Molecular Analysis of Alpha Hemoglobin Stabilizing Protein (AHSP) in Caucasian Patients with different Beta-Thalassemia Phenotypes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3770-3770 ◽  
Author(s):  
M. Domenica Cappellini ◽  
Chiara Refaldi ◽  
Daniela Bignamini ◽  
Laura Zanaboni ◽  
Gemino Fiorelli
Keyword(s):  
High Performance ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Clinical Severity ◽  
Beta Thalassemia ◽  
Red Cell ◽  
Globin Genes ◽  
Nucleotide Polymorphisms ◽  
Thai Population ◽  
Globin Chains

Abstract Beta-thalassemia is a inherited hemoglobin disorder characterized by absent or reduced synthesis of the b globin chains. The pathophysiology and the severity of b-thalassemias reflect the degree of globin chain imbalance and the excess of free a globin chains that precipitate and cause oxidative damage in red cell precursors inducing their premature destruction in the bone marrow (ineffective erythropoiesis). Although the phenotype of b thalassemias can be modified by inherited factors such as different number of a globin genes or increased fetal hemoglobin production, other mechanisms appear to be involved. Recently, a protein, named alpha hemoglobin stabilizing protein (AHSP), that acts as a molecular chaperone specifically for free a globin chains, preventing their precipitation in red cell precursors, has been identified. To establish whether AHSP might have a role in modifying the clinical outcome of b thalassemias, we have analyzed the AHSP gene in 70 Caucasian b thalassaemic subjects: 26 patients with b°/b° genotype (Thalassaemia Major),24 patients with Thalassemia Intermedia (b°/b+ or b+/b+) and 20 patients with a Thalassaemia Intermedia phenotype but with only one mutation in the b globin gene, a normal a globin genotype and no other causes of anemia. In all the subjects, we have performed Denaturing High-Performance Liquid Chromatography (DHPLC) of the three exons and the direct genomic sequencing of coding and noncoding regions (~ 1.5 kb) of AHSP gene. No mutations able to modify the structure or function of AHSP have been found, however we identified eight single nucleotide polymorphisms (SNPs) spanned along the whole gene that segregate in four different aplotypes. To evaluate a possible relationship between a particular aplotype and b thalassemia severity, the allele frequency of each single aplotype in the tree groups has been established and compared to that of 33 Caucasian normal controls: no statistically significant association has been proved. Even though the loss of AHSP aggravates the b thalassaemia phenotype in mice, in Thalassemic Caucasian population the AHSP apparently doesn’t make changes in the clinical severity of b thalassemia confirming the results recently found in Thai population.

scholarly journals Molecular analysis of Japanese delta beta-thalassemia

Blood ◽  
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.

Haemoglobinopathies that occur with decreased HbA2 levels: a gene mutation set involving the δ gene at a Spanish centre

2016 ◽  
Vol 70 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Ana Villegas ◽  
Fernando Ataúlfo González ◽  
Jorge M Nieto ◽  
Félix de la Fuente-Gonzalo ◽  
Rafael Martínez ◽  
...  
Keyword(s):  
High Performance ◽  
Globin Gene ◽  
Reversed Phase ◽  
Globin Genes ◽  
Reversed Phase Hplc ◽  
Structural Variants ◽  
Globin Chains ◽  
Zone Electrophoresis ◽  
Essential Parameter ◽  
Capillary Zone

AimsHaemoglobin A2 (HbA2) consists of two globin chains, α and β. Alterations in any of these genes influences the level of HbA2. Here, we present cases of structural Hb variants and thalassaemias which present either alone or together and reduce the level of HbA2 at varying degrees. Furthermore, we present a novel structural mutation in the δ globin gene, called Hb A2-Madrid.MethodsThe levels of HbA2 and HbF and the different haemoglobin variants were measured and analysed by ion exchange high performance liquid chromatography (HPLC, VARIANT II), the types of haemoglobins were determined by capillary zone electrophoresis (CZE) (Sebia) and the globin chains were determined by reversed-phase HPLC. Genetic analysis was performed by automatic sequencing of the α and δ genes as well as by multiple PCRs for the α globin genes.ResultsIn α thalassaemia (n=94), the HbA2 levels ranged from 1.39% to 2.43%. Among individuals with δ thalassaemia (n=5), the HbA2 level of those with δ+ thalassaemia was 1.77%, and that of those with δ0 thalassaemia was 1.70%. Among the individuals with δβ thalassaemia (n=13), those who were homozygous lacked HbA2. All structural haemoglobinopathies (n=97) were heterozygous; the α chain variants (n=84) presented with an HbA2 level of 1.76%, while the δ chain variants (n=13) presented with a level of 1.75%.ConclusionHbA2 is an essential parameter in the diagnostics of haemoglobinopathies. HPLC-EC and CZE allow the quantification of HbA2. Here, we show that quantification of HbA2 is critical for the identification of α, δ and βδ thalassaemias. Structural variants are discovered by HPLC. Molecular genetics is required for the proper identification of the mutations. Only with this knowledge is genetic counselling possible.

scholarly journals Molecular analysis of Japanese delta beta-thalassemia

Blood ◽  
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

Abstract 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.

scholarly journals Genetic Engineering in Hematopoietic Stem Cells for Gene Therapy of Hemoglobinopathies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-23-SCI-23
Author(s):  
Giuliana Ferrari
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Genetically Modify ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Hematopoietic Stem

Beta-thalassemia and sickle cell disease (SCD) are congenital anemias caused by mutations in the beta-globin gene, resulting in either reduced/absent production of globin chains or abnormal hemoglobin structure. At present, the definitive cure is represented by allogeneic hematopoietic stem cell transplantation, with a probability to find a well-matched donor of <25%. Experimental gene therapy for hemoglobinopathies is based on transplantation of autologous hematopoietic stem cells genetically modified to express therapeutic hemoglobin levels. Approaches to genetically modify HSCs for treatment of hemoglobinopathies include: 1) the addition of globin genes by lentiviral vectors and 2) gene editing by nucleases to reactivate fetal hemoglobin either through inhibition of repressors or by reproducing mutations associated with high fetal hemoglobin levels. The outcomes of early clinical trials are showing the safety and potential efficacy, as well as the hurdles still limiting a general application.Current challenges and improved strategies will be presented and discussed. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Plerixafor

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

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 983-988 ◽  
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.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3193-3193
Author(s):  
Swee Lay Thein ◽  
Chad P. Garner ◽  
Tim D Spector ◽  
Stephan Menzel
Keyword(s):  
Gene Expression ◽  
Peripheral Blood ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Nucleotide Polymorphisms ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Genome Wide ◽  
F Cells

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.

scholarly journals Fetal hemoglobin silencing in humans

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2081-2086 ◽  
Author(s):  
Patricia A. Oneal ◽  
Nicole M. Gantt ◽  
Joseph D. Schwartz ◽  
Natarajan V. Bhanu ◽  
Y. Terry Lee ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Postnatal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Age Related

Abstract Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

scholarly journals Prediction of RNA Secondary Structure at IVS1 Mutation of Beta Globin Gene

2020 ◽  
Vol 13 (1) ◽  
pp. 247-252
Author(s):  
Nur Imaniati Sumantri ◽  
Dian Rachma Wijayanti
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Globin Chains ◽  
Intronic Mutation

Background: Beta globin gene is responsible for producing beta globin chains that stabilize the structure and function of hemoglobin. This gene expression is controlled by complex interactions of transcriptions factors and its regulatory elements in a specific manner. Disturbed beta globin genes may result in hemoglobinopathies, mainly sickle cell disease and beta thalassemia. It seems interesting that several mutations occurring in intronic region results in severe symptoms to beta thalassemia patients, such an IVS1nt5 G>C. This research aimed to analyze RNA structural alteration effected by intronic mutation of beta thalassemia. Methods: The most prevalent mutation of beta thalassemia in Indonesia was obtained from Ithanet. The RNA secondary structure of IVS1nt5 G>C and beta globin gen (HBB) wildtype were performed by RNAStructure, along with probknot prediction. Results: The result showed that intronic mutation caused conformational change in beta globin secondary structure, either for max expect or base pairing probability approach. The mutant had bigger and more loops that diminished the protein stability. Thus, the structure might undergo dysfunction. Conclusion: The comprehensive structural-functional significance of these findings needs further study.

scholarly journals The 18- to 23-kb deletion of the Macedonian delta beta-thalassemia includes the entire delta and beta globin genes

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 971-974
Author(s):  
GD Efremov ◽  
N Nikolov ◽  
Y Hattori ◽  
I Bakioglu ◽  
TH Huisman
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Restriction Sites ◽  
Endonuclease Mapping

Restriction endonuclease mapping analyses were made of DNA from a few members of a Macedonian family with hematological characteristics of delta beta-thalassemia, ie, microcytosis, normal HbA2 levels, and elevated levels of HbF (7% to 14%) with G gamma (average 40.5%) and A gamma T chains (average 59.5%). A large deletion of 18 to 23 kb was present with a 5′ breakpoint within a 670-bp segment of DNA between the HpaI and NcoI restriction sites 5′ to the delta globin gene, and a 3′ breakpoint between the BamHI and HpaI restriction sites located some 9 to 13 kb 3′ to the beta globin gene. This deletion is different from those present in other types of G gamma A gamma(delta beta)zero- thalassemia. The similarity of the hematological expression of these delta beta-thalassemic conditions which have somewhat comparable 5′ breakpoints supports the idea that an important fetal hemoglobin- controlling region lies between the psi beta and delta globin genes.

scholarly journals The 18- to 23-kb deletion of the Macedonian delta beta-thalassemia includes the entire delta and beta globin genes

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 971-974 ◽  
Author(s):  
GD Efremov ◽  
N Nikolov ◽  
Y Hattori ◽  
I Bakioglu ◽  
TH Huisman
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Restriction Sites ◽  
Endonuclease Mapping

Abstract Restriction endonuclease mapping analyses were made of DNA from a few members of a Macedonian family with hematological characteristics of delta beta-thalassemia, ie, microcytosis, normal HbA2 levels, and elevated levels of HbF (7% to 14%) with G gamma (average 40.5%) and A gamma T chains (average 59.5%). A large deletion of 18 to 23 kb was present with a 5′ breakpoint within a 670-bp segment of DNA between the HpaI and NcoI restriction sites 5′ to the delta globin gene, and a 3′ breakpoint between the BamHI and HpaI restriction sites located some 9 to 13 kb 3′ to the beta globin gene. This deletion is different from those present in other types of G gamma A gamma(delta beta)zero- thalassemia. The similarity of the hematological expression of these delta beta-thalassemic conditions which have somewhat comparable 5′ breakpoints supports the idea that an important fetal hemoglobin- controlling region lies between the psi beta and delta globin genes.

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