Increased autophagy leads to decreased apoptosis during ß-thalassaemic mouse and patient erythropoiesis.

ß-Thalassaemia results from defects in ß-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both ßIVS2-654-thalassaemic mice and ß-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in ß-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both ß-thalassaemic mice and ß-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in ß-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in ß-thalassaemia.

Thalassemia in Thailand.

Thailand has a population of 66.2 million with 30.0-40.0% of them carrying thalassemia genes. Interaction of these thalassemia genes lead to more than 60 genotypes with a wide spectrum of clinical severity from asymptomatic to lethal. Estimation based on gene frequencies and number of babies born each year, there will be about 1.2% babies born with severe cases of thalassemia each year. Further estimation revealed that 1.0% of the Thai population have thalassemia disease, which is a big health problem for the country. Thalassemia prevention and control programs were introduced using post conception screening in couples and prenatal diagnosis (PND) for the prevention of new thalassemic births. Moreover, the majority of existing cases are undergoing supportive treatment with regular blood transfusions and iron chelation. Curative treatment by hematopoietic stem cell transplantation (HSCT) is available but is limited to a minority of the patients.

Impaired Terminal Erythroid Maturation in ß0-Thalassemia/HbE Patients with Different Clinical Severity.

Anemia in ß-thalassemia is associated with ineffective erythropoiesis and a shortened lifespan of erythroid cells. The limited differentiation of ß-thalassemic erythroblasts has been documented, but the characteristic feature of terminal erythroid maturation and its physiological relevance are not clearly described in ß-thalassemias. Here, the red blood cell and reticulocyte cellular characteristics were determined in patients with ß0-thalassemia/HbE in comparison to patients with iron deficiency anemia and healthy normal subjects. Severely affected ß0-thalassemia/HbE patients showed the highest increase in immature reticulocytes, but the number of total erythrocytes was the lowest. Despite similar ranges of hemoglobin levels, ß0-thalassemia/HbE patients had a higher number of reticulocytes and a greater proportion of immature fraction than patients with iron deficiency anemia did. In vitro CD34+ hematopoietic progenitor cells' culture and flow cytometry analysis were conducted to investigate the erythroid maturation and mitochondrial clearance in ß0-thalassemia/HbE erythroid cells as compared to normal cells. The delayed erythroid maturation and evidence of impaired mitochondria clearance were observed in ß0-thalassemia/HbE cells at the terminal stage of differentiation. Additionally, increased transcript levels of genes related to erythroid mitophagy, BNIP3L and PINK1, were revealed in ß0-thalassemia/HbE erythroblasts. The findings indicate that the erythroid maturation is physiologically relevant, and that the restoration of terminal maturation represents a potential therapeutic target for ß-thalassemias.

Update in Laboratory Diagnosis of Thalassemia.

Alpha- and ß-thalassemias and abnormal hemoglobin (Hb) are common in tropical countries. These abnormal globin genes in different combinations lead to many thalassemic diseases including three severe thalassemia diseases, i.e., homozygous ß-thalassemia, ß-thalassemia/Hb E, and Hb Bart's hydrops fetalis. Laboratory diagnosis of thalassemia requires a number of tests including red blood cell indices and Hb and DNA analyses. Thalassemic red blood cell analysis with an automated hematology analyzer is a primary screening for thalassemia since microcytosis and decreased Hb content of red blood cells are hallmarks of all thalassemic red blood cells. However, these two red blood cell indices cannot discriminate between thalassemia trait and iron deficiency or between α- and ß-thalassemic conditions. Today, Hb analysis may be carried out by either automatic high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) system. These two systems give both qualitative and quantitative analysis of Hb components and help to do thalassemia prenatal and postnatal diagnoses within a short period. Both systems have a good correlation, but the interpretation under the CE system should be done with caution because Hb A2 is clearly separated from Hb E. In case of α-thalassemia gene interaction, it can affect the amount of Hb A2/E. Thalassemia genotypes can be characterized by the intensities between alpha-/beta-globin chains or alpha-/beta-mRNA ratios. However, those are presumptive diagnoses. Only DNA analysis can be made for specific thalassemia mutation diagnosis. Various molecular techniques have been used for point mutation detection in ß-thalassemia and large-deletion detection in α-thalassemia. All of these techniques have some advantages and disadvantages. Recently, screening for both α- and ß-thalassemia genes by next-generation sequencing (NGS) has been introduced. This technique gives an accurate diagnosis of thalassemia that may be misdiagnosed by other conventional techniques. The major limitation for using NGS in the screening of thalassemia is its cost which is still expensive. All service labs highly recommend to select the technique(s) they are most familiar and most economic one for their routine use.

Development of DNA controls for detection of ß-thalassemia mutations commonly found in Asian.

INTRODUCTION: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of ß-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe ß-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for ß-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. METHODS: Four recombinant ß-globin plasmids, including a normal sequence and three different mutant panels covering 10 common ß-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis. RESULTS: We demonstrated the applicability of established positive DNA controls for ß-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant ß-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant ß-globin plasmids, are required for detecting 10 common ß-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. CONCLUSION: The ß-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of ß-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.

Elevated levels of miR-210 correlate with anemia in ß-thalassemia/HbE patients.

Ineffective erythropoiesis in ß-thalassemia patients is caused by the premature death of red blood cell precursors due to excess α-globin chains. As a consequence, patients develop chronic anemia and hypoxia. Upregulation of miR-210, a hypoxia-induced miRNA, has been shown to regulate globin gene expression and erythroid differentiation in ß-thalassemia/HbE erythroid progenitor cell culture. The present study examined whether the expression of miR-210 in circulation reflects the anemic condition in these patients. The level of miR-210 expression was directly examined from red blood cells and plasma of ß-thalassemia/HbE patients. Transferrin receptor, a marker of erythropoiesis activity, was also analyzed. Increased expression of both red blood cells and plasma miR-210 as well as elevated levels of serum transferrin receptor in ß-thalassemia/HbE patients were observed when compared to those of normal individuals (p < 0.05). In addition, red blood cell miR-210 level was inversely correlated with hemoglobin levels (r = -0.7054, p < 0.01) and hematocrit (r = -0.6017, p < 0.05). The higher expression of miR-210 in these patients may be the consequence of hypoxia occurring from the lower hemoglobin level. Thus, analysis of red blood cell miR-210 may be useful as a method for assessing hypoxia in ß-thalassemia patients.

Molecular Epidemiology of Hemoglobinopathies in Cambodia.

Determining the magnitude of the thalassemia problem in a country is important for implementing a national prevention and control program. In order to acquire accurate thalassemia prevalence data, the gene frequency of α- and ß-thalassemia (α- and ß-thal) in different regions of a country should be determined. The molecular basis of thalassemia in Cambodia was performed by polymerase chain reaction (PCR)-based techniques in a community-based cross-sectional survey of 1631 unrelated individuals from three regions, Battambang, Preah Vihear and Phnom Penh. Thalassemia mutations were detected in 62.7% of the three studied population of Cambodia. Hb E (HBB: c.79G > A) was the most common ß-globin gene mutation with a frequency ranging from 0.139 to 0.331, while the most frequent α-globin gene mutation was the -α(3.7) (rightward) deletion (0.098-0.255). The other frequencies were 0.001-0.003 for ß-thal, 0.008-0.011 for α-thal-1 (- -(SEA)), 0.003-0.008 for α-thal-2 [-α(4.2) (leftward deletion)], 0.021-0.044 for Hb Constant Spring (Hb CS, HBA2: c.427T > C) and 0.009-0.036 for Hb Paksé (HBA2: c.429A > T). A regional specific thalassemia gene frequency was observed. Preah Vihear had the highest prevalence of Hb E (55.9%), α-thal-2 (24.0%) and nondeletional α-thal (15.1%), whereas Phnom Penh had the lowest frequency of thalassemia genes. Interestingly, in Preah Vihear, the frequency of Hb Paksé was extremely high (0.036), almost equivalent to that of Hb CS (0.044). Our results indicate the importance of micromapping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in Cambodia.

Accelerated telomere shortening in ß-thalassemia/HbE patients.

ß-Thalassemia/HbE disease is caused by a defective ß-globin synthesis that leads to accumulation of excess unbound α-globins, and consequently oxidative stress, ineffective erythropoiesis and chronic anemia. Cell replication and oxidative stress are factors contributing to erosion of telomeres responsible for maintaining genomic stability and cell replication capability. In this study, the rate of telomere shortening in ß-thalassemia/HbE patients was compared to the rate of telomere shortening in normal individuals. Telomere length was determined from peripheral blood mononuclear cells of 43 ß-thalassemia/HbE patients and 22 normal controls using Flow-FISH analysis. The telomere length was shown to be age-dependent in normal group (rs = 0.715, P = 0.002), whereas severity-dependent telomere shortening was observed in the patients. The telomere length of patients who had severe clinical symptoms (10.07 ± 2.15%) was shorter than that of patients who showed mild symptoms (15.59 ± 2.27%), moderate symptoms (14.50 ± 1.41%) and those in the normal group (14.75 ± 3.11%, P < 0.05). Additionally, reticulocyte count and oxidative stress were correlated with telomere length. This indicates that increased oxidative stress and markedly enhanced erythropoiesis in ß-thalassemia/HbE patients leads to accelerated telomere erosion in clinically severe patients.

Discrimination of various thalassemia syndromes and iron deficiency and utilization of reticulocyte measurements in monitoring response to iron therapy.

Decreased hemoglobinization of red cells resulting in hypochromia and microcytosis are the main features of thalassemia syndromes, and also of iron deficiency anemia (IDA). A simple and reliable method is required to distinguish the two conditions in the routine laboratories. In this study we analyzed the red cell and reticulocyte parameters from 414 samples of various types of thalassemias and IDA and discovered a variety of discriminating criteria including a discrimination index (DI) which should be useful for differential diagnosis. Slightly decreased MCV and CH are suggestive of α-thalassemia 2, Hb CS, and Hb E heterozygotes whereas the increased Rbc counts are obvious in α-thalassemia 1 and ß-thalassemia. In Hb E, the number of microcytic red cells was greater than the number of hypochromic red cells resulting in an increased M/H ratio. Hb H diseases are characterized by a higher number of hypochromic red cells and decreased CHCM, while broadening of hemoglobin concentration histogram results in increased HDW in ß-thalassemia diseases. Iron deficiency anemia results in hypochromic-microcytic red cells and increased RDW. The number of reticulocyte with %High Retic and CHr value were increased in the first month of iron supplementation indicating the response to iron therapy.

Validation of the immunochromatographic strip for α-thalassemia screening: a multicenter study.

α(0)-Thalassemia occurs from a deletion of 2 linked α-globin genes and interaction of these defective genes leads to hemoglobin (Hb) Bart's hydrops fetalis, the most severe and lethal thalassemia syndrome. Identification of α(0)-thalassemia carriers is thus essential for the prevention and control program. An immunochromatographic (IC) strip test was developed for rapid screening of α(0)-thalassemia by testing for Hb Bart's in the blood samples using a specific monoclonal antibody against Hb Bart's. To evaluate its sensitivity and specificity, the IC strip test was assessed in a cohort with various thalassemia genotypes from 4 different laboratories in Thailand and Australia. The result showed 97% sensitivity in α-thalassemia carriers with 2 α-globin genes deletion and Hb H disease. This is, in particular, the useful rapid screening test for regions where ß-thalassemia and homozygous Hb E are also common. Similar hematologic and Hb data make it impossible to address the concomitant inheritance of α(0)-thalassemia in these samples without polymerase chain reaction (PCR)-based techniques, leading to misdiagnosis of the risk of having Hb Bart's hydrops fetalis. However, α-globin genotyping should be carried out in samples with positive IC strip as positive reactivity was also observed in homozygous α(+)-thalassemia carriers who have 2 trans α-globin gene deletions. These results indicate that in combination with red blood cell indices, the IC strip test could rule out mass populations for further α(0)-thalassemia detection by PCR-based analysis. The Alpha Thal IC strip also has the potential to replace testing for Hb H inclusion bodies, as it appears to be more sensitive, specific, and less labor intensive.

Cardiomyocyte ultrastructural damage in ß-thalassaemic mice.

ß-thalassaemia is a hereditary anaemia resulting from the absence or reduction in ß-globin chain production. Heart complications related to iron overload are the most serious cause of death in these patients. In this report cardiac pathology of ß-thalassaemic mice was evaluated by light and electron microscopy. The study was carried out in thalassaemic mice carrying human ß-thalassaemia mutation, IVSII-654 (654), transgenic mice carrying human ß(E) -globin transgene insertion (E4), thalassaemic mice with human ß(E) -globin transgene insertion (654/E4) and homozygous thalassaemic mice rescued by the human ß(E) -globin transgene (R), which is generated by cross-breeding between the 654 and E4 mice. Histology showed iron deposition in cardiac myocytes of 654 and R mice, but the ultrastructural damage was observed only in the R mice when compared with the wild type, 654, E4 and 654/E4 mice. Histopathological changes in the cardiomyocytes of the R mice included mitochondrial swelling, loss of myofilaments and the presence of lipofuscin, related to the increased level of tissue iron content. The progressive ultrastructural pathology in R mice cardiomyocytes is consistent with the ultrastructural pathology previously studied in patients with thalassaemia. Thus, this R thalassaemic mouse model is suitable for in vivo pathophysiological study of thalassaemic heart.

A reduced curcuminoid analog as a novel inducer of fetal hemoglobin.

Thalassemia is an inherited disorder of hemoglobin molecules that is characterized by an imbalance of α- and ß-globin chain synthesis. Accumulation of unbound α-globin chains in erythroid cells is the major cause of pathology in ß-thalassemia. Stimulation of γ-globin production can ameliorate disease severity as it combines with the α-globin to form fetal hemoglobin. We examined γ-globin-inducing effect of curcuminoids extracted from Curcuma longa L. and their metabolite reduced forms in erythroid leukemia K562 and human primary erythroid precursor cells. The results showed that curcuminoid compounds, especially bisdemethoxycurcumin are potential γ-globin enhancers. We also demonstrated that its reduced analog, hexahydrobisdemethoxycurcumin (HHBDMC), is most effective and leads to induction of γ-globin mRNA and HbF in primary erythroid precursor cells for 3.6 ± 0.4- and 2.0 ± 0.4-folds, respectively. This suggested that HHBDMC is the potential agent to be developed as a new therapeutic drug for ß-thalassemia and related ß-hemoglobinopathies.

Haemoglobinopathies in southeast Asia.

In Southeast Asia α-thalassaemia, ß-thalassaemia, haemoglobin (Hb) E and Hb Constant Spring (CS) are prevalent. The abnormal genes in different combinations lead to over 60 different thalassaemia syndromes, making Southeast Asia the locality with the most complex thalassaemia genotypes. The four major thalassaemic diseases are Hb Bart's hydrops fetalis (homozygous α-thalassaemia 1), homozygous ß-thalassaemia, ß-thalassaemia/Hb E and Hb H diseases. α-Thalassaemia, most often, occurs from gene deletions whereas point mutations and small deletions or insertions in the ß-globin gene sequence are the major molecular defects responsible for most ß-thalassaemias. Clinical manifestations of α-thalassaemia range from asymptomatic cases with normal findings to the totally lethal Hb Bart's hydrops fetalis syndrome. Homozygosity of ß-thalassaemia results in a severe thalassaemic disease while the patients with compound heterozygosity, ß-thalassaemia/Hb E, present variable severity of anaemia, and some can be as severe as homozygous ß-thalassaemia. Concomitant inheritance of α-thalassaemia and increased production of Hb F are responsible for mild clinical phenotypes in some patients. However, there are still some unknown factors that can modulate disease severity in both α- and ß-thalassaemias. Therefore, it is possible to set a strategy for prevention and control of thalassaemia, which includes population screening for heterozygotes, genetic counselling and foetal diagnosis with selective abortion of affected pregnancies.

Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of ß(E) pre-mRNA splicing determine disease severity.

Thalassaemia is characterized by the reduced or absent production of globins in the haemoglobin molecule leading to imbalanced α-globin/non α-globin chains. HbE, the result of a G to A mutation in codon 26 of the HBB (ß-globin) gene, activates a cryptic 5' splice site in codon 25 leading to a reduction of correctly spliced ß(E) -globin (HBB:c.79G>A) mRNA and consequently ß(+) -thalassaemia. A wide range of clinical severities in bothα- and ß-thalassaemia syndromes, from nearly asymptomatic to transfusion-dependent, has been observed. The correlation between clinical heterogeneity in various genotypes of thalassaemia and the levels of globin gene expression and ß(E) -globin pre-mRNA splicing were examined using multiplex quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and allele-specific RT-qPCR. The α-globin/non α-globin mRNA ratio was demonstrated to be a good indicator for disease severity among different thalassaemia disorders. However, the α-globin/non α-globin mRNA ratio ranged widely in ß-thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced ß(E) -globin mRNA ratio in 30% of mild ß-thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of ß(E) -globin pre-mRNA differs among ß-thalassaemia/HbE patients and serves as one of the modifying factors for disease severity.

Quantitative analysis of Hb Bart's in cord blood by capillary electrophoresis system.

It has long been recognized that the presence of hemoglobin (Hb) Bart's in newborn's blood is associated with α-thalassemia. However, the automated high-performance liquid chromatography or low-performance liquid chromatography system is unable to quantify the amount of Hbs Bart's and H, which are eluted at the retention time close to 0 min. This study used automatic capillary electrophoresis (CE) system to diagnose various types of α-thalassemia in 587 cord blood samples, including 429 normal α-globin genotype, 120 cases of thalassemia with one α-globin gene defect, 34 cases with two α-globin genes defect, and four cases with three α-globin genes defect. The result showed that the level of Hb Bart's in cord blood was increased accordingly with the increasing numbers of the defective α-globin genes. In addition, Hb Bart's level at 0.2%, as measured by CE, can be used as a cut-off point for α-thalassemia diagnosis in newborns.

Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand.

Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies.

Effect of human beta-globin bacterial artificial chromosome transgenesis on embryo cryopreservation in mouse models.

The purpose of the present study was to investigate the efficiency of embryo cryopreservation for four transgenic (TG) thalassaemic mouse strains, which is a key element of the ongoing gene banking efforts for these high-value animals. Heterozygous TG embryos were produced by breeding four lines of TG males to wild-type (WT) females (C57BL/6J). Intact two-cell embryos were cryopreserved by vitrification in straws using 35% ethylene glycol. Survival rates of cryopreserved embryos ranged between 91.1% (102/112) and 93.6% (176/188) without significant differences between the lines. In contrast, the paternal line had a significant effect on the development of these embryos to the blastocyst stage, which ranged from 50.6% (92/182) to 77.5% (79/102). This effect was also noted following embryo transfers, with implantation rates varying from 17.3% (19/110) to 78.1% (35/45). The results demonstrate that the in vivo developmental potential is significantly influenced by TG line and reveal a specific line effect on cryosurvival. All bacterial artificial chromosome transgenic fetuses developed from vitrified-warmed embryos showed expression of the human beta-globin transgene. In conclusion, the present study shows a strong TG line effect on developmental competence following cryopreservation and the vitrification method was successful to bank the human beta-globin TG-expressing mouse strains.

Expression of microRNA-451 in normal and thalassemic erythropoiesis.

MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis. Thalassemia, a defective globin synthesis leading to precipitate of excess unbound globins in red blood cell precursors, results in defective erythroid precursors and ineffective erythropoiesis. Expression pattern of miR-451, an erythroid-specific miRNA, was analyzed during differentiation of erythroid progenitors derived from normal and thalassemic peripheral blood CD34-positive cells, after 14 days of culture. A biphasic expression with transient up-regulation of miRNA-451 on day 3 of cultures was observed during thalassemic erythroid differentiation. In contrast, the expression pattern of the miR-451 in erythroid cells obtained from the other extravascular hemolytic anemia, i.e., hereditary spherocytosis patients showed no transient up-regulation of miR-451 on day 3 of cultures. Our results suggest that early erythroid progenitors in beta-thalassemia have a dysregulated miRNA-451 expression program, and analysis of microRNA is a relevant approach to determine abnormalities of erythropoiesis.

Genetic modifiers of Hb E/beta0 thalassemia identified by a two-stage genome-wide association study.

BACKGROUND: Patients with Hb E/beta0 thalassemia display remarkable variability in disease severity. To identify genetic modifiers influencing disease severity, we conducted a two-stage genome scan in groups of 207 mild and 305 severe unrelated patients from Thailand with Hb E/beta0 thalassemia and normal alpha-globin genes. METHODS: First, we estimated and compared the allele frequencies of approximately 110,000 gene-based single nucleotide polymorphisms (SNPs) in pooled DNAs from different severity groups. The 756 SNPs that showed reproducible allelic differences at P < 0.02 by pooling were selected for individual genotyping. RESULTS: After adjustment for age, gender and geographic region, logistic regression models showed 50 SNPs significantly associated with disease severity (P < 0.05) after Bonferroni adjustment for multiple testing. Forty-one SNPs in a large LD block within the beta-globin gene cluster had major alleles associated with severe disease. The most significant was bthal_bg200 (odds ratio (OR) = 5.56, P = 2.6 x 10(-13)). Seven SNPs in two distinct LD blocks within a region centromeric to the beta-globin gene cluster that contains many olfactory receptor genes were also associated with disease severity; rs3886223 had the strongest association (OR = 3.03, P = 3.7 x 10(-11)). Several previously unreported SNPs were also significantly associated with disease severity. CONCLUSIONS: These results suggest that there may be an additional regulatory region centromeric to the beta-globin gene cluster that affects disease severity by modulating fetal hemoglobin expression.

Rapid diagnosis of alpha-thalassemia by melting curve analysis.

alpha-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of alpha-globin protein. Couples who both carry the alpha-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the alpha-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study, a new and accurate method for alpha-thalassemia diagnosis was developed by genotyping alpha-thalassemia-1, the Southeast Asian type (--(SEA)) and Thai type (--(THAI)) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments, the --(SEA) and --(THAI) deletions and two normal fragments, psizeta- and alpha2-globin gene. The primers were capable of distinguishing alpha-thalassemia 1 heterozygotes from alpha-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the --(THAI), --(SEA), psizeta-globin, and alpha2-globin gene fragments were 79.9 +/- 0.2, 89.4 +/- 0.5, 92.8 +/- 0.2, and 85.0 +/- 0.2 degrees C, respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis, and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process, which is laborious, and allows high throughput screening suitable for large population screening for prevention and control of thalassemia.