No abstract

We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha- globin gene expression.

We describe a family in which alpha-thalassemia occurs in association with a deletion of 62 kilobases from a region upstream of the alpha globin genes. DNA sequence analysis has shown that the transcription units of both alpha genes downstream of this deletion are normal. Nevertheless, they fail to direct alpha globin synthesis in an interspecific hybrid containing the abnormal (alpha alpha)RA chromosome. It seems probable that previously unidentified positive regulatory sequences analogous to those detected in a corresponding position of the human beta globin cluster are removed by this deletion.

Fetal hemoglobin (HbF) levels vary widely among individuals with sickle cell anemia (SS). Previous studies have suggested that HbF levels in SS individuals with alpha-thalassemia (two or three functional alpha- globin genes) are lower than HbF levels in SS individuals with four normal alpha-globin genes. Using immunocytochemical techniques, we studied F cell production as measured by % F reticulocytes, the amount of HbF per F cell, and the preferential survival of F cells versus non- F cells in 51 subjects with four alpha genes, 32 subjects with three alpha genes, and 18 subjects with two alpha genes. Comparison between alpha-globin gene groups was performed for the total sample as well as for a subset of 82 individuals who had replicate samples and a further subset of 39 age-matched individuals. %HbF levels were 6.8, 4.9, and 4.5 percent for the total four-, three-, and two-alpha-globin-gene groups, respectively. The percentage of F reticulocytes, percentage HbF per F cell, and the enrichment ratio (% F cell/% F reticulocytes) did not change significantly with alpha-globin gene number. Moreover, no correlation existed between alpha-globin gene number and the absolute number of F cells in any group studied. However, there was a strong inverse correlation (r = -0.407, P = .0001) between non-F cell levels (1.7 +/- 2, 2.2 +/- 5, 3.0 +/- 1.0 X 10(12)/L) and decreasing alpha- globin gene number. These data suggest that falling HbF levels among SS individuals with lessened numbers of alpha-globin genes reflect prolonged survival of non-F cells and are not due to intrinsic differences in F cell production or in the amount of HbF per F cell. The improved survival of non-F cells in SS alpha-thalassemia is presumed to be due to the lower MCHC observed in such individuals.

zeta-Globin chain expression in carriers of a number of deletional alpha-thalassemias is investigated by radioimmunoassay. In a few cases, zeta-globin mRNAs are also studied. zeta-Globin chains are detected in (--SEA/), (--MED/), and (--SPAN/) deletions, but not in six other deletional mutations. These results suggest that the DNA element capable of suppressing zeta-globin expression in adult erythroid cells is present within the (--SPAN/) deletion, while the DNA fragment between the 5′ breakpoints of the (--SA/) and the (--SEA/) deletions may contain sequences necessary for augmenting zeta-globin expression in adult erythroid cells. Furthermore, zeta-globin chains are shown by an immunocytologic technique to be present in all circulating erythrocytes in carriers of the (--SEA/) and (--MED/) deletions. This simple immunocytologic test is highly sensitive and specific to detect adult carriers of either the (--SEA/) or (--MED/) deletions, and can be used for the detection of couples at risk of pregnancies involving fetuses with homozygous alpha-thalassemia.