Molecular Characterization of Three Novel Large Deletions Causing α0-Thalassemia

Abstract

Alpha-thalassemia is most often caused by large deletions within the α-globin gene cluster which reduce or abolish α-globin chain synthesis. Several common deletions are well described, but atypical structural variants remain underdiagnosed. In this study, we report three novel large heterozygous deletions of the α-globin cluster. The variants were identified in unrelated patients who presented with persistent microcytosis and hypochromia in the absence of iron deficiency or structural hemoglobin variants. A stepwise molecular diagnostic approach was applied. It combined commercial deletion screening assays, Sanger sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA), and targeted Next-Generation Sequencing (NGS) with the Devyser Thalassemia panel. MLPA detected three deletions ranging from ~17 kb to ~360 kb. All involved the critical HS-40 regulatory region and both HBA1 and HBA2 structural genes, consistent with α0-thalassemia alleles. Next-Generation Sequencing confirmed the extent of each deletion and refined their genomic boundaries. Comparative genomic mapping showed that these deletions are distinct from classical variants such as --SEA or --MED, indicating novel structural configurations. Clinically, all patients displayed a carrier phenotype, with normal HbF levels (<1%) and normal or slightly reduced HbA2 values. This study broadens the mutational spectrum of α0-thalassemia and demonstrates the diagnostic value of combining MLPA and NGS in patients with unexplained microcytosis. By enabling accurate distinction from iron-deficiency anemia and other microcytic disorders, these findings have direct translational implications for improving diagnostic precision and genetic counseling in clinical practice.