Large-Scale Screening of T halassemia in Ji’an, P.R. China

Background: To evaluate the prevalence of alpha-and beta-thalassemia in Ji'an City, Jiangxi Province, 28,941 residents in the region were genetically screened to identify various thalassemia genotypes. Methods: High-throughput amplicon sequencing was used to screen 301 thalassemia alleles in 28,941 people in the region. Pregnant women were the focus of this screening, and if a pregnant woman had mutations in a thalassemia-causing gene, her spouse was also genetically screened. Results: Of the participants, 2,380 people were carriers of thalassemia, with at least one thalassemia allele, including 1,694 alpha-thalassemia carriers, 638 beta-thalassemia carriers and 48 composite alpha and beta-thalassemia carriers. In Ji‘an, the total carrying rate of thalassemia was 8.22%, while alpha-and beta-thalassemia were 5.85% and 2.20%, respectively. In addition, the first measured carrier rate of composite alpha- and beta-thalassemia in Ji'an was 0.17%. According to the geographical distribtion of the 1,694 alpha -thalassemia carriers, the city with the highest carrier rate was Suichuan, followed by Wan’an and Taihe. According to the geographical distribution of the 638 beta-thalassemia carriers, the top three cities with high carrier rates were Suichuan, Wan'an and Xiajiang, sequentially. Conclusions: This research demonstrates the critical nature of large-scale population screening and that comprehensive molecular epidemiology data are necessary for the proper prevention and treatment of thalassemia. The updated epidemiological data from this study may help the local government to focus on the severity of this disease and find some methods for resource allocation that is successful under limited resource conditions.


Background
Hemoglobinopathy is a term that refers to any hereditary blood disease caused by an abnormal hemoglobin molecular structure or an abnormal rate of globin peptide synthesis (thalassemia) [1].
According to the affected globin gene, thalassemia is classified as alpha-thalassemia and beta-thalassemia.
Clinically, there are many symptoms of thalassemia, from asymptomatic to lethal. Patients with such symptoms might be classified as having thalassemia minor, intermedia and major according to the clinical severity. Thalassemia is a condition that affects patients with the later two categories. The severity of this condition is primarily determined by the degree to which the α:non-α chain is imbalanced [2]. This monogenic disease is one of the most prevalent and harmful in the world, affecting the greatest number of people [3,4]. It is estimated that 1-5% of the global population are carriers of thalassemia mutations [5].
Alpha-and beta-thalassemia are a growing health burden in a number of Asian nations [6]. Plans aimed at preventing and effectively managing these diseases have the potential to considerably improve health indexes in many developing countries [7]. Accurate population frequency data are necessary for development of these programs.
In China, the high-incidence areas of thalassemia include Guangdong, Guangxi, Hainan, Yunnan, Guizhou, Sichuan, Chongqing, Hunan, Jiangxi and other southern regions [8,9]. At present, apart from hematopoietic stem cell transplantation, there is no effective treatment for thalassemia major. Numerous individuals require lifelong blood transfusions and chelation therapy, and the only effective preventive methods for thalassemia major and thalassemia intermedia are carrier screening and prenatal diagnosis [10]. For the correct prevention and treatment of thalassemia illness, comprehensive molecular epidemiology data are required. Thus far, significant volumes of genomic data have been generated by next-generation sequencing (NGS) in order to characterize people's genetic makeup and assess potential health concerns. The scope of variation in thalassemia appears to be more than previously described [11], and NGS is an excellent approach for screening for thalassemia-related variation.
Although a large-scale investigation of thalassemia has been conducted in recent years in various regions of China [4,8,12], the epidemiologic characteristics of thalassemia in people from Jiangxi Province, one of the high-prevalence areas, and particularly in those from Ji'an City, remain unknown [13]. Ji'an is located

Participants
In this study, our strategy was to screen pregnant women in the Ji'an region with high-throughput amplicon sequencing for the 301 thalassemia alleles (Table S1). This is a government-funded public health service program, and all pregnant women in the jurisdiction chose to participate in the free testing.
If a pregnant woman is found as a thalassemia carrier, her spouse must also consent to a genetic test for thalassemia. The screening procedure in detail is depicted in Figure 1.

NGS library preparation
As previously described, a series of primers based on the features of three genes associated with alpha-and beta-thalassemia, HBA1, HBA2 and HBB, were created to facilitate gene amplification [4].

Bioinformatics analysis
We employed an in-house pipeline that includes HbVar [12] and IthaGenes [14] Database was used to annotate the detected SNPs and Indels. The mutations were named in accordance with the published literature [15]. The complete procedure is depicted in Figure 2.

Thalassemia carriers identified by NGS
A total of 28,941 people were involved in thalassemia gene screening via NGS. Among these participants, 2,380 people were diagnosed as carriers of thalassemia, including 1,694 carriers of α-thalassemia, 638 carriers of beta-thalassemia and 48 carriers of composite alpha-and beta-thalassemia (Table S2).
The total carrier rate of thalassemia in Ji'an was 8.22%, and the carrier rates of alpha-and betathalassemia were 5.85% and 2.20%, respectively. In addition, the first measured incidence rate of composite alpha-and beta-thalassemia in Ji'an was 0.17%.   (Table 4).

Geographical distribution of thalassemia carriers in Ji'an
The total carrier rate of thalassemia in Ji'an was 8.22%, and the carrier rate in the southern counties was the highest (Figure 3a). Suichuan has the highest carrier rate of the 1,694 alpha-thalassemia carriers, followed by Wan'an and Taihe (Figure 3b). Suichuan, Wan'an, and Xiajiang were the top three cities with the highest carrier rates, sequentially, according to the geographical distribution of the 638 betathalassemia carriers (Figure 3c). Moreover, we also counted the carriers of αα/--SEA , the most prevalent genotype of alpha-thalassemia. As illustrated in     were carriers of alpha-and beta-thalassemia, respectively, and that the incidence rate of composite alpha-and beta-thalassemia was 0.17%. Ji'an's southern counties had a high proportion of thalassemia carriers and varied geographically.
The carrier rate of alpha-thalassemia has been found to be substantially higher than in Nanchang (1.49%) and Xinyu (2.2%), but slightly lower than in Ganzhou (7.19%) in Jiangxi [13]. The carrier rate of beta-thalassemia is higher than in Nanchang (1.14%), Xinyu (1.7%) and also slightly lower than Ganzhou (2.3%) [13]. Furthermore, the alpha-thalassemia and beta-thalassemia composite carrier rates in Jiangxi are similar to those in Ganzhou(0.18%) [13]. The most prevalent alpha-thalassemia mutation was αα/--SEA , which was consistent with earlier observations [16,17]. Codons 41/42 (-TTCT) and IVS-II-654 (C> T) were the two most prevalent beta-thalassemia alleles. The ranking of the two main alleles differed from those found in prior Jiangxi Province studies [13]. We think this may be caused by the new genetic screening method. Our research indicated that the carrier rates of alpha-and beta-thalassemia in this area were significantly lower than those in Guangdong Province (11.07%) [16] and Guangxi Zhuang Autonomous Region (24.51%) [18], two high-incidence areas of alpha/beta-thalassemia. The frequency spectrum of alpha-and beta-thalassemia mutations in this study is similar to that previously described in South China, e.g., Chenzhou [12]. For the composite genotypes, αα/--SEA and IVS-II-654 (C>T) /β N were more than other genotypes. The thalassemia carrier rate in South China was higher than that in North China, similar to the general geographical distribution of people with thalassemia in China. We also found a significant difference between the common mutation of alpha-thalassemia, αα/--SEA , and the common mutation of beta-  Hospital of Ji'an, and all the participants signed written informed consent. Written consent was obtained from parents/legal guardians of any participants under 16 years of age.

Consent to publish
Not applicable

Competing interests
The authors declare that they have no competing interests.

Funding
No funding was obtained for this study.

Authors' Contributions
LWM performed the data generation, processing, and analysis and prepared the paper and figures; YQ,SPC,HL and HRW contributed to data analysis and interpretation; LPG, JH, XW, YL contributed to result interpretation; JX, YF designed the study and carried out sample collection, storage and transport; All authors have read and approved the manuscript.