Co-inheritance of α0 -thalassemia elevates Hb A2 level in homozygous Hb E: Diagnostic implications.

INTRODUCTION: Differentiation of homozygous hemoglobin (Hb) E with and without α0 -thalassemia is subtle on routine hematological ground. We examined in a large cohort of homozygous Hb E if the level of Hb A2 is helpful. METHODS: A total of 592 subjects with homozygous Hb E were recruited from ongoing thalassemia screening program. Additionally, five couples at risk of having fetuses with Hb Bart's hydrops fetalis who were homozygous Hb E were also investigated. Hb analysis was performed using capillary electrophoresis system. Globin genotypes were defined by DNA analysis. RESULTS: Subjects were classified into four groups including pure homozygous Hb E (n=532), homozygous Hb E/α0 -thalassemia (n=48), Hb Constant Spring EE Bart's disease (n=8), and Hb EE Bart's disease (n=4). The levels of Hb A2 were found, respectively, to be 4.97±0.69, 6.64±1.02, 4.86±0.87, and 7.60±1.04%. Among five couples at risk, α0 -thalassemia was identified in three subjects with Hb A2 >6.0%. CONCLUSIONS: Increased Hb A2 level is a useful marker for differentiation of homozygous Hb E with and without α0 -thalassemia. This should lead to a significant reduction in number of referral cases of homozygous Hb E for molecular testing of α0 -thalassemia in routine practice.

Diagnosis of common hemoglobinopathies among South East Asian population using capillary isoelectric focusing system.

INTRODUCTION: We have evaluated an automated capillary isoelectric focusing (cIEF)-based Hb analyzer in diagnosis of hemoglobinopathies commonly found among South East Asian population. METHODS: Study was performed on a cohort of 665 adult Thai subjects and 13 fetal blood specimens obtained at routine thalassemia diagnostic laboratory. Hb analysis was performed using the cIEF system. Thalassemia genotypes were defined by DNA analysis. RESULTS: The system revealed satisfactorily within-run and between-run precision for quantitation of Hb A2 and Hb E (CV: 0.02-0.09%). The reference ranges of Hb A2 and Hb E were 2.6-4.0% and 25.7-33.1%, respectively. The system identified the cases of ß-thalassemia and Hb E disorders correctly. Several thalassemia genotypes and Hb variants were identifiable. However, Hb Constant Spring was separated closely to Hb A2 and Hbs Bart's and H were relatively difficult to be reported due to interfering peaks separating at the same regions. Prenatal diagnosis by fetal blood analysis was found to be accurate for Hb Bart's hydrops fetalis and Hb E-ß0 -thalassemia disease. CONCLUSIONS: The cIEF system could accurately diagnose ß-thalassemia and Hb E carriers and demonstrate many Hb variants found in the region. The system cannot report Hb A2 in the presence of Hb E whereas Hbs Lepore and F are comigrated. Diagnosis of α-thalassemia disease based on Hb H and Hb Bart's might be difficult.

Presumptive diagnosis of common haemoglobinopathies in Southeast Asia using a capillary electrophoresis system.

INTRODUCTION: This study was conducted to examine ability of the Capillarys 2 haemoglobin (Hb) testing system to assist in presumptive diagnosis of common Hb variants found in Southeast Asia including five α-chain and nine ß-chain variants. METHODS: Blood samples with unknown Hb variants sent from other hospitals to our centre for identification were re-analysed using the Capillarys 2 Hb analyser (SEBIA). DNA analyses by allele specific PCR assays were used to establish the final diagnoses. RESULTS: Five α-globin chain variants including Hbs Q-Thailand, Queens, Siam, Constant Spring and Paksé were detected. In heterozygous form, the machine demonstrated clearly two abnormal derivatives of Hbs A and A(2) for the former three variants. Small peaks of Hb Constant Spring and Hb Paksé were observed but could not be differentiated. In contrast, only one abnormal peak of Hb A was observed for ß-globin chain variants including those with more negative charge (Hb J-Bangkok, Hb Hope and Hb Pyrgos) and less negative charge (Hb D-Punjab, Hb S, Hb Korle-Bu and Hb C). Hb Tak, an elongated ß-chain variant was co-separated with Hb F whereas the Hb Malay co-migrated with Hb A in a subject with high Hb A(2) ß- thalassaemia trait. CONCLUSION: The capillary electrophoresis system could clearly demonstrate the presence of abnormal Hbs and provide useful information for presumptive diagnoses in most cases. The Hb analysis results could help in selection of appropriate DNA testing for final diagnoses of these variants.

Molecular and haematological characterization of compound Hb E/Hb Pyrgos and Hb E/Hb J-Bangkok in Thai patients.

We describe haematological and DNA characterization of haemoglobinopathies in Thai adolescents caused by compound heterozygosities for Hb E [beta26(B8) Glu-Lys] and two other beta-globin chain variants, Hb Pyrgos [beta83(EF7) Gly-Asp] and Hb J Bangkok [beta56(D7) Gly-Asp]. Hb analysis demonstrated that although these two beta-chain variants have separated elution profiles on liquid chromatography-based Hb analysis, they have similar alkaline electrophoretic mobilities on cellulose acetate electrophoresis. Haematological data associated with these two previously undescribed conditions were compared with those of pure carriers of the variants found in other unrelated Thai individuals. beta-Globin gene haplotypes linked to these two beta-chain variants and a simple DNA testing based on multiplex allele-specific polymerized chain reaction for differential diagnosis are presented.

Laboratory diagnosis of a compound heterozygosity for Hb Hekinan [alpha27(B8) Glu-Asp] and a deletional alpha-thalassaemia 2 in Thailand.

We report the haematological and molecular characterization of a previously undescribed condition of compound heterozygosity for haemoglobin (Hb) Hekinan [alpha27(B8) Glu-Asp] and a deletional alpha-thalassaemia 2 detected in a Thai individual. Hb analysis demonstrated that although this Hb variant co-migrates with Hb A on cellulose acetate electrophoresis and cation-exchange high-performance liquid chromatography (HPLC), the HPLC procedure using a weak cation-exchange material with polyaspartic acid could clearly differentiate the two Hb. The variant could then be confirmed using the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis of the amplified alpha1-globin gene.

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

We describe the hematological and DNA characterization of hemoglobin (Hb) E heterozygote with various forms of alpha-thalassemia in Thai individuals. Altogether, 202 unrelated adult subjects with Hb E heterozygotes either with or without alpha-thalassemia determinant were studied. The most prevalent interaction was found to be a double heterozygote for Hb E/alpha-thalassemia 2, followed by a double Hb E/alpha-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 alpha-thalassemia 2 and Hb E with a compound alpha-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 alpha-thalassemia 1/alpha-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 alpha-thalassemia. The hematological expression of these Hb E heterozygotes with various forms of alpha-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.

Compound heterozygosity for Hb Korle-Bu (beta(73); Asp-Asn) and Hb E (beta(26); Glu-Lys) with a 3.7-kb deletional alpha-thalassemia in Thai patients.

Hemoglobin (Hb) Korle-Bu (beta73; Asp-Asn) is the most frequent of the rare beta-chain variants in the population of West Africa whereas Hb E (beta26; Glu-Lys) is common among the Southeast Asian population. We report a hitherto undescribed condition in which these two beta-chain variants co-segregate. The proband was a 19-year-old Thai pregnant woman in her second trimester of pregnancy who visited our thalassemia screening unit. Cellulose acetate electrophoresis and high-performance liquid chromatography (HPLC) analysis of Hb detected one abnormal Hb in addition to the Hb E. Analysis of DNA sequences revealed a GAT-AAT mutation at codon 73 in trans to a GAG-AAG mutation at codon 26 of the beta-globin gene. Polymerase chain reaction (PCR) analysis of the alpha-globin gene cluster of the patient detected a 3.7-kb deletional alpha-thalassemia 2. Family study identified that her mother had the same genotype and her father was a simple Hb E carrier. The hematological data of these unusual cases of hemoglobinopathy are presented and compared with a simple heterozygote for Hb Korle-Bu found in another unrelated Thai family. beta-Globin gene haplotype linked to the Thai beta(Korle-Bu) and a simple DNA assay based on allele-specific PCR for rapid diagnosis of Hb Korle-Bu are also described.

Simultaneous PCR detection of beta - thalassemia and alpha - thalassemia 1 (SEA type) in prenatal diagnosis of complex thalassemia syndrome.

OBJECTIVE: To establish a rapid PCR method for simultaneous detection of beta-thalassemia and alpha-thalassemia 1 genes for diagnosis of complex alphabeta-thalassemia syndrome. DESIGN AND METHODS: Using multiplex allele specific PCR approach, we evaluated a simultaneous detection of the SEA type alpha-thalassemia 1 and the common Southeast Asian beta-thalassemia and hemoglobin E genes. The system was tested on known cases of double heterozygote for alpha- and beta-thalassemias and in a prenatal diagnosis of complex alphabeta-thalassemia syndrome. RESULTS: Co-inheritance of alpha-thalassemia 1 (SEA type) with each of the common beta-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 alpha-thalassemia 1 and beta-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 alpha- and beta-thalassemias and hemoglobin E are common.

Molecular characterization of hemoglobin C in Thailand.

We describe hematologic and DNA characterization of 12 hemoglobin C heterozygotes and three compound heterozygotes for hemoglobin C and hemoglobin E found in Thailand. Amplification and DNA analysis of genomic DNA by the polymerase chain reaction procedure permitted the identification of the beta(C) mutation at codon 6 of beta-globin gene (beta 6; GAG-AAG). beta-Globin gene haplotype analysis demonstrated that all beta(C) globin genes detected in these Thai individuals were associated with the haplotype (+ - - - - - +), indicating a non-African origin of this abnormal hemoglobin in Thailand. On routine hemoglobin typing, hemoglobin C is usually mistakenly identified as hemoglobin E because of theirs similar mobilities on cellulose acetate electrophoresis. The simple DNA assay for hemoglobin C based on an allele-specific polymerase chain reaction for accurate diagnosis of hemoglobin C was therefore developed.

Atypical hemoglobin H disease in a Thai patient resulting from a combination of alpha-thalassemia 1 and hemoglobin Constant Spring with hemoglobin J Bangkok heterozygosity.

A case of hemoglobin H disease in combination with hemoglobin Constant Spring and a beta-globin chain variant is reported in a 3-yr-old Thai girl. On routine cellulose acetate electrophoresis, one abnormal band in addition to the hemoglobins A, A2, H, Bart's and Constant Spring was detected. The amount of this abnormal band movement towards more anodic to the hemoglobin A was 35.7%. DNA analysis of the alpha-globin gene cluster by polymerase chain reaction (PCR) revealed a combination of defects caused by the SEA-type alpha-thalassemia 1 and the alpha-Constant Spring gene. Analysis of beta-globin gene by PCR and DNA sequencing also detected the heterozygosity for the GGC-GAC mutation at codon 56, leading to a substitution of aspartic acid for glycine resulting in the hemoglobin J Bangkok. The hematologic data of this unusual case of hemoglobin H disease are presented and compared with two compound heterozygotes for hemoglobin J Bangkok and alpha-thalassemia 1 found in the patient's father and grandfather. A simple DNA assay based on an allele-specific PCR for rapid diagnosis of the hemoglobin J Bangkok is also described.

Mitochondrial DNA polymorphisms in Thailand.

Nucleotide sequences of the D-loop region of human mitochondrial DNA from six small ethnic groups of Thailand i.e., Hill tribes (Lisu and Mussur), Phuthai, Lao Song, Chong, and aboriginal Sakai, were analyzed. The sequences were compared with those of native Thai populations from two provinces, Chiang Mai and Khon Kaen. Based on a comparison of the 563-bp sequences in 215 Thai individuals, 137 different sequence types were observed. Of these, 124 were unique to their respective populations, whereas 13 were shared between two to five populations. The intergenic COII/tRNALys 9-bp deletion was observed in every Thai population examined, except for the Sakai, with varying frequencies ranging from 18% to 40%. The D-loop sequences variation, and phylogenetic analysis, suggested that the 9-bp deletion had occurred in a very ancient ancestry of Southeast Asians, although multiple origins of the deletion cannot be ruled out. Genetic distances, based on net nucleotide diversities, between populations revealed that the Sakai were distantly related to the other Thai populations, while the Lao Song and Chong were closely related to each other. Close genetic affinities were also observed among the Hill tribes, Phuthai, and native northeast Thai (Khon Kaen), indicating that they may share some degree of the common ancestral maternal lineages.

Molecular characterization of (deltabeta)(0)/beta(0)-thalassemia and (deltabeta)(0)-thalassemia/hemoglobin E in Thai patients.

Two cases of the Thai thalassemia patients with compound heterozygosities for (deltabeta)(0)/beta(0)-thalassemia and (deltabeta)(0)-thalassemia/hemoglobin E have been reported. The first case was a 8-yr-old boy who had the following hematologic data: Hb 6.5 g/dL, Hct 20.5%, MCV 70.4 fL, MCH 22.3 pg and MCHC 31.7 g/dL. Hemoglobin analysis revealed 1.9% hemoglobin A2 and 91.7% hemoglobin F. The second case, with Hb 13.9 g/dL, Hct 41.5%, MCV 69.5 fL, MCH 22.5 pg and MCHC 32.2 g/dL, was a 16-yr-old male who had 46.1% hemoglobin E and 49.8% hemoglobin F. Globin gene analyses showed that both probands carried the same deletional type (deltabeta)(0)-thalassemia trans to the 4 bp deletions in codons 41/42 beta(0)-thalassemia and to the betaE-globin gene, respectively. Polymerase chain reaction and DNA sequence analyses demonstrated that the 5' breakpoint of the (deltabeta)(0)-thalassemia deletion was located in the second intron of the delta-globin gene and that the 3' breakpoint lay within a cluster of LI repetitive sequences at 4.7 kb 3' to the beta-globin gene.

Molecular and hematological characterization of HbE heterozygote with alpha-thalassemia determinant.

Hemoglobin E and alpha-thalassemia are prevalent in Thailand. The chance that an individual heterozygous for HbE also carries an alpha-thalassemia determinant is high. In this individual, the amount of HbE and other hematological parameters may be differed from that of usual observation. In this study, a total of 132 HbE heterozygotes were screened for alpha-thalassemia 1 gene deletion by the polymerase chain reaction. Out of 132 cases, 71 could be completely analyzed for hematologic parameters. Forty-three of 88 cases with HbE less than 25% as measured using microcolumn chromatography were positive for this gene deletion. In twenty of these 43 alpha-thalassemia 1 positive cases, the average values of Hb, Hct, MCV, MCH, MCHC, RDW and HbE were 10.6 g/ dl, 33.1%, 64.8 fl, 21.0 pg, 32.3 pg/dl, 18.6% and 17.4%, respectively. Eight of 9 alpha-thalassemia 1 negative cases were positive for alpha-thalassemia 2 gene deletion in Southern blot analysis. In this later group, hematological parameters were similar to that of the former. Co-inheritance of the Hb Constant Spring gene has no direct effect on the level of HbE. No alpha-thalassemia 1 gene was detected in the remaining 34 cases whose HbE were above 25%. The average amount of Hb, Hct, MCV, MCH, MCHC, RDW and HbE were 12.4 g/dl, 37.7%, 79.7 fl, 26.2 pg, 32.7 pg/dl, 25.8% and 28.5%, respectively. Therefore, screening for HbE level below 25% may be a convenient way of identifying parents of carrying alpha-thalassemia 1 determinant.

Molecular and hematological characterization of Hb Tak and Hb Pyrgos in Thailand.

Two hemoglobin variants that migrate abnormally on gel electrophoresis were found in four unrelated Thai individuals. One variant that migrate faster than HbA but more slowly than Hb Bart's was detected in two heterozygotes. Another abnormal Hb migrating between HbA2 and HbF was found in one heterozygote and one compound heterozygote with HbE. In all cases, no microcytic anemia was observed. PCR amplification and direct DNA sequencing established that the first variant was caused by a missense mutation at codon 83 (GGC-GAC) that leads to Gly to Asp substitution previously described as the Hb Pyrgos in a Greek boy. The second variant was caused by an AC insertion at the termination codon that leads to synthesis of elongated beta-globin chain known as the Hb Tak. Beta globin gene haplotype analysis demonstrated that each variant was found on the same chromosome background in Thai individuals. The simple non-radioactive DNA assays based on allele specific polymerase chain reaction for the detection of these two Hb mutations in a routine laboratory are described.

Beta-globin gene haplotypes in some minor ethnic groups in Thailand.

In order to provide population genetic data of various ethnic groups in Thailand, we have determined the type of hemoglobin by electrophoresis and the beta-globin gene haplotypes by PCR followed by restriction digestion in five small ethnic groups namely hill tribes, PhuTai, Chong, Lao Song and Sakai inhabiting in the north, northeast, east, central and south of Thailand, respectively. In each group, in addition to HbA and HbA2, the HbE, the most common hemoglobinopathy in Southeast Asia was detected at 2.5%, 51.6%, 84.0%, 8.6% and 11.8%, respectively. Haplotype analysis demonstrated that in all groups the beta A-globin gene was associated with various haplotypes and beta-globin gene frameworks. However, beta E -globin gene was associated with haplotypes ((-)+(-)+ + +(-)) and ((+)-(-)-(-)+(-)) on the beta-globin gene framework 2 in all ethnic groups except in Chong people whose the beta E-globin gene was mostly linked to haplotype ((-)+(-)++(-)+) and beta-globin gene framework 3 which was commonly found among Cambodian. It appears therefore that the Chong population is more related to Cambodian than Thai.

mtDNA polymorphism in East Asian Populations, with special reference to the peopling of Japan.

Nucleotide sequences of the major noncoding (D-loop) region of human mtDNA from five East Asian populations including mainland Japanese, Ainu, Ryukyuans, Koreans, and Chinese were analyzed. On the basis of a comparison of 482-bp sequences in 293 East Asians, 207 different sequence types were observed. Of these, 189 were unique to their respective populations, whereas 18 were shared between two or three populations. Among the shared types, eight were found in common between the mainland Japanese and Koreans, which is the largest number in the comparison. The intergenic COII/tRNA(Lys) 9-bp deletion was observed in every East Asian population with varying frequencies. The D-loop sequence variation suggests that the deletion event occurred only once in the ancestry of East Asians. Phylogenetic analysis revealed that East Asian lineages were classified into at least 18 monophyletic clusters, though lineages from the five populations were completely intermingled in the phylogenetic tree. However, we assigned 14 of the 18 clusters for their specificity on the basis of the population from which the maximum number of individuals in each cluster was derived. Of note is the finding that 50% of the mainland Japanese had continental specificity in which Chinese or Koreans were dominant, while < 20% of either Ryukyuans or Ainu possessed continental specificity. Phylogenetic analysis of the entire human population revealed the closest genetic affinity between the mainland Japanese and Koreans. Thus, the results of this study are compatible with the hybridization model on the origin of modern Japanese. It is suggested that approximately 65% of the gene pool in mainland Japanese was derived from the continental gene flow after the Yayoi Age.

Expression of hemoglobin E in newborn.

Hemoglobin E(HbE) is an abnormal hemoglobin resulted from a point mutation in codon 26 (GAG-AAG) of beta-globin gene. The mutation causes an amino acid substitution (Glu-Lys) and abnormal splicing in exon 1 that reduce the amount of beta E chain synthesis. While in adult, the HbE can easily be diagnosed, its level in newborn is usually underrepresent. In this study, we examined a relationship between genotype of HbE and the amount of HbE in cord blood. 145 Cord blood specimens were analysed by starch gel electrophoresis and the amounts of HbE were determined by microcolumn chromatography. The zygosity of beta E globin gene was determined by the polymerase chain reaction. The levels of HbE were 3.17 +/- 1.79% for 59 heterozygotes, 8.55 +/- 2.52% for 3 homozygotes and 0.48 +/- 0.22% in 83 normal newborns. This result provides useful data for a neonatal screening program and implies that expression of HbE in newborn dependent on a copy number of beta E globin gene in each individual.