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

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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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.bloodjournal713766 ◽

1988 ◽

Vol 71 (3) ◽

pp. 766-770

Author(s):

PT Curtin ◽

YW Kan

Keyword(s):

Globin Gene ◽

Large Deletion ◽

Beta Thalassemia ◽

Ribonuclease Protection Assay ◽

Beta Globin ◽

Gamma Delta ◽

Beta Globin Gene ◽

Thalassemia Minor

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

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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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Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.

Genes & Development ◽

10.1101/gad.9.24.3083 ◽

1995 ◽

Vol 9 (24) ◽

pp. 3083-3096 ◽

Cited By ~ 142

Author(s):

J Bungert ◽

U Dave ◽

K C Lim ◽

K H Lieuw ◽

J A Shavit ◽

...

Keyword(s):

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Beta Globin ◽

Beta Globin Gene ◽

Synergistic Regulation ◽

Gene Switching

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

Blood ◽

10.1182/blood.v83.3.822.822 ◽

1994 ◽

Vol 83 (3) ◽

pp. 822-827 ◽

Cited By ~ 17

Author(s):

AJ Dimovski ◽

V Divoky ◽

AD Adekile ◽

E Baysal ◽

JB Wilson ◽

...

Keyword(s):

Control Region ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Locus Control Region ◽

Regulatory Sequence ◽

Beta Globin ◽

Gamma Chain ◽

Beta Globin Gene ◽

Gamma Globin

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

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Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region.

Genes & Development ◽

10.1101/gad.6.6.928 ◽

1992 ◽

Vol 6 (6) ◽

pp. 928-938 ◽

Cited By ~ 71

Author(s):

C G Kim ◽

E M Epner ◽

W C Forrester ◽

M Groudine

Keyword(s):

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Beta Globin ◽

Beta Globin Gene

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Molecular analysis of Japanese delta beta-thalassemia

Blood ◽

10.1182/blood.v72.5.1771.1771 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1771-1776 ◽

Cited By ~ 9

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.

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The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene

Development ◽

10.1242/dev.122.12.3991 ◽

1996 ◽

Vol 122 (12) ◽

pp. 3991-3999 ◽

Cited By ~ 1

Author(s):

R. Tewari ◽

N. Gillemans ◽

A. Harper ◽

M. Wijgerde ◽

G. Zafarana ◽

...

Keyword(s):

Expression Pattern ◽

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Developmental Expression ◽

Chromatin Conformation ◽

Lacz Gene ◽

Beta Globin ◽

Beta Globin Gene ◽

Hypersensitive Sites

The beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5′HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the beta-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic epsilon-globin and fetal gamma-globin promoters, resulting in the almost exclusive transcription of the beta-globin gene in adult erythropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5′HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The results show that the muLCR and 5′HS3 direct erythroid-specific, embryonic expression of this construct, while 5′HS1, 5′HS2 and 5′HS4 are inactive at any stage of development. Expression of the muLCR and 5′HS3 transgenes is repressed during fetal stages of development. The transgenes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybridization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a beta-globin gene is required for LCR function as conditions become more stringent during development.

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New Concepts in Genome Regulation

Blood ◽

10.1182/blood.v128.22.sci-47.sci-47 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-47-SCI-47

Author(s):

Ann Dean ◽

Jongjoo Lee ◽

Ryan Dale ◽

Ivan Krivega

Keyword(s):

Sickle Cell Disease ◽

Long Range ◽

Control Region ◽

Sickle Cell ◽

Globin Gene ◽

Locus Control Region ◽

Beta Thalassemia ◽

Beta Globin ◽

Cell Disease ◽

Gamma Globin

Abstract Manipulating gene regulation to favor gamma-globin transcription over beta-globin transcription has been a goal of research in erythropoiesis for decades because of its relevance to amelioration of the pathophysiology of sickle cell disease and beta-thalassemia. A fundamental unanswered question in biology is how the unique pattern of gene expression, the transcriptome, of the many different individual mammalian cell types arises from the same genome blueprint and changes during development and differentiation. There is a growing appreciation that genome organization and the folding of chromosomes is a key determinant of gene transcription. Within this framework, enhancers function to increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding, or looping. These long range interactions are orchestrated by cell type specific proteins and protein complexes that bind to enhancers and promoters and stabilize their interaction with each other. We have been studying LDB1, a member of an erythroid protein complex containing GATA1, TAL1 and LMO2. The LDB1complex activates erythroid genes through occupancy of virtually all erythroid enhancers. LDB1engages in homo- and heterotypic interactions with proteins occupying the promoters of erythroid genes to bring them into proximity with their enhancers. We find that enhancer long range looping activity can be redirected. Both targeting of the beta-globin locus control region to the gamma-globin gene in adult erythroid cells by the tethering of LDB1 or epigenetic unmasking of a silenced gamma-globin gene lead to increased locus control region (LCR)/gamma-globin contact frequency and reduced LCR/beta-globin contact. The outcome of these manipulations is robust, pan-cellular gamma-globin transcription activation with a concomitant reduction in beta-globin transcription. These examples suggest that chromosome looping can be considered a therapeutic target for gene activation or gene silencing to ameliorate genetic diseases such as sickle cell disease and beta-thalassemia. Disclosures No relevant conflicts of interest to declare.

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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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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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