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 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.

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.

A silencer element from the alpha-globin gene inhibits expression of beta-like genes

1988 ◽  
Vol 8 (11) ◽  
pp. 5047-5051
Author(s):  
G F Atweh ◽  
J M Liu ◽  
H E Brickner ◽  
X X Zhu
Keyword(s):  
Globin Gene ◽  
Expression System ◽  
Globin Genes ◽  
Beta Globin ◽  
Base Pair Fragment ◽  
Alpha Globin ◽  
In Trans ◽  
Cis And Trans ◽  
In Cis ◽  
Silencer Element

We have studied the cis and trans interactions of the alpha- and beta-globin genes in a transient expression system. We found that the alpha-globin gene inhibited beta-globin expression in cis but not in trans. The silencer element responsible for this inhibition was localized to a 259-base-pair fragment at the 5' end of the alpha-globin gene.

scholarly journals 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 ◽  
1995 ◽  
Vol 85 (4) ◽  
pp. 1111-1117 ◽  
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).

scholarly journals The detection and use of hemoglobin mutants in the direct analysis of human globin genes

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 1060-1062 ◽  
Author(s):  
PF Little ◽  
E Whitelaw ◽  
G Annison ◽  
R Williamson ◽  
JM Kooter ◽  
...  
Keyword(s):  
Restriction Site ◽  
Globin Gene ◽  
Direct Analysis ◽  
Globin Genes ◽  
Beta Globin ◽  
Gene Sequences ◽  
Gamma Globin ◽  
Alpha Globin ◽  
Specific Restriction ◽  
Alpha Beta

Abstract Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma- , and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.

scholarly journals Hypomethylation of DNA derived from purified human erythroid cells correlates with gene activity of the beta-globin cluster

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1202-1207 ◽  
Author(s):  
A Oppenheim ◽  
Y Katzir ◽  
E Fibach ◽  
A Goldfarb ◽  
E Rachmilewitz
Keyword(s):  
Peripheral Blood ◽  
Globin Gene ◽  
Genetic Regulation ◽  
Inverse Correlation ◽  
Thalassemia Major ◽  
Gene Activity ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
A Site

Abstract Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3′ to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5′ to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual patients. A site 5′ to the delta gene was unmethylated in normoblasts as well as in WBC. No apparent relation between hypomethylation and gene activity was observed for two additional sites. The results suggest that methylation at specific chromosomal locations participate in genetic regulation of the beta- like globin genes in humans.

scholarly journals Developmental switch in the relative expression of the alpha 1- and alpha 2-globin genes in humans and in transgenic mice

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2471-2474 ◽  
Author(s):  
M Albitar ◽  
FE Cash ◽  
C Peschle ◽  
SA Liebhaber
Keyword(s):  
Transgenic Mice ◽  
Globin Gene ◽  
Gene Activation ◽  
Experimental System ◽  
Gene Clusters ◽  
Globin Genes ◽  
Beta Globin ◽  
Relative Expression ◽  
Structural Identity ◽  
Alpha Globin

Abstract Human alpha-globin is encoded by two adjacent genes, alpha 2 and alpha 1. Despite their remarkable level of structural identity, the more 5′ (alpha 2) gene is the major alpha-globin locus in the normal adult, expressed at 2.6-fold higher levels than the adjacent and more 3′ (alpha 1) globin gene. In light of the well-characterized pattern of gene activation in the human alpha- and beta-globin gene clusters during development, we considered the possibility that the relative expression of these two alpha-globin loci might be developmentally controlled. Analysis of human embryonic and early fetal erythroid RNA samples confirmed this possibility; levels of mRNA encoded by the two alpha-globin loci are equal in the embryo and subsequently shift to dominant expression of the alpha 2-globin locus at week 8 in utero. In transgenic mice carrying the entire human alpha-globin cluster (except for the theta gene) we show the same shift from equal expression of the alpha 1- and alpha 2-globin loci at the embryonic stage to predominance of the alpha 2-globin locus in the adult. These data demonstrate a switch in the expression of the two adjacent alpha-globin genes during the embryonic-to-fetal switch in erythroid development and provide an experimental system for its further characterization.

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.

Alpha-thalassemia caused by a large (62 kb) deletion upstream of the human alpha globin gene cluster

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 221-227
Author(s):  
CS Hatton ◽  
AO Wilkie ◽  
HC Drysdale ◽  
WG Wood ◽  
MA Vickers ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Dna Sequence ◽  
Interspecific Hybrid ◽  
Globin Gene ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Beta Globin ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Globin Synthesis

We describe a family in which alpha-thalassemia occurs in association with a deletion of 62 kilobases from a region upstream of the alpha globin genes. DNA sequence analysis has shown that the transcription units of both alpha genes downstream of this deletion are normal. Nevertheless, they fail to direct alpha globin synthesis in an interspecific hybrid containing the abnormal (alpha alpha)RA chromosome. It seems probable that previously unidentified positive regulatory sequences analogous to those detected in a corresponding position of the human beta globin cluster are removed by this deletion.

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.

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