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.     

Physical mapping of the globin gene deletion in (delta beta (0)) -thalassaemia.

We have constructed a physical map of restriction endonuclease cleavage sites in the (delta (+) beta)-globin gene region in the DNA of patients with (delta beta(0))-thalassaemia. This map shows that a 10 kb deletion has occured in (delta beta (0))-thalassaemia to remove the entire beta-globin gene and the 3' portion of the delta-globin gene. The 5' terminus of the deletion is in the large intron of the delta-globin gene and the 3' terminus 1.8 kb to the 3'-side of the beta-globin gene. A similar deletion of about 7 kb has been described previously in the DNA of patients with Hb Lepore; the 5' terminus of the deletion is also in the delta-globin gene but the 3' terminus is in the beta-globin gene. Comparison of the foetal (gamma) globin gene expression in adults with (delta beta(0))-thalassaemia and Hba Lepore suggests that the 3' extragenic regions of the beta-globin gene contain DNA sequences involved in the regulation of gamma-globulin gene expression.     

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.     

A Chinese G gamma + (A gamma delta beta)zero thalassemia deletion: comparison to other deletions in the human beta-globin gene cluster and sequence analysis of the breakpoints.

A clone was isolated that contains the deletion junction region from an individual with a deletion associated with Chinese G gamma + (A gamma delta beta)zero thalassemia. A clone containing the normal DNA corresponding to the 3' breakpoint of this deletion was also isolated. Portions of these two clones were sequenced and compared to the region in the A gamma-globin gene where the 5' breakpoint occurs. This comparison reveals that the breakage and reunion event was nonhomologous and that it probably involved the insertion of 36-41 bases of DNA belonging to the L1 (KpnI) family of repetitive DNA. Genomic mapping revealed that the DNA on the 3' side of this deletion is closely linked in normal DNA to the 3' breakpoints of two different large deletions that are associated with hereditary persistence of fetal hemoglobin (HPFH). We cloned and mapped 35 kbp of normal DNA from this region (greater than 45 kbp downstream of the human beta-globin gene) that contains the 3' breakpoints of the Chinese thalassemia and the two HPFH deletions. An endogenous retrovirus-like element and several other repetitive sequences are located within this region. We show that the Chinese thalassemia deletion is greater than 80 kbp in length and differs in size from the two HPFH deletions by less than 6%. We also show that the Chinese thalassemia deletion is at least 40 kbp larger than several other deletions associated with a very similar phenotype.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

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.     

与胎儿珠蛋白基因持续表达有关的(A_γδβ)°—地贫纯合子及杂合子的分子鉴别

我们分子鉴别了一个缺失型中国(A_γδβ)°-地贫家系。先证者为这一缺失的纯合子,具有中度贫血症状。家系的另五个成员均为这一缺失的杂合子,其胎儿血红蛋白(HbF)为16—21％,接近或达到HPFH杂合子的HbF水平,并且几乎不表现贫血症状。限制性内切酶图谱分析证明了β-珠蛋白基因簇内的DNA顺序缺失,缺失的5′端点位于Aγ基因IVSⅡ内,3′端点在β-珠蛋白基因下游区远端,距HPFH-2的3′缺失端点上游区约11kb。缺失的总长度约为80kb。本文讨论了这一缺失导致胎儿血红蛋白在成人中持续活跃表达的可能机制。     

A G gamma type of the hereditary persistence of fetal hemoglobin with beta chain production in cis.

In a new subclass of G gamma HPFH which has been detected in a black family, beta A chains are produced in cis to the HPFH determinant (the G gamma-beta+ HPFH). No other instance of beta chain production in cis to HPFH has been reported. All individuals in this family are well even if Hb S is produced in trans to HPFH. Genetically, this new subclass requires a slightly smaller deletion in the gamma, delta, and beta complex of genes than do other forms of HPFH. It is speculated that a subclass (the G gamma-(G gamma A gamma)-beta+ HPFH) in which beta S chains are produced in cis to HPFH in conjunction with true beta S genes in trans may be responsible for "mild" cases of sickle cell anemia.     

Molecular characterization of a novel form of (A gamma delta beta)zero-thalassemia deletion with a 3'' breakpoint close to those of HPFH-3 and HPFH-4: insights for a common regulatory mechanism.

We have molecularly characterized a novel (A gamma delta beta)zero-thalassemia associated with increased synthesis of HbF in three members of a German family. The levels of HbF in the peripheral blood red cells of the heterozygotes ranged between 9.9% and 12.5% with a heterocellular distribution in the red cells, as detected by immunofluorescence. The mutation resulted from a deletion starting about 1.5 to 1.9 kb from the 3' end of the G gamma-gene and ending 27 +/- 0.5 kb 3' to the beta-globin gene. Thus, the total deletion is 52 +/- 0.5 kb. Its 5' breakpoint is similar to that of the previously described (A gamma delta beta)zero-thalassemias, while the location of the 3' breakpoint is placed very close to the 3' breakpoints of HPFH-4 and HPFH-3 deletions. The proximity of the 3' breakpoint of the German (A gamma delta beta)zero-thalassemia to those of HPFH-3 and HPFH-4 deletions raises the possibility that a common mechanism, such as the juxtaposition of an enhancer, might underlie the activation of the gamma-globin genes in these three mutants.     

An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene.

A 7.5 kb Hsu I restriction fragment of genomic DNA containing a beta-globin gene has been isolated from a patient doubly heterozygous for beta + thalassaemia and a delta beta (Lepore globin fusion gene. This fragment must be derived from the chromosome carrying the beta +-thalassaemia determinant. The gross structure of the cloned gene plus flanking sequences is indistinguishable from that of a normal beta-globin gene. Within in 1606 base-pair transcribed region of the gene there is only one nucleotide difference from the normal beta-globin gene sequence. This is a G leads to A replacement 21 nucleotides upstream from the 3' terminus of the small intron. This nucleotide lies within a 10 base-pair sequence repeated in an inverted configuration near the 5' terminus of the small intron. The nucleotide replacement may result in a precursor mRNA less amenable to RNA splicing than its normal counterpart.     

Analysis of an inversion within the human beta globin gene cluster.

We have cloned and sequenced the DNA from two regions of the defective beta-globin gene cluster from a patient with Indian A gamma delta beta thalassaemia, and confirmed the complex and unusual pattern of rearrangement involving two separate deletions (0.8 kb and 7.5 kb) the inversion of the 15.5 kb segment separating them, as previously proposed from gene mapping studies [1]. All four breakpoints occur within the transcribed region of the globin genes and at one junction are found six nucleotides of unknown origin. This unique rearrangement results in enhanced expression of the upstream fetal gene, and is therefore is pertinent to the localisation of any putative control region involved in the coordinate expression of fetal and adult genes.     

Demonstration of an enhancer activity in a fragment of DNA located at the 3' of the adult alpha globin gene in the duck

We found an enhancer element placed at the 3' side of the adult duck alpha A globin gene. The duck alpha globin gene cluster contains three genes from the 5' to 3' side: the pi embryonic gene, the alpha D minor adult gene and the alpha A adult major gene. We analyzed a 16 kb genomic domain extending from 2 kb upstream of the pi gene to 5 kb downstream of the alpha A gene. This enhancer is active in AEV transformed chicken erythroblasts. Its is inactive both in HeLa cells and in the human erythroid cells K562 which express only embryonic genes. These findings are discussed in relation to previous results concerning the duck beta globin enhancer located at the 3' side of the beta A globin gene.     

Hereditary persistence of fetal hemoglobin, beta thalassemia, and the hemoglobin delta-beta locus: further family data and genetic interpretations.

Three Negro kindreds with hereditary persistence of fetal hemoglobin (HPFH) alone and in combination with various other hemoglobin abnormalities including beta thalassemia are presented. Among 11 offspring of two women heterozygous for both HPFH and the delta chain mutation Hb B2, five inherited the HPFH gene and six inherited the Hb B2 gene. In another kindred, a man inferred to be heterozygous for both HPFH and Hb C had six children; three offsprivg obtained the Hb C gene and three the HPFH gene. Similarly, a woman heterozygous for both Hb S and HPFH transmitted the Hb S gene to one of her two children and the HPFH gene to the other. Thus among 19 offspring, no crossovers between the HPFH locus or the Hb delta-beta locus were observed. These and earlier data are compatible with deletion of the Hb beta and delta loci as the primary event to explain the genetic origin of HPFH. Genetic considerations indicate that the finding of a single person with a hematologically normal phenotype among offspring of heterozygotes for both the African type of HPFH and a Hb beta or Hb delta structural abnormality would invalidate the deletion model.