Hemoglobinopathies in Southeast Asia: molecular biology and clinical medicine.
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Asia, Southeastern↗
Biomedical subjects
P Winichagoon
Publications and source records attributed to P Winichagoon.
Hydroxyurea increases hemoglobin F levels and improves the effectiveness of erythropoiesis in beta-thalassemia/hemoglobin E disease.
Hydroxyurea (HU) is one of several agents that have been shown to enhance hemoglobin (Hb) F levels in patients with sickle cell disease and may be useful as a therapy for beta-globinopathies. However, limited information exists on the effects of HU in patients with thalassemia. Accordingly, we examined the hematologic effects of orally administered HU in 13 patients with beta-thalassemia/Hb E, including four patients who had been splenectomized. These patients were treated with escalating doses (final range, 10 to 20 mg/kg/d) for 5 months and were observed in the outpatient hematology clinic every 2 to 4 weeks. Complete blood counts including reticulocyte counts, amounts of Hb E and Hb F, G gamma:A gamma and alpha:non-alpha globin biosynthetic ratios were evaluated before and during treatment. Almost all patients responded with an average increase of 33% in Hb F levels, from a mean (+/- SD) of 42% +/- 11% to 56% +/- 8% (P < .0001), and a reciprocal decline in the percentage of Hb E from 59% +/- 9% to 49% +/- 8% (P < .001). Reticulocytosis was decreased from a mean (+/- SD) of 18.0% +/- 15.6% to 11.7% +/- 9.1% (P < .05); there was also a slight (10%) but statistically significant increase in hemoglobin levels and an improved balance in alpha:non-alpha globin chains ratios. The side effects were minimal in most patients, although these patients tended to tolerate a lower dose of HU before significant myelosuppression than has been our previous experience in sickle cell disease. One splenectomized patient died of sepsis during the trial. We conclude that increased Hb F production in beta-thalassemia/Hb E patients, with an improvement in the alpha:non-alpha globin ratios and, probably, the effectiveness of erythropoiesis, can be achieved using HU. Longer trials of HU in this population, including at other doses and in combination with other agents, appear warranted.
Adolescent↗
Reverse dot-blot detection of Thai beta-thalassaemia mutations.
Pending curative therapy, newborn screening and prenatal diagnosis are essential to the management of beta thalassaemia. Diagnosis using electrophoretic methods is difficult in the presence of composite phenotypes and high Hb F levels. Direct DNA detection of mutant alleles circumvents both problems, but the enormous diversity of beta-thalassaemia mutations poses challenges for this approach. Among PCR-based tests, the reverse dot-blot method enables screening several mutations with a single hybridization reaction. Unfortunately it has often been targeted to only the common mutations of a particular ethnic population, necessitating the use of more arduous detection methods for the less common mutations. We developed a reverse dot-blot strip for the 10 beta-thalassaemia mutations, including the beta-thalassaemic haemoglobinopathies Hb E and Hb Malay, that account for 96% of beta thalassaemia in Thailand, and another strip for six less common Thai mutations. The second strip precludes the need for more technically challenging methods. To avoid problems associated with secondary structure of amplified full-length target DNA, we amplified and labelled beta-globin DNA as two shorter fragments that encompassed all known Thai mutations. Reverse dot-blotting is a rapid, accurate method for detecting beta-thalassaemia mutations.
Base Sequence↗
Detection of alpha-thalassemia-1 (Southeast Asian type) and its application for prenatal diagnosis.
A simple non-radioactive method based on the polymerase chain reaction was used to detect the Southeast Asian type of alpha-thalassemia 1 (--). Three oligonucleotide primers, one of which was adjacent to the breakpoint of the alpha-thalassemia-1 allele, were used to amplify the 570 and 194 bp DNA fragments. The 570 bp product was specific to the alpha-thalassemia-1 determinant and the 194 bp fragment was amplified from either the alpha-thalassemia-2 (-alpha) or normal alpha-globin (alpha alpha) determinants. In Hb Bart's hydrops fetalis (--/--), only the 570 bp fragment was obtained, whereas the 194 bp fragment was amplified in normal individual (alpha alpha/alpha alpha) and alpha-thalassemia-2 trait (-alpha/alpha alpha). Both 570 and 194 bp fragments were detected in alpha-thalassemia-1 trait (--/alpha alpha) and Hb H patients (--/-alpha). This procedure is useful for the rapid screening of alpha-thalassemia-1 trait and prenatal diagnosis of Hb Bart's hydrops fetalis in populations with a high frequency of the Southeast Asian Type of alpha-thalassemia-1.
Asia, Southeastern↗
Molecular mechanisms of thalassemia in southeast Asia.
Hemoglobinopathies are the most common genetic disorders in Southeast Asia. alpha-Thalassemia is most often due to a alpha-globin gene deletion. Hb Constant Spring (CS) occurs from the mutation at the termination codon of the alpha-globin gene resulting in an elongated polypeptide; alpha(CS)-globin mRNA is also unstable and only small amounts of Hb CS are produced. Thus Hb CS has an alpha-thalassemia 2-like effect. beta-Thalassemia results from a variety of molecular mechanisms, most of which are single base substitutions or deletions or insertions of one to four nucleotides. Hemoglobin E occurs from a Glu --> Lys substitution at position 26 of the beta-globin chain. The abnormal gene also results in reduced amounts of beta E-mRNA and hence of beta E-globin chains. Therefore, Hb E has a mild beta + thalassemia phenotype. Homozygous beta-thalassemia and beta-thalassemia/Hb E are the major beta-thalassemic syndromes in Southeast Asia. In spite of seemingly identical genotypes, severity of beta-thalassemia/Hb E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. A number of genetic factors have been shown to determine the differences in severity of anemia in beta-thalassemia/Hb E, including co-inheritance of alpha-thalassemia determinants and co-inheritance of other determinants which elevate Hb F expression. A correlation between the extent of beta E-globin mRNA cryptic splicing and the severity of anemia in beta(zero)-thalassemia/Hb E patients has been observed. Complete characterization of mutations causing hemoglobinopathies will help to bolster the establishment of prenatal diagnosis of these genetic disorders in the region.
Alternative Splicing↗
Role of alternatively spliced beta E-globin mRNA on clinical severity of beta-thalassemia/hemoglobin E disease.
In spite of seemingly identical genotypes, severity of beta-thalassemia/hemoglobin (Hb) E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. Since mutation in codon 26 of the beta E-globin gene can lead to an alternative splicing, Hb E acts like a mild beta(+)-thalassemia. Variation in the amount of beta E-globin mRNA may also govern the difference in severity of anemia in beta-thalassemia/Hb E patients who otherwise have the same genetic determinants. We have determined the percentage of the alternatively spliced beta E-globin mRNA by the RT-PCR technique in 14 patients and found that the amount of abnormal spliced beta E-globin mRNA in those patients with severe symptoms ranged between 2.9 to 6.1%, whereas those with milder symptoms had the values which ranged between 1.6 to 2.6%. The extent of beta E-globin mRNA cryptic splicing was better associated with clinical severity of the patients than did the patterns of the Xmn I polymorphism at position -158 of the G gamma-globin gene or levels of Hb F.
Adolescent↗
Prenatal diagnosis for beta-thalassemia syndromes using HRP-labeled oligonucleotide probes at Siriraj Hospital.
Characterization of the molecular defect of beta-thalassemia in Thais has enabled us to establish prenatal diagnosis for homozygous beta-thalassemia and beta-thalassemia/Hb E. The nature of the beta-thalassemia mutation of each high risk couple or of the previous affected child was firstly identified after counseling. Detection of beta-thalassemia mutations was performed by dot-blot hybridization of the amplified DNA with a set of HRP-labeled ASO-probes specific for the common mutations. If the mutation could be characterized, prenatal diagnosis (PND) would be performed by using DNA extracted either from the chorionic villi (CVS) or amniotic fluid fibroblast in the first trimester of pregnancy or from fetal blood in the second trimester. DNA analysis was carried out in 23 couples at risk of having homozygous beta-thalassemia and 88 couples at risk for beta-thalassemia/Hb E. However, PND was performed by this technique in 22 pregnancies from 21 couples at risk of having homozygous beta-thalassemia children and 86 pregnancies from 71 couples at risk for beta-thalassemia/Hb E; 9 couples underwent more than one prenatal diagnosis. The results showed that, although there are more than 20 beta-thalassemia mutations in the Thai population, PND by DNA analysis could be carried out in more than 95% of the risk couples by using beta(E) and 10 different HRP-labeled ASO probes. This technique was simple, economic and avoided the use of radioactive isotope.
Amniocentesis↗
Nondeletional type of hereditary persistence of fetal haemoglobin: molecular characterization of three unrelated Thai HPFH.
The beta-globin gene clusters of three unrelated Thai families with a nondeletional type of hereditary persistence of fetal haemoglobin (HPFH) were studied using polymerase chain reaction-related techniques. All appeared to have normal nucleotide sequences from the Cap site to position -400 of both the G gamma- and A gamma-globin genes. Two individuals suspected of having a beta-thalassaemia gene linked to the high HbF condition also had a normal beta-globin gene sequence, spanning from position -108 from the Cap site to the polyadenylation site. Deletion of four nucleotides, AGCA, at positions -225 to -222 of one A gamma-globin allele was detected in one subject and was confirmed by dot-blot hybridization. Restriction fragment length polymorphisms in the beta-globin gene cluster showed that the 5' haplotype (-+-++) and the presence (+) of an Xmm 1 polymorphic site at -158 of the G gamma-globin gene are associated with the high F phenotype in these families. Direct sequencing of the 5' hypersensitive-2 (5' HS-2) site of the locus control region (LCR) showed that this Xmn 1 (+) site is also linked to a specific rearrangement of TA repeats (TA)9CACATATACG(TA)10, in HS-2 segment.
Adult↗
Severity differences in beta-thalassaemia/haemoglobin E syndromes: implication of genetic factors.
Genetic factors determining the difference in severity of anaemia in beta-thalassaemia/HbE disease were studied in 90 patients who had haemoglobin levels, at steady state, ranging from 4.2 to 12.6 g/dl. Co-inheritance of alpha-thalassaemia 2 and haemoglobin Constant Spring could significantly decrease the severity of the disease. Inheritance of a beta-thalassaemia chromosome with Xmn I cleavage site at position -158 of the G gamma-globin gene which was linked to the haplotype -+-++ or ++-++, was associated with a milder anaemia. Two copies of these alleles were necessary to produce a significant clinical effect. Increased expression of the G gamma-globin gene and higher production of haemoglobin F, which could reduce the overall globin chain imbalance, were also associated with homozygosity for the Xmn I cleavage site and thus with less severe anaemia. However, this effect was not seen in Xmn I site heterozygotes. Whether the effects of the Xmn I polymorphism, HbF concentration and G gamma/A gamma ratio act separately or through common mechanisms in reducing anaemia remains to be ascertained.
Adolescent↗
Identification of Hb C [beta 6(A3)Glu-->Lys] in a Thai male.
The propositus was a 29-year-old Thai male, whose electrophoretic pattern showed Hb A (58%) plus an abnormal hemoglobin (42%) with mobility identical to Hb A2 and Hb E. Protein sequencer analysis and tryptic peptide mapping of the beta chain indicated that the abnormal hemoglobin was Hb C [beta 6(A3)Glu-->Lys], rather than Hb E which is more commonly found in South East Asia. This conclusion was confirmed by direct sequence analysis of the propositus' DNA, which showed AAG as well as GAG at codon 6 of the beta gene, in agreement with heterozygosity for Hb C and Hb A. Furthermore, the beta gene framework (Ava II-, Bam HI+) of the propositus suggested that the beta C gene may have arisen from an independent mutation. Since Hb C and Hb E have the same mutation (Glu-->Lys) in the beta chain, although at different positions, and behave similarly in electrophoresis, cases of Hb C and Hb E may sometimes have been mistakenly identified for each other, based on whichever variant is most prevalent in the particular population.
Adult↗
Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease.
6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.
Adult↗
Thalassemia in SouthEast Asia: problems and strategy for prevention and control.
In Southeast Asia alpha-thalassemia, beta-thalassemia, hemoglobin (Hb) E and Hb Constant Spring are prevalent. The gene frequencies of alpha-thalassemia reach 30-40% in Northern Thailand and Laos. beta-Thalassemia gene frequencies vary between 1 and 9%. Hb E is the hallmark of Southeast Asia attaining a frequency of 50-60% at the junction of Thailand, Laos, and Cambodia. Hb Constant Spring gene frequencies vary between 1 and 8%. These abnormal genes in different combinations lead to over 60 different thalassemia syndromes. The four major thalassemic diseases are Hb Bart's hydrops fetalis (homozygous alpha-thalassemia 1), homozygous beta-thalassemia, beta-thalassemia/Hb E and Hb H diseases. The molecular basis of most of these abnormal genes have been recently described. Therefore, it is possible to set a strategy for prevention and control of thalassemia which includes population screening for heterozygotes, genetic counseling and fetal diagnosis with selective abortion of affected pregnancies.
Asia, Southeastern↗
Hypoxemia in thalassemia.
Data are reviewed describing hypoxemia, a newly identified feature in thalassemia. Evidence indicates platelet aggregation in the pulmonary circulation as being a key factor leading to hypoxemia and cor-pulmonale with right heart failure.
Aspirin↗
The molecular basis of alpha-thalassemia in Thailand.
Alpha thalassemia is the most common single gene mutation worldwide. In Thailand there exists 15-30% alpha-thalassemia carriers distributed throughout the country. DNA analysis by Southern blot hybridization reveals that the two major alpha-thalassemia alleles, alpha-thalassemia 1 and alpha-thalassemia 2 have different extents of alpha-globin gene deletion. In alpha-thalassemia 1, approximately 20 kb of DNA including the two linked alpha 1-and alpha 2-genes are removed and only the alpha-globin gene is intact. Total deletion of the alpha-globin gene cluster is rarely observed. In contrast, only one alpha-globin gene is deleted in alpha-thalassemia 2 of which two types have been detected, one involving a deletion of 4.2 kb of DNA (leftward type, -alpha 4.2) and another of 3.7 kb (rightward type, -alpha 3.7); the latter being more common than the former in Thailand. Compound heterozygosity for alpha-thalassemia 1 and alpha-thalassemia 2 results in HbH disease while homozygosity for alpha-thalassemia 1 leads to Hb Bart's hydrops fetalis, the most severe form of thalassemic disease. Three alpha-thalassemic hemoglobinopathies have been detected in Thailand, two of which produce a remarkable reduction in gene product. Upon interacting with alpha-thalassemia 1 gene they can lead to HbH disease. The most common in this group is Hb Constant Spring which arises from mutation of the termination codon in the alpha 2-gene resulting in an elongation of the alpha-globin chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood Protein Electrophoresis↗
Hemoglobin constant spring in Bangkok: molecular screening by selective enzymatic amplification of the alpha 2-globin gene.
Hemoglobin Constant Spring (Hb CS) is a hemoglobin variant with an elongated alpha-globin chain secondary to a chain termination mutation. The diagnosis of HbCS by electrophoresis is difficult because it is present in very low amounts in the red cells of heterozygotes. Selective enzymatic amplification of the alpha 2-globin gene and allele-specific hybridization for Hb CS gene provided accurate diagnosis of Hb Constant Spring. We have used this approach to detect the alpha cs mutation in the cord blood that contained all four alpha-globin genes but had Hb Bart on electrophoresis. The alpha cs mutation was found in six subjects whose Hb Bart levels were 3.0, 3.2, 3.7, 4.0, 4.9, and 9.8%. The latter also had -alpha mutation on the other chromosome, giving rise to the genotype alpha cs alpha/-alpha, which produced high Hb Bart. The gene frequency for alpha cs in the Thai calculated from a total of 406 cord blood studied in Bangkok was found to be approximately 0.008.
Base Sequence↗
Prenatal diagnosis of thalassemia and hemoglobinopathies in Thailand: experience from 100 pregnancies.
In this review, we describe a simple strategy to detect the three severe thalassemic diseases commonly found in Thailand. Hb Bart's hydrops fetalis can be detected unambiguously by ultrasonography at 18-20 weeks of gestation or detected early in the first trimester by the gene amplification technique. Prenatal diagnosis for homozygous beta-thalassemia is better performed in the second trimester by in vitro protein synthesis. This is because the molecular defects of some beta-thalassemias are still unknown and homozygosity of the same mutation is low. In contrast, beta-thalassemia/Hb E is easily detected, in the first trimester, by direct visualization on electrophoresis or by dot blot analysis of enzymatically amplified DNA with a set of nonradioactively labeled oligonucleotide probes complementary to the most common mutations. We also found that the beta/gamma synthesis ratio in homozygous Hb E is similar to that of beta-thalassemia/Hb E and DNA analysis is the only method to distinguish these two conditions in the couple at risk of having either beta-thalassemia/Hb E or asymptomatic homozygous Hb E. In 100 pregnancies studied, the diagnoses were achieved in 96 pregnancies. Complications leading to fetal loss were found in 3 pregnancies: one woman developed amnionitis after fetal blood sampling; one had amniotic fluid leakage after the biopsy, and the third, carrying a normal fetus, aborted 10 days after fetal blood sampling with urinary tract infection and high fever. However, these figures are compatible with other reports and the risks are significantly lower than that of thalassemic disease the fetus is facing. One case of beta-thalassemia/Hb E was incorrectly diagnosed prenatally as being Hb E trait. In twenty-five pregnancies (25%) prenatally diagnosed to carry affected fetuses it was decided to have abortion. This study shows the feasibility of prenatal diagnosis for thalassemic diseases in Thailand which, in addition to screening and genetic counseling, can support prevention and control programs for thalassemia.
Female↗
Hemoglobin Dhonburi alpha 2 beta 2 126 (H4) Val----Gly: a new unstable beta variant producing a beta-thalassemia intermedia phenotype in association with beta zero-thalassemia.
While investigating the mechanism of a beta-thalassemia intermedia phenotype in a 34 year old Thai male, a new Hb variant beta 126 Val----Gly named Hb Dhonburi was discovered. Genetic and structural studies revealed the existence of a beta zero-thalassemia genotype in association with the beta variant. The new variant is unstable but exhibits normal oxygen binding properties. Hb Dhonburi was also discovered in the mother of the propositus in association with Hb E.
1-Propanol↗
Ultrasonographic method for detection of haemoglobin Bart's hydrops fetalis in the second trimester of pregnancy.
In nine pregnant women at risk for fetal alpha-thalassaemia, the two affected fetuses were diagnosed by ultrasonography at 18-20 weeks' gestation. In countries with limited resources, ultrasonography provides a cost-effective method of prenatal screening for this condition.
Female↗