Hb H diseases with the clinical features of thalassemia are found in many parts of the world, including Southeast Asia and southern China. There are limitations in molecular data from the population of Thailand, which includes multiple ethnic groups. Here, we characterized the molecular basis of the disease among a large cohort from this region. A total of 479 unrelated Thai patients with Hb H disease were studied. Mutations of the α-globin gene were characterized by conventional gap-PCR and rare genotypes were identified by MLPA analysis and direct DNA sequencing. The molecular characterization showed five common Hb H genotypes (472/479; 98.54%), including three deletional types (–^SEA/-α^3.7; n = 312), (–^SEA/-α^4.2; n = 26), (–^THAI/-α^3.7; n = 1) and two non-deletional types (–^SEA/α^CSα; n = 131), (–^SEA/α^Pakséα; n = 2). Herein, we firstly report a rare genotype of Hb H disease with (–^SA/-α^3.7; n = 1) that has not been documented in Thailand, and rare genotypes related to (–^SEA/-α^16.6; n = 1), and (–^SEA/α^QSα; n = 3) as well. The remaining two cases could not be characterized. The hematological parameters demonstrated that the clinical phenotype of non-deletional Hb H diseases is more severe than the deletional type of α⁺-thalassemia. The molecular spectrum of α-thalassemia is useful for prevention and thalassemia control and genetic counseling for couples at risk in this region.

We aimed to evaluate the efficiency of identification and quantification of hemoglobin (Hb) Constant Spring (CS) and Hb Paksé by capillary electrophoresis (CE). Blood samples collected from 2057 patients were used for identifying and quantifying Hb by CE. Molecular analysis of α- and β-thalassemia, Hb CS, and Hb Paksé was performed. Hb CS and Hb Paksé were identified in 573 samples (27.86%) with diverse genotypes. Thirty-eight samples (6.6%) showed no Hb CS peak. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of Hb CS by CE were 93.37, 95.96, 89.92, 97.40, and 95.24%, respectively. The amount of Hb CS in those carrying Hb CS was 0.2–6.5% which showed an increasing trend according to the number of defective α-globin genes, in contrast to Hb A₂ levels, which decreased. Hb CS level ≥1.0% accurately excluded heterozygotes and that of ≥2.0% could identify homozygotes. CE has the high potential for identifying and quantifying Hb CS and Hb Paksé, especially in an area with a high prevalence of thalassemia. Hb CS levels can be used as a potential marker to distinguish the genotype of individuals carrying Hb CS.

Elevated Hb A₂ 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 A₂ levels are below 4.0%. In addition, coinheritance of α-thalassemia (α-thal) and β-thal might affect Hb A₂ levels. Therefore, the aim of this study was to investigate the mutations of β- and α-globin genes in individuals with borderline Hb A₂ levels in Thailand. Three hundred samples from individuals with Hb A₂ levels of 3.5–3.9% were collected for molecular diagnosis of β-globin gene mutations. In addition, the α⁰-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 α⁰-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 A₂ 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 A₂ levels, leading to a misdiagnosis of β-thal in analysis programs.

Hemoglobin (Hb) D-Punjab [β 121(GH4) Glu→ln; HBB: C.364G>C] and β⁰-thalassemia 3.4 kb deletion are very rare in the Thai population. For the first time, the coinheritance of HbD-Punjab with β⁰-thalassemia 3.4 kb deletion was reported in a 7-year-old Thai girl. She had mild anemia (Hb 115.0 g/L and mean corpuscular hemoglobin 18.1 pg) with red blood cell microcytosis (mean corpuscular volume 52.5 fL). By capillary electrophoresis (CE), HbD-Punjab was found at a migration position of 180 s with the value of 81.9% while the level of HbA₂was 7.3%. Based on the elevated HbA₂, the molecular analysis for detection of β⁰-thalassemia mutations was performed. The 490 bp amplified fragments from β⁰-thalassemia 3.4 kb deletion was observed. Thus, the coinheritance of HbD-Punjab with β⁰-thalassemia can be found in the Thai population. The HbA₂measured on CE is a reliable parameter for differentiating the homozygote of HbD-Punjab and compound heterozygote of HbD-Punjab and β⁰-thalassemia.

Objectives: Accurate quantification of hemoglobin (Hb) A₂ is vital for diagnosing β-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA₂ 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 HbA₂ 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: HbA₂ 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 HbA₂ 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.

To determine the molecular basis, genotype - phenotype relationship, and genetic origin of Hemoglobin (Hb) Hekinan associated with several forms of α-thalassemia and other hemoglobinopathies for a better understanding of its diverse clinical phenotypes. Seventeen participants with suspected abnormal Hb were studied. Hb analysis was performed using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Mutational and α-haplotypic and structural analyses were conducted, and the effects of mutations on globin-chain stability were determined. All participants harbored Hb Hekinan II (HBA1:c.84 G>T) co-inherited with another α-globin gene anomaly. Three novel genotypes, (αα^Hekinan/α^CSα), (αα^Hekinan/α^CSα,β^A/β^E), and (αα^Hekinan/α^CSα,β^E/β^E), were characterized. Despite being co-inherited with both α- and β-Hb variants Hb Hekinan II led to minimal changes in erythrocyte parameters, suggesting a non-pathological nature. HPLC but not CE revealed a distinct small shoulder-like Hb pattern. Thai Hb Hekinan II was strongly associated with haplotype [+ - S + - - -] and the possibility of four different haplotypes, while two Burmese Hb Hekinan II were associated with haplotypes [± - S + - + -] and [± - S + - - -]. The novel genotypes identified provide a fresh perspective on Hb Hekinan II diversity. HPLC has superior identification capabilities for samples of Hb Hekinan II co-inherited with α-thalassemia. Thai and Burmese Hb Hekinan II have diverse origins.