The Georgia type of nondeletional hereditary persistence of fetal hemoglobin has a C---T mutation at nucleotide-114 of the A gamma-globin gene [letter]

Hematological and hemoglobin composition data, and results from extensive gene mapping, using a battery of restriction enzymes and probes, have been used to distinguish different types of hereditary persistence of fetal hemoglobin (HPFH) (or delta beta-thal) among three Chinese families from the southern part of China. The first (Family Z) is an A gamma-(delta beta)+-HPFH without a detectable deletion and may be the same as, or similar to, that described by Farquhar et al (Am J Hum Genet 35:611, 1983). The second (Family C) resembles a G gamma(A gamma delta beta)o-thalassemia and is characterized by a large deletion of DNA originating 3′ to the G gamma globin gene and extending beyond sequences recognized by the pRK28 probe. Data from various digests indicate possible differences in the 3c′ end of the deletion when compared with data for some other types of G gamma(A gamma delta beta)o- thalassemia, described by Trent et al (Br J Haematol 57:279, 1984). The third (Family Zh) concerns a G gamma A gamma(delta beta)+-HPFH, which is characterized in heterozygotes by a fetal hemoglobin level of 20% to 25% with a G gamma value averaging 60% and by the absence of any DNA deletion detectable by extensive gene mapping analyses. The C----G mutation at position 202 5c′ to the G gamma globin gene [characteristic for the high G gamma-(delta beta)+-HPFH (Proc Natl Acad Sci USA 81:4894, 1984; Blood 64:1292, 1984)] was absent, but the Xmn I site at position 158 5c′ to the G gamma globin gene [characteristic for a modest increase in G gamma values and thus and increased G gamma to A gamma ratio (Blood)] was present. No indication has yet been obtained explaining the elevation in both G gamma and A gamma chains; haplotyping showed that the chromosome carrying this G gamma A gamma(delta beta)+ determinant is unusual among the Chinese population.

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A fetal globin gene mutation in A gamma nondeletion hereditary persistence of fetal hemoglobin increases promoter strength in a nonerythroid cell.

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.713 ◽

1988 ◽

Vol 8 (2) ◽

pp. 713-721 ◽

Cited By ~ 22

Author(s):

M W Rixon ◽

R E Gelinas

Keyword(s):

Transient Expression ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Type A ◽

Base Substitution ◽

Globin Genes ◽

Wild Type ◽

Gamma Globin ◽

Base Substitutions ◽

Hereditary Persistence

Single base substitutions have been identified in the promoter regions of A gamma-globin genes from individuals with certain types of nondeletion A gamma hereditary persistence of fetal hemoglobin (HPFH). The presence of these mutations is closely associated with the A gamma HPFH phenotype, but proof that they are the nondeletion HPFH determinants is lacking. To test directly whether these base substitutions can result in an increase in A gamma-globin gene transcription, we studied cosmid clones containing the G gamma- through beta-globin gene regions from individuals with Greek-type (G-to-A base substitution at -117) and Chinese-type (C-to-T base substitution at -196) A gamma HPFH in a transient expression assay. When tested as part of a cosmid clone, the Greek HPFH A gamma-globin gene consistently produced about 1.4 times as much RNA as the wild-type A gamma-globin gene when standardized against RNA transcribed from the G gamma genes in cis. The relative strengths of the normal and HPFH A gamma-globin gene promoters were also compared in transient expression assays with plasmids containing the A gamma-globin genes. Pseudo-wild-type A gamma-globin genes containing a short, transcriptionally neutral deletion were used so that two A gamma-globin genes that differed in their promoter sequences could be compared in the same transfection. The plasmid transient expression results indicated a 1.3- to 1.4-fold increase in steady-state RNA levels from the Greek-type A gamma HPFH promoter compared with the wild-type A gamma promoter, while no difference was documented between the Chinese-type A gamma HPFH promoter and the wild-type A gamma promoter.

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Expression of the affected A gamma globin gene associated with Greek nondeletion hereditary persistence of fetal hemoglobin.

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2999 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2999-3003 ◽

Cited By ~ 13

Author(s):

C J Stoeckert ◽

J E Metherall ◽

M Yamakawa ◽

J M Eisenstadt ◽

S M Weissman ◽

...

Keyword(s):

Transcription Initiation ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Mutant Gene ◽

Retroviral Vector ◽

Base Substitution ◽

Gamma Globin ◽

Erythroleukemia Cell ◽

Hereditary Persistence ◽

Globin Gene Expression

The overexpressed A gamma globin gene in the Greek type of nondeletion hereditary persistence of fetal hemoglobin has a unique single-base substitution located at position -117 relative to the site of transcription initiation. This gene and its normal counterpart were transferred into cultured cell lines by using a retroviral vector. The only difference in expression between the transferred normal and mutant gamma genes was observed in the human erythroleukemia cell line KMOE after exposure of the cells to cytosine arabinoside, a condition that resulted in an adult pattern of endogenous globin gene expression by the cells and was associated with increased expression of the mutant gene.

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Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter

Blood ◽

10.1182/blood.v71.3.815.815 ◽

1988 ◽

Vol 71 (3) ◽

pp. 815-817 ◽

Cited By ~ 33

Author(s):

S Ottolenghi ◽

S Nicolis ◽

R Taramelli ◽

N Malgaretti ◽

R Mantovani ◽

...

Keyword(s):

Globin Gene ◽

Single Point ◽

Point Mutations ◽

Fetal Hemoglobin ◽

Regulatory Elements ◽

Globin Genes ◽

Gamma Globin ◽

New Type ◽

Hereditary Persistence ◽

Globin Promoter

Abstract A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3′ end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

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Hereditary persistence of fetal hemoglobin or (delta beta)o- thalassemia: three types observed in South-Chinese families

Blood ◽

10.1182/blood.v66.6.1430.1430 ◽

1985 ◽

Vol 66 (6) ◽

pp. 1430-1435 ◽

Cited By ~ 19

Author(s):

YT Zeng ◽

SZ Huang ◽

B Chen ◽

YC Liang ◽

ZM Chang ◽

...

Keyword(s):

Gene Mapping ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Restriction Enzymes ◽

Large Deletion ◽

Gamma Delta ◽

Chinese Families ◽

Gamma Globin ◽

Hereditary Persistence ◽

Fetal Hemoglobin Level

Abstract Hematological and hemoglobin composition data, and results from extensive gene mapping, using a battery of restriction enzymes and probes, have been used to distinguish different types of hereditary persistence of fetal hemoglobin (HPFH) (or delta beta-thal) among three Chinese families from the southern part of China. The first (Family Z) is an A gamma-(delta beta)+-HPFH without a detectable deletion and may be the same as, or similar to, that described by Farquhar et al (Am J Hum Genet 35:611, 1983). The second (Family C) resembles a G gamma(A gamma delta beta)o-thalassemia and is characterized by a large deletion of DNA originating 3′ to the G gamma globin gene and extending beyond sequences recognized by the pRK28 probe. Data from various digests indicate possible differences in the 3c′ end of the deletion when compared with data for some other types of G gamma(A gamma delta beta)o- thalassemia, described by Trent et al (Br J Haematol 57:279, 1984). The third (Family Zh) concerns a G gamma A gamma(delta beta)+-HPFH, which is characterized in heterozygotes by a fetal hemoglobin level of 20% to 25% with a G gamma value averaging 60% and by the absence of any DNA deletion detectable by extensive gene mapping analyses. The C----G mutation at position 202 5c′ to the G gamma globin gene [characteristic for the high G gamma-(delta beta)+-HPFH (Proc Natl Acad Sci USA 81:4894, 1984; Blood 64:1292, 1984)] was absent, but the Xmn I site at position 158 5c′ to the G gamma globin gene [characteristic for a modest increase in G gamma values and thus and increased G gamma to A gamma ratio (Blood)] was present. No indication has yet been obtained explaining the elevation in both G gamma and A gamma chains; haplotyping showed that the chromosome carrying this G gamma A gamma(delta beta)+ determinant is unusual among the Chinese population.

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Sequence analysis of the gamma-globin gene locus from a patient with the deletion form of hereditary persistence of fetal hemoglobin

Blood ◽

10.1182/blood.v75.2.499.499 ◽

1990 ◽

Vol 75 (2) ◽

pp. 499-504 ◽

Cited By ~ 3

Author(s):

CA Stolle ◽

LA Penny ◽

S Ivory ◽

BG Forget ◽

EJ Jr Benz

Keyword(s):

Allelic Variation ◽

Polymerase Chain Reaction Amplification ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Chromosomal Dna ◽

Gamma Globin ◽

Allele Specific ◽

Reaction Amplification ◽

Hereditary Persistence

Abstract The gamma-globin genes from a patient homozygous for a deletion form of hereditary persistence of fetal hemoglobin (HPFH-1) have been cloned and sequenced. The DNA sequence of the patient's gamma-globin genes corresponds to a previously identified sequence framework (chromosome A) with the exception of 10 base changes. Seven of these base changes can be attributed to normal allelic variation generated by small gene conversion events. The remaining three base changes are present in a 0.76 kb HindIII fragment containing a putative enhancer located 3′ to the A gamma-globin gene. The same three base changes have also been described in the Seattle variant of nondeletion HPFH. We have analyzed 16 alleles from non-HPFH individuals and five alleles from individuals with nondeletion or deletion HPFH for the presence of these base changes by polymerase chain reaction amplification of cloned or chromosomal DNA and hybridization to allele-specific oligonucleotide probes. Although these base changes were found in an individual with HPFH-2, they were not found in the DNA from two patients with nondeletion HPFH. More importantly, all three base changes were detected in DNA from five non-HPFH individuals and appear to be common in blacks. We conclude that these base changes do not correlate with an HPFH phenotype and that the significant mutation in HPFH-1 is the deletion of over 100 kb of genomic DNA.

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Italian type of deletional hereditary persistence of fetal hemoglobin

Blood ◽

10.1182/blood.v68.3.646.646 ◽

1986 ◽

Vol 68 (3) ◽

pp. 646-651 ◽

Cited By ~ 25

Author(s):

G Saglio ◽

C Camaschella ◽

A Serra ◽

T Bertero ◽

G Rege Cambrin ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Type I ◽

Italian Population ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

New Type ◽

Hereditary Persistence ◽

Chain Synthesis

Abstract We report a new type of deletion of the beta globin gene cluster in the Italian population that confers a phenotype of hereditary persistence of fetal hemoglobin (HPFH) to the carriers. This deletion begins approximately 5 kilobases (kb) 5′ to the delta globin gene and ends approximately 30 kb 3′ to the beta globin gene, in close proximity to the 3′ end of an Indian HPFH. In all four previously described HPFH, a repetitive Alu I region 5′ to the delta globin gene is largely or completely deleted; the 5′ end of the new HPFH is consistent with this common feature. In addition, the finding that Italian and Indian HPFHs, as reported for other groups of deletions, have very close 3′ ends, strengthens the idea that common mechanisms may operate in generating these deletions. Finally, we show that, in spite of similar 5′ breakpoints, the deletion of Spanish delta beta degrees-thalassemia is at least 8 kb longer than that of Negro HPFH type I, thus ruling out the hypothesis that the overall extent of the deletion might influence the level of gamma globin chain synthesis.

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A fetal globin gene mutation in A gamma nondeletion hereditary persistence of fetal hemoglobin increases promoter strength in a nonerythroid cell

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.713-721.1988 ◽

1988 ◽

Vol 8 (2) ◽

pp. 713-721

Author(s):

M W Rixon ◽

R E Gelinas

Keyword(s):

Transient Expression ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Type A ◽

Base Substitution ◽

Globin Genes ◽

Wild Type ◽

Gamma Globin ◽

Base Substitutions ◽

Hereditary Persistence

Single base substitutions have been identified in the promoter regions of A gamma-globin genes from individuals with certain types of nondeletion A gamma hereditary persistence of fetal hemoglobin (HPFH). The presence of these mutations is closely associated with the A gamma HPFH phenotype, but proof that they are the nondeletion HPFH determinants is lacking. To test directly whether these base substitutions can result in an increase in A gamma-globin gene transcription, we studied cosmid clones containing the G gamma- through beta-globin gene regions from individuals with Greek-type (G-to-A base substitution at -117) and Chinese-type (C-to-T base substitution at -196) A gamma HPFH in a transient expression assay. When tested as part of a cosmid clone, the Greek HPFH A gamma-globin gene consistently produced about 1.4 times as much RNA as the wild-type A gamma-globin gene when standardized against RNA transcribed from the G gamma genes in cis. The relative strengths of the normal and HPFH A gamma-globin gene promoters were also compared in transient expression assays with plasmids containing the A gamma-globin genes. Pseudo-wild-type A gamma-globin genes containing a short, transcriptionally neutral deletion were used so that two A gamma-globin genes that differed in their promoter sequences could be compared in the same transfection. The plasmid transient expression results indicated a 1.3- to 1.4-fold increase in steady-state RNA levels from the Greek-type A gamma HPFH promoter compared with the wild-type A gamma promoter, while no difference was documented between the Chinese-type A gamma HPFH promoter and the wild-type A gamma promoter.

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The -195C→G Substitution In Brazilian Hereditary Persistence of Fetal Hemoglobin Decreases NF-E1/YY1 Binding and Increases PAX1 Binding to the A Gamma Globin Promoter Region

Blood ◽

10.1182/blood.v116.21.2066.2066 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2066-2066 ◽

Cited By ~ 3

Author(s):

Fernanda Marconi Roversi ◽

Anderson Ferreira Cunha ◽

Carolina Lanaro ◽

Ana Flavia Brugnerotto ◽

Maria Emília Favero ◽

...

Keyword(s):

Dna Binding ◽

Promoter Region ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Dna Array ◽

Gamma Globin ◽

Nuclear Extracts ◽

Hereditary Persistence ◽

Globin Promoter

Abstract Abstract 2066 Hereditary persistence of fetal hemoglobin (HPFH) is a condition that prevents hemoglobin switching and the consequent silencing of the gamma globin genes, resulting in continued hemoglobin (Hb) F synthesis in adults. Two types of HPFH are responsible for this phenotype: deletional HPFH – deletions in the end of the beta globin locus – and non-deletional HPFH (ndHPFH) – single point mutations in the proximal promoter of both gamma globin genes. Sickle cell anemia patients or beta-thalassemia patients that present HPFH show high levels of HbF that are associated with less severe clinical course in these diseases. The development of new therapies based on the reactivation of gamma globin expression may be important for the treatment of these patients. The Brazilian ndHPFH type is characterized as a C→G substitution in the A gamma globin promoter at position –195 and the molecular mechanism responsible for the reactivation of this gene in the Brazilian ndHPFH type remains unclear. In contrast to the British ndHPFH type (-198), where the mechanism responsible for the increase of HbF levels is mediated by the raising in the affinity for the Sp1 transcription factor (TF), the Brazilian ndHPFH mutation does not affect Sp1 binding. Thus, other TF may be involved in the reactivation of the A gamma globin gene in the Brazilian ndHPFH type. The aim of this study was to investigate the mechanism involved in the reactivation or repression of the A gamma globin gene in the Brazilian ndHPFH type and identify possible TF responsible for this phenotype. In vitro primary human erythroblast cultures, derived from human CD34+ hematopoietic cells from 4 Brazilian ndHPFH type subjects and 4 control subjects, were proliferated and differentiated into late stage erythroblasts. The nuclear extracts from predominantly basophilic and polychromatic erythroblasts were used to profile TF activity using Protein-DNA Array method. The analysis of the array densitometry identified a number of TF whose DNA binding activities were either enhanced or repressed in the Brazilian ndHPFH cultures. Among the TF analyzed, the NF-E1/YY1 and the PAX-1 were selected for this study. Since this assay requires a secondary method to confirm these results, nuclear extracts were used to conduct chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). ChIP was carried out using antibodies against NF-E1/YY1 and PAX-1 to quantify the binding to these TF to the –195 A gamma globin promoter region. EMSA was performed using probes with the same sequence spotted on the array membrane to analyze the activity of NF-E1/YY1 and PAX-1. Both methods confirmed and validated the previous array results. NF-E1/YY1 is a transcription factor that represses embryonic (epsilon) and fetal (gamma) globin genes. Protein-DNA array and EMSA showed a decreased binding of NF-E1/YY1 in Brazilian ndHPFH nuclear extracts and ChIP analysis revealed diminished NF-E1/YY1 occupancy at the –195 A gamma globin promoter region of Brazilian ndHPFH. The consensus binding site for NF-E1/YY1 is a CCAN motif that is observed between the –195 and –192 position in the A gamma globin promoter region. The C→G substitution at –195 position may disrupt this DNA binding site, cause decreased NF-E1/YY1 interaction and probably allows the binding of PAX-1, a transcriptional activator with a paired box DNA-binding domain that has as a DNA binding core motif, the sequence TTCCGC. This sequence, located between the –199 and –194 position in the A gamma globin promoter, is only presente in the Brazilian type of ndHPFH. Our protein-DNA array and EMSA results showed an increased binding of PAX-1 in the Brazilian ndHPFH nuclear extracts and quantitative ChIP analysis with anti-PAX-1 antibody showed that PAX-1 binds to the –195 A gamma globin promoter region only in the presence of this C→G substitution. These results suggest that the –195 site (C→G) in the A gamma globin promoter region may decrease NF-E1/YY1 binding and increase PAX-1 binding in this DNA region, probably resulting in the reactivation of the A gamma globin gene. The increase in the HbF levels in the Brazilian ndHPFH occurs differently from the British ndHPFH type and represents a novel mechanism of A gamma globin reactivation. Such findings may lead to the development of future therapeutic strategies for HbF induction in the treatment of other hemoglobinopathies. Support by FAPESP and CNPq. Disclosures: No relevant conflicts of interest to declare.

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