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

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.