Detection of a major gene for heterocellular hereditary persistence of fetal hemoglobin after accounting for genetic modifiers.

"Heterocellular hereditary persistence of fetal hemoglobin" (HPFH) is the term used to describe the genetically determined persistence of fetal hemoglobin (Hb F) production into adult life, in the absence of any related hematological disorder. Whereas some forms are caused by mutations in the beta-globin gene cluster on chromosome 11, others segregate independently. While the latter are of particular interest with respect to the regulation of globin gene switching, it has not been possible to determine their chromosomal location, mainly because their mode of inheritance is not clear, but also because several other factors are known to modify Hb F production. We have examined a large Asian Indian pedigree which includes individuals with heterocellular HPFH associated with beta-thalassemia and/or alpha-thalassemia. Segregation analysis was conducted on the HPFH trait FC, defined to be the percentage of Hb F-containing cells (F-cells), using the class D regressive model. Our results provide evidence for the presence of a major gene, dominant or codominant, which controls the FC values with residual familial correlations. The major gene was detected when the effects of genetic modifiers, notably beta-thalassemia and the XmnI-G gamma polymorphism, are accounted for in the analysis. Linkage with the beta-globin gene cluster is excluded. The transmission of the FC values in this pedigree is informative enough to allow detection of linkage with an appropriate marker(s). The analytical approach outlined in this study, using simple regression to allow for genetic modifiers and thus allowing the mode of inheritance of a trait to be dissected out, may be useful as a model for segregation and linkage analyses of other complex phenotypes.     

A novel C-T transition within the distal CCAAT motif of the G gamma-globin gene in the Japanese HPFH: implication of factor binding in elevated fetal globin expression.

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by the continued expression of the fetal globin gene in adulthood. Both deletional and nondeletional forms have been described. We studied one Japanese family with two different nondeletional forms of HPFH. Analysis of polymorphic restriction sites in the beta-globin gene cluster suggested that one affecting both G gamma and A gamma globin expression in two members of the family could be associated with unknown conditions not linked to the beta-globin gene loci. Characterization by the polymerase chain reaction (PCR) of another form producing a G gamma-HPFH phenotype in two other members demonstrated a novel C-T transition at the nucleotide -114 within the distal CCAAT motif of the G gamma-globin gene. Using gel retardation assays on various nuclear extracts, we also demonstrated that this novel mutation abolishes the binding of the ubiquitous CCAAT binding factor, CP1 to the distal CCAAT motif of the gamma-globin gene but does not affect the binding of any erythroid specific factor, thereby suggesting a possible role for CP1 in the developmental regulation of fetal globin expression.     

Physical mapping of the globin gene deletion in hereditary persistence of foetal haemoglobin (HPFH).

We have mapped the globin gene region in the DNA of two HPFH patients. In a patient homozygous for the G gamma A gamma type of HPFH at least 24 kb of DNA in the globin gene region has been deleted to remove most of the gamma-delta intergenic region and the delta and beta globin genes. The 5' break point of the deletion is located about 9 kb upstream from the delta globin gene. The 3' break point has not been precisely located but is at least 7 kb past the beta globin gene. DNA from an individual heterozygous for the Greek (A gamma) type of HPFH, however, shows no detectable deletion in the entire gamma delta beta-globin gene region. HPFH, therefore, appears to occur in different molecular forms. These results are discussed in terms of a model for the regulation of globin gene expression in man.     

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.     

A novel deletion in delta beta-thalassemia found in Japan

High molecular weight DNA from a Japanese individual homozygous for delta beta-thalassemia was analyzed by the blot hybridization technique of Southern. Results indicated a large deletion of the non-alpha-globin gene cluster, starting in the vicinity of 3' to the A gamma-globin gene and extending through the 3' side of the beta-globin gene. Persistent expression of the gamma-globin gene in adult life has been supposed to be caused by loss of a region located about 3-4 kb 5' to the delta-globin gene from comparison of the extents of deletions in several different forms of delta beta-thalassemia and HPFH (hereditary persistence of fetal hemoglobin). But the novel deletion found in the present case of delta beta-thalassemia suggests that the above putative regulatory region does not have this effect on expression of the gamma-globin gene. Some explanations of expression of fetal type globin genes in this delta beta-thalassemia are discussed.     

A gene controlling fetal hemoglobin expression in adults is not linked to the non-alpha globin cluster.

The possible linkage between a gene causing heterocellular hereditary persistence of fetal hemoglobin (HPFH) and human non-alpha globin loci has been studied in a large Sardinian family. In this family a homozygous beta o-thalassemic patient was found, with an unusually mild form of this disease, which was ascribed to the co-existence of a gene causing heterocellular HPFH. DNA polymorphisms in the non-alpha globin cluster were analyzed by restriction enzyme digestion with HincII, HindIII and BamHI and with epsilon-, gamma-and beta-globin probes; the pattern of inheritance of these polymorphisms indicates that the HPFH gene is transmitted with one beta o-thalassemic gene in a single instance, with the second beta o-thalassemic gene in three instances and with a normal beta-globin gene in two cases. These data indicate that this HPFH gene is not linked to the non-alpha globin gene cluster, in contrast to previous observations with different HPFH genes, and suggest that this gene might code for diffusible substances acting, directly or indirectly, on gamma-globin gene expression.     

Translocation of an erythroid-specific hypersensitive site in deletion-type hereditary persistence of fetal hemoglobin.

Hereditary persistence of fetal hemoglobin (HPFH) can involve large deletions which eliminate the 3' end of the beta-like globin gene cluster and more than 70 kilobases (kb) of flanking DNA. Blot hybridization revealed a DNase I-hypersensitive site extending from 1.1 to 1.4 kb downstream of the HPFH-1 3' deletion endpoint. The site was found in normal fetal and adult nucleated erythroid cells and in two erythroleukemia cell lines but not in nonerythroid cells and tissues. Simian virus 40 core enhancer-like sequences were found nonrandomly distributed within the boundaries of the site, which is contained in a fragment of known enhancer activity (E. A. Feingold and B. G. Forget, Blood, in press). A second hypersensitive site was found 0.5 kb upstream of the HPFH-1 3' deletion endpoint but was not erythroid specific. A third site, most prominent in fetal liver-derived erythroid cells, was found 1 kb upstream of the HPFH-2 deletion endpoint. As predicted by the locations of the deletion endpoints, the first two sites were translocated to within 12 kb of the A gamma gene in erythroid colonies derived from an HPFH-2 heterozygote and in hybrid mouse-human erythroid cells carrying the HPFH-2 deletion chromosome. Further analysis of this region showed that it was DNase I sensitive in erythroid and myeloid cells, indicating that it resides in an open chromatin domain. These observations suggest that alterations of chromatin structure flanking the fetal globin genes may contribute to abnormal gene regulation in deletion-type 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.     

Analyses of linked beta-globin genes suggest that nondeletion forms of hereditary persistence of fetal hemoglobin are bona fide switching mutants.

The analysis of nondeletion forms of hereditary persistence of fetal hemoglobin (ndHPFH) has led to the identification of cis-acting elements, located in the promoter regions of the fetal genes, that appear to be involved in the process of fetal to adult hemoglobin switching. Individuals with these disorders demonstrate elevated levels of fetal hemoglobin and lowered levels of adult hemoglobin during adult life. This phenotype could be either the result of an abnormality in the switching process or the result of two independent mutations: one mutation increasing the level of fetal (gamma) gene expression and another mutation decreasing the level of adult (beta) globin gene expression. Here we demonstrate that the adult beta genes linked to two different forms of ndHPFH, G gamma beta + HPFH and Greek ndHPFH, produce normal levels of correctly processed mRNA in transient-expression systems. We also report that the nucleotide sequences of the beta genes are normal. These results indicate that these gamma gene promoter mutations are linked to functionally normal beta-globin genes and are consistent with the hypothesis that these mutations interfere with the normal switching process.     

野生型及启动子区突变的HPFHAγ基因在MEL细胞中的表达

研究了野生型Ａγ珠蛋白基因、中国型胎儿血红蛋白持续存在综合症（ＨＰＦＨ）Ａγ基因（启动子区─１９６Ｃ→Ｔ突变）及希腊型ＨＰＦＨＡγ基因（─１１７Ｇ→Ａ突变）在鼠红白血病（ＭＥＬ）ＧＭ９７９细胞中的表达。比较每个整合的Ａγ基因表达的ｍＲＮＡ量,─１９６Ｃ→Ｔ突变Ａγ基因未显示比野生型Ａγ基因明显增加的表达水平,而─１１７Ｇ→Ａ突变Ａγ基因在未分化的及ＨＭＢＡ或丁酸钠诱导分化的ＭＥＬＧＭ９７９细胞中的表达水平增加到野生型Ａγ基因相应水平的２．３倍至３．１倍。此结果提供了又一个实验证据,说明启动子─１１７Ｇ→Ａ突变是希腊型ＨＰＦＨ中胎儿Ａγ基因在成人中持续活跃表达的原因,这也为β地中海贫血和镰刀性贫血的基因治疗提供了新途径,即可以考虑用转移─１１７Ｇ→Ａ突变Ａγ基因的方法改善患者的贫血症状。     

Nucleotide sequence of the Belgian G gamma+(A gamma delta beta)0-thalassemia deletion breakpoint suggests a common mechanism for a number of such recombination events

Various types of thalassemia or hereditary persistence of fetal hemoglobin (HPFH) are caused by deletions at the human beta-globin gene cluster. Many of these molecular lesions show a clear clustering as far as size and location of their breakpoints are concerned. This might indicate common recombination mechanisms responsible for the generation of these deletions. The Belgian G gamma+(A gamma delta beta)zero-thalassemia results from a large deletion spanning the beta-globin gene cluster 3' of the A gamma gene. The extent of this deletion, analyzed by field-inversion gel electrophoresis, is approximately 50 kb and is very similar to that of the Indian HPFH (G gamma A gamma HPFH III) previously characterized by P. S. Henthorn et al. (1986). Proc. Natl. Acad. Sci. USA 83: 5194-5198. Isolation of the deletion junction of the Belgian G gamma+(A gamma delta beta)zero-thalassemia by means of inverse polymerase chain reaction confirmed a very close relationship between these two independent deletions. The 3' breakpoint of the Belgian deletion is located at the midpoint of a 160-bp palindrome, only four nucleotides 5' from the correspondent endpoint of the Indian HPFH.     

HbF in the adult: could its composition discriminate normal from abnormal foetal globin gene expression?

The relative proportions of the two gamma chain species (G gamma and A gamma) have been reinvestigated in newborns, during the physiological switch from foetal to adult haemoglobin, and in adults with some persistent expression of HbF. In newborns, with about 80% HbF, the G gamma percentage was close to 70% while in adult RBC, with less than 0.5% HbF, the G gamma chain was almost non-detectable and may reflect the completion of the foetal to adult switch. Conversely, in adult patients with HbF above 0.6%, usually accompanying some degree of marrow stimulation, the relative ratio of G gamma varied between 40 and 60%, independently of HbF level. This ratio corresponds to what has been described in the literature as being the adult type of HbF. In all the cases where we found higher levels of G gamma, the results could be explained by the presence of a specific genetic background such as the Senegalese haplotype in sickle cell disease.     

δβ-Thalassemia is due to a gene deletion

DNA has been prepared from peripheral blood or cultured skin fibroblasts obtained from three Sicilian and one Greek δβ-thalassemia homozygotes. Globin-gene analysis was carried out using a cDNA_β probe, and the results indicate that δβ-thalassemia has arisen from a deletion of the β-globin genes. A similar result was obtained using DNA prepared from cultured skin fibroblasts from an individual homozygous for the Negro form of hereditary persistence of fetal hemoglobin (HPFH). In both cases, the deletion has spared the ^Gγ and ^Aγ loci directing the γ chains of hemoglobin F, but it has not been possible to demonstrate any difference between the size of the deletion involved in the production of δβ-thalassemia and that which gave rise to HPFH. These experiments provide further direct evidence that deletions of critical areas of the γ-δ-β gene cluster result in persistent γ chain synthesis in adult life.     

High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin.

Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.     

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.