Increased expression of the Gγ and Aγ globin genes associated with a mutation in the Aγ enhancer

Abstract

We have previously described a unique type of δβ-thalassemia in a Chinese family characterized by increased expression of the Gγ and Aγ fetal globin genes in the absence of a large deletion in the β-globin gene cluster. Our earlier study of the β-globin gene on this δβ-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this δβ-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the Gγ and the Aγ genes and the 3′ Aγ enhancer element of this δβ-thalassemia chromosome. DNA sequence analysis of the Gγ and Aγ-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element down-stream from the Aγ-globin gene showed a C to T substitution 2,401 nucleotides downstream from the Aγ cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant Aγ enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the Aγ enhancer on a chromosome that carries a partially inactivated β-globin gene may be responsible for the increased expression of both γ-globin genes seen in this condition. © 1994 by The American Society of Hematology.