The DNA polymorphisms of the beta-globin gene cluster and the arrangements of the alpha- and the gamma-globin genes in Koreans

The DNA polymorphisms at the seven restriction sites in the beta-globin gene cluster in healthy Koreans were examined using four restriction endonucleases, Hinc II, Hind III, Ava II, and Bam HI. Seven (f = 0.326) and four individuals (f = 0.246) were homozygous for [+-----+] and [+----+-], respectively, among 66 individuals examined. As to the subhaplotypes 5' to the delta-globin gene, 25 (f = 0.615) and three individuals (f = 0.213) were homozygous for [+----] and [-+-++], respectively. The frequency of [-++-+], which carries the A gamma T-globin gene, may be low in this population. It was recognized again that [+/+] at the Hinc II site 5' to the epsilon-globin gene always accompanied [--+--] at the Hinc II sites in and 3' to the psi beta 1-globin gene. The Korean, Japanese, and Chinese populations were not significantly different from each other in their haplotypes (subhaplotypes). The frequencies of the abnormal alpha- and gamma-globin gene arrangements in Koreans were low. alpha-Thalassemia may occur at low frequency in Koreans.     

DNA polymorphism in the beta-globin gene cluster in Saudi Arabs: relation to severity of sickle cell anaemia.

Significant DNA polymorphisms have been reported in the beta-globin gene cluster of epsilon-G gamma-A gamma-psi beta-delta-beta-gene region, in normal (Hb AA) individuals and in patients with sickle cell anaemia (SCA). Investigations of the extent of the DNA polymorphisms in the beta A- and beta S-globin gene cluster using Hind III, Hinc II, Ava II, Xmn I, and Hpa I, revealed several associations with mild SCA. The correlation of the presence (+) or absence (-) of the restriction endonuclease site to clinical severity in patients homozygous for beta S-gene showed that the mild form of SCA was associated mainly (> 90%) with the Xmn I polymorphic site 5' to G gamma, and to a lesser extent with Hinc II polymorphic site 5' to epsilon and in the psi beta-gene, with Hind III polymorphic site in G gamma and Hpa I polymorphic site 3' to the beta-globin gene, while in the severe form of SCA these polymorphic sites were absent in most patients. The polymorphism in the beta-globin gene cluster was significantly related to the expression of the beta S-gene and clinical severity of SCA.     

Diversity of human gamma-globin gene loci including a quadruplicated arrangement

A quadruplicated gamma-globin gene as a (5'-G gamma-gamma-gamma-A gamma- 3') was detected in an adult Chinese during a survey designed to detect G gamma- and A gamma-globin genes in Japanese. Five triplicated (-G gamma-gamma-A gamma-) and two single (-gamma-) haplotypes were also detected in 103 healthy adult Japanese. All of the unusual chromosomes appeared to reflect an unequal but homologous crossover between G gamma- and A gamma-globin genes. A new Bgl II polymorphic site located around the 3' terminal region of the G gamma-globin gene was also discovered.     

The Xmn I site (-158, C----T) 5' to the G gamma gene: correlation with the Senegalese haplotype and G gamma globin expression.

There are three major African haplotypes associated with the sickle mutation: Benin (#19), Senegalese (#3), and Central African Republic (#20). Previous studies have suggested that the Xmn I site (-158 bp 5' to the G gamma gene) is associated with elevated levels of G gamma and with the Senegalese haplotype, while other investigators questioned this association. In order to clarify the issue, we have determined beta haplotypes, tested for the presence of the Xmn I site, and measured Hb F and G gamma expression levels in 143 American Black patients with sickle cell anemia. Haplotypes were determined using eight polymorphic sites in the beta-like globin gene cluster: Hinc II 5' to epsilon, Hind III in IVS-II G gamma and A gamma, Hinc II within and 3' to psi beta, Ava II in IVS-II of beta, and Hpa I and Bam HI 3' to beta. The G gamma /A gamma ratio was analyzed by high performance liquid chromatography using a C18 column. The Xmn I site was present in all 31 chromosomes with the Sengalese haplotype. Of the remaining 255 chromosomes with other haplotypes, only 2 (0.8%) had the Xmn I site present. There was significant correlation between the presence of the Xmn I site and increased G gamma /A gamma ratio in a dose-dependent manner. The Hb F level was not significantly increased in the presence of the Xmn I site. The data indicate that the Xmn I site maintains a G gamma /A gamma ratio typical of fetal life but does not necessarily cause elevation of Hb F. The latter seems to depend on factors other than the Xmn I site.     

Association of the level of G gamma chain in the fetal hemoglobin of normal adults with specific haplotypes

The levels of G gamma chain in the fetal hemoglobin of more than 40 Black and Caucasian females were determined with a sensitive high performance liquid chromatography procedure and were correlated with their haplotypes, defined by the presence or absence of 10 different restriction sites. Blood was collected during the 16th and 31st week of pregnancy because of a slightly elevated level of Hb F which facilitated the isolation of this protein from a relatively small sample. Four distinct G gamma levels were observed, each being associated with a specific haplotype. Homozygosity for sub-haplotype A [- + + - + +] is associated with high G gamma values (60-70%); that for sub-haplotype B [- - - - - +] with low levels (25-30%); and that for sub-haplotype C [+ - - - - -] with very low levels (10-15%) (restriction sites listed are Hinc II at epsilon; Xmn I 5' to G gamma; Hind III at G gamma and A gamma; Hinc II at psi beta and 3' to it). Sub-haplotype D [(14)- + - - +] with a rare polymorphism 5' to epsilon is associated with extremely high G gamma values. Hb F levels were low (less than 2.5%) and were independent of the haplotype. It is speculated that, yet unknown, variations in the DNA of gene activity controlling regions are responsible for the differences in G gamma value.     

β-Thalassaemia in Campania: DNA polymorphism analysis in βA and βthat chromosomes and its usefulness in prenatal diagnosis

In order to evaluate the feasibility of first trimester prenatal diagnosis of beta-thalassaemia by restriction fragment length polymorphism (RFLP) in Campania, one of the most affected regions in Southern Italy, DNA polymorphism analysis was performed on 40 unrelated patients, affected with homozygous beta-thalassaemia, and on their parents. Frequency of the presence of the Hinc II epsilon, Hind III G gamma and A gamma, Hinc II psi beta and 3' psi beta, Ava II psi beta, Ava II beta and Bam HI 3' beta sites have been determined in the beta A and beta thal chromosome samples. In 31 families (over 75%), RFLPs enabled tracing the beta-thalassaemia mutations in both father and mother (100% diagnosis). In the remaining nine families, RFLPs enabled tracing only one of the two mutations (50% diagnosis) because the other parent was found to be homozygous in all the analysed polymorphic sites. Restriction haplotypes, assembled on the basis of linkage analysis, were most heterogeneous, hence a wide heterogeneity of mutations is expected.     

Non-random association of the polymorphic Taq I restriction site, located 3 KB 5' to the human delta-globin gene, with the polymorphic Hind III sites within the G gamma- and A gamma-globin genes

During the study of the DNA from 25 beta-thalassemic subjects from mediterranean origin the polymorphic Taq I restriction site located 3-kb 5' to the human delta-globin gene, was found non-randomly associated to the polymorphic Hind III sites within the G gamma- and A gamma-globin genes. This indicates that the 3' limit of the linkage group of polymorphic restriction sites including the gamma-globin genes is located downstream to the polymorphic Taq I site.     

Application of DNA polymorphisms for prenatal diagnosis of beta thalassemia in Chinese

Forty-seven Chinese suffering from beta thalassemia major and their parents were studied to establish linkage of the beta thal and beta A genes with 11 restriction site polymorphisms. There is marked linkage disequilibrium at the BamH I site 3' to the beta globin gene, such that, in 31% of pregnancies, absence of the site in the fetus can exclude beta thalassemia major. Using four restriction sites (Hinc II psi beta, Ava II beta, Hind III beta, and BamH I beta), prenatal diagnosis is feasible in all families. In 46% of all cases, a definitive diagnosis can be made, and in the remaining cases, a 50% chance of exclusion is possible. Fetal blood globin chain analysis would be required for the failures. Our experience in nine successive beta thalassemia prenatal diagnosis is also reported.     

Human fetal to adult hemoglobin switching: changes in chromatin structure of the beta-globin gene locus.

We have investigated the chromatin structure of the chromosomal DNA regions containing the human G gamma-, A gamma-, delta-, and beta-globin structural genes in both fetal and adult erythropoietic tissues and in two human erythroleukemia cells lines before and after induction. Our results indicate that DNase I introduces specific cuts into the beta-globin gene cluster in erythroid cells but not in leukocytes. The predominant sites are located at the 5' sides of the G gamma-, A gamma-, delta-, and beta-globin genes, within 200 base pairs of the respective cap sites. Examination of fetal liver cells has revealed the presence of hypersensitive sites at the 5' side of all four genes, whereas analysis of adult bone marrow has revealed the characteristic sites near the delta- and beta-globin genes but no hypersensitive sites at the 5' termini of the G gamma- or A gamma-globin genes. The presence of delta and beta hypersensitive sites in fetal cells suggests that the increment in expression of the delta and beta genes during development most likely involves the modulation of another pathway to gene expression. Using isolated nuclei from HEL and K562 cells, we have found that the G gamma, A gamma, delta, and beta genes are preferentially sensitive [relative to the pro-alpha2(I) collagen gene] to mild digestion with DNase I, whereas these genes are as resistant as collagen genes in cells that do not express globin. These findings are discussed within the context of chromatin structural correlates of hemoglobin switching.     

Two independent genetic factors in the beta-globin gene cluster are associated with high G gamma-levels in the HbF of SS patients

The gamma-chains of fetal hemoglobin (HbF) of newborn babies are composed of about 70% G gamma and 30% A gamma. In most babies, the G gamma value declines postnatally to 40%, but in about 20% of black SS patients from Georgia, 5 years and older, the G gamma level remains high at 60%. Moreover, some 3% to 4% of black newborns have high G gamma values of 85%. PstI digestion of DNA of one such high G gamma baby and of one normal newborn showed the former to be heterozygous for the -G gamma-G gamma- and -G gamma-A gamma-chromosomes. Only about one fourth of high G gamma SS patients were such heterozygotes, while three fourths were -G gamma-A gamma-/-G gamma-A gamma-homozygotes. Analysis of DNA of 38 SS patients without the -G gamma-G gamma-chromosome showed a correlation of G gamma values with genotype at one polymorphic restriction site: at the HincII site in the psi beta gene, all -G gamma- A gamma-/-G gamma-A gamma-homozygotes with high G gamma were +/- or +/+, while low G gamma individuals were all -/-. Family studies, involving analyses at four polymorphic sites (HindIII sites in the G gamma and A gamma genes and HincII sites in the psi beta gene and 3' to it), suggested the association of an unidentified high G gamma genetic determinant with haplotype + - + +. This indicates that a genetic factor causing high G gamma levels in SS patients is closely linked to the -G gamma-A gamma-psi beta region of the beta-globin gene cluster.     

Effect of 5-aza-2'-deoxycytidine (Dacogen) on covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin promoters in Papio anubis

OBJECTIVE: Treatment with the DNA demethylating drug 5-aza-2'-deoxycytidine (Dacogen; DAC) increased fetal hemoglobin and F cells to therapeutically significant levels in patients with sickle cell disease. To gain more insight into the mechanism of action of this drug and to increase our understanding of the relationship between DNA methylation and chromatin structure, we have determined the effect of DAC on covalent histone modifications of chromatin associated with the epsilon, gamma-, and beta-globin promoters in purified bone marrow erythroid cells of four baboons (P. anubis) pre- and posttreatment. RESULTS: Fetal hemoglobin increased from 6.45%+/-1.75% in pretreatment samples to 62.1%+/-7.94% following DAC. DNA methylation of three CpG sites within the epsilon-globin promoter and 5 CpG sites within the gamma-globin promoter decreased more than 50% following DAC treatment. Levels of RNA polymerase II, acetyl-histone H3, acetyl-histone H4, dimethyl-histone H3 (lys4), dimethyl-histone H3 (lys36), and dimethyl-histone H3 (lys79) associated with the epsilon-, gamma-, and beta-globin promoters were determined by chromatin immunoprecipitation of formaldehyde-fixed chromatin followed by real-time PCR. Dacogen treatment increased the association of RNA polymerase II, acetyl-histone H3, and acetyl-histone H4 with the gamma-globin promoter but did not significantly affect the association of dimethyl-histone H3 (lys4), dimethyl-histone H3 (lys36), and dimethyl-histone H3 (lys79) with the epsilon-, gamma-, and beta-globin gene promoters. CONCLUSION: These experiments illustrate the usefulness of the baboon model to investigate the mechanism of pharmacologic reactivation of fetal hemoglobin synthesis at the molecular level.     

Prenatal diagnosis of beta-thalassemias by amniocentesis: linkage analysis using multiple polymorphic restriction endonuclease sites

In order to assess the applicability of multiple restriction endonuclease analyses of amniocyte DNA to the prenatal diagnosis of beta-thalassemias in general, we studied 12 consecutive couples at risk. DNA of both members of the 12 couples and a previous offspring of each was analyzed for the presence of 4 polymorphic restriction endonuclease sites: the Hpa I site 3' to the beta-globin gene, the Hind III site in the G gamma gene, the Hind III site in the A gamma gene, and the Bam HI site 3' to the beta-gene. Linkage disequilibrium between these sites and beta A or beta thal genes was not found, presumably due to the heterogeneity of beta thal genes. However, the high frequency of polymorphism at these sites allowed differentiation of beta A-bearing chromosomes from beta thal or beta S-bearing chromosomes in both members of 6 couples. In these couples, complete prenatal diagnosis by linkage analysis of amniocyte DNA would be possible. In the remaining 6 couples, beta A and beta thal chromosomes could be discriminated in one member. In about 50% of the pregnancies of these couples, exclusion of beta-thalassemia is possible by this analysis. These data suggest that when linkage analysis of polymorphic restriction endonuclease sites is carried out, prenatal diagnosis of beta-thalassemia states can be accomplished by amniocentesis alone in 75% of pregnancies at risk.     

Effect of deletion of 5''HS3 or 5''HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.     

Molecular analysis of Japanese delta beta-thalassemia

A DNA fragment containing the deletion junction region from a Japanese individual with homozygous delta beta-thalassemia has been cloned. A clone containing the normal DNA surrounding the 3' breakpoint of this deletion and a clone carrying the G gamma- and A gamma-globin genes of this patient were also isolated. Sequences of the deletion junction and both gamma-globin genes were determined. A comparison of these sequences with previously determined sequences of the normal counterparts revealed that the 5' breakpoint is located between 2,134 and 2,137 base pairs (bp) 3' to the polyA site of the A gamma-globin gene, the 5' breakpoint is located just downstream of the 3' border of the fetal gamma-globin duplication unit, and no molecular defects are evident within the gamma-globin gene region. A comparison between the sequences of the normal DNA surrounding the 3' breakpoint and the normal DNA surrounding the 5' breakpoint shows that deletion is the result of a nonhomologous recombination event. There are A+T-rich stretches near the 5' and 3' breakpoints in the normal DNA, and a portion of an Aly repeat is located in the region 3' to the 3' breakpoint. Southern blot analysis using probes 3' to the beta-globin gene showed that the deletion extends in the 3' direction further than any other deletions associated with delta beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) heretofore reported. These results are discussed in terms of the mechanism generating large deletions in mammalian cells and three models for the regulation of gamma-globin and beta-globin gene expression in humans.     

Cloning and characterization of DNA sequences surrounding the human gamma-, delta-, and beta-globin genes.

The human gamma-, delta-, and beta-globin genes are located within a 30-kilobase (kb) region of DNA, of which only 20% represents the globin genes. We have attempted to define the nature of flanking and intergenic sequences by isolating recombinants containing the human epsilon, both gamma-, or the 3' end of the beta-globin gene from a bacteriophage library of cloned human DNA. Comparison of these recombinants and a recombinant containing the delta- and beta-globin genes (H beta G1) has provided the following results. The epsilon-globin gene is located 14 kb 5' to the G gamma gene. DNA sequence homology between the region containing the two G gamma genes and the delta nd beta gene region is limited to only a few hundred nucleotides which include the globin coding sequences. Repetitive DNA sequences have been found in the region 3' to the beta-globin gene. Sequences located adjacent to the beta-globin gene are repeated in the globin gene region. A repetitive DNA sequence more than 3.2 kb long is repeated frequently in the human genome but is not repeated in the globin gene region in the clones examined.     

Deletion of the human beta-globin LCR 5'HS4 or 5'HS1 differentially affects beta-like globin gene expression in beta-YAC transgenic mice

A 213 kb human beta-globin locus yeast artificial chromosome (beta-YAC) was modified by homologous recombination to delete 2.9 kb of cross-species conserved sequence similarity encompassing the LCR 5' hypersensitive site (HS) 4 (Delta5'HS4 beta-YAC). In three transgenic mouse lines, completion of the gamma- to beta-globin switch during definitive erythropoiesis was delayed relative to wild-type beta-YAC mice. In addition, quantitative per-copy human beta-like globin mRNA levels were similar to wild-type beta-YAC transgenic lines, although beta-globin gene expression was slightly decreased in the day 12 fetal liver of Delta5'HS4 beta-YAC mice. A 0.8 kb 5'HS1 fragment was similarly deleted in the YAC. Three Delta5'HS1 beta-YAC transgenic lines were established. epsilon-globin gene expression was markedly reduced, approximately 16 fold, during primitive erythropoiesis compared to wild-type beta-YAC mice, but gamma-globin expression levels were unaffected. However, during the fetal stage of definitive erythropoiesis, gamma-globin gene expression was decreased approximately 4 fold at day 12 and approximately 5 fold at day 14. Temporal developmental expression profiles of the beta-like globin genes were unaffected by deletion of 5'HS1. Decreased expression of the epsilon- and gamma-globin genes is the first phenotype ascribed to a 5'HS1 mutation in the human beta-globin locus, suggesting that this HS does indeed have a role in LCR function beyond simply a combined synergism with the other LCR HSs.     

Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants.

To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of beta-globin locus YAC developmental mutants: (i) mice carrying a G-->A transition at position -117 of the A gamma gene, which is responsible for the Greek A gamma form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) beta-globin locus YAC transgenic lines carrying delta- and beta-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and delta beta-thalassemia syndromes. The mice carrying the -117 A gamma G-->A mutation displayed a delayed gamma- to beta-globin gene switch and continued to express A gamma-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the gamma-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development.