GENETIC CONTROL OF FETAL Y-GLOBIN IN ADULT RED CELLS

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Fetal hemoglobin (Hb F) is present in fetuses and newborn, and a small amount (less than 1%) is found in normal adults. The two types of Gamma chains of Hb F (Ggamma and Agamma) are found in a 70:30 ratio in most newborn and 40:60 in most adults. However, about 20% of Black SS patients have a high (60:40) ratio of G-gamma to A-gamma chains. A specific haplotype for polymorphic restriction endonuclease sites in the Beta-globin gene cluster is found only in SS individuals with G-gamma greater than or equal 50% of total Gamma chain; this suggests close linkage of a high G-gamma genetic determinant to the Beta-globin gene cluster. Three observations suggest that this determinant is near (and possibly 5' to), the G-gamma and/or A-gamma genes: 1) A subhaplotype, + - + +, for sites in the Gamma genes and near the PSBeta gene, is associated with high G-gamma in Black SS, in the Saudi Arabian form of SS, and in low HbF G-gamma-B+-HPFH, although the full haplotypes (for 7 sites) differ. 2) In the newborn baby, the hybrid gene -Ggamma Agamma-produces Agamma polypeptide at levels expected for the G-gamma gene. 3) A mutation 5' to the Ggamma gene is hypothesized as the cause of high Ggamma levels in high Hb F Ggamma-B+-HPFH. This proposal presents the hypothesis that DNA sequence variation near Ggamma and/or Agamma genes changes their relative rates of transcription, as the result of altered interactions of specific DNA sequences with adult erythroid cell factors. To test this, comparisons will be made of the high G-gamma haplotype of Black SS with a low G-gamma haplotye, and two types of high Ggamma HPFH (high and low HB F Ggamma-Beta+-HPFH) with a low Ggamma HPFH (Agamma-B+-HPFH), for the following: 1) DNA sequence of Ggamma and Agamma genes, including flanking regions; this will also be done for three other high Ggamma haplotypes - two seen in Black Beta-thalassemia, and that of Saudi SS. 2) Levels of mature and precursor Ggamma and Agamma mRNA, in reticulocytes and bone marrow cells, and the extent of methylation and DNase I hypersensitivity of DNA of the latter. 3) mRNA expression of cloned Ggamma and Agamma genes in stably-transfected non-erythroid (L) cells and erythroid (mouse erythroleukemia) cells. 4) The effect of in vitro alterations of DNA sequence on gamma-gene expression of cloned DNA, to confirm the effects on expression of mutations found in aim #1. Data obtained in testing this hypothesis will provide important information on the nature of genetic determinants controlling adult G-gamma:A-gamma ratios and HB F levels.