Cytokine Dependent Hematopoietic Cell Linker (CLNK) is Highly Elevated in Blood Transfusion Dependent Beta-Thalassemia Major Patients

Beta-thalassemia major (β-TM) is a severe form of thalassemia caused by mutations in the β-globin gene, resulting in partial or complete deficiency of β-globin chains. This deficiency results in oxidative stress, dyserythropoiesis, and chronic anemia. Cytokine dependent hematopoietic cell linker (CLNK) belongs to the adaptor protein family and has the capacity to interact with multiple signaling proteins thereby modulating signal transduction. The aim of the present study was to examine CLNK in sera of β-TM patients and examine its association with iron overload biomarkers. Sixty β-TM patients, aged 3–12 years old and undergoing blood transfusions, and 30 healthy control children were recruited and CLNK, ferritin and iron status parameters were measured. The results showed a significant increase (p < 0.001) in serum CLNK levels in β-TM patients as compared with normal controls. The increased levels of CLNK were significantly associated with increased ferritin levels. Increased CLNK levels in β-TM may be explained by reciprocal effects between immune signaling and immature erythrocytes, which, release soluble receptors and signaling molecules, including CLNK, in the blood.

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Evaluation of Erythroferrone, Hepcidin, and Iron Overload Status in Iraqi Transfusion-dependent β-Thalassemia Major Patients

10.20944/preprints201912.0356.v1 ◽

2019 ◽

Author(s):

Hasan Smesam ◽

Hasan Qazmooz ◽

Sareh Arjmand ◽

Hussein Kadhem Al-Hakeim ◽

Seyed Omid Ranaei‐Siadat,

Keyword(s):

Iron Overload ◽

Iron Status ◽

Iron Chelation ◽

Fold Increase ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Control Group ◽

Beta Globin ◽

Beta Thalassemia Major ◽

Globin Chains

Beta thalassemia major (β-TM) disorder characterized by the lack, or severe reduction in the production of hemoglobin β-globin chains. The standard protocol for the management of β-TM is blood transfusion and iron chelation therapy to reduce the iron overload state. The present study aimed to investigate the relationships between two iron regulatory hormones, hepcidin (HEPC) and erythroferrone (ERFE) levels and iron status parameters (ISPs) in Iraqi patients with β-TM. ISPs and hormones were measured in sixty patients and compared with thirty healthy controls. The results indicated significant changes in different iron status parameters, while ferritin (FRT) with the ~11 fold increase showed the most change. Significant reduction in HEPC and increase in ERFE levels were detected in patients as compared to the control group, while no direct correlation was identified with the other measured ISPs. Receiver operating characteristic (ROC) analysis showed that the z-score of the composite of ERFE+FRT has a full diagnostic ability for β-TM. In conclusion, our finding indicated the correlation between different ISPs, FRT as the leading predictor of iron overload and tow main iron regulatory hormones.

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A first case of hemoglobin Castilla [Beta 32(B14) Leu>Arg; HBB: c.98T>G] associated with [IVS-I-1 (G>A); HBB:c.92+1G>A] mutation found in a Syrian betathalassemia patient

Thalassemia Reports ◽

10.4081/thal.2020.8396 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ahmad Shoujaa ◽

Yasser Mukhalalaty ◽

Hossam Murad ◽

Faizeh Al-Quobaili

Keyword(s):

Molecular Analysis ◽

Clinical Case ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Blood Transfusions ◽

Beta Globin ◽

First Report ◽

Beta Thalassemia Major ◽

First Case

Beta thalassemia (β-thal) is one of the most common worldwide inherited hemoglobinopathies. Proper identification and diagnosis of hemoglobin (Hb) variants provide a major challenge. In this report, we describe a 1-year-old boy, presented with the diagnosis of β-TM (beta thalassemia major), has received regular blood transfusions. The molecular analysis revealed the presence of rare Hb Castilla [Beta 32(B14) Leu>Arg; HBB: c.98T>G] variant associated with β0 [IVS-I-1 (G>A); AG^GTTGGT- >AGATTGGT beta0] (HBB:c.92+1G>A) Mutation in beta-globin (β-globin) gene. To our knowledge, this is the first report of Hb Castilla [Beta 32(B14) Leu>Arg] in ExonII of β-globin gene which were found in Syrian male proband. However, we should investigate abnormal hemoglobins in patients with beta thalassemia to determine whether they have involvement with β-thalassemia mutations in the clinical case of the patients or not.

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Investigation of beta globin gene mutations in Syrian refugee patients with thalassemia major

Turkish Journal of Biochemistry ◽

10.1515/tjb-2018-0492 ◽

2019 ◽

Vol 44 (2) ◽

pp. 126-129

Author(s):

Hatice Çevirici ◽

Can Acıpayam ◽

Ebru Dündar Yenilmez ◽

Fatma Burcu Belen ◽

Esra Pekpak ◽

...

Keyword(s):

Sequence Analysis ◽

Globin Gene ◽

Gene Mutations ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Amplification Refractory Mutation System ◽

Beta Globin ◽

Beta Globin Gene ◽

Beta Thalassemia Major ◽

Mutation System

Abstract Objectives This study, detection of beta globin gene mutations in thalassemia major patients who migrated from Syria to Kahramanmaraş region were planned. Materials and methods The study included 35 Syrian national beta thalassemia major patients. Beta globin gene mutations were detected by ARMS (Amplification Refractory Mutation System) method, RFLP (Restriction Fragment Length Polymorphism) method and DNA sequence analysis. Codon 15, codon 9/10, codon 5 and codon 8 mutations, which we could not detect with other methods in our study, were detected by sequence analysis. Results In beta thalassemia major patients, 16 types of mutations were detected, the most common being IVS-I-110 (n=8). Other mutations are according to frequency order IVS-II-745 (n=3), codon 44 (n=3), codon 15 (n=3), IVS-I-110/IVS-I-1 (n=3), codon 5 (n=2), IVS-I-1 (n=2), codon 8/IVS-II-1 (n=2), codon 44/codon 15 (n=2), IVS-II-1 (n=1), codon 39 (n=1), IVS-I-6/codon 5 (n=1), codon 9/10 (n=1), IVS-I-110/codon 39 (n=1), IVS-I-5/IVS-II-1 (n=1), codon 39/IVS-II-745 (n=1). Conclusions According to the results of our study beta-thalassemia mutations in Syrian immigrant groups show heterogeneity and mutation types of mutation map is similar to Turkey. The conclusion is to prevent families to have a second patient child by genetic counseling.

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Beta-globin gene mutations in children with beta-thalassemia major from Sanliurfa province, Turkey

Turkish Journal of Hematology ◽

10.5152/tjh.2011.86 ◽

2011 ◽

Vol 2011 (4) ◽

pp. 264-268 ◽

Cited By ~ 2

Author(s):

Ali Aycicek ◽

Ahmet Koc ◽

Zeynep Canan Ozdemir ◽

Hasan Bilinc ◽

Abdurrahim Kocyigit ◽

...

Keyword(s):

Globin Gene ◽

Gene Mutations ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Beta Globin Gene ◽

Beta Thalassemia Major

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Current Perspective of Beta Thalassemia and Its Treatment Strategies

10.20944/preprints201907.0277.v1 ◽

2019 ◽

Author(s):

Shaukat Ali ◽

Shumaila Mumtaz ◽

Hafiz Abdullah Shakir ◽

Hafiz Muhammad Tahir ◽

Tafail Akbar Mughal

Keyword(s):

Genetic Testing ◽

Blood Transfusion ◽

Dna Analysis ◽

Complete Blood Count ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Hematopoietic Stem ◽

Thalassemia Minor

Thalassemia is genetic blood disease cause by absence or decrease of one or more of the globin chain synthesis. Beta thalassemia is characterized by one or more mutations in beta globin gene. Absence or reduced amount the of beta globin chains cause ineffective erythropoiesis which leads to anemia. Beta thalassemia has been further divided into three main forms: Thalassemia minor/silent carrier, major and intermedia. More severe form is thalassemia major in which patients depend upon blood transfusion for survival and high level of iron occur as a consequence of consistent blood transfusion. Over loaded iron invokes the synthesis of reactive oxygen species that are toxic in redundancy and triggering the impairment to vascular, endocrine and hepatic system. Thalassemia can be diagnosed and detected through various laboratory tests such as blood smear, prenatal testing (genetic testing of amniotic fluid), DNA analysis (genetic testing) and complete blood count. Treatment of thalassemia intermedia is symptomatic but it can also be managed by splenectomy and folic supplementation. While thalassemia major can be treated by transplantation of bone marrow, regular transfusion of blood and iron chelation treatment, stimulation of fetal hemoglobin production, hematopoietic stem cell transplantation and gene therapy.

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

Blood ◽

10.1182/blood.v66.5.1202.1202 ◽

1985 ◽

Vol 66 (5) ◽

pp. 1202-1207 ◽

Cited By ~ 19

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.

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Molecular analysis of beta zero-thalassemia intermedia in Sardinia

Blood ◽

10.1182/blood.v74.2.823.823 ◽

1989 ◽

Vol 74 (2) ◽

pp. 823-827 ◽

Cited By ~ 42

Author(s):

R Galanello ◽

E Dessi ◽

MA Melis ◽

M Addis ◽

MA Sanna ◽

...

Keyword(s):

Nonsense Mutation ◽

Frameshift Mutation ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Thalassemia Intermedia ◽

Mild Phenotype ◽

Beta Globin Gene ◽

Alpha Thalassemia

Abstract In this study we have carried out alpha- and beta-globin gene analysis and defined the beta-globin gene polymorphisms in a group of patients with thalassemia intermedia of Sardinian descent. A group of patients (109) with thalassemia major of the same origin served as control. Characterization of the beta-thalassemia mutation showed either a frameshift mutation at codon 6 or a codon 39 nonsense mutation. We found that homozygotes for the frameshift mutation at codon 6 or compound heterozygotes for this mutation and for the codon 39 nonsense mutation develop thalassemia intermedia more frequently than thalassemia major. The frameshift mutation at codon 6 was associated with haplotype IX that contains the C-T change at position -158 5′ to the G gamma globin gene implicated in high gamma chain production and thus the mild phenotype. In patients' homozygotes for codon 39 nonsense mutation, those with thalassemia intermedia more frequently had the two- gene deletion form of alpha-thalassemia, or functional loss of the alpha 2 gene as compared with those with thalassemia major. In a few siblings with thalassemia major and intermedia, the thalassemia intermedia syndrome correlated with the presence of the -alpha/-alpha genotype. No cause for the mild phenotype was detected in the majority of patients who had not inherited either haplotype IX or alpha- thalassemia.

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Growth Differentiation Factor 15 (GDF15) and Erythropoietin (EPO) Levels in Beta Talassemia Major Patients.

Blood ◽

10.1182/blood.v112.11.1881.1881 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1881-1881

Author(s):

Ilaria Salussoglia ◽

Gisella Volpe ◽

Silvia Fracchia ◽

Simona Roggero ◽

Filomena Longo ◽

...

Keyword(s):

Iron Status ◽

Clinical Status ◽

Transferrin Saturation ◽

Iron Chelation ◽

Serum Levels ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Transfusion Therapy ◽

Beta Thalassemia Major ◽

The Mean

Abstract Background: The serum level of GDF15 has been recently indicated as a possible marker of erythropoiesis (Tanno et al., Nature 2007) suggesting a role of its over-expression in contributing to iron overload in thalassemia syndromes by inhibiting hepcidin expression. The aim of present study has been to evaluate GDF15 serum levels in a homogeneous series of thalassemia patients and the relationship with transfusional parameters and iron status markers. Methods: A group of consecutive patients with beta thalassemia major followed at our institution were included in the study. All patients were on regular transfusion and iron chelation treatment. Quantification of GDF15 on serum samples was performed with DuoSet ELISA for human GDF15 (R&D Systems) following the manufacturer’s protocol (Tanno et al., Nature 2007). Each patient had also a blood test for haemoglobin (Hb), serum iron, ferritin, transferrin, transferrin saturation and EPO levels. Liver Iron Concentration by SQUID and cardiac iron by MRI T2* have been assessed. The mean hemoglobin levels of the previous year (pre-transfusional, post-transfusional and mean) have been calculated for each individual. The presence of mild thalassemic mutations was used to classify mild or severe genotype. Clinical status has been assessed on the presence/absence of main complications (heart disease, liver disease, diabetes, hypothyroidism). Statistical analysis was performed using the software Statistica (StatSoft). Results: One hundred-forty patients (73 male, 67 females) were studied. The mean age was 27.9 ± 9.0 years (range: 3.5–42). One hundred (71%) were splenectomised. Betathalassemia major patients had elevated GDF15 serum levels (mean 6892 ± 6894 pg/mL; range 720–52521) in comparison with healthy volunteers (273 ± 104 pg/mL; range 129–401). GDF 15 levels were strongly related to EPO levels (r=0,81; p<0,001). GDF15 levels were not related with age, gender, spleen, clinical status and iron markers. Patients with a severe genotype had higher GDF15 levels than mild genotype patients. GDF15 levels had a negative correlation with Hbs (p<0,05 for actual Hb and pre-transfusional Hb; p<0,001 for post-transfusional Hb and mean Hb). In thalassemia major patients with a severe genotype, GDF15 levels within thrice the normal range have been observed only in patients with pre-transfusional Hb above 9,6, post-transfusional Hb above 12,5 and a mean Hb above 11,3. Conclusions: In beta thalassemia major patients on regular transfusion and iron chelation, serum GDF15 levels are high, inversely related to the haemoglobin levels maintained. Further studies of this marker may lead to a rethinking of the optimal transfusion therapy in these conditions.

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Ineffective Erythropoiesis and Production of Normoblasts with a Beta Thalassemia Major Phenotype Using CD34+ Cells From Healthy Donors

Blood ◽

10.1182/blood.v118.21.1085.1085 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1085-1085

Author(s):

Y.Terry Lee ◽

Colleen Byrnes ◽

Emily Riehm Meier ◽

Antoinette Rabel ◽

Jeffery L. Miller

Keyword(s):

Ex Vivo ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Ineffective Erythropoiesis ◽

Globin Mrna ◽

Cell Counts ◽

Cd34 Cells ◽

Gene And Protein Expression

Abstract Abstract 1085 Reversal of anemia is the major target of thalassemia research, but studies of the molecular and cellular basis of the ineffective erythropoiesis of thalassemia are limited by access to donor progenitor cells. Here we demonstrate that thalassemic erythropoiesis may be recapitulated ex vivo by reducing the expression of hemoglobin in cultured CD34+ cells. Using lentiviral transduction of progenitor cells obtained from three healthy adult human donors, shRNA molecules were screened for their ability to reduce beta-globin gene and protein expression over 21 days in culture. Cells transduced with a scrambled vector served as donor-matched controls. Among the screened shRNA, one named HBB caused a consistent and significant reduction in beta-globin mRNA and protein. Beta-globin mRNA was reduced to levels <10% (p<0.001) compared to that of the controls (day14/21), while maintaining expression of gamma- and alpha-globin mRNA. HPLC was performed on an equivalent number of cells sampled on culture day 21 for hemoglobin type (HbA vs. HbF) and quantitation (area under each HPLC peak). The HbA peak was reduced by 96%, and there was a minor increase in the HbF peak (1.6 fold) after HBB transduction. Based upon these quantitative changes in hemoglobin, HbF represented 49.3±9.3% in the HBB transduced population compared with 2.9±0.7% (p<0.01) in controls. On culture day 14, there was no significant difference in glycophorin A (CD235), transferrin receptor (CD71), or cellular morphology despite the reduction in beta-globin mRNA. However, impaired terminal differentiation was detected by retainment of surface CD71 and a lack of enucleation during the third week of culture. Cell counts were lower in HBB transduced cells during the final stages of erythroid differentiation with a 61% (p=0.03) reduction in total cell counts by day 21 when compared to controls. Annexin V assay on day 21 also demonstrated increased phosphatidylserine expression in the HBB transduced cells [HBB=55.7±14.4% vs. Control=25.0±3.0%] in association with the decreased terminal differentiation. GDF15 quantitation demonstrated a significant (p=0.006) increase in the culture supernatants of HBB transduced cells. Sorted cytospin preparations revealed a distinct population of mature normoblasts containing a highly condensed nucleus surrounded by a thin ring of hypochromic cytoplasm. Reduction of erythroblast beta-globin gene and protein expression to levels associated with beta thalassemia major in humans causes ineffective erythropoiesis ex vivo by reducing cell production, increasing surface expression of phosphatidylserine, and impairing enucleation during terminal maturation. Efforts are now underway to use the culture system to explore mechanisms whereby reduced hemoglobin synthesis causes normoblast defects, and for screening of chemical and genetic rescue therapies for the thalassemic erythroid phenotype. Disclosures: No relevant conflicts of interest to declare.

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In silico study on RNA structures of intronic mutations of beta-globin gene

F1000Research ◽

10.12688/f1000research.21953.3 ◽

2020 ◽

Vol 9 ◽

pp. 49

Author(s):

Nur Imaniati Sumantri ◽

Kenny Lischer ◽

Dian Rachma Wijayanti ◽

Tomy Abuzairi

Keyword(s):

In Silico ◽

Rna Structure ◽

Tertiary Structure ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Rna Structures ◽

Beta Globin Gene ◽

Mrna Structure

Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G>T) and 147 (IVS1nt5G>C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and 3dRNA v2.0, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G>T and IVS1nt5G>C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.

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