Context.—

Thalassemia is the most widely distributed monogenic autosomal recessive disorder in the world. Accurate genetic analysis of thalassemia is crucial for thalassemia prevention.

Objective.—

To compare the clinical utility of a third-generation sequencing–based approach termed comprehensive analysis of thalassemia alleles with routine polymerase chain reaction (PCR) in genetic analysis of thalassemia and explore the molecular spectrum of thalassemia in Hunan Province.

Design.—

Subjects in Hunan Province were recruited, and hematologic testing was performed. Five hundred four subjects positive on hemoglobin testing were then used as the cohort, and third-generation sequencing and routine PCR were used for genetic analysis.

Results.—

Of the 504 subjects, 462 (91.67%) had the same results, whereas 42 (8.33%) exhibited discordant results between the 2 methods. Sanger sequencing and PCR testing confirmed the results of third-generation sequencing. In total, third-generation sequencing correctly detected 247 subjects with variants, whereas PCR identified 205, which showed an increase in detection of 20.49%. Moreover, α triplications were identified in 1.98% (10 of 504) hemoglobin testing–positive subjects in Hunan Province. Seven hemoglobin variants with potential pathogenicity were detected in 9 hemoglobin testing–positive subjects.

Conclusions.—

Third-generation sequencing is a more comprehensive, reliable, and efficient approach for genetic analysis of thalassemia than PCR, and allowed for a characterization of the thalassemia spectrum in Hunan Province.

Thalassemia is an inherited disorder characterized by defects in globin synthesis. It is the most widely distributed monogenic autosomal recessive disorder in the world.13  In China, thalassemia is most frequent in southern provinces such as Guangdong, Guangxi, and Hainan.47  It is mainly classified into α-thalassemia, β-thalassemia, and α+β-thalassemia. At the molecular level, depending on whether one or both of the α-globin genes are deleted or reduced in activity, α-thalassemia variants can be categorized as α+ and α0. β-globin gene variants range from mild variants that cause a partial reduction in β-globin synthesis (β+) to severe variants that lead to no β-globin synthesis (β0).8 

The genotypes of thalassemia are region specific. In China, the 25 most common variants in HBA and HBB genes are routinely screened by polymerase chain reaction (PCR) in diagnostic laboratories of thalassemia.9  Recently, increasing detection of rare thalassemia variants such as α duplications, HKαα, and large deletions has suggested the inclusion of these relatively rare variants in genetic testing.10,11  For example, the incidence of α duplications in Sri Lanka12  is 2%, and in the Yunnan, Hainan, Guangxi, Guizhou, and Guangdong provinces of southern China, the incidences are 2.25%, 0.92%, 1.28%, 1.79%, and 1.99%, respectively.13,14  Currently reported duplications of an α-globin gene include αααanti3.7, αααanti4.2, αααα282, and αααα204, among which αααanti3.7 and αααanti4.2 are the most common in the Chinese population. The reported α-globin duplications in Guangxi and Guizhou provinces include αααanti3.7 and αααanti4.2, and Guangdong, Hainan, and Yunnan provinces additionally identified αααα121.2, αααα20.9, and αααα69.4, respectively.13 

Phenotypes of thalassemia range from asymptomatic to severe anemia, with disease severity well correlated with the imbalance of α/β-globins.15  Patients with α-thalassemia major (α00) experience in utero or early postnatal death, and patients with α-thalassemia intermedia (α0+) may develop transfusion-dependent anemia. β-thalassemia major and some of the β-thalassemia intermedia patients (β++, β0+, or β00) require frequent blood transfusions.8  In addition, triplications of α-globin genes can aggravate the symptoms of β-thalassemia because of the increased imbalance of α/β-globin chains. Thus, α-globin triplications combined with heterozygous β0 or β+ can cause relatively severe symptoms, such as hepatosplenomegaly and a need for blood transfusions, and lead to thalassemia intermedia.16,17 

There is no effective way to cure thalassemia. Therefore, it is extremely important to conduct carrier screening for thalassemia prevention.18  The detection of the thalassemia variants is included in premarital examination in areas endemic for thalassemia in China.14  Screening begins with hemoglobin (Hb) testing, followed by PCR for one of the HBA or HBB genes. This screening fails to detect all cases. One of the reasons is that genetic diagnostics for thalassemia is particularly challenging because of the complex structures of globin gene clusters. The routine PCR technique possesses several limitations, as it detects only common variants, and thus rare, complex variants are missed. In addition, it cannot determine whether 2 or more variants are in cis or trans configuration.1921 

Recently, next-generation sequencing (NGS) and third-generation sequencing (TGS) have been emerging as new techniques in genetic diagnostics for thalassemia.13,2225  In 2017, carrier screening of thalassemia conducted by NGS was first reported in China.20  Although it is high throughput and can detect both known and unknown variants, the short read lengths of NGS make it inaccurate in thalassemia diagnostics because of highly homologous regions and repeat regions in Hb genes. Compared with NGS, TGS has the advantage of long reads, and there is no amplification during the sequencing process, which results in minimum GC bias. In addition, it can identify both known and unknown variants and determine whether 2 or more variants are in cis or trans configurations.26  In 2021, Liang et al25  assessed the clinical feasibility of a TGS-based approach termed comprehensive analysis of thalassemia alleles (CATSA) for carrier screening of thalassemia.

In this study, CATSA and routine PCR were simultaneously conducted in the genetic analysis of thalassemia among subjects of Hunan Province and the efficacy of the 2 methods was compared. The incidence of α-globin duplications and other common and rare variants in Hunan Province was explored.

Study Participants

Subjects from Hunan Province were recruited at Changsha Hospital for Maternal & Child Health Care Affiliated to Hunan Normal University (Changsha, China). Hb testing was conducted for these subjects. CATSA and routine PCR were simultaneously performed for 504 Hb testing–positive subjects. Ethics approval was given by the institutional review board (No. 2021097). All subjects provided informed written consent.

Hb Testing

Hb testing was carried out by standard blood assays and Hb electrophoresis. Standard blood assays were conducted with an automated cell counter (Sysmex XN-1000, Sysmex Co, Ltd, Kobe, Japan). Blood indexes included mean corpuscular volume, mean corpuscular Hb, and Hb. An Hb electrophoresis system (Capillarys 2 Flex Piercing, Sebia, Evry Cedex, France) was used to analyze the Hb components, which included HbA2 and HbF. Normal ranges were mean corpuscular volume 80 fL or higher, mean corpuscular Hb 27 pg or higher, HbA2 levels between 2.5% and 3.5%, and HbF 5% or lower.

Genetic Analysis by Routine PCR

PCR was performed to test α-thalassemia variants including –SEA (Southeast Asia), -α3.7 (rightward), -α4.2 (leftward), HBA2:c.427T>C, HBA2:c.377T>C, and HBA2:c.369C>G and β-thalassemia variants including HBB:c.126_129delCTTT, HBB:c.130G>T, HBB:c.316-197C>T, HBB:c.52A>T, HBB:c.45_46insG, HBB:c.-76A>G, HBB:c.-79A>G, HBB:c.216_217insA, HBB:c.79G>A, HBB:c.92+1G>T, HBB:c.92+1G>A, HBB:c.84_85insC, HBB:c.92+5G>C, HBB:c.-11_-8delAAAC, HBB:c.-50A>C, HBB:c.2T>G, HBB:c.94delC, HBB:c.-80T>C, and HBB:c.-82C>A. The experiment was performed according to the manufacturer’s protocol (Kaipu Bioscience, Chaozhou, China).

Genetic Analysis by CATSA

CATSA was performed at Berry Genomics as described previously.24  Briefly, multiplex PCR was conducted to amplify the genomic regions covering the majority of known structural variations, single-nucleotide variants (SNVs), and indels for the HBA1, HBA2, and HBB genes. Bar-coded adaptors were ligated to the PCR products by a 1-step end-repair and ligation reaction. Unligated products were removed by exonucleases (Enzymatics), and libraries were pooled together by equal mass. The pooled library was converted to SMRT bell library by Sequel Binding and Internal Ctrl Kit 3.0 (Pacific Biosciences) and sequenced with Sequel II Sequencing Kit 2.0 using the CCS mode on the Sequel II platform (Pacific Biosciences). The raw reads were converted to CCS reads by CCS software (Pacific Biosciences) and demultiplexed by bar codes using lima in the Pbbioconda package (Pacific Biosciences). Then, the processed reads were then aligned to genome build hg38 with pbmn2. FreeBayes1.3.4 (https://www.geneious.com/plugins/freebayes; Biomatters Inc, San Diego, California) was used to call SNVs, and indels and structural variations were identified based on HbVar, Ithanet, and LOVD databases.

Confirmation of Discordant Variants

Discordant structural variants identified by CATSA but missed by routine PCR were verified by specially designed PCR. Discordant SNVs and indels between CATSA and routine PCR were validated by Sanger sequencing.

Comparison Between CATSA and Routine PCR

Subjects in Hunan Province were recruited and hematologic testing was performed. CATSA and routine PCR were conducted for 504 Hb testing–positive subjects side by side (Figure 1; see Supplemental Table in the supplemental digital content, containing 1 table and 3 figures, at https://meridian.allenpress.com/aplm in the March 2024 table of contents). Of these subjects, 462 (91.67%) had the same results whereas 42 (8.33%) exhibited discordant results between the 2 methods. Among the 42 discordant results, 4 variants were within the PCR detection range (common thalassemia variants), and 38 variants were outside the PCR detection range. Of those variants outside the PCR detection range, 19 were rare thalassemia variants and 19 had unknown pathogenicity. Sanger sequencing and specially designed PCR confirmed that the results of CATSA were all correct. In total, CATSA correctly detected 247 subjects with variants, whereas PCR correctly identified 205 subjects with variants, which showed an increase of 20.49%.

Figure 1

Flowchart of the study. Abbreviations: CATSA, comprehensive analysis of thalassemia alleles; Hb, hemoglobin; PCR, polymerase chain reaction.

Figure 1

Flowchart of the study. Abbreviations: CATSA, comprehensive analysis of thalassemia alleles; Hb, hemoglobin; PCR, polymerase chain reaction.

Close modal

Among 228 subjects diagnosed as carriers of thalassemia with common or rare variants by CATSA, 103 (45.18%) were diagnosed as carriers of α-thalassemia, 110 (48.25%) were carriers of β-thalassemia, and 15 (6.58%) had coinheritance of α- and β-thalassemia (Table 1). Among the 103 α-thalassemia carriers, 62 (60.19%) carried –SEA/αα, which was the most common genotype. The next 4 most common genotypes were -α3.7/αα (12.62%), -α4.2/αα (5.83%), HBA2:c.427T>C Hete (3.88%), and HBA2:c.369C>G Hete (2.91%). Among the 110 β-thalassemia carriers, HBB:c.316-197C>T Hete was the most common genotype (39 subjects; 35.45%). The next 4 most common genotypes were codons HBB:c.126_129delCTTT Hete (25.45%), HBB:c.52A>T Hete (15.45%), HBB:c.216_217insA Hete (4.55%), and HBB:c.84_85insC Hete (3.64%). In the 15 α+β-thalassemia carriers, there were 14 different genotypes. Two subjects had the genotype of -α3.7/αα compound with HBB:c.316-197C>T Hete.

Table 1

Distribution of α- and β-Thalassemia Genotypes by Comprehensive Analysis of Thalassemia Alleles

Distribution of α- and β-Thalassemia Genotypes by Comprehensive Analysis of Thalassemia Alleles
Distribution of α- and β-Thalassemia Genotypes by Comprehensive Analysis of Thalassemia Alleles

Discordant Results Within the PCR Detection Range

Of the 504 subjects, 4 subjects had discordant results between routine PCR and CATSA within the PCR detection range (Table 2). Sanger sequencing and specially designed PCR verified the results of the CATSA were correct (Supplemental Figure 1). Sequencing reads generated by CATSA for discordant variants were displayed in Integrative Genome Viewer (Figure 2, A through D). Two subjects had discordant variants in HBA genes, and 2 had discordant variants in HBB genes. For subject 21KDP33313, PCR could not determine the correct variant but CATSA confirmed she did not possess -α3.7. For subjects 21KDP33003, 21KDP33032, and 21KDP33371, the routine screening protocol tested only HBA genes or HBB genes by PCR based on the results of the hematologic testing. The results of PCR were normal, whereas CATSA identified thalassemia variants that were within the PCR detection range. Subject 21KDP33371 had hypochromic microcytosis, and subject 21KDP33003 had moderate anemia. The results of CATSA were more consistent with their phenotypes. To further confirm the results of CATSA, routine PCR was performed again for the missing alleles. The results of the second round of PCR were the same with CATSA.

Table 2

Discordant Results at Common Thalassemia Variant Sites

Discordant Results at Common Thalassemia Variant Sites
Discordant Results at Common Thalassemia Variant Sites
Figure 2

Integrative Genomics Viewer plots display comprehensive analysis of thalassemia alleles (CATSA) reads of discordant results between routine polymerase chain reaction and CATSA for common thalassemia variants (A through D) and rare thalassemia variants (E through I). Blue and yellow areas indicate 2 chromosomes. Red rectangles indicate the positions of discordant variants.

Figure 2

Integrative Genomics Viewer plots display comprehensive analysis of thalassemia alleles (CATSA) reads of discordant results between routine polymerase chain reaction and CATSA for common thalassemia variants (A through D) and rare thalassemia variants (E through I). Blue and yellow areas indicate 2 chromosomes. Red rectangles indicate the positions of discordant variants.

Close modal

Additional Thalassemia Variants Detected by CATSA

In addition to the thalassemia variants tested by routine PCR, CATSA can also detect rare thalassemia variants in α- and β-globin genes. Of the 504 subjects, 19 subjects had rare thalassemia variants detected by CATSA (Table 3). Sanger sequencing and specially designed PCR verified that the results of the CATSA were correct (Supplemental Figure 2). Sequencing reads generated by CATSA of the representative variants were displayed in Integrative Genome Viewer (Figure 2). Eighteen subjects had discordant variants in HBA genes, and 1 had a discordant variant in HBB genes. Among these 18 subjects, 10 subjects had additional α triplications identified by CATSA and 8 subjects had additional insertions, deletions, and SNVs detected by CATSA. In the 10 subjects with α triplications, 6 were also compounded with β-thalassemia. Most of the 6 subjects with α triplications and β-thalassemia variants had hypochromic microcytosis and anemia. For subject 21KDP33130, the genotype determined by PCR was HBB:c.316-197C>T Hete, whereas CATSA identified additional αααanti3.7/αα (Figure 2, E). Her Hb was only 92 g/L when she was 6 months old, which was more consistent with the result of CATSA. For subject 21KDP33235, PCR detected only HBB:c.126_129delCTTT Hete, whereas CATSA identified additional αααanti4.2/αα (Figure 2, F). She suffered from hypochromic microcytosis and her Hb level was only 98 g/L. For subject 21KDP33087, the genotype determined by PCR was αα/-α4.2, which predicted silent thalassemia. However, that determined by CATSA was -α4.2/HBA1:c.223G>C, which predicted thalassemia trait. For subject 21KDP33442, the genotype determined by PCR was αα/-α3.7 whereas that determined by CATSA was -α3.7/HBA1:c.364G>A. This subject had hypochromic microcytosis, low HbA2, and mild anemia, which were more consistent with the result of CATSA. For subjects 21KDP33033, 21KDP33075 (Figure 2, G), 21KDP33206, 21KDP33339, and 21KDP33472 (Figure 2, I), the results of PCR were normal, whereas CATSA detected rare thalassemia SNVs and indels. These subjects had either hypochromic microcytosis or low HbA2, which was more consistent with the result of CATSA. For subject 21KDP33057, PCR only detected 1 thalassemia variant in the HBB gene, whereas CATSA detected 2 thalassemia variants in the HBB gene. Particularly, CATSA revealed that the variants were in trans configurations (Figure 2, H), which predicted β-thalassemia intermedia/major.

Table 3

Discordant Results at Rare Thalassemia Variant Sites

Discordant Results at Rare Thalassemia Variant Sites
Discordant Results at Rare Thalassemia Variant Sites

Discordant Results of Unknown Pathogenicity

Nineteen subjects had discordant results of unknown pathogenicity between CATSA and PCR (Table 4; Figure 2). Sanger sequencing confirmed that the results of CATSA were correct (Supplemental Figure 3). Five subjects had discordant SNVs in HBA genes. Fourteen subjects had discordant SNVs in HBB genes. Among these discordant variants, the most frequent one was HBB:c.315+180T>C Hete.

Table 4

Discordant Single-Nucleotide Variants of Unknown Pathogenicity

Discordant Single-Nucleotide Variants of Unknown Pathogenicity
Discordant Single-Nucleotide Variants of Unknown Pathogenicity

Potential Pathogenic Variants Identified by CATSA in the Screening

In the subjects with positive results for Hb testing, 9 did not possess thalassemia variants but had other SNVs in their HBA and HBB genes. Three subjects had SNVs in HBA genes. Six subjects had SNVs in HBB genes, among whom 3 harbored HBB:c.315+180T>C Hete. Because these subjects all exhibited at least 1 abnormal hematologic parameter, these 7 SNVs may have potential pathogenicity of thalassemia (Table 5).

Table 5

Single-Nucleotide Variants With Potential Pathogenicity of Thalassemia

Single-Nucleotide Variants With Potential Pathogenicity of Thalassemia
Single-Nucleotide Variants With Potential Pathogenicity of Thalassemia

In this study, the comparison of CATSA and routine PCR of samples with positive results for Hb testing was carried out on a large scale. Among the 504 subjects, 462 (91.67%) had the same results between CATSA and PCR, whereas 42 (8.33%) had discordant results. Specially designed PCR or Sanger sequencing verified the results of CATSA. In this study, routine PCR had several false-positive and false-negative results. PCR could not detect -α3.7 and did not detect α triplications in 10 subjects (of 504; 1.98%). The incidence of αααanti3.7 and αααanti4.2 was as high as 1% to 2% among the general population in southern China.13  Because α-globin triplications can cause β-thalassemia intermedia when compounded with β0 or β+ heterozygotes, it would be critical to include these variants in thalassemia carrier screening and prenatal diagnosis. In addition, routine PCR missed rare SNVs and indels, and other Hb variants in 9 additional subjects. Because these subjects all exhibited at least 1 abnormal hematologic parameter, these SNVs may have potential pathogenicity of thalassemia.

Recently, the clinical utility of TGS in the diagnosis of thalassemia was validated, and TGS was demonstrated as a novel and powerful tool in the genetic analysis of thalassemia. Zhuang et al27  performed TGS in 70 suspected carriers of rare thalassemia variants and found that TGS yielded a 7.14% (5 of 70) increment of rare α- and β-globin gene variants as compared with PCR and Sanger sequencing. Peng et al28  conducted TGS for 100 cases of suspected thalassemia and identified an additional 10 cases of rare thalassemia variants compared with traditional thalassemia testing. Long and Liu14  carried out TGS in 4 samples that showed abnormalities in the traditional genetic tests and demonstrated that they carried complex α-globin gene variants. Jiang et al29  conducted TGS in 9 samples that showed abnormalities in the traditional genetic tests and found that they possessed various complex variants. However, the comparison between TGS and routine methods in Hunan Province has not been explored.

In China, the routine strategy for thalassemia screening is Hb testing. Then, PCR for one or both of the HBA and HBB genes is performed based on the result of Hb testing. In this research, 8 subjects did not have their HBA genes and 280 subjects did not have their HBB genes tested by routine PCR. However, CATSA found that 1 of the 8 subjects possessed an HBA variant within the PCR detection range and 2 of the 280 subjects possessed HBB variants within the PCR detection range. To further confirm the results of CATSA, routine PCR was performed again for the missing alleles in 288 subjects. The results of the second round of routine PCR were the same with CATSA. This means all the HBA and HBB alleles should be tested by genetic analysis.

In conclusion, CATSA is a more comprehensive, reliable, and effective approach in genetic analysis of thalassemia compared with PCR. In terms of cost, previous research30,31  demonstrated that TGS based on the PacBio platform could competitively deliver a test as low as around $20. However, there are still potential drawbacks of CATSA relative to other methods. The PacBio sequencing platform Sequel II is expensive and hundreds of samples need to be pooled together for sequencing in one flow cell to reduce the cost per sample, which makes it suitable only for large centers at present. Thus, developing a benchtop PacBio sequencing platform with lower cost and lower throughput would be more clinically feasible. Currently, large deletions are often identified by multiple-ligation probe amplification, which determines the copy number of each probe using quantitative analysis. Unlike multiple-ligation probe amplification, CATSA relies on the combination of primers to directly identify the breakpoints of deletions, which is more straightforward but might miss novel deletions that are not covered by the primer set. Thus, it is important to review and optimize the primer set of CATSA periodically for comprehensive coverage of rare deletions. There are also certain limitations in the present study. First, the current method only covered HBA and HBB genes. In the future, it is technically possible to include other clinically significant regions, such as the HS40 region and modifier genes. Second, this study evaluated the clinical utility of CATSA only in samples with positive results for Hb testing. Future experiments can be conducted in Hb testing–negative samples to compare the effectiveness of PCR and CATSA. Third, this was a retrospective study with a limited sample size. A large-scale and multicenter prospective study can be directed to fully explore the molecular spectrum of the subjects in Hunan Province.

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Author notes

Funding of this research was supported by Project of Changsha Hospital for Maternal & Child Health Care in 2021 (2021YJ04) and Natural Science Foundation of Hunan Province (2021JJ40620).

Supplemental digital content is available for this article at https://meridian.allenpress.com/aplm in the March 2024 table of contents.

The authors have no relevant financial interest in the products or companies described in this article.

Xu, Hu, and Liu contributed equally to this work.

Supplementary data