Kanokwan Sanchaisuriya

A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia

OBJECTIVE To evaluate a simple screening strategy for thalassaemia and haemoglobin (Hb) E in a prevention and control programme for thalassaemia in rural communities with limited resources. METHODS Blood samples from 301 Thai-Khmer participants were screened for thalassaemia and Hb E using a combined modified one-tube osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) precipitation test. Results were evaluated with standard haematological analyses including erythrocyte indices, Hb typing and quantification and polymerase chain reaction (PCR) analysis of alpha-globin and beta-globin genes. FINDINGS Participants were divided into four groups according to the results of the combined tests. Altogether, 104 of 301 participants (34.6%) had negative results on both tests; 48 (15.9%) were positive on the OF test but not the DCIP test; 40 (13.3%) were negative on the OF test but positive on DCIP test; and 109 (36.2%) were positive on both tests. No carrier of clinically significant forms of thalassaemia (alpha(o)-thalassaemia, beta-thalassaemia) or Hb E was found among the group that had negative results for both tests. All participants with Hb E had positive DCIP tests. Carriers of alpha+-thalassaemia or Hb Constant Spring could generate either positive or negative OF test results but they all had negative DCIP tests. Using both tests as a preliminary screening for the three important groups of carriers gave a sensitivity of 100% and a specificity of 69.8%. The positive predictive value of the combined test was 77.2%. The negative predictive value was 100%. Further evaluation of the screening system by local staff at three community hospitals found a sensitivity of 98.1-100% and a specificity of 65.4-88.4% with positive predictive values of 75.0-86.9% and negative predictive values of 98.1-100%. CONCLUSION A combined test using OF and DCIP could be used as an effective preliminary screening alternative to an electronic blood cell count for identifying carriers with alpha(o)-thalassaemia, beta-thalassaemia and Hb E. The strategy should prove useful for population screening in prevention and control programmes in rural communities in south-east Asia where laboratory facilities and economic resources are limited.

Molecular and hematologic features of hemoglobin E heterozygotes with different forms of α-thalassemia in Thailand

We describe the hematological and DNA characterization of hemoglobin (Hb) E heterozygote with various forms of α-thalassemia in Thai individuals. Altogether, 202 unrelated adult subjects with Hb E heterozygotes either with or without α-thalassemia determinant were studied. The most prevalent interaction was found to be a double heterozygote for Hb E/α-thalassemia 2, followed by a double Hb E/α-thalassemia 1 and a Hb E/Hb Constant Spring (CS), even though the Hb CS was not detected. Double heterozygotes for Hb E and homozygous α-thalassemia 2 and Hb E with a compound α-thalassemia 2/Hb CS were also encountered with lower frequencies. Unexpectedly, as many as 18 cases previously diagnosed as Hb E carriers at routine Hb analysis were indeed Hb E heterozygotes with compound α-thalassemia 1/α-thalassemia 2, indicating a need for globin genotyping for accurate diagnosis. A change in Hb E level was observed which was related to a concomitant inheritance of α-thalassemia. The hematological expression of these Hb E heterozygotes with various forms of α-thalassemia, including a hitherto undescribed condition of double heterozygosity for Hb E/Hb Paksé identified in two subjects, is presented comparatively with those of the 80 cases of pure Hb E carriers. A multiplex allele-specific polymerase chain reaction (PCR) assay for simultaneous detection of Hb E and Hb CS genes is also described.

A reliable screening protocol for thalassemia and hemoglobinopathies in pregnancy: an alternative approach to electronic blood cell counting.

Primary screening for thalassemia and hemoglobinopathies usually involves an accurate blood count using an expensive electronic blood cell counter A cheaper alternative method was tested by using a modified osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) test. Altogether 423 pregnant Thai women participated in this project. Hemoglobin patterns and globin genotypes were determined using an automated high-performance liquid chromatography analyzer and polymerase chain reaction analysis of alpha- and beta-globin genes. Among the 423 subjects, 264 (62.4%) carried thalassemia genes. The combined OF and DCIP tests detected all pregnant carriers of the 3 clinically important thalassemias, ie, alpha0-thalassemia, beta-thalassemia, and hemoglobin E with a sensitivity of 100.0%, specificity of 87.1%, positive predictive value of 84.5%, and negative predictive value of 100.0%, which show more effectiveness than these values for the standard method based on RBC counts. A combination of modified OF and DCIP tests should prove useful and applicable to prenatal screening programs for thalassemia and hemoglobinopathies in communities with limited facilities and economic resources.

The diverse molecular basis and hematological features of Hb H and AEBart's diseases in Northeast Thailand

We defined the molecular basis and correlated the hematological phenotypes with the globin genotypes in 52 patients with Hb H disease and 29 patients with AEBart’s disease of northeast Thailand. Among the former group, the most prevalent molecular defect was found to be the interaction of α-thalassemia 1 (SEA type) with the Hb Constant Spring (Hb CS; 35 of 52 patients), followed by the deletion of three α-globin genes with the SEA type α-thalassemia 1 and the 3.7- or 4.2-kb deletion of α-thalassemia 2 (14 of 52 patients) and the interaction of the SEA α-thalassemia 1 with the Hb Paksé which was found in the remaining 3 patients. Among the 29 patients of the latter group, in 18 disease was caused by interactions of Hb E heterozygotes with the SEA α-thalassemia 1 and Hb CS. Interaction of Hb E heterozygotes with a deletional form of Hb H disease was detected in 7 patients and the Hb Paksé AEBart’s disease was found in another 3 patients. A remaining patient with an unusually severe form of AEBart’s disease with a lower Hb E level and observable Hb H was associated with a hitherto undescribed condition, the interaction of Hb E heterozygote with α-thalassemia 1 and an α2 codon 30 (GAG) deletion. Hematological characterization of the patients demonstrated that although disease in most of them was associated with thalassemia intermedia phenotypes, it was apparent that association with the nondeletional form of α-thalassemia 2 produced a more severe phenotype than that of the deletional one. Therefore, α-globin gene analysis of Hb H and AEBart’s disease patients would be useful for predicting the clinical outcome and improving genetic counseling.

Hb Paksé [(alpha2) codon 142 (TAA-->TAT or Term-->Tyr)J in Thai patients with EAbart's disease and Hb H Disease.

Hb Paksé is caused by an α2-globin gene termination codon mutation, TA A→TA T or Term→Tyr, initially described in a Laotian family. We now report for the first time that the same mutation has been found in 14 Thai patients, seven with EABarts disease, four with Hb H disease, and three with α-thalassemia trait who were initially diagnosed as having Hb Constant Spring (Hb CS; α2-globin gene termination codon mutation T AA→C AA or Term→Gln). Co-inheritance of this mutation with α-thalassemia-1 (SEA type) leads to Hb H disease (hereafter designated as Hb H-Paksé disease) and to a complex thalassemia syndrome, namely EABarts-Paksé disease. Hematological data of these patients were compared with those of classical Hb H-CS and the EABarts patients. To facilitate epidemiological and diagnostic screening of Hb Paksé, a simple assay procedure based on allele specific polymerase chain reaction (PCR) amplifications was developed and validated. Using this allele specific PCR as a screening method, five additional individuals with Hb Paksé were found among 71 Thai subjects previously thought to have Hb CS.

Prevention of severe thalassemia in northeast Thailand: 16 years of experience at a single university center.

To demonstrate the performance of thalassemia prevention in northeast Thailand during 1993–2008.

Frequency Distribution and Haplotypic Heterogeneity of βE-Globin Gene among Eight Minority Groups of Northeast Thailand

The frequencies of hemoglobin E and βE-globin gene haplotypes were determined in eight minority groups living in the northeastern part of Thailand. A total of 478 samples of eight minority groups, namely Soui, Thai Khmer, So, Yor, Phuthai, Thai Puan, Thai Loei and Thai Dam, were examined. High prevalences of hemoglobin E (>50%) were observed in Soui, Thai Khmer, So, Yor and Phuthai inhabiting the region near Cambodia and Laos. Thai Puan, Thai Loei, Thai Dam and native Thai living in the same geographical area had prevalences of 42.6, 35.9, 21.4 and 27.9%, respectively. A prevalence of 9.5% was found among the Thai with Chinese background living in the same area. β-Globin gene haplotypes analysis demonstrated that most of the βE-globin genes in these Thai populations were associated with two haplotypes: (– + – + + + –) and (+ – – – – + –) on chromosomes with framework 2 variety. Some βE-globin genes in Soui and Thai Khmer groups were associated with the framework 3 chromosome. Genetic distances based on the β-globin gene haplotypes between minority groups revealed that Soui and Thai Khmer were closely related to each other. This finding has a valuable implication for study of the origin and spread of hemoglobin E in the region.

Molecular, hematological and clinical aspects of thalassemia major and thalassemia intermedia associated with Hb E-β-thalassemia in Northeast Thailand

Hb E-beta-thalassemia is the most common form of beta-thalassemia found in Thailand. The disease exhibits a varied clinical expression ranging from severe transfusion dependence to relatively mild thalassemia intermedia. We evaluated the effects of primary and secondary genetic factors in modulating the hematological and clinical presentation of 148 northeast Thai patients including 103 severe thalassemia major (TM) and 45 thalassemia intermedia (TI). Among 148 cases examined, eleven different mutations including two novel ones; (beta(33/34 (-G)) and beta(IVS2#815 C-T)) were identified in trans to the beta(E) gene in two TM cases. The other 9 known mutations included beta(41/42), beta(17), beta(IVS2#654), beta(-28), beta(71/72), beta(35), beta(IVS1#5), beta(IVS1#1) and beta(41). Except for the beta(-28) mutation which was found only in the TI group, others mutations were identified in both TM and TI. Co-inheritance of alpha-thalassemia as a phenotype modulating factor was not evident in this study, nor was the presence of the -158 (G)gamma-globin Xmn I polymorphism. Further analysis of the polymorphic (TG)n(CG)m repeats within the IVS2 of the two gamma-globin genes revealed no different proportions of the polymorphic patterns among TM and TI groups of patients either. Our data reveals that in the majority of these Hb E-beta-thalassemia patients, it is very hard to predict the clinical phenotype of the patients from the beta-globin mutations and these secondary genetic modifiers.

Simultaneous PCR detection of β – thalassemia and α – thalassemia 1 (SEA type) in prenatal diagnosis of complex thalassemia syndrome

Abstract Objective: To establish a rapid PCR method for simultaneous detection of β-thalassemia and α-thalassemia 1 genes for diagnosis of complex αβ-thalassemia syndrome. Design and methods: Using multiplex allele specific PCR approach, we evaluated a simultaneous detection of the SEA type α-thalassemia 1 and the common Southeast Asian β-thalassemia and hemoglobin E genes. The system was tested on known cases of double heterozygote for α- and β-thalassemias and in a prenatal diagnosis of complex αβ-thalassemia syndrome. Results: Co-inheritance of α-thalassemia 1 (SEA type) with each of the common β-thalassemia genes in Southeast Asian and with hemoglobin E could be identified in a single PCR reaction. A successful application of this simultaneous detection system in prenatal diagnosis of a complex thalassemia syndrome caused by an EFBart’s disease was demonstrated in a Thai family. Conclusion: We have shown that correct diagnosis of double heterozygosity for α-thalassemia 1 and β-thalassemia or hemoglobin E could be obtained using a simultaneous multiplex PCR. These rapid PCR assays would facilitate characterization and prenatal diagnosis of complex thalassemia syndromes in the regions where both α- and β-thalassemias and hemoglobin E are common.

α⁰-Thalassemia and Related Disorders in Northeast Thailand: A Molecular and Hematological Characterization

α⁰-Thalassemia is the most severe form of α-thalassemia commonly encountered in Asians. To provide relevant information for effective prevention and control of this disorder, we have examined the molecular basis and hematological features of α⁰-thalassemia-related disorders in northeast Thailand. A multiplex polymerase chain reaction for simultaneous detection of the southeast Asian (SEA) and the THAI α⁰-thalassemia determinants was developed and used for screening of 1,541 Thai individuals who were positive at the preliminary screening in an ongoing thalassemia control program. α⁰-Thalassemia deletions were detected in 397 (25.8%) cases, 396 with the SEA deletion and 1 with the THAI deletion. While the latter was found in association with the Hb Constant Spring in a patient with severe Hb H disease, the former was encountered in as many as 12 thalassemia genotypes whose hematological features were comparatively presented. The results obtained should prove useful in carrier screening and prenatal diagnosis programs of this common genetic disorder in the region.