Identification of novel candidate genes for globin regulation in erythroid cells containing large deletions of the human β-globin gene cluster

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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β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia

Asia Pacific Journal of Molecular Biology and Biotechnology ◽

10.35118/apjmbb.2021.029.1.02 ◽

2021 ◽

pp. 10-18

Author(s):

Siti Nor Assyuhada Mat Ghani ◽

Rozieyati Mohamed Saleh ◽

Wan Suriana Wan Ab Rahman ◽

Mohd Nazri Hassan ◽

Wan Zaidah Abdullah ◽

...

Keyword(s):

Gene Cluster ◽

Teaching Hospital ◽

Globin Gene ◽

Single Mutation ◽

Globin Gene Cluster ◽

Large Deletions ◽

Arms Pcr ◽

Tertiary Teaching ◽

Tertiary Teaching Hospital ◽

Hbf Level

Mutations in the β-globin gene cluster can lead to β-thalassaemia, δβ-thalassaemia, hereditary persistence of foetal haemoglobin (HPFH) and some of the haemoglobin variants. The clinical and haematological spectrum of thalassaemia range from benign to pathogenic conditions including severe transfusion dependent thalassaemia. Therefore, it is important to accurately diagnose β-globin gene cluster mutations to prevent thalassaemia major or intermedia offspring. The objective of this study is to detect β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia. This study involved 144 anaemic patients with HbF level >1.0%. High-Performance Liquid Chromatography (HPLC) was used to determine the HbF and HbA2 levels. Multiplex-ARMS (ARMS)-PCR and gap-PCR were performed for those patients with high HbA2 level (>3.2%) and normal HbA2 level (≤3.2%) to detect β-globin gene cluster mutations and large deletions respectively. The majority of patients were Malays (99.3%) with mean age of 19.99 ± 1.64 years and female 61.1% predominance. Out of 87 samples tested using multiplex ARMS-PCR against eight targeted single mutation; Cd 41/42, IVS 1–5, Cd 26, Cd 17, Cd 71/72, IVS 1–1, Cd 8/9 and -28 mutations, 65 (74.7%) patients were detected which comprises of Cd 26 (56.3%), Cd 41/42 (11.5%), compound Cd 26 and Cd 41/42 (3.4%) and IVS 1–1 (3.4%). Meanwhile, for multiplex gap-PCR which detect four types of large deletions; Thai (δβ)o-thalassaemia, HPFH-6, Siriraj J and Hb Lepore, one out of 57 patients (1.8%) was found positive with Thai (δβ)o-thalassaemia. There was a significant difference between the mean of HbF level, MCV level as well as MCH level of patients with and without β-globin gene mutations and large deletions (P<0.05). This study highlighted the presence of various types of β-globin gene cluster mutations detection in establishing a definitive diagnosis among this selected group of patients for the large-scale screening of the thalassaemia gene.

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Identification and molecular characterization of four new large deletions in the β-globin gene cluster

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2009.01.017 ◽

2009 ◽

Vol 43 (1) ◽

pp. 53-57 ◽

Cited By ~ 14

Author(s):

Philippe Joly ◽

Philippe Lacan ◽

Caroline Garcia ◽

Nicole Couprie ◽

Alain Francina

Keyword(s):

Gene Cluster ◽

Molecular Characterization ◽

Globin Gene ◽

Globin Gene Cluster ◽

Large Deletions

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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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