Thongperm Munkongdee

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.

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.

Simple method for screening of α-thalassaemia 1 carriers

Abstractα-Thalassaemia 1 genetic disorder occurs when there is a deletion of two linked α-globin genes. The interaction between these abnormal genes leads to the most severe type of thalassaemia disease, haemoglobin (Hb) Bart’s hydrops fetalis. The identification of α-thalassaemia 1 carriers and genetic counselling are essential for the prevention and control of severe thalassaemia diseases. In this study, we have developed a rapid screening method for identifying α-thalassaemia 1. A sandwich-type immunochromatographic (IC) strip test was developed, using the generated monoclonal anti-Hb Bart’s antibody, to trace the Hb Bart’s in haemolysates. When assayed by our IC strip test, all α-thalassaemia 1, HbH disease, HbH-Constant Spring (H-CS) disease, HbH-CS and heterozygous HbE (CSEA) Bart’s disease, and homozygous α-thalassaemia 2 showed positive results. No false negative results were observed in these blood samples. In α-thalassaemia 2 heterozygotes, 83% of them showed positive reactivity. Among HbE (both homozygotes and heterozygotes), β-thalassaemia (heterozygotes, homozygotes and β-thalassaemia/HbE) and normal subjects, the IC strip test revealed negative reactivity of 100, 85 and 97%, respectively. These results indicate that this novel immunodiagnostic kit, in combination with red blood cell indices, is suitable for screening and ruling out mass populations for the presence of α-thalassaemia 1.

Coinheritance of the different copy numbers of α-globin gene modifies severity of β-thalassemia/Hb E disease

Abstractβ-Thalassemia/Hb E patients show a range of clinical severities, from nearly asymptomatic to transfusion-dependent thalassemia major. This study investigated the clinical heterogeneity and hematologic parameters obtained in the large cohort of 925 Thai β0-thalassemia/Hb E patients. Coinheritance of α-thalassemia with β0-thalassemia/Hb E produces a milder clinical phenotype in contrast to an interaction of α-globin gene triplication in severe thalassemia. The mean steady-state Hb was also higher, whereas the mean corpuscular volume and the percentage of Hb F were markedly lower in the former group. This finding demonstrates that the genetic combination leading to the more/less degree of α- to non-α-globin chains imbalance is indeed the cause of the severe/mild thalassemia phenotype.

Association of SNP in exon 1 of HBS1L with hemoglobin F level in β0-thalassemia/hemoglobin E

Increase in fetal hemoglobin (Hb F) reduces globin chain imbalance in β-thalassemia, consequently improving symptoms. QTL mapping together with previous genome-wide association study involving approximately 110,000 gene-based SNPs in mild and severe β0-thalassemia/Hb E patients revealed SNPs in HBS1L significantly associated with severity and Hb F levels. Given its potential as binding site for transcription factor activator protein 4, HBS1L exon 1 C32T polymorphism was genotyped in 455 cases, providing for the first time evidence that C allele is associated with elevated Hb F level among β0-thalassemia/Hb E patients with XmnI-Gγ−/−and XmnI-Gγ+/−polymorphisms.

Genetic Analysis of Candidate Modifier Polymorphisms in Hb E‐β0‐Thalassemia Patients

Abstract: Hemoglobin E (Hb E)‐β‐thalassemia patients display a range of clinical severities, from nearly asymptomatic to transfusion‐dependent thalassemia major. Given this clinical heterogeneity, additional genetic factors modifying disease severity remain to be discovered. Association studies are being conducted to elucidate the role of genetic polymorphisms as disease severity modifiers in Hb E‐β‐thalassemia patients. Using strict scoring criteria, 1060 Hb E‐β‐thalassemia patients were categorized into mild, moderate, and severe groups. Taking a candidate gene approach, we found no statistically significant differences between the mild and severe patients groups in allelic or genotypic frequencies for single nucleotide polymorphisms within five genes known to influence globin gene expression and erythropoiesis.

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.

Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of βE 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 (β‐globin) gene, activates a cryptic 5′ splice site in codon 25 leading to a reduction of correctly spliced βE‐globin (HBB:c.79G>A) mRNA and consequently β+‐thalassaemia. A wide range of clinical severities in bothα‐ and β‐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 βE‐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 β‐thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced βE‐globin mRNA ratio in 30% of mild β‐thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of βE‐globin pre‐mRNA differs among β‐thalassaemia/HbE patients and serves as one of the modifying factors for disease severity.

Molecular Epidemiology of Hemoglobinopathies in Cambodia

Abstract 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 β-thalassemia (α- and β-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 β-globin gene mutation with a frequency ranging from 0.139 to 0.331, while the most frequent α-globin gene mutation was the –α3.7 (rightward) deletion (0.098–0.255). The other frequencies were 0.001–0.003 for β-thal, 0.008–0.011 for α-thal-1 (– –SEA), 0.003-0.008 for α-thal-2 [–α4.2 (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.

Establishment of MUi009 – A human induced pluripotent stem cells from a 32 year old male with homozygous β°-thalassemia coinherited with heterozygous α-thalassemia 2

The thalassemias are a group of genetic disorders characterized by a deficiency in the synthesis of globin chains. In this study the MUi009-A human induced pluripotent stem cell line was successfully generated from peripheral blood CD34+ haematopoietic progenitors of a 32year old male who had coinherited a homozygous β°-thalassemia mutation at codon 41/42 (-TCTT) and a heterozygous α-thalassemia 4.2 deletion. The MUi009-A cell line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into the three germ layers. The cell line may provide a tool for drug testing and gene therapy studies.