Search

Beta-thalassemia is an inherited disorder prevalent in Thailand and Southeast Asia. Several molecular techniques for identifying β-thalassemia mutations have been reported. Next-generation sequencing (NGS) is a type of effective molecular testing with high throughput and accuracy. Hence, this study aims to evaluate a novel barcode-tagged NGS approach based on a short-read assay. A total of 258 samples with 54 different β-thalassemia genotypes related to 32 mutations were gathered and evaluated. A library was constructed with the BTSeq TM kit and sequencing was performed on the Illumina NGS machine. The validation results showed 98.45% concordance with conventional genotypes. Less discordant results (1.55%) were limited to insertional mutations and included one case of each of the following: HBB :c.27dupG, HBB :c.85dupC, HBB :c.216dupT, and HBB :c.440_441dupAC. Five single-nucleotide polymorphisms that derived from the NGS results were also analyzed in terms of allele frequency and revealed significant differences between the wild types and other β-genotypes. Furthermore, this paper is the first to describe rare single-nucleotide polymorphisms including IVS II-109 (C/T), IVS II-258 (G/A), IVS II-613 (T-C), and IVS II-806 (G/C). Interestingly, the C allele of IVS II-806 was found to have 100% linkage with two cases of Hb Tak. The haplotype and phylogenetic analysis was also constructed based on variants and revealed three clusters in the Hb variants, which represented their evolution and genetic background. Finally, NGS with the barcode-tagged method has a high throughput, which is suitable for large population screening. Its cost effectiveness and less complicated process promote its application in further works.

The aim of this study was to determine the molecular spectrum of β-thalassemia (β-thal) mutations in eastern Thailand. We identified β-thal mutations using allele specific-polymerase chain reaction (ASPCR) and direct DNA sequencing. We found 18 different β-thal mutations in a total of 191 unrelated subjects. Six common β-thal mutations comprised 86.91% of all the mutations, including codons 41/42 (-TTCT) ( HBB : c.126_129delCTTT) (35.60%), codon 17 (A>T) ( HBB : c.52A>T) (18.85%), -28 (A>G) ( HBB : c.-78A>G) (15.71%), IVS-II-654 (C>T) ( HBB : c.316-197C>T) (6.28%), IVS-I-1 (G>T) ( HBB : c.92+1G>T) (5.76%) and codon 19 (A>G) ( HBB :(c.59A>G) (4.71%). In addition, a novel 60 kb deletion in two unrelated cases was characterized and initially suspected to originate from eastern Thailand. Moreover, we demonstrated the molecular spectrum of recent β-thal mutations in Thailand, and data from this study were compared with five reference laboratory centers in Thailand. This study is the first to identify the comprehensive molecular spectrum of β-thal mutations in eastern Thailand, information that may be essential for screening, genetic counseling and prenatal diagnosis (PND) in this region.

The α 0 -thalassemia (α 0 -thal) [- - SEA (Southeast Asian) deletion] is highly prevalent in Southeast Asia and South China. The linkage between the single nucleotide polymorphism (SNP) rs77308790 and the - - SEA deletion was reported in the Chinese population. This study reported the genotype of SNP rs77308790 using the high resolution melting (HRM) curve analysis in the Thai population and the application for double-checking diagnosis of Hb Bart's (γ4) hydrops fetalis syndrome. A total of 202 samples, including α 0 -thal carriers (- - SEA /αα) ( n  = 99) and wild-type ( n  = 103), was recruited. Minor allele frequency (MAF) of SNP rs77308790 (T allele) represented a significant difference ( p <0.001) between carrier (- - SEA deletion) (MAF 0.455) and wild-type (MAF 0.039). The T allele of SNP rs77308790 showed a strong linkage with the - - SEA deletion allele [correlation coefficient between pairs of loci (D' = 1)] based on constructed random samples (CRSs) in Thais. Moreover, worldwide populations, based on the 1000Genomes database, also found the T allele to be less than 1.0%. For providing a double-checked diagnosis, two SNP (rs3760053, rs77308790) genotypes showed 100.0% concordance with a conventional gap-polymerase chain reaction (gap-PCR) method in nine families at-risk for Hb Bart's hydrops fetalis. The double-checked diagnosis based on the two SNPs (rs3760053, rs77308790) is suitable for implementation in routine diagnosis of Hb Bart's hydrops fetalis syndrome. Furthermore, our HRM analysis system can be amplified with a small amount of fetal DNA and could avoid allele dropouts.

We report the molecular and hematological identifications of a Hb A 2 variant [coinheritance of Hb A 2 -Melbourne ( HBD : c.130G>A) and Hb E ( HBB : c.79G>A)] found for the first time in the Lao People's Democratic Republic (PDR). The subject was a 29-year-old pregnant Laotian woman who was a foreign worker in Thailand and was diagnosed with thalassemia and hemoglobinopathies. Capillary electrophoresis (CE) demonstrated 1.6% of Hb A 2 , with a minor unknown peak at the initial Z1 zone (1.7%). Identification of abnormal hemoglobin (Hb) using direct DNA sequencing showed a genetic defect causing a δ-globin gene missense mutation at codon 43 ( G AG> A AG) causing a glutamic acid to lysine substitution corresponding to Hb A 2 -Melbourne. The origin of Hb A 2 -Melbourne in Lao PDR may be similar to a case found in Thailand with the [+ - - - - + +] haplotype. We developed a method that could clearly detect Hb A 2 -Melbourne and Hb A 2 -Lampang ( HBD : c.142G>A) mutations in a single tube using high resolution melt (HRM) analysis. The HRM analysis is a more effective method for rapid detection than conventional polymerase chain reaction (PCR), as there is no need for a post-PCR step, and no exposure to ethidium bromide. This new method would be a useful addition for the first investigation of a suspected Hb A 2 variant in the routine molecular setting.