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. 2024 Jan 21;12(1):e2365. doi: 10.1002/mgg3.2365

Third‐generation sequencing identified two rare α‐chain variants leading to hemoglobin variants in Chinese population

Jianlong Zhuang 1,✉, Yuying Jiang 1, Yu'e Chen 2, Aiping Mao 3, Junwei Chen 4,✉, Chunnuan Chen 5,✉
PMCID: PMC10801340  PMID: 38284449

Abstract

Background

Rare and novel variants of HBA1/2 and HBB genes resulting in thalassemia and hemoglobin (Hb) variants have been increasingly identified. Our goal was to identify two rare Hb variants in Chinese population using third‐generation sequencing (TGS) technology.

Methods

Enrolled in this study were two Chinese families from Fujian Province. Hematological screening was conducted using routine blood analysis and Hb capillary electrophoresis analysis. Routine thalassemia gene testing was carried out to detect the common mutations of α‐ and β‐thalassemia in Chinese population. Rare or novel α‐ and β‐globin gene variants were further investigated by TGS.

Results

The proband of family 1 was a female aged 32, with decreased levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Hb A2, and abnormal Hb bands in zone 5 and zone 12. No common thalassemia mutations were detected by routine thalassemia analysis, while a rare α‐globin gene variant Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] was identified by TGS. Subsequent pedigree analysis showed that the proband's son also harbored the Hb Jilin variant with slightly low levels of MCH, Hb A2, and abnormal Hb bands. The proband of family 2 was a male at 41 years of age, exhibiting normal MCV and MCH, but a low level of Hb A2 and an abnormal Hb band in zone 12 without any common α‐ and β‐thalassemia mutations. The subsequent TGS detection demonstrated a rare Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] variant in HBA2 gene.

Conclusion

In this study, for the first time, we present two rare Hb variants of Hb Jilin and Hb Beijing in Fujian Province, Southeast China, using TGS technology.

Keywords: hemoglobinopathy, Sanger sequencing, single‐molecule real‐time sequencing, thalassemia, third‐generation sequencing

Two rare α‐chain variants that causing hemoglobinopathy were first identified in Fujian Province, Southeast China, using third‐generation sequencing technology. Abnormal hemoglobin (Hb) bands in zone 12 may be associated with Hb Beijing and Hb Jilin variants.

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1. INTRODUCTION

The hemoglobin (Hb) disorders are a group of autosomal recessive disorders caused by human globin gene variants (Huang et al., 2019; Zaino & Tien, 1981). Commonly, defects in Hb structure and number of globin chains would give rise to Hb variants and thalassemia, respectively (Iyer et al., 2015). Among them, thalassemia is an inherited genetic disorder, of which α‐ and β‐thalassemia were the most common genotypes (Weatherall, 2001). To date, over 1000 Hb variants have been identified and the number is continuously increasing. Thalassemia and Hb variants were highly prevalent in Southern China, including Guangdong, Guangxi, Fujian, Hainan, and other provinces (He et al., 2018; Li et al., 2014; Lu et al., 2021; Yao et al., 2014; Yin et al., 2014; Zhang et al., 2010). A previous study conducted by Huang et al. (2019) indicated that the overall prevalence of thalassemia and Hb variants in Fujian Province, Southeast China, was 6.8% and 0.26%, respectively. The Quanzhou region of Fujian Province, Southeast China, which is located along the southeastern coastal regions of China, manifested greater diversity and complexity of thalassemia gene mutations (Huang et al., 2019; Zhuang et al., 2020, 2021).

High‐throughput sequencing technologies are more appropriate and valuable for thalassemia genetic analysis. Among them, next‐generation sequencing is superior at variant calling to third‐generation sequencing (TGS) because of its lower error rates, while longer reads nature of the TGS permits haplotype phasing that is superior for variant discovery on the homologous genes and CNV calling (Hassan et al., 2023). The TGS based on Pacific Bioscience (PacBio) and Oxford Nanopore Technologies has been gradually utilized in genetics etiology diagnosis. With long reads, single‐molecule resolution, and the ability to distinguish variants in cis or trans, TGS has showed obvious advantages in identifying single‐nucleotide variations, indels, and structural variants (Cretu Stancu et al., 2017; Loose, 2017; Xu et al., 2020). In recent years, several findings highlight the valuable application of TGS technology in investigating thalassemia and Hb variants (Long et al., 2022; Xu et al., 2020; Zhuang et al., 2023).

In this study, TGS was used for identification of two rare α‐globin gene Hb variants in Chinese population. Both two Hb variants were identified in Fujian Province, Southeast China, for the first time. In addition, Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] variant was the second reported case, while with slightly decreased hematological results. This finding further strengthened the application value of TGS in the genetic diagnosis of thalassemia and Hb variants.

2. MATERIALS AND METHODS

2.1. Subjects

In our clinical practice, prevention and control of thalassemia, both routine blood analysis and Hb capillary electrophoresis analysis are used for hematological screening, and samples that had positive screening results from either test would be subjected to further thalassemia genetic analysis (Figure 1). Enrolled in this study were two Chinese families from Quanzhou region of Southeast China, with three members in family 1 and two members in family 2. All of the members in the two enrolled families denied a history of blood transfusion. Peripheral blood samples from the two families were collected and stored for further investigation.

FIGURE 1.

FIGURE 1

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The screening flow of thalassemia in this study.

2.2. Hematological screening

Approximately 4 mL of peripheral blood was collected from each subject and anticoagulated with EDTA‐K2 for routine blood analysis and Hb capillary electrophoresis analysis. An automated cell counter was used for routine blood analysis (Sysmex XS‐1000i; Sysmex Co., Ltd., Kobe, Japan) including mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), and Hb capillary electrophoresis was used to detect Hb A, Hb A2, and Hb F and other abnormal Hb bands (Sebia, Evry Cedex, France). A MCV of <82 fL, and/or a MCH concentration of <27 pg, and/or Hb A2 >3.4% or Hb A2 <2.6% or Hb F > 2.0% or abnormal Hb bands were defined as positive thalassemia screening results.

2.3. Routine thalassemia genetic testing

The genomic DNA of the subjects in two Chinese families were extracted via an automatic nucleic acid extractor (Ruibao Biological Co., Ltd). The PCR reverse dot hybridization technique (PCR‐RDB) was used to detect the 23 common α‐thalassemia and β‐thalassemia variants (Yaneng Biological Technology Co., Ltd., Shenzhen) (Liu et al., 2019), including −α3.7, −α4.2, −SEA, Hb Constant Spring, Hb Quong Sze, Hb Westmead, CD41‐42(‐TCTT), IVS‐II‐654(C>T), −28(A>G), CD71/72(+A), CD17(AAG>TAG), CD26(GAG>AAG), CD43(GAG>TAG), −29(A>G), CD31(‐C), −32(C>A), IVS‐I‐1(G>T), CD27/28(+C), −30(T>C), CD14‐15(+G), Cap+40–43(–AAAC), initiation codon(ATG>AGG), and IVS‐I‐5(G>C). The experimental procedures were performed strictly according to the manufacturer's protocol.

2.4. Thalassemia genetic testing by TGS

Genomic DNA was obtained and then sent to an independent laboratory (Berry Genomics, Beijing) for TGS on the PacBio Sequel II platform. The TGS‐based thalassemia detection was conducted according to the manufacture protocol described previously (Zhuang et al., 2023). In summary, we employed optimized primers to generate specific amplicons that encompass well‐established structural variation regions and single nucleotide variations within the HBA1/2 and HBB globin genes. These designs were based on information retrieved from reputable databases such as HbVar, ITHANET, LOVD, and LOVD‐China. After purification and end repair, double barcode adapters were ligated to the 5′ and 3′ ends, and Sequel Binding and Internal Ctrl Kit 3.0 (PacBio) was used to prepare SMRT bell libraries. Sequencing was performed on the PacBio Sequel II System after primed DNA‐polymerase complexes were loaded onto SMRT cells (PacBio).

2.5. Data analysis

After alignment of the subreads, the consensus circular sequence was mapped to the GRCh38 reference and variants called (FreeBayes software, version 1.2.0). Linkage analysis (in cis or trans) in the long‐read‐based phasing was conducted using WhatsHap (version 0.18) software. The Integrative Genomics Viewer was used to display the alignments of variant and wild‐type reads. In addition, for large deletion variants Gap‐PCR was further conducted for confirmation with specific primers. Rare or novel variants in α‐ and β‐globin genes were confirmed by Sanger sequencing.

3. RESULTS

3.1. Hematological screening results

The hematological screening results of family 1 are listed in Table 1. Routine blood analysis elicited decreased levels of MCV (79.9 fl) and MCH (21.6 pg) in the proband of family 1. Hb capillary electrophoresis results can be divided into 15 zones according to Hb electric charge, as delineated in Figure 2. A decreased level of Hb A2 (1.6%) and two abnormal Hb bands in zone 5 (0.4%) and zone 12 (28.7%) were identified in the proband using Hb capillary electrophoresis analysis, which indicated the proband maybe a α‐thalassemia carrier. In addition, the proband's son also had α‐thalassemia trait with low levels of Hb A2 (2.1%), MCV (26.3 pg), and the similar abnormal Hb bands (Hb zone 5: 0.5% and zone 12: 28.9%). However, the proband's husband showed normal hematological analysis results.

TABLE 1.

The hematological screening and molecular analysis results in family 1.

Parameters Proband Proband's husband Proband's son
Sex‐age F‐32 M‐31 M‐7
RBC (1012/L) 5.51 5.34 5.06
Hb (g/L) 119 166 133
MCV (fl) 79.9 90.9 85.4
MCH (pg) 21.6 31.1 26.3
Hb A (%) 69.3 97.1 68.5
Hb A2 (%) 1.6 2.9 2.1
Hb F (%) 0 0 0
Hb zone 5 (%) 0.4 0 0.5
Hb zone 12 (%) 28.7 0 28.9
Thalassemia genotype αα/αα, βN /βN αα/αα, βN/βN αα/αα, βN /βN
Hb variants HBA2:c.418A>C N HBA2:c.418A>C
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Abbreviations: F, female; Hb, hemoglobin; M, male; MCH, mean corpuscular hemoglobin; CV, mean corpuscular volume; N, normal; RBC, red blood cells.

FIGURE 2.

FIGURE 2

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The hematological screening results in probands of family 1 and family 2 using Hb capillary electrophoresis analysis. (a) Two abnormal Hb bands in zone 5 (0.4%) and zone 12 (28.7%) were identified in the proband of family 1 using Hb capillary electrophoresis analysis. (b) An abnormal Hb band was presented in the proband of family 2 in Hb zone 12 (26.2%).

As delineated in Table 2, both of the proband and his wife in family 2 had decreased levels of Hb A2, and both of them were suspected as α‐thalassemia carriers. In addition, an abnormal Hb band in zone 12 (26.2%) was also identified in the proband of family 2 using Hb capillary electrophoresis analysis (Figure 2).

TABLE 2.

The hematological screening and molecular analysis results in family 2.

Parameters Proband Proband's wife
Sex‐age M‐41 F‐36
RBC (1012/L) 5.28 4.75
Hb (g/L) 175 105
MCV (fl) 91.8 66.2
MCH (pg) 33.1 22.2
Hb A (%) 72 97.6
Hb A2 (%) 1.8 2.4
Hb F (%) 0 0
Hb zone 12 (%) 26.2 0
Thalassemia genotype αα/αα, βN/βN αα/αα, βN/βN
Hb variants HBA2:c.51G>T N
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Abbreviations: F, female; Hb, hemoglobin; M, male; MCH, mean corpuscular hemoglobin; CV, mean corpuscular volume; N, normal; RBC, red blood cells.

3.2. Routine thalassemia genetic testing results

The PCR‐RDB was used to detect the 23 common α‐ and β‐thalassemia variants in the Chinese population in both of the enrolled families. However, as demonstrated in Tables 1 and 2, no common mutations and deletions of α‐ and β‐thalassemia were observed. Based on the above results, the enrolled members with α‐thalassemia traits may harbor rare or novel globin genes variants. Subsequently, TGS technology was carried out to investigate the possibly HBA1/2 and HBB globin genes variants in the suspected subjects.

3.3. Thalassemia genetic testing results by TGS

As shown in Figure 3, a rare Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] variant in HBA2 gene was identified in the proband of family 1 by TGS and further confirmed by Sanger sequencing. The same variant was also presented in the proband's son of family 1, who exhibiting decreased levels of Hb A2 and abnormal Hb screening results. In addition, no variants in HBA1/2 and HBB globin genes were observed in the proband's husband using TGS technology who manifest normal hematological screening results.

FIGURE 3.

FIGURE 3

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Two rare hemoglobin (Hb) variants were identified by third‐generation sequencing (TGS) and verified by Sanger sequencing. (a and b) Two rare Hb variants of Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] and Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] were identified by TGS. (c and d) The Sanger sequencing technology was further performed and confirmed the variants identified by TGS.

In the proband of family 2, a rare Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] variant in HBA2 gene was identified by TGS technology and confirmed by Sanger sequencing (Figure 3). However, none of HBA1/2 or HBB globin gene variants were identified by TGS in the proband's wife who had low levels of MCV, MCH, and Hb A2. Regrettably, no other members were available for further genetic investigation in this family.

4. DISCUSSION

Thalassemias and Hb variants are widespread inherited genetic disorders in South China. Only few Hb variants can lead to abnormal hematological results and causing thalassemia, such as Hb E and Hb CS. In the clinical practice, combined use of routine blood analysis and Hb electrophoresis analysis is effective for screening of thalassemia and Hb variants, and the subsequent routine thalassemia genetic testing was used for genetic diagnosis in subjects with positive hematological screening results (Wu et al., 2016). Our previous study indicated a diversity of Hb variants in Quanzhou region, including Hb Q‐Thailand, Hb G‐Honolulu, Hb Owari, Hb New York, Hb J‐Bangkok, Hb Miyashiro, Hb G‐Coushatta, and other Hb variants (Zhuang et al., 2021). In the present study, two rare α‐chain variants of Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] and Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] causing Hb variants were identified, which was the first report in Fujian Province, Southeast China.

The Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] is an extremely rare α‐chain variant. In the literature, only one report was available that conducted by Xu et al. (2019). It was suggested to be a stable Hb variant, which results from a single base substitution in the HBA2 globin gene in the exon 3 and would replace the protein residue lysine (Lys) as glutamine (Glu) at codon 139. Finally, it was named as Hb Jilin according to the region where the patient originated from and interpreted as Hb variant with only one abnormal Hb band in zone 12 using Hb capillary electrophoresis analysis. Interestingly, as delineated in Figure 2, the proband and the proband's son of family 1 showed two abnormal Hb bands of HbA' and HbA2' in zone 12 and zone 5, respectively, which indicated the existence of α‐chain variant. The thalassemia gene testing based on TGS revealed a rare variant of Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] in the proband and the proband's son who had decreased levels of MCV and/or MCH.

Iron deficiency anemia (IDA) is similar to α‐thalassemia in terms of symptoms, which usually exhibits microcytic hypochromic anemia and decreased level of Hb A2. IDA carriers typically exhibit low level of serum ferritin and increased erythrocyte distribution width (RDW) value. In contrast, both of the indexes are normal in pure thalassemia‐only carriers (Çil et al., 2020). The proband in family 1 who harbored Hb Jilin variant exhibited low levels of MCV and MCH and also had a low level of serum ferritin (6 μg/L) and increased RDW value (19.5%), which may explain his abnormal hematological screening results. In addition, the proband's son in family 1 also had Hb Jilin variant with slightly decreased level of MCH (26.3 pg), but normal level of serum ferritin (48 μg/L). However, a low neutrophil percentage (40.2%) and high lymphocyte percentage (45.3%) were also observed in the patient, which indicated an infection in the proband's son of family 1 and may explain his slightly low level of MCH.

The other rare Hb variant identified in this study was Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T], which was the first case described in Fujian Province, Southeast China. The Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] variant was suggested as a fast‐moving Hb variant with protein residue replaced lysine (Lys) by asparagine (Asn) at codon 16 and originally found in a Chinese individual reported by Liang et al. (1982). In 2013, it was secondly reported in a Chinese individual with normal hematological screening results from Jiangsu Province, Eastern China, by Lin et al. (2013). Similar to the previous studies, the proband of family 2 had the same Hb variant, which also exhibits normal hematological screening results. At present, only four references are available in the literature to our best knowledge, and the other two reports are from Thai population (Fucharoen et al., 2005; Panyasai et al., 2016). One of them was from a large cohort retrospective study that enrolled 23,914 subjects from Thai population and first identified the Hb Beijing in Thailand (Panyasai et al., 2016). In addition, the other study presented a complex hemoglobinopathies of Hb Beijing with the beta‐E‐globin chains in a Thai patient and indicated that it may lead to a new Hb E Beijing variant (Fucharoen et al., 2005).

To date, TGS has been gradually used in identifying thalassemia and Hb variants with high accuracy and efficiency (Long et al., 2022; Xu et al., 2020; Zhuang et al., 2023). In the present study, two rare α‐chain variants that causing Hb variants were successfully identified in Chinese population. However, there are also some limitations of this study, including limit enrolled families and lack of further functional analysis.

In conclusion, for the first time, we presented two rare Hb variants of Hb Jilin and Hb Beijing in Fujian Province, Southeast China. Our findings further enhanced the application value of TGS in the identification of thalassemia and Hb variants. Additionally, this was the 2nd report of Hb Jilin, which may provide additional reference data for clinical counseling.

AUTHOR CONTRIBUTIONS

JZ designed the study and wrote the article. YJ and YC contributed in patients' recruitment and clinical consultation; JZ and AM performed routine thalassemia analysis, TGS and analyzed the data; and CC and JC revised and polished the paper. All authors approved the final article.

FUNDING INFORMATION

This research was supported by Quanzhou City Science and Technology Project (no. 2020N049s); Huaqiao University Joint of Hospital and University Innovation Project (no. 2021YX005); and Fujian Provincial Key Laboratory of Prenatal Diagnosis and Birth Defects open project (no. cqzdsys‐2022‐01).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interests.

ETHICS STATEMENT

This study was approved by the ethics committee of The Women's and Children's Hospital of Quanzhou (2021 No. 61). All procedures performed involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all the participants and from the legal guardians of the participants who were below 16 years of age.

ACKNOWLEDGMENTS

We wish to express our appreciation to Quanzhou Science and Technology, Fujian Provincial Key Laboratory of Prenatal Diagnosis and Birth Defects, and Huaqiao University Joint of Hospital and University projects for funding this work. We also express our appreciation to the patients who participated in this study.

Zhuang, J. , Jiang, Y. , Chen, Y. , Mao, A. , Chen, J. , & Chen, C. (2024). Third‐generation sequencing identified two rare α‐chain variants leading to hemoglobin variants in Chinese population. Molecular Genetics & Genomic Medicine, 12, e2365. 10.1002/mgg3.2365

Contributor Information

Jianlong Zhuang, Email: 415913261@qq.com.

Junwei Chen, Email: 2365573595@qq.com.

Chunnuan Chen, Email: chenchunnuan1983@aliyun.com.

DATA AVAILABILITY STATEMENT

The datasets used and analyzed in the current study were obtained from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and analyzed in the current study were obtained from the corresponding author on reasonable request.

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