Identification of a novel complex variant in a patient involving the α-globin gene cluster by third-generation sequencing

Abstract

Various methods are available to detect common deletions and mutations of genes related to thalassemia, including gap-polymerase chain reaction (Gap-PCR), next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), and quantitative real-time polymerase chain reaction (qRT-PCR). Unequal crossover during the recombination of α1 and α2 hemoglobin can be detected but hardly accurately defined by above-mentioned technologies. A couple with abnormal hematological test results arrived at our department for genetic consultation. Preliminary analysis using NGS revealed a 3.7 kb (chr16:223462-227311) heterozygote deletion in the wife and nearly six copies in the chr16:223462-227311 (GRCh37/hg19) region of the husband. Further Gap-PCR results for the wife were consistent with the NGS results. MLPA and qRT-PCR were performed to detect the potential extra copies of the α-globin gene of the husband. The analyses simultaneously showed that the copy numbers of HBA1/HBA2 genes were nearly six. A specialized primer of the α-globin gene was designed to elucidate the structure of the 4.2 or 3.7 kb repeats of the husband. Third-generation sequencing (TGS) revealed the existence of the four extra tandem duplications of 3.7 kb in DNA strand 1 (chr16:173302-177106, hg38) of the α-globin gene and the existence of a heterozygous c.301-31_301-24delinsG insertion and deletion (InDel) in DNA strand 2 in the HBA1 gene (chr16:177252-177259, hg38). These results were confirmed by Sanger sequencing. Integrative Genomics Viewer analysis of the BAM files of NGS detected a low-level (reference/alternative, 0.19%) heterozygous InDel (c.301-31_301-24delinsG) in HBA1. Compared to traditional methods, TGS was better able to detect variants accurately and find rare genotypes and rearrangements of the α-globin gene cluster.

Keywords: Multiple copies of α-globin gene cluster; Thalassemia; Third-generation sequencing; Traditional methods.

Fig. 1

The result of Gap-PCR. M: marker; NTC: no template control; F: female sample; PC: positive control(-α^3.7/αα); NC: negative control

Fig. 2

A Copy numbers of HBA1/HBA2 of the female sample by NGS. B Copy numbers of HBA1/HBA2 of the male sample by NGS. C Copy numbers of HBA1/HBA2 of the negative control by NGS

Fig. 3

The result of qRT-PCR. M: male sample; NC: negative control; PC: positive control, --^SEA/αα

Fig. 4

The result of MLPA. Analysis of the multiple copies of α- globin cluster of the male. The y-axis represents the ratio signal as compared to the negative control (ratio 0.8–1.2); on the x-axis, the MLPA-probe numbers are ordered chronologically in the 5’ to 3’ direction along the region

Fig. 5

TGS results of the male sample. A After analysis using TGS, the gene structures of two DNA strands were detected, where chain 1 had a complex structure and chain 2 has a heterozygous c.301−31_301-24delinsG InDel. According to the homology of the α-globin gene cluster, we divided it into X, Y, and Z segments^[16]. According to the sequencing results, the structure of chain 1 is seen as α2-α^anti3.7-α^anti3.7-α^anti3.7-α^anti3.7-α^anti3.7-α1. B TGS double-stranded DNA results, corresponding to the structure in A

Fig. 6

Sanger sequencing verification results of mutation. A DNA sequencing results of HBA1 (chr16:227251–227258) c.301−31_301−24. B DNA sequencing results of HBA2 (chr16:223440–223447) c.301−31_301−24

Fig. 7

The NGS results of the male sample. A low-level (reference/alternative, 0.19%) heterozygous InDel (c.301−31_301-24delinsG) in the HBA1