Prevalence and molecular characterization of common thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province in Southwestern China

ABSTRACT Objectives: Thalassemia, the most common global monogenetic disorder, is highly prevalent in southern China. Epidemiological and molecular characterization of thalassemia is important for designing appropriate prevention strategies in high-risk areas, especially the border area of Guangxi-Yunnan-Guizhou province in southwestern China. Methods: We recruited 38812 reproductive age couples and screened them for thalassemia. Routine blood tests as well as hemoglobin components and levels were evaluated. In addition, suspected thalassemia were identified by gap polymerase chain reaction (Gap-PCR) and PCR-based reverse dot blot (PCR-RDB). Results: The overall prevalence of thalassemia was 26.76%. Specifically, incidences of α-thalassemia, β-thalassemia, and concurrent α- and β-thalassemia were 17.52%, 6.92%, and 2.32%, respectively. The diagnosed α-thalassemia anomalies were associated with six gene mutations and 25 genotypes. The β-thalassemia anomalies were associated with 12 gene mutations and 15 genotypes. Moreover, among the 1799 concurrent mutated α- and β-thalassemia genes, 95 different genotypes were identified. Couples in which both partners were positive for α-thalassemia and β-thalassemia isotypes were 8.80% and 2.08%, respectively. The proportion of couples at a risk of having children with thalassemia major or intermedia was high. Conclusions: This study elucidates on the prevalence and molecular characterization of thalassemia in the border area of Guangxi-Yunnan-Guizhou provinces. These findings provide valuable baseline data for genetic counseling and prenatal diagnosis, with the overarching goal of preventing and controlling severe thalassemia.


Introduction
Thalassemia, an autosomal-recessive inherited disorder, is characterized by reduced or absent synthesis of one or several globin peptide chains, which may result in hemolytic anemia [1]. The degree of anemia varies depending on imbalances in production of αand β-like chains. Thalassemia minor may be asymptomatic or may present mild symptoms, whereas thalassemia intermedia is commonly heterogeneous with symptoms varying between severe transfusion-dependent thalassemia major to the mild thalassemia intermedia form. Babies with thalassemia major either die before birth or are born with serious complications that cause significant psychosocial and economic burden to their families and society [2,3]. Thalassemia major is refractory but preventable, with the only effective interventions involving precision genetic analysis on thalassemia intermediate, and preventing the birth of babies reproductive age in the border area of Guangxi-Yunnan-Guizhou provinces in southwestern China. Collectively, our findings provide critical insights for genetic counseling, prenatal diagnosis, and prevention of severe thalassemia in this region.

Ethical statement
This study was reviewed and approved by the Ethical Review Committee of the Affiliated Hospital of You-Jiang Medical College for Nationalities (YYFY-LL-2022-001). Prior to being enrolled in the study, signed informed consents were obtained from all participants. All experiments were performed in accordance with the relevant institutional guidelines and regulations.

Study participants
During their antemarital and prenatal examinations, 38812 couples were recruited in this study and screened for thalassemia in the period from January 2017 to December 2021. These participants were from the border area of Guangxi-Yunnan-Guizhou provinces. This border area, which is a multi-ethnic district, includes the Baise city, the northwest region of Guangxi Province, Wenshan Zhuang, and Miao Autonomous Prefecture in southeastern Yunnan Province, as well as Qianxinan Buyi and Miao Autonomous Prefecture in Guizhou Province.

Statistical analyses
All statistical analyses were performed using SPSS 22.0 software. Gene mutations, frequency, and spectrum of thalassemia were analyzed by a descriptive method. Given that the obtained data did not conform to normal distribution, hematological parameters data are presented as M (P25, P75) and were compared in different genders and genotypes using the non-parametric Wilcoxons rank sum test. p ≤ 0.05 was considered to be statistically significant.

Characteristics and hematological parameters of α-thalassemia
Hematological parameters and molecular characteristics of the top 18 frequent α-thalassemia mutations are shown in Table 5. Due to the low number of cases, the remaining seven genotypes were not included in the analysis. Among participants of different genders and genotypes, hematological and electrophoretic parameters included MCV (fl), MCH (Pg), Hb (g/dl), HbA2 (%), and HbF (%) are shown in Figure 2. A significant difference between genders was observed for Hb, with males exhibiting higher Hb levels compared to females (p < 0.001) ( Figure 2(C)). In partial genotypes, MCV and MCH levels were significantly higher in males than in females (p < 0.05) ( Figure 2(A,B)). Interestingly, in a small number of genotypes, females had higher HbA2 and HbF levels than males ( Figure 2(D,E)).

Characteristics and hematological parameters of β-thalassemia
Characteristics and hematological parameters, including MCV, MCH, Hb, HbA2, and HbF, of the top nine frequent types of β-thalassemia are shown in Table 6.

Discussion
The high frequency of thalassemia in the border area of Guangxi-Yunnan-Guizhou provinces of southwestern China has become a major public health concern. It is associated with a considerable socioeconomic burden. However, currently, data on the prevalence and molecular characterization of thalassemia in this region are not conclusive. The prevalence of thalassemia in this study was higher than that reported in a previous large population-based study conducted in Guangxi, Yunnan, Guizhou, Guangdong, and Hainan provinces [7]. It was also higher than the 24.51% total heterozygote frequency of thalassemia and other haemoglobinopathies reported in the 2010 sampling survey performed in Guangxi province [8]. Moreover, our data showed higher frequencies than those in previous reports [9,10], which may be attributed to the large sample size. Collectively, thalassemia is highly prevalent in the border area of Guangxi-Yunnan-Guizhou provinces, Southwestern China, suggesting that premarital genetic counseling should be performed to prevent the birth of babies with thalassemia major. We identified 25 α-thalassemia genotypes, including 79.13% deletional mutations, 16.96% non-deletional mutations, and 3.91% concurrent deletional  (E) HbF levels among participants with different genders and genotypes. # Males had significantly higher levels (p < 0.05) than females in the same genotypic group, * Males had significantly lower levels (p < 0.05) than females in the same genotypic group. P values for significant differences (p < 0.05) between genotypes of α-thalassaemia are shown. Non-listed comparisons are not significant. and non-deletional mutations ( Table 1). The five most frequent α-thalassemia genotypes were --SEA /αα, -α 3.7 / αα, α CS α/αα, -α 4.2 /αα, and α WS α/αα genotypes, which accounted for 89.83% of all α-thalassemia genotypes. The spectrum of mutations detected in this study was similar to the findings observed in southern regions of China [9,11,12]. These results form the basis for designing efficient and cost-effective prevention interventions for α-thalassemia major and intermedia.
Patients with mild concurrent αand β-thalassemia mostly exhibited elevated HBA2 and/or HbF levels and had characteristics of β-thalassemia heterozygotes (Figure 4(D,E)), therefore, the characteristics of α-thalassemia were masked. The high proportions (2.32%) of concurrent αand β-thalassemia suggests that careful screening of α-thalassemia should be performed in all β-thalassemia patients to obtain accurate genotypes, thereby avoiding misinterpreting the appropriate risk assessment and counseling, especially in prenatal diagnosis [17,18]. In this study, concurrent αand β-thalassemia that compounded with α + were associated with significantly low MCV, MCH and Hb levels, compared to α-thalassemia alone group.
However, most of the genotypes that combined with α 0 had significantly elevated MCV, MCH and Hb levels. Eighteen cases of concurrent αand β-thalassemia were characterized with intermediate disease phenotypes, including three cases of -α 3.7 /αα/β CD26 /β N , two cases of -α 3.7 /αα/β IVS-II-654 /β N and α CS α/αα/β IVS-I-1 / β N respectively, and --SEA /αα/β CD41-42 /β N , α 3.7 /αα/ β CD17 /β N , -α 4.2 /αα/β CD17 /β N , --SEA /αα/β IVS-I-1 /β N , α CS α/ αα/β IVS-II-654 /β N , etc. other 6 genotypes of one case each. However, the reason is not yet clear. Notably, the βthalassemia phenotype was not exacerbated in the rest of concurrent αand β-thalassemia individuals apart from genotype --SEA /αα/β CD26 /β N , compared to the β-thalassemia alone group, nearly all genotypes have significant higher value of MCV and MCH, and parts genotypes have significant higher value of Hb. In the same β-thalassemia genotype, individuals with α 0 had higher MCV, MCH and Hb levels, relative to those with α + . The MCV, MCH and Hb levels increased with decreasing copy numbers of the functional αglobin gene. This is because, the synthesis of both α and β peptide chains is reduced in concurrent αand β-thalassemia, and the severity of clinical symptoms in thalassemia patients mainly depend on imbalances of α/β peptide chains [19][20][21]. Differences in levels of some hematological parameter indicators between concurrent αand β-thalassemia and α-thalassemia or β-thalassemia alone group were significant. However, it is challenging to strictly distinguish the single types or compound types of thalassemia for the detection of a single sample. Therefore, differentiating between the two cannot be reliably done by using hematological phenotypes alone, it must be based on accurate molecular diagnosis.
A limitation of this study is that we only analyzed common thalassemia. Given the rapid development of molecular diagnostic techniques in recent years, deficiencies and inadequacies of conventional thalassemia gene screening methods have gradually emerged. Table 6. Characteristics and hematological parameters of β-thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province, Southwestern China, M (P25, P75). The HbA2% level of genotype β CD26 /β N was actually the sum of HbA2% and HbE%. (E) HbF levels among participants with different genders and genotypes. # Males had significantly higher levels (p < 0.05) than females in the same genotypic group, * Males had significantly lower levels (p < 0.05) than females in the same genotypic group. P values for significant differences (p < 0.05) between genotypes of β-thalassaemia are shown. Non-listed comparisons were not significant. The HbA2% level of genotype β CD26 /β N was the sum of HbA2% and HbE%, and was not compared with other genotypes.
According to the HbVar Database of Hemoglobin Variants and Thalassemia Mutations (http://globin.bx.psu. edu/hbvar/), as of April 2022, 536 different mutations of thalassemia have been globally characterized. Conventional commercial kits fail to detect rare types of thalassemia, resulting in misdiagnosis. Among the 38812 couples involved in this study, 3830 cases were positive for thalassemia screen but non-iron deficiency, suggesting rare types of thalassemia and/or hemoglobinopathies. From the 3830 cases, we detected 100 samples with rare thalassemia gene mutations using a next-generation sequencing-based (NGS-based) approach 'Dx0593 thalassemia gene detection (α +β)508plus' by Shenzhen Huada Gene Co., Ltd. Specifically, 25 cases had rare thalassemia gene mutations, including --THAI /αα, HKαα/-α 4.2 , -α 21.9 /αα, HBA2:c.184A > T, SEA-HPFH, β −86 /β N , and Chinese G γ + ( A γδβ) 0 among others, while 17 cases had hemoglobinopathies, including HBA2:c.344C > A, HbJ-Wenchang-Wuming, HbG-Coushatta, and Hb G-Honolulu among others. These results suggest that a constant number of rare thalassemias and abnormal hemoglobinopathies exist in the 3830 samples. The NGS-based approach identified both annotated and novel variants, and common and rare mutations in carriers with or without thalassemic trait phenotypes, thereby significantly improving the detection of carrier status, and ultimately decreasing the risk of missed diagnoses and misdiagnoses [7,[22][23][24]. Nevertheless, taking the cost implications and clinical significance into account, screening the priority population for NGS is necessary and there should be economic and policy support. Priority population for NGS testing are: Couples who previously had a child with thalassemia major or intermedia, but either one or both husband and wife were negative for common thalassemia; One of the couples positive for the common thalassemia; Individuals with obvious phenotypes, such as anemia or with abnormal hemoglobin band in hemoglobin analysis but negative for common thalassemia.
Given that there is no effective treatment option for thalassemia, there is an urgent need to prioritize thalassemia prevention and control in mainland China [25,26]. Immediate and concerted actions for thalassemia prevention and control should be mandatory in high prevalence areas [27,28]. Provision of appropriate genetic counselling, prenatal diagnosis and interventions to thalassemia carriers can prevent and control severe thalassemia. In this study, the proportions of couples found to be at risk of having children with thalassemia major and thalassemia intermedia were high (Table 4). Therefore, individuals in this high prevalence region should be subjected to premarital thalassemia major and intermedia screening. Since thalassemia intermedia patients have a high genetic heterogeneity and broad phenotypic diversity [29,30], they should be carefully screened for all known disease-causing modifier variants and mutations, with the overarching goal of providing critical clinical information insights into their potential offsprings, and facilitating precise diagnosis and genetic counselling.
Currently, thalassemia diagnosis relies on routine blood examination combined with blood hemoglobin electrophoresis and thalassemia genetic gene detection. Characteristics and hematological parameters have a potential role in thalassemia genotyping [13,31]. The characteristics and hematological parameters in thalassemia patients vary with lifestyle, race, and altitude [32]. Nevertheless, a limited number of studies have described routine blood parameters, electrophoretic detection and quantification for screening thalassemia in the Chinese population. In this study, Hb levels was significantly different between males and females in the same genotype, whether α-thalassemia or β-thalassemia. The Hb reference value for males was higher than that of females. Table 7. Characteristics and hematological parameters of concurrent αand β-thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province, Southwestern China, M (P25, P75).  (E) HbF levels among participants with different genders and genotypes. # Males had significantly higher levels (p < 0.05) than females in the same genotypic group; △ Compared to β-thalassemia alone group in the same gender, concurrent αand β-thalassemia group had significantly higher levels (p < 0.05); ⋆ compared to α-thalassemia alone group in the same gender, concurrent αand β-thalassemia group had significantly higher levels (p < 0.05). ▴ Compared to β-thalassemia alone group in the same gender, concurrent αand β-thalassemia group had significantly lower levels (p < 0.05); ★ compared to the αthalassemia alone group in the same gender, concurrent αand β-thalassemia group had significantly lower levels (p < 0.05). Nonlisted comparisons were not significant. The HbA2% level of genotype --SEA /αα/β CD26 /β N was the sum of HbA2% and HbE%, and was not compared with other genotypes except, β CD26 /β N .
Pregnant women, especially those with thalassemia, were more likely to be anaemic, the reasons and mechanisms, however, should be further investigated. In our study, pregnant women accounted for the majority of females. These may explain why males had significantly higher Hb levels, relative to females with the same genotype. Furthermore, males and females with partial genotypes had different MCV, MCH, HbA2, and HbF levels. The higher Hb levels in males were comparable to those found by Zhang et al. [32]. But, differences in MCV, MCH, HbA2, and HbF levels between males and females were inconsistent with results reported by Zhang et al. [32]. In this study, the prevalence of β CD26 /β N was 0.42% (328/77624). However, HPLC could not differentiate between HbA2 and HbE because they had the same elution time, hence, capillary electrophoresis techniques are recommended in future studies. Our findings form the basis for future studies on thalassemia.
In summary, this large-scale analysis of the prevalence and molecular characterization of common thalassemia provides valuable knowledge on thalassemia in China. Thalassemia is highly prevalent in the border area of Guangxi-Yunnan-Guizhou provinces, Southwestern China, and the thalassemia genotypes are characterized by a huge genetic diversity. Collectively, these findings provide valuable baseline data for prenatal diagnosis and genetic counseling in the region.

Disclosure statement
No potential conflict of interest was reported by the author(s).