Case report: Long-read sequencing identified a novel 14.9-kb deletion of the α-globin gene locus in a family with α-thalassemia in China

Abstract

Background: Thalassemia is a hereditary blood disease resulting from globin chain synthesis impairment because of α- and/or β-globin gene variants. α-thalassemia is characterized by non-deletional and deletional variants in the HBA gene locus, of which rare deletional variants are difficult to detect by conventional polymerase chain reaction (PCR)-based methods. Case report: We report the case of a one-month-old boy, who and his mother had abnormal hematological parameters, while his father had normal hematology. Conventional PCR-reverse dot blot (RDB) was performed for all family members to analyze the 23 most common thalassemia variants in China, but did not identify any pathologic variants. Single-molecule real-time (SMRT) long-read sequencing (LRS) technology was then performed and identified an unreported 14.9-kb large deletion (hg38 chr16:168,803-183,737) of the α-globin gene locus, which disrupted both HBA1 and HBA2 genes in the proband and his mother. The exact breakpoints of the deletion were confirmed by gap-PCR and Sanger sequencing. Conclusion: We have detected a novel large deletion in α-globin gene locus in China, which not only enriches the variant spectrum of thalassemia, but also demonstrates the accuracy and efficiency of LRS in detecting rare and novel deletions.

Keywords: large deletion; long-read sequencing; rare variant; thalassemia; α-globin gene locus.

FIGURE 1

Thalassemia genetic testing by LRS technology. (A) Pedigree of the family. (B, C) Integrative Genomics Viewer plot displayed the CCS reads of SMRT sequencing for the proband (Ⅱ-1) and mother (I-2). The pink area showed the αα allele, and the green area showed the other allele with 14.9-kb deletion. (D) Validation of the large deletion by gap-PCR. The primers were designed flanking hg38 chr16:168803-183737 to detect the 14.9-kb deletion. (E,F) Confirmation of the exact breakpoints of deletions by Sanger sequencing for the proband (II-1) and mother (I-2). (G) Sequence of breakpoint junctions of the 14.9-kb deletion. Reference sequences encompassing the breakpoints were shown above and below the sequences with deletion. The sequences with microhomology in the breakpoint junctions were underlined. (H) Display of the 14.9-kb deletion in related to other recurrent deletions in the α-globin gene cluster.