Absence of messenger RNA and gene DNA for β-globin chains in hereditary persistence of fetal hemoglobin

The relative amounts of α- and β-globin mRNA and globin gene DNA were measured in reticulocyte RNA and lymphocyte DNA of an individual with homozygous hereditary persistence of fetal hemoglobin whose red blood cells contain 100% fetal hemoglobin (Hb F: α₂γ₂). Molecular hybridization assays used as probes full-length DNA copies of human α- and β-globin messenger RNA. The results of these hybridization assays demonstrated the expected amounts of α-globin mRNA and gene DNA, but absence of β-globin mRNA and absence of β-globin gene DNA. In the individual studied, hereditary persistence of fetal hemoglobin is associated with total deletion of the β-globin structural gene.     

Synergistic effect of two β globin gene cluster mutations leading to the hereditary persistence of fetal hemoglobin (HPFH) phenotype

Co-inheritance of gamma and beta globin gene mutations in a compound heterozygous state is rare but of clinical interest as it provides an important data on understanding the HbF expression. Hematological analysis was carried out (Sysmex KX-21). F-cells were enumerated using flow cytometry. Beta globin gene was analysed by CRDB technique and by DNA sequencing. Gamma globin promoter region was sequenced and expression studies were carried out using real time Taqman assay. We report a family, where two inherited defects of the β globin gene cluster segregate. The proband and her sibling were compound heterozygotes for a novel ^Gγ promoter mutation and the 619 bp deletion a common Indian β thalassemia mutation. Molecular characterization revealed that the father (HbA₂ 5.1%, HbF 5.4%), proband (HbA₂ 3.6%, HbF 31.7%) and her brother (HbA₂ 3.9%, HbF 23.6%) were heterozygous for the 619 bp deletion. The mother (HbA₂ 2.1%, HbF 3.4%) had a normal β globin gene. As both the children showed high HbF levels, the γ globin gene work up was carried out. The ^Gγ-globin gene promoter analysis revealed that the mother and the two children were heterozygous for a 5 bp deletion -ATAAG (-533 to -529) that resides in the GATA binding site. These findings suggest that the 5 bp deletion in the ^Gγ globin promoter has a functional role in silencing the γ-globin gene expression in adults by disrupting GATA-1 binding and the associated repressor complex and results in the up-regulation of gamma globin gene expression. When co-inherited with β -thalassemia trait it leads to a phenotype of HPFH.     

Contribution of β-globin cluster polymorphisms to raise fetal hemoglobin levels in normal adults

Hereditary persistence of fetal hemoglobin (HPFH) is a group of genetically heterogeneous conditions characterized by continued expression of fetal hemoglobin (HbF) in adulthood. HPFH may be due not only to point mutations or large deletions in different regions of the cluster β globin, but also to variations in several polymorphic sequences in this cluster. The objective of this work was to evaluate effects of polymorphic markers within cluster β globin on HbF expression. For the purpose, we have explored in this first study of Tunisian HPFH four polymorphic regions of β globin cluster in 68 healthy adults (34 subjects with high levels of HbF and 34 with normal HbF levels). Our results showed that the increase of HbF levels is associated with the −158 Gγ C → T polymorphism, the TG₁₈CG₂CACG, TC TG₉AG TG₂CG₂ and TG₁₁CG₄ configurations in the second intron of Gγ gene and the −540 β (AT)₆T₉ and (AT)₇T₈ repeated sequences. Among the 34 subjects with raised levels of HbF, approximately 97% carried one or more of these six markers. This study suggests that there is a significant association between certain polymorphic configurations of the β globin cluster and the increase of HbF levels in healthy individuals.     

Heterocellular hereditary persistence of fetal hemoglobin (HPFH). Molecular mechanisms of abnormal γ-gene expression in association with β thalassemia and linkage relationship with the β-globin gene cluster

Summary We report a study of four families of Italian origin in which heterocellular HPFH is inherited linked to thalassemia over two or three generations. The HPFH + thalassemia carriers showed thalassemic blood pictures and elevated HbF and F-cell number without increase in the HbF/F-cell content. Association of this gene complex with a second thalassemia trait gives rise to a mild clinical picture characterized by 9–12 g/dl of mainly HbF in peripheral blood and no transfusion requirement. In two families, independent segregation of the HPFH or -thal trait was observed, and in one case the study of the DNA polymorphisms within the gene cluster indicated that the HPFH mutation lies outside that DNA region. In one family the coexistence of a polymorphic variant of the ^A chain (the ^AT chain) allowed us to demonstrate that the increased chain synthesis caused by the heterocellular HPFH determinant is directed by both chromosomes.     

A β-thalassemia mutant caused by a 300-bp deletion in the human β-globin gene

Summary Larger deletions are a rare cause of -thalassemia. We report a further instance of a deletion comprising about 300 bp in a female heterozygote. Exon 1, part of IVS-1 and the 5 -globin gene promoter region are lost.     

Hemoglobin F production in heterocellular hereditary persistence of fetal hemoglobin and its linkage to the β globin gene complex

Summary Some types of nondeletional heterocellular hereditary persistence of fetal hemoglobin (HPFH) appear to be caused by mutations in the globin gene cluster near the globin genes, while in other cases the condition is associated with a gene or genes outside the globin gene complex. We have used DNA probes for chromosome 11 markers to localize the HPFH determinant in a large Australian kindred with nondeletional heterocellular HPFH. In 13 of the 58 family members studied the Hb F levels are increased to between 1.8% and 7.9%, the Hb F being composed predominantly of ^A chains. All family members were typed for restriction fragment length polymorphisms detected by probes from the globin gene complex, and the nearby genetic markers D11S12, INS, and HRAS. Linkage analysis showed HPFH is closely linked to the globin gene cluster (confidence limits of 0,0.0-0.19), D11S12 (0, 0.0-0.23) and the insulin gene (0,0.0-0.11). These data and the chain composition are consistent with HPFH in this family being caused by a mutation within the globin gene cluster.     

Homozygotes for the hereditary persistence of fetal hemoglobin: The ratio of Gγ to Aγ chains and biosynthetic studies

Two sons of a previously reported Ghanaian homozygote for the hereditary persistence of fetal hemoglobin (HPFH) (Ringelhann et al., 1970) also are HPFH homozygotes. In addition, another unrelated adult Ghanaian homozygote has been detected. All of these Ghanaian homozygotes as well as three American Black HPFH homozygotes have the G gamma A gamma type of HPFH with a G gamma to A gamma ratio of about 3:2, in contrast to an Asiatic Indian homozygote who has the G gamma type. Globin chain synthesis in HPFH homozygotes is unbalanced, with a gamma/alpha ratio of 0.6 or less, whereas it is balanced in heterozygotes according to most reports.     

Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

A case of homozygous δ thalassemia not due to a deletion of the δ globin structural gene

We have used restriction endonucleases for mapping the δ globin gene within the genomic DNA obtained from an individual homozygous for δ thalassemia. The results of our analyses indicate that δ thalassemia is not due to a detectable structural gene deletion as found in α thalassemia, δβ thalassemia or hereditary persistance of fetal hemoglobin, but probably consist of molecular lesions similar to those found in the β^° or β⁺ thalassemias.     

The Cretan type of non-deletional hereditary persistence of fetal hemoglobin [Aγ–158C→T] results from two independent gene conversion events

We report a new type of non-deletional hereditary persistence of fetal hemoglobin that is due to a C→T transition at position –158, relative to the Cap site of the human Aγ-globin gene. This mutation was identified in three unrelated adult cases presenting slightly elevated levels of fetal hemoglobin (Hb F), i.e. 2.9–5.1%, and normal hematological indices. Our sequencing results, from both polymerase chain reaction-amplified and subcloned DNA fragments, indicate that the Aγ–158C→T mutation occurred by two independent gene conversion events in the three cases studied. In addition, hematological and molecular data, including restriction fragment length polymorphism haplotyping in the β-globin gene cluster, extended haplotype analysis inside the γ-globin gene region and routine analysis of three tandem repeat loci (D1S80, 3′-HVR/apoB and F8vWf), led us to conclude that the Aγ–158C→T mutation in one of the three cases occurred recently in the parental germ line (P=99.47%), representing the first example of a de novo gene conversion event identified in humans. Received: 10 November 1997 / Accepted: 10 February 1998     

Comparison of theα-globin gene cluster structure in Perissodactyla

Summary To investigate molecular evolution in a mammalian order with a comprehensive fossil record, we have constructed-globin-like gene cluster maps for members of the order Perissodactyla. Although the arrangement of genes is the same in the five Equidae examined, the tapir and rhinoceros differ from each other and the horse in the position and number of their genes, but not in the arrangement of their and genes. In contrast to morphological work, a dendrogram derived from restriction site maps associates the tapir with the horse rather than with the rhinoceros; however, this phylogeny is not statistically significant. Among the Equidae,Equus caballus emerges as an outgroup, in agreement with data from other disciplines.     

Heterogeneity in the molecular basis of three types of hereditary persistence of fetal hemoglobin and the relative synthesis of the Gγ and Aγ types of γ chain

Restriction endonuclease analyses of DNA from one Black ^GA-HPFH homozygote and four Black and one Indian ^GA-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the and genes. Two of the types are comparable to those characterized previously, but the third, which is present in the Indian heterozygote, shows a distinct difference in the size of the deletion. The 5 end point of the deletion in this type III ^GA-HPFH extends 0.5–1.0 kb beyond the 5 end point of one of the Black types of HPFH (type I). Each of the three types is associated with a distinct ratio between the ^G and the ^A chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III ^GA-HPFH, with 69.3% ^G chains, while the averages for the other types were 50.7% ^G (type I) and 32.3% ^G (type II).This research was supported in part by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution 0764 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912. P. K. Sukumaran was on leave from the Bai Jerbai Wadia Hospital for Children, Parel, Bombay, India.     

Generation of stable recombinant retroviruses containing the β-globin genes linked to complex regulatory elements by using transient transfection

A transient packaging method for a retroviral vector containing β-globin sequences, mediated by Ca₃(PO₄)₂ precipitation, generated stable recombinant β-globin retroviruses with intact viral genomes. We suggest the use of this method as an alternative means to overcome the production of β-globin encoding retroviruses with aberrant viral genomes using the routine packaging procedure.     

Population study of a sequence polymorphism in intron 2 of the human β-globin gene

The distribution of a nucleotide polymorphism in intron 2 of the -globin gene (IVS-2 nt 666 C > T was examined in populations in southern Germany and Cameroon. The allelic frequencies were 0.86 for T and 0.14 for C in southern Germany and 0.87 for T and 0.13 for C in Cameroon, respectively.