Accurate genotype diagnosis of Hong Kongαα thalassemia based on third-generation sequencing

Introduction

Thalassemia is one of the most common genetic diseases worldwide, characterized by a variable degree of chronic progressive hemolytic anemia (1). According to hemoglobin chain damage, thalassemia can be categorized into α-, β-, δ-, γ-, δβ-, and εγδβ-thalassemias (2). Thalassemia is highly prevalent in the Mediterranean, central Asia, Middle East, India, and southern China (3). Of all thalassemia types, α-thalassemia is the most widely distributed and is common in Southeast Asia and China (2,4,5). This thalassemia type is caused by disturbances in α-globin chain synthesis. The gene for synthesizing α-like globin peptides is located on 16p13.3. Normally, with 2 α-globin genes on each chromosome, an individual would have 2 pairs of α-globin genes, defined as αα/αα. In the α-globin gene cluster, HBA1 and HBA2 have a high degree of homology (homologous X, Y, Z regions), which can lead to unequal crossover.

The most common type of α-thalassemia is caused by deletions of different lengths in the α-globin genes, with -α^3.7 and -α^4.2 variants being the more common forms (5,6). The reciprocal recombination between Z segments during meiosis leads to the -α^3.7 and ααα^{anti 3.7} chromosomes. The crossover between impaired X-boxes leads to -α^4.2 and ααα^{anti 4.2} chromosomes (4). On this basis, further misalignment and unequal crossover of homologous regions in α-globin gene clusters may generate other special crossover events. Wang et al. (7) first reported a complex structural rearrangement containing both -α^3.7 and ααα^{anti 4.2} crossover junctions on the same chromosome, designated as Hong Kongαα (HKαα). The HKαα allele contains neither a single gene deletion nor triplication. Several studies have reported that individuals with the HKαα allele present with normal hematological phenotype (8-10). Individuals with compound heterozygote of HKαα and --^SEA showed a typical α-thalassemia trait (11). Although the HKαα allele does not have any deleterious effect on the clinical phenotype of carriers, genetic testing of the HKαα allele has important implications for genetic counseling and prenatal diagnosis. Because routine molecular testing reagents for deletional α-thalassemia cannot directly detect ααα^{anti 4.2} and ααα^{anti 3.7}, it is easy to misjudge HKαα/αα or anti-HKαα/αα as -α^3.7/αα or -α^4.2/αα. Such misjudgments may lead to incorrect genetic counseling by clinical geneticists and may lead to incorrect prenatal diagnoses for patients.

In recent years, researchers have developed several molecular detection methods to detect HKαα thalassemia. Wang et al. (7) first developed a two-round nested PCR strategy to detect the presence of the novel HKαα allele. Long et al. (12) designed a qPCR system to screen for ααα^{anti 3.7}, ααα^{anti 4.2}, and HKαα genes. Huang et al. (13) also proposed that PCR-based multicolor melting curve analysis could identify HKαα-derived genotypes. However, these methods have certain limitations.

In clinical practice, the use of conventional thalassemia gene analysis methods to detect the HKαα allele presents certain challenges. In the α-globin gene cluster, HBA1 and HBA2 are highly homologous, and the fragment of the homologous region is relatively long, thus the sequence detection of highly homologous regions of α-globin genes remains a major problem for short-read technology. Long reads can resolve tandem repeats and complex structural rearrangements (14). Long-molecule sequencing (LMS) is the dominant feature of third-generation sequencing (TGS). Studies have proven that the LMS-based PacBio TGS platform is an accurate test with potential clinical utility as an alternative for screening thalassemia carriers (15,16). In this study, TGS based on single-molecule real-time (SMRT) technology was performed to detect the HKαα allele, and this was compared with the other molecular techniques. We present the following article in accordance with the MDAR reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-4309/rc).

Methods

Patients

Between January 2015 and June 2021, 32 individuals suspected of having HKαα thalassemia at the Affiliated Hospital of Guilin Medical University (Guilin, People’s Republic of China) were included in the study. Fresh peripheral blood samples were collected and then stored at 4 ℃. The basic information, routine hematology examination, and hemoglobin electrophoresis of all patients were collected from medical records. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of the Affiliated Hospital of Guilin Medical University (No. 2020GZRLL-46) and informed consent was obtained from all individual participants.

DNA extraction

Genomic DNA was extracted using the Nucleic Acid Extraction System (Zeesan Biotech, Xiamen, China). DNA concentration and quality were determined by the NanoDrop 2000 spectrophotometer. DNA concentration ranged from 40 to 60 ng/µL, and the A260/A280 ranged from 1.80 to 1.88. DNA was stored at −20 ℃ in a freezer until use.

Gap-PCR

Gap-PCR and agarose gel electrophoresis for -α^3.7, -α^4.2, --^SEA, and --^THAI were performed using a commercial deletional α-thalassemia gene detection kit (Yishengtang Biological Products Co. Ltd., Shenzhen, China). The two-round nested PCR was performed to determine the presence of the HKαα allele as our previously described methods (10).

PCR-reverse dot blot (RDB) assay

PCR-RDB analysis was used to detect 3 common point mutations in α-thalassemia (α^CSα, α^QSα, and α^WSα) and 17-point mutations in β-thalassemia using a commercial kit (Yaneng BioSciences Co., Shenzhen, China).

Multiplex ligation-dependent probe amplification (MLPA) assay

MLPA was used to detect copy number variations of α-globin genes. The MLPA-PCR reaction was performed in a 50 µL reaction system with the SALSA MLPA Probemix P140-C1 kit (MRC-Holland, Amsterdam, Netherlands). In short, the thermocycler program for the MLPA reaction was as follows: (I) DNA denaturation; (II) hybridization reaction; (III) ligation reaction; and (IV) PCR reaction. The PCR products were analyzed using the 3500Dx Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

TGS and data analysis

The long-read sequencing-based comprehensive analysis of thalassemia alleles (CATSA) assay was carried out similarly as previously described (16). In brief, genomic DNA was extracted and subjected to multiplex long-range PCR with primers covering the full length of the HBA and HBB genes and the majority of known structural variations (SVs). End-repair and ligation reactions were added to PCR amplicons with unique PacBio barcoded adaptors, and the exonucleases were added to digest failed ligation products to obtain an individual prelibrary for each sample. After purification and quantification, the individual prelibrary was pooled by equal mass and converted to an SMRT dumbbell library with the Sequel Binding and Internal Ctrl Kit 3.0 (Pacific Biosciences, Menlo Park, CA) and sequenced on the Sequel II platform (Pacific Biosciences) for 30 h. High fidelity circular consensus sequencing (CCS) reads were generated from raw subreads, demultiplexed by barcodes, and aligned to genome build hg38 in the SMRT Link system (Pacific Biosciences). Single-nucleotide variations (SNVs) and indels were called by FreeBayes1.3.4 (Biomatters, Inc., San Diego, CA), and SVs were called according to read length. SNVs, indels, and SVs were annotated according to HbVar, Ithanet, and LOVD databases. CCS reads generated by CATSA were displayed in the Integrative Genomics Viewer (IGV) to show different variants.

Results

HKαα allele detection by gap-PCR and two-round nested PCR

By using the deletional α-thalassemia gene detection kit, the 32 samples were detected by the gap-PCR method. The test results showed that individuals with -α^3.7/αα, HKαα/αα, and HKαα/-α^3.7 were positive for the -α^3.7 allele and the normal allele (Figure 1). The electrophoresis bands of HKαα/αα and HKαα/-α^3.7 were similar to -α^3.7/αα. However, for HKαα/αα, the electrophoresis band of the -α^3.7 allele was significantly weaker than that of the normal allele. Individuals with HKαα/-α^4.2 were positive for the -α^3.7 allele, -α^4.2 allele, and the normal allele, while individuals with HKαα/--^SEA were positive for the -α^3.7 allele, --^SEA allele, and the normal allele (Figure 1). Two-round nested PCR was performed to confirm the HKαα allele, which was published by our research group in previous papers (10).

Figure 1 Molecular detection of HKαα alleles by gap-PCR. HKαα, Hong Kongαα; PCR, polymerase chain reaction.

HKαα allele detection by the MLPA assay

The MLPA assay can be used to detect the deletion/duplication of large fragments in the HBA gene region. The copy number of α-globin genes in individuals with the HKαα allele was detected using MLPA (Figure 2). For the HKαα/αα genotype, the probe signals in the -α^3.7 region were decreased (ratio 0.5), while the probe signals in the -α^4.2 region were increased (ratio 1.5) (Figure 2A). For the HKαα/-α^3.7 genotype, the probe signals in the -α^3.7 region were completely absent (ratio 0), while the probe signals in the -α^4.2 region were increased (ratio 1.5) (Figure 2B). In samples with HKαα/-α^4.2, only the probe signals in the -α^3.7 region were reduced, which would cause the sample to be misdiagnosed as -α^3.7/αα (Figure 2C). In samples with HKαα/--^SEA, the signal ratio of the probe in the HBA gene region was the result of the sum of -α^3.7, --^SEA, and ααα^{anti 4.2} (Figure 2D). It should be noted that a single MLPA test is not sufficient to identify the HKαα allele.

Figure 2 Molecular detection of HKαα alleles by MLPA. (A) HKαα/αα. (B) HKαα/-α^3.7. (C) HKαα/-α^4.2. (D) HKαα/--^SEA. The x-axis represents MLPA probe location (hg18). The y-axis represents the final ratio. HKαα, Hong Kongαα; MLPA, multiplex ligation-dependent probe amplification.

HKαα allele detection by TGS

TGS based on long-read SMRT technology was used to detect the complex structural rearrangements of α-globin genes. The results of TGS technology for individuals with the HKαα allele are shown in Figure 3. Recombinant SVs involving -α^3.7, -α^4.2, --^SEA, and HKαα variants on 2 DNA strands were well identified. The TGS technology could directly detect the HKαα allele and correctly distinguish various types of genotypes. In addition, 2 rare variants in HBB [-50 (G>A) and -198A>G] were discovered in 2 HKαα samples by the TGS assay.

Figure 3 Molecular detection of HKαα alleles by TGS. (A) HKαα/αα. (B) HKαα/-α^3.7. (C) HKαα/-α^4.2. (D) HKαα/--^SEA. HKαα, Hong Kongαα; TGS, third-generation sequencing.

In this study, the contiguous sequence of the HKαα allele was detected using long reads of TGS technology. Sequence data are provided in the Appendix 1.

Hematological characteristics of individuals with the HKαα allele

The hematological characteristics of individuals with the HKαα allele are shown in Table 1. Most individuals with HKαα/αα and β^N/β^N genotypes had normal hematological phenotypes. Only 2 of 18 individuals with HKαα/αα and β^N/β^N genotypes manifested obvious microcytic hypochromic anemia, which might be related to the combination of iron deficiency. Individuals with HKαα/-α^4.2 or HKαα/α^WSα genotypes showed silent α-thalassemia, while individuals with the HKαα/--^SEA genotype showed mild α-thalassemia. HKαα/αα and HKαα/-α^3.7 combined with the HBB variant displayed obvious hematological features of β-thalassemia (Table 1).

Discussion

HKαα is a rare complex crossover event of the a-globin gene cluster, containing a cis-recombination of -α^3.7 and ααα^{anti 4.2}. Research has reported that the carrier rates of the HKαα allele ranged from 0.04% to 0.33% in southern China (8,12,17-20). Furthermore, the frequency of HKαα ranged from 2.27% to 8.81% in -α^3.7 carriers (9,20,21). The carrying rate of HKαα is significantly different in different regions, which might be related to different detection methods and ancestral effects of population distribution. HKαα is a rearrangement of the a-globin gene. As shown in Figure 3, Integrative Genomics Viewer plots of HKαα allele reveal that there is virtually no deletion or duplication of the gene. Therefore, individuals with HKαα/αα presented a normal hematological phenotype, and this structural variant does not aggravate the clinical phenotype of patients even when combined with other α/β-mutations, which is consistent with previously reported results (7,8,11,19,21). Although the carrier rate of the HKαα allele is low and the carriers do not have any clinical manifestations, its molecular diagnosis has important clinical significance.

At present, DNA testing is a regular part of the management of thalassemia. In clinical practice, the most common DNA testing methods for thalassemia are gap-PCR and PCR-RDB analysis with hot-spot mutation detection kits. The gap-PCR-based deletional α-thalassemia gene detection kit used in clinical practice cannot detect triplication, which will result in missed diagnosis or misdiagnosis of the HKαα allele. In this study, the results of gap-PCR showed that electrophoresis bands of HKαα/αα and HKαα/-α^3.7 were the same as -α^3.7/αα, resulting in both HKαα/αα and HKαα/-α^3.7 being misdiagnosed as -α^3.7/αα. Such misjudgments can lead clinicians to provide incorrect genetic counseling and prenatal diagnosis. Furthermore, α⁰-thalassemia coinherited with -α^3.7 (--/-α^3.7) can result in hemoglobin H (HbH) disease, while α⁰-thalassemia coinherited with the HKαα allele (--/HKαα) presents as mild α-thalassemia.

The MLPA technique is also a common clinical method for detecting copy number changes when rare variations in the α-globin gene are suspected. Most of the deletion subtypes of α-thalassemia can be detected by MLPA, including -α^3.7, -α^4.2, --^SEA, --^THAI, --^FIL, and (-α^20.5), among others. The α-triplication (ααα^{anti 3.7} and ααα^{anti 4.2}) can also be detected by MLPA with the P140 Probemix. However, copy number variations of the HBA gene detected by MLPA should be confirmed using a different technique. As shown in Figure 2A, both -α^3.7 and ααα^{anti 4.2} were detected in a single sample, but it was impossible to determine whether the genotype was an HKαα/αα or ααα^{anti 4.2}/-α^3.7 compound heterozygous mutation. Additional experiments or pedigree analysis are required to determine the correct genotype, as genetic counseling for HKαα/αα and ααα^{anti 4.2}/-α^3.7 compound heterozygous mutation is completely different. In the case of ααα^{anti 4.2}/-α^3.7 compound heterozygosity, inheritance to the offspring could only be ααα^{anti 4.2} or -α^3.7. In addition to compound heterozygosity of -α^3.7 and α⁰ mutations leading to HbH disease, α-triplication might exacerbate the proportional imbalance of α and β chains and produce more severe phenotypes of β-thalassemia when α-triplication is coinherited with HBB variants (12,22). In the case of HKαα/αα, it would be passed to the offspring as the αα allele or HKαα allele. Genetic modification of the HKαα allele to other thalassemia subtypes was not obvious. In this study, MLPA analysis showed no net copy number change in the 4.2 region for the HKαα/-α^4.2 genotype due to a -α^4.2 deletion on one chromosome and a similarly sized ααα^{anti 4.2} duplication on the other chromosome, which might result in a false result (Figure 2C). Therefore, it is difficult to identify the HKαα allele using the MLPA technique.

Two-round nested PCR is a classical method to detect the HKαα allele (7). However, HKαα/αα, HKαα/HKαα, HKαα/-α^3.7, and HKαα/ααα^{anti 4.2} cannot be distinguished by two-round nested PCR. Chen et al. (21) suggested that the combination of gap-PCR, MLPA, and nested PCR could be used to detect HKαα. MLPA and the two-round nested PCR method can verify each other and help differentiate HKαα/αα, HKαα/HKαα, HKαα/-α^3.7, HKαα/-α^4.2, HKαα/HKαα, HKαα/ααα^{anti 3.7}, and HKαα/ααα^{anti 4.2}. However, this combination involves multiple sets of experiments, and the process is cumbersome and time-consuming, making it difficult to achieve large-scale population screening in clinical practice.

Recently, TGS technology based on LMS has become an efficient, cost-effective, and accurate approach for comprehensive thalassemia carrier screening (15,16). Using long-read single-molecule sequencing technology, TGS can accurately distinguish the highly homologous sequences of the HBA1 and HBA2 genes, and correctly discern the real genetic carrier status of complex structural variants and homologous recombination. For samples with compound heterozygotes, TGS can directly determine whether the genotypes are in cis or in trans without pedigree analysis. In this study, the subtypes of α-thalassemia with the HKαα allele were correctly detected by the TGS approach, including HKαα/αα, HKαα/-α^3.7, HKαα/-α^4.2, and HKαα/--^SEA. In addition, a recent study also showed that more complex α-globin gene cluster variants, such as αα^anti3.7α^anti3.7α^17.2/αα and --^SEA/α^Westmeadα^{anti3.7+Westmead}α, were identified by TGS testing (23). Therefore, long-read TGS can greatly benefit resolving complex structural rearrangements and help improve the accuracy of genetic testing.

The HKαα allele was confirmed by Sanger sequencing in our previous research (10). As described by our research group (10), the HKαα allele sequence is divided into 3 regions: -α^3.7, ααα^{anti 4.2}, and a special region about 802 base pairs long. Interestingly, in the special 802 base pairs region, the upstream area is identical to the sequence of the α-globin gene ranging from 171,116 to 171,235, the downstream area is the same as that on the α-globin gene and ranges from 171,223 to 171,882, but the part that crosses them is the undetected sequence (NCBI Reference Sequence: Ng_0000016.10), which might be caused by the shortcomings of short reads. The PacBio long-read sequencing approach can overcome the limitations (14). In this study, the contiguous sequence of the HKαα allele was detected using long reads of TGS technology. Moreover, we discovered that the sequence not detected in the previous study contained 17 T, which shared the same sequence with the α-globin gene ranging from 171,219-171,235 (hg38). The sequence was lost in the splicing, so that it could not be detected by Sanger sequencing. By using long-read contiguous sequences, TGS technology greatly makes up for the limitation of short-read sequencing and provides full-length coverage of thalassemia alleles, including HBA and HBB genes. Surprisingly, 2 rare variants in HBB [-50 (G>A) and -198A>G] were discovered when HKαα samples were subjected to the TGS assay. The mutation of -50 (G>A) (HBB: c.-100G>A) was first reported by Li et al. in 2009 (24). Consistent with previous studies (25,26), our result suggested that individuals with the -50 (G>A) heterozygous variant had a normal hematological phenotype. Unexpectedly, our study showed that an individual with the -198 (A>G) (HBB: c.-248A>G) mutation exhibited microcytic hypochromic anemia, which was inconsistent with a previously reported result that the -198 (A>G) heterozygous variant has a normal hematological phenotype with decreased HbA2 level (26). The reason was that the individual in our study had concomitant iron deficiency anemia with a serum ferritin of 9.73 ng/mL. In short, long-read TGS technology can identify both common and rare mutations in the HBA and HBB genes, thus improving the detection rate of rare thalassemia.

With sequencing of long reads, the biggest advantage of TGS is to directly identify the junction site of complex structural variants like HKαα, α-globin gene triplications and even quadruplications. The accuracy of PacBio sequencing was guaranteed by two levels. First, the PacBio platform utilized a hairpin library structure which enables it to sequence the same insert (subread) for multiple passes to generate high fidelity CCS reads. Second, a minimum 60× coverage depth of α-globin and β-globin genes was sequenced, which further proofed any sequencing errors in CCS reads. To reduce the cost per test, hundreds of barcoded samples need to be pooled together for sequencing in one flowcell, which might hinder the clinical application of TGS in small centers with limited number of samples. Pooling samples with different TGS-based tests like congenital adrenal hyperplasia, spinal muscular atrophy and fragile-X syndrome could be a solution. In addition, TGS cannot detect all the deletional thalassemia, so some difficult cases require the combination of clinical phenotype and multiple detection techniques to make a correct diagnosis.

Besides the methods mentioned above, researchers have reported that qPCR system and multicolor melting curve analysis can be used to detect the HKαα thalassemia (12,13). However, they cannot directly determine whether the genotype is in cis or in trans, and further experiments are needed to verify. Next-generation sequencing (NGS) is an effective means of detecting rare thalassemia, but it is difficult to detect HKαα due to the limitation of short-read length.

In conclusion, long-read TGS technology has significant advantages in detecting the HKαα allele, and can accurately distinguish various subtypes of HKαα thalassemia, thus achieving accurate detection. The TGS method can help to comprehensively improve the detection level of thalassemia mutant genes in the population, and will be beneficial for clinical geneticists in carrying out genetic counseling for patients and making correct prenatal diagnoses.

Acknowledgments

We thank Aiping Mao, Jiaqi Li, and Tiantian Xie from Berry Genomics Corporation for their assistance with TGS technology.

Funding: This study was supported by the National Natural Science Foundation of China (No. 82060037), the Natural Science Foundation of Guangxi Province (No. 2018JJA140062), and Guangxi Ba Gui Scholars Special Project.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-4309/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-4309/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-4309/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of the Affiliated Hospital of Guilin Medical University (No. 2020GZRLL-46) and informed consent was obtained from all individual participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: C. Betlazar-Maseh)