Screening for the carriers of thalassemias and abnormal hemoglobins at the community level.

Thalassemia and abnormal hemoglobins are common genetic disorders in Southeast Asia. Thalassemia is not only an important public health problem but also a socio-economic problem of many countries in the region. The approach to deal with the thalassemic problem is to prevent and control births of the new cases. This requires an accurate identification of couple at high risk to have a thalassemic child. The diagnosis of thalassemia carriers need several tests that are not practical for screening the population at large. In this study we used two simple laboratory tests to screen for potential thalassemia carriers and hemoglobin E individuals. Three-hundred pregnant women and 40 spouses were recruited in this study. The methods were the red cell osmotic fragility (OF) screening test with 0.36% NaCl and the dichlorophenolindophenol (DCIP) precipitation test to detect Hb E and unstable hemoglobins. Standard methods for red cell indices, hemoglobin analysis and detection of alpha-thalassemia by immunological method were also performed to confirm genotypes of thalassemia. The results showed that 98 women (32.7%) were carriers of thalassemias and hemoglobin E. The number of false positive by OF test was 3.2% and by DCIP test was 0.6%. Sensitivity and specificity of OF test were 89.5% and 93.3%, respectively whereas those of DCIP test were 100%. Of the 40 couples investigated, one was found to be at risk of having beta-thalassemia/Hb E fetus. Screening techniques including one tube osmotic fragility and DCIP precipitation tests are sensitive and specific for the detection of thalassemia and unstable hemoglobins such as Hb E. The techniques are also simple, economic, rapid, and give minimal false negative result.

Standardization on laboratory diagnosis of thalassemia and abnormal hemoglobin.

Thalassemia is one of the most common single gene disorders. The geographic distribution of thalassemia and abnormal hemoglobin has been known for many years. A worldwide significant spread of these abnormal genes, especially from Southeast Asia, occurred in the last two decades. This has resulted in a dramatic increase of Hb E disorders and various Southeast Asian thalassemia genotypes, which means that requests for hemoglobinopathy investigations are likely to increase in many laboratories worldwide. Hemoglobinopathy screening and diagnosis may need to be undertaken antenatally, neonatally and in certain hematological situations. The introduction of automation for hemoglobinopathy screening, including the automated cell counting and HPLC system, is an important advance in technology for hematology laboratories. The instruments need to be calibrated and standardized to get an accurate data for interpretation. Internal and external control samples are also needed. Combination of test results is usually required to achieve a proper diagnosis, which in turn, provide a self-check for each laboratory test.

Detection and structural analysis of abnormal hemoglobins found in Thailand.

In Thailand, some 20 different abnormal hemoglobins have been found in the last 30 years. Most are rare except for Hb E and Hb Constant Spring, found with frequencies of 10-53% and 1-8% respectively in different parts of the country. Most mutations are point mutations, but C-terminal elongations and crossing-over are also found. Most mutations do not cause clinical problems, but some can give rise to mild thalassemia syndromes, or cause problems in association with thalassemia. Abnormal hemoglobins may often be diagnosed by electrophoresis, but some variants have the same mobility, so that other techniques are required, such as HPLC and the use of allele-specific polymerase reaction or oligonucleotide probes. Novel variants, not previously described in Thailand, require structural analysis at the protein and DNA level.

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.

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.

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.

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.

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.

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)

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.

Increased circulating platelet aggregates in thalassaemia.

Examination for circulating platelet aggregates according to Wu and Hoak revealed increased circulating platelet aggregates in 71% of splenectomized and 35% of nonsplenectomized patients with beta (0)-thalassaemia/Hb E disease. This may be causally related to the newly observed high incidences of pulmonary artery thrombosis and hypoxaemia in splenectomized thalassaemic patients. It is recommended that anti-platelet aggregation drugs such as aspirin and/or dipyridamole are given to thalassaemic patients after splenectomy.