COMPARATIVE-STUDIES OF NONDELETIONAL HPFH GAMMA-GLOBIN GENE PROMOTERS

The -198 T-->C and the -175 T-->C transitions involving the proximal g amma-globin gene promoter are associated with the hereditary persisten ce of fetal hemoglobin (HPFH) phenotype and have been demonstrated to increase promoter activity in erythroid cells using transient and stab le transfection systems. The above base changes are thought to alter t he binding of different transcription regulatory proteins. Another mut ation of the proximal gamma-globin promoter, -158 C-->T, has been less clearly linked to the HPFH phenotype but has been associated with inc reased Gy activity. In the present paper, the -198 T-->C, -175 T-->C a nd -158 C-->T mutations both singly and in various combinations were e valuated by an in vitro expression assay. gamma-Globin promoters were transfected by electroporation into K562 human erythroleukemia cells a nd their activity measured in a human growth hormone (hGH) reporter ge ne assay. A novel cotransfectant was used to assess transfection effic iency. Results confirmed the previously reported upregulation of gamma -globin activity with the -198 T-->C and -175 T-->C HPFH mutations and a cooperative effect on promoter activity when both these mutations a re present in cis. No effect of the -158 C-->T mutation was seen eithe r alone or in combination with the -175 T-->C and -198 T-->C mutations .