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.

KEYWORDS:

• Thalassemia
• common thalassemia
• prevalence
• molecular characterization
• people of reproductive age
• spectrum
• genotype
• gene mutation

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 α- and 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 with thalassemia major by detection of carriers and prenatal genetic diagnosis [4,5].

Globally, thalassemia is common in Mediterranean countries, Middle East, Indian subcontinent, and Southeast Asia [6]. In China, thalassemia is prevalent in southern regions, with Guangxi, Yunnan, Guizhou, Guangdong, and Hainan provinces being the most affected [7]. However, currently, there is little information on epidemiology of thalassemia in the border area of Guangxi-Yunnan-Guizhou provinces, an underdeveloped minority nationality region, which greatly affects genetic counseling and prenatal diagnosis. Therefore, we aimed at exploring thalassemia gene mutations, and uncovering the characteristics as well as hematological parameters of thalassemia genotypes in the border area of Guangxi-Yunnan-Guizhou provinces, southwest China, during a 5-year period ranging from 2017 to 2021. We found a high prevalence of thalassemia, with diversity mutation spectrums among people of 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.

Materials and methods

Ethical statement

This study was reviewed and approved by the Ethical Review Committee of the Affiliated Hospital of YouJiang 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.

Thalassemia screening

EDTA-K2-anticoagulated peripheral blood samples were collected from each participant. Next, blood cell parameters were determined by full automatic blood cell analysis (XN-1000, Sysmex, Japan), followed by analysis of hemoglobin components and levels by high-performance liquid chromatography (Variant II, BIO-RAD, USA). In conjunction with the local thalassemia prevention and control program, thalassemia screening was positive if it met any one of the following criteria: (1) Corpuscular volume (MCV) <82.5fL; (2) Mean corpuscular Hb (MCH) levels <27pg; (3) Hemoglobin A2 (HbA2) >3.5%; (4) HbA2 < 2.5%; (5) Fetal hemoglobin (HbF) >2.0%; and (6) Abnormal hemoglobin peaks. All individuals who were positive for thalassemia during screening were subjected to genetic testing.

Genetic analysis

Genomic DNA was extracted from peripheral blood using a genomic DNA isolation kit (Yaneng Biological Technology Co., Ltd., Shenzhen, China). The three common types of deletional α-thalassemia (-α^3.7, -α^4.2, and --^SEA) were detected by gap polymerase chain reaction (Gap-PCR). In addition, the three non-deletional types of α-thalassemia [Hb Constant Spring (Hb CS), Hb Quong Sze (Hb QS), and Hb Westmead (Hb WS)] and the 17 β-globin gene mutations [CD41/42 (-TTCT), CD43 (GAG-TAG), IVS-II-654 (C-T), -28 (A-G), -29 (A-G), -32 (C-A), CD71/72 (+A), CD26 (GAG-AAG), CD17 (AAG-TAG), CD31 (-C), CD14/15 (+G), CD27/28 (+C), IVS-I-1 (G-T), IVS-I-5 (G-C), 5'UTR;+40-43 (-AAAC), initiation codon (ATG-AGG), and -30 (T-C)] were genotyped by PCR-based reverse dot-blot (PCR-RDB) (Yaneng Biological Technology Co., Ltd., Shenzhen, China). All procedures were performed in accordance with the manufacturer’s instructions.

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.

Results

Prevalence of common thalassemia

Among the 38812 couples, 15231 couples had one or both sides of the couple being positive for thalassemia during screening, and thus, further genetic testing was performed. Among them, 20770 cases were diagnosed with thalassemia, 5862 cases had iron deficiency, while 3830 cases were retained for further analysis. The overall incidence of thalassemia was 26.76% (20770/77624) (Figure 1(A)), while incidences of α-thalassemia, β-thalassemia, and concurrent α- and β-thalassemia were 17.52% (13596/77624), 6.92% (5375/77624), and 2.32% (1799/77624), respectively (Figure 1(B)). Moreover, the ratios of α-thalassemia, β-thalassemia, and concurrent α- and β-thalassemia were 65.46%, 25.88%, and 8.66%, respectively (Figure 1(C)).

Figure 1. (A) The prevalence of common thalassemia; (B) The prevalence of α-thalassemia, β-thalassemia, and concurrent α- and β-thalassemia; (C) The constituent ratio of α-thalassemia, β-thalassemia, and concurrent α- and β-thalassemia.

α-thalassemia genotypes

Twenty-five genotypes of α-thalassemia, which were highly abundant and diverse, were identified and are presented in order of abundance in Table 1. Genotype --^SEA/αα (46.76%) was the most frequent deletional mutation, followed by −α^3.7/αα (20.49%) and −α^4.2/αα (7.16%) genotypes. In order of decreasing frequency, the three non-deletional mutations were α^CSα/αα (10.54%), α^WSα/αα (4.88%), and α^QSα/αα (0.94%) genotypes. The top five most frequent double heterozygotes or homozygous genotypes were ^--SEA/-α^3.7 (2.24%), ^--SEA/α^CSα (1.15%), -α^3.7/-α^3.7 (0.91%), ^--SEA/-α^4.2 (0.88%), and ^--SEA/α^WSα (0.85%).

Table 1. Genotyping of α-thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province, Southwestern China.

Genotype	Phenotype	No. patients detected	Constituent ratio (%)
^--^SEA/αα	α^0/α	6357	46.76
-α^3.7/αα	α^+/α	2786	20.49
α^CSα/αα	α^+/α	1433	10.54
-α^4.2/αα	α^+/α	973	7.16
α^WSα/αα	α^+/α	664	4.88
^--^SEA/-α^3.7	α^0/α^+	304	2.24
^--^SEA/α^CSα	α^0/α^+	157	1.15
α^QSα/αα	α^+/α	128	0.94
-α^3.7/-α^3.7	α^+/α^+	124	0.91
^--^SEA/-α^4.2	α^0/α^+	119	0.88
^--^SEA/α^WSα	α^0/α^+	116	0.85
-α^3.7α/α^CSα	α^+/α^+	102	0.75
-α^3.7/-α^4.2	α^+/α^+	82	0.60
-α^3.7α/α^WSα	α^+/α^+	59	0.43
α^CSα/α^CSα	α^+/α^+	42	0.31
-α^4.2α/α^WSα	α^+/α^+	39	0.29
-α^4.2α/α^CSα	α^+/α^+	35	0.26
α^WSα/α^CSα	α^+/α^+	29	0.21
-α^3.7α/α^QSα	α^+/α^+	12	0.09
-α^4.2/-α^4.2	α^+/α^+	12	0.09
^--^SEA/α^QSα	α^0/α^+	8	0.06
α^CSα/α^QSα	α^+/α^+	8	0.06
-α^4.2α/α^QSα	α^+/α^+	4	0.03
α^WSα/α^WSα	α^+/α^+	2	0.01
α^QSα^/α^QSα	α^+/α^+	1	0.01
Subtotal	–	13596	100

α⁰, indicates absent synthesis of α-globin peptide chain; α⁺, indicates reduced synthesis of α-globin peptide chain; α, indicates no mutation.

β-thalassemia genotypes

Fifteen different genotypes were found in 5375 β-thalassemia subjects. Among them, β^CD17/β^N and β^CD41-42/β^N were the dominant genotypes with frequencies of 43.39% and 33.38%, respectively, accounting for the vast majority of β-thalassemia patients (76.77%). In addition, β^CD26/β^N, β^IVS-I-1/β^N, β^CD71-72/β^N, β^-28/β^N, and β^IVS-II-654/β^N genotypes accounted for 6.10%, 4.91%, 3.91%, 3.80%, and 2.83% of β-thalassemia patients, respectively. Proportions of the other eight genotypes were all less than 1% (Table 2).

Table 2. Genotyping of β-thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province, Southwestern China.

Genotype	Phenotype	No. patients detected	Constituent ratio (%)
β^CD17/β^N	β^0/β^N	2332	43.39
β^CD41-42/β^N	β^0/β^N	1794	33.38
β^CD26/β^N	Hb E	328	6.10
β^IVS-I-1/β^N	β^0/β^N	264	4.91
β^-28/β^N	β^+/β^N	210	3.91
β^CD71-72/β^N	β^0/β^N	204	3.80
β^IVS-II-654/β^N	β^+/β^N	152	2.83
β^CD43/β^N	β^0/β^N	44	0.82
β^-29/β^N	β^+/β^N	36	0.67
β^CD 27-28/β^N	β^0/β^N	6	0.11
β^CD14-15/β^N	β^0/β^N	1	0.02
β^CAP/β^N	β^+/β^N	1	0.02
β^-28/β^CD17	β^+/β^0	1	0.02
β^-28/β^CD41-42	β^+/β^0	1	0.02
β^-28/β^IVS-I-1	β^+/β^0	1	0.02
Subtotal	–	5375	100

β⁰, absent synthesis of β-globin peptide chain; β⁺, reduced synthesis of β-globin peptide chain; N, no mutation.

Genotypes of concurrent α- and β-thalassemia

Among the 1799 concurrent mutated α- and β-thalassemia genes, 95 different genotypes were identified. Table 3 shows the top 35 frequent types, which accounted for 92.50% of all concurrent α- and β-thalassemia genotypes. It was found that ^--SEA/αα/β^CD17/β^N was the most prevalent type, accounting for 14.06%. The next five frequent mutations were ^--SEA/αα/β^CD41-42/β^N (12.06%), -α^3.7/αα/β^CD17/β^N (10.12%), -α^3.7/αα/β^CD41-42/β^N (9.78%), α^CSα/αα/β^CD17/β^N (5.61%), and α^CSα/αα/β^CD41-42/β^N (4.56%). The remaining 60 genotypes, which all accounted for less than seven cases, are not shown in Table 3. Among them, -α^3.7/-α^3.7/β^CD41-42/β^N, -α^3.7α/α^WSα/β^CD17/β^N, -α^4.2/αα/β^-28/β^N, and α^QSα/αα/β^CD41-42/β^N genotypes were all found in six cases, whereas ^--SEA/α^WSα/β^CD17/β^N and α^CSα/αα/β^CD26/β^N genotypes were both found in five cases.

Table 3. Genotyping of concurrent α- and β-thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province, Southwestern China.

Genotype	Phenotype	No. patients detected	Constituent ratio (%)
^--^SEA/αα/β^CD17/β^N	α^0/α/β^0/β^N	253	14.06
^--^SEA/αα/β^CD41-42/β^N	α^0/α/β^0/β^N	217	12.06
-α^3.7/αα/β^CD17/β^N	α^+/α/β^0/β^N	182	10.12
-α^3.7/αα/β^CD41-42/β^N	α^+/α/β^0/β^N	176	9.78
α^CSα/αα/β^CD17/β^N	α^+/α/β^0/β^N	101	5.61
α^CSα/αα/β^CD41-42/β^N	α^+/α/β^0/β^N	82	4.56
-α^4.2/αα/β^CD17/β^N	α^+/α/β^0/β^N	76	4.22
α^WSα/αα/β^CD41-42/β^N	α^+/α/β^0/β^N	71	3.95
α^WSα/αα/β^CD17/β^N	α^+/α/β^0/β^N	65	3.61
^--^SEA/αα/β^CD26/β^N	α^0/α/Hb E	50	2.78
-α^4.2/αα/β^CD41-42/β^N	α^+/α/β^0/β^N	48	2.67
^--^SEA/αα/β^-28/β^N	α^0/α/β^+/β^N	31	1.72
^--^SEA/αα/β^IVS-I-1/β^N	α^0/α/β^0/β^N	28	1.56
-α^3.7/αα/β^CD26/β^N	α^+/α/Hb E	26	1.45
-α^3.7/αα/β^IVS-I-1/β^N	α^+/α/β^0/β^N	24	1.33
^--^SEA/αα/β^CD71-72/β^N	α^0/α/β^0/β^N	22	1.22
-α^3.7/αα/β^CD71-72/β^N	α^+/α/β^0/β^N	22	1.22
^--^SEA/-α^3.7/β^CD17/β^N	α^0/α^+/β^0/β^N	18	1.00
^--^SEA/αα/β^IVS-II-654/β^N	α^0/α/β^+/β^N	16	0.89
-α^3.7/αα/β^IVS-II-654/β^N	α^+/α/β^+/β^N	15	0.83
-α^3.7/αα/β^-28/β^N	α^+/α/β^+/β^N	14	0.78
α^WSα/αα/β^CD71-72/β^N	α^+/α/β^0/β^N	13	0.72
α^WSα/αα/β^CD26/β^N	α^+/α/Hb E	12	0.67
^--^SEA/-α^3.7/β^CD41-42/β^N	α^0/α^+/β^0/β^N	11	0.61
α^CSα/αα/β^IVS-I-1/β^N	α^+/α/β^0/β^N	11	0.61
-α^4.2/αα/β^CD26/β^N	α^+/α/Hb E	9	0.50
-α^4.2/αα/β^CD71-72/β^N	α^+/α/β^0/β^N	9	0.50
α^CSα/αα/β^-28/β^N	α^+/α/β^+/β^N	9	0.50
-α^3.7/-α^3.7/β^CD17/β^N	α^+/α^+/β^0/β^N	8	0.44
-α^3.7α/α^CSα/β^CD41-42/β^N	α^+/α^+/β^0/β^N	8	0.44
α^CSα/αα/β^CD71-72/β^N	α^+/α/β^0/β^N	8	0.44
α^CSα/αα/β^IVS-II-654/β^N	α^+/α/β^+/β^N	8	0.44
-α^3.7α/α^CSα/β^CD41-42/β^N	α^+/α^+/β^0/β^N	7	0.39
-α^3.7α/α^WSα/β^CD41-42/β^N	α^+/α^+/β^0/β^N	7	0.39
-α^3.7α/α^CSα/β^IVS-I-1/β^N	α^+/α^+/β^0/β^N	7	0.39
Remaining 60 genotypes	–	135	7.50
Subtotal	–	1799	100

Forms of thalassemia for which fetuses would be at risk among couples in which both partners were positive for thalassemia isotypes

Among the 38812 couples, couples in which both partners were positive for α-thalassemia isotype were 3414/38812 (8.80%), and those positive for β-thalassemia isotype were 807/38812 (2.08%). The proportion of couples found to be at risk of having a baby with fetal Hb Bart’s disease, HbH disease, HbH or α-thalassemia minor, α-thalassemia minor, and β-thalassemia major or intermedia were 808/38812 (2.08%), 1700/38812 (4.38%), 149/38812 (0.38%), 757/38812 (1.95%), and 807/38812 (2.08%), respectively (Table 4).

Table 4. Forms of thalassemia for which fetuses would be at risk among couples in which both partners were positive for thalassemia in isotype among people of reproductive age in the Border Area of Guangxi-Yunnan-Guizhou province, Southwestern China.

Major classes	Genotypes	No. couples detected
Hb Bart's	--/--	808
HbH	--/-α	1019
HbH	--/α^Tα	681
HbH or TMN	α^Tα/α^Tα	149
TMN	-α/-α	350
TMN	-α/α^Tα	407
TM or TI	β^0/β^0	627
TM or TI	β^+/β^0	97
TM or TI	β^+/β^+	6
TM or TI	HbE/HbE	4
TM or TI	HbE/β^0	65
TM or TI	HbE/β^+	8

Abbreviations: Hb Bart’s, Hb Bart’s hydrops fetalis; HbH, Hb H disease; TMN, thalassemia minor; TM, thalassemia major; TI, thalassemia intermedia.

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)). Figure 2 shows pairwise comparison results for each parameter between different genotypes. For MCV, MCH, Hb, HbA2, and HbF values, 114, 119, 105, 98, and 7 out of 153 pairwise comparisons were significantly different in males, and 116, 128, 108, 89, and 33 out of 153 pairwise comparisons were significantly different in females, respectively (Figure 2).

Figure 2. Hematological characterization of α-thalassemia among different genders and genotypes. (A) MCV levels among participants with different genders and genotypes; (B) MCH levels among participants with different genders and genotypes; (C) Hb levels among participants with different genders and genotypes; (D) HbA2 levels among participants with different genders and genotypes; (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.

Table 5. Characteristics and hematological parameters of α-thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province, Southwestern China, M (P25, P75).

Genotype	Gender (cases)	MCV (fl)	MCH (Pg)	Hb (g/dl)	HbA2 (%)	HbF (%)
^--^SEA/αα	M (3187)	69.80 (67.50,72.50)	21.70 (20.80, 22.60)	13.80 (13.20, 14.50)	2.40 (2.30, 2.60)	0.40 (0.23, 0.80)
	F (3170)	68.30 (66.00, 70.80)	21.30 (20.50, 22.10)	11.00 (10.40, 11.60)	2.50 (2.30, 2.60)	0.50 (0.30, 0.79)
-α^3.7/αα	M (1367)	83.65 (81.00, 86.27)	26.55 (25.82, 27.50)	14.80 (14.10, 15.30)	2.60 (2.40, 2.80)	0.30 (0.20, 0.55)
	F (1419)	82.00 (79.85, 84.80)	26.60 (25.67, 27.30)	11.90 (11.20, 12.50)	2.60 (2.40, 2.80)	0.40 (0.30, 0.60)
α^CSα/αα	M (669)	82.70 (80.42, 84.90)	26.30 (25.60, 27.00)	14.60 (14.00, 15.20)	2.40 (2.30, 2.50)	0.40 (0.30, 0.70)
	F (764)	82.40 (80.00, 85.00)	26.20 (25.30, 27.10)	11.60 (11.10, 12.40)	2.40 (2.20, 2.60)	0.50 (0.40, 0.90)
-α^4.2/αα	M (492)	83.40 (81.90, 85.17)	26.70 (26.10, 27.40)	14.80 (14.20, 15.40)	2.50 (2.40, 2.60)	0.40 (0.30, 0.50)
	F (481)	82.03 (79.40, 85.00)	26.50 (25.50, 27.24)	11.90 (11.31, 12.50)	2.60 (2.40, 2.80)	0.40 (0.30, 0.73)
α^WSα/αα	M (340)	85.30 (81.90, 88.00)	27.36 (26.20, 28.80)	15.00 (14.43, 15.80)	2.75 (2.43, 3.00)	0.40 (0.30, 0.60)
	F (324)	83.40 (80.45, 86.60)	27.00 (26.00, 28.00)	12.00 (11.20, 12.60)	2.80 (2.70, 3.00)	0.40 (0.30, 0.70)
^--^SEA/-α^3.7	M (141)	60.60 (58.80, 67.00)	17.90 (17.00, 19.00)	11.00 (10.35, 11.50)	1.60 (1.40, 1.80)	0.50 (0.20, 0.70)
	F (163)	63.00 (57.38, 66.15)	18.30 (17.10, 19.03)	8.70 (8.13, 9.30)	1.65 (1.35, 1.95)	0.30 (0.20, 0.98)
^--^SEA/α^CSα	M (62)	75.05 (70.00, 78.18)	19.60 (19.00, 20.85)	9.40 (8.37, 10.05)	1.15 (0.88, 1.45)	0.35(0.20, 0.43)
	F (95)	74.60 (69.70, 76.90)	18.90 (17.80, 19.75)	7.70 (6.95, 8.17)	1.30 (1.20, 1.85)	0.70 (0.35, 1.05)
α^QSα/αα	M (72)	81.20 (78.00, 84.18)	25.40 (24.4, 25.65)	14.40 (13.47, 14.88)	2.55 (2.50, 2.70)	0.40 (0.33, 0.78)
	F (56)	75.40 (73.50, 77.00)	23.81 (23.10, 25.00)	11.60 (11.00, 12.31)	2.60 (2.50, 2.80)	0.50 (0.40, 0.90)
-α^3.7/-α^3.7	M (61)	77.05 (73.72, 80.58)	23.40 (22.60, 26.30)	13.60 (12.90, 14.70)	2.65 (2.47, 2.70)	0.45 (0.38, 0.73)
	F (63)	72.00 (68.10, 73.80)	22.60 (21.00, 23.75)	11.70 (10.95, 12.05)	2.60 (2.50, 2.75)	0.70 (0.55, 0.90)
^--^SEA/-α^4.2	M (55)	61.20 (60.40, 64.30)	18.50 (18.35, 18.85)	11.60 (10.45, 12.00)	1.60 (1.50, 1.75)	0.60 (0.35, 0.75)
	F (64)	60.80 (59.38, 63.88)	18.00 (17.28, 18.78)	8.65 (7.98, 9.33)	1.55 (1.50, 1.80)	0.70 (0.38, 1.18)
^--^SEA/α^WSα	M (59)	66.80 (66.00, 69.23)	20.95 (19.80, 22.00)	13.15 (12.75, 13.55)	2.40 (2.20, 2.68)	0.35 (0.23, 0.50)
	F (57)	67.30 (63.52, 70.90)	19.90 (18.70, 22.00)	11.30 (10.20, 11.70)	2.60 (2.45, 2.70)	0.40 (0.25, 0.60)
-α^3.7α/α^CSα	M (49)	78.95 (75.48, 84.23)	23.50 (22.80, 25.18)	13.00 (12.58, 13.50)	2.60 (2.35, 2.60)	0.30 (0.20, 0.93)
	F (53)	76.00 (68.30, 81.00)	22.50 (21.30, 24.90)	10.05 (9.90, 10.60)	2.50 (2.30, 2.80)	0.70 (0.40, 1.30)
-α^3.7/-α^4.2	M (48)	78.00 (71.10, 83.25)	24.00 (22.85, 26.70)	14.30 (13.45, 15.25)	2.50 (2.20, 2.60)	0.40 (0.25, 0.65)
	F (34)	73.50 (71.23, 76.00)	23.15 (22.43, 23.97)	11.03 (9.98, 11.29)	2.50 (2.40, 2.58)	0.40 (0.33, 0.50)
-α^3.7α/α^WSα	M (24)	78.00 (70.83, 83.90)	26.00 (21.00, 26.28)	14.10 (13.43, 14.48)	2.30 (2.18, 2.55)	0.40 (0.20, 0.60)
	F (35)	68.50 (66.88, 72.03)	21.80 (20.68, 22.68)	11.50 (10.38, 12.23)	2.50 (2.28, 2.70)	0.40 (0.28, 0.75)
α^CSα/α^CSα	M (22)	84.00 (77.80, 85.40)	24.20 (22.80, 26.10)	11.60 (10.20, 13.20)	1.60 (1.50, 1.80)	0.70 (0.20, 1.10)
	F (20)	81.40 (78.48, 86.08)	25.50 (23.98, 25.88)	9.20 (8.88, 10.65)	1.60 (1.25, 2.13)	0.60 (0.38, 1.20)
-α^4.2α/α^WSα	M (22)	79.60 (77.40, 81.25)	25.00 (23.28, 25.60)	14.05 (13.55, 14.63)	2.35 (2.23, 2.58)	0.55 (0.30, 1.05)
	F (17)	72.90 (71.39, 74.00)	22.40 (22.30, 23.00)	10.80 (10.60, 10.90)	2.50 (2.40, 2.60)	0.60 (0.30, 1.10)
-α^4.2α/α^CSα	M (13)	80.50 (79.00, 81.33)	24.10 (23.33, 24.60)	13.60 (12.75, 14.50)	2.30 (2.18, 2.43)	0.60 (0.35, 0.93)
	F (22)	72.14 (70.65, 80.48)	22.35 (20.98, 24.50)	10.75 (10.35, 10.97)	2.30 (2.02, 2.73)	0.75 (0.33, 1.03)
α^WSα/α^CSα	M (14)	80.40 (79.18, 81.78)	25.50 (23.50, 26.68)	14.65 (13.58, 15.20)	2.45 (2.18, 2.58)	0.45 (0.23, 0.98)
	F (15)	80.50 (73.40, 86.40)	24.50 (23.30, 26.20)	11.45 (10.80, 12.10)	2.60 (2.20, 2.70)	0.65 (0.20, 1.10)

Abbreviations: M, Male; F, Female.

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. Notably, due to the low number of cases, β^CD27-28/β^N, β^CD14-15/β^N, β^CAP/β^N, β^-28/β^CD17, β^-28/β^CD41-42, and β^-28/β^IVS-I-1 were not included in the analysis. The MCV, MCH, Hb, HbA2, and HbF values for participants with different genders and genotypes are shown in Figure 3. Compared to females, males in each genotypic group had significantly higher Hb levels (p < 0.001) (Figure 3(C)), significantly higher MCV levels in β^CD41-42/β^N, β^CD26/β^N, and β^-28/β^N genotypic groups (p < 0.05) (Figure 3(A)), significantly higher MCH levels in β^-28/β^N and β^CD43/β^N genotypic groups (p < 0.05) (Figure 3(B)), and significantly lower HbF levels in β^CD17/β^N, β^CD41-42/β^N, β^IVS-I-1/β^N β^CD71-72/β^Nβ^-28/β^N, and β^IVS-II-654/β^N genotypic groups (p < 0.05) (Figure 3(E)). However, differences in HbA2 levels between genders were not significant. Pairwise comparisons for each parameter between different genotypes are shown in Figure 3. For MCV, MCH, Hb, HbA2, and HbF values, 24, 27, 19, two, and five out of 36 pairwise comparisons were significantly different in males, and 24, 20, 22, five, and 17 out of 36 pairwise comparisons were significantly different in females, respectively (Figure 3).

Figure 3. Hematological characterization of β-thalassemia among different genders and genotypes. (A) MCV levels among participants with different genders and genotypes; (B) MCH levels among participants with different genders and genotypes; (C) Hb levels among participants with different genders and genotypes; (D) HbA2 levels among participants with different genders and genotypes; (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.

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).

Genotype	Gender (cases)	MCV (fl)	MCH (Pg)	Hb (g/dl)	HbA2 (%;)	HbF (%)
β^CD17/β^N	M (1135)	64.30 (61.50, 67.91)	20.10 (19.20, 21.30)	13.10 (12.40, 13.87)	5.40 (5.10, 5.70)	1.00 (0.60, 1.39)
	F (1197)	64.60 (61.90, 67.40)	20.20 (19.40, 21.20)	10.40 (9.62, 11.10)	5.40 (5.10, 5.70)	1.40 (0.80, 2.39)
β^CD41-42/β^N	M (881)	65.20 (62.71, 68.78)	20.30 (19.40, 21.40)	13.20 (12.40, 13.90)	5.40 (5.10, 5.70)	0.80 (0.40, 1.40)
	F (913)	65.00 (62.10, 67.70)	20.30 (19.40, 21.23)	10.30 (9.60, 11.00)	5.30 (5.05, 5.60)	1.00 (0.70, 1.90)
β^CD26/β^N	M (153)	78.00 (73.35, 80.15)	25.27 (24.60, 26.25)	14.20 (13.28, 14.86)	25.35 (23.53, 26.90)	0.90 (0.55, 1.55)
	F (175)	76.35 (68.43, 78.55)	25.25 (23.90, 26.30)	11.50 (10.75, 12.25)	26.00 (22.10, 28.05)	0.90 (0.58, 1.45)
β^IVS-I-1/β^N	M (134)	65.53 (63.08, 67.93)	20.70 (19.80, 21.85)	13.20 (12.68, 13.85)	5.20 (4.90, 5.40)	1.00 (0.40, 1.30)
	F (130)	65.70 (63.60, 69.07)	20.45 (19.70, 21.95)	10.70 (9.85, 10.98)	5.20 (4.80, 5.60)	1.20 (0.80, 1.85)
β^CD71-72/β^N	M (90)	64.60 (63.40, 69.15)	19.70 (19.00, 21.05)	12.60 (12.00, 13.35)	5.20 (4.90, 5.50)	0.80 (0.40, 0.90)
	F (114)	65.70 (62.30, 68.00)	20.60 (19.20, 21.33)	10.50 (9.70, 11.10)	5.50 (5.10, 5.60)	0.70 (0.60, 1.00)
β^-28/β^N	M (109)	73.75 (71.00, 76.00)	23.30 (22.50, 23.72)	14.30 (13.28, 14.90)	5.30 (4.90, 5.50)	0.90 (0.60, 1.81)
	F (101)	72.00 (69.13, 74.63)	23.00 (21.68, 23.55)	10.95 (10.48, 11.65)	5.40 (4.90, 5.60)	1.60 (1.20, 2.30)
β^IVS-II-654/β^N	M (81)	63.80 (62.45, 68.45)	19.70 (19.05, 21.25)	13.40 (12.70, 13.90)	5.50 (4.50, 5.80)	0.70 (0.50, 0.90)
	F (71)	64.15 (61.10, 66.13)	20.08 (18.93, 21.15)	10.60 (9.58, 11.50)	5.00 (4.90, 5.30)	1.00 (0.70, 1.50)
β^CD43/β^N	M (19)	65.00 (63.80, 68.00)	21.65 (20.70, 22.40)	13.25 (12.90, 13.60)	5.25 (5.00, 6.10)	0.65 (0.60, 0.90)
	F (25)	65.90 (60.47, 66.50)	19.50 (18.48, 21.20)	10.20 ( (9.00, 10.70)	5.50 (5.30, 5.70)	0.90 (0.50, 1.10)
β^-29/β^N	M (18)	69.30 (67.80, 70.00)	23.90 (22.70, 24.00)	13.30 (13.10, 13.70)	5.30 (5.10, 5.60)	0.70 (0.40, 0.90)
	F (18)	67.90 (64.20, 75.20)	24.30 (21.00, 25.60)	11.65 (10.80, 12.40)	5.65 (4.90, 5.90)	0.80 (0.60, 1.20)

The HbA2% level of genotype β^CD26/β^N was actually the sum of HbA2% and HbE%.

Characteristics and hematological parameters of concurrent α- and β-thalassemia

Clinical features of the top 10 frequent concurrent α- and β-thalassemia mutations are shown in Table 7. The MCV, MCH, Hb, HbA2, and HbF levels for participants from different genders with different genotypes are shown in Figure 4. Compared to females, males in each genotypic group had significantly higher Hb levels (p < 0.001) (Figure 4(C)); compared to the α-thalassemia alone group, the concurrent α- and β-thalassemia group had significantly higher HbA2 levels (p < 0.001) (Figure 4(D)); Compared to the β-thalassemia alone group, the concurrent α- and β-thalassemia group had significantly higher MCV and MCH levels, apart from females with genotype -α^3.7/αα/β^CD17/β^N and both males and females with genotype ^--SEA/αα/β^CD26/β^N. Differences in MCV levels for males with genotype -α^4.2/αα/β^CD17/β^N were insignificant. The remaining relationships are illustrated in Figure 4. Pairwise comparisons for each parameter between different genotypes are shown in Figure 4. For the MCV, MCH, Hb, HbA2, and HbF values, 16, 15, 14, three, and six out of 45 pairwise comparisons were significantly different in males, and 20, 15, six, 10, and one out of 45 pairwise comparisons were significantly different in females, respectively.

Figure 4. Hematological characterization of concurrent α- and β-thalassemia among different genders and genotypes. (A) MCV levels among participants with different genders and genotypes; (B) MCH levels among participants with different genders and genotypes; (C) Hb levels among participants with different genders and genotypes; (D) HbA2 levels among participants with different genders and genotypes; (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 α- and β-thalassemia group had significantly higher levels (p < 0.05); ^⋆compared to α-thalassemia alone group in the same gender, concurrent α- and β-thalassemia group had significantly higher levels (p < 0.05). ^▴Compared to β-thalassemia alone group in the same gender, concurrent α- and β-thalassemia group had significantly lower levels (p < 0.05); ^★compared to the α-thalassemia alone group in the same gender, concurrent α- and β-thalassemia group had significantly lower levels (p < 0.05). Non-listed 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.

Table 7. Characteristics and hematological parameters of concurrent α- and β-thalassemia among people of reproductive age in the border area of Guangxi-Yunnan-Guizhou province, Southwestern China, M (P25, P75).

Genotype	Gender	MCV (fl)	MCH (Pg)	Hb (g/dl)	HbA2 (%;)	HbF (%;)
^--^SEA/αα/β^CD17/β^N	M (118)	70.15 (68.15, 72.35)	22.00(21.48, 23.18)	14.00 (13.55, 14.75)	5.60 (4.95, 6.00)	0.60 (0.30, 1.30)
	F (135)	69.20 (67.80, 71.35)	21.90 (21.40, 22.65)	11.20 (10.65, 11.70)	5.50 (5.20, 6.00)	0.70 (0.60, 1.06)
^--^SEA/αα/β^CD41-42/β^N	M (113)	72.55 (69.10, 77.03)	22.85 (21.95, 24.13)	14.50 (13.78, 14.98)	5.40 (5.03, 5.70)	0.55 (0.33, 0.78)
	F (104)	71.90(67.58, 75.28)	22.35 (21.34, 23.71)	11.10 (10.56, 11.90)	5.30 (5.13, 5.63)	0.90(0.60, 1.45)
-α^3.7/αα/β^CD17/β^N	M (97)	68.00 (64.53, 71.28)	21.00 (19.85, 22.55)	13.30 (12.90, 13.78)	5.50 (5.40, 5.60)	0.90 (0.65, 1.10)
	F (85)	65.20 (63.75, 66.55)	20.45 (19.38, 21.18)	10.60 (9.80, 11.00)	5.80 (5.30, 5.90)	1.00 (0.60, 1.40)
-α^3.7/αα/β^CD41-42/β^N	M (87)	69.25 (65.15, 73.35)	21.30 (20.30, 22.60)	13.55 (12.98, 14.50)	5.35(5.18, 5.50)	0.80 (0.60, 1.18)
	F (89)	66.95 (64.53, 69.42)	20.90 (20.40, 21.38)	10.85(10.00, 11.19)	5.45 (5.30, 5.90)	0.85 (0.60, 1.35)
α^CSα/αα/β^CD17/β^N	M (58)	69.10 (66.05, 72.28)	21.45 (20.38, 22.53)	13.60 (12.98, 14.00)	5.20 (4.73, 5.48)	0.85 (0.60, 1.15)
	F (43)	67.20 (65.80, 70.33)	20.90 (20.10, 21.70)	10.90 (9.80, 11.20)	5.25 (4.85, 5.50)	1.05 (0.88, 1.23)
α^CSα/αα/β^CD41-42/β^N	M (36)	70.65 (66.58, 73.25)	21.95 (20.35, 22.95)	13.80 (13.53, 14.08)	5.10 (4.83, 5.38)	0.75 (0.53, 0.90)
	F (46)	68.70 (67.90, 70.80)	21.20 (20.80, 22.00)	10.90 (9.60, 11.50)	5.40 (5.10, 5.60)	0.90 (0.70, 1.10)
-α^4.2/αα/β^CD17/β^N	M (42)	66.90 (63.10, 69.83)	20.81 (19.78, 22.05)	13.27 (12.73, 13.58)	5.15 (4.63, 5.55)	0.70 (0.58, 1.10)
	F (34)	67.40 (66.30, 69.90)	21.10 (20.80, 22.20)	10.90 (10.40, 11.40)	5.40 (4.97, 5.50)	0.80 (0.60, 1.10)
α^WSα/αα/β^CD41-42/β^N	M (41)	68.60 (63.70, 70.70)	21.50 (20.00, 22.40)	13.40 (12.90, 13.80)	5.20 (5.00, 5.50)	0.70 (0.40, 0.80)
	F (30)	68.55 (64.39, 71.63)	21.25 (19.94, 22.52)	10.80 (9.69, 12.12)	5.10 (4.90, 5.50)	0.80 (0.50, 1.10)
α^WSα/αα/β^CD17/β^N	M (40)	68.55 (63.15, 72.28)	21.65 (20.03, 22.28)	13.60 (13.30, 14.30)	5.20(5.00, 5.50)	0.60 (0.30, 0.80)
	F (25)	68.57 (63.35, 70.70)	21.30 (19.65, 22.53)	10.90(10.35, 11.30)	5.30 (5.15, 5.55)	0.80 (0.45, 0.90)
^--^SEA/αα/β^CD26/β^N	M (23)	73.95 (70.50, 76.25)	23.80 (22.50, 25.48)	13.95(12.08, 14.53)	22.50 (22.20, 23.40)	0.50 (0.35, 0.85)
	F (27)	70.40 (68.00, 72.70)	22.40 (21.30, 24.59)	11.17 (10.50, 12.10)	22.60 (20.60, 24.60)	0.90 (0.60, 1.10)

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 socio-economic 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 and non-deletional mutations (Table 1). The five most frequent α-thalassemia genotypes were ^--SEA/αα, -α^3.7/αα, α^CSα/αα, -α^4.2/αα, and α^WSα/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.

We also identified 15 different β-thalassemia genotypes, with β^CD17/β^N, β^CD41-42/β^N, and β^CD26/β^N being the three most frequent genotypes, followed by β^IVS-I-1/β^N, β^CD71-72/β^N, β^-28/β^N, and β^IVS-II-654/β^N. These seven genotypes accounted for 98.32% of all β-thalassemia genotypes. These results indicate a high genetic heterogeneity for β-thalassemia in the border area of Guangxi-Yunnan-Guizhou provinces, Southwestern China. Even though these results differed from the spectrum of β-thalassemia gene mutations in Guangdong, Guangxi, Yunnan, and Guizhou provinces [13–16], the most common genotype (β^CD17/β^N) was consistent with previous reports [15,16]. Our results indicate a high genetic diversity and significant genetic variations of β-thalassemia in the border area of Guangxi-Yunnan-Guizhou provinces, Southwestern China.

Patients with mild concurrent α- and 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 α- and 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 α- and 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 α⁰ had significantly elevated MCV, MCH and Hb levels. Eighteen cases of concurrent α- and 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^CSα/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^CSα/αα/β^IVS-II-654/β^N, etc. other 6 genotypes of one case each. However, the reason is not yet clear. Notably, the β- thalassemia phenotype was not exacerbated in the rest of concurrent α- and 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 α⁰ 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 α-globin gene. This is because, the synthesis of both α and β peptide chains is reduced in concurrent α- and β-thalassemia, and the severity of clinical symptoms in thalassemia patients mainly depend on imbalances of α/β peptide chains [19–21]. Differences in levels of some hematological parameter indicators between concurrent α- and 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. 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 ^Gγ⁺(^Aγδβ)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 [7,22–24]. 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. 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.

Institutional review board statement

This study was approved by the Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China.

Data sharing statement

Readers can get the data of this paper by contacting us via E-mail: 1123049646 @qq.com.

ARRIVE guidelines statement

The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.

Disclosure statement

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

Additional information

Funding

This study was supported by Natural Science Foundation of GuangXi Province [Grant/Award Number: 2020GXNSFBA297047]; The First Batch of Highlevel Talent Scientific Research Projects of the Affiliated Hospital of Youjiang Medical University for Nationalities [Grant/Award Number: Y202011708]; The Science and Technology Plan Project of Baise [Grant/Award Numbers: BaiKe20211815].