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Spanish delta beta zero-thalassemia, a mild thalassemic condition characterized by increased level of hemoglobin (Hb) F production during adult life, is known to be due to a large deletion starting within the beta globin gene cluster and extending beyond the 3' breakpoint of any other similar deletional defects so far identified. By molecular cloning and by genomic mapping we now demonstrate that the deletion of Spanish delta beta zero-thalassemia ends at approximately 11 and 17 kilobases (kb) downstream to the 3' endpoints of black hereditary persistence of fetal hemoglobin (HPFH) type 1 and 2, respectively. As suggested by the complete characterization of this and other deletional defects involving the beta globin gene cluster, the 5' and 3' breakpoints of several deletions cluster in rather restricted DNA areas, further strengthening the idea that common molecular mechanisms may operate in causing these deletions.

We report that bromodeoxyuridine (BrdU) addition in semi-solid cultures of normal adult erythroid progenitors causes a sharp rise of gamma- globin gene expression in erythroid colonies. Control studies were carefully carried out to exclude the possibility of toxic effects exerted by the drug in these experimental conditions. In particular, BrdU addition induces a sharp increase in the level of relative gamma- globin synthesis and content in pooled BFU-E-derived colonies: this rise is clearly observed in single bursts of the mature type (largely composed of late erythroblasts) but not in immature ones (essentially comprising early erythroblasts). Furthermore, it is associated with an increase of the G gamma/G gamma + A gamma synthetic ratio from adult up to fetal like values. Reactivation of gamma-synthesis was observed even if BrdU was added to colonies composed essentially of early erythroblasts, ie, when BrdU was added to either bursts at day 10 of culture or late CFU-E-derived clones at day 1. These in vitro observations indicate modulation of gamma-synthesis at the stage of erythroblasts from normal adults. At the molecular level we suggest that BrdU, by replacing thymidine in DNA, may inhibit the switch from a fetal-like biosynthetic program expressed in early erythroblastic differentiation to the adult program expressed in later stages of maturation.

Restriction enzymes analysis of the DNA from two unrelated Italian families with Hb Hasharon, a variant Hb (α 47asp → his) frequently occurring in the Polesine area in Italy, indicates that this variant is associated to an α globin gene deletion. The αHashgenotype most likely results from a mutation on an α thal2genotype.

Restriction enzymes analysis of the DNA from two unrelated Italian families with Hb Hasharon, a variant Hb (alpha 47asp replaced by his) frequently occurring in the Polesine area in Italy, indicates that this variant is associated to an alpha globin gene deletion. The alpha Hash genotype most likely results from a mutation on an alpha thal2 genotype.

Isoelectric focusing, cellulose acetate electrophoresis, and carboxymethylcellulose chromatography in the presence of Nonidet P-40 allow the separation of pure gamma chains into two fractions. Amino acid analysis of their cyanogen bromide fragment 3 (gamma CB3) identifies these fractions as the separated G gamma (Gly-136) and A gamma (Ala-136) globin chains. Fingerprint and amino acid analyses of the gamma Tp9 tryptic peptide from the purified A gamma and G gamma fractions from two different patients demonstrate that the commonly occurring gamma Sardinia variant (gamma 75 isoleucine leads to threonine), also known as T gamma chain, has alanine in position 136. From this analysis we suggest that the T gamma gene is an allele of the A gamma locus (A gamma Sardinia) rather than a third gamma locus.

In cases of β0- thalassaemia from Ferrara, Italy, the β-globin gene is transcribed into mRNA but no protein is synthesised. For these cases there is no hybridisation data suggesting a globin gene structural mutation. This again demonstrates the diverse molecular events which may cause this prevalent hereditary disease.

The GATA-1 gene encodes a transcription factor expressed in early multipotent haemopoietic progenitors, in more mature cells of the erythroid, megakaryocytic and other lineages, but not in late myeloid precursors; its function is essential for the normal development of the erythroid and megakaryocytic system. To define regulatory elements of the mouse GATA-1 gene, we mapped DNaseI-hypersensitive sites in nuclei of erythroid and haemopoietic progenitor cells. Five sites were detected. The two upstream sites, site 1 and site 2, represent a new and a previously defined erythroid enhancer respectively. The site 1 enhancer activity depends both on a GATA-binding site (also footprinted in vivo) and on several sites capable of binding relatively ubiquitous factors. A DNA fragment encompassing site 1, placed upstream of a GATA-1 minimal promoter, is able to drive expression of a simian virus 40 (SV40) T-antigen in the yolk sac, but not bone-marrow cells, obtained from mice transgenic for this construct, allowing in vitro establishment of immortalized yolk-sac cells. A similar construct including site 2, instead of site 1, and previously shown to be able to immortalize adult marrow cells is not significantly active in yolk-sac cells. Sites 4 and 5, located in the first large intron, have no enhancer activity; they include a long array of potential Ets-binding sites. MnlI restriction sites, overlapping some of the Ets sites, are highly accessible, in intact nuclei, to MnlI. Although these sites are present in all GATA-1-expressing cells studied, they are the only strong sites detectable in FDCP-mix multipotent progenitor cells, most of which do not yet express GATA-1. The data indicate that appropriate GATA-1 regulation may require the co-operation of different regulatory elements acting at different stages of development and cell differentiation.

During embryonic development there is a transition from embryonic and fetal to adult beta-type globin chains. The high-molecular-weight RNA found in nuclei from embryonic and adult human erythropoietic tissues, fetal liver, and bone marrow, have been investigated for the presence of gamma(fetal)- and beta(adult)-globin messenger RNA sequences by molecular hybridization. Unlike alpha- and beta-globin mRNA sequences, gamma-globin mRNA sequences are absent from both total and high-molecular-weight nuclear RNA isolated from adult bone marrow. The amount of cytoplasmic gamma-globin mRNA is proportional to the level of gamma-chain synthesis, demonstrating that translational control is not a major control mechanism in the expression of globin genes. Since the gamma-, delta-, and beta-globin genes are known to be closely linked genetically, transcriptional control can discriminate between similar gene sequences that are spatially adjacent to one another.

Gamma-globin chain synthesis has been evaluated in individual bursts and subcolonies that were generated by normal adult blood BFU-Es in methylcellulose cultures containing semipurified erythropoietin (Ep) and then analyzed via either isoelectric focusing (IEF) of globin chains or immunofluorescence techniques. At variance with previously reported results, based on plasma clot culture and immunofluorescence, all bursts and subcolonies analyzed synthesize γ-globin chains. Identification of γ-chains has been confirmed by preparative IEF of HbF, followed by either carboxymethylcellulose chromatography or IEF analysis of the resulting globin chains. In all bursts analyzed, the relative synthesis of the two types of γ-globin chains (Gγ and Aγ) shows an adult ratio (i.e., approximately 1:1). The results obtained via IEF have been confirmed by immunofluorescence studies, which apparently showed presence of at least some HbF-positive cells within all scrutinized bursts or subcolonies. The significance of these studies is discussed in the light of current hypotheses on mechanism(s) underlying HbF synthesis in normal adults.

Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene.

During a study of Sardinian families with hereditary persistence of fetal hemoglobin (HPFH), two unrelated subjects with unusually elevated Hb F levels were identified. By selective amplification of the Aygene promoter and hybridization to synthetic oligonucleotides, we demonstrate that these subjects are homozygous for the -117 Aγ G → A substitution that is responsible for a form of nondeletional HPFH. The hemoglobin synthetic pattern of these patients is discussed.© 1989 by Grune & Stratton, Inc. 0006-4971/89/7307-0033$3.00/0

THE thalassaemias are a group of inherited disorders characterised by the defective production of either α(αthalassaemias) or non-α(βand δ°β° thalassaemias) globin chains of haemoglobins (Hb)1. In βthalassaemias the decreased synthesis of β-globin chains is only partially compensated by the increased production of γchains, which probably reflects2the massive hypertrophy of the erythron with selective survival of the clones of adult haemoglobin F-producing cells (F cells3,4). The situation is very different in other genetic disorders of the non-αgene cluster, known as δ°β° thalassaemias and Negro type of hereditary persistence of fetal haemoglobin (HPFH). In these two forms there is a genuine increase of γ-chain production, as shown by the high level of HbF found in heterozygotes. Although a clearcut distinction from both the clinical and haematological point of view cannot be traced between these two forms, the HPFH differs from the δ°β° thalassaemia in having a higher degree of γ-chain synthesis and a more homogeneous distribution of HbF within red cells. Recently, it has been possible to carry out gene analysis on DNA prepared from β°, δ°β° thalassaemic and HPFH patients. The β-globin gene is present in β° thalassaemias5–9, but in δ°β° thalassaemias and HPFH a major deletion, possibly involving both δand βgenes, has been demonstrated by hybridisation studies8,10–12. To characterise the molecular defect in these genetic disorders more precisely, we have hybridised DNA from homozygotes with HPFH, δ°β° thalassaemia and Hb Lepore disease (in which non-α-chains are a δβfusion product13). For this we used a pure full-size cDNAβprobe and specific 5′ end and 3′ end cDNA fragments (we designate as 5′ end cDNA the portion corresponding to the 5′ end of the mRNA; the same for the 3′ end). Our results, reported here, show that in contrast to HPFH, where a complete δand βgene deletion occurs, in δ°β° thalassaemia a 5′-end fragment of the δgene is present.

Summary A survey of hemoglobinopathies in northern Sardinia revealed a high frequency (0.3%) of carriers of a hematologic condition characterized by increased expression of fetal hemoglobin during adult life (hereditary persistence of fetal hemoglobin or HPFH). In spite of a normal hematologic phenotype, the heterozygous carriers for this condition display about 12% HbF, almost exclusively of the Aγ type; compound heterozygotes with β-thalassemia have 20%–26% HbF and run a very mild clinical course. The sequence analysis of the cloned Aγ gene linked to the HPFH determinant revealed the presence of a G→A substitution at position-117 of the Aγ- gene promoter; the same mutation occurs also in Greek HPFH, although associated with different restriction polymorphisms. Another hereditary condition characterized by increased HbF (α2Aγ2) level and a mild thalassemic phenotype in Sardinia is associated with the-196 C→T substitution in the Aγ-globin gene promoter (Sardinian δβ-thalassemia). Population studies using oligonucleotides complementary both to the-117 G→A and-196 C→T mutations and the corresponding normal sequences confirm the presence of these mutations only in HPFH and δβ-thalassemia chromosomes and exclude these changes being common DNA polymorphisms.

Erythroid bursts from cord or adult blood were grown in methylcellulose cultures (3 international units of erythropoietin per plate). On day 13, single bursts were picked up and reincubated for 16-24 hr with [3H]leucine. Radioactive globin chains [alpha,beta,G gamma, and A gamma (Ala-136)] were analyzed by either isoelectric focusing on polyacrylamide gels and fluorography or carboxymethylcellulose chromatography. In all cases, alpha to non-alpha globin radioactivity ratios were close to 1. In single cord blood bursts, the values of both gamma-to-beta and G gamma-to-A gamma ratios were spread over a large spectrum and further characterized by a continuous rather than a bimodal distribution. Morever, the G gamma-to-A gamma ratios demonstrated in single bursts appeared to be directly correlated with the respective gamma-to-beta ratios. These data suggest that both the gamma leads to beta and the G gamma leads to A gamma switches are mediated via mechanisms modulating the relative activities of the different genes in the non-alpha globin gene cluster rather than via selection of clones committed to the preferential synthesis of beta and A gamma globins. In contrast with the results obtained with cord blood, individual adult blood bursts synthesize a lower and hence relatively more uniform amount of gamma globin chains.

The hematological phenotypes of several Mediterranean patients with delta beta-thalassemia and hereditary persistence of fetal hemoglobin have been characterized. Although clinical and hematological characteristics are essentially superimposable in all heterozygous delta beta-thalassemics, these patients show typical G gamma/A gamma ratios in their Hb F, ranging from approximately 0.07 in Sardinian to approximately 0.15 in Sicilian and approximately 0.35 in Spanish patients. A gamma Sardinian-(isoleucine-75 leads to threonine) is found in Spanish patients and accounts for all of the A gamma production in heterozygotes, indicating that persistent production of gamma chains occurs cis to the delta beta-thalassemia gene. The molecular heterogeneity of these conditions is demonstrated by restriction enzyme mapping of DNA; Sicilian and Calabrian patients show a deletion starting from the delta-globin intron and extending several kilobases 3' to the beta-globin gene; in Spanish patients the deletion starts approximately 2-3 kilobases 5' to the delta-globin gene and extends well beyond the beta-globin gene. Comparison of these deletions with previously described ones in Negro and in a new Southern Italian case of hereditary persistence of fetal hemoglobin suggests that the deletion of a region centered at a cluster of repetitive sequences approximately 3.5 kilobases 5' to the delta-globin gene may be critical for the persistent expression of high levels of gamma-globin in hereditary persistence of fetal hemoglobin compared to delta beta-thalassemia. The concept that the deletion or mutation of specific areas (rather than nonspecific changes brought about by large deletions in the globin cluster) is important in determining the persistent expression of gamma-globin genes is supported by the finding of a nondeletion type of delta beta-thalassemia in Sardinians.

Spanish δβ°-thalassemia, a mild thalassemic condition characterized by increased level of hemoglobin (Hb) F production during adult life, is known to be due to a large deletion starting within the β globin gene cluster and extending beyond the 3’ breakpoint of any other similar deletional defects so far identified. By molecular cloning and by genomic mapping we now demonstrate that the deletion of Spanish δβ°-thalassemia ends at approximately 11 and 17 kilobases (kb) downstream to the 3’ endpoints of black hereditary persistence of fetal hemoglobin (HPFH) type 1 and 2, respectively. As suggested by the complete characterization of this and other deletional defects involving the β globin gene cluster, the 5’ and 3’ breakpoints of several deletions cluster in rather restricted DNA areas, further strengthening the idea that common molecular mechanisms may operate in causing these deletions.

Thermosensitive (dna) mutants of Bacillus subtilis defective in deoxyribonucleic acid replication can be divided into two groups on the basis of their ability to spontaneously yield secondary mutants with an HDS phenotype (thermoin-sensitivity and resistance to aryl-azo-pyrimidines) at frequencies higher than 10(-8). Such a phenotype is due to alleles at the hds locus (mapping close to cysA), which act as extragenic pleiotropic suppressors. HDS suppressibility has been used as a screening tool to identify new dna strains among uncharacterized temperature-sensitive mutants.

We report a new type of deletion of the 0 globin gene cluster in the Italian population that confers a phenotype of hereditary persistence of fetal hemoglobin (HPFH) to the carriers. This deletion begins ~5 kilobases (kb) 5’ to the δglobin gene and ends ~30 kb 3’ to the ß globin gene, in close proximity to the 3’ end of an Indian HPFH. In all four previously described HPFH, a repetitive Alu I region 5’ to the δglobin gene is largely or completely deleted; the 5’ end of the new HPFH is consistent with this common feature. In addition, the finding that Italian and Indian HPFHs, as reported for other groups of deletions, have very close 3’ ends, strengthens the idea that common mechanisms may operate in generating these deletions. Finally, we show that, in spite of similar 5’ breakpoints, the deletion of Spanish δß°-thalassemia is at least 8 kb longer than that of Negro HPFH type I, thus ruling out the hypothesis that the overall extent of the deletion might influence the level of γ globin chain synthesis.