Analysis of genotype distribution of thalassemia and G6PD deficiency among Hakka population in Meizhou city of Guangdong Province

Abstract Objective The aim of the study was to explore genotype distribution thalassemia and G6PD deficiency in Meizhou city, China. Methods A total of 16 158 individuals were involved in thalassemia genetic testing. A total of 605 subjects were screened for common Chinese G6PD mutations by gene chip analysis. Genotypes and allele frequencies were analyzed. Results A total of 5463 cases carried thalassemia mutations were identified, including 3585 cases, 1701 cases, and 177 cases with α‐, β‐, and α + β‐thalassemia mutations, respectively. ‐‐SEA (65.12%), ‐α3.7 (19.05%), and ‐α4.2 (8.05%) deletion were the main mutations of α‐thalassemia, while IVS‐II‐654(C → T) (40.39%), CD41‐42(‐TCTT) (32.72%), ‐28(A → G) (10.11%), and CD17(A → T) (9.32%) mutations were the principal mutations of β‐thalassemia in Meizhou. There were significant differences in allele frequencies in some counties. Genetic testing for G6PD deficiency, six mutation sites, and one polymorphism were detected in our study. A total of 198 alleles with the mutation were detected among 805 alleles (24.6%). G6PD Canton (c.1376 G → T) (45.96%), G6PD Kaiping (c.1388 G → A) (39.39%), and G6PD Gaohe (c.95 A → G) (9.09%) account for 94.44% mutations, followed by G6PD Chinese‐5 (c.1024 C → T) (4.04%), G6PD Viangchan (c.871G → A) (1.01%), and G6PD Maewo (c.1360 C → T) (0.51%). There were some differences of the distribution of G6PD mutations among eight counties in Meizhou. Conclusions The ‐‐SEA, ‐α3.7, and ‐α4.2 deletion were the main mutations of α‐thalassemia, while IVS‐II‐654(C → T), CD41‐42(‐TCTT), ‐28(A → G), and CD17(A → T) mutations were the principal mutations of β‐thalassemia in Meizhou. G6PD c.1376 G → T, c.1388 G → A, and c.95 A → G were the main mutations of G6PD deficiency. There were some differences of the distribution of thalassemia and G6PD mutations among eight counties in Meizhou.


| INTRODUC TI ON
Hemoglobin disease and glucose-6-phosphate dehydrogenase (G6PD) deficiency are widespread human erythrocytogenetic diseases that affect millions of population. 1,2 The geographical distribution of G6PD deficiency and thalassemia is closely related to the past and present epidemic of malaria, because it has the selective advantage of anti-malaria infection. 3,4 Thalassemia is an inherited autosomal recessive disease with microcytic hypochromic anemia resulting from reduced or absent synthesis of one or more of the globin chains of hemoglobin. As one of the commonest monogenic disorder in the world, thalassemia assumes diversity in clinical phenotypes varying from almost asymptomatic to lethal hemolytic anemia. [5][6][7] Thalassemia is the most common monogenic disorder in the world and is especially prevalent in Mediterranean countries, Southeast Asia, Africa, Middle East, and in the Indian subcontinent. There are two main types of thalassemia, α and β. 8,9 According to the reports of previous researches, thalassemia was mainly prevalent in the population of southern areas of the Yangtze River in southern China, [10][11][12][13] especially in three most southerly provinces of Guangxi, [14][15][16] Guangdong, 17 and Hainan, 18,19 in which individuals seems to be selective advantages to carry such mutations.
Hereditary G6PD deficiency is one of the most common genetic enzyme deficiency diseases in the world. G6PD deficiency is an X-linked incomplete dominant inherited disease. The G6PD gene is located on chromosome Xq28 which consists of 13 exons and 12 introns, encoding 515 amino acids. The deficiency is widely distributed and occurs in about 400 million people worldwide. 20 G6PD deficiency has an obvious geographical distribution in the mainland China, and it is higher in the provinces south of the Yangtze River, including Guangdong, Hainan, Guangxi, Yunnan, Guizhou, and Sichuan provinces. [21][22][23] Meizhou is a city located in the northeast of Guangdong Province, and most of the residents living in this area are Hakka peoples. Hakka is an intriguing Han Chinese population that mainly inhabit in southern China who migrated to south originally from northern China. 24 Meizhou city consists of eight counties including Wuhua, Fengshun, Dabu, Jiaoling, Meijiang, Meixian, Pingyuan, and Xingning, where custom, lifestyle, and diet manifest some distinctions in certain extent.
Population screening and genetic counseling are important to prevent the birth of children with thalassemia major. Using genetic analysis for prenatal diagnosis can diagnose thalassemia major fetuses in early pregnancy and terminate pregnancy in time, so as to avoid the birth of thalassemia major patients, which is an effective method to prevent this disease at present. Precise mutation frequencies studies in different populations will help healthcare programs to control thalassemia. 25,26 G6PD deficiency causes neonatal hyperbilirubinemia and chronic hemolytic anemia. Although most affected individuals are asymptomatic, exposure to oxidative stressors, such as certain drugs or infection, can elicit acute hemolysis. 27,28 Meizhou is regarded as underdeveloped and backward city in Guangdong Province. As a social medical and health problem, thalassemia has brought great challenges to the development of Meizhou region.
Here, we perform a survey of thalassemia and G6PD deficiency to analyze the feature of genotypes distribution and frequencies among eight counties of Meizhou area.

| Genetic testing for G6PD deficiency
Genomic DNA was extracted from EDTA anticoagulant blood of subjects using the QIAamp DNA Blood Mini Kit (Qiagen).
Amplification was performed using the G6PD Gene Typing Detection Kit (gene chip assay) (Sinochips Bioscience Co.). We

| Statistical analysis
This study used SPSS statistical software version 20.0 (International Business Machines Corporation) to analyze data, and the results would be displayed with corresponding proportion. Descriptive analysis and Pearson chi-square test were used to compare the frequencies of genotype and allele among different counties in Meizhou region. P < .05 was considered to statistical difference. The result of the incidence rate of α-and β-thalassemia of eight counties in Meizhou area is shown in Table 1. A total of 5463 cases of thalassemia were identified including 3585 cases of α-thalassemia, 1701 cases of β-thalassemia, and 177 cases of α-compound β-thalassemia. The total prevalence of thalassemia in Meijiang (31.84%, P = .043) was lower than other counties in Meizhou area. However, the relative proportions of α-and β-thalassemia did not differ greatly among eight counties. The prevalence of α-thalassemia in Meijiang (20.73%, P = .046) was lower than other counties in Meizhou area.

| RE SULTS
The prevalence of Hb H disease in Wuhua (2.31%, P = .008) was higher than other counties in Meizhou area, while in Jiaoling (0.89%, P = .015) was lower than other counties.
Allele frequencies of α-and β-thalassemia of eight counties in Meizhou area are exhibited in Table 2. A total of 5952 mutant chromosomes including 4063 α-thalassemia mutant chromosomes and 1889 β-thalassemia mutant chromosomes were identified.
Compared with other counties, Wuhua assumed higher allele frequencies in both α-thalassemia (13.39%, P = .032) and -α 3.7 deletion (2.75%, P = .045), and higher frequencies in α WS α allele of Xingning  Table 3, showing subtle differences among each county. There were higher genotype frequencies in -  The shaded cells show that there are statistically significant differences compared with other counties.
a Manifests that compared with other counties, P < .05.         The shaded cells show that there are statistically significant differences compared with other counties.

TA B L E 2
a manifests that compared with other counties, P < .05.
thalassemia mutations were identified, and 13 cases with both thalassemia and G6PD deficiency mutations and 46 cases did not carry thalassemia and G6PD deficiency mutations. Due to the small number of cases, we cannot analyze the relationship of thalassemia and G6PD deficiency. Of course, further studies are required to reveal the relationship, which is one of our next major research goals.

| D ISCUSS I ON
Thalassemia is an inherited autosomal recessive disease that is mainly prevalent in Guangdong, Guangxi, and Hainan provinces.
Thalassemia major places a heavy financial burden on some families. [10][11][12]29 Meizhou located in the northeastern part of Guangdong Province is an underdeveloped city, in which the vast majority of permanent residents are Hakka. In result of geography, culture, and customs, the proportion of intermarriage between Hakka people in Meizhou and other regions is relatively small, 24 which consequently triggered higher genetic frequency of thalassemia. In broad terms, due to the higher prevalence and some differences from other cities of Guangdong Province, to make sure whether the eight counties of Meizhou city have some differences is necessary.
In this study, the total incidence of thalassemia in Meizhou region It is reported that the G6PD enzyme activity in patients with G6PD deficiency compound thalassemia may be higher than that of G6PD deficiency patients, due to an increase in the number of newborn erythrocytes and increase in the activity of G6PD in the chronic hemolysis of the G6PD deficiency compound thalassemia patients. [38][39][40] The patients with G6PD deficiency compound thalassemia may have normal G6PD activity, so such patients may be missed in the screening of G6PD activity. We are collecting data from different types of thalassemia genotypes with G6PD gene mutations, and next study may reveal the correlation between thalassemia and G6PD deficiency.
The genotype distribution and frequencies of thalassemia and G6PD deficiency in Meizhou area have regional characteristics, and there are also significant differences in different counties. Local governments can formulate corresponding measures and detection projects to prevent and control thalassemia major and G6PD deficiency according to the genotype distribution and frequencies, effectively saving costs and enhancing social benefits.

| CON CLUS ION
In conclusion, --SEA , -α 3.7 , and -α 4.2 deletion were the main mutations of α-thalassemia, while IVS-II-654(C → T), CD41-42(-TCTT), -28(A → G) and CD17(A → T) mutations were the principal mutations of β-thalassemia in Meizhou. Genetic testing for G6PD deficiency, c.1376 G → T, c.1388 G → A, and c.95 A → G were the main mutations of G6PD deficiency in this region. In addition, there were some differences of the distribution of thalassemia and G6PD mutations among eight counties in Meizhou.

ACK N OWLED G M ENTS
The author would like to thank other colleagues who were not listed