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Objectives: Accurate quantification of hemoglobin (Hb) A 2 is vital for diagnosing β-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA 2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE)., Methods: We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA 2 cutoff value for β-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the β- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing., Results: HbA 2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias, -0.33; r, 0.991), β-thalassemia (bias, -0.27; r, 0.927), and non-β-thalassemia carriers (bias, -0.36; r, 0.928). An HbA 2 cutoff value of ≥4.0 % for β-thalassemia screening achieved 100 % sensitivity and 99.6 % specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3 , 99.8, 97.3, 99.8, and 99.7 %, respectively. We also identified a rare β-Hb variant, Hb La Desirade [ HBB :c.389C>T], associated with β-thalassemia and co-inherited with a single α-globin gene., Conclusions: The Premier Resolution HPLC is a reliable and accurate method for routine β-thalassemia carrier screening, aligning with existing CE methods., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

The interactions of δ-globin variants with α- and β-thalassemia or other hemoglobinopathies cause complex thalassemic syndromes and potential diagnostic problems. Understanding the molecular basis and phenotypic expression is crucial. Four unrelated Thai subjects with second hemoglobin (Hb) A2 fractions were studied. A standard automated cell counter was used to acquire initial hematological data. Hb analysis was carried out by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assays. Globin gene mutations and haplotype were identified by appropriate DNA analysis. An allele-specific polymerase chain reaction method was developed to provide a simple molecular diagnostic test. Hb analysis revealed a Hb A2 variant in all cases. DNA analysis of the δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu > Lys] variant in combination with Hb E in three cases. Analysis of the remaining case identified a novel δ-Hb variant, namely Hb A2-Mae Phrik [δ52(D3)GAT > GGT; Asp > Gly], found in association with Hb E and α+-thalassemia, indicative of the as yet undescribed combination of triple heterozygosity of globin gene defects. An allele-specific PCR-based assay was successfully developed to identify this variant. The β-haplotype of the Hb A2 Mae-Phrik allele was strongly associated with haplotype [+ − − − − ± +]. This study advanced our understanding of the phenotypic expression of known and novel δ-Hb variants coinherited with other globin gene defects, routinely causing problems with diagnosis. Therefore, knowledge and recognition of this Hb variant and molecular assessments are crucial to improving diagnosis.

Background: Delta-chain (δ-chain) variants are a group of rare hemoglobin (Hb) variants resulting from mutations within the δ-globin gene. Although quantification of Hb A2 levels is a useful screening tool for the beta-thalassemia trait, the coinheritance of a δ-globin gene mutation can lead to misinterpretation of diagnostic results. Objective: To identify an unreported Hb A2 variant in Thailand and to develop a high resolution melting (HRM) curve assay for the four δ-globin chain variants found in the Thai population. Materials and Methods: Allele-specific polymerase chain reaction (ASPCR) was used to analyze a total of 18 DNA samples for Hb variants comprising 10 wild-type controls, 4 Hb A2-Melbourne, 1 Hb A2-Lampang, 2 Hb A2-Kiriwong, and an unknown variant via HRM assays. Results: The unreported Hb A2 variant in Thailand was found to be Hb A2-Walsgrave resulting from δ-globin gene mutation at codon 52 (GAT>CAT). This was also confirmed using ASPCR. In addition, we demonstrated that the HRM curve profile for Hb A2-Melbourne, Hb A2-Lampang, Hb A2-Walsgrave, and Hb A2-Kiriwong could be identified so as to distinguish the mutant alleles from one another and from wild-type alleles. Conclusion: This HRM assay detected both known and unknown mutations with simultaneous differentiation between heterozygous and homozygous alleles on a polymerase chain reaction fragment spanning four of the δ-globin variants found in Thailand. This assay may help to support the prevention and control of thalassemias and hemoglobinopathies in Thailand.

Elevated Hb A 2 level (≥4.0%) is considered to be reliable parameter to identify β-thalassemia (β-thal) carriers. However, some β-thal carriers have been misdiagnosed as their Hb A 2 levels are below 4.0%. In addition, coinheritance of α-thalassemia (α-thal) and β-thal might affect Hb A 2 levels. Therefore, the aim of this study was to investigate the mutations of β- and α-globin genes in individuals with borderline Hb A 2 levels in Thailand. Three hundred samples from individuals with Hb A 2 levels of 3.5-3.9% were collected for molecular diagnosis of β-globin gene mutations. In addition, the α 0 -thal, α + -thal, Hb Constant Spring (Hb CS, HBA2 : c.427T>C), and Hb Paksé ( HBA2 : c.429A>T) diagnostics were also performed. Sixteen samples (5.33%) had β-globin gene mutations, and codon 41/42 (-TTCT) ( HBB : c.126_129delCTTT) was the most prevalent mutation. Ninety-eight samples (32.67%) had α-globin gene mutations including four Hb H (β4)-Hb CS disease, two Hb H disease, 13 heterozygous α 0 -thal, 11 homozygous α + -thal, two α + -thal/Hb CS, one α + -thal/Hb Paksé, 61 heterozygous α + -thal, and four Hb CS. Furthermore, seven cases of β-thal carriers coinheriting α-thal were observed, and five of them carried Hb H disease. High prevalence of both α- and β-thal in subjects with borderline Hb A 2 levels suggested that molecular diagnosis of α- and β-thal should be performed, especially in a high prevalence area of thalasssemia carriers, for accurate diagnosis and genetic counseling to prevent and control new severe thalassemia cases. Moreover, β-thal carriers who coinherited Hb H disease might have reduced Hb A 2 levels, leading to a misdiagnosis of β-thal in analysis programs.

In this study, Hb A 2 variants and their association with α- and β-thalassemia (α- and β-thal) were analyzed. We performed molecular analyses to identify α-thal [- - SEA (Southeast Asian), - - THAI (Thai), -α 3.7 (rightward) and -α 4.2 (leftward)] deletions, and Hb Constant Spring (Hb CS; HBA2 : c.427T>C), Hb A 2 -Melbourne ( HBD : c.130G>A), Hb A 2 ' ( HBD : c.49G>C), Hb A 2 -Lampang ( HBD : c.142G>A). β 0 -Thalassemia mutations included codon 17 (A>T) ( HBB : c.52A>T), codons 41/42 (-TCTT) ( HBB : c.126_129delCTTT), codons 71/72 (+A) ( HBB : c.216_217insA) and IVS-I-1 (G>T) ( HBB : c.92+1G>T) in 23 samples which had a Hb A 2 variant peak in zone 1 of the capillary electrophoresis (CE) electropherogram. Results showed that 20 patients (87.0%) carried Hb A 2 -Melbourne with seven different genotypes for α- and β-thal, two (8.7%) carried Hb A 2 ' and one (4.3%) carried Hb A 2 -Lampang. All three samples doubly heterozygous for Hb A 2 -Melbourne/β 0 -thal had Hb A 2 levels lower than 4.0%, while summation of Hb A 2 and Hb A 2 -Melbourne ranged from 4.9-5.3%, reaching the accepted range (4.0-10.0%) for β-thal trait. Hb A 2 -Melbourne is the most common δ-globin variant in the Thai population. Hb A 2 variant and Hb A 2 levels must be combined in order to diagnose carriers of β-thal. β-Globin haplotype analysis showed an association with a single β-globin haplotype [+ - - - - + +] of Hb A 2 -Melbourne, Hb A 2 ' and Hb A 2 -Lampang, indicating that they were of the same origin. We developed a multiplex allele-specific polymerase chain reaction (ASPCR) for simultaneous detection of these three Hb A 2 variants.

The capillary electrophoresis (CE) system allows the quantification of Hb Bart's (γ4) and Hb H (β4) that is used for screening of Hb H disease. However, Hb Bart's hydrops fetalis and Hb H are not always codetected in patients with Hb H disease. In this study, 35 samples were analyzed for the α 0 -thalassemia (α 0 -thal) [- - SEA (Southeast Asian) and - - THAI (Thailand)] deletions and the α + -thal [-α 3.7 (rightward) and -α 4.2 (leftward)] type deletions using real time-polymerase chain reaction (real time-PCR) with SYBR Green1 and high-resolution melting (HRM) analysis and conventional gap-PCR techniques, respectively. Results showed that 28 of 29 (96.6%) samples with the Hb A 2 -Hb H phenotype on CE electrophoregrams presented the genotype of - - SEA /-α 3.7 , while the - - SEA /-α 4.2 made up the remainder. The - - SEA /-α 3.7 genotype was also found in all six samples (100.0%) with Hb A 2 -Hb Bart's on CE electrophoregrams. Thus, for genetic counseling, prevention and control programs of Hb Bart's hydrops fetalis and Hb H disease, α-thal genotype analysis is required.

Hb A' 2 (or Hb B 2 ) ( HBD : c.49G>C) is the most frequent δ chain variant that has been described in Africa but not in Thailand. We report here a 10-month-old Thai infant with compound heterozygosity for β 0 codon 17 (A>T; HBB : c.52A>T) and β + IVS II-654 (C>T; HBB : c.316-197C>T). Under diagnosed β-thalassemia (β-thal) in her father, who carries Hb A' 2 and a heterozygous β 0 codon 17 mutation, and the mother, who carries a heterozygous β + IVS II-654 mutation, was noted. Although Hb A' 2 does not cause any problems, heterozygosity for Hb A' 2 can lead to under diagnosis of β-thal in Hb A' 2 samples. This case highlights the importance of Hb A' 2 in prenatal diagnosis (PND). Thus, molecular analysis for β-thal mutations should be carried out when a small peak presents at the retention time (RT) of 4.71 min. on high performance liquid chromatography (HPLC) and the summation level of this peak and Hb A 2 was equal or higher than 4.0%.

Background: We reported molecular and hematological characteristics of δ-globin chain variants and addressed diagnostic consideration of complex hemoglobinopathies caused by their interactions with α- and β-thalassemias., Methods: Study was done on four unrelated Thai subjects with second Hb A2 fractions. Hb analysis was carried out using automated HPLC and capillary electrophoresis. Mutations were identified by DNA analysis. Novel diagnostic methods based on PCR-RFLP and allele specific PCR were developed., Results: Hb analysis revealed Hb A2 variant in all cases. DNA analysis of δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu→Lys] in combination with α(+)-thalassemia, α(0)-thalassemia and β(0)-thalassemia in the first three cases, respectively. Analysis of the remaining case identified a novel δ-Hb variant namely the Hb A2-Lampang [δ47(CD6)GAT→AAT; Asp→Asn] found in association with Hb E and α(+)-thalassemia. These mutations could be identified using PCR-RFLP and allele specific PCR assays developed., Conclusions: It is necessary to recognize the Hb A2 variant and to combine the amounts of Hb A2 and Hb A2-variant for a total Hb A2 value to make better diagnostic of these complex syndromes. Co-inheritance of these multiple globin gene defects could lead to complex hemoglobinopathies requiring comprehensive Hb and molecular assessments., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report the molecular and hematological feature of a Thai woman who had clinical diagnosis of β-thalassemia intermedia (β-TI). Hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified Hb A (64.4%), Hb F (12.3%) and Hb A2/E (15.9%) with small peaks of Hb Bart's (γ4) and Hb H (β4). She was initially diagnosed as EA Bart's disease, which occurs from combination of Hb H disease and Hb E (HBB: c.79G > A) trait. However, the Hb analysis using capillary electrophoresis (CE) demonstrated no Hb E, 68.5% Hb A, 15.5% Hb F and 16.0% Hb A2. DNA analysis showed a compound heterozygosity for (β(+)) -31 (A > G) (HBB: c.-81A > G) and (β(0)) codon 17 (A > T) (HBB: c.52A > T) mutations and deletional Hb H (- -(SEA)/-α(3.7)). Thus, she was finally diagnosed with a combination of Hb H disease and compound heterozygosity of β(+)/β(0)-thalassemia (β(+)/β(0)-thal). The β-globin mutations could affect not only hematological parameters but also elevate the Hb A2 levels. These effects could not be ameliorated by the coinheritance of Hb H disease. Therefore, a better understanding of the effects of this combination on hematological analysis data will be useful for providing accurate diagnosis, genetic counseling, prevention and control programs of β-thalassemia major (β-TM).