RESUMO
BACKGROUND AND OBJECTIVES: Analysis of DNA polymorphic sites is a powerful tool for detection of gene flow in human evolutionary studies and to trace genetic background associated with abnormal genes. The beta-globin locus contains more than 20 single-base restriction fragment length polymorphism (RFLP) sites spanning over 80 kb on chromosome 11. Far downstream of the expressed genes, there is a hypersensitive site (HS). The function of the 3'-HS remains unknown. As an approach to the understanding of the 3'-HS region in sickle cell anemia we searched for sequence polymorphism in the AT-rich region, using a non-radioactive polymerase chain reaction (PCR)-single strand conformational polymorphism (SSCP) technique. DESIGN AND METHODS: A 460 bp fragment located at the 3' of the b globin gene was amplified from patients (with sickle cell anemia and HbSC disease), and from AS individuals. Standard RFLP-haplotyping was performed and compared with the PCR-SSCP screening strategy. RESULTS: Two distinct band patterns were revealed by SSCP testing, each one in strict linkage disequilibrium with either Benin or Bantu haplotypes. Direct sequencing of the amplified segment revealed a TAA insertion in the AT-rich region, in all 121 beta(S) Benin chromosomes tested, but not in other beta(S) haplotypes from the total of 380 beta(S) chromosomes typed. INTERPRETATION AND CONCLUSIONS: SSCP analysis could easily distinguish sequence variations in the 3'AT-rich region of the beta-globin cluster, and a TAA insertion in this region seems to be specific for the Benin-beta(S) chromosome.
