Two Distinct Genetic Mechanisms Modify the Level of HbA2 in Peripheral Blood,

Abstract Abstract 3193 The switch from embryonic to fetal hemoglobin (HbF, α2γ2) in utero, and from fetal to adult hemoglobin at birth is well documented and achieved by the sequential activation of ε, γ and δ/β genes at the β globin gene (HBB) cluster. A change in the expression of hemoglobin genes also take place in adult erythropoiesis: earlier erythroid progenitors have been shown to produce significant amounts of fetal hemoglobin, while the more mature progenitors contain essentially none. In keeping with the sequential activation of β-like globin genes, δ globin chain synthesis also declines as maturation in erythroid progenitors progresses. Understanding the developmental changes of gene expression at the beta globin locus is not purely of academic interest, since a therapeutic induction of HbF or HbA2 (α2δ2) production would be of significant clinical benefit for patients with a defect of HbA (α2β2) function or abundance, such as sickle cell disease or β thalassemia. We have previously studied the genetic regulation of fetal hemoglobin persistence in a genome-wide association study (GWAS) in healthy volunteers, and are now extending this approach to the study of HbA2. Our study population is the 'Twins UK' twin registry of healthy Europeans, mostly female adult individuals, with genome-wide single polymorphisms (SNP) data and hemoglobin phenotypes for a primary study group (n=2,340) and a second replication group (n=1,880). A quantitative trait GWAS analysis was carried out to assess the relationship between SNPs and the HbA2 trait. We found that HbA2 (as a percentage of total hemoglobin) was weakly, but significantly, correlated with the amount of fetal hemoglobin carrying cells (F cells) an individual possesses (r = 0.14, p < 0.01). This suggests the existence of some common biological process that influences both hemoglobin species. We also found that the same SNP alleles at chromosome 6q23.3 (HBS1L-MYB, peak signal rs7775698, p = 2.51×10−9) that are associated with a boost in the prevalence of F cells and larger red blood cells (denoted by the mean cell volume or MCV) also promote HbA2 levels, again pointing to some common biological factor connected with the erythropoietic maturation process. Interestingly, neither of the other two major HbF loci, BCL11A on chromosome 2p, or the HbF-promoting regions within the HBB cluster (at the β LCR and the γ globin genes) on chromosome 11p, showed association with HbA2 levels. Instead, SNPs around the β globin gene itself (clearly separate also from the delta gene) exert a significant influence on HbA2 levels (peak association rs12793110, p=5.11×10−12) (see Figure 1). In contrast to the HBS1L-MYB region on chromosome 6, the HbA2-boosting alleles at these SNPs do not increase red blood cell MCV. We propose that the SNPs around HBB influences HbA2 (ie. δ globin gene) expression via a mechanism that is related to the competitive process between the β and δ gene expression that might mimic a very mild β thalassemic effect. Figure 1: Association with single-nucleotide polymorphisms (SNP) near the beta globin gene cluster on chromosome 11p15.4 with abundance of HbA2 (filled circles ¥) and F cells (empty circles ○) in the peripheral blood of Northern European adults (Twins UK). Figure 1:. Association with single-nucleotide polymorphisms (SNP) near the beta globin gene cluster on chromosome 11p15.4 with abundance of HbA2 (filled circles ¥) and F cells (empty circles ○) in the peripheral blood of Northern European adults (Twins UK). We propose that the systematic genetic study of specialized hematological traits in healthy volunteers can help to understand the biology of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age

Blood ◽

10.1182/blood.v85.4.1111.bloodjournal8541111 ◽

1995 ◽

Vol 85 (4) ◽

pp. 1111-1117 ◽

Cited By ~ 79

Author(s):

YC Chang ◽

KD Smith ◽

RD Moore ◽

GR Serjeant ◽

GJ Dover

Keyword(s):

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Beta Globin ◽

Gene Number ◽

Cohort Group ◽

Five Factors ◽

F Cells ◽

Alpha Globin

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

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Molecular analysis of the erythroid phenotype of a patient with BCL11A haploinsufficiency

Blood Advances ◽

10.1182/bloodadvances.2020003753 ◽

2021 ◽

Vol 5 (9) ◽

pp. 2339-2349

Author(s):

Marja W. Wessels ◽

Marjon H. Cnossen ◽

Thamar B. van Dijk ◽

Nynke Gillemans ◽

K. L. Juliëtte Schmidt ◽

...

Keyword(s):

Gene Expression ◽

De Novo ◽

Fetal Hemoglobin ◽

Chromatin Accessibility ◽

Neuronal System ◽

Globin Genes ◽

Erythroid Progenitors ◽

Genome Wide ◽

Localization Signal ◽

Significant Expression

Abstract The BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient’s cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in β-hemoglobinopathy patients. Our data strongly support this approach.

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Laotian (delta beta) degree-thalassemia: molecular characterization of a novel deletion associated with increased production of fetal hemoglobin

Blood ◽

10.1182/blood.v72.3.983.983 ◽

1988 ◽

Vol 72 (3) ◽

pp. 983-988 ◽

Cited By ~ 1

Author(s):

JW Zhang ◽

G Stamatoyannopoulos ◽

NP Anagnou

Keyword(s):

Globin Gene ◽

Repetitive Sequences ◽

Adult Life ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Mild Anemia ◽

F Cells

Abstract We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that increases fetal hemoglobin (HbF) synthesis in a 24-year-old Laotian man who is heterozygous for this mutation. The patient is asymptomatic with a mild anemia, hypochromia, and microcytosis (Ht = 39%, MCH = 22.8 pg, MCV = 71 fl), normal levels of HbA2 (3.0%) and 11.5% HbF (G gamma A gamma ratio 60 to 40), with heterocellular distribution (52% F cells). Extensive restriction endonuclease mapping defined the 5′ breakpoint within the IVS II of the delta-globin gene, between positions 775 to 781 very similar to the 5′ breakpoint of the Sicilian delta beta-thalassemia. However, the 3′ breakpoint was localized between two Pst I sites 4.7 kb 3′ of the beta- globin gene, thus ending about 0.7 kb upstream from the 3′ breakpoint of the Sicilian delta beta-thalassemia. This results in a 12.5 kb deletion of DNA. It is of interest that the 5′ breakpoint of the deletion residues within an AT-rich region which has been proposed as a specific recognition signal for recombination events, while the 3′ breakpoint lies within a cluster of L1 repetitive sequences (formerly known as Kpn I family repeats). The presence of the 3′ breakpoints of several other deletions within this region of L1 repeats also suggests that such sequences might serve as hot spots for recombination and eventually lead to thalassemia deletions. The similarity of the 5′ and 3′ breakpoints of these delta beta-thalassemias underscores the putative regulatory role of the deleted and juxtaposed sequences on the expression of the gamma-globin genes in adult life.

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“Definitive” Erythropoiesis Has Distinct Developmental Origins and Globin Expression Patterns in the Mammalian Embryo.

Blood ◽

10.1182/blood.v114.22.2539.2539 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2539-2539

Author(s):

Kathleen E. McGrath ◽

Jenna M Frame ◽

George Fromm ◽

Anne D Koniski ◽

Paul D Kingsley ◽

...

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Low Levels ◽

Globin Gene Expression

Abstract Abstract 2539 Poster Board II-516 A transient wave of primitive erythropoiesis begins at embryonic day 7.5 (E7.5) in the mouse as yolk sac-derived primitive erythroid progenitors (EryP-CFC) generate precursors that mature in the circulation and expand in numbers until E12.5. A second wave of erythroid progenitors (BFU-E) originates in the yolk sac beginning at E8.25 that generate definitive erythroid cells in vitro. These BFU-E colonize the newly forming liver beginning at E10.5, prior to the initial appearance there of adult-repopulating hematopoietic stem cells (HSCs) between E11.5-12.5. This wave of definitive erythroid yolk sac progenitors is proposed to be the source of new blood cells required by the growing embryo after the expansion of primitive erythroid cells has ceased and before HSC-derived hematopoiesis can fulfill the erythropoietic needs of the embryo. We utilized multispectral imaging flow cytometry both to distinguish erythroid lineages and to define specific stages of erythroid precursor maturation in the mouse embryo. Consistent with this model, we found that small numbers of definitive erythrocytes first enter the embryonic circulation beginning at E11.5. All maturational stages of erythroid precursors were observed in the E11.5 liver, consistent with these first definitive erythrocytes having rapidly completed their maturation in the liver. The expression of βH1 and εy-beta globin genes is thought to be limited to primitive erythroid cells. Surprisingly, examination of globin gene expression by in situ hybridization revealed high levels of βH1-, but not εy-globin, transcripts in the parenchyma of E11.5-12.5 livers. RT-PCR analysis of globin mRNAs confirmed the expression of βH1- and adult β1-, but not εy-globin, in E11.5 liver-derived definitive (ckit+, Ter119lo) proerythroblasts sorted by flow cytometry to remove contaminating primitive (ckit-, Ter119+) erythroid cells. A similar pattern of globin gene expression was found in individual definitive erythroid colonies derived from E9.5 yolk sac and from early fetal liver. In vitro differentiation of definitive erythroid progenitors from E9.5 yolk sac revealed a maturational “switch” from βH1- and β1-globins to predominantly β1-globin. βH1-globin transcripts were not observed in proerythroblasts from bone marrow or E16.5 liver or in erythroid colonies from later fetal liver. ChIP analysis revealed that hyperacetylated domains encompass all beta globin genes in primitive erythroid cells but only the adult β1- and β2-globin genes in E16.5 liver proerythroblasts. Consistent with their unique gene expression, E11.5 liver proerythroblasts have hyperacetylated domains encompassing the βh1-, β1- and β2-, but not εy-globin genes. We also examined human globin transgene expression in mice carrying a single copy of the human beta globin locus. Because of the overlapping presence and changing proportion of primitive and definitive erythroid cells during development, we analyzed sorted cell populations whose identities were confirmed by murine globin gene expression. We confirmed that primitive erythroid cells express higher levels of γ- than ε-globin and little β-globin. E11.5 proerythroblasts and cultured E9.5 progenitors express γ- and β-, but not ε-globin. E16.5 liver proerythroblasts express β- and low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. In summary, two forms of definitive erythropoiesis emerge in the murine embryo, each with distinct globin expression patterns and chromatin modifications of the β-globin locus. While both lineages predominantly express adult globins, the first, yolk sac-derived lineage uniquely expresses low levels of the embryonic βH1-globin gene as well as the human γ-globin transgene. The second definitive erythroid lineage, found in the later fetal liver and postnatal marrow, expresses only adult murine globins as well as low levels of the human γ-globin transgene only in the fetus. Our studies reveal a surprising complexity to the ontogeny of erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Laotian (delta beta) degree-thalassemia: molecular characterization of a novel deletion associated with increased production of fetal hemoglobin

Blood ◽

10.1182/blood.v72.3.983.bloodjournal723983 ◽

1988 ◽

Vol 72 (3) ◽

pp. 983-988 ◽

Cited By ~ 14

Author(s):

JW Zhang ◽

G Stamatoyannopoulos ◽

NP Anagnou

Keyword(s):

Globin Gene ◽

Repetitive Sequences ◽

Adult Life ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Mild Anemia ◽

F Cells

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that increases fetal hemoglobin (HbF) synthesis in a 24-year-old Laotian man who is heterozygous for this mutation. The patient is asymptomatic with a mild anemia, hypochromia, and microcytosis (Ht = 39%, MCH = 22.8 pg, MCV = 71 fl), normal levels of HbA2 (3.0%) and 11.5% HbF (G gamma A gamma ratio 60 to 40), with heterocellular distribution (52% F cells). Extensive restriction endonuclease mapping defined the 5′ breakpoint within the IVS II of the delta-globin gene, between positions 775 to 781 very similar to the 5′ breakpoint of the Sicilian delta beta-thalassemia. However, the 3′ breakpoint was localized between two Pst I sites 4.7 kb 3′ of the beta- globin gene, thus ending about 0.7 kb upstream from the 3′ breakpoint of the Sicilian delta beta-thalassemia. This results in a 12.5 kb deletion of DNA. It is of interest that the 5′ breakpoint of the deletion residues within an AT-rich region which has been proposed as a specific recognition signal for recombination events, while the 3′ breakpoint lies within a cluster of L1 repetitive sequences (formerly known as Kpn I family repeats). The presence of the 3′ breakpoints of several other deletions within this region of L1 repeats also suggests that such sequences might serve as hot spots for recombination and eventually lead to thalassemia deletions. The similarity of the 5′ and 3′ breakpoints of these delta beta-thalassemias underscores the putative regulatory role of the deleted and juxtaposed sequences on the expression of the gamma-globin genes in adult life.

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A β-Globin Locus-Intrinsic Epigenetic Mechanism Underlies Fetal Globin Production in F-Cells

Blood ◽

10.1182/blood-2020-137646 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 16-17

Author(s):

Eugene Khandros ◽

Peng Huang ◽

Scott A. Peslak ◽

Malini Sharma ◽

Osheiza Abdulmalik ◽

...

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Epigenetic Mechanism ◽

Chromatin Domain ◽

Globin Genes ◽

Chromatin Architecture ◽

A Cells ◽

F Cells ◽

Globin Gene Expression

Upregulation of fetal hemoglobin (HbF, α2γ2) by reversing the developmental switch to adult HbA (α2β2) is a key approach for both pharmacologic and gene targeting therapies in the treatment of sickle cell disease (SCD) and β-thalassemia. HbF expression in healthy individuals, patients with SCD, and those treated with hydroxyurea is restricted to a subset of red blood cells known as F-cells; effective SCD therapy requires increasing the proportion of F-cells expressing sufficient HbF to block sickling. Although these cells have been observed since the 1950s, there have not been previous direct comparisons of F-cells to matched HbF-low A-cells from the same individual. Fetal erythroblasts have distinct global transcriptional programs and distinct long-range chromatin looping at the β-globin locus when compared to adult erythroblasts. An important question is therefore whether F-cells are formed through reversion to a fetal-like state at the transcriptional and epigenetic level. To address this clinically important question, we previously reported development of new techniques for the purification of stage-matched F- and A-erythroblasts from primary human CD34+ cell erythroid cultures and their downstream analysis (Khandros et al, Blood 2020). We demonstrated that F-cells in primary erythroid cultures have minimal transcriptional differences with A-cells and that the few differentially expressed transcripts do not overlap with fetal-specific transcripts. Furthermore, treatment with hydroxyurea or pomalidomide did not enhance transcriptional differences between F- and A-cells. Surprisingly, we did not find differences in the expression of any known HbF regulators such as BCL11A, LRF, or NuRD complex members that would account for differential HbF expression. Based on these findings, we hypothesized that F-cells are distinguished by epigenetic variation specifically at the β-globin locus. Given that fetal erythroblasts differ from adult erythroblasts in the chromatin architecture of the β-globin locus (e.g. Huang et al, Genes and Development 2017), we compared the higher order chromatin organization of the β-globin locus between F- and A-cells by Capture-C, a next-generation sequencing-adapted form of chromatin conformation capture. We found that in F-cells, contacts between the distal enhancer and the promoters of the fetal globin genes HBG1 and HBG2 were increased, while those between the enhancer and adult globin genes (HBB and HBD) were reduced. Other architectural changes associated with fetal globin gene expression, including fetal specific contacts of an intergenic non-coding gene with chromatin domain boundaries at the β-globin locus were also partially enriched in F-cells. We also did not find any differences in promoter-enhancer contacts between F- and A-cells for other developmentally regulated genes BCL11A, LIN28B, and THRB. Together these results are consistent with the concept that epigenetic changes associated with nuclear architecture that occur specifically at the β-globin locus underlie the difference in globin gene expression profiles between F- and A-cells. In sum our data demonstrate that in adult erythropoiesis, F-cells do not arise through either a wholesale reversion to a fetal-like genetic program or through variation in any known HbF regulators. Instead, modulation of chromatin architecture intrinsic to the β-globin locus, perhaps in a stochastic manner, accounts for elevated fetal globin expression in F-cells. We are currently performing mechanistic studies to elucidate the basis for the epigenetic regulation of the β-globin locus in F-cells. These studies will further our understanding of fetal hemoglobin regulation in adult cells and might inform new therapeutic approaches for SCD and β-thalassemia. Disclosures Blobel: Fulcrum Therapeutics: Consultancy; Pfizer: Research Funding.

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A Mutation in a GATA-1 Binding Site 5′ to the Gγ-Globin Gene (nt -567, T>G) May Be Associated with Increased Levels of Fetal Hemoglobin.

Blood ◽

10.1182/blood.v104.11.500.500 ◽

2004 ◽

Vol 104 (11) ◽

pp. 500-500 ◽

Cited By ~ 1

Author(s):

H.Y. Luo ◽

D. Mang ◽

G.P. Patrinos ◽

C.J.Y. Wu ◽

S.H. Eung ◽

...

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Family Members ◽

Point Mutations ◽

Fetal Hemoglobin ◽

Proximal Promoter ◽

Globin Genes ◽

Nucleotide Polymorphisms ◽

Mobility Shift ◽

Globin Gene Expression

Abstract The identification of single nucleotide polymorphisms (SNPs) associated with increased fetal hemoglobin (Hb F) levels in adults provide important insights to the regulation of γ-globin gene expression, and the modulation of Hb F production in severe β hemoglobinopathies. Fourteen known point mutations located between nucleotide (nt) −110 to −205 5′ to the Gγ- and Aγ-globin genes are associated with hereditary persistence of fetal hemoglobin (HPFH). These likely affect the interactions between transcription factors and proximal promoter elements. We investigated both parents and 2 sons of an Iranian-American family, in whom the father and his younger son had elevated Hb F levels (See table). The mother and her older son were heterozygous for the (−α3.7) single α-globin gene deletion, likely accounting for their borderline microcytosis. Extensive nt sequencing of the β-globin gene and promoters of Gγ- and Aγ-globin genes was carried out. No known β-thalassemia mutation was detected in any of the 4 family members. None of the known HPFH point mutations was present. The C>T SNP (Xmn I) at nt −158 5′ to the Gγ-globin gene that has been associated with increased Gγ-globin gene expression was also not found. However, a novel T>G substitution was detected at nt −567 5′ to the Gγ-globin gene in the father and his younger son, but not in the mother and her older son. This SNP alters a putative GATA-1 binding sequence, AGATAA to AGAGAA. Haplotyping of the Gγ Aγβ region in the family showed that the T>G SNP in the father and his younger son resides on the same GγAγβ haplotype. This SNP was not present in 15 individuals of diverse racial and ethnic origins, in 186 Thai individuals, and in 133 of 134 Iranians living in Tehran. To our knowledge, this SNP has not been previously reported in the literature. To begin to study the functional significance of this SNP, gel mobility shift analysis was done with two 40 nt long oligomers, one with the wild type GATA sequence and the other with the mutated GAGA sequence, using uninduced mouse erythroleukemia (MEL) cell nuclear extracts. The mutant GAGA sequence results in a complete loss of GATA-1 binding. The region from nt −382 to −730 5′ to the Aγ-globin gene was reported to be related to Aγ-globin gene silencing (Stamatoyannopoulos et al, Mol Cell Biol 13:7636, 1993). The nt −567 T>G SNP is located within the comparable region of the Gγ-globin gene, which is highly homologous to the Aγ-globin gene. Among the 4 family members under study, no other SNPs are found in the same region. Taken together, these observations raise the possibility that the T>G SNP at nt −567 5′ to the Gγ-globin gene is associated with elevated Hb F, that might be caused by a novel mechanism, i.e., incomplete silencing of the Gγ-globin gene, resulting from the abolished GATA-1 binding. Additional clinical and functional studies will be needed to further document the effect of this SNP upon Gγ-globin gene expression and to ascertain that this SNP represents a HPFH mutation. Hematological Data of Family Father Mother Son Son Age 52 44 13 9 Hb 15.7 14.1 13.2 13.8 MCV 82 77 75 75 Hb A2 2.5 % 3.0 % 3.4 % 3.3 % Hb F 10.2 % 0.7 % 0.7 % 5.9 %

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High Throughput Screen To Identify Small Molecules That Differentially Regulate Expression of the Globin Genes.

Blood ◽

10.1182/blood.v106.11.3632.3632 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3632-3632

Author(s):

Benjamin L. Ebert ◽

Raymond Mak ◽

Jennifer L. Pretz ◽

David Peck ◽

Stephen Haggerty ◽

...

Keyword(s):

Gene Expression ◽

High Throughput ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Globin Genes ◽

Beta Globin ◽

Diversity Oriented Synthesis ◽

Gamma Globin ◽

Globin Gene Expression

Abstract Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.

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Molecular analysis of Japanese delta beta-thalassemia

Blood ◽

10.1182/blood.v72.5.1771.bloodjournal7251771 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1771-1776

Author(s):

S Shiokawa ◽

H Yamada ◽

Y Takihara ◽

E Matsunaga ◽

Y Ohba ◽

...

Keyword(s):

Mammalian Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

Large Deletions ◽

Deletion Junction

A DNA fragment containing the deletion junction region from a Japanese individual with homozygous delta beta-thalassemia has been cloned. A clone containing the normal DNA surrounding the 3′ breakpoint of this deletion and a clone carrying the G gamma- and A gamma-globin genes of this patient were also isolated. Sequences of the deletion junction and both gamma-globin genes were determined. A comparison of these sequences with previously determined sequences of the normal counterparts revealed that the 5′ breakpoint is located between 2,134 and 2,137 base pairs (bp) 3′ to the polyA site of the A gamma-globin gene, the 5′ breakpoint is located just downstream of the 3′ border of the fetal gamma-globin duplication unit, and no molecular defects are evident within the gamma-globin gene region. A comparison between the sequences of the normal DNA surrounding the 3′ breakpoint and the normal DNA surrounding the 5′ breakpoint shows that deletion is the result of a nonhomologous recombination event. There are A+T-rich stretches near the 5′ and 3′ breakpoints in the normal DNA, and a portion of an Aly repeat is located in the region 3′ to the 3′ breakpoint. Southern blot analysis using probes 3′ to the beta-globin gene showed that the deletion extends in the 3′ direction further than any other deletions associated with delta beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) heretofore reported. These results are discussed in terms of the mechanism generating large deletions in mammalian cells and three models for the regulation of gamma-globin and beta-globin gene expression in humans.

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Concordance of a point mutation 5' to the G gamma globin gene with G gamma beta +. Hereditary persistence of fetal hemoglobin in the black population

Blood ◽

10.1182/blood.v64.6.1292.1292 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1292-1296 ◽

Cited By ~ 14

Author(s):

FS Collins ◽

CD Boehm ◽

PG Waber ◽

CJ Jr Stoeckert ◽

SM Weissman ◽

...

Keyword(s):

Point Mutation ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Black Population ◽

Globin Genes ◽

Beta Globin ◽

Restriction Endonuclease Site ◽

Benign Condition ◽

The Third ◽

Hereditary Persistence

Abstract Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5′ to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.

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