scholarly journals Suppression of human alpha-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors.

1994 ◽  
Vol 179 (2) ◽  
pp. 733-738 ◽  
Author(s):  
S Ponnazhagan ◽  
M L Nallari ◽  
A Srivastava
Keyword(s):  
Globin Gene ◽  
Gene Promoter ◽  
Early Gene ◽  
Beta Thalassemia ◽  
Transcriptional Level ◽  
Globin Genes ◽  
Antisense Gene ◽  
Adeno Associated Virus ◽  
Bacterial Gene ◽  
Alpha Globin

We sought to investigate the usefulness of the adeno-associated virus 2 (AAV)-based vectors to suppress the excess production of the human alpha-globin gene product towards developing a treatment modality for beta-thalassemia since accumulation of free alpha-globin reduces the lifespan of red blood cells in these patients. We constructed recombinant AAV virions containing the human alpha-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (TK) promoter, the SV40 early gene promoter, and the human alpha-globin gene promoter, respectively, as well as a bacterial gene for resistance to neomycin (neoR) as a selectable marker. These recombinant virions were used to infect a human erythroleukemia cell line (K562) that express high levels of alpha-globin mRNA. Clonal populations of neoR cells were obtained after selection with the drug G418, a neomycin analogue. Total genomic DNA samples isolated from these cells were analyzed on Southern blots to document stable integration of the transduced neo and alpha-globin genes. Total cellular RNA samples isolated from mock-infected and recombinant virus-infected cultures were also analyzed by Northern blots. Whereas the TK promoter-driven antisense alpha-globin sequences showed no inhibition of expression of the endogenous alpha-globin gene, the SV40 promoter and the alpha-globin gene promoter-driven antisense alpha-globin sequences suppressed the expression of this constitutively over-expressed gene by approximately 29 and 91%, respectively, at the transcriptional level. These studies suggest the feasibility of utilizing the AAV-based antisense gene transfer approach in the potential treatment of beta-thalassemia.

scholarly journals Beta zero-thalassemia in association with a gamma-globin gene quadruplication

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1394-1397
Author(s):  
KG Yang ◽  
JZ Liu ◽  
F Kutlar ◽  
A Kutlar ◽  
C Altay ◽  
...  
Keyword(s):  
Clinical Condition ◽  
Globin Gene ◽  
Normal Chromosome ◽  
Gene Arrangement ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Gamma Globin ◽  
Alpha Globin

We have studied the hematology, hemoglobin composition, and globin gene arrangements in one young Turkish boy with a beta zero-thalassemia homozygosity and in 11 of his relatives. Evidence is presented that the chromosome with the beta zero-thalassemia determinant carries a gamma- globin gene quadruplication, perhaps in a -G gamma-G gamma-G gamma-A gamma-gene arrangement. The eight gamma-globin genes in this patient produced G gamma and A gamma chains in a 95 to 5 ratio, and nearly 99% of the patient's hemoglobin was of the fetal type. The clinical condition resembled that of a thalassemia intermedia. HbF levels in eight beta-thalassemia heterozygotes varied between 0.5 and 4.2% and the percentages of G gamma in this HbF averaged at 87% or 95%; this level is to some extent related to the haplotype of the normal chromosome. All subjects carried four alpha-globin genes; a new BglII polymorphism was observed within the psi alpha-globin gene.

Phenotypic effect of heterozygous alpha and beta 0-thalassemia interaction

Blood ◽  
1983 ◽  
Vol 62 (1) ◽  
pp. 226-229 ◽  
Author(s):  
MA Melis ◽  
M Pirastu ◽  
R Galanello ◽  
M Furbetta ◽  
T Tuveri ◽  
...  
Keyword(s):  
Globin Gene ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Phenotypic Effect ◽  
Screening Programs ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Red Blood Cell Indices ◽  
Endonuclease Mapping ◽  
Glucose 6 Phosphate Dehydrogenase

In this study, we carried out restriction endonuclease mapping in order to characterize the alpha-globin genotype of 10 Sardinian beta 0- thalassemia heterozygotes, all of whom presented with normal red blood cell indices and increased HbA2 levels. In 8 of these subjects, we found the deletion of two alpha-globin genes (-alpha/-alpha), and in the remaining two the deletion of a single alpha-globin gene (- alpha/alpha alpha). In three of these carriers with the (-alpha/-alpha) alpha-globin genotype and in one with the (-alpha/alpha alpha) genotype, we also found the glucose-6-phosphate dehydrogenase (G6PD) defect of the Mediterranean type. On the basis of these findings, we may conclude that the interaction of heterozygous beta 0-thalassemia with alpha-thalassemia, due to the deletion of either one or two alpha- globin genes, may lead to the production of red blood cells with normal indices. The association of the G6PD defect with this thalassemia gene complex may eventually contribute to this effect. We suggest, therefore, that screening programs for heterozygous beta-thalassemia in populations where alpha-thalassemia is also prevalent, should incorporate the determination of HbA2 in the first set of tests.

scholarly journals Developmental silencing of the embryonic zeta-globin gene: concerted action of the promoter and the 3'-flanking region combined with stage-specific silencing by the transcribed segment.

1996 ◽  
Vol 16 (6) ◽  
pp. 2637-2646 ◽  
Author(s):  
S A Liebhaber ◽  
Z Wang ◽  
F E Cash ◽  
B Monks ◽  
J E Russell
Keyword(s):  
Gene Silencing ◽  
Fetal Liver ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Promoters ◽  
Globin Genes ◽  
Alpha Globin ◽  
Flanking Region ◽  
Zeta Globin Gene ◽  
The Individual

Globin gene switching is a well-described model of eucaryotic developmental control. In the case of the human alpha-globin gene cluster, migration of erythropoietic activity from the embryonic yolk sac to the fetal liver is parallaled by the zeta-globin gene silencing and enhanced expression of the alpha-globin genes. To map critical cis determinants of this switch, the human zeta-globin gene, the alpha-globin gene, and chimeric recombinants were introduced into the mouse genome. Consistent with previous studies, expression of the individual alpha- and zeta-globin transgenes was found to be developmentally appropriate. Contrary to current models, however, the alpha- and zeta-globin gene promoters were not sufficient to establish this control. Instead, full silencing of the zeta-globin gene required the combined activities of this promoter, transcribed region, and 3'-flanking sequences. Individually, the silencing activities of the zeta-globin gene promoter and 3'-flanking region were minimal but increased markedly when both regions were present. The zeta-globin transcribed region appeared to contribute to gene silencing by a mechanism specifically activated in definitive erythroblasts in the fetal liver. These data demonstrate that a complex set of controls, requiring at least three determinants and involving at least two independent mechanisms, is necessary for full developmental silencing of the human zeta-globin gene.

scholarly journals Usefulness of NGS for Diagnosis of Dominant Beta-Thalassemia and Unstable Hemoglobinopathies in Five Clinical Cases

2021 ◽  
Vol 12 ◽  
Author(s):  
Valeria Rizzuto ◽  
Tamara T. Koopmann ◽  
Adoración Blanco-Álvarez ◽  
Barbara Tazón-Vega ◽  
Amira Idrizovic ◽  
...  
Keyword(s):  
De Novo ◽  
Globin Gene ◽  
Clinical Manifestations ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Alpha Globin ◽  
Abnormal Hemoglobin ◽  
High Index Of Suspicion ◽  
Clinical Cases

Unstable hemoglobinopathies (UHs) are rare anemia disorders (RADs) characterized by abnormal hemoglobin (Hb) variants with decreased stability. UHs are therefore easily precipitating, causing hemolysis and, in some cases, leading to dominant beta-thalassemia (dBTHAL). The clinical picture of UHs is highly heterogeneous, inheritance pattern is dominant, instead of recessive as in more prevalent major Hb syndromes, and may occur de novo. Most cases of UHs are not detected by conventional testing, therefore diagnosis requires a high index of suspicion of the treating physician. Here, we highlight the importance of next generation sequencing (NGS) methodologies for the diagnosis of patients with dBTHAL and other less severe UH variants. We present five unrelated clinical cases referred with chronic hemolytic anemia, three of them with severe blood transfusion dependent anemia. Targeted NGS analysis was performed in three cases while whole exome sequencing (WES) analysis was performed in two cases. Five different UH variants were identified correlating with patients’ clinical manifestations. Four variants were related to the beta-globin gene (Hb Bristol—Alesha, Hb Debrousse, Hb Zunyi, and the novel Hb Mokum) meanwhile one case was caused by a mutation in the alpha-globin gene leading to Hb Evans. Inclusion of alpha and beta-globin genes in routine NGS approaches for RADs has to be considered to improve diagnosis’ efficiency of RAD due to UHs. Reducing misdiagnoses and underdiagnoses of UH variants, especially of the severe forms leading to dBTHAL would also facilitate the early start of intensive or curative treatments for these patients.

scholarly journals Phenotypic effect of heterozygous alpha and beta 0-thalassemia interaction

Blood ◽  
1983 ◽  
Vol 62 (1) ◽  
pp. 226-229 ◽  
Author(s):  
MA Melis ◽  
M Pirastu ◽  
R Galanello ◽  
M Furbetta ◽  
T Tuveri ◽  
...  
Keyword(s):  
Globin Gene ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Phenotypic Effect ◽  
Screening Programs ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Red Blood Cell Indices ◽  
Endonuclease Mapping ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract In this study, we carried out restriction endonuclease mapping in order to characterize the alpha-globin genotype of 10 Sardinian beta 0- thalassemia heterozygotes, all of whom presented with normal red blood cell indices and increased HbA2 levels. In 8 of these subjects, we found the deletion of two alpha-globin genes (-alpha/-alpha), and in the remaining two the deletion of a single alpha-globin gene (- alpha/alpha alpha). In three of these carriers with the (-alpha/-alpha) alpha-globin genotype and in one with the (-alpha/alpha alpha) genotype, we also found the glucose-6-phosphate dehydrogenase (G6PD) defect of the Mediterranean type. On the basis of these findings, we may conclude that the interaction of heterozygous beta 0-thalassemia with alpha-thalassemia, due to the deletion of either one or two alpha- globin genes, may lead to the production of red blood cells with normal indices. The association of the G6PD defect with this thalassemia gene complex may eventually contribute to this effect. We suggest, therefore, that screening programs for heterozygous beta-thalassemia in populations where alpha-thalassemia is also prevalent, should incorporate the determination of HbA2 in the first set of tests.

Identification of a Point Mutation in Position – 83 (G>A) of the β Globin Gene Promoter and the Influence of Globin Genes Mutation on the HbA2 Level

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5422-5422
Author(s):  
Estelle Cadet ◽  
Karine Foulon ◽  
Jean-Francois Claisse ◽  
Jacques Rochette
Keyword(s):  
Iron Deficiency ◽  
Direct Sequencing ◽  
Globin Gene ◽  
Clinical Signs ◽  
Gene Promoter ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Alpha Thalassemia ◽  
Normal Ranges ◽  
Iron Deficient

Abstract Carriers of typical β thalassemia alleles have microcytic hypochromic red blood cells with elevated Hb A2 values and a normal or slightly elevated level of Hb F. The increased level of Hb A2 is a reliable marker for heterozygous beta-thalassemia while Hb A2 levels within normal ranges do not exclude heterozygous beta-thalassemia. A male patient of Gabonese ancestry with both parents belonging to the Obamba sub-population was referred to us because of persistent microcytosis with anisopoikylocytosis and spherocytosis in the absence of clinical signs. The Hb separation pattern on alkaline electrophoresis and on ion exchange HPLC was normal. The Hb A2 level was 3.5% and the HbF level was 0.8%. Direct sequencing of the β globin genes revealed a G>A transition in position − 83 upstream of the cap site. The − 83 G>A substitution is located in a region between the CACCC motif (from − 90 to − 86) and the CCAAT motif (from − 76 to − 72) and has not been reported before. An alpha thalassemia associated with the β mutation was considered as well as a 𝛉 gene defect. Both hypotheses were based on the Hb A2 level. The Gap-PCR revealed the presence of − α3.7 (rightward) deletion in the heterozygous state in the proband while the 𝛉 genes did not show any mutation when compared to the normal sequence. In some instances, carriers for β thalassemia mutation may have normal Hb A2 level and can be mistaken for α-thal and/or 𝛉-thal or iron deficiency. We have revisited the Hb A2 level in different genotype conditions by using the VARIANT TM HPLC system with the β-Thalassemia short program (Bio-Rad Laboratories, Hercules, CA, USA). We have established thal carriers of typical nondeletional β-Thal alleles with no alpha thalassemia and in the absence of iron deficiency have a remarkable uniform level of Hb A2, rarely more than 6% and with an average value of 4.82 % ± 0.67( n=214). Point mutations located within the β globin gene promoter gives rise to an average of Hb A2 level of 5.42% ± 0.76 (n= 27). When a premature termination codon is present the Hb A2 percentage reaches 5.90 % ± 0.32 (n=12). The Hb A2 level drops to an average of 2.54 % ± 0.40 (n=108) in individuals carrying the − α3.7/αα genotype only. The association of the − α3.7/αα genotype with a non deletional β thalassemic allele shows an average of Hb A2 at 3.30 % ± 0.32 (n=134). Thus we explain the normal Hb A2 level in our patient by the co-inheritance of the β mutation together with the common heterozygous deletional α thalassemia (− α3.7/αα). Also, it is very unlikely that the − α3.7/αα genotype by its own may explain the red cell indices found in our patient (MCV=73 ; Hb = 12g/dl ; MCH = 23 pg) Co-inheritance of α and β thalassemia should always be considered when non iron deficient hypochromic microcytic anemia is diagnosed together with normal Hb A2 level, especially in regions with a high prevalence of both types of thalassemia. DNA analyses are needed for accurate diagnosis as this combination of genotypes can have implications for genetic counselling and prenatal diagnosis.

scholarly journals Beta zero-thalassemia in association with a gamma-globin gene quadruplication

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1394-1397 ◽  
Author(s):  
KG Yang ◽  
JZ Liu ◽  
F Kutlar ◽  
A Kutlar ◽  
C Altay ◽  
...  
Keyword(s):  
Clinical Condition ◽  
Globin Gene ◽  
Normal Chromosome ◽  
Gene Arrangement ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Gamma Globin ◽  
Alpha Globin

Abstract We have studied the hematology, hemoglobin composition, and globin gene arrangements in one young Turkish boy with a beta zero-thalassemia homozygosity and in 11 of his relatives. Evidence is presented that the chromosome with the beta zero-thalassemia determinant carries a gamma- globin gene quadruplication, perhaps in a -G gamma-G gamma-G gamma-A gamma-gene arrangement. The eight gamma-globin genes in this patient produced G gamma and A gamma chains in a 95 to 5 ratio, and nearly 99% of the patient's hemoglobin was of the fetal type. The clinical condition resembled that of a thalassemia intermedia. HbF levels in eight beta-thalassemia heterozygotes varied between 0.5 and 4.2% and the percentages of G gamma in this HbF averaged at 87% or 95%; this level is to some extent related to the haplotype of the normal chromosome. All subjects carried four alpha-globin genes; a new BglII polymorphism was observed within the psi alpha-globin gene.

scholarly journals Nuclear factor I (NF I) binds to an NF I-type site but not to the CCAAT site in the human alpha-globin gene promoter.

1992 ◽  
Vol 267 (12) ◽  
pp. 8478-8484 ◽  
Author(s):  
H Zorbas ◽  
T Rein ◽  
A Krause ◽  
K Hoffmann ◽  
E.L. Winnacker
Keyword(s):  
Globin Gene ◽  
Gene Promoter ◽  
Nuclear Factor ◽  
Factor I ◽  
Alpha Globin ◽  
Nuclear Factor I ◽  
Type Site

Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells

1987 ◽  
Vol 7 (1) ◽  
pp. 398-402
Author(s):  
T Rutherford ◽  
A W Nienhuis
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Promoters ◽  
Globin Genes ◽  
Specific Expression ◽  
Tissue Specific ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.

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