Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing

Abstract Objectives Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we...

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Published in:American journal of clinical pathology 2024-04, Vol.161 (4), p.411-417
Main Authors: Xu, Anping, Ye, Yinghui, Huang, Yueying, Huang, Yun, Guo, Hui, Ji, Ling
Format: Article
Language:English
Subjects:
Breakpoints
Capillary electrophoresis
DNA sequencing
Fusion protein
Gene fusion
Genes
Hemoglobin
Hemoglobins, Abnormal - analysis
Hemoglobins, Abnormal - genetics
Humans
India
Meiosis
Multiplex Polymerase Chain Reaction
Polymerase chain reaction
Thalassemia
Thalassemia - genetics
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Summary:Abstract Objectives Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. Methods Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. Results The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. Conclusions Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.
ISSN:0002-9173
1943-7722
DOI:10.1093/ajcp/aqad155