phenotype without any haematological abnormalities noted.6 Moreover, it seems likely that 8p23 locus is not associated with anaemia. Families with 8p23-ter deletion have been described with symptoms very similar to DBA - postnatal growth retardation, congenital heart defects (CHD), dysmorphic faces, urogenital malformation, severe to mild mental retardation and others, but without anaemia or other haematological disorders.8,9 Similarly, CHD patients with a linkage to 8p23 did not suffer from anaemia.10 From this point of view it is likely that the 8p23 locus might be rather involved in congenital heart defects and other abnormalities found in CHD/DBA patients and in patients with 8p deletion. Yet another factor(s) outside the 8p23 region must be therefore employed to develop anaemia in DBA patients with a linkage to 8p23, or a dominant mutation in another gene from the 8p23 locus would be anticipated.

Funding

The work was supported by grant NE 6689-3 from the Internal Grant Agency of the Czech Ministry of Health, and by grant 301/00/1061 from the Grant Agency of the Czech Republic

Acknowledgements

We thank to Dr. M. Stimac for co-operation, D. Zakova for excellent technical help with sequencing, and Dr. J. Jelinek for reading the manuscript.

References

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