Molecular Characterization of Complex Thalassemia with Multiple Variants in β-Globin Gene Cluster and the Identification of a Novel Structural Rearrangement in γ-Globin Gene.

Molecular characterization was performed for investigation of β-globin gene cluster in a pregnant Chinese female with mild microcytic hypochromic anemia accompanied with complicated hemoglobin fractions. Routine hematological parameters and hemoglobin analyses were conducted using an automated cell counter and capillary electrophoresis, separately. Long-read single molecule real time (SMRT) sequencing was employed to molecularly characterize this individual. Hematological indices showed mild microcytic hypochromic anemia, and hemoglobin analyses demonstrated normal HbA2 percentage of 2.3% and increased HbF value of 13.1% in this female. SMRT thalassemia genetic testing showed a heterozygous β⁺ mutation HBB:c0.316-197C > T (β^{IVS-II-654 (C>T)}) and heterozygous HBG2:c.-211C > T (-158Gγ (C > T)), which has independently been reported to result in elevated HbF levels. A variant HBD: c.-127T > C (-77 (T > C)) was also identified in the promoter region, which has been frequently reported to result in normal HbA2 levels in patients with β-thalassemia. All the three variants were further validated by Sanger sequencing. Moreover, SMRT analysis unraveled a novel duplicated structural variation of HBG1/HBG2 (^Gγ^Aγ/^{-158(C>T)GγGγGγAγ}), a rearrangement of four γ-globin genes including one entire HBG1 and three entire HBG2 in one chromosome. We herein first described a novel structural quadruplet γ-globin genes of HBG2 by SMRT and reported the molecular characterization of a complex thalassemia with variants involving HBG1/HBG2, HBD and HBB genes. Our work may facilitate genetic counseling and bring insight into future diagnosis of complex thalassemia.