Prevalence and molecular characterization of alpha and beta-Thalassemia mutations among Hakka people in southern China

Abstract Our aim was to investigate molecular features of thalassemia for proper clinical consultation and prevention in Heyuan. In our research, a total of 25,437 positive screening subjects were further subjected to a genetic analysis of α-thalassemia (α-thal) and β-thalassemia (β-thal). The deletion of α-thal mutation was tested by Gap-PCR, while the non-deletion of α-thal and β-thal mutation were identified by the PCR-reverse dot blot (PCR-RDB) technique. Nested PCR detected Hkαα/-- SEA and Hkαα/αα. Among the 25,437 positive screening subjects, 44.09% (11216/25437) subjects were bearers of thalassemia variations, and 30.85% (7847/25437) subjects showed α-thal changes alone. Among the 23 genotypes with α-thal mutation alone, the three common genotypes were --SEA/αα(68.34%), -α3.7/αα(16.44%), and -α4.2/αα(6.38%). Of the 11.50% (2924/25437) subjects and 29 genotypes with β-thal mutation alone, the three common genotypes were βCD41-42/βN(36.22%), βIVS-II-654/βN(30.88%), and β-28/βN(13.47%). Additionally, of the 1.75% (445/25437) subjects and 55 genotypes showed both α- and β-thal mutations. We also identified 269 cases of Hb H and six patients of Hkαα. Furthermore, the common genotypes of α-thal and β-thal mutations were consistent with allele frequencies of mutations. Our study establishes molecular features of thalassemia among Hakka people in Heyuan. It will be useful for developing strategies to prevent thalassemia.


Introduction
Thalassemia is an inherited chronic hemolytic disease on account of the deletion or point mutation of the human globin gene. According to the type of defective gene, thalassemias are divided into α-, β-, γ-, δ-, δβ-, and εγδβ-thalassemias. Among them, α-thalassemia (α-thal) and β-thalassemia (β-thal) were the two main types (Barnett, 2019;Shafique et al., 2021;De Simone et al., 2022). Carriers of thalassemia mutations account for about 5% of the global population, particularly in tropical and subtropical areas, for instance, North Africa, the Mediterranean region, India, Southeast Asia, Pakistan, and the Middle East (Merkeley and Bolster, 2020). In China, thalassemia is mainly found in the south of the Yangtze River, especially in the Guangxi, Hainan, and Guangdong regions (Yang et al., 2019;Cheng et al., 2022).
Human hemoglobin has three different developmental stages in the embryo, fetus, and adult. At the molecular level, hemoglobin synthesis is determined by two multigene clusters which are located in chromosome 16 and chromosome 11, respectively (Merkeley and Bolster, 2020;Shafique et al., 2021). The underlying molecular defects in the α-globin or β-globin gene clusters constitute the foundation of hemoglobin synthesis defects and the various genetic forms of α-or β-thal (Taher et al., 2018;Viprakasit and Ekwattanakit, 2018). According to the severity of clinical symptoms, thalassemia is primarily divided into thalassemia trait (TT), thalassemia intermedia (TI), and thalassemia major (TM). TI and TM are generally identified as thalassemia patients (Taher et al., 2018;Viprakasit and Ekwattanakit, 2018). The heterozygous state of α⁺-thal (αα T /αα,-α/αα,α T α/αα) or α 0 -thal (-α/-α, --/αα,-α/αα T ) is general clinically microcytic anemia or asymptomatic. While α-TI also called hemoglobin H disease (Hb H) (--/-α, --/α T α) is usually due to the compound heterozygous types for α⁺-thal and α 0 -thal. The homozygous types for α 0 -thal lead to hemoglobin Bart's (--/--), which gives rise to death in utero or shortly after birth (Farashi and Harteveld, 2018;Kalle Kwaifa et al., 2020). The β-thal heterozygous state (β + /β N , β 0 /β N ), which is called β-thalassemia trait or β-thalassemia minor, usually presents as asymptomatic microcytic anemia. Both β-thalassemia intermedia (β-TI) and β-thalassemia major (β-TM) arise from compound heterozygotes or homozygous β-globin mutations. Patients with β-TM usually depend on blood transfusion throughout life because they suffer from severe anemia since infancy. In contrast, patients with β-TI show considerable heterogeneity and may have mild or moderate anemia, and thus have different blood transfusion requirements (Jaing et al., 2021, Taher et al., 2021. The clinical phenotype of thalassemia ranges from asymptomatic to fatal hemolytic anemia. So far, studies have shown that hematopoietic stem cell transplantation (HSCT) can cure thalassemia. Due to family economic conditions, post-transplant responses, and donor sources, a large number of patients still choose to receive long-term, massive blood transfusion therapy to inhibit the production of erythropoietin, reduce the incidence of skeletal deformities, and maintain the normal growth and development of patients with TM Zeng et al. 2 (De Simone et al., 2022). For that matter, it is significant to implement the genetic testing of thalassemia in the population of childbearing age and prenatal diagnosis for high-risk groups of thalassemia in the first and second trimesters to prevent the birth of the fetus with TM. As of May 2022, more than 948 mutant forms of the β-gene and 827 mutant forms of the α-gene have been identified, which are described in detail in the HbVar database (https://globin.bx.psu.edu/) According to investigation, the Hakkas with unique genetic characteristics are considered Han Chinese people that primarily live in southern China. Many studies have shown significant genetic heterogeneity among Hakka people in different regions (Zhao et al., 2018;Ma et al., 2021). As one of the primary residences of the Hakkas, Heyuan City is situated in the northern mountainous area of Guangdong Province, China ( Figure 1). Knowingly, the occurrence of thalassemia in Guangdong Province is so high. However, the prevalence and molecular characterization of α-thal and β-thal mutations among Hakka people in Heyuan so far have not been reported.
In this study, we retrospectively performed a survey on thalassemia genotypes among the Hakka population in Heyuan. This study aims to reveal the prevalence and molecular characterization of α-and β-thal mutations among Hakka people, provide the scientific basis for the prevention and control of thalassemia, and construct a detailed frequency map of the spectrum of thalassemia mutation in Heyuan. This study will provide comprehensive data on the prevalence of thalassemia in Heyuan, providing a valuable reference for the government to formulate thalassemia prevention and control policies.

Study population
From October 2010 to August 2020, a total of 25,437 participants who came to the Heyuan Women and Children Hospital carried out α-thal and/or β-thal genetic testing. We eliminated 118 patients who were repeatedly tested in the inpatient or outpatient department. The age of the patients varied from a few days to 91 years old (median age: 24 years), and all the participants or their families obtained informed consent. This study was endorsed by the Medical Ethics Committee of the Heyuan Women and Children Hospital.

Hematological analysis
The blood samples were obtained from the peripheral venous system of all subjects, which were collected in an anticoagulation tube containing ACD or EDTA-k 2 . Routine hematological examinations were performed using a hemocytometer (Sysmex XS-1800i, Japan). A capillary electrophoresis system (Sebia, France) was applied to analyze the levels of hemoglobin components. The subjects who met the following criteria were considered as suspected carriers of thalassemia and checked for further genetic analysis: (1) mean corpuscular volume (MCV) <82 fL and/or mean corpuscular hemoglobin (MCH) <27 pg; (2) HbA 2 <2.5% or HbA 2 >3.5% or HbF >2.0%; (3) abnormal haemoglobin zone; (4) parents or siblings carried the thalassemia gene mutation (Huang et al., 2019;Yang et al., 2019;Cai et al., 2021).

DNA extraction and molecular analysis
The genomic DNA of suspected thalassemia subjects was obtained by the extraction kit (Zee san, China) and stored at -20 °C for subsequent tests. The concentration and purity of DNA were assessed by the NanoDrop 2000c UV-Vis spectrophotometer (Thermo Fisher, USA). The protocols are strictly following the manufacturer's instructions. The deletion of α-thal mutation (--SEA /, -α 4.2 /, and -α 3.7 /) was tested by Gap-PCR. The PCR-RDB technique was applied to test the non-deletion α-thal mutations (α CS α/, α QS α/ and α WS α/), and 17 types of β-thal mutations,  (Munkongdee et al., 2020;Wang et al., 2021;Zhuang et al., 2021). Hkαα/--SEA and Hkαα/αα was detected by nested PCR which was confirmed by Shenzhen Yaneng Biotechnology Co. Ltd.

Statistical analyses
We used SPSS19.0 (IBM, USA) and Excel (Microsoft, USA) software to analyze the data. We used n% to express the constituent ratio. P<0.05 was used for a statistical cut-off.