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

Thalassemia is an autosomal recessive disease and one of the most common monogenic diseases worldwide [1]. Thalassemia is a hemoglobinopathy caused by variations (including mutations, deletions, duplications, and gene rearrangements) in alpha (α)- and beta (β)-globin gene clusters that disrupt the balance of synthesis between the α- and β-globin chains which compose hemoglobin [2]. The α-globin gene cluster lies within chromosome 16 [3]. The HBA locus contains two almost identical genes, HBA1 and HBA2, which encode the α-hemoglobin chain [3, 4]. The clinical presentations of α-thalassemia vary widely [5, 6]. There are three types of α-thalassemia carrier states (silent, mild, and intermedia) and one disease state (major), depending on the number of globin chains and the disease state [7].

Approximately 5% and 1.5% of the global population carry mutations in α- and β-thalassemia genes, respectively [4, 8]. As a result, a large number of children are born each year with hemoglobin disorders which can lead to serious birth defects and place a heavy burden on society and families [9]. According to literature reports, the frequency of thalassemia gene carriage in southern China was 3–24%, the genotype of α^{Hb Westmead}α/αα carrying rate was 3.41% [10] in Guangxi, China, and the prevalence of triplicated alpha thalassemia was 1.99% in Guangdong and Hunan province of China [11, 12]. Thus, accurate diagnosis of thalassemia in patients and carriers remains challenging owing to the complexity of thalassemia genetics and genotype-phenotype correlations.

Traditional DNA analysis methods for the diagnosis of thalassemia include gap polymerase chain reaction (Gap PCR), PCR reverse dot blot (PCR-RDB) hybridization, multiple ligation-dependent probe amplification, and Sanger sequencing [13, 14]. More recently, next-generation sequencing (NGS) methods have been used for genetic screening for thalassemia [15, 16]. 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]. 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.

The copy number of HBA genes (3, 2, 1, or none) in Asian patients with thalassemia results in four different α-thalassemia syndromes [25]. Three normal HBA genes result in a silent carrier state, usually without clinical symptoms [1, 2, 25]. Two normal HBA genes result in mild α-thalassemia, usually with Hypochromic microcytosis but presenting as asymptomatic, without anemia [1, 2, 4, 25]. One normal HBA gene results in Hb H disease, in which the clinical manifestations and the degree of anemia vary greatly. Patients with mild symptoms only had mild thalassemia without obvious clinical symptoms. In severe cases, regular blood transfusion is required, and obvious thalassemia features such as hepatosplenomegaly, thalassemia-like skeletal changes, jaundice, and others are present [1, 2, 4, 25]. The absence of a normal HBA gene results in homozygous α-thalassemia, which manifests as fatal hydrops fetalis. Hemoglobin (Hb) Bart’s edematous fetus is characterized by severe anemia, jaundice, systemic edema, hepatosplenomegaly, dysplasia, short limbs, and giant placenta, among other features, and is a fatal blood disease. The affected fetus usually dies in utero at 23–40 weeks of gestation or within half an hour after birth as a result of severe anemia and hypoxia [1‐4, 25, 26].

Molecular diagnosis of thalassemia carrier states is challenging, especially because of the complexity of the HBA gene [9, 15]. Most thalassemia diagnostic laboratories use gap PCR and PCR-RDB to identify the most prevalent pathogenic HBA1/2 and HBB variants [6, 15]. These methods are mainly used to detect four common α-thalassemia deletions (--^SEA, -α^3.7, -α^4.2, --^Thai), three non-deletion α-thalassemia variants (α^{Hb Constant Spring}α, α^{Hb Quong Sze}α, and α^{Hb Westmead}α), and 17 common β-thalassemia variants in the Chinese population [1, 15], and are economical and practical testing methods. Nevertheless, in addition to the 23 common thalassemia variants, there are hundreds of rare and emerging genotypes. In these cases, gap PCR and PCR-RDB cannot meet the detection requirements, and more accurate and efficient screening programs are needed. Recently, NGS has emerged as an alternative molecular method for the genetic detection of thalassemia. This method has the advantages of simple sample collection and highly accurate results [15‐17]. Compared to traditional diagnostic methods, NGS can generate a large amount of genomic data and provide abundant genetic information. However, it may not be able to detect polystructures, tandem repeats, GC-rich regions, and other special structural regions, as well as highly homologous sequences [14, 15]. TGS, also called SMRT, was developed and validated using PacBio Sequel II [18]. Unlike NGS, which targets only exonic regions or selected intronic regions, TGS is used to generate longer PCR fragments that include both intergenic and intragenic regions improving the ability to identify variants [27‐31]. 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]. TGS can detect all mutation types of α-thalassemia and β-thalassemia genes [18, 30, 31]. However, TGS is currently not widely used in clinical testing owing to its high cost.

In this study, different methods were used for screening thalassemia. First, the routine hematological phenotypes of the patient were detected, finding both MCV and MCH showed a mild decrease. Subsequent analysis of 23 common thalassemia variations showed that the patient carried the -α^3.7 heterozygous deletion and Hb Westmead (c.369 C > G) homozygous variation. In our study, targeted NGS showed that the patient carried the homozygous variant c.369 C > G in HBA2, but the copy number of α^3.7 was between normal, 1, and loss of heterozygosity, 0.5, which cannot be determined accurately. Finally, TGS analysis showed a novel genotype in the α-globin gene cluster: one chromosome carried the -α^3.7 deletion, and the other carried the ααα^anti3.7, with two copies of the triplet carrying the HBA2: c.369 C > G (Hb Westmead) variant. Therefore, the intermediate copy number identified through NGS can be understood in the context of the TGS results. The TGS results also intuitively showed that the Hb Westmead homozygous variation detected by PCR-RDB was not from the chromosome with the -α^3.7 deletion. Compared with GAP-PCR, NGS has a wider detection range and can detect deletion types, point mutations, and other rare variants in common thalassemia screening. However, owing to the limitation of NGS testing over the highly homologous regions of HBA1 and HBA2 genes, there may be false positives and false negatives in the detection of α-deletion thalassemia. It is necessary to use other methods, such as TGS, for verification and typing. TGS uses long reads that could cover many rare gene loci, and its PCR-free characteristic means it is possible to reflect real arrangements in the genome [30, 31]. To the best of our knowledge, the genotypes identified in this study have not been reported previously. The genotype -α^3.7/ααα^anti3.7 has been reported in neonates from Mazandaran; however, the phenotype of the newborn has not been specifically described [32]. There are many types of variation in thalassemia-related genes, some of which are complex. When the clinical phenotype is inconsistent with the laboratory molecular test results, TGS should be performed to help accurately identify genetic variations. Accurate genetic test results are a prerequisite for the accurate assessment of reproductive risk.

In summary, a novel genotype in the α-globin gene cluster was confirmed by TGS in a Chinese female with mild decreases in MCV and MCH. Our study showed that TGS technology has the potential to detect novel variants which may be beyond the scope of traditional analytical methods. Therefore, TGS can be an effective and reliable approach for thalassemia screening in individuals suspected to carry rare mutations or complex variants. In addition, TGS analysis should be considered for the accurate diagnosis of uncertain cases of thalassemia, which could also improve the accuracy of genetic counseling. Couples who are thalassemia carriers have the opportunity to seek prenatal diagnosis and even preimplantation genetic testing services to reduce their risk of having a child with thalassemia.