Genotypes and phenotypes of thalassemia: A discussion.
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Animals↗
Biomedical subjects
Suthat Fucharoen
Publications and source records attributed to Suthat Fucharoen.
Noninvasive prenatal diagnosis for hemoglobin Bart's hydrops fetalis.
Hemoglobin (Hb) Bart's hydrops fetalis, the most severe thalassemic disease, occurs from homozygosity of alpha-thalassemia 1. Deletion of all 4 alpha-globin genes (- -/- -) in this condition results in the absence of alpha-globin chains, and the physiologic dysfunction of Hb Bart's (gamma4) leads to lethality, either in utero or soon after birth. The best strategy for prevention and control of the disease is prenatal diagnosis in the mothers at risk. However, conventional prenatal diagnosis involves invasive procedures that may result in infection or abortion. In this study, a simple technique was developed to identify the presence or absence of alpha-globin chains in fetal nucleated red blood cells (NRBCs) enriched from maternal blood. Mononuclear cells including fetal NRBCs were isolated from maternal blood at 10 to 26 weeks of pregnancy by density-gradient centrifugation. Immunomagnetic separation with anti-CD71 antibody was employed to enrich fetal NRBCs, whose numbers increase with increasing gestational age. For the unaffected fetus, fetal NRBCs were detected by immunofluorescence microscopy after staining with rabbit antihuman alpha-globin antibody. In contrast, fetal red blood cells homozygous for alpha-thalassemia 1, which were identified from their size and morphology, did not stain for alpha-globin antibody. In this study, 3 affected fetuses were detected from 10 pregnancies at risk of Hb Bart's hydrops fetalis, and the results were confirmed by DNA analysis. In the remaining cases, all fetal NRBCs were positive for immunofluorescence staining. DNA analysis revealed that 2 cases were normal, 1 was heterozygous for alpha-thalassemia 2, and the other 4 cases were heterozygous for alpha-thalassemia 1.
Erythroblasts↗
Hb Kurosaki [alpha7(A5)Lys -->Glu (AAG --> GAG)]: an alpha2-globin gene mutation found in Thailand.
Hb Kurosaki [alpha 7(A5)Lys --> Glu (AAG --> GAG)], has been found for the first time in Thailand. The 30-year-old Thai male had a normal hematological profile at the steady state, but showed an abnormal hemoglobin (Hb) present at a level of 28%. Protein characterization was performed by automated sequencer analysis of the abnormal alpha-globin chain and amino acid analysis of the abnormal alphaT-1,2 peptide. Direct DNA sequence analysis of selectively amplified segments of the alpha1 and alpha2 genes showed that codon 7 of the alpha2-globin gene was heterozygous for AAG (Lys) and GAG (Glu). This was confirmed by restriction endonuclease digestion with Eco31I.
Adult↗
Flow cytometric quantitation of red blood cell vesicles in thalassemia.
BACKGROUND: Thalassemia is a hereditary hemolytic anemia caused by mutations in the globin gene complex. Circulatory disturbances including arterial and venous thrombosis have also been noted in these patients. Aggregability of abnormal RBC and the high level of membrane-derived microparticles stemming from activated platelets and other blood cells are thought to be responsible for the associated thrombotic risk. Destruction of RBC is also thought to be an important pathophysiological consequence, particularly through the formation of circulating vesicles. To our knowledge, there has been no attempt to quantitatively evaluate the number of RBC vesicles in thalassemia. This prompted us to study the level of RBC vesicles in the peripheral blood of thalassemia patients using quantitative flow cytometry. METHODS: Whole blood from each subject was doubly stained for RBC and platelet or annexin V markers, together with the known density TruCount beads. RBC vesicles were gated according to their forward/side scatter and RBC marker. Percentage of RBC vesicles and their absolute number were analyzed by flow cytometry. RESULTS: Our data indicated that RBC vesicles were annexin V-positive. The number of annexin V-positive events was higher than their intact RBCs. RBC vesicles were present in both normal and thalassemic blood samples, but the numbers of RBC vesicles were significantly higher in thalassemia. Both the percentage and the absolute number of RBC vesicles were especially marked in splenectomized subjects with beta-thalassemia/Hemoglobin E. When clinical and hematological indices were compared with RBC vesicles, there was an inverse relationship between the degree of severity in thalassemia patients and the number of RBC vesicles. CONCLUSION: Flow cytometric quantitation of RBC vesicles is simple, reliable and may offer new insights in to study of the relationship between defective hemoglobin synthesis, RBC perturbation and pathophysiological complications in thalassemia.
Adolescent↗
Two cases of compound heterozygosity for Hb Hekinan [alpha27(B8)Glu-->Asp (alpha1)] and alpha-thalassemia in Thailand.
Two unrelated cases of compound heterozygosity for Hb Hekinan [alpha27(B8)Glu-->Asp (alpha1) and alpha-thalassemia have been found in Thailand. Mutations were established at protein level by peptide mapping and at the DNA level by direct sequence analysis. Proband S.S. had genotype - -SEA/alpha2(A)alpha1Hekinan, betaA/betaE, while an unrelated proband, S.J., is the first case described with the genotype - -SEA/alpha2(A)alpha1Hekinan, betaA/betaA. Both alpha1Hekinan mutations were located in the alpha1 locus. Hb Hekinan could not be accurately estimated by HPLC, since it was poorly separated from Hb A. However IEF gave good separation of Hb Hekinan and Hb A, leading to estimates of Hb Hekinan (alpha Hekinan 2/beta A 2 and alpha Hekinan 2/beta E 2) level as 40-43% of total Hb.
Adult↗
Insertion of common mutations into the human beta-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette.
A large number of mutations have been described in the human beta-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human beta-globin locus. We report here the use of the GET Recombination system with an EcoRI/Kan(R) counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG-->AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human beta-globin locus. The counterselection cassette was first inserted into the target sequence in the beta-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI(q). Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.
Beta-Globulins↗
Prevalence and clinical significance of hepatitis C virus infection in Thai patients with thalassemia.
Hepatitis C virus (HCV) infection is a common cause of liver disease in thalassemia major patients in Western, especially Mediterranean, countries. Its significance in thalassemic patients from Southeast Asia has not been critically evaluated. In this report, we describe our study of the prevalence of HCV infection among Thai patients with thalassemia. The relationships of the infection to blood transfusion and the infection's effects on liver function have also been determined. Of the 104 patients studied, 21 (20.2%) tested positively by enzyme immunoassay for anti-HCV antibody, whereas only 2 patients (2%) had the hepatitis B surface antigen. There was no significant relationship between the presence of anti-HCV antibodies and the number and frequency of blood transfusions. In fact, 2 patients (10%) who tested positive for anti-HCV antibodies had never received transfusions. Patients with anti-HCV antibodies had significantly abnormal liver functions, such as higher levels of serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) and lower levels of serum albumin, compared with patients without anti-HCV antibodies (P = .021, .017, and .004, respectively). However, there were also significant correlations between iron status as indicated by transferrin saturation or serum ferritin levels and SGOT, SGPT, and gamma-glutamyltransferase (GGT) levels. Moreover, abnormal liver function as represented by elevated levels of SGOT, SGPT, GGT, and serum alkaline phosphatase was observed more frequently in patients with iron overload than in patients with a lower degree of iron burden. The presence of HCV did not alter the effects of iron overload on liver function. The findings suggest that both HCV and iron overload are the main causes of abnormal liver function in Thai patients with thalassemia. The treatment of both problems, if coexisting in patients with thalassemia, is required to prevent progression to chronic liver disease.
Adolescent↗
The signaling pathways of erythropoietin and interferon-gamma differ in preventing the apoptosis of mature erythroid progenitor cells.
Interferon (IFN)-gamma is a survival factor for mature erythroid progenitor cells. To elucidate related survival mechanisms, we compared the role of phosphatidylinositol 3-kinase (PI3-kinase) in the survival signals of IFN-gamma and erythropoietin (EPO). Human erythroid colony-forming cells (ECFCs) purified from peripheral blood were used, and Ly294002 was used as a PI3-kinase inhibitor. Treating ECFCs with a high concentration of Ly294002 (50 micromol/L) in the presence of EPO and/or IFN-gamma reduced cell viability by inducing apoptosis. However, treating cells with a lower concentration of Ly294002 (10 micromol/L) did not affect the antiapoptotic function of IFN-gamma and abolished the antiapoptotic effect of EPO. Adding IFN-gamma or EPO induced Bcl-x expression in ECFCs, as determined by Western blotting, and expression was suppressed in the presence of Ly294002. We also examined the phosphorylation of the protein kinase Akt, the downstream target of PI3-kinase. EPO stimulation significantly increased the level of Akt phosphorylation, but IFN-gamma did not. These results suggest that IFN-gamma plays a role in preventing the apoptosis of erythroid progenitor cells by affecting Bcl-x expression, thereby reducing the disruption of the mitochondrial transmembrane potential via PI3-kinase pathways that are related to but distinct from the EPO pathway.
Apoptosis↗
Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette.
There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human beta-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I-SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I-SceI site in the counterselection cassette is cut by I-SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants.
Base Sequence↗
Clinical trial of deferiprone iron chelation therapy in beta-thalassaemia/haemoglobin E patients in Thailand.
Nine patients with either beta-thalassaemia/haemoglobin E (7) or homozygous beta-thalassaemia (2) not requiring regular transfusions were treated with the oral iron chelator, deferiprone 25-50 mg/kg/d for between 17 and 86 weeks (mean 49 weeks). There were significant decreases in serum ferritin (initial mean +/- standard deviation 2168 +/- 1142, final 418 +/- 247 micro g/l; t-test for paired samples, P = 0.005), hepatic iron (initial 20.3 +/- 6.26, final 11.7 +/- 4.83 mg/g/dry weight; P = < 0.02), red cell membrane iron (initial 76.2 +/- 3.64, final 7.2 +/- 0.56 mmol/mg protein; P = < 0.0005) and serum non-transferrin bound iron (initial 9.0 +/- 0.56, final 5.9 +/- 0.89 micro mol/l; P = < 0.0005). There was also a significant rise in serum erythropoietin (initial 240 +/- 195.1, final 433.2 +/- 269.2 U/l; P = 0.034). The haemoglobin level rose in three patients and transfusion requirements were reduced substantially in four patients. Serum thiobarbituric acid reactive substance (TBARS) also fell in six of eight patients. Patients generally improved clinically, with weight gain observed. Side-effects were mild and included gastrointestinal symptoms (6) and arthralgia (1), not requiring withdrawal of the drug. One patient died at 17 weeks of therapy as a result of an intercurrent infection. His neutrophil count was normal. We conclude that deferiprone is an effective, well-tolerated iron chelator for patients with thalassaemia intermedia. Further studies are needed to determine the optimum dose and length of treatment needed to reduce iron burden to a safe level in these patients.
Administration, Oral↗
The effects of vitamin E on platelet activity in beta-thalassaemia patients.
A double-blind, crossover, placebo-controlled study of the effect of vitamin E on platelet functions was performed on nine splenectomized and 16 non-splenectomized beta-thalassaemia/haemoglobin E (beta-thalassaemia/HbE) patients. The patients were supplemented with a daily dose of vitamin E (525 IU) for 3 months. The functions of platelets were assessed by adenosine diphosphate (ADP)-induced platelet aggregation and adenosine triphosphate release. Plasma alpha-tocopherol, plasma thiobarbituric reactive substances (TBARs) and serum ferritin levels represented patients' antioxidant status, lipid peroxidation status and iron status respectively. Before experimentation, all patients had low plasma alpha-tocopherol levels. The splenectomized patients showed severe iron overload iron, had higher plasma TBAR levels and their platelets were more reactive to ADP than those of non-splenectomized patients. Three months of daily vitamin E supplementation resulted in a significant increase in plasma alpha-tocopherol levels and reduction in plasma TBAR levels in all patients. Serum ferritin levels of the patients were not altered; however, vitamin E reduced the platelet reactivity of the splenectomized patients towards normal levels. The influence of vitamin E on platelet reactivity may result in delaying hypoxaemia and pulmonary occlusion that commonly occurs in splenectomized beta-thalassaemia/HbE patients.
Adult↗
Hemin: a possible cause of oxidative stress in blood circulation of beta-thalassemia/hemoglobin E disease.
A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of beta-thalassemia/hemoglobin E disease (beta-thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 microM in serum of beta-thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of beta-thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of beta-thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum heroin, r = 0.778 (p < 0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p = 0.259) and 0.519 (p = 0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of beta-thal/Hb E.
Adult↗
Hb Kodaira II [beta146(HC3)His --> Gln] detected in Thailand.
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Adult↗
Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments.
We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system. Recombinant BAC clones are identified by PCR, using allele-specific amplification for the mutated sequences. We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, C-->T) into the human beta-globin locus. We have observed the frequency of recombinant clones to be as high as 1 in 100-200 clones. Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications.
Base Sequence↗
Hemoglobin H disease: not necessarily a benign disorder.
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Comorbidity↗
Increased circulating activated endothelial cells, vascular endothelial growth factor, and tumor necrosis factor in thalassemia.
An increased number of circulating endothelial cells (CECs) was demonstrated in alpha- and beta-thalassemic patients, beta-thalassemia/hemoglobin E (BE), both splenectomized (BE[S]) and non-splenectomized (BE[NS]), had higher numbers of CECs than alpha-thalassemia, both HbH (alpha-thal l/alpha-thal 2; H) and HbH with hemoglobin Constant Spring (alpha-thal 1/CS; H/CS). CECs were also increased in heterozygous HbE (EA) and homozygous HbE (EE). The highest level of tumor necrosis factor-alpha (TNF-alpha) was found in HbH/CS patients, whereas the highest levels of vascular endothelial growth factor (VEGF) was observed in BE[S] patients. Significant decreases, in protein C and protein S levels were found in both alpha- and beta-thalassemia compared with normal. Good correlations between the numbers of CEC and TNF-alpha, VEGF, protein C, and protein S levels were demonstrated in this study. In addition, markers for endothelial cell activation and injury (intercellular adhesion molecule-1, ICAM-1/CD54; vascular cell adhesion molecule-1, VCAM-1/CD106; and E-selectin, ELAM-1/CD62E) were detected on the surface of isolated CECs using immunofluorescence technique. Appearance of CECs with markers for endothelial cell activation, together with increased levels of TNF-alpha and VEGF and decreased levels of protein C and protein S in the circulation, may account for the propensity of vascular perturbation in thalassemic subjects.
Adult↗
Molecular analysis of beta-thalassemia in South Vietnam.
In Vietnam, the carrier rate for beta-thalassemia varies from 1.5% to 25% depending on the ethnic groups of the population. The molecular basis of beta-thalassemia in South Vietnam was studied in 50 unrelated beta-thalassemia patients. Of these, 31 had beta-thalassemia/Hb E, 18 were homozygous for beta-thalassemia, and 1 carried the beta-thalassemia trait. The majority of the patients were Kinh, four were Chinese, and two were Kinh-Chinese. All had severe anemia and received blood transfusions regularly, every 1-3 months. Hepatosplenomegaly was found in all patients, and splenectomy had been done in six patients. Normal alpha-globin genotype (alpha alpha/alpha alpha) was found in all subjects. Reverse dot-blot hybridization using oligonucleotide probes specific for Southeast Asian mutations can detect beta-thalassemia in 60 chromosomes in addition to 31 chromosomes with beta(E) mutation. Excluding the beta(E) gene, six previously reported Thai and Chinese beta-thalassemia mutations were found, including codons 41/42 (-TCTT) 35.3%, codon 17 (A-->T) 25.0%, -28 (A-->G) 7.3%, codons 71/72 (+A) 7.3%, IVS-II nt 654 (C-->T) 7.3%, and IVS-I nt 1 (G-->T) 6.0%. The Vietnamese frameshift mutation at codon 95 (+A) was detected by ARMS in seven chromosomes (10.3%). DNA polymorphism of the beta-globin gene cluster carrying the codon 95 mutation was - + - - - - - + for (G)gamma/XmnI, epsilon/HincII, (G)gamma/HindIII, (A)gamma/HindIII, psi beta/HincII, 3' psi beta/HincII, beta/AvaII, and 3'beta/BamHI, respectively. The remaining mutation detected by the gap PCR was a large deletion known as the Chinese (G)gamma((A)gamma delta beta)(0)-thalassemia. The two most common mutations were the frameshift at codons 41/42 (-TCTT) and the nonsense mutation in codon 17 (A-->T). Thus beta-thalassemia mutations in South Vietnam is similar to the previous report from the North, although at different frequencies. This result will help to establish a center for prenatal diagnosis and for prevention and control of thalassemia in Vietnam.
Adolescent↗
Repair of a splicing defect in erythroid cells from patients with beta-thalassemia/HbE disorder.
A HeLa cell line stably expressing the human beta-globin gene carrying thalassemic mutations beta(E)/IVS1-6 served as a thalassemia model for repair of aberrant splicing of beta(E)-globin pre-mRNA with antisense oligonucleotides. Treatment of beta(E)/IVS1-6 HeLa cells with a morpholino oligonucleotide targeted immediately upstream of the aberrant 5' splice site activated by the mutations resulted in an increase in the amount of correctly spliced beta(E)-globin mRNA in a dose-dependent and sequence-specific fashion. The repaired beta(E)-globin mRNA was stable and could be translated into full-length beta(E)-globin polypeptide. Application of the same oligonucleotide to erythroid progenitor cells from two beta-thalassemia/HbE patients resulted in an approximately 70% increase in correct beta(E)-globin mRNA and 36% increase in hemoglobin E. The erythroid progenitor cells represent the actual targets for the clinical application of antisense repair of defective pre-mRNAs.
Alternative Splicing↗