Pranee Winichagoon

Hemoglobinopathies in Southeast Asia: Molecular Biology and Clinical Medicine

Thalassemia (thal) and hemoglobinopathies are widespread, recessive inherited diseases. Approximately 250 million people (4.5% of the world population) carry abnormal hemoglobin (Hb) genes. Southeast Asia consists of 10 countries, i.e. Brunei, Cambodia, Indonesia, Laos, Malaysia, Myanmar, the Philippines, Singapore, Thailand, and Vietnam, with a total population of more than 400 million. In most of these countries the population is ethnically heterogeneous. Thalassemias are common in Southeast Asia. α-Thalassemias are very prevalent, attaining frequencies of 20–40%. β-Thal, Hb Constant Spring (CS) and Hb E are also common, the latter attaining a frequency as high as 50–60% among certain populations (1). Abnormal genes in different combinations lead to more than 60 different thalassemic syndromes. The thalassemia disorders were originally confined to the tropics and subtropics. However, Southeast Asians are now scattered around the world and have carried with them most of the complex thalassemic conditions...

Clinical and hematologic aspects of hemoglobin E β-thalassemia

Hemoglobin E β-thalassemia is an important cause of childhood chronic disease in Southeast Asia. It is characterized by the presence of hemoglobin E and F, and the amount of hemoglobin E ranges from 35% to 75%. The patients are generally classified as having thalassemia intermedia because they have

A scoring system for the classification of β-thalassemia/Hb E disease severity

β‐Thalassemia intermediate patients show a remarkable clinical heterogeneity. We examined the phenotypic diversity of 950 β‐thalassemia/Hb E patients in an attempt to construct a system for classifying disease severity. A novel scoring system based on six independent parameters, hemoglobin level, age at disease presentation, age at receiving first blood transfusion, requirement for transfusion, spleen size, and growth and development, was able to separate patients into three distinctive severity categories: mild, moderate, and severe courses. This system, therefore, can increase the accuracy of studies of genotype–phenotype interactions and facilitate decisions for appropriate patient management. Am. J. Hematol. 2008.

Genetic factors affecting clinical severity in beta-thalassemia syndromes.

Purpose Heterogeneity in the clinical manifestation of &bgr;-thalassemic diseases may occur from the nature of &bgr;-globin gene mutations, &agr;-thalassemia gene interaction, or differences in the amount of hemoglobin (Hb) F production. This study was conducted to determine whether these genetic determinant factors can predict phenotypic severity of patients with &bgr;-thalassemia and to assess the relationship between the genotype and phenotype of the disease. Materials and Methods A total of 144 patients with &bgr;-thalassemia were divided into mild (46 patients), intermediate (55 patients), and severe groups (43 patients). DNA analysis based on polymerase chain reaction technique was performed to characterize types of &bgr;-thalassemia mutation, interaction of &agr;-thalassemia, and Xmn I polymorphism 5´ to G&ggr;-globin gene. Results Two alleles of mild &bgr;-thalassemia mutation (&bgr;+/&bgr;+-thalassemia or &bgr;+-thalassemia/Hb E) resulted in a mild clinical symptom whereas two alleles of severe &bgr;-thalassemia mutation (&bgr;o/&bgr;o) produced a severe clinical phenotype. Compound heterozygosity for mild and severe alleles of &bgr;-thalassemia (&bgr;o/&bgr;+-thalassemia or &bgr;o-thalassemia/Hb E) led to variable severity of anemia. Coinheritance of &agr;-thalassemia alleviated the severity of &bgr;-thalassemia disease in those patients with at least one allele of the mild &bgr;-thalassemia genotype. DNA polymorphism at position -158 nt 5´ to the G&ggr;-globin gene was demonstrated by Xmn I restriction enzyme. Homozygote of the Xmn I site, +/+, was found to have a strong linkage with high Hb F levels and high hemoglobin production in two patients who had mild clinical symptoms. However, some patients who had Xmn I site −/− also had mild clinical symptoms because the Xmn I− was found to be associated with &bgr;+-thalassemia mutation. Conclusion Types of &bgr;-thalassemia mutation and coinheritance of &agr;-thalassemia in the patient who has at least one allele of the mild &bgr;-thalassemia genotype are predictive for the clinical severity of the disease. However, a mild clinical symptom in some patients with &bgr;o/&bgr;+-thalassemia or &bgr;o-thalassemia/Hb E who do not have a detectable &agr;-thalassemia haplotype and no linkage with Xmn I++ suggests that there are other confounding factors responsible for the severity differences of the disease.

A MULTI-CENTER STUDY IN ORDER TO FURTHER DEFINE THE MOLECULAR BASIS OF β-THALASSEMIA IN THAILAND, PAKISTAN, SRI LANKA, MAURITIUS, SYRIA, AND INDIA, AND TO DEVELOP A SIMPLE MOLECULAR DIAGNOSTIC STRATEGY BY AMPLIFICATION REFRACTORY MUTATION SYSTEM-POLYMERASE CHAIN REACTION

The spectrum of the β-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 β-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (−A) and IVS-I-129 (A → C), both found in Sri Lankan patients. Two β-thalassemia mutations were found to coexist in one β-globin gene: Sri Lankan patients homozygous for the β0 codon 16 (−C) frameshift were also homozygous for the β+ codon 10 (C → A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C → A) mutation is just a rare polymorphism on an ancestral allele, on which the β0 codon 16 (−C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the β-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.

Prenatal diagnosis of β-thalassaemia by reverse dot-blot hybridization

Thalassaemia is the most common genetic disease and is a public health problem of Thailand. Prevention and control of β‐thalassaemia diseases need accurate diagnosis of carriers and proper genetic counselling. Prenatal diagnosis is needed to prevent birth of the thalassaemic offspring in the couple at risk. This can be performed in the first trimester of pregnancy by DNA analysis using the polymerase chain reaction (PCR). Since there are more than 20 mutations causing β‐thalassaemia in Thailand, the point mutation detection by reverse dot‐blot allele‐specific oligonucleotide (ASO) hybridization was developed using two sets of ASO probes. The first battery of ASO probes has been designed to detect 10 common β‐globin gene mutations including codon 26, G→A (Hb E); codons 41/42, ‐TCTT; codon 17, A→T; IVS 2 nt 654, C→T; IVS 1 nt 1, G→T; IVS 1 nt 5, G→C; codon 19, A→G (Hb Malay); codon 35, C→A; codons 71/72, +A and ‐28 ATA, A→G. The second set of ASO probes detect 14 uncommon β‐thalassaemia mutations.

Detection of α-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 α‐thalassemia 1 (–). Three oligonucleotide primers, one of which was adjacent to the breakpoint of the α‐thalassemia‐1 allele, were used to amplify the 570 and 194 bp DNA fragments. The 570 bp product was specific to the α‐thalassemia‐1 determinant and the 194 bp fragment was amplified from either the α‐thalassemia‐2 (‐α) or normal α‐globin (αα) determinants. In Hb Barts hydrops fetalis (–/–), only the 570 bp fragment was obtained, whereas the 194 bp fragment was amplified in normal individual (αα/αα) and α‐thalassemia‐2 trait (‐α/αα). Both 570 and 194 bp fragments were detected in α‐thalassemia‐1 trait (–/αα) and Hb H patients (–/‐α). This procedure is useful for the rapid screening of α‐thalassemia‐1 trait and prenatal diagnosis of Hb Barts hydrops fetalis in populations with a high frequency of the Southeast Asian Type of α‐thalassemia‐1.

Thalassaemia classification by neural networks and genetic programming

This paper presents the use of a neural network and a decision tree, which is evolved by genetic programming (GP), in thalassaemia classification. The aim is to differentiate between thalassaemic patients, persons with thalassaemia trait and normal subjects by inspecting characteristics of red blood cells, reticulocytes and platelets. A structured representation on genetic algorithms for non-linear function fitting or STROGANOFF is the chosen architecture for genetic programming implementation. For comparison, multilayer perceptrons are explored in classification via a neural network. The classification results indicate that the performance of the GP-based decision tree is approximately equal to that of the multilayer perceptron with one hidden layer. But the multilayer perceptron with two hidden layers, which is proven to have the most suitable architecture among networks with different number of hidden layers, outperforms the GP-based decision tree. Nonetheless, the structure of the decision tree reveals that some input features have no effects on the classification performance. The results confirm that the classification accuracy of the multilayer perceptron with two hidden layers can still be maintained after the removal of the redundant input features. Detailed analysis of the classification errors of the multilayer perceptron with two hidden layers, in which a reduced feature set is used as the network input, is also included. The analysis reveals that the classification ambiguity and misclassification among persons with minor thalassaemia trait and normal subjects is the main cause of classification errors. These results suggest that a combination of a multilayer perceptron with a blood cell analysis may give rise to a guideline/hint for further investigation of thalassaemia classification.

Rapid diagnosis of thalassemias and other hemoglobinopathies by capillary electrophoresis system

Basic diagnosis of hemoglobinopathies can be performed by analysis of erythrocyte indices as well as by the separation and quantification of the common hemoglobin (Hb) fractions Hb A(2), Hb S, Hb C, Hb D, Hb E, and Hb F. This study used an automatic capillary zone electrophoresis system to diagnose various types of hemoglobinopathies common in the Thai population. A total of 459 adults were recruited, which consisted of normal, various types of thalassemia carriers, and thalassemia patients with different genotypes. Hb types and quantification of all Hb components were determined by an automated capillary zone electrophoresis. The automatic capillary electrophoresis system can separate and quantitate Hbs A, F, E, A(2), Constant Spring (CS), H, and Barts in a way that is comparable with other Hb analysis methods. Moreover, the Hb A(2) peak can be distinguished clearly from the Hb E peak in individuals who carry Hb E. The slightly increased levels of Hb A(2), 3.5% +/- 0.4%, which is shown in the carriers of Hb E, confirm that Hb E is the silent phenotype of beta(+)-thalassemia.

Reverse dot‐blot detection of Thai β‐thalassaemia mutations

Summary. Pending curative therapy, newborn screening and prenatal diagnosis are essential to the management of (3 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 β‐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 β‐thalassaemia mutations, including the β‐thalassaemic haemoglobino‐pathies Hb E and Hb Malay, that account for 96% of j3 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 β‐globin DNA as two shorter fragments that encompassed all known Thai mutations. Reverse dot‐blotting is a rapid, accurate method for detecting β‐thalassaemia mutations.