2 MUTATIONS IN THE LOCUS-CONTROL REGION HYPERSENSITIVITY SITE-2 (5'HS-2) OF HAPLOTYPE 19 BETA(S) CHROMOSOMES ALTER BINDING OF TRANS-ACTING FACTORS

There are five major haplotypes associated with sickle cell anemia (SS ), Individuals homozygous for haplotypes 3 (Senegal) and 31 (Saudi Ara bian) have high fetal hemoglobin (HbF) levels (15 to 30% of total hemo globin) whereas individuals homozygous for haplotypes 17 (Cameroon), 1 9 (Benin), and 20 (Bantu) have low HbF levels (1 to 10%). We previousl y identified several point mutations in the LCR 5'HS-2 that were speci fic for haplotype 19 PS chromosomes (compared to the GenBank HUMHBB re ference sequence, T-->G at position 8580, A-->G at position 8598, and A-->T at position 9114). We postulated that one or more of these mutat ions may alter the binding of specific trans-acting factors and ultima tely affect the expression of HbF in these sickle cell patients, We pe rformed gel mobility shift assays using P-32-end-labeled double-strand ed 19mers corresponding to each of the LCR 5'HS-2 normal (GenBank) and mutant sequences, Nuclear extracts prepared from HeLa and HEL cells w ere used in our experiments and neither the normal nor mutant sequence at position 8580 bound trans-acting factors in either nuclear extract . The 8598 mutant increased binding of Spl; using purified protein and both nuclear extracts, HEL extracts were used to quantify the increas e in Spl binding to the 8598 mutation and we found an increase in bind ing of 66 and 47%, respectively, in two shifted bands, The 9114 mutati on sharply decreased binding of an unknown trans-acting factor by 74%, This factor was present in both HeLa and HEL nuclear extracts. (C) 19 96 Wiley-Liss, Inc.