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In order to investigate the origin(s) of the mutation(s) leading to the beta S-globin gene in North American populations of African ancestry, we analyzed DNA polymorphisms in the beta-globin gene cluster in a large number of both beta A- and beta S-globin gene-bearing chromosomes in U.S. and Jamaican Blacks. We found 16 different haplotypes of polymorphic sites associated with 170 beta S-globin gene-bearing chromosomes. The three most common beta S haplotypes, which account for 151/170 of the beta S-globin gene-bearing chromosomes, are only rarely seen in the chromosomes bearing the beta A-globin gene in these populations (6/47). Two observations suggest multiple origins or interallelic gene conversion, or both, of the beta S mutation. First, the mutation is present in all three beta-globin gene frameworks. Second, the beta S haplotypes can be divided into four groups, each of which cannot be derived from any other by less than two crossing-over events. In summary, our observation of the beta S mutation on 16 different haplotypes in African populations can be best explained by (i) a number of simple recombination events 5' to the beta-globin gene and (ii) up to four independent mutations and/or interallelic gene conversions.

The present study was undertaken to determine whether a cAMP pathway mediates the mobility of CD3, CD4, and CD8 within the membrane. Crosslinking CD3, CD4, and CD8 with monoclonal antibody and anti-antibody induced rapid accumulation of intracellular cAMP, occupancy of cAMP receptors, and was temporally associated with the mobilization and directed movement of these molecules to a pole of the cell. This capping process could be partially inhibited in a dose-dependent manner by treatment of T cells with 2',5'-dideoxyadenosine, a ribose-modified adenosine analogue that binds to the P site of the catalytic subunit of adenylate cyclase and reduces adenylate cyclase activity. Furthermore, inhibition of cAMP-dependent endogenous phosphorylation of 17.5-kDa, 23/25-kDa, and 33.5-kDa bands in intact T cells by N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide, a cell-permeable inhibitor of cyclic nucleotide-dependent protein kinase, blocked the capping event. Data support the conclusion that crosslinking of CD3, CD4, and CD8 activates a cAMP-dependent pathway that mediates the mobilization and directed movement of these molecules. cAMP-dependent protein phosphorylation is an integral step leading to the capping process.

Polymorphism for a Hpa I restriction endonuclease site associated with about 60% of beta S genes in American Blacks allows exact prenatal diagnosis of sickle cell anemia by amniocentesis in 36% of couples at risk. In three families in whom exact diagnosis by Hpa I sites was impossible, we found analysis for the presence of polymorphic HindIII sites in the G gamma and A gamma intervening sequences would allow an exact prenatal diagnosis of sickle cell status in all three. In one of these families, the presence of an A gamma HindIII site in amniocyte DNA confirmed the diagnosis (sickle cell trait) made by synthetic studies using fetal erythrocytes obtained at fetoscopy. Studies of other Black families and individuals provide evidence for linkage disequilibrium in the G gamma-A gamma-delta-beta gene complex involving the four sites, G gamma HindIII, A gamma HindIII, beta S, and Hpa I, which span 33 kilobases (kb). Ten of 14 chromosomes bearing a beta S gene in a 7.6-kb Hpa I fragment contained a G gamma but not an A gamma HindIII site, whereas 16 of 16 chromosomes bearing a beta S gene in a 13-kb Hpa I fragment lacked both the G gamma and A gamma HindIII sites. Two-thirds of beta A-bearing chromosomes lacked both G gamma and A gamma sites, whereas one-third contained either the G gamma or both G gamma and A gamma sites. These data demonstrate that combined analysis of both Hpa I and HindIII polymorphisms and verification of their linkage phase should increase the fraction of couples for whom amniocentesis can provide an exact diagnosis of sickle cell status from 36% to greater than 80%.

Seven beta-thalassemia genes were characterized after they were identified as candidates for previously undescribed mutations based upon the close association of DNA polymorphism haplotypes in the beta-globin gene cluster with specific ethnic mutations. The molecular defect in four of these genes was identical, a frameshift deletion of four nucleotides (-CTTT) within codons 41 and 42. This gene represents a common Southeast Asian mutation shared by a Laotian beta-thalassemia gene, [framework 1 (FR1)], a Vietnamese (FR1), and two Chinese patients (FR3 Asian and FR1). The deletion has been observed previously in Chinese (FR1) and Asian Indians (FR2) and is an example of independent origins of the same molecular defect, possible interallelic gene conversion (as it is seen on two different beta-globin gene frameworks in Chinese), and mutant gene migration in the Asian countries. A second example of mutant gene migration was identified in an Iranian patient with a nucleotide insertion (G) between codons 8 and 9, the same mutation previously found in an Asian Indian in the same chromosomal background. The last two genes examined represent further strong evidence for independent origins of mutation. A C-to-T substitution at position -88 in an Asian Indian has been identified previously in an American Black on a different beta-globin gene framework, and a G-to-A transition at nucleotide 1 of intervening sequence 2 found in an American Black has been observed previously on a different chromosome background in Mediterraneans. This study suggests that there are not many common beta-thalassemia mutations remaining to be discovered. It also suggests that certain sequences in the beta-globin gene are relatively mutation sensitive.

To investigate whether recurrent mutation has contributed to the high frequency of the beta E-globin gene in Southeast Asia, we used the haplotypes at three polymorphic restriction sites within and to the 3' side of the beta-globin gene to predict the framework of 23 beta E-globin genes. These haplotypes suggested that beta E-globin genes are present in two different beta-globin gene frameworks. DNA sequence determination of one gene representing each framework demonstrated that the same mutation (GAG leads to AAG at codon 26) was present in both frameworks. Moreover, the frameworks differed at three nucleotide positions known to be polymorphic in Mediterraneans. These polymorphic sites are located 70 nucleotides to the 5' side of the beta E mutation and 382 and 1032 nucleotides to the 3' side of it. The existence of the beta E mutation in these two beta-globin gene frameworks can be explained by (i) recurrent mutation giving rise to beta E-globin, (ii) a double crossing-over event, or (iii) two single crossing-over events. Mathematical analysis suggests that the first alternative, recurrent mutation of G leads to A at the first nucleotide of codon 26, is most likely.