Epigenetic Analysis of Beta-Globin Gene Cluster during Hematopoiesis.

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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Extensive methylation of a part of the CpG island located 3.0–4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells 1 1Edited by M. Yaniv

Journal of Molecular Biology ◽

10.1006/jmbi.2000.3775 ◽

2000 ◽

Vol 299 (4) ◽

pp. 845-852 ◽

Cited By ~ 14

Author(s):

Sergey V Razin ◽

Elena S Ioudinkova ◽

K Scherrer

Keyword(s):

Gene Cluster ◽

Cpg Island ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Cells ◽

Globin Gene Cluster ◽

Alpha Globin

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Severe Thalassemia Intermedia Resulting From Coinheritance of IVSI-110 G>A Beta Gene Mutation and Duplication of Alpha-Globin Gene Cluster in Homozygosis.

Blood ◽

10.1182/blood.v114.22.2011.2011 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2011-2011 ◽

Cited By ~ 1

Author(s):

Chiara Refaldi ◽

Wilma Barcellini ◽

Elena Cassinerio ◽

Giovanna Graziadei ◽

Maria Domenica Cappellini

Keyword(s):

Gene Cluster ◽

Molecular Mechanisms ◽

Globin Gene ◽

Clinical Severity ◽

Globin Genes ◽

Beta Globin ◽

Thalassemia Intermedia ◽

Globin Gene Cluster ◽

Increased Risk ◽

Alpha Globin

Abstract Abstract 2011 Poster Board I-1033 Introduction The clinical severity of thalassemia intermedia depends on the degree of a/non-a-chains iimbalance. Among the molecular mechanisms responsible for thalassemia intermedia is the coinheritance of excessive a-globin gene production with a defective beta-globin gene. Materials and Methods: we describe an Italian family where thalassemia intermedia apparently segregates as a dominant form but it tourned out to be due to the coinheritance of a beta-globin mutation and a duplication of the alpha-globin gene cluster. The father (aged 51yrs) showed a well tolerated severe chronic hemolytic anemia (Hb 7.5-8.5 g/dL) not transfusion dependent, jaundice, splenomegaly and leg ulcers:The mother (aged 46yrs) has a completely normal hematological and hemoglobin pattern. Two sons (19 and 14 yrs) showed more severe clinical manifestations than the father. They underwent splenectomy at 12 and 13 years respectively without any benefit and afterwards they become transfusion dependent. Results: The hemoglobin analysis revealed that the father and the sons were heterozygotes for the beta mutation IVSI-110 G>A. MLPA analysis of the alpha-globin gene cluster disclosed a full duplication of the alpha-globin locus, spanning a 175 kb from the telomere to the 3'HVR downstream of the alpha-globin gene and including the upstream regulatory element HS-40. This rearrangement increases the number of the active alpha-globin genes in cis from 2 to 4.Surprisengly it was found in heterozygosis in both parents and in homozygosis in both sons. The hematological and molecular data of the family are reported in the table. In the father the 6 alpha-globin genes led to increased synthesis of alpha-chains; the coinheritance with a beta-thalassemia mutation causes a moderate/severe thalassemia intermedia phenotype. The presence of 8 alpha-globin genes in the sons raises further the degree of globin-chains imbalance and exacerbates the clinical phenotype. It is important to note that splenectomy worsened the clinical course.in the 2 homozygotes for the alpha duplication. Conclusions: Based also on previous experience we suggest that splenectomy in patients with a real excess of alphaa chain production is unconvenient since a large amount of circulating red cells with precipitated alpha chains may be responsible for increased hemolysis as well as increased risk of thrombosis This family moreover raises concerns regarding genetic counselling, suggesting that whenever one of the partner is affected by TI it is advisable a complete molecular screening of the couple in order to exclude any possible alpha gene defects interaction Disclosures: No relevant conflicts of interest to declare.

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

Hemoglobin ◽

10.3109/03630268908998063 ◽

1989 ◽

Vol 13 (2) ◽

pp. 137-146 ◽

Cited By ~ 2

Author(s):

K. Shimizu ◽

K. S. Park ◽

K. Omoto

Keyword(s):

Gene Cluster ◽

Globin Gene ◽

Dna Polymorphisms ◽

Globin Genes ◽

Globin Gene Cluster

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Methylation of α-type embryonic globin gene απrepresses transcription in primary erythroid cells

Blood ◽

10.1182/blood-2002-02-0457 ◽

2002 ◽

Vol 100 (12) ◽

pp. 4217-4222 ◽

Cited By ~ 22

Author(s):

Rakesh Singal ◽

Jane M. vanWert ◽

Larry Ferdinand

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Luciferase Reporter ◽

Globin Genes ◽

Erythroid Cells ◽

Histone H4 ◽

Luciferase Reporter Gene ◽

Cpg Dinucleotides ◽

Expression Construct ◽

Globin Promoter

The inverse relationship between expression and methylation of β-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian α-type globin genes. The embryonicαπ-globin promoter was unmethylated, andαπ-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the απ promoter associated with loss of expression of απ-globin gene was seen during development in primary erythroid cells. A 315-bpαπ-globin promoter region was cloned in an expression construct (απpGL3E) containing a luciferase reporter gene and SV40 enhancer. The απpGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of απpGL3E plasmid andαπ-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bpαπ-globin gene promoter fragment formed amethyl cytosine-binding proteincomplex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with theαπ-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian α-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.

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<i>ß</i>-Globin Gene Cluster Haplotypes and Clinical Severity in Sickle Cell Anemia Patients in Southern Brazil

Open Journal of Blood Diseases ◽

10.4236/ojbd.2014.42003 ◽

2014 ◽

Vol 04 (02) ◽

pp. 16-23 ◽

Cited By ~ 1

Author(s):

Maria A. L. da Silva ◽

João R. Friedrisch ◽

Christina M. Bittar ◽

Meide Urnau ◽

Jóice Merzoni ◽

...

Keyword(s):

Gene Cluster ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Globin Gene ◽

Clinical Severity ◽

Southern Brazil ◽

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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An Olfactory Receptor Gene Is Located in the Extended Human β-Globin Gene Cluster and Is Expressed in Erythroid Cells

Genomics ◽

10.1006/geno.1999.5935 ◽

1999 ◽

Vol 61 (1) ◽

pp. 15-23 ◽

Cited By ~ 56

Author(s):

Elise A. Feingold ◽

Laura A. Penny ◽

Arthur W. Nienhuis ◽

Bernard G. Forget

Keyword(s):

Gene Cluster ◽

Olfactory Receptor ◽

Receptor Gene ◽

Globin Gene ◽

Olfactory Receptor Gene ◽

Erythroid Cells ◽

Globin Gene Cluster ◽

In Erythroid Cells

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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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