B Locus YAC Mice Carrying G Gene Promoter Mutations: Insights into the Mechanism of Action of the -175 HPFH Mutation.

Non-deletional HPFHs are g gene promoter mutations, which are associated with HbF elevation in heterozygous adult individuals. To investigate the mechanism whereby these mutations result in g gene activation, the −198 (T→C) and −175 (T→C) mutations were introduced into the promoter of the ^Ag gene in the context of a 213 kb b-globin locus YAC. The mutated YAC DNA was then used to produce transgenic mice. Expression of the human g and b globin gene was examined by RNase protection assay in blood of the adult transgenic mice carrying either −198 or −175 mutations. The level of g gene expression ranged from 4.0 to 8.5% of the human b mRNA in the −198 mutant transgenic mice, and from 25 to 41% in the −175 mutant mice. In contrast, there was no detectable level of g gene expression in the blood of wild type transgenic mice. The ratios of g/b mRNA in the mutant transgenic mice correspond to the levels of Hb F in adult individuals carrying the corresponding mutations: Hb F in heterozygotes for HPFH −198 and −175 mutations ranges from 3.5 to 10% and from 36 to 41%, respectively. To delineate the mechanism of g gene reactivation by the HPFH mutations, the CACCC box in the g gene promoter was deleted in the context of the mutant YAC. In contrast to the −175 HPFH bYAC mice, in the doubly mutated −175 HPFH D^AgCACCC bYAC mice, expression of the g gene in the adult blood of the transgenic mice was undetectable. These results provide strong evidence that an intact CACCC box is required for g gene activation in the −175 HPFH mutation. We conclude that YAC mice can faithfully reproduce the phenotypes of nondeletional HPFH mutations and be used for the investigation of the mechanisms by which these mutations activate the g gene.