Role of the Krüppel-Type Zinc Finger Transcription Factor ZBP-89 In Human Globin Gene Regulation and Erythroid Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2067-2067
Author(s):  
Andrew J. Woo ◽  
Jonghwan Kim ◽  
Jian Xu ◽  
Hui Huang ◽  
Alan Cantor
Keyword(s):  
Globin Gene ◽  
Regulatory Elements ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Histone 3 ◽  
Cd34 Cells ◽  
Alpha Globin ◽  
Globin Gene Regulation ◽  
Erythroid Development

Abstract Abstract 2067 The molecular mechanisms underlying developmental globin gene regulation remain incompletely understood. Prior studies have identified key cis-regulatory elements within the beta globin locus that contain core regions of closely spaced functional binding sites for GATA, NF-E2p45/maf and GT/GC box binding transcription factors. We recently identified the GT/GC-box binding transcription factor ZBP-89 as a novel GATA-1 interacting partner, and showed that it is involved in erythroid development in mice (Woo et al. 2008. Mol. Cell Bio. 28:2675-2689). Brand et al. independently isolated ZBP-89 in NF-E2p45/mafk complexes from induced mouse erythroid leukemia (MEL) cells (Brand et al. 2004. Nat. Struct. Mol Biol. 11:73-80). In the current study, we show that ZBP-89 protein levels increase during in vitro erythroid differentiation of human bone marrow derived CD34+ cells. This correlates with the onset of alpha and beta globin gene transcription. ChIP-chip studies using ENCODE v2.0 arrays demonstrate that ZBP-89 occupies key cis-regulatory elements within both the beta globin (locus control regions HS3, HS2; delta and beta proximal promoters; and an intergenic region between gamma1 and delta globin) and alpha globin (HS-48, HS-40, HS-10 and alpha globin proximal promoters) loci in primary human erythroid precursors. Comparative analysis across the entire ENCODE array reveals a strong positive correlation between ZBP-89 occupancy, RNA polymerase II occupancy, and the activating histone marks acetylated histone 3 (AcH3) and trimethylated histone 3 lysine 4 (H3K4me3); and a negative correlation with the repressive mark trimethylated histone 3 lysine 27 (H3K27me3). Motif analysis under the ZBP-89 occupancy peaks indicates a preference for GGGG(G/A)NGGGG in vivo binding sites. Lentiviral shRNA mediated knock down of ZBP-89 in the in vitro differentiated CD34+ cells results in 30–50% reduction of alpha-, gamma-, and beta-globin gene expression, as well as modestly decreased expression of a number of additional erythroid-specific genes. Co-immunoprecipitation experiments demonstrate physical association between ZBP-89 and the GCN5/Trapp histone acetyltransferase complex. Based on these findings, we propose that ZBP-89 participates with GATA-1 and NF-E2 in the final epigenetic changes required for high-level expression of globin and other erythroid genes in terminally differentiating human erythroid cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Adult Hemoglobin Production, Chain Rebalance, and Splice Correction in IVS2-745 Beta-Thalassemia Patient Cells Using 2'-O-Methoxyethyl Splice-Switching Oligos

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1014-1014
Author(s):  
Alisa Cheung Dong ◽  
Valentina Ghiaccio ◽  
Irene Motta ◽  
Shuling Guo ◽  
Raechel Peralta ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Globin Gene ◽  
Model System ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Advisory Committees ◽  
Beta Globin Gene ◽  
Cd34 Cells ◽  
Adult Hemoglobin

Abstract Hemoglobinopathies are the most common inherited blood disorders. World Health Organization statistics show that in the Mediterranean, Eastern European, and Middle Eastern regions, frequencies range from 0.1 to 4.9/1000 of live births. The mutation known as IVS2-745 is relatively common in the regions of Spain, Jordan, Romania, and Serbia (Ithanet Database, http://www.ithanet.eu/db/ithamaps), reaching as high as 15-20% of beta-thalassemia mutations in these regions. The IVS2-745 is a splicing mutation that occurs in intron 2 of the beta-globin gene and results in an aberrantly spliced mRNA that incorporates an extra exon and premature stop codon. Here we report novel uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) that reverse the aberrant splicing and restore up to 80% adult hemoglobin (HbA) production in vitro. Uniform 2'-MOE SSOs do not mediate RNase H degradation when they bind their targets; therefore, they can be used to redirect the splicing machinery and restore WT splicing. After generating mouse erythroleukemia cells that carry the human IVS2-745 mutated beta-globin gene, lead 2'-MOE SSOs targeting the 745 mRNA were raised against these cell lines. With these lead SSOs we have demonstrated aberrant 745 to WT splice switching in 5 patient samples. CD34+ cells were isolated from the blood of four 745/β0 compound heterozygotes and one 745/ 745 homozygote (Breda et al, PloS One 2012). After CD34+ expansion, cells were differentiated to the red cell lineage and treated via syringe loading or lipofectamine transfection with 2'-MOE SSOs. Up to 80% HbA protein production was restored with 2'MOE-SSO treatment in the 745 homozygote patient sample (Figure 1), and up to 60% HbA in multiple 745 compound heterozygote specimens. Compared to 3-6% HbA in scramble treated controls, this represents up to a 20-fold increase in HbA with treatment. In addition to HbA production, we have shown improvement in other parameters characteristic in beta-thalassemia, such as the imbalance of alpha and beta chains and the accumulation of toxic alpha-only homotetramers. 2'MOE SSOs are able to reinstate balance of beta- to alpha-like chains, which resulted in a near elimination of toxic alpha-only homotetramers in the homozygote cell lysate as detected by HPLC (Figure 1). We further proved the benefit of 2'MOE SSOs in a 745/Sickle model system, where in vitro sickling was significantly reduced as a result of increased levels of HbA. To create this model system, we transduced CD34+ cells from a homozygous sickle patient specimen with a lentivirus expressing human IVS2-745 beta-globin. With vector copy numbers ~2, this system replicates what a single allele would do, as the 2 endogenous sickle alleles are equally matched. Upon differentiation and exposure to hypoxia, in vitro sickling was reduced by 50% in 2'MOE-SSO treated samples as compared to scramble controls (Figure 2). In summary, 2'MOE-SSOs are a promising therapy for certain splicing forms of beta-thalassemia. Their ability to correct the underlying splicing cause offers a pharmacological treatment that is both direct and specific. As such, this therapy could help patients reduce their transfusion dependence or even reach transfusion independence. Disclosures Guo: Ionis Pharmaceuticals: Employment, Equity Ownership. Peralta:Ionis Pharmacueticals: Employment. Cappellini:Celgene: Membership on an entity's Board of Directors or advisory committees; Genzyme-Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

1993 ◽  
Vol 13 (8) ◽  
pp. 4844-4851 ◽  
Author(s):  
G Zitnik ◽  
Q Li ◽  
G Stamatoyannopoulos ◽  
T Papayannopoulou
Keyword(s):  
Globin Gene ◽  
Cumulative Number ◽  
Beta Globin ◽  
Serum Factors ◽  
Beta Globin Gene ◽  
Cell Divisions ◽  
Growth Promoting ◽  
Gene Switching ◽  
Mouse Erythroleukemia

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.

scholarly journals The inactive beta globin gene on a gamma delta beta thalassemia chromosome has a normal structure and functions normally in vitro

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 766-770
Author(s):  
PT Curtin ◽  
YW Kan
Keyword(s):  
Globin Gene ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Ribonuclease Protection Assay ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Thalassemia Minor

We have previously described an English family with gamma delta beta- thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5′ to the cap site to 350 basepairs 3′ to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.

scholarly journals Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2781-2790
Author(s):  
DE Fleenor ◽  
RE Kaufman
Keyword(s):  
Topoisomerase Ii ◽  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Beta Globin Gene

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

1987 ◽  
Vol 7 (11) ◽  
pp. 4024-4029
Author(s):  
M Trudel ◽  
J Magram ◽  
L Bruckner ◽  
F Costantini
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Base Pairs ◽  
Beta Globin Gene ◽  
Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

scholarly journals A novel deletion of approximately 27 kb including the beta-globin gene and the locus control region 3'HS-1 regulatory sequence: beta zero- thalassemia or hereditary persistence of fetal hemoglobin?

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 822-827 ◽  
Author(s):  
AJ Dimovski ◽  
V Divoky ◽  
AD Adekile ◽  
E Baysal ◽  
JB Wilson ◽  
...  
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Locus Control Region ◽  
Regulatory Sequence ◽  
Beta Globin ◽  
Gamma Chain ◽  
Beta Globin Gene ◽  
Gamma Globin

Abstract A novel deletion of approximately 27 kb with the 5′ breakpoint 1.5 to 2.2 kb upstream of the beta-globin gene, and the 3′ breakpoint approximately 24 kb downstream of the beta-globin gene, has been found in five members of two families from Southeast Asia (Vietnam and Cambodia). Six members of another family from China, previously reported from our laboratory, have also been shown to carry this deletion. The patients presented with mild hypochromia and microcytosis, a hemoglobin (Hb) A2 level of approximately 4.0%, and a markedly increased, heterocellularly distributed, Hb F level (14.0 to 26.0%). In vitro globin-chain synthesis showed a mild imbalance with appreciable gamma-chain compensation (alpha/beta + gamma ratio of 1.46). The 3′ end of this deletion includes the 3′HS-1, and we hypothesize that removal of this region results in the loss of its gamma-globin gene-silencing effect, which causes a markedly elevated Hb F level with a modest increase in Hb A2 levels, unlike the situation in other deletional beta zero-thalassemias. The possible influence of particular sequence variations in the locus control region 5′HS-2 and the G gamma promoter, present on the chromosome with this deletion, on the overall gamma-globin gene should also be considered.

scholarly journals IGF2BP1 Reverses Hemoglobin Switching in Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 639-639
Author(s):  
Laxminath Tumburu ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Jaira F. de Vasconcellos ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adult Stage ◽  
Globin Gene ◽  
Expression Patterns ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Empty Vector ◽  
Cd34 Cells ◽  
Gene And Protein Expression

Abstract During human ontogeny, high-level transcription within the beta-globin gene cluster switches sequentially from embryonic-to-fetal-to-adult genes. Beta-thalassemias and sickle-cell disease are manifested by reduced or mutated expression of the adult-stage, beta-globin gene. Research is aimed toward the eventual therapeutic goal of safely preventing or reversing the fetal-to-adult hemoglobin switch among these patient populations. To identify genes that may be involved in regulation of the fetal-to-adult erythroid switch, purified CD34(+) cells from six umbilical cord (fetal) and six adult peripheral blood samples were cultured in serum-free medium, and gene expression libraries were prepared and sequenced from CD71(+), CD235a(+) erythroblast mRNA. In total, 546 million paired-end reads with a length of 101bp were generated for a comparison of cord and adult erythroblast transcriptomes. Reads were aligned to the human reference genome (hg19), and differential gene expression was identified [false discovery rate ≤ 0.05, fold change ≥ 1.5, and reads per kilobase per million mapped reads (RPKM) ≥ 0.5]. A total of 145 genes were differentially expressed according to these criteria, with four of the top five encoding targets of the let-7 family of microRNAs. The topmost gene was insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which is normally involved in transcriptome regulation and developmental timing. IGF2BP1 expression was 770-fold increased in the fetal erythroblasts (RPKM > 3.0) compared with low background levels in adult erythroblasts (RPKM < 0.01). IGF2BP1 protein is present in fetal tissues including fetal liver; however, it is not detected in adult human bone marrow. A potential role for adult-stage IGF2BP1 over-expression (IGF2BP1-OE) in the regulation of globin genes and proteins was explored using lentiviral vectors designed for let-7 resistant, erythroid-specific expression of IGF2BP1 protein. IGF2BP1-OE transduced CD34(+) cells expressed the transgenic protein and maintained their ability to differentiate, accumulate hemoglobin, and enucleate ex vivo in the presence of erythropoietin. Globin mRNA and protein levels were investigated. While alpha-globin mRNA remained unchanged, gamma-globin mRNA became predominant [90% of (gamma + beta) mRNA] in IGF2BP1-OE samples [Control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p < 0.05], and beta-globin mRNA decreased to minor levels [Control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p < 0.05]. IGF2BP1-OE caused a pan-cellular HbF distribution by flow cytometry. Cellular fetal hemoglobin percentages [HbF/(HbF + HbA)] were measured as 5.3 ± 0.4% in donor matched control cells versus 80.3 ± 3.7% in IGF2BP1-OE cells (p < 0.05). HPLC tracings revealed that the minor HbA2 peak, composed of alpha and delta globin chains, was reduced or absent in IGF2BP1-OE. Also, IGF2BP1-OE suppressed the expression of related genes including the transcription factor BCL11A. These data demonstrate that erythroblast IGF2BP1 is silenced in humans during fetal-to-adult ontogeny, and that IGF2BP1 in adult erythroblasts reverses the developmentally related switch in beta-like globin gene and protein expression patterns. Disclosures No relevant conflicts of interest to declare.

scholarly journals Eastern European (delta beta) zero-thalassemia: molecular characterization of a novel 9.1-kb deletion resulting in high levels of fetal hemoglobin in the adult [see comments]

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3738-3745 ◽  
Author(s):  
A Palena ◽  
A Blau ◽  
G Stamatoyannopoulos ◽  
NP Anagnou
Keyword(s):  
Globin Gene ◽  
Adult Life ◽  
Fetal Hemoglobin ◽  
Direct Repeat ◽  
Regulatory Elements ◽  
Negative Control ◽  
Beta Thalassemia ◽  
Eastern European ◽  
Beta Globin ◽  
Beta Globin Gene

A novel deletion in the human beta-globin gene cluster associated with increased levels of fetal hemoglobin (HbF) in adult life was molecularly characterized in a member of a family of Eastern European descent. The phenotype of the deletion, documented in five members of the family, shows mild hypochromia and microcytosis (mean corpuscular Hb, 24 to 25.9 pg; mean corpuscular volume, 74 to 78.5 fL) but high production of HbF (13% to 24%) with heterocellular distribution (36% to 86% F cells). Extensive restriction enzyme mapping of the beta-globin cluster and sequencing of the region encompassing the breakpoints showed that the deletion starts 1,612 bp upstream of the cap site of the delta-globin gene, and terminates within the first intron of the beta-globin gene, deleting 9.1 kb of DNA. This length is definitely shorter than the average 12.0 kb of the previously characterized (delta beta) zero-thalassemias. The 5′ breakpoint of the new deletion is close to that of the Yugoslavian delta beta-thalassemia deletion, whereas the 3′ breakpoint is very close to those of the Turkish and the Greek beta zero-thalassemia deletions. The breakpoints of the deletion occur within a direct repeat containing a tetranucleotide exhibiting homology to a donor-splice site, and is symmetrically flanked by a set of 13- and 14-bp homologous complementary sequences, respectively. It is likely that the deletion may be the result of an “illegitimate” or “nonhomologous” recombination event to which these two short sequences may have contributed. It is of interest that the novel deletion (9.1 kb) is comparable to the Italian HPFH-5 deletion (12.9 kb), regarding both the size and the position of the breakpoints. However, the HPFH-5 deletion includes sequences flanking the breakpoints that are preserved in the new deletion. Considering the resulting two discrete phenotypes (ie, delta beta-thalassemia v HPFH), it can be hypothesized that the deleted sequences in the Italian HPFH-5 mutation may harbor regulatory elements that exert a negative control on the gamma-globin gene expression.

Eastern European (delta beta) zero-thalassemia: molecular characterization of a novel 9.1-kb deletion resulting in high levels of fetal hemoglobin in the adult [see comments]

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3738-3745
Author(s):  
A Palena ◽  
A Blau ◽  
G Stamatoyannopoulos ◽  
NP Anagnou
Keyword(s):  
Globin Gene ◽  
Adult Life ◽  
Fetal Hemoglobin ◽  
Direct Repeat ◽  
Regulatory Elements ◽  
Negative Control ◽  
Beta Thalassemia ◽  
Eastern European ◽  
Beta Globin ◽  
Beta Globin Gene

Abstract A novel deletion in the human beta-globin gene cluster associated with increased levels of fetal hemoglobin (HbF) in adult life was molecularly characterized in a member of a family of Eastern European descent. The phenotype of the deletion, documented in five members of the family, shows mild hypochromia and microcytosis (mean corpuscular Hb, 24 to 25.9 pg; mean corpuscular volume, 74 to 78.5 fL) but high production of HbF (13% to 24%) with heterocellular distribution (36% to 86% F cells). Extensive restriction enzyme mapping of the beta-globin cluster and sequencing of the region encompassing the breakpoints showed that the deletion starts 1,612 bp upstream of the cap site of the delta-globin gene, and terminates within the first intron of the beta-globin gene, deleting 9.1 kb of DNA. This length is definitely shorter than the average 12.0 kb of the previously characterized (delta beta) zero-thalassemias. The 5′ breakpoint of the new deletion is close to that of the Yugoslavian delta beta-thalassemia deletion, whereas the 3′ breakpoint is very close to those of the Turkish and the Greek beta zero-thalassemia deletions. The breakpoints of the deletion occur within a direct repeat containing a tetranucleotide exhibiting homology to a donor-splice site, and is symmetrically flanked by a set of 13- and 14-bp homologous complementary sequences, respectively. It is likely that the deletion may be the result of an “illegitimate” or “nonhomologous” recombination event to which these two short sequences may have contributed. It is of interest that the novel deletion (9.1 kb) is comparable to the Italian HPFH-5 deletion (12.9 kb), regarding both the size and the position of the breakpoints. However, the HPFH-5 deletion includes sequences flanking the breakpoints that are preserved in the new deletion. Considering the resulting two discrete phenotypes (ie, delta beta-thalassemia v HPFH), it can be hypothesized that the deleted sequences in the Italian HPFH-5 mutation may harbor regulatory elements that exert a negative control on the gamma-globin gene expression.

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