Evaluation of a Customized RDB-FTH Panel for β-Globin Variants in a Malaysian Tertiary Center.

Abstract

β-Thalassemia is one of the most prevalent autosomal recessive genetic disorders in Malaysia, with an estimated prevalence of 4.5%, encompassing asymptomatic carriers, intermedia and transfusion-dependent β-thalassemia. Molecular diagnosis has become increasingly important to correlate phenotype to genotype, guiding management, and facilitating genetic counseling, particularly in the prenatal diagnostic setting. This study evaluated a rapid reversed dot-blot-based flow-through-hybridization (RDB-FTH) assay for the molecular detection for β-thalassemia and related variants. A total of 112 DNA samples from patients with β-thalassemia and other hemoglobinopathies were analyzed, comprising thalassemia trait (n = 74), HbE trait (n = 15), HbE/β-thalassemia (n = 10), compound heterozygous β-thalassemia (n = 6), homozygous HbE (n = 4), homozygous HbS (n = 2), and HbS trait (n = 1). Initial diagnoses were based on full blood count and hemoglobin analysis. Genomic DNA was subsequently analyzed using a customized RDB-FTH assay targeting 23 β-globin mutations and two β-globin deletions. The detected point mutations were confirmed by Sanger sequencing, while deletions were validated using multiplex gap-PCR. Reversed dot-blot-based flow-through-hybridization successfully genotyped all samples (100%). Out of the 224 alleles examined, 134 β-thalassemia alleles were identified, representing 14 mutations, two Hb-variant, and one β-globin deletion. The predominant mutation was Cd26(G > A) (24.6%), followed by IVS-I-5(G > C) (12.7%), Cd41/42(-TTCT) (11.9%), while the 45 kb-Filipino deletion accounted for 6.7%. The assay demonstrated 100% concordance in selected cases whose HBB genes were sequenced and had gap-PCR. Reversed dot-blot-based flow-through-hybridization is a rapid and accurate method for β-thalassemia molecular diagnosis, enabling simultaneous detection and genotyping of multiple mutations on a single membrane, thereby improving precision and efficiency in high workload diagnostic settings.