Variants of uncertain significance (VUS) are often challenging for genetic counseling and require additional data for accurate variant classification. This study aims to describe the genotype-phenotype correlation of the seven β-globin gene variants found in Thailand. Retrospective data in a total of 45,914 subjects encountered at our diagnostic laboratory from January 2012 to December 2024 were reviewed. A total of 33 leftover EDTA blood specimens, suspected of having β-globin gene defects, were included. Eighty-nine normal subjects were also analyzed to confirm phenotypic expression of the variants. The whole β-globin and Krüppel-like factor 1 (KLF1) genes were examined using PCR-based methods. Seven nucleotide variants were identified among 33 suspected subjects, including a novel (β^−206(C>G)), four hitherto undescribed in Thailand [β^−198(A>G), β^{IVSII−180(T>C)}, β^{IVSII−337(A>G)}, and β^*233(G>C)], and two known variants [β^−50(G>A) and β^{IVSII−258(G>A)}]. The β^−198(A>G) and β^*233(G>C) variants were also identified in 1.69% of normal subjects, indicating neutral DNA polymorphisms. All subjects of β^−198(A>G), β^{IVSII−180(T>C)}, β^{IVSII−258(G>A)}, and β^{IVSII−337(A>G)} with borderline Hb A₂ levels had KLF1 mutations. Compound heterozygous β^−206(C>G) and known β⁺-thalassemia trait revealed β-thalassemia trait phenotype. In silico pathogenicity prediction showed that the β^−206(C>G), β^−198(A>G), β^{IVSII−180(T>C)}, β^{IVSII−258(G>A)}, β^{IVSII−337(A>G)}, and β^*233(G>C) were associated with benign variants. It was found that heterozygous β^−50(G>A) had elevated Hb A₂ levels resembling those of β-thalassemia trait. However, the association of the β^−50(G>A) and Hb E or β-thalassemia revealed a phenotype of Hb E or β-thalassemia trait. Most prediction tools indicate that the β^−50(G>A) is associated with benign variants; however, PromoterAI revealed that the β^−50(G>A) is associated with under-expression of the β-globin gene with high sensitivity. Based on these findings, the β^−50(G>A) is most likely a very mild β⁺-thalassemia allele. This study described the genotype-phenotype correlation of known and novel β-globin gene variants found in Thailand. The data should prove useful for accurate variant classification, genetic counseling, and a prevention and control program of severe thalassemia diseases in Thailand.

Laos, Khmer, Kui, and Yer ethnic populations had a high prevalence of α⁺-thalassemia, but the genetic background remains poorly understood. This study examined genetic variation at the α-globin gene cluster and three VNTR loci (D1S80, D17S5, and TPO) in these ethnic populations. For α-globin haplotype analysis, 110 subjects with normal α-globin and 232 subjects with α⁺-thalassemia were selected to analyze six polymorphic sites using the PCR-RFLP technique. For VNTR loci analysis, 447 subjects were examined for D1S80, D17S5, and TPO allele frequencies using a PCR-based method. The results of this study revealed that the most frequent haplotypes found in Laos, including (framework 1; + M - + − 0) linked to (-α^3.7) and (framework 3; - S - + + -) related to (α^CSα) were different from those found in Khmer and Kui [(framework 2; + S - + − 0) linked to (-α^3.7) and (framework 1; + M - + + -) related to (α^CSα)]. For the (α^PSα) gene, the haplotype (+ S - + - -) of framework 2 was found in all ethnic groups, and the haplotype (- M - + + -) of framework 1 in the Yer only. The distribution of allele frequencies for the D1S80, D17S5, and TPO VNTR loci showed extensive genetic variation in the ethnic population studied. The number of alleles is higher than that of the previously reported populations. Based on D17S5 analysis, the phylogenetic tree suggested that Khmer and Kui ethnic groups had a close relationship but were distant from Laos. In addition, ethnic relationships were observed in the Yer and Kui populations. In contrast, consistent results were not obtained based on D1S80 and TPO analysis. The findings indicate genetic variation in these ethnic populations, but the conclusion remains tentative. However, this study provides useful information to better understand genetic origins and ethnic relationships in the region.

The Southeast Asian deletion (--^SEA), the most prevalent α⁰-thalassemia mutation in Southeast Asia, is a major target of regional thalassemia prevention programs. Coinheritance of α⁰-thalassemia with other hemoglobinopathies, particularly hemoglobin E (Hb E), complicates routine hematological screening and requires reliable molecular confirmation. This study developed and validated a duplex nanoplate digital polymerase chain reaction (dPCR) assay for simultaneous identification of the wild-type α-globin and --^SEA deletion alleles in a single reaction. The assay was established on the QIAcuity nanoplate platform and evaluated using 171 blinded leftover DNA specimens from individuals with Hb E. Diagnostic performance was compared with conventional gap-PCR. Distinct fluorescence amplitude patterns enabled discrimination among normal, heterozygous, and homozygous --^SEA genotypes in a single closed tube. Among the 171 specimens, 70 were identified as --^SEA carriers and 101 as non-carriers, showing 100% concordance with gap-PCR. All subjects with Hb E + A₂ levels ≥ 25% were negative for the --^SEA deletion, whereas 56.0% of those with levels < 25% carried the deletion. In the representative prenatal case, duplex dPCR accurately identified parental carrier status and a homozygous --^SEA fetus. This assay provides an accurate and practical molecular confirmatory assay for thalassemia screening and prenatal diagnosis in Southeast Asia.