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

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.

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

Blood ◽

10.1182/blood.v71.3.766.766 ◽

1988 ◽

Vol 71 (3) ◽

pp. 766-770 ◽

Cited By ~ 12

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

Abstract 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.

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The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽

10.1182/blood.v70.5.1470.bloodjournal7051470 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1470-1474 ◽

Cited By ~ 1

Author(s):

GF Atweh ◽

XX Zhu ◽

HE Brickner ◽

CH Dowling ◽

HH Jr Kazazian ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Beta Globin ◽

Promoter Mutation ◽

Beta Globin Gene ◽

Gamma Globin ◽

New Type

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

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

Blood ◽

10.1182/blood.v68.4.971.bloodjournal684971 ◽

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.

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

Blood ◽

10.1182/blood.v68.4.971.971 ◽

1986 ◽

Vol 68 (4) ◽

pp. 971-974 ◽

Cited By ~ 18

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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The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽

10.1182/blood.v70.5.1470.1470 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1470-1474 ◽

Cited By ~ 6

Author(s):

GF Atweh ◽

XX Zhu ◽

HE Brickner ◽

CH Dowling ◽

HH Jr Kazazian ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Beta Globin ◽

Promoter Mutation ◽

Beta Globin Gene ◽

Gamma Globin ◽

New Type

Abstract A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

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A novel mutation in the TATA box in a Japanese patient with beta +- thalassemia

Blood ◽

10.1182/blood.v67.2.547.547 ◽

1986 ◽

Vol 67 (2) ◽

pp. 547-550 ◽

Cited By ~ 39

Author(s):

Y Takihara ◽

T Nakamura ◽

H Yamada ◽

Y Takagi ◽

Y Fukumaki

Keyword(s):

Novel Mutation ◽

Globin Gene ◽

Japanese Woman ◽

Tata Box ◽

Proximal Promoter ◽

Base Substitution ◽

Beta Thalassemia ◽

Beta Globin ◽

Beta Globin Gene

Abstract A single base substitution (A-G) at position -31 within the highly conserved proximal promoter element, the TATA box, was identified in the beta-globin gene cloned from a Japanese woman with beta +- thalassemia. It appears that she is homozygous for this specific allele, as determined by haplotype analysis using seven different polymorphic sites in the beta-globin gene cluster. Transient expression of the mutant gene in COS cells revealed a 45% reduction in beta-globin RNA production, relative to normal. These results establish the functional significance of the second base of the TATA box for in vivo transcription of the human beta-globin gene.

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The entire beta-globin gene cluster is deleted in a form of gamma delta beta-thalassemia

Blood ◽

10.1182/blood.v61.6.1269.bloodjournal6161269 ◽

1983 ◽

Vol 61 (6) ◽

pp. 1269-1274

Author(s):

ER Fearon ◽

HH Jr Kazazian ◽

PG Waber ◽

JI Lee ◽

SE Antonarakis ◽

...

Keyword(s):

Gene Cluster ◽

Mexican American ◽

Globin Gene ◽

Dna Polymorphisms ◽

Beta Thalassemia ◽

Beta Globin ◽

Gamma Delta ◽

Beta Globin Gene ◽

Globin Gene Cluster ◽

Endonuclease Mapping

We have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA fragment that maps greater than 40 kilobases (kb) 5′ to the epsilon-gene as a probe, reduced amounts of normal fragments were found in the DNA of affected family members. Similar analysis using radiolabeled DNA fragments located 3′ to the beta-globin cluster has shown that the deletion extends more than 17 kb 3′ to the beta-gene, but terminates before the 3′ endpoint of the Ghanian HPFH deletion. Hence, this gamma delta beta-thalassemia deletion eliminates over 105 kb of DNA and is the first report of a deletion of the entire beta-globin gene cluster.

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Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia

Blood ◽

10.1182/blood.v83.8.2031.bloodjournal8382031 ◽

1994 ◽

Vol 83 (8) ◽

pp. 2031-2037 ◽

Cited By ~ 4

Author(s):

GW Hall ◽

S Thein

Keyword(s):

Globin Gene ◽

Beta Thalassemia ◽

Beta Globin ◽

Terminal Exon ◽

Mrna Accumulation ◽

Beta Globin Gene ◽

Globin Chains ◽

Nonsense Codon ◽

Nonsense Codons

We present in vivo evidence that there is no reduction in beta-mRNA accumulation in patients with nonsense codons in the terminal exon of the beta-globin gene. Using reverse transcriptase/polymerase chain reaction (RT-PCR), beta-globin cDNA was isolated from the reticulocytes of individuals heterozygous for nonsense codon mutations in exons II and III of the beta-globin gene. Clinically asymptomatic individuals heterozygous for mutations causing premature termination of translation in exon II [beta(0)39(C-T) and F/S71/72(+A)] were found to have almost no mutant beta-cDNA, whereas patients with nonsense codon mutations in exon III [beta 121(G-T) and beta 127(C-T)] with the clinical phenotype of thalassemia intermedia had comparable levels of mutant and normal beta-cDNA. Translation of the mutant beta-mRNA from patients with nonsense codon mutations in exon III would give rise to truncated beta- globin chains, which could explain the more severe phenotype seen in these individuals.

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Large Deletions Involving the Beta Globin Gene Complex: Genotype-Phenotype Correlation of 119 Cases

Blood ◽

10.1182/blood.v126.23.3374.3374 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3374-3374

Author(s):

Molly Susan Hein ◽

Jennifer L Oliveira ◽

Kenneth C Swanson ◽

Patrick A Lundquist ◽

Joella A Yungerberg ◽

...

Keyword(s):

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Microcytic Anemia ◽

Beta Thalassemia ◽

Gene Complex ◽

Beta Globin ◽

Gamma Delta ◽

Beta Globin Gene ◽

Large Deletions

Abstract Background: Large deletions involving the beta globin complex are relatively rare. They can be categorized generally into five groups by deletion size and/or location: 1) beta zero thalassemia (BZT); 2) delta beta thalassemia (DBT); 3) hereditary persistence of fetal hemoglobin (HPFH); 4) gamma delta beta thalassemia (GDBT); and 5) epsilon gamma delta beta thalassemia (EGDBT). These deletions are not well understood but often have significant clinical impact, either when present alone or in combination with other hemoglobin mutations. In this study, we analyze phenotypic and molecular data on a large number of cases with deletions in the beta globin gene complex to better classify these five groups of deletions as they occur in isolation. Methods: A query of the routine clinical testing patient files from the Mayo Clinic Metabolic Hematology and Molecular Genetics Laboratories from 2010 to 2015 identified 179 patients with a deletion confirmed by a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. Twenty-four probes sets were placed from the 5' locus control region (LCR) to the 3' hypersensitivity region, spanning the beta globin gene complex. Using a Luminex LX200 flow cytometer, a gene dosage ratio was calculated for each probe set using the median fluorescent intensity value collected. The size and location of the deletion and patient phenotype were compared. Results: Of the 179 total cases, the following large deletions were identified: beta gene (HBB) (n = 47), delta (HBD) through HBB (n = 105), A-gamma (HBG2) through HBB (n = 20), and locus control region (LCR) through HBB (n = 7). One case had a deletion involving the LCR epsilon with the rest of the complex left intact. A subset (n = 60) of cases had compound hemoglobin mutations that altered the phenotype. The BZT cases had relatively high Hb A2 levels and variable Hb F levels consistent with promotor region loss. The main differences between DBT and HPFH included Hb F and Hb A2 levels. GDBT cases presented with median Hb F levels higher than that observed in DBTs, normal Hb A2, and microcytic anemia. EGDBT cases had variable features according to age of the patient and Hb F level; severe microcytic anemia was observed in neonates, milder microcytic anemia in young children, and microcytosis without anemia in an adult case. The phenotypic features of 119 patients with isolated large deletions are compiled in table 1. Conclusion: In general, all five categories of large deletions in an isolated heterozygous state can present with microcytic anemia and are typically benign with the exception of transient severe microcytic anemia in neonatal EGDBT cases. Although phenotypes associated with large deletions involving the beta globin gene complex are frequently distinctive, significant phenotypic overlap can be seen in a subset of cases. These cases require molecular analysis due to their clinical importance when in combination with another beta globin gene complex mutation for an adequate diagnosis and treatment approach. Table 1. Deletion type Age n HbF (%) HbA2 (%) Hb (g/dL) MCV (fL) RBC (10^12/L) RDW (%) MCH (pg/cell) BZT 20 6.3 (0.6-94.4) 6.8 (3.4-11.6) 11.1 (8.3-14.5) 65.4 (60.8-77.2) 5.4 (4.2-6.2) 19.2 (16.6-21.2) 20.9 (18.3-25.7) DBT 56 10.6 (2.7-22.4) 2.7 (2.5-3.1) 11.7 (8.6-14.4) 68.9 (61.3-83.5) 5.3 (4.1-7.3) 21.4 (18.2-26.8) 21.6 (19.9-39.2) HPFH 23 25.9 (17.6-39.7) 2.0 (1.5-2.4) 11.6 (8.1-16.7) 78.4 (60.2-101.9) 4.4 (3.0-6.3) 17.5 (14.1-22.3) 25.4 (17.6-29.7) GDBT 14 13.3 (8.2-19.0) 2.6 (1.8-2.7) 11.0 (8.6-14.1) 72.5 (57.9-82.1) 5.1 (3.5-6.2) 20.6 (17.4-23.5) 22 (17.9-25.1) EGDBT* 28 Y 1 0.3 3 13.3 59.4 6.9 15.4 19.2 1-4 Y 3 0.9 (0-1.6) 3.2 (2.9-3.5) 9.5 (8.8-13.3) 57.8 (57.6-59.4) 5.2 (4.9-6.9) 16.6 (15.4-17.4) 18.5 (18.1-19.2) <6 month 2 21.4 (14.8-27.9) 2.6 (2.2-2.9) 6.3 (6.0-6.6) 61.3 (59.9-62.6) 3.4 (3.3-3.3) 21.5 (21.2-21.7) 18.4 (18.1-18.7) medians, (min, max); *stratified by age Disclosures No relevant conflicts of interest to declare.

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