Analysis of genotypic distribution and rare variants of patients with α/β-thalassemia screened in one hospital in Beijing, China

Abstract

Objective

Thalassemia is among the most common inherited diseases worldwide. We aimed to analyze the genotype and frequency distribution of thalassemia in a general hospital in Beijing and provide a reference for genetic counseling and prenatal diagnosis.

Methods

A total of 3196 cases of thalassemia screened at Peking Union Medical College Hospital (PUMCH) between January 2018 and January 2022 were collected. Thalassemia genotypes were tested using gap polymerase chain reaction (gap-PCR), PCR, reverse dot blot (RDB), and Sanger sequencing analyses. The pathogenicity of the rare variants was analyzed using bioinformatics approaches.

Results

Total of 1936 positive routine α/β-thalassemia were detected from 3196 blood samples, including 733 α-thalassemia variants, 1170 β-thalassemia variants, and 33 cases with concurrent α- and β-thalassemia variants. Two novel variants, HBA2:c.300+82G>C and HBB:codon85(-T), were identified in HBA2 and HBB genes, respectively, and were not detected in the ExAC, gnomAD, HbVar, and HGMD databases.

Conclusions

The genotype distribution of thalassemia in a general hospital in Beijing is complex and heterogeneous. The novel variants in HBA2 and HBB are likely to underlie α/β-thalassemia in these patients.

Introduction

Thalassemia, a congenital hemolytic anemia, is among the most common single gene and inherited autosomal recessive genetic diseases. It is a hereditary chronic hemolytic anemia caused by a reduction or complete loss of globin peptide chain synthesis due to point mutations or deletion of hemoglobin’s globin peptide chain gene [1,2,3]. The clinical manifestations of thalassemia vary according to severity, ranging from asymptomatic to anemia, hepatosplenomegaly, and severe anemia (hemoglobin H disease, α-thalassemia major and β-thalassemia major). Patients with severe thalassemia typically undergo blood transfusion and iron chelation therapy. Screening and genetic diagnosis of thalassemia can be performed by routine blood examination, hemoglobin analysis, osmotic fragility tests, and molecular biology [2].

According to the different types of globin peptide chain deficiency, thalassemia can be divided into α-thalassemia (α-thal), β-thalassemia (β-thal), δβ-thalassemia (δβ-thal), δ-thalassemia (δ-thal), and εβγδ-thalassemia (εβγδ-thal). There are two major types of thalassemia in clinical settings, α-thal and β-thal, which are caused by the loss or defect of α-globin genes (HBA1 and HBA2) or β-globin gene (HBB), respectively. This results in an imbalance in globin synthesis, ineffective erythropoiesis, and extravascular hemolysis [2, 3].

Approximately 5% of the global population carries at least one variant allele of thalassemia, with up to 900,000 individuals projected to manifest significant clinical symptoms by the early 21st century [4, 5]. Thalassemia cases are more common in countries bordering the Mediterranean, the Middle East, India, and Southeast Asia. Thalassemia is more prevalent in the southern provinces of Guangdong, Guangxi, Hainan, and Sichuan in China, with fewer cases reported in the northern regions [6,7,8]. Historically, the incidence of thalassemia has been low in Beijing, northern China. However, Beijing is an open international metropolis, and the increased migrant population in recent years has increased the incidence of thalassemia. Effective treatments for severe thalassemia are lacking; therefore, carrier screening, prenatal diagnosis, and gene screening before embryo implantation are the only effective measures to prevent the birth of children with severe thalassemia and improve the quality of this population [9, 10].

In this study, we analyzed the samples of thalassemia gene screening at PUMCH from January 2018 to January 2022 and preliminarily obtained the genotype and frequency distributions of thalassemia gene variants in Beijing. Our findings can enrich the relevant information on thalassemia in Beijing and provide an important basis for genetic counseling and prenatal diagnosis of thalassemia in Beijing.

Materials and methods

Research subjects

Specifically, 3196 thalassemia screening samples were obtained from PUMCH between January 2018 and January 2022. There was no genetic relationship between these patients, and there were no restrictions by sex or age. These subjects underwent routine examinations at the hospital, and discarded blood samples were used for further research. All procedures were conducted following the ethics guidelines for research on human subjects. Ethics approval was granted by the institutional review board of PUMCH (I-22PJ1013).

Routine hematological analysis

Peripheral venous blood (2 mL) was collected in EDTA anticoagulant tubes. Routine blood examinations were performed on an automatic blood cell analyzer (XN-9100, Sysmex, Kobe, Japan). Hemoglobin components were quantitatively analyzed using an automatic capillary electrophoresis apparatus (Sebia Minicap-FP, Bole Company, France).

Whole blood gDNA extraction

Genomic DNA was extracted from peripheral venous blood using a DNA Rapid Extraction Kit (Tianlong Technology Co., Ltd., Xi’ an, China). DNA concentration and purity were determined by ultraviolet spectrophotometer (Multiskan Go, Thermo Fisher Technologies, USA), and qualified samples met the following criteria: (1) concentration > 20 ng/µL; (2) A260/280 ratios between 1.8 and 2.0.

Analysis of routine α/β-thalassemia genotypes

The experiment was conducted following the instructions specified in the thalassemia gene detection kit (Yaneng Biotechnology Co., Ltd., Shenzhen, China). Three common deletion types of α -thalassemia (--^SEA, -α^3.7, and -α^4.2) in Chinese were detected by gap-PCR. PCR-RDB analysis revealed the presence of three common non-deletion types of α-thalassemia (α^WSα, α^CSα, and α^QSα), and 17 common non-deletion types of β-thalassemia, including a range of mutations, such as CD41-42(-TTCT), IVS-II-654(C>T), -28(A>G), CD17(A>T), CD71/72(+A), βE(G>A), -29(A>G), CD43(G>T), CD27/28(+C), CD14/15(+G), CD31(-C), -32(C>A), -30(T>C), IVS-I-1(G>T), IVS-I-5(G>C), Int(ATG>AGG), and 5’UTR +40-43(-AAAC).

Sanger sequencing in patients with rare thalassemia

Sanger sequencing has been utilized for patients initially identified with normal thalassemia gene profiles through routine screening but displaying abnormal hemoglobin electrophoresis or distinct clinical phenotypes indicative of rare forms of thalassemia. Gene sequence information was obtained from the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu), and Primer3 Input (version 4.1.0) was used to design primer sequences. Primers were synthesized by Tianyi Huiyuan Biotech Co., Ltd. (Beijing, China). HBA1, HBA2, and HBB genes of patients were amplified by PCR, and the products were sent to Nuosai Gene Company (Beijing, China) for sequencing. The results were analyzed using Chromas sequencing software. The detection of copy number variations within the globin gene cluster involved the Multiplex Ligation-dependent Probe Amplification (MLPA) method using the SALSA MLPA kit P102 HBB and the SALSA MLPA kit P140 HBA (MRC-Holland, Amsterdam, Netherlands) was employed following the manufacturer’s instructions. The HbVar (http://globin.cse.psu.edu/hbvar/), HGMD (http://www.biobase-international.com/product/hgmd), and PubMed databases were used to verify whether the variants were previously documented. DNAMAN software was used to analyze the interspecies conservation of amino acids at rare variant sites, and the in silico prediction tools, namely VarCards, MutationTaster2, CADD, Polyphen 2, and SIFT were used to predict variant pathogenicity.

Statistical analysis

Microsoft Excel 2007 and GraphPad Prism 9 software were used for statistical analyses. First, the normality of the distributions was analyzed. The Mann-Whitney U test was used to analyze non-normal distributions. The genotype, frequency, hematological characteristics, and geographical distribution of thalassemia genes in Beijing were analyzed using descriptive methods.

Result

Genotype distribution of routine α/β-thalassemia

Routine thalassemia was diagnosed in 1936 of the 3196 samples, with 733 cases of α-thalassemia (37.86%), 1170 of β-thalassemia (60.43%), and 33 cases of compound heterozygous α-/β-thalassemia (1.70%). The study population comprised 635 males (32.80%) and 1301 females (67.20%) (Fig. 1A), with the age distribution primarily concentrated in the 20–29 and 30–39 age groups, accounting for 1375 patients (71.02%) (Fig. 1B).

Among 733 patients with α-thalassemia, 679 (92.63%) carried heterozygous variants, and 54 (7.37%) had homozygous or compound heterozygous variants. --^SEA/αα was the most common genotype, with 550 cases, accounting for more than half (75.03%) of all α-thalassemia genotypes. Other common genotypes of α-thalassemia included -α^3.7/αα, -α^3.7/--^SEA, and -α^4.2/ αα (Table 1).

Fig. 1

Constituent ratios of different gender, age groups and basic hematological red blood cell parameters of α/β-thalassemia patients (Beijing, China). A Gender distribution among of α/β-thalassemia patients. B Age demographics among α/β-thalassemia patients. C Proportions of mean corpuscular volume (MCV, including 379 alpha-thalassemia samples and 611 beta-thalassemia samples), mean corpuscular hemoglobin concentration (MCHC, including 365 alpha-thalassemia samples and 588 beta-thalassemia samples), and mean corpuscular hemoglobin (MCH, including 365 alpha-thalassemia samples and 592 beta-thalassemia samples) among α/β-thalassemia patients. Note: **** means P < 0.0001

Table 1 Genotypes and genotype frequencies of α-thalassemia, β-thalassemia, and α-/β-thalassemias (Beijing, China)

Among 1170 patients with β-thalassemia, 1164 (99.49%) carried heterozygous variants, and 6 (0.51%) had homozygous or compound heterozygous variants. The most frequent genotypes were β^{IVS-II-654(C>T)}/β^N, β^{CD41-42(-TCTT)}/β^N, and β^CD17(A>T)/β^N, collectively representing 91.28% of all β-thalassemia genotypes. Other common genotypes of β-thalassemia included β^-28(A>G)/β^N, β^CD27-28(+C)/β^N, and β^E(GAG>AAG)/β^N (Table 1).

Thirty-three patients carried the compound heterozygous α-/β-globin variants. Seventeen genotypes were detected in these patients, and the most common type was -α^3.7/αα combined with β^{IVS-II-654(C>T)}/β^N (Table 1).

There were significant differences in the basic hematological parameters erythrocyte mean corpuscular volume (MCV, P < 0.0001), erythrocyte mean corpuscular hemoglobin concentration (MCHC, P < 0.0001), and erythrocyte mean corpuscular hemoglobin (MCH, P < 0.0001) between patients with α- and β-thalassemia (Fig. 1C).

Geographic distribution of patients with routine α/β-thalassemia

A cohort of 1936 patients diagnosed with routine thalassemia hailing from various regions in China, including North China, Southwest China, and the middle and lower reaches of the Yangtze River, including 310 in Beijing, 180 in Hunan, 182 in Sichuan, 142 in Guangxi, and 129 in Hebei (Fig. 2).

Fig. 2

Geographic distribution of α/β-thalassemia. Red represents α-thalassemia, green represents β-thalassemia, blue represents Concurrent α-/β-thalassemias, and the size of the colour of the circle represents the number of people affected (Beijing, China). The southern regions, delineated by the Qinling Mountains to the north and the Huai River to the south, are highlighted in a soft pink shade. With the exception of Tibet, the unshaded areas are classified as part of the northern region

Identification of rare α/β-thalassemia genotypes

Rare globin gene variants were detected in 31 patients (cases 1–31, Table 2), including 4 patients with rare α-thalassemia (case 4–7), 24 patients with rare β-thalassemia (cases 8–31) and 3 patients with rare deletion (cases 1–3, Fig. 3A; Table 2). In this study, two new pathogenic gene variants of α/β-thalassemia cases were identified, including case 4: HBA2:c.300+82G>C; and case 8: HBB:codon85(-T). These variants have not been reported in HbVar, HGMD, and PubMed databases.

Fig. 3

Multiplex Ligation-dependent Probe Amplification (MLPA) analysis, Sanger sequencing and Hemoglobin electrophoresis results of rare cases (Beijing, China). The ordinate represents the gene copy number, the abscissa represents the chromosomal position of the gene. The probe ratio within the red box, indicating a deletion, showed a value of only 0.5 (3 A-C). A MLPA result showed the deletion across 5 probes targeting HBB gene (deletion probes involved 5 probes between hg18 loc.11p15.4:5177860-5203330, including HBB-3, HBB-down and ORF51V1-1 regions) (Case 1). B MLPA result showed the deletion across 8 probes targeting HBB gene (deletion probes involved 8 probes between hg18 loc.11p15.4:5202720-5204970, including HBB-1, HBB-1 (WT) c, HBB-Intr1, HBB-Intr. 2, HBB-3 and HBB-down regions) (Case 2). C MLPA result showed the deletion across 11 probes targeting HBB gene (deletion probes involved 11 probes between hg18 loc.11p15.4:5202711-5210637, including HBB-up, HBB-1, HBB-1 (WT) c, HBB-Intr1, HBB-Intr. 2, HBB-3 and HBB-down regions) (Case 3). D and E Two new pathogenic variants in the HBA2 gene and HBB (Cases 4 and 8)

Table 2 Rare gene variants with α-thalassemia (n = 4), and β-thalassemia (n = 25) (Beijing, China)

Case 4, Male, 1-year-old, ancestral home in Inner Mongolia with mild anemia. The patient exhibited no jaundice and had no history of blood transfusion. Hemoglobin (HGB, 9.1 g/dL) was detected on physical examination at another hospital, and the patient was prescribed enhanced iron supplementation. HGB (9.4 g/dL) was reexamined one month later. After the patient regained strength with supplementary iron feeding, HGB (9.7 g/dL) was reexamined. On November 11, 2021, he visited the Pediatric Outpatient Department of PUMCH for thalassemia screening. A comprehensive panel of 23 routine thalassemia gene tests yielded negative results in laboratory analysis. The hemoglobin profile was as follows: HbA, 97.1%; HbA2, 2.3%, and HbF, 0.6%. Sanger sequencing revealed a novel heterozygous mutation in the α-globin gene HBA2:c.300+82G>C(Fig. 3B). HBA2:c.300+82G>C was predicted as polymorphism by MutationTaster2.

Case 8, Female, 34 years from Shandong, 24 weeks of gestation, presented with mild anemia. She had been experiencing anemia for more than ten years. The patient was administered iron supplementation. During the previous pregnancy, the hemoglobin level was 9 g/dL, which decreased to 7.2 g/dL in the current pregnancy. Additionally, the MCV was 64 fL, reticulocyte count (RET) was 3.56%, serum ferritin (SF) was 128 µg/L, and levels of bilirubin and lactate dehydrogenase (LDH) were within normal ranges. Iron supplementation was briefly administered during the pregnancy. The mother was anemic. Her three brothers had no anemia, and the MCV decreased in the first baby boy. Laboratory assessments revealed negative results for 23 routine thalassemia gene tests. The hemoglobin contents were HbA 93.3%, HbA2 5.4%, and HbF 1.3%, respectively. Sanger sequencing detected a novel heterozygous mutation of the β-globin gene, HBB:Codon85(-T) (Fig. 3C). HBB:Codon85(-T) was predicted as disease causing by MutationTaster2.

Thirteen cases of anemia with abnormal hemoglobin electrophoresis due to HBD, HBG1, and HBG2 gene mutations were screened, including two cases of HBD:c.-127T>C; a case of HBD:c.-80T>C; a case of HBG1:-314(G>T); 5 case of HBG1:+25(G>A) combined with HBG2:-158(C>T); 2 cases of HBG1:c.-272_-275dupAGCA; a case of HBB:c.91A>G combined with HBG1:c.-272_-275dupAGCA; a case of HBG2:398(A>G).

Discussion

In terms of detection rate, among the 3196 samples in this study, the detection rate of thalassemia was over 60%. Of these cases, α-thalassemia accounted for approximately 40%, β-thalassemia accounted for over 60%, and both α-/β-thalassemia accounted for less than 2%. In our study, out of the 1936 patients, 1301 (67.20%) were female, and 635 (32.80%) were male. Specifically, more than 60% patients were aged between 20 and 40 years, and most were young or middle-aged women. The majority of female patients are aged between 20 and 40 years, a demographic in which females are more likely to focus on anaemia compared to males, with an increased propensity to seek medical attention, primarily due to menstrual blood loss. In 733 patients with α-thalassemia, heterozygous variants accounted for more than 90%, of which --^SEA/αα was the most common. Among 1170 patients with β-thalassemia, heterozygous variants accounted nearly 99.5%, of which β^{IVS-II-654(C>T)}/β^N was the most common, and there was no significant difference compared to previous reports [11, 12]. Although the number of routine α-thalassemia patients in our study was lower than that of routine β-thalassemia, Guo et al. report that α-thalassemia is highly prevalent in Fujian Province, China, with 67.4% of cases being α⁰-thalassemia [13]. Given China’s vast territory, large population, and diverse ethnic groups add complexity to the thalassemia variants. Additionally, the number of patients included may have affected our conclusions. Prospective multicenter large-sample studies are needed to confirm these findings.

There were apparent differences in geographical distribution in this study. Diagnoses of routine thalassemia were mainly from North China, Southwest China, and the middle and lower reaches of the Yangtze River, including 310 cases in Beijing, 180 cases in Hunan, 182 cases in Sichuan, 142 cases in Guangxi, and 129 cases in Hebei. There are some differences between this result and those of other studies, mainly reflected in the high incidence rate in northern areas, such as Beijing and Hebei, and the relatively low incidence rate in high-incidence areas, such as Guangxi. This may be attributed to the fact that most individuals who visit PUMCH for medical care are from northern regions. However, with population migration, the number of patients with thalassemia in northern China has also increased [14, 15].

In general, the majority of thalassemia carriers can be screened by routine hematologic analysis of MCV, MCHC, and MCH, combined with hemoglobin electrophoresis. Therefore, routine α/β-thalassemia genotype examination can help confirm the diagnosis. However, some thalassemia gene variants cannot be detected using typical hematological analyses. Therefore, for suspected cases, further gene sequencing is needed to avoid missed diagnoses, laying the foundation for genetic counseling and prenatal diagnosis [16]. Owing to the lack of effective treatments, enhanced carrier screening and prenatal genetic diagnosis are the most effective measures for reducing the incidence, especially for those with a family history [9, 16].

Rare globin gene variants were detected in 31 patients, including four patients with rare α-thalassemia, 24 with rare β-thalassemia and three with rare deletion. In this study, two new gene variants of α/β-thalassemia cases were identified, including case 4: HBA2: c.300+82G>C; and case 8: HBB:codon85(-T). Which were not been reported in HbVar, HGMD, and PubMed databases. Importantly, two patients presented with low hemoglobin concentrations and ineffective iron supplementation.

Thalassemia is highly heterogeneous. Therefore, studying the incidence, genotype, and frequency of thalassemia in Beijing, China is important. Public education, population screening, and prenatal diagnosis should be the focus for thalassemia prevention and control in Beijing. More than 2,000 variants in the α/β-globin gene have been reported in HGMD and HbVar databases, including SNPs, deletion or insertion and CNVs, and this number will continue to grow as detection techniques continue to improve [17, 18].

In conclusion, we analyzed the hematological characteristics and genotypes of patients with thalassemia in PUMCH, and through the Sanger sequencing, confirmed α/β-globin gene variants HBA2:c.300+82G>C, and HBB:codon85(-T) for the first time. Our findings complement existing data on rare alpha-thalassemia gene types. The results of this study also revealed gene deletions and variants in patients with thalassemia in Beijing, providing valuable basic information for genetic counseling and prenatal diagnosis of thalassemia in Beijing. However, the sample size of this study was small, regional, and mainly concentrated in North China, leading to certain inherent limitations that should be addressed in the future.