Analysis of rare thalassemia genetic variants based on third-generation sequencing

Abstract

Thalassemia is a group of common hereditary anemias that cause significant morbidity and mortality worldwide. However, precisely diagnosing thalassemia, especially rare thalassemia variants, is still challenging. Long-range PCR and long-molecule sequencing on the PacBio Sequel II platform utilized in this study could cover the entire HBA1, HBA2 and HBB genes, enabling the diagnosis of most of the common and rare types of thalassemia variants. In this study, 100 cases of suspected thalassemia were subjected to traditional thalassemia testing and third-generation sequencing for thalassemia genetic diagnosis. Compared with traditional diagnostic methods, an additional 10 cases of rare clinically significant variants, including 3 cases of structure variants and 7 cases of single nucleotide variations (SNVs) were identified, of which a case with − α3.7 subtype III (− α3.7III) was first identified and validated in the Chinese population. Other rare variants of 11.1 kb deletions (− 11.1/αα), triplicate α-globin genes (aaa3.7/αα) and rare SNVs have also been thoroughly detected. The results showed that rare thalassemia variants are not rare but have been misdiagnosed by conventional methods. The results further validated third-generation sequencing as a promising method for rare thalassemia genetic testing.

Third-generation sequencing identified a novel complex variant in a patient with rare alpha-thalassemia

“…Each DNA molecule is sequenced separately in TGS [ 18 , 19 ]. TGS has multiple advantages including extremely long reads and being PCR-free [ 18 – 20 ]. Recently, TGS technology has become popular for the genetic detection of thalassemia [ 27 – 29 ].…”

“…The experiments were conducted as previously described [ 20 ]. gDNA was amplified using PCR with primers targeting the majority of known structural variation regions, single nucleotide variations (SNVs), and insertions and deletions (indels) in the HBA1 , HBA2 , and HBB genes.…”

“…In addition to discovering novel gene variants, these methods can effectively detect genotypes [ 16 , 17 ]. In recent years, third-generation sequencing (TGS) technologies have been applied to detect thalassemia genes [ 18 – 20 ]. These technologies can generate ultra-long reads and achieve high sequence precision and are characterized by the absence of GC preference and single-molecule resolution [ 18 , 19 ].…”

“…These technologies can generate ultra-long reads and achieve high sequence precision and are characterized by the absence of GC preference and single-molecule resolution [ 18 , 19 ]. Such methods are helpful for the accurate diagnosis and subsequent treatment of disease and minimize the risk of missed diagnosis [ 18 – 20 ]. This study utilized TGS to identify a novel genotype of α-thalassemia.…”

Thalassemia screening by third-generation sequencing: Pilot study in a Thai population

“…The molecular diagnostic methods involved various assays 2 namely allele-specific polymerase chain reaction, reverse dot-blot hybridization, Gap-PCR, multiplex ligation-dependent probe amplification (MLPA), and Sanger sequencing. 3,4 However, these methods have limited use in clinical practice due to their high cost, time-consuming nature, and labor intensiveness. In our setting, multiplex PCR with high-resolution melting (HRM) analysis was adopted to identify common alpha-thalassemia (– SEA , –THAI ) and common beta-thalassemia mutations.…”