Elevated fetal hemoglobin (Hb F) ameliorates diseases of adult hemoglobin, such as sickle cell anemia (SS) and beta-thalassemia. This laboratory has demonstrated mutations associated with increased expression of gamma genes in vivo, at -158 (C to T ) and -161 (G to A) of the G gamma gene and -202 ( C to T) of A gamma. A 4 base pair deletion at -222 to -225 of the A gamma gene is associated with decreased A gamma expression in Mediterranean haplotype II betao-thalassemia. The Benin betaS haplotype is generally associated with low Hb F, but preliminary data show high Hb F determinants linked to it in certain families. Literature data suggest that the Asian BS (Saudi) haplotype may also be associated with unidentified high Hb- F determinants. Part of this proposal aims to clarify the relationship between haplotype and Hb F expression in SS and beta- thal. We will test the related hypotheses that certain occurrences of the Benin and Asian betaS haplotypes have closely-linked but unidentified high Hb F mutations. For selected cases (high Hb F Benin and Asian betaS), amplified genomic DNA will be sequenced in the G gamma and A gamma promoters. If no mutations are found, then the gamma enhancer will be tested in the Bodine & Ley expression system and sequenced if activity is elevated. We will also test the hypothesis that the 4 bp deletion is a major factor the severity of betao -thal by using oligonucleotide probes to screen mild and severe cases. The second part of this proposal will test the hypothesis that the -158, -161, -202 mutations and the 4 bp deletion act by altering affinity for transcription-regulating proteins. Our model suggests possible protein-binding sites at several sequence motifs, including """"""""TC"""""""" (198 to -192), """"""""G-C rich"""""""" (-208 to -200), SphI (- 226 to -219) and """"""""enhancer core"""""""" (-270 to -264). Mutant promoters will be placed 5' to a """"""""marked"""""""" gamma globin gene in the presence and absence of gamma enhancer, and expression tested in K562 erythroleukemia cells by mRNA assays. Deletions 5' to the -200 and -158 mutations will test the importance of 5' sequences to elevated expression of those mutations. Comparison of protein binding to these motifs in normal and mutant promoter fragments will be done by gel retardation assays. Competition between normal and mutant oligomers will show the effects of mutations on protein binding. Footprinting and methylation interference data will precisely define binding sites, test the hypothesis that the -158 and -161 mutations affect binding to known motifs, and aid in characterizing partially purified proteins of K562 and bone marrow erythroid cells. These data will contribute to the mapping of structure- function relationships of gamma globin transcription in red cells of human adults.
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