Functional analysis of a novel KLF1 gene promoter variation associated with hereditary persistence of fetal hemoglobin

Abstract During a study of Sardinian families with hereditary persistence of fetal hemoglobin (HPFH), two unrelated subjects with unusually elevated Hb F levels were identified. By selective amplification of the A gamma gene promoter and hybridization to synthetic oligonucleotides, we demonstrate that these subjects are homozygous for the -117A gamma G---- A substitution that is responsible for a form of nondeletional HPFH. The hemoglobin synthetic pattern of these patients is discussed.

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Sardinian delta beta zero-thalassemia: a further example of a C to T substitution at position -196 of the A gamma globin gene promoter

Blood ◽

10.1182/blood.v69.4.1058.bloodjournal6941058 ◽

1987 ◽

Vol 69 (4) ◽

pp. 1058-1061

Author(s):

S Ottolenghi ◽

B Giglioni ◽

A Pulazzini ◽

P Comi ◽

C Camaschella ◽

...

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Indirect Evidence ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

Functional Defect ◽

Hereditary Persistence ◽

Hereditary Conditions

Selective overexpression (50- to 100-fold) in adult erythroid cells of either G gamma or A gamma fetal globin gene is observed in hereditary conditions known as delta beta zero-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C-T change at position -196 of an overexpressed A gamma globin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed A gamma-globin gene from a Sardinian patient with a different syndrome (delta beta zero- thalassemia). The Sardinian A gamma globin gene differs from both the HPFH and the normal A gamma globin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant -196 A gamma-globin gene is linked to a normal beta globin gene in HPFH, and to a beta-thalassemic gene (beta 39CAG-TAG) in delta beta zero-thalassemia. These data strengthen the suggestion that -196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the -196 mutation in different syndromes.

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Expressions of KLF1, FOXO3a, HOOK3 and MIER1 genes Are Associated with Fetal Hemoglobin Levels in Hereditary Persistence of Fetal Hemoglobin,

Blood ◽

10.1182/blood.v118.21.3195.3195 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3195-3195

Author(s):

Fernanda Marconi Roversi ◽

Anderson Ferreira Cunha ◽

Ana Flavia Brugnerotto ◽

Regiane Ferreira ◽

Dulcinéia Martins Albuquerque ◽

...

Keyword(s):

Transcription Factor ◽

Western Blotting ◽

Globin Gene ◽

Gene Promoter ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

Hereditary Persistence ◽

Cellular Modifications

Abstract Abstract 3195 The hereditary persistence of fetal hemoglobin (HPFH) is the consequence of impaired switching in adult life, which results in the continued expression of gamma globin gene. The Brazilian HPFH type is characterized by a C → G substitution at the –195 position of the A gamma globin gene promoter, and associated with HbF levels ranging from 6 to 16% in the heterozygote state. This study was undertaken to identify genes that may be involved in hemoglobin switching and/or maintenance of elevated HbF levels in Brazilian HPHF subjects. The Suppressive Subtractive Hybridization libraries were constructed using a pool of RNA extracted from reticulocytes of peripheral blood in individuals with normal hematological data and from subjects with Brazilian and 55 and 57 overexpressed genes were identified in the normal and Brazilian HPFH library, respectively; findings were validated by qRT-PCR and Western blotting. One transcription factor identified was FOXO3a, whose expression was increased in Brazilian HPFH subjects, compared to control subjects. Moreover, the non-phosphorylated Foxo3a protein that interacts with DNA, was detected only in Brazilian HPFH when analyzed by Western blotting. FOXO3a binds to PAX1 promoter, a transcription factor whose activity was higher in Brazilian HPFH. The FOXO3a/PAX1 complex may be important in the mechanism responsible for increasing HbF levels in this genetic disorder. Another gene analyzed was KLF1, a transcription factor known as a regulator of switching from the gamma to beta globin gene. This gene was underexpressed in the reticulocytes of HPFH subjects. Klf1 protein activity in erythroid cells of healthy donors was also increased compared to Brazilian HPFH, when analyzed by DNA/protein array. Results suggest that the decreased KLF1 levels in Brazilian HPFH favors the interaction between the A gamma globin gene and the Locus Control Region, agreeing with previous studies that demonstrated that knockdown of KLF1 in adult erythroid progenitors favors the formation of complexes that bind to the gamma globin gene promoter. MIER1 expression was also found to be decreased in Brazilian HPFH reticulocytes, compared to controls. The MIER1 gene is able to recruit chromatin remodeling-enzymes leading, to the formation of heterochromatin and, consequently, silencing genes. This MIER1 may be an important gene in gamma to beta globin gene switching, where it could help in the maintenance of a closed chromatin structure in the gamma globin gene in individuals with low HbF levels. The expression of the HOOK3 gene was decreased in Brazilian HPFH reticulocytes, compared to controls. The HOOK3 encodes a protein that interacts with a GTPase protein, stimulating INF-gamma, responsible for the phosphorylation of p65/p50 and RXR beta like proteins which regulate the transcription of the beta globin gene. The reduced beta globin gene expression in Brazilian HPFH resulting from the reactivation of gamma globin gene may be responsible for a decrease in the HOOK3 expression. These results suggest that, in the HPFH Brazilian type, the FOXO3a/PAX1 complex could contribute to the continued expression of the A gamma globin gene. Additionally, some cellular modifications, such as low Klf1 activity and decreased HOOK3 and MIER1 expression, could participate in the maintenance of HbF levels. These genes could be important in therapeutic approaches for the development of new HbF induction agents for the hemoglobinopathies. Support by FAPESP, CNPq.and INCTS Disclosures: No relevant conflicts of interest to declare.

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Sardinian delta beta zero-thalassemia: a further example of a C to T substitution at position -196 of the A gamma globin gene promoter

Blood ◽

10.1182/blood.v69.4.1058.1058 ◽

1987 ◽

Vol 69 (4) ◽

pp. 1058-1061 ◽

Cited By ~ 23

Author(s):

S Ottolenghi ◽

B Giglioni ◽

A Pulazzini ◽

P Comi ◽

C Camaschella ◽

...

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Indirect Evidence ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

Functional Defect ◽

Hereditary Persistence ◽

Hereditary Conditions

Abstract Selective overexpression (50- to 100-fold) in adult erythroid cells of either G gamma or A gamma fetal globin gene is observed in hereditary conditions known as delta beta zero-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C-T change at position -196 of an overexpressed A gamma globin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed A gamma-globin gene from a Sardinian patient with a different syndrome (delta beta zero- thalassemia). The Sardinian A gamma globin gene differs from both the HPFH and the normal A gamma globin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant -196 A gamma-globin gene is linked to a normal beta globin gene in HPFH, and to a beta-thalassemic gene (beta 39CAG-TAG) in delta beta zero-thalassemia. These data strengthen the suggestion that -196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the -196 mutation in different syndromes.

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The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Annals of Hematology ◽

10.1007/s00277-008-0643-0 ◽

2008 ◽

Vol 88 (6) ◽

pp. 549-555 ◽

Cited By ~ 4

Author(s):

Christos Chassanidis ◽

Angelos Kalamaras ◽

Marios Phylactides ◽

Farzin Pourfarzad ◽

Sophia Likousi ◽

...

Keyword(s):

Novel Mutation ◽

Gene Promoter ◽

Fetal Hemoglobin ◽

Hereditary Persistence

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Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Journal of Biological Chemistry ◽

10.1074/jbc.m610404200 ◽

2006 ◽

Vol 282 (2) ◽

pp. 853-862 ◽

Cited By ~ 8

Author(s):

Ivan A. Olave ◽

Catalin Doneanu ◽

Xiangdong Fang ◽

George Stamatoyannopoulos ◽

Qiliang Li

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Fetal Hemoglobin ◽

Hereditary Persistence

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Chinese A gamma fetal hemoglobin: C to T substitution at position-196 of the A gamma gene promoter

Blood ◽

10.1182/blood.v67.6.1777.1777 ◽

1986 ◽

Vol 67 (6) ◽

pp. 1777-1779 ◽

Cited By ~ 31

Author(s):

R Gelinas ◽

M Bender ◽

C Lotshaw ◽

P Waber ◽

H Jr Kazazian ◽

...

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Fetal Hemoglobin ◽

Nucleotide Sequence Analysis ◽

Globin Genes ◽

Gamma Globin ◽

Hemoglobin C ◽

Hereditary Persistence ◽

Gene Switching ◽

Chinese Individual

Abstract The molecular basis for the hereditary persistence of fetal hemoglobin (HPFH) phenotype was studied in a Chinese individual who was heterozygous for a nondeletion form of A gamma-HPFH. Both allelic A gamma-globin genes were isolated by molecular cloning and subjected to nucleotide sequence analysis. One A gamma gene promoter showed a cytosine to thymine transition at position -196, whereas the other promoter was normal. This mutation at position -196 has now ben found in unrelated individuals with the A gamma-HPFH phenotype from Italy, Sardinia, and China, suggesting that it may have arisen independently. The implications of this mutation for models of fetal globin gene switching are discussed.

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Chinese A gamma fetal hemoglobin: C to T substitution at position-196 of the A gamma gene promoter

Blood ◽

10.1182/blood.v67.6.1777.bloodjournal6761777 ◽

1986 ◽

Vol 67 (6) ◽

pp. 1777-1779

Author(s):

R Gelinas ◽

M Bender ◽

C Lotshaw ◽

P Waber ◽

H Jr Kazazian ◽

...

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Fetal Hemoglobin ◽

Nucleotide Sequence Analysis ◽

Globin Genes ◽

Gamma Globin ◽

Hemoglobin C ◽

Hereditary Persistence ◽

Gene Switching ◽

Chinese Individual

The molecular basis for the hereditary persistence of fetal hemoglobin (HPFH) phenotype was studied in a Chinese individual who was heterozygous for a nondeletion form of A gamma-HPFH. Both allelic A gamma-globin genes were isolated by molecular cloning and subjected to nucleotide sequence analysis. One A gamma gene promoter showed a cytosine to thymine transition at position -196, whereas the other promoter was normal. This mutation at position -196 has now ben found in unrelated individuals with the A gamma-HPFH phenotype from Italy, Sardinia, and China, suggesting that it may have arisen independently. The implications of this mutation for models of fetal globin gene switching are discussed.

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The homozygous state of G to A--117A gamma hereditary persistence of fetal hemoglobin

Blood ◽

10.1182/blood.v73.7.1999.bloodjournal7371999 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1999-2002

Author(s):

C Camaschella ◽

L Oggiano ◽

M Sampietro ◽

E Gottardi ◽

A Alfarano ◽

...

Keyword(s):

Gene Promoter ◽

Fetal Hemoglobin ◽

Homozygous State ◽

Selective Amplification ◽

Hereditary Persistence ◽

Synthetic Oligonucleotides

During a study of Sardinian families with hereditary persistence of fetal hemoglobin (HPFH), two unrelated subjects with unusually elevated Hb F levels were identified. By selective amplification of the A gamma gene promoter and hybridization to synthetic oligonucleotides, we demonstrate that these subjects are homozygous for the -117A gamma G---- A substitution that is responsible for a form of nondeletional HPFH. The hemoglobin synthetic pattern of these patients is discussed.

Download Full-text