Two mutations in the locus control region hypersensitivity site‐2 (5′HS‐2) of haplotype 19 β 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 Arabian) have high fetal hemoglobin (HbF) levels (15 to 30% of total hemoglobin) whereas individuals homozygous for haplotypes 17 (Cameroon), 19 (Benin), and 20 (Bantu) have low HbF levels (1 to 10%). We previously identified several point mutations in the LCR 5′HS‐2 that were specific for haplotype 19 β s chromosomes (compared to the GenBank HUMHBB reference 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 mutations may alter the binding of specific trans ‐acting factors and ultimately affect the expression of HbF in these sickle cell patients. We performed gel mobility shift assays using 32 P‐end‐labeled double‐stranded 19mers corresponding to each of the LCR 5′HS‐2 normal (GenBank) and mutant sequences. Nuclear extracts prepared from HeLa and HEL cells were 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 Sp1; using purified protein and both nuclear extracts. HEL extracts were used to quantify the increase in Sp1 binding to the 8598 mutation and we found an increase in binding of 66 and 47%, respectively, in two shifted bands. The 9114 mutation sharply decreased binding of an unknown trans ‐acting factor by 74%. This factor was present in both HeLa and HEL nuclear extracts. © 1996 Wiley‐Liss, Inc.