Nattaya Sae-ung

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.

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.

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.

Analysis of fetal blood using capillary electrophoresis system: a simple method for prenatal diagnosis of severe thalassemia diseases

Introduction:  Prenatal diagnosis of severe α‐ and β‐thalasssemia diseases is usually performed by DNA analysis.

Genotype and phenotype characterizations in a large cohort of β-thalassemia heterozygote with different forms of α-thalassemia in northeast Thailand

In order to update the molecular basis of β-thalassemia and describe hematological features among different mutations and the concurrent of α- and β-thalassemias, 849 unrelated β-thalassemia heterozygotes recruited in northeast Thailand during a prevention and control program were studied. β- and α-thalassemia mutations were investigated using the polymerase chain reaction (PCR)-based technologies and hematological parameters were recorded using standard methods. Seventeen different mutations including both β(0)- and β(+) -thalassemias were identified. Eight of these 17 β-thalassemia alleles accounted for 97.4%, others were found at lower frequencies (<1.0%). Of the 849 cases, 626 were investigated for common α-thalassemia mutations and 155 (24.8%) were found to be co-inherited with different forms of α-thalassemia. Comparison of the hematological parameters among different β-thalassemia mutations revealed an increasing trend of MCV and MCH in a group of heterozygous states for the 3.4kb deletion and the A-G substitution at nucleotide (NT) -28. Hb A(2) and Hb F levels in individuals with the 3.4kb deletion were significantly higher than those with other mutations. Interaction of each β-thalassemia mutation with α-thalassemia did not affect the diagnostic ranges of Hb A(2) and Hb F, though the significantly increased MCV and MCH was noted. These findings underline the heterogeneity of β-thalassemia and the importance of hematological and molecular analyses of both α-and β-thalassemias in the diagnosis and genetic counseling of the couples at-risk of having babies with severe thalassemia diseases in the region.

Phenotypic expression of hemoglobins A2, E and F in various hemoglobin E related disorders

Study on the phenotypic expression of hemoglobin (Hb) A(2) and Hb E in Hb E disorders has been difficult due to the co-separation of Hb A(2) and Hb E in most Hb analysis assays. Because these two Hbs are separated on capillary electrophoresis, we studied phenotypic expression of Hbs A(2), E and F in various Hb E disorders using this system. This was done on 362 subjects with several Hb E disorders including heterozygous Hb E, homozygous Hb E, β-thalassemia/Hb E, δβ-thalassemia/Hb E, and Hb Lepore/Hb E and those of these disorders with several forms of α-thalassemia. Normal controls showed Hb A(2) of 2.7 ± 0.3%. Heterozygous Hb E and homozygous Hb E had elevated Hb A(2) i.e. 3.8 ± 0.3% and 4.8 ± 0.5%, respectively. Further elevations were observed for β(0)-thalassemia/Hb E (6.1 ± 1.9%) and β(+)-thalassemia/Hb E (7.1 ± 1.2%). Interestingly, no elevation of Hb A(2) was found in the δβ-thalassemia/Hb E, and Hb Lepore/Hb E (2.3 ± 0.3%) but higher Hb F levels were noted which could be useful diagnostic markers. The levels of Hb E were variable. Co-inheritance of these Hb E disorders with α-thalassemia were associated with lower outputs of Hb E and Hb F but the levels of Hb A(2) were not altered. Different phenotypic expression of Hb A(2), Hb E and Hb F could help in differential diagnosis of these Hb E related disorders commonly encountered in the regions where access to molecular techniques is limited.

Thalassemia and hemoglobinopathies in pregnant Lao women: carrier screening, prevalence and molecular basis

To provide relevant evidence base for implementation of a prevention and control program for thalassemia in the Lao People’s Democratic Republic (Lao PDR), we have evaluated a simple screening protocol and examined the prevalence and the molecular basis of thalassemia in pregnant Lao women. The study was conducted on 307 pregnant women attending the Mother and Child Health Hospital, Vientiane. Initial screening was performed locally, applying a combined osmotic fragility (OF) and dichlorophenolindophenol (DCIP) test. Erythrocyte counts were recorded. The remaining blood specimens were transferred to Thailand for further hemoglobin (Hb) and DNA analyses. Subjects were divided into four groups according to the results of the screening tests. Among 307 participants examined, 154 (50.2%) had negative results on both tests (−/−), 58 (18.8%) were positive on the OF test but not the DCIP test (+/−), 22 (7.1%) were negative on the OF test but positive on the DCIP test (−/+), and 73 (23.7%) were positive on both tests (+/+). As many as 25 thalassemia genotypes including various complex syndromes were observed. Three clinically important forms of thalassemia including αo-, β-thalassemia, and Hb E were identified in 39 (12.7%), 11 (3.6%), and 93 (30.2%) subjects, respectively. The performance characteristic of the initial screening for these three forms of thalassemia was determined. The sensitivity, specificity, positive, and negative predictive values were found to be 99.2%, 85.5%, 83.0% and 99.4%, respectively. Therefore, thalassemia and hemoglobinopathies are prevalent and heterogeneous among the Lao population. Implementation of a simple carrier screening in pregnancy is practicable in the Lao PDR.

Hemoglobin Q-Thailand related disorders: Origin, molecular, hematological and diagnostic aspects

We describe the molecular and hematological profiles of thalassemia syndromes caused by interactions of hemoglobin (Hb) Q-Thailand [α74(EF3) Asp-His] and various hemoglobinopathies found in 52 unrelated adult Thai subjects. Ten genotypes including several previously undescribed conditions were observed, which were classified into 4 groups. Group I included 26 Hb Q-Thailand heterozygotes and a homozygotous subject. Group II included subjects with Hb Q-Thailand and other α-thalassemia alleles in trans including 1 compound Hb Q-Thailand/α(+)-thalassemia (-α(3.7)), 2 Hb Q-Thailand/Hb Constant Spring disease and 6 Hb H/Q-Thailand disease. The average levels of Hb Q-Thailand were found to be 29.8%, 82.3%, 34.7%, 49.2-49.3% and 79.4%, respectively. Both Hbs Barts and H were observed in addition to Hb Q-Thailand in all 6 cases with Hb Q-H disease but not in a homozygous Hb Q-Thailand. Group III included 7 double heterozygotes for Hb Q-Thailand/Hb E, 3 Hb Q-Thailand/Hb E/α(+)-thalassemia (-α(3.7)), 3 heterozygous Hb Q-Thailand/homozygous Hb E and 1 triple heterozygote for Hb Q-Thailand/Hb Constant Spring/Hb E. In this group, Hbs E (α(A)(2)β(E)(2)), Q-Thailand (α(QT)(2)β(A)(2)) and QE (α(QT)(2)β(E)(2)) were observed on both HPLC and capillary electrophoresis. The Hb QE, rather than Hb Q-Thailand, was detected in all 3 cases with heterozygous Hb Q-Thailand and homozygous Hb E. The remaining two cases in group 4 were double heterozygotes for Hb Q-Thailand and β(0)-thalassemia in which Hb Q-Thailand, elevated Hb A(2) (α(A)(2)δ(2)), and Hb QA(2) (α(QT)(2)δ(2)) were detected. DNA analysis identified the Hb Q-Thailand mutation (α74: GAC-CAC) and the linked (-α(4.2)) in all cases. Analysis of α-globin gene haplotype provided the first evidence of a single origin of this Hb variant in Thai population.