The DNA Polymorphisms of the β-Globin Gene Cluster and the Arrangements of the α- and the γ-Globin Genes in Koreans

Abstract Hemoglobin (Hb) switching is described as temporal, tissue- and stage-specific patterns of globin gene expression; from embryonic to fetal and adult Hb in parallel to developmental stages of erythropoiesis. DNA methylation, one of the epigenetic mechanisms, was associated with inactivated chromatin domain and repressive transcription. To study the role of the DNA methylation on the beta (β)-globin genes, we analyzed CpG dinucleotides in 87 kb regions around β-globin gene cluster, including 5’upstream locus control regions (LCR; DNAse I Hypersensitive site (HS) 1–5), 3’HS1, the promoter regions of the G-and A-gamma (Gγ and Aγ), and β-globin genes, in several representative cells. These cells were primary adult erythroid cells culture (three different stages: early, intermediate, and late), fetal cord blood DNA, and neutrophil cell line (non-erythroid). Using bisulphite modification, followed by nested PCR and in vitro translation, the cleavage products were analysed by MALDI-TOF Mass Spectrometry to quantify the DNA methylation level. The results were consistent with bisulphite sequencing. We found that the promoters of Gγ and Aγ-globin genes were significantly hypomethylated in fetal cells (44% and 47% global methylation), when γ-globin genes were fully expressed, while they were heavily methylated in non-erythroid (86% and 95%). There was also a decreasing trend of the DNA methylation level at Gγ and Aγ-globin genes during adult erythroid differentiation from 80% and 82%, in early stage, to 67% and 66% in late stage (p=0.12 and 0.04). At β-globin promoter, the global methylation level changed from 90% in non-erythroid to 81%, 42%, and 26% in fetal, early and late adult erythroid cells, respectively. Moreover, we found the significant changes at 5’HS4, 3, and 1 as all erythroid cells were hypomethylated compare to non-erythroid. While at the insulators, 5’HS5 and 3’HS1, all tested CpG dinucleotides were heavily methylated in all cells. This is the first report that demonstrates the differences in DNA methylation at β-globin LCR between erythroid and non-erythroid cells. These epigenetic marks were associated with globin genes expression and might be useful to predict clinical severity in patients with β-thalassemia intermedia.

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Expression of Human Globin Genes in Transgenic Mice Carrying the ?-Globin Gene Cluster with a Mutation CausingG??+Hereditary Persistence of Fetal Hemoglobin

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1990.tb24303.x ◽

1990 ◽

Vol 612 (1 Sixth Cooley') ◽

pp. 167-178 ◽

Cited By ~ 3

Author(s):

MINORU TANAKA ◽

JUDITH A. NOLAN ◽

AJAY K. BHARGAVA ◽

KIRSTEN ROOD ◽

FRANCIS S. COLLINS ◽

...

Keyword(s):

Transgenic Mice ◽

Gene Cluster ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Globin Gene Cluster ◽

Hereditary Persistence

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Genetic distance analysis using DNA polymorphisms in the α-globin gene cluster

Annals of Human Biology ◽

10.1080/03014468700009211 ◽

1987 ◽

Vol 14 (5) ◽

pp. 393-404

Author(s):

K.M. Summers ◽

P. Yenchitsomanus ◽

R.M. Chapple

Keyword(s):

Genetic Distance ◽

Gene Cluster ◽

Globin Gene ◽

Dna Polymorphisms ◽

Distance Analysis ◽

Globin Gene Cluster ◽

Genetic Distance Analysis

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The entire beta-globin gene cluster is deleted in a form of gamma delta beta-thalassemia

Blood ◽

10.1182/blood.v61.6.1269.bloodjournal6161269 ◽

1983 ◽

Vol 61 (6) ◽

pp. 1269-1274

Author(s):

ER Fearon ◽

HH Jr Kazazian ◽

PG Waber ◽

JI Lee ◽

SE Antonarakis ◽

...

Keyword(s):

Gene Cluster ◽

Mexican American ◽

Globin Gene ◽

Dna Polymorphisms ◽

Beta Thalassemia ◽

Beta Globin ◽

Gamma Delta ◽

Beta Globin Gene ◽

Globin Gene Cluster ◽

Endonuclease Mapping

We have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA fragment that maps greater than 40 kilobases (kb) 5′ to the epsilon-gene as a probe, reduced amounts of normal fragments were found in the DNA of affected family members. Similar analysis using radiolabeled DNA fragments located 3′ to the beta-globin cluster has shown that the deletion extends more than 17 kb 3′ to the beta-gene, but terminates before the 3′ endpoint of the Ghanian HPFH deletion. Hence, this gamma delta beta-thalassemia deletion eliminates over 105 kb of DNA and is the first report of a deletion of the entire beta-globin gene cluster.

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Evidence that Microdeletions in the α Globin Gene Protect Against the Development of Sickle Cell Glomerulopathy in Humans

Journal of the American Society of Nephrology ◽

10.1681/asn.v1051014 ◽

1999 ◽

Vol 10 (5) ◽

pp. 1014-1019

Author(s):

ANTONIO GUASCH ◽

CARLOS F. ZAYAS ◽

JAMES R. ECKMAN ◽

KASINATHAN MURALIDHARAN ◽

WEI ZHANG ◽

...

Keyword(s):

Gene Cluster ◽

Sickle Cell ◽

Gene Locus ◽

Globin Gene ◽

Renal Involvement ◽

Hemoglobin Concentration ◽

Mean Corpuscular Hemoglobin ◽

Globin Genes ◽

Large Variability ◽

Globin Gene Cluster

Abstract. There is a large variability in the severity of the clinical manifestations of sickle cell anemia (SSA), including renal involvement. Haplotypes in the β-globin gene cluster associated with the geographical origin of the sickle mutation, as well as microdeletions in the α-globin genes, could provide an epigenetic influence on the heterogeneous outcome in SSA. It has been determined that the cause of progressive renal insufficiency in SSA is a glomerulopathy, clinically detected by the presence of macroalbuminuria (albumin excretion rate >300 mg/g creatinine). To investigate the role of the α-globin gene microdeletion and β-globin gene cluster haplotypes on the degree of glomerular involvement, 76 adult SSA patients (hemoglobin SS) were studied to determine the relationship between these genetic markers and the development of sickle cell glomerulopathy. Macroalbuminuria was present in 22 (29%) of 76 adult SSA patients. The coinheritance of microdeletions in one or two of the four α-globin genes (α-thalassemia) was associated with a lower prevalence of macroalbuminuria (13%) versus patients with intact α-globin genes (40%, P = 0.01). By contrast, there was no association between albuminuria and β-globin gene haplotypes (Central African Republic [CAR] versus non-CAR haplotypes). Patients with α-globin gene microdeletions had lower mean corpuscular volumes and mean corpuscular hemoglobin concentration than patients with all four α genes (86 ± 2 versus 99 ± 3 fl, and 33.9 ± 0.2 versus 34.9 ± 0.2%, respectively, P < 0.05). There were no such hematologic differences between CAR and non-CAR β-globin haplotypes. There were no differences in duration of disease (age), hemoglobin levels, reticulocyte index, and lactate dehydrogenase levels between those with and without glomerulopathy, but the mean arterial pressure was higher (87 ± 1 mmHg) in patients with intact α gene locus versus those with microdeletions (80 ± 2 mmHg, P < 0.05). It is concluded that the coinheritance of microdeletions in the α-globin gene locus in SSA patients confers “renoprotection” by mechanisms not related to the degree of anemia or the severity of hemolysis, but could be related to a reduced mean corpuscular volume or to a lower erythrocyte hemoglobin concentration.

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Novel Rearrangements and Mutations in the Alpha-Globin Gene Cluster Detected by a New Alpha-Globin Dosage Assay, Cation Exchange HPLC and Comprehensive DNA Sequencing.

Blood ◽

10.1182/blood.v108.11.1595.1595 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1595-1595

Author(s):

Feras M. Hantash ◽

Monica V. Gallivan ◽

Mikula Mario ◽

Starn Kelsey ◽

Sheng-Biao Wang ◽

...

Keyword(s):

Dna Sequencing ◽

Gene Cluster ◽

Gene Dosage ◽

Globin Gene ◽

Point Mutations ◽

Globin Genes ◽

Globin Gene Cluster ◽

Large Deletions ◽

Alpha Globin ◽

Small Base

Abstract The alpha globin gene cluster contains two highly homologous alpha globin genes, HBA1 and HBA2, that code for identical proteins. Mutations in the alpha globin gene cluster are predominantly large deletions causing the loss of either one copy of the alpha-globin gene (e.g. -α3.7 and -α4.2 deletions) or both copies (e.g. --THAI, --FIL or --MED). A few large deletions encompassing the regulatory HS-40 region have also been described in alpha-thalassaemia patients. Point mutations and small base pair insertions or deletions have also been detected in HBA1 and HBA2 genes. Seven common large deletions in the alpha globin gene cluster are detected by a gapped-PCR assay. These common mutations and some other types of rearrangements can be detected by Southern blot, a laborious and time consuming method. However, these methods may not accurately identify the total number of copies of alpha globin like genes. We designed a single-tube alpha globin gene dosage assay (αGDA) using semi-quantitative fluorescent PCR (SQF PCR) for detecting the total number of alpha globin genes. Primers that amplify specific fragments from HBA1 and HBA2 genes, a fragment between the alpha globin pseudogenes, and three fragments flanking and including the HS-40 regulatory region were included in a single PCR reaction together with primers that amplify fragments from 3 different normalization genes. Using the αGDA, we were able to detect in patient samples varying copy numbers of alpha globin genes and to identify the nature of DNA rearrangements between HBA1 and HBA2. We also identified novel alpha globin conversion events that were verified by DNA sequencing. We also designed a complimentary comprehensive DNA sequencing assay to detect point mutations and small base pair insertions or deletions in the HBA1 and HBA2 genes. Using this method, and in combination with cation exchange HPLC and agarose gel electrophoresis, novel mutations in alpha globins were identified and submitted to the globin gene server, including Hb Linwood (α2 40 Lys>Gln), Hb Creve Coeur (α2 24 Tyr>Asp), and Hb Westborough (α-3.7 130 Ala>Val). The simplicity of αGDA will allow the replacement of the laborious Southern blot analysis to detect large deletions in the alpha globin gene cluster and to provide accurate information of total a-globin gene dosage, while the DNA sequencing assay will allow the detection of known and novel variants.

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Linkage of β-thalassaemia mutations and β-globin gene polymorphisms with DNA polymorphisms in human β-globin gene cluster

Nature ◽

10.1038/296627a0 ◽

1982 ◽

Vol 296 (5858) ◽

pp. 627-631 ◽

Cited By ~ 625

Author(s):

Stuart H. Orkin ◽

Haig H. Kazazian ◽

Stylianos E. Antonarakis ◽

Sabra C. Goff ◽

Corinne D. Boehm ◽

...

Keyword(s):

Gene Cluster ◽

Gene Polymorphisms ◽

Globin Gene ◽

Dna Polymorphisms ◽

Globin Gene Cluster

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Homology of a 130-kb region enclosing the ?-globin gene cluster, the ?-locus controlling region, and two non-globin genes in human and mouse

Mammalian Genome ◽

10.1007/bf00357090 ◽

1993 ◽

Vol 4 (6) ◽

pp. 314-323 ◽

Cited By ~ 11

Author(s):

Menno F. Kielman ◽

Ron Smits ◽

Tara S. Devi ◽

Riccardo Fodde ◽

Luigi F. Bernini

Keyword(s):

Gene Cluster ◽

Globin Gene ◽

Globin Genes ◽

Globin Gene Cluster ◽

Human And Mouse

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G-Quadruplex Forming Sequences Found In The Beta-Globin Gene Cluster Regulate Erythroid Differentiation Of K562 Cells

Blood ◽

10.1182/blood.v122.21.1203.1203 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1203-1203

Author(s):

Francine Rezzoug ◽

Shelia Thomas ◽

Donald Max Miller

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

Gene Cluster ◽

Globin Gene ◽

K562 Cells ◽

Erythroid Differentiation ◽

Globin Genes ◽

Beta Globin ◽

Globin Gene Cluster ◽

G Quadruplex

Abstract Introduction Guanine-rich DNA sequences can form stable four stranded structures by folding of the DNA strands, forming stacks of G-tetrads called G-quadruplex. G-quadruplex forming sequences are found in eukaryotic telomeres, in promoter regions and in noncoding regions of the genome. Although the function of these structures has not yet been identified it has been suggested that they play a negative regulatory role at the transcription level. The most studied G-quadruplex sequences (AS1411 and PU27) show activity in reducing growth and invasiveness of malignant cells, as well as increasing cell death of a large array of tumors with promising therapeutic applications. We identified two G-quadruplex forming sequences in the human beta globin cluster (Hbd and Hbg2). We confirmed quadruplex formation by these sequences using circular dichroism spectroscopy. We also observed in Southern blot assay that these oligo-sequences bind to specific β-globin gene fragments after EcoRI digestion. Finally we demonstrate that neither Hbd nor Hbg2 (5 or 10µM) were growth inhibitory to four different leukemia cell lines after 6 days culture measured by MTT. Results The β-globin cluster contains 5 functional globin-like genes and long range regulator elements, the expression of each of these genes is tightly regulated during development (from embryo to fetus to adult), in cell type and in the course of differentiation/maturation of the erythroid lineage. Recent studies have demonstrated the importance of folding and looping of the DNA in the control of globin genes expression. We hypothesized that the G-quadruplex forming sequences within the β-globin gene cluster participate in regulation and splicing of the different globin genes during erythroid maturation. The erythroleukemia cell line K562 was used in this study because of its ability to differentiate into erythrocytes under Hemin stimulation. K562 cells were divided into 6 groups of treatment and cultured for 3 days in the presence or absence of Hemin (30µM) with or without either Hbd or Hbg2 oligonucleotides (5µM). Erythroid differentiation was evaluated by quantitative QRT-PCT for the expression of β-, γ- and ε-globin genes as well as α-globin and by flow-cytometry for the presence of β-, γ- and ε-globin; α-globin and CD235a (Glycophorin A) were used as controls. The data reveal a difference in gene expression for all globin genes when K562 were exposed to Hbd or Hbg2 only, interestingly, a significant up-regulation of β-globin was observed only in Hbg2 treated cells. Similarly in K562 induced to differentiate with Hemin show up-regulation of γ-, ε- and α- globin, the addition of Hbd or Hbg2 oligonucleotides to Hemin at the beginning of the culture slightly improve the expression of γ- and ε- however there was no difference for the expression of β- globin in the hemin +/- Hb oligonucleotides treated cells compared to control untreated cells. Flow-cytometry experiments confirmed the increase in the expression of each globin gene in all treatments compared to control. Altogether the data suggest a role for Hbd and Hbg2 in the regulation of β-globin gene transcription and therefore in the differentiation of K562 in erythroid lineage. Conclusions The implications of our findings are multiple. 1) the presence of G-quadruplex sequences in a gene complex not involved in growth regulation or oncogenesis; 2) to our knowledge this is the first time that G-quadruplex sequences are shown to specifically alter the expression of the gene in which they are located; and 3) the potential use of globin G-quadruplex to modify the globin gene expression therapeutically. Disclosures: No relevant conflicts of interest to declare.

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The entire beta-globin gene cluster is deleted in a form of gamma delta beta-thalassemia

Blood ◽

10.1182/blood.v61.6.1269.1269 ◽

1983 ◽

Vol 61 (6) ◽

pp. 1269-1274 ◽

Cited By ~ 28

Author(s):

ER Fearon ◽

HH Jr Kazazian ◽

PG Waber ◽

JI Lee ◽

SE Antonarakis ◽

...

Keyword(s):

Gene Cluster ◽

Mexican American ◽

Globin Gene ◽

Dna Polymorphisms ◽

Beta Thalassemia ◽

Beta Globin ◽

Gamma Delta ◽

Beta Globin Gene ◽

Globin Gene Cluster ◽

Endonuclease Mapping

Abstract We have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA fragment that maps greater than 40 kilobases (kb) 5′ to the epsilon-gene as a probe, reduced amounts of normal fragments were found in the DNA of affected family members. Similar analysis using radiolabeled DNA fragments located 3′ to the beta-globin cluster has shown that the deletion extends more than 17 kb 3′ to the beta-gene, but terminates before the 3′ endpoint of the Ghanian HPFH deletion. Hence, this gamma delta beta-thalassemia deletion eliminates over 105 kb of DNA and is the first report of a deletion of the entire beta-globin gene cluster.

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