Exome sequencing identifies GATA1 mutations resulting in Diamond-Blackfan anemia

Abstract

Diamond-Blackfan anemia (DBA) is a hypoplastic anemia characterized by impaired production of red blood cells, with approximately half of all cases attributed to ribosomal protein gene mutations. We performed exome sequencing on two siblings who had no known pathogenic mutations for DBA and identified a mutation in the gene encoding the hematopoietic transcription factor GATA1. This mutation, which occurred at a splice site of the GATA1 gene, impaired production of the full-length form of the protein. We further identified an additional patient carrying a distinct mutation at the same splice site of the GATA1 gene. These findings provide insight into the pathogenesis of DBA, showing that the reduction in erythropoiesis associated with the disease can arise from causes other than defects in ribosomal protein genes. These results also illustrate the multifactorial role of GATA1 in human hematopoiesis.

Figure 1. Identification of GATA1 mutations in DBA.

(A) Pedigree of a family with DBA in 2 male children and normal hematological findings in the parents. (B) Sanger sequencing chromatograms from the 3′ end of exon 2 and 5′ end of intron 2 on GATA1 in the mother (I-2) and 3 children from the family shown in A. (C) RT-PCR analysis of GATA1 exons 1 and 3 from peripheral blood RNA samples from the patients (II-1 and II-3); their mother (I-2), who carries the GATA1 mutation; and a normal control. FL, full-length. (D and E) Bone marrow core sections from II-1 and II-3 shown at ×100 magnification demonstrate erythroid hypoplasia. (F) Rare erythroblasts with normal morphology were noted on bone marrow aspirates (from II-1; shown at ×100 magnification). (G) Sanger sequencing chromatograms show a GATA1 G nucleotide deletion (at the exon 2–intron 2 junction) in an independent male patient with DBA.

Figure 2. GATA1 exon 2 mutation results in trace amounts of GATA1 mRNA containing exon 2.

Quantitative RT-PCR was performed on peripheral blood RNA samples from individuals I-2, II-1, and II-3, as well as a normal control individual. Data are shown as the mean ± SD. Trace amounts of exon 2 are present in the male carriers of the GATA1 exon 2 mutation (3% and 5%, respectively), and a female carrier (I-2) with this mutation has half the level of mRNA compared with controls (53%).

Figure 3. A model of how GATA1 mutations in DBA favor production of GATA1s alone.

(A) Normal splicing pattern of GATA1 in humans and the resulting mRNA and protein products. (B) Aberrant splicing of exons 2 and 3 (highlighted by red star at mutation sites) leads to production of GATA1s alone. For the protein products, the transactivation domain (TD), N-terminal zinc finger (NF), and C-terminal zinc finger (CF) are shown.