The term, hereditary persistence of fetal hemoglobin (HPFH), describes a hereditary benign disease, characterized by an increase in fetal hemoglobin (HbF) during adult life. Non-deletional forms of HPFH are characterized by single-base mutations in the promoter region (most of them between −114 and −202 from the cap site) of either the Gγor Aγ-globin gene, resulting in an increase of HbF ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the Aγ-195 C →G mutation that causes the Brazilian type of HPFH (HPFH-B). A previous study showed that the -195 mutations alone was not able to increase gene expression in vitro and a modest increase was observed when a LCR element fragment (HS2) was introduced in the construction. This study showed too, that the the mechanism of HbF elevation by the -195 mutation was neither mediated by the Sp-1 transcription factor nor by the creation of a CACCC box, as described for the -198 mutation. Thus, other proteins may be involved in the over-expression of the γ-globin chain and/or may depend on DNA structure and these mechanisms remain to be clarified. To better understand this mechanism we have developed HPFH-B transgenic mice. The promoter with mutation was amplified using the genomic DNA of a HPFH-B patient and cloned in a μLCRAγψβδβ cosmid. This construct, containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into single cell mouse embryos. To detect the differences in developmental regulation of the human gamma-globin gene expression in the transgenic mice, we analyzed the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5 of both mutated and non-mutated transgenic mice by RNAse Protection Assay (RPA) and Real time PCR (RT-PCR). Levels of expression of murine alpha-globin mRNA were used as internal controls in the RPA experiment and levels of murine-GAPDH were used in RT-PCR. mRNA levels of human gamma-globin in fetal liver of transgenic mice containing mutation were clearly higher, as compared with control transgenic mice bearing cosmid construct with wild a type sequence gamma promoter. Additionally a nearly 10-fold increase in human gamma-globin expression was detected in fetal livers of HPFH transgenic mice at E13.5 compared to the yolk sac. Thus, our data indicate that the - 195 mutation is the unique cause of elevation of HbF in Brazilian HPFH, but the exact mechanism needs to be clarified.

Author notes

Disclosure: No relevant conflicts of interest to declare.