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. Objective: To establish a simple method, we have evaluated the reliability of prenatal diagnosis by fetal blood analysis using automated capillary electrophoresis system. Methods: Forty‐seven fetal blood specimens collected by cordocentesis at 18–28 wk of gestation were analyzed by the capillary electrophoresis system (Sebia). Fetal DNA was analyzed for respective thalassemia alleles by PCR. Results: Among 47 fetuses, 20 were at risk for the Hb Bart’s hydrops fetalis. DNA analysis identified four cases of homozygous α°‐thalassemia (SEA type). Hb analysis by the capillary electrophoresis demonstrated a major peak of Hb Bart’s (78.4–81.3%), Hb H (0.8–1.4%) and minor peaks of presumably embryonic Hbs. No Hb F and Hb A was observed. The level of Hb Bart’s was found to be 3.4–5.8% in unaffected heterozygote whereas normal fetus had no Hb Bart’s. Among the remaining 27 fetuses at risk for Hb E‐β‐thalassemia, DNA analysis identified 12 affected fetuses. Hb analysis showed Hb F (94.9–98.9%) and Hb E (1.1–1.8%) without Hb A in all cases. The levels of Hb A were found to be (4.3–7.2%), (1.0–5.5%) and (2.1–3.9%) in normal, heterozygous Hb E and heterozygous β‐thalassemia fetuses, respectively. Affected and unaffected fetuses could be easily distinguished. Conclusion: Capillary electrophoresis system is a simple and automated procedure for accurate prenatal diagnosis of severe thalassemia diseases which could readily be performed in routine setting.     

Molecular and hematological characterization of HPFH-6/Indian deletion-inversion Ggamma(Agammadeltabeta)0-thalassemia and Ggamma(Agammadeltabeta)0-thalassemia/HbE in Thai patients

The hereditary persistence of fetal hemoglobin (HPFH)-6 is sporadically found in Thailand whereas the deletion-inversion type (G)gamma((A)gamma delta beta)(0)-thalassemia is described among Indians. We report a hitherto un-described case in which these two defects co-segregate. He was a 3-year-old Thai boy who had a feature of thalassemia intermedia phenotype with the following hematologic data; Hb 8.8 g/dL, Hct 29.2%, MCV 66.9 fL, MCH 20 pg, and MCHC 30.1 g/dL. Hemoglobin analysis revealed 100% Hb F with only (G)gamma-globin chain. Globin gene analyses demonstrated that he carried the HPFH-6 deletion in trans to the Indian deletion-inversion (G)gamma((A)gamma delta beta)(0)-thalassemia. Hematologic data of the patient was compared to those of the HPFH-6 heterozygote found in his father, to (G)gamma((A)gamma delta beta)(0)-thalassemia heterozygotes detected in his mother and sister, and to that of an unrelated Thai patient who was a compound heterozygote for the deletion-inversion (G)gamma((A)gamma delta beta)(0)-thalassemia and HbE.     

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 erythroid transcription factor KLF1 mutations associated with borderline hemoglobin A2 in the Thai population

IntroductionElevated hemoglobin (Hb) A₂ is an important diagnostic marker for β-thalassemia carriers. However, diagnosis of cases with borderline Hb A₂ may be problematic. We described the molecular characteristics found in a large cohort of Thai subjects with borderline Hb A₂.Material and methodsExamination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb A₂ (3.5–4.0%) were selectively recruited and hematological parameters were recorded. DNA variants in α-, β-, δ-globin, and Krüppel-like factor 1 (KLF1) genes were examined using PCR.ResultsAmong 202 subjects, DNA analysis identified carriers of α⁺-thalassemia (n = 48; 23.8%), β-thalassemia (n = 22; 10.9%) and KLF1 mutations (n = 48; 23.8%). No molecular defect was observed in the remaining 84 (41.5%) subjects. Interaction of KLF1 and α-thalassemia was observed in 10 cases. Of the 22 β-thalassemia carriers, five β⁺-thalassemia mutations were identified with lower MCV and higher Hb A₂. Seven KLF1 mutations were detected in 10 genotypes in subjects with higher MCV and Hb F. No β⁰-thalassemia, α-globin gene triplication or δ-globin gene mutation was detected.ConclusionsA large proportion of subjects with borderline Hb A₂ are not β-thalassemia carriers and for those with β-thalassemia, only mild β⁺-thalassemia mutations were detected. Evaluation of the patients using Hb A₂, Hb F and MCV values will help in selecting cases for further molecular analysis. The results should explain the unusual phenotype of the cases and facilitate a thalassemia screening program in the region.     

Compound heterozygote states for Hb C/Hb Malay and Hb C/Hb E in pregnancy: a molecular and hematological analysis

Hemoglobin (Hb) C (alpha2beta(2)6Glu-Lys) is a variant Hb found mainly in West Africa where individuals carrying both Hb C and Hb S (alpha2beta(2)6Glu-Val) usually have a disease similar to sickle cell disease. The Hb C molecule has reduced solubility leading to crystal formation and hemolytic anemia. We report a hitherto undescribed interaction of Hb C and Hb Malay (alpha2beta(2)19Asn-Ser) in a Thai individual. She was a 24-year-old pregnant woman with moderate anemia who had the following hematologic data; Hb 8.9 g/dl, Hct 30.0%, MCV 81.0 fl, MCH 24.1 pg, MCHC 29.7 g/dl, RDW 17.1% and instead of Hb crystal a marked number of target cell in peripheral blood was observed. Hb analysis revealed 22.5% Hb Malay, 64.6% Hb C and 4.5% Hb A2. Globin gene analyses demonstrated that she carried the betaC mutation (beta6: GAG-AAG) in trans to the betaMalay mutation (beta19: AAC-AGC). Hematologic data of the patient were compared to those of the compound heterozygote for Hb C and Hb E (alpha2beta(2)26Glu-Lys) found in 5 other unrelated Thai pregnant women and 11 pregnant women with Hb C heterozygote with or without co-inheritance of alpha-thalassemia who had much lower Hb C levels and the non-pregnant women with Hb C heterozygote and a compound Hb E/Hb Malay syndrome. Different genotype-phenotype correlations observed in these Thai patients with Hb C disorders are illustrated.     

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 α^o-, 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.     

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.     

Thalassemia intermedia associated with the Hb Constant Spring EE Bart's disease in pregnancy: a molecular and hematological analysis

We defined the molecular basis and analyzed hematological phenotype associated with an unusual form of thalassemia intermedia caused by interaction of the hemoglobin Constant Spring (Hb CS), homozygous Hb E and alpha degrees -thalassemia found in two unrelated pregnant Thai women. Both patients had moderate anemia and characteristic of thalassemia intermedia. Hb-HPLC analysis demonstrated in both cases, Hb E and Hb Constant Spring with 3-4% Hb Bart's. Hb F was marginally elevated (3-5%). Both of them were diagnosed hematologically as the Hb CS EE Bart's disease. DNA analysis revealed the homozygosity for Hb E in both cases and identified the Hb CS mutation in trans to the alpha degrees -thalassemia allele with the SEA deletion in one case and with the Thai deletion in another. The appearance of Hb-HPLC peak resembling the Hb CS in peripheral blood of the two cases indicated the ability to form a tetrameric Hb molecule between alpha(CS) and beta(E) chains leading to a hybrid Hb namely the Hb E-CS (alpha2(CS)beta2(E)) with similar characteristics to Hb CS (alpha2(CS)beta2(A)). Hematological data of the patients were presented comparatively with other forms of related disorders in our series including 2 Hb H/Hb EE diseases, 16 homozygous Hb CS with and without Hb E, 14 Hb H diseases and 35 Hb H-CS diseases. Different genotype-phenotype correlations observed in these Thai patients with these disorders are illustrated.