Molecular cloning of the breakpoints of the hereditary persistence of fetal hemoglobin type-6 (HPFH-6) deletion and sequence analysis of the novel juxtaposed region from the 3′ end of the β-globin gene cluster

Abstract: Naturally occurring deletion mutations within the human β-globin cluster lead to specific, phenotypically discrete syndromes (i.e., δβ-thalassemias and hereditary persistence of fetal hemoglobin, HPFH), characterized by increased production of fetal hemoglobin in adult life. We have previously characterized an enhancer element, which is juxtaposed to the fetal Gγ-gene, by means of a deletion first described in a Thai family. To obtain further insights into the mechanisms involved in this deletion, we have now characterized several of its novel features. Following amplification by the polymerase chain reaction and sequencing of the 1.5-kb bridging fragment, we have shown that the 5′ breakpoint of the deletion occurs 1260 bp 3′ of the fetal Gγ-globin gene, whereas the 3′ breakpoint lies 521 bp upstream of the EcoRI site of the enhancer element and 2845 bp upstream of the 3′ breakpoint of the Chinese (Aγδβ)°-thalassemia deletion. The total length of the deletion is 101 kb, which resembles that of HPFH-1 and HPFH-2 deletions and a set of two γδβ-thalassemia deletions. Our data further support the hypothesis that these sets of large deletions with almost identical lengths are generated via the loss of a complete chromatin loop. To elucidate further the mechanisms leading to the deletion, we have sequenced the novel 0.5-kb region residing immediately 3′ to the breakpoint and shown that it contains putative binding sites for several transcription factors, such as HNF-1, AP-1, and TFIID. Sequence comparison of the deletion breakpoints reveals no junctional homology, indicating an end-to-end joining of blunted ends; a pair of 7-nt complementary repeats adjacent to a set of a direct CCCT repeat flanks the breakpoints. This limited homology constitutes a frequent characteristic of a non-homologous recombination mechanism. All these features of the HPFH-6 deletion suggest that this mutation has resulted from a non-homologous recombination event.