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

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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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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The Organization in Micro-Loops of an Extended Fragment of Chicken Chromosome 14, Including the Alpha Globin Gene Cluster in the Erythroid Cells

Acta Naturae ◽

10.32607/20758251-2009-1-1-105-108 ◽

2009 ◽

Vol 1 (1) ◽

pp. 105-108

Author(s):

E S Philonenko ◽

A A Gavrilov ◽

S V Ravin ◽

O V Iarovaia

Keyword(s):

Gene Cluster ◽

Globin Gene ◽

Chicken Chromosome ◽

Erythroid Cells ◽

Chromosome 14 ◽

Globin Gene Cluster ◽

Alpha Globin

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Developmental switching of messenger RNA expression from the human alpha-globin cluster: fetal/adult pattern of theta-globin gene expression

Blood ◽

10.1182/blood.v80.6.1586.1586 ◽

1992 ◽

Vol 80 (6) ◽

pp. 1586-1591 ◽

Cited By ~ 10

Author(s):

M Albitar ◽

A Care ◽

C Peschle ◽

SA Liebhaber

Keyword(s):

Steady State ◽

Gene Cluster ◽

Messenger Rna ◽

Globin Gene ◽

Mrna Level ◽

Globin Genes ◽

Globin Mrna ◽

Globin Gene Cluster ◽

Adult Pattern ◽

Alpha Globin

Abstract The alpha-globin gene cluster contains four functional globin genes, zeta, alpha 2, alpha 1, and theta. The developmental regulation of the embryonic zeta and fetal/adult alpha 2- and alpha 1-globin genes is well characterized at the level of protein synthesis. The developmental pattern of the theta-globin gene is not well characterized due to the inability to detect its encoded protein. Direct analysis of the globin switching at the steady-state messenger RNA (mRNA) level has been hampered by the difficulty in obtaining quantities of embryonic and early fetal mRNA sufficient for analysis. We analyzed the relative levels of the steady-state zeta-, alpha-, and theta-globin mRNAs in yolk sac in 5-, 6-, 7-, and 8-week postconception embryonic liver, and in cord and adult blood reticulocytes. We show that the switch in the alpha-globin gene cluster from the embryonic to fetal/adult pattern of expression begins at 5 to 6 weeks of gestation. Both the theta- and alpha-globin genes show similar patterns of developmental control that are reciprocal to zeta. alpha-globin RNA is barely detectable or undetectable at 5 weeks, and increases in the 6- to 8-week period, while theta-globin mRNA shows a parallel increase at 5 to 8 weeks postconception and is expressed in cord blood and adult reticulocytes. These data show that the theta-globin gene represents a fetal/adult gene, albeit expressed at a low level.

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Isolation and nucleotide sequence of the rabbit globin gene cluster psi zeta-alpha 1-psi alpha. Absence of a pair of alpha-globin genes evolving in concert.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36172-0 ◽

1986 ◽

Vol 261 (2) ◽

pp. 839-848

Author(s):

J F Cheng ◽

L Raid ◽

R C Hardison

Keyword(s):

Nucleotide Sequence ◽

Gene Cluster ◽

Globin Gene ◽

Globin Genes ◽

Globin Gene Cluster ◽

Alpha Globin

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Developmental switching of messenger RNA expression from the human alpha-globin cluster: fetal/adult pattern of theta-globin gene expression

Blood ◽

10.1182/blood.v80.6.1586.bloodjournal8061586 ◽

1992 ◽

Vol 80 (6) ◽

pp. 1586-1591

Author(s):

M Albitar ◽

A Care ◽

C Peschle ◽

SA Liebhaber

Keyword(s):

Steady State ◽

Gene Cluster ◽

Messenger Rna ◽

Globin Gene ◽

Mrna Level ◽

Globin Genes ◽

Globin Mrna ◽

Globin Gene Cluster ◽

Adult Pattern ◽

Alpha Globin

The alpha-globin gene cluster contains four functional globin genes, zeta, alpha 2, alpha 1, and theta. The developmental regulation of the embryonic zeta and fetal/adult alpha 2- and alpha 1-globin genes is well characterized at the level of protein synthesis. The developmental pattern of the theta-globin gene is not well characterized due to the inability to detect its encoded protein. Direct analysis of the globin switching at the steady-state messenger RNA (mRNA) level has been hampered by the difficulty in obtaining quantities of embryonic and early fetal mRNA sufficient for analysis. We analyzed the relative levels of the steady-state zeta-, alpha-, and theta-globin mRNAs in yolk sac in 5-, 6-, 7-, and 8-week postconception embryonic liver, and in cord and adult blood reticulocytes. We show that the switch in the alpha-globin gene cluster from the embryonic to fetal/adult pattern of expression begins at 5 to 6 weeks of gestation. Both the theta- and alpha-globin genes show similar patterns of developmental control that are reciprocal to zeta. alpha-globin RNA is barely detectable or undetectable at 5 weeks, and increases in the 6- to 8-week period, while theta-globin mRNA shows a parallel increase at 5 to 8 weeks postconception and is expressed in cord blood and adult reticulocytes. These data show that the theta-globin gene represents a fetal/adult gene, albeit expressed at a low level.

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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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Analysis of the human alpha-globin gene cluster in transgenic mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.11262 ◽

1993 ◽

Vol 90 (23) ◽

pp. 11262-11266 ◽

Cited By ~ 38

Author(s):

J A Sharpe ◽

D J Wells ◽

E Whitelaw ◽

P Vyas ◽

D R Higgs ◽

...

Keyword(s):

Transgenic Mice ◽

Gene Cluster ◽

Copy Number ◽

Globin Gene ◽

Globin Genes ◽

Regulation Of Expression ◽

Globin Gene Cluster ◽

Hypersensitive Sites ◽

Alpha Globin ◽

High Level

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.

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