Screening and Diagnosis of Rare Thalassemia Variants: Is Third-Generation Sequencing Enough?

The study protocol was approved by the ethics committee of Bo’ai Hospital of Zhongshan (Guangdong Province, China; Approval No. KY- 2020-012-86) and conducted in accordance with the ethical principles for medical research involving human subjects described in the Declaration of Helsinki. Prior to inclusion in this study, written informed consent was obtained from all subjects or legal guardians.

Initially, 200 patients who underwent genetic testing for diagnosis of thalassemia were screened. Further screening was conducted for those who met the following inclusion criteria: (1) mean corpuscular volume (MCV) less than 82 μm³(fL) and/or mean corpuscular hemoglobin (MCH) less than 27 pg/cell; or (2) conventional genetic testing was insufficient to explain the clinical phenotype (MCV and MCH). In total, 128 cases with IDA were excluded. The final study cohort included 72 patients (age range, 2–51 years) with suspected mutations to thalassemia-related genes.

Routine blood tests were conducted using an automated cell counter (Auto Hematology Analyzer BC-6800; Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China). Positive screening for thalassemia was defined as MCV < 82 μm³ and/or MCH < 27 pg/cell. Those with positive test results were selected for further screening of thalassemia-related genes.

Single-tube multiplex Gap-PCR was used for the detection of 3 common α-thalassemia deletion mutations, which included -α^3.7, -α^4.2, and the Southeast Asian deletion (–^SEA). A PCR-RDB assay (Yaneng BIOscience Co Ltd, Shenzhen, China) was used to detect 3 common α-thalassemia nondeletion mutations, which included Hemoglobin Constant Spring (Hb CS, HBA2: c.427T>C), Hemoglobin Quong Sze (Hb QS, HBA2: c.377T>C), and Hemoglobin Westmead (Hb WS, HBA2: c.369C>G), in addition to 17 β-thalassemia mutations, which included −32 (HBB: c. −82C>A), −30 (HBB: c. −80T>C), −29 (HBB: c. −79A>G), −28 (HBB: c. −76A>G), CAP (HBB: c. −11_ −8delAAAC), Int (T>G) (HBB: c.2T>G), 14–15 (HBB: c.45_46insG), 17 (HBB: c.52A>T), 26 (or Hb E; HBB: c.79G>A), 27–28 (HBB: c.84_85insC), 31 (HBB: c.94delC), 41–42 (HBB: c.126_129delCTTT), 43 (HBB: c.130G>T), 71–72 (HBB: c.216_217insA), IVS-I-1 (HBB: c.92 + 1G>T), IVS-I-5 (HBB: c.92 + 5G>C), and IVS-II-654 (HBB: c.316–197C>T).

Variants of the α- and β-globin genes (HBA1, HBA2, and HBB), 1 rare α-globin gene deletion (–^THAI), 3 β-globin gene deletions (^Gγ⁺(^Aγδβ)⁰, SEA-HPFH, and Taiwanese), an α-globin gene triplet mutation (ααα^anti3.7 or ααα^anti4.2), and 1 fusion gene were detected among the 72 individuals with suspected rare mutations to thalassemia-related genes. A tag sequence was introduced for identification. The sequences were amplified and enriched by multiplex PCR with primers described in a previous report.⁷  The PCR products (≤96) were pooled for library preparation. For sequencing and library identification, the DNA sequences were supplemented with a linker sequence. The detailed experimental protocol and bioinformatics analysis were conducted as previously described.²  A next-generation sequencing library protocol was used for library construction, which included the purification of genomic DNA, quantification, fragmentation, blunt-ended fragmentation, addition of 3'-dA overhangs, paired-end adapter ligation, DNA fragment separation (CNVs not included), and size selection with magnetic beads. Finally, sequencing was performed with the paired end tag (PE100) on a MGISEQ-2000 chip. For bioinformatics analyses, a bioinformatics pipeline focused on detecting Hb gene deletions and point mutations. First, low-quality reads and reads with sequencing adaptor contamination were removed. Then, the clean data were classified using adapter information (index primer). Finally, the clean data were aligned to the human genome reference sequence (hg19) using BWA software.⁸  The final bam files were generated with SAMtools. The length, coverage, and depth of each consensus sequence were recorded with ReSeqTools.²  Then, the detected mutation types were annotated in reference to the HbVar (http://globin.bx.psu.edu/hbvar) and Ithanet (https://www.ithanet.eu/) databases. Positive NGS results were validated by Gap-PCR and Sanger sequencing. Gap-PCR was employed to validate 1 rare deletion associated with α-thalassemia (–^THAI) and 3 deletion mutations of the β-globin gene (^Gγ⁺(^Aγδβ)⁰, SEA-HPFH, and Taiwanese). All single-nucleotide variants of the α- and β-globin genes were confirmed by Sanger sequencing. The NGS results suggested high risk of suspected triplication among participants, as further confirmed by an anti-3.7/4.2 multiplex-PCR method.⁹ 

For samples that were negative after NGS, purified DNA samples were quantified and subsequently sent to an independent laboratory (Berry Genomics, Beijing, China) for comprehensive analysis of thalassemia alleles, which was performed with the PacBio Sequel II platform (Pacific Biosciences, Menlo Park, California), as previously described¹⁰  but with additional primers to further increase the coverage of deletion arrangements. Briefly, genomic DNA samples were subjected to multiplex long-molecule PCR using optimized primers to generate specific amplicons that encapsulate currently known SV regions (35 and 28 SVs of α- and β-thalassemia, respectively) as well as single-nucleotide variations (903 and 1135 mutations to the α- and β-globin genes, respectively) within the HBA1, HBA2, and HBB genes based on data from the HbVar database, Ithanet database, Leiden Open Variation Database (LOVD; https://www.lovd.nl/), and LOVD-China database (http://www.genomed.zju.edu.cn/LOVD3/genes). After purification and end repair, double bar code adapters were ligated to the 5'; and 3'; ends for construction of SMRTbell libraries with the Sequel Binding and Internal Ctrl Kit 3.0 (Pacific Biosciences). Primed DNA-polymerase complexes were loaded onto SMRT cells (Pacific Biosciences) and sequenced with the PacBio Sequel II System to generate 10 to 25 subreads per molecule. Following alignment of the subreads, the consensus circular sequence was mapped to the GRCh38 reference genome and variants using FreeBayes software (version 1.2.0; https://github.com/freebayes/freebayes). WhatsHap software (version 0.18; https://github.com/whatshap/whatshap/) was used for linkage analysis (cis or trans) by long read–based phasing.¹¹  The Integrative Genomics Viewer (https://software.broadinstitute.org/software/igv/) was used to obtain alignments of variant and wild-type molecules, whereas Gap-PCR was employed for further confirmation of large deletion variants. DNA sequencing was conducted to confirm mutations to the α- and β-globin genes.

For the samples that were negative after TGS, CNVs, with a particular focus on chromosomes 16 and 11, were identified using the CYTOSCAN 750K chip (Affymetrix Inc, Santa Clara, California) or by CNV-seq analysis.

Of 200 individuals with small cell hypochromic anemia that could not be explained by common thalassemia genes, 128 (64.0%) received a diagnosis of IDA, whereas 72 (36%) were highly suspected to carry rare HBA1, HBA2, or HBB gene variants. NGS, TGS, and CMA/CNV-seq analyses were sequentially applied for confirmation of rare thalassemia variants. Among the 72 patients, 49 (68.1%) were positive for either the α- or β-gene variant. As shown in Table 1, a total of 45 patients tested positive for rare thalassemia variants, and 4 others were positive for rare Hb variants. The detection rate for rare thalassemia based on MCV and MCH levels to rule out IDA was 68.1% (49 of 72). Among the 49 positive cases, there were 24 rare α-gene variants, 22 rare β-gene variants, 1 microdeletion of chromosome 16p13.3, 1 microdeletion of chromosome 11, and 1 β41-42/βN mutation with βN/βN mosaicism. Table 2 presents the spectrum of α-globin gene mutations, whereas Table 3 shows the spectrum of β-globin gene mutations. The hematologic data and instances of the rare α-globin genotype are shown in Table 4, whereas the hematologic data and instances of the rare β-globin genotype are presented in Table 5.

NGS and Gap-PCR analyses of all 72 individuals identified 1 rare α‐globin gene deletion (–^THAI; Figure 2, A), 3 β‐globin gene deletions (^Gγ⁺(^Aγδβ)⁰, SEA-HPFH, and Taiwanese; Figure 2, A), α-globin gene triplet (Figure 2, B), and a fusion gene (Figure 3), as well as sequencing of the HBA1, HBA2, and HBB genes. In addition, of the 72 patients, 20 (27.8%) tested positive for the rare α-gene variant and 22 (30.6%) tested positivefor the β-gene variant (Table 1).

For 30 suspected thalassemia samples with no abnormalities detected by combined NGS and Gap-PCR analyses, the genomic DNA was sent to Berry Genomics Laboratory for comprehensive analysis of thalassemia alleles based on TGS technology and data analysis. Of these 30 samples, 4 (13.3%) carried α-globin genetic deletions, as determined by TGS analysis, but not NGS. The SVs included –11.1, -α27.6, -α2.4, and -α21.9 (Figure 4).

In addition, 26 samples with no signs of abnormality after NGS and TGS were subjected to chromosome CNV analysis using the CYTOSCAN 750K chip or CNV-seq. One sample was confirmed to carry a microdeletion of chromosome 16, arr[GRCh37] 16p13.3(85 880–282 157)x1, which spanned approximately 196 kb at the 16p13.3-pter position of chromosome 16 and involved 11 genes, including HBA1 and HBA2 (Figure 5, A). Another individual carried a microdeletion of chromosome 11, arr[GRCh37] 11p15.4(5188944_5277199)x1, which involved deletion of approximately 88 kb at position 11p15.4 on chromosome 11 and involved 6 genes, including HBB (Figure 5, B).

Among the 24 samples with rare α-gene variants, 5 (20.8%) were positive for the –^THAI/αα variant and 5 (20.8%) others were positive for the fusion gene/αα. The second most common variants were α^{CD 30}α and ααα^anti4.2, with 4 carriers each. Additionally, there were 2 carriers of the Hb Hekinan II variant. Furthermore, 4 rare α-thalassemia deletion types (–^11.1, -α^27.6, -α^2.4, and -α^21.9) were identified in the Zhonshan region.

Among the 22 rare β-gene variants, ^Gγ⁺(^Aγδβ)⁰ and SEA-HPFH were the most common, with 7 carriers each, followed by the β^Taiwanese and β^{CD 37 (TGG>TAG)} variants, with 2 carriers each. Among the remaining 4 patients, 1 carried both Hb S (HBB: c.20A>T) and Hb Zengcheng (HBB: c.343C>A). Additionally, the β^IVS-II-2(-T) mutation was identified in Guangdong Province, whereas the β^{IVS-II-5(G>C)} and Hb Ube-1(HBB: c.295G>A) mutations were confirmed in the Zhonshan region.

One case was heterozygous for β^41–42 by PCR-RDB with normal hematologic and ferroprotein data (Hb, 15.3 g/dL; MCV, 82.3 μm³; MCH, 27.8 pg/cell; Hb A2, 3.0%). The initial results of NGS were β^41–42/β^N, whereas TGS showed β^N/β^N. Upon reanalysis, 1307 sequencing reads were selected for NGS, with 243 sequencing reads specifically targeting β41–42 (HBB: c.126_129delCTTT), resulting in a ratio of 18.6% (243 of 1307). For TGS, 932 sequencing reads were obtained, with 169 specifically targeting β41–42 (HBB:c.126_129delCTTT), resulting in a ratio of 18.1% (169 of 932). Sanger sequencing detected a short peak for HBB:c.126_129delCTTT and a tall peak for normal β-globin genes. The results of NGS, TGS, and Sanger sequencing were in agreement, indicating that this case is a mosaic of β^41-42/β^N and β^N/β^N.

Thalassemia intermedia and major are especially burdensome to the patient, patient’s family, and society because of the current lack of effective treatment.^12,13  In China, traditional 3-level hematologic and genetic screening for thalassemia can identify most carriers. However, conventional genetic testing methods, such as Gap-PCR and RDB, can detect only 6 common α-thalassemia mutations and 17 common β-thalassemia mutations in the Chinese population, resulting in misdiagnosis of rare thalassemia gene mutations. More than 2000 thalassemia- or hemoglobin-related variant sites have been reported, and thus an economical and effective model is needed for screening and diagnosis of rare variants. In this study, the parameters of MCV less than 82 μm³ and/or MCH less than 27 pg/cell were adopted to rule out IDA and common thalassemia. NGS is increasingly used to amplify all exons and introns of the HBA1, HBA2, and HBB genes, thereby enabling identification of most thalassemia-associated mutations in the HbVar database.^2–4  NGS has the advantages of high throughput and relatively low cost, but it is limited for the identification of rare SVs of thalassemia genes. Recently, TGS technology, also known as long-molecule sequencing, has emerged as a useful method for genetic diagnosis because of the advantages of long reads, high accuracy, single-molecule resolution, and no GC preference.¹⁴  TGS is particularly useful for detection of rare thalassemia deletion mutations, but it is relatively expensive and time-consuming, and it cannot detect chromosomal CNVs.

Screening for thalassemia is mainly performed by hematologic analysis. However, MCV and MCH are influenced by various factors, especially iron deficiency. In this study, of 200 cases of hypochromic microcytic anemia (MCV <82 μm³ and/or MCH <27 pg/cell) with normal common thalassemia genes, conventional genetic testing was insufficient to explain the clinical phenotype, and 128 cases were eventually classified as IDA. Thus, IDA was the most common cause of hypochromic microcytic anemia in this study, with an incidence of 64.0% (128 of 200). Of the 72 individuals with suspected rare thalassemia, 49 (68.1%) carried rare α- or β-gene variants. After ruling out IDA, a screening strategy for rare thalassemia based on MCV and MCH levels was implemented, resulting in a positive detection rate of 68.1% (49 pf 72). In a recent study,⁵  100 cases of suspected rare thalassemia were based on (1) routine hematologic examinations showing abnormal hemoglobin (HbA2 <2.3% or HbA2 ≥3.2%) or elevated HbF, (2) blood tests showing abnormal levels of MCV ≤80 μm³ for adults and/or MCH ≤27 pg/cell, or (3) conventional genetic testing that could not adequately explain the clinical phenotype. TGS analysis for genetic diagnosis of thalassemia identified 10 rare clinically significant variants, resulting in a detection rate of 10% (10 of 100). In another recent study,⁶  routine Gap-PCR, DNA sequencing, and TGS technology were used to identify rare thalassemia variants in 72 individuals (MCV <82 μm³ and/or MCH <27 pg/cell, and/or HbA2 >3.4% or HbA2 <2.6%, or HbF >2.0%). As a result, 29 individuals were found to harbor rare α- or β-globin gene variants, resulting in a detection rate of 40.3% (29 of 72). The detection rate of rare thalassemia in the present study was higher than in previous reports, possibly due to the exclusion of IDA and the use of multiple assays, including TGS. In this study, only MCV and MCH were used as indicators for screening of rare thalassemia variants, without HbA2 and HbF measurements, which could also explain the higher detection rate. Nonetheless, the combination of MCV and MCH levels was the most valuable indicator for screening of thalassemia.

In this study, four rare deletions of HBA (–^11.1, -α^2.4, -α^27.6, and -α^21.9) were identified via TGS analysis, because conventional methods and NGS are insufficient, which increased the variant detection rate by 5.6% (4 of 72). Although all 4 deletions have been previously reported,^15–17  this is the first report of each in the Zhongshan region. A rare 11.1-kb deletion involving the HBA2 and HBA1 genes (–^11.1) led to a type of α0-thalassemia. The 2.4-kb deletion of the α-globin gene cluster (-α^2.4) is an α⁺-thalassemia allele, which results from a deletion spanning from nucleotide position 36 860 to 39 251 of the α-globin gene. This genotype contains a complete α₂ fragment. The -α^27.6 and -α^21.9 genotype heterozygotes are the α⁺ genotype, because each has a complete α₁ fragment, which allows for the propositus to maintain an active α₁ genotype. In this study, the combination of -α^27.6 and –^SEA resulted in Hb H disease, with a common genotype of –^SEA/–^SEA identified during routine analysis. This finding is consistent with that observed for −α^21.9 combined with –^SEA. Therefore, TGS is recommended for the detection of rare deletions when routine analysis shows common α-thal genotypes of –^SEA/–^SEA but phenotypic analysis indicates Hb H disease.

TGS, or long-molecule sequencing, is particularly useful for the detection of rare SVs of thalassemia genes, but it cannot detect chromosomal CNVs. In this study, 2 samples with a microdeletion of chromosome 16 or 11 were detected by CMA, which increased the detection rate of rare thalassemia by 2.8% (2 of 72). So, for confirmation of suspected rare thalassemia variants, chromosomal CNV should also be considered in addition to identification of deletions and point mutations of thalassemia-related genes.

For a case that was heterozygous for β^41–42 by PCR-RDB with normal hematologic data, the initial results of NGS confirmed β^41–42/β^N, whereas TGS showed β^N/β^N. Upon reanalysis, both NGS and TGS suggested a mosaic pattern of β^41–42/β^N and β^N/β^N, demonstrating that a combination of multiple methods is required for detection of rare thalassemia variants, because NGS or TGS alone is insufficient. Also, the genetic results must be analyzed in conjunction with hematologic phenotypes.

Four cases of α-globin gene triplication (ααα^anti4.2) were identified by NGS in this study and confirmed by specific PCR and agarose gel electrophoresis.⁷  It is evident that α-globin gene triplication is not uncommon, as evidenced by 2 carriers with heterozygous α-globin gene triplication exhibiting slightly lower MCV or MCH. One patient with coinheritance of ααα^anti4.2 and –^SEA had only slightly lower MCV and MCH values, indicating a phenotype similar to silent α-thalassemia. Another patient with coinheritance of ααα^anti4.2 and -α^3.7 had normal hematologic results. The tristrain of the α-globin gene is known to exacerbate symptoms of anemia and cause β-thalassemia intermedia when combined with β-thalassemia. When 1 member of a couple has β-thalassemia, it is important to consider whether the other carries α-globin gene triplication. Additionally, if the blood test results do not align with typical α thalassemia gene patterns, the presence of α-globin gene triplication should be suspected. Notably, the presence of α-globin gene triplication may explain a milder hematologic phenotype, which cannot be attributed to common α thalassemia. In this study, 4 cases of α-globin gene triplication were identified as ααα^anti4.2, whereas there was no case of ααα^anti3.7, which may be due to the presence of an additional complete α₂ gene in ααα^anti4.2, whereas an additional fusion gene would comprise a partial segment of both the α₁ and α₂ genes in ααα^anti3.7. Although it is possible that the function of an additional α₂ gene in ααα^anti4.2 is more potent than that of the fusion gene in ααα^anti3.7, further confirmation is required.

The hematologic phenotypes of cases 11, 12, and 13 resembled Hb H disease, which is characterized by significant reductions in MCV, MCH, and Hb levels, as well as abnormal hemoglobin (Hb Bart and Hb H). Despite screening for common thalassemia deletions or mutations, only the heterozygous Southeast Asian deletion (–^SEA/αα) was identified, which may not fully account for the observed abnormal hematologic phenotype and Hb electrophoresis. NGS analysis detected the presence of α₂ codon 30 (−GAG), which was subsequently confirmed by DNA sequencing. Some researchers have suggested that children with the α^{CD 30}α/–^SEA phenotype exhibit more severe clinical symptoms, and the α₂ codon 30 (−GAG) mutation should be considered a severe form of the α⁺-thal mutation, similar to the α^QSα mutation,¹⁸  which differs from the mild α-thal Hb variant.¹⁹  Three patients in this study exhibited coinheritance of –^SEA and the α₂ codon 30 (−GAG), which resulted in nondeletional Hb H disease with varying clinical phenotypes. Among them, 1 patient experienced severe symptoms and required blood transfusion, whereas the other 2 presented with mild to moderate anemia. Accordingly, although phenotypic analysis indicates Hb H disease, routine analysis can only detect common genotypes of –^SEA/αα. Therefore, NGS should be included for the detection of rare point mutations. If the fetus inherits both gene defects from parents with the α^CD30α/–^SEA genotype, the parents should receive counseling to clarify the potential clinical outcomes associated with nondeletional Hb H disease, which can range from mild to severe anemia requiring periodic transfusions.

Patient 44 had the β^{CD 17}/β^IVS-II-5 phenotype associated with severe transfusion-dependent β-thalassemia. Prenatal diagnosis was insufficient for this patient because her mother carried the β^IVS-II-5 mutation but had a normal blood phenotype (Hb, 12.8 g/dL; MCV, 88.3 μm³; MCH, 27.1 pg/cell), demonstrating challenges in the detection of the β^IVS-II-5 mutation.

Patient 46 had normal results for common testing of thalassemia-related genes but presented with pancytopenia (red blood cell count, 3.9 × 10⁶/μL; Hb, 9.7 g/dL; white blood cell count, 2.68 × 10³/μL; platelet count, 92 × 10³/μL) with suspected leukemia, aplastic anemia, or hemophilic syndrome, although further tests were inconclusive. Meanwhile, oxygen saturation (SpO₂) was low on pulse oximetry, causing great concern. A pedigree investigation revealed that the mother also had thrombocytopenia, leading to suspicion of a rare hereditary hemoglobinopathy. NGS testing revealed the presence of Hb Ube-1 (HBB: c.295G>A), which is an unstable hemoglobin variant that is prevalent in Europe and the United States but rare in China. Hence, an unstable hemoglobin variant, such as Hb Ube-1, should be suspected for patients presenting with pancytopenia, in addition to leukemia, aplastic anemia, or hemophilic syndrome.

Previous reports of fusion genes are limited, particularly regarding hematologic phenotypes. In this study, the fusion gene was the most prevalent rare α-globin gene variant (20%; 5 of 25 cases). It is evident that fusion genes are not uncommon, but rather often undetected. NGS analysis revealed a mutation resulting from the fusion of the α₂ and ψα₁ genes. The recombination was a cross at exon 3 of the α₂ gene with exon 3 of the ψα₁ gene. In this study, all adults who were heterozygous for the fusion gene exhibited a mild hypochromic and microcytic red cell phenotype, whereas hemoglobin and HbA2 levels were normal. The hematologic phenotype was similar to that of silent α-thalassemia. Huang et al²⁰  identified a patient with classic Hb H disease and compound heterozygosity for the –^SEA deletion and fusion gene.

The β^IVS-II-2 mutation was initially reported in Guangdong Province, China. In 2000, Ma et al²¹  first described this variant in the Chinese population in Hong Kong. This study identified anther individual who carried the rare β-gene variant, which is the second report in the Chinese population. Two mutations to the β-gene, β^IVS-II-5 (HBB: c.315 + 5G>C) and Hb Ube-1 (HBB: c.295G>A), were initially discovered in the Zhonshan region, in addition to 4 rare deletions of HBA (–^11.1, -α^27.6, -α^2.4, and -α^21.9).

Thalassemia variants are not necessarily rare, but rather often misdiagnosed using conventional methods. Despite the introduction of TGS, there is no available method to detect all types of thalassemia, so it is especially important to be aware of the limitations of testing methods. A comprehensive approach combining hematologic and genetic testing is necessary for accurate screening and diagnosis. If an abnormal blood test result is detected, the possibility of IDA should be ruled out first. Once small cell low pigment caused by IDA has been excluded, various methods must be employed to identify the underlying causes and prevent misdiagnosis, while providing guidance for fertility. The screening and diagnosis of thalassemia require comprehensive hematologic and genetic testing to fully understand the hematologic phenotypes of all rare cases. It is important for doctors to possess a solid understanding of the basic theory and clinical knowledge of thalassemia in order to improve patient outcomes and enhance consultation skills.