The population incidence of thalassemia gene variants in Baise, Guangxi, P. R. China, based on random samples

ABSTRACT

Objective

Thalassemia is a monogenic genetic disorder with a high prevalence in populations in the southern region of China. The thalassemia gene prevalence rate in the Baise population in China is high, and several rare gene variants have been detected in the population of this region during routine testing by our study group. To accurately reveal the thalassemia gene variants carried by the population in Baise, and to provide a basis for the formulation of thalassemia prevention and control policies in the region, we conducted a more comprehensive study in a randomly selected population.

Results

In all, 4,800 randomized individuals were recruited for testing from Baise, and the detection of hot spot thalassemia genetic variants were performed by Gap-PCR and PCR-RDB methods, combined with the relative quantification of homologous fragments and AS-PCR to expand the detection range. The prevalence of thalassemia variants in this population was 24.19%, among which 16.69% of individuals carried α-thalassemia gene variants alone, 5.62% carried β-thalassemia gene variants alone, and 1.88% carried both variants.

Conclusions

The use of positive primary screening combined with hot spot gene variant detection alone can result in a certain degree of missed detection. In the prevention and control of thalassemia in the region, testing institutions need to pay attention to the detection of rare thalassemia gene variants such as ααα^anti4.2, ααα^anti3.7, –α^2.4, –α^21.9, β⁻⁵⁰, β⁻⁹⁰, and β^IVS-II−5, to provide more accurate genetic counseling advice to subjects.

KEYWORDS:

• Thalassemia
• genetic variants
• Baise
• Guangxi

1. Introduction

Thalassemia is a monogenic genetic disease with a high prevalence in southern China. The molecular mechanisms mainly involve variants in the α-globin gene cluster (NG_000006.1) and the β-globin gene cluster (NG_000007.3)[1, 2]. According to the distribution of gene variations, thalassemia is mainly divided into α-, β- and γ- forms. The carriers of α- and β- thalassemia are the most numerous and these forms are most harmful to the individuals, so the targets tested by genetic testing institutions at all levels are mainly α- and β- thalassemia variants[3]. The most severe cases in patients with α-thalassemia are mainly those that lead to hemoglobin (Hb) Bart’s hydrops fetalis and Hb H disease. Pregnant women showing Hb Bart’s hydrops fetalis may experience postpartum hemorrhage during labor, endangering maternal life, while some individuals with Hb H disease can develop moderate/severe anemia and hepatosplenomegaly, endangering the patient’s health[4]. Patients with moderate/severe β-thalassemia can develop transfusion dependence, and the symptoms of anemia usually appear around the age of 1 year in affected individuals[5]. Conventional transfusion with iron removal therapy can cause a heavy financial burden and mental stress to patients, while the high cost of hematopoietic stem cell transplantation is difficult for some families to afford. Therefore, the current prevention and control strategy of thalassemia is mainly based on prenatal genetic diagnosis[6].

At present, China has established a three-level prevention and control system for thalassemia[7]. Secondary prevention and control mainly entail the genetic diagnosis of thalassemia in individuals who screen positive at primary institutions, and is therefore crucial in the prevention and control of the disease. The current prevention and control strategy is mainly a model in which tested individuals first complete hematological screening, and positive individuals then undergo genetic testing using hotspot mutation detection kits, which has also achieved some success. Baise City is located in southern China, in the northwestern part of Guangxi region, and is a multi-ethnic area with a high incidence of thalassemia genes in the population. Different research groups have studied the detection of thalassemia gene variants in the population of Baise region, but our research group has found a certain number of rare thalassemia gene variants in our daily work[8–10]. To define the exact prevalence of thalassemia genetic variants in the population of Baise region and provide a basis for the formulation of thalassemia prevention and control policies in the region, we aimed to conduct a more comprehensive study of thalassemia variants in a random population in this region.

2. Materials and methods

2.1. Materials

The study was approved by the Ethics Committee of the Baise People’s Hospital. Samples were obtained from patients at the same hospital, with a total of 4800 participants. Of these, 2341 were female and 2459 were male. All samples were obtained from healthy individuals participating in annual physical examinations in the medical examination center without any selection for thalassemia-related phenotypes. The individuals from whom the samples were obtained were informed of the study aims. All samples were of venous blood, and EDTA was used as anticoagulant. Blood was stored in a refrigerator at 4–8 °C for backup, and DNA extraction was completed within 3 days of collection. DNA extraction was done by the magnetic bead method, and the DNA concentration was detected using an UV spectrophotometer. Samples were then stored at –20 °C.

2.2. Analysis of thalassemia hot-spot genetic variants

Gap-PCR and PCR-RDB methods were used to complete the analysis of thalassemia hot-spot genetic variants. The deletional α thalassemia hot-spot variants were detected using specific test kits (Yilifang Bio, Gap-PCR method) for –^THAI, –^SEA, –α^3.7 and –α^4.2. Nondeletional α thalassemia hot-spot allele variants were detected using specific test kits (Yaneng Bio, PCR-RDB method) for α^CSα, α^QSα, and α^Westmeadα. Nondeletional β thalassemia hotspot variants were completed using specific gene test kits (produced by Yaneng Bio, PCR-RDB method), which detect β^CD41/42, β^IVS-II−654, β^CD17, β⁻²⁸, β⁻²⁹, β⁻³⁰, β⁻³², β^CD26, β^CD71/72, β^CD43, β^IVS-I−1, β^27/28, β^{Int CD}, β^CD31, β^Cap+40−43, β^CD14/15, and β^IVS-I−5. The detection ranges of the above three thalassemia gene variant detection kits are consistent with the detection ranges of the main detection reagents currently used in the laboratories of the secondary prevention and control institutions in mainland China.

2.3. Quantification of α genes and analysis of rare thalassemia variants

To improve the detection range of thalassemia gene variants, we used the homologous fragment relative quantification detection system and AS-PCR assay system designed by our research group for the rapid detection of rare thalassemia gene variants.

Quantification of homologous gene fragments was adopted from the published assay protocol of our research group, which focused on the relative quantification of the A1/A2 region and Y1/Y2 region with high homology in the α globin gene cluster, combined with the results of the deletional α-thalassemia gene detection kits[11, 12]. When the sample is of a wild-type, A1:A2 = 1, Y1:Y2 = 1; when the sample carries a single alpha gene deletion or duplication, the result of A1:A2 or Y1:Y2 ≠ 1, combined with the results of the deletion α gene test kit, we can determine the possibility that the sample carries a copy number variation of the gene fragment. After comprehensive analysis of the results, individuals that may carry ααα^anti3.7, ααα^anti4.2, HKαα, –α^2.4, –α^21.9 or other rare thalassemia variants can be screened out and conclusions can be drawn after validation using Gap-PCR, MLPA and other methods.

For the detection of rare thalassemia gene variants, direct Sanger sequencing would be too time-consuming and complex. We used the AS-PCR-based real-time PCR detection system designed by our group to rapidly screen the tested samples for HPFH-SEA, ^Gγ⁺(^Aγδβ)⁰, β⁻⁵⁰, β⁻⁹⁰, β^IVS-II−5, β^CD37, and other variants that were detected occasionally in our previous studies[7, 8, 13].

3. Results

3.1. Alpha-thalassemia gene variants

Gap-PCR, PCR-RDB and AS-PCR techniques were used to complete the analysis of all samples, and 891 individuals were detected carrying α-thalassemia gene variants. The test results are shown in Table 1. The prevalence of these variants in the Baise population was 18.57% (Table 2).

Table 1. Prevalence of α-thalassemia genotypes in the population of Baise, Guangxi.

Genotype	Detection cases	Carriage rate (%)	Female	Male
αα/αα	3909	81.43	1904	2005
–^SEA/αα	297	6.18	145	152
-α^3.7/αα	215	4.48	109	106
α^CSα/αα	98	2.04	52	46
α^Westmeadα/αα	91	1.90	47	44
-α^4.2/αα	83	1.73	36	47
–^SEA/-α^3.7	20	0.42	9	11
ααα^anti4.2/αα	11	0.23	4	7
ααα^anti3.7/αα	10	0.21	4	6
-α^3.7/-α^3.7	8	0.17	5	3
–^SEA/α^Westmeadα	8	0.17	4	4
-α^2.4/αα	7	0.15	4	3
α^QSα/αα	7	0.15	6	1
-α^3.7/α^CSα	6	0.13	2	4
-α^3.7/α^Westmeadα	6	0.13	3	3
HKαα/αα	5	0.10	0	5
–^SEA/-α^4.2	4	0.08	1	3
–^THAI/αα	4	0.08	1	3
–^SEA/α^CSα	3	0.06	0	3
-α^4.2/α^CSα	2	0.04	1	1
α^CSα/α^Westmeadα	1	0.02	1	0
-α^3.7/-α^4.2	1	0.02	1	0
Hb Q-thailand/αα	1	0.02	0	1
-α^4.2/α^Westmeadα	1	0.02	0	1
-α^21.9/αα	1	0.02	1	0
-α^2.4/α^Westmeadα	1	0.02	1	0

A total of 4800 individuals aged 20–60 years participated in the study, including 2341 female and 2459 male subjects. All samples were collected at our medical examination center (for a healthy population), and the individuals tested had no indication of any hematological disease and were not screened using other laboratory indicators.

Table 2. Proportions of α-thalassemia gene variants in the Baise population.

α-thal variants	Cases	Percentage (%)
–^SEA	332	34.87
-α^3.7	264	27.73
α^CSα	110	11.55
α^Westmeadα	108	11.34
-α^4.2	91	9.56
ααα^anti4.2	11	1.15
ααα^anti3.7	10	1.05
-α^2.4	8	0.84
α^QSα	7	0.74
HKαα	5	0.53
–^THAI	4	0.42
Q-Thailand	1	0.11
-α^21.9	1	0.11

The numbers in the table indicate the total number of α gene variants. For example, the total number of –α^3.7 variants for individuals with the –α^3.7/–α^3.7 genotype was 2.

3.2. Beta-thalassemia gene variants

PCR-RDB and AS-PCR methods were used to test the collected samples for β-thalassemia pathogenic variants, and 360 affected individuals were detected (Table 3). The prevalence of β-thalassemia gene variants was 7.50% in the samples we collected, and the percentages of variant types are shown in Table 4.

Table 3. Prevalence of β-thalassemia genotype in the population of Baise, Guangxi.

Genotype	Detection cases	Carriage rate (%)
β^N/β^N	4440	92.50
β^CD17/β^N	145	3.02
β^CD41/42/β^N	106	2.21
β^CD26/β^N	20	0.42
β^IVS-I−1/β^N	18	0.38
β^−28/β^N	17	0.36
β^−50/β^N	16	0.33
β^CD71/72/β^N	15	0.31
β^IVS-II−654/β^N	11	0.23
β^−29/β^N	3	0.06
β^IVS-II−5/β^N	2	0.04
β^−90/β^N	2	0.04
β^CD37/β^N	1	0.02
β^CD27/28/β^N	1	0.02
β^CD43/β^N	1	0.02
β^IVS-I−130/β^N	1	0.02
β^CD17/β^IVS-I−1	1	0.02

Table 4. Proportion of β-thalassemia gene variants in the population of Baise, Guangxi.

β-thal variants	Cases	Percentage (%)
β^CD17	146	40.44
β^CD41/42	106	29.36
β^CD26	20	5.54
β^IVS-I−1	19	5.26
β^−28	17	4.71
β^−50	16	4.43
β^CD71/72	15	4.16
β^IVS-II−654	11	3.05
β^−29	3	0.83
β^IVS-II−5	2	0.55
β^−90	2	0.55
β^CD37	1	0.28
β^CD27/28	1	0.28
β^CD43	1	0.28
β^IVS-I−130	1	0.28

3.3. Individuals carrying both α- and β-thalassemia gene variants

In all, 90 samples were detected to carry both α- and β-thalassemia gene variants (Table 5), and the prevalence of such genotypes in the Baise population was 1.88%.

Table 5. Genotypes and number of detected individuals carrying both α- and β- thalassemia variants in the population of Baise, Guangxi.

α-thal genotype	β-thal genotype	Cases
–^SEA/αα	β^CD17/β^N	14
–^SEA/αα	β^CD41/42/β^N	14
α^Westmeadα/αα	β^CD17/β^N	6
-α^4.2/αα	β^CD17/β^N	5
-α^3.7/αα	β^CD17/β^N	7
-α^3.7/αα	β^CD41/42/β^N	4
–^SEA/αα	β^IVS1−1/β^N	3
–^SEA/αα	β^CD26/β^N	3
–^SEA/-α^3.7	β^CD41/42/β^N	3
α^CSα/αα	β^CD17/β^N	5
α^CSα/αα	β^CD26/β^N	2
-α^4.2/αα	β^CD41/42/β^N	3
α^CSα/αα	β^CD41/42/β^N	2
-α^3.7/αα	β^IVS1−1/β^N	2
α^Westmeadα/αα	β^IVS1−1/β^N	1
α^Westmeadα/αα	β^CD41/42/β^N	1
α^Westmeadα/αα	β^CD26/β^N	1
α^Westmeadα/αα	β^−28/β^N	1
–^SEA/α^Westmeadα	β^−28/β^N	1
–^SEA/α^Westmeadα	β^CD17/β^N	1
–^SEA/αα	β^−28/β^N	1
–^SEA/αα	β^CD17/β^IVS1−1	1
–^SEA/αα	β^CD71/72/β^N	1
α^QSα/αα	β^IVS1−1/β^N	1
α^CSα/αα	β^IVS1−1/β^N	1
α^CSα/αα	β^CD71/72/β^N	1
-α^4.2/αα	β^−50/β^N	1
-α^3.7/αα	β^CD71/72/β^N	1
-α^3.7/αα	β^−50/β^N	1
-α^3.7/αα	β^−28/β^N	1
-α^3.7/α^CSα	β^CD71/72/β^N	1

Comprehensive analysis of the data showed that the proportion of individuals carrying thalassemia gene variants in this population was 24.19%.

4. Discussion

Thalassemia is a monogenic genetic disorder with a clear pathogenic mechanism, and genetic diagnosis and prenatal diagnosis are the main methods of prevention and control in regions with high prevalence[14]. A thalassemia three-level prevention and control strategy is widely implemented in the southern regions of China. For this, primary control facilities usually perform routine hematology tests and hemoglobin electrophoresis for subjects (fees of approximately $4 and $13 in 2022), and individuals with positive screening results are then tested for hot spot genetic variants by secondary testing laboratories (fees of approximately $80 in 2022). In regions with a high incidence of the thalassemia gene variant in the population, the government usually implements various exemption policies to increase the testing rate. Thanks to the effective implementation of the three-level prevention and control system, birth defects caused by thalassemia have decreased year by year[15].

For rapid genotyping, testing laboratories often prioritize the use of thalassemia gene hotspot variant detection kits for large-scale screening[16]. However, the limited detection range of these kits can lead to some missed detection. In our thalassemia prevention and control system, the primary screening facility provides the hematology test results of the subject, and when the hematology test results are inconsistent with the results of the hotspot gene variant test, further analysis is required to screen for the presence of rare variants. However, such a testing process can still lead to certain omissions. Therefore, when implementing three-level prevention and control of thalassemia, a comprehensive genetic analysis of thalassemia in the target population should be conducted to clarify the types of gene variants carried, and the detection rate of secondary prevention and control can be improved by using appropriate hotspot variant detection kits selected for the test results.

Baise City is located in northwestern Guangxi region, adjacent to Vietnam, and has a high rate of thalassemia variants in the population. The prevention and control of thalassemia in this city has been carried out for many years, but since the test data published by most testing laboratories in the past were based on the results of thalassemia gene hotspot variant testing in the hematology screening positive population, their conclusions had some limitations. Because some of the carriers of the silent α thalassemia variant and part of the β+ thalassemia gene variant have negative hematological screening[17], this group of individuals might be recorded as negative and be ignored during primary screening. The detection range of the kits used by secondary testing institutions only includes 24 relatively common thalassemia gene hotspot variants, and many rare thalassemia gene variants reported by other research groups in Guangxi are not included in the detection range. Therefore, the results of previous studies are mostly limited to the exploration of hotspot thalassemia gene variants.

Lu et al. conducted a retrospective analysis of the results of genetic testing for thalassemia in 3482 pregnant women from Baise and their spouses[18]. They used the Gap-PCR and PCR-RDB methods, which are commonly used in hotspot thalassemia gene variant detection, to perform the tests. They detected 2,260 samples carrying thalassemia gene variants. The main α gene variants detected were –^SEA/αα (43.66%), –α^3.7/αα (20.97%), α^CSα/αα (9.80%), –α^4.2/αα (8.50%), and α^Westmeadα/αα (5.27%). The results did not include rare thalassemia gene variants. Compared with the present study, a higher proportion of the silent α-thalassemia variants were detected and 3.79% of the rare α-thalassemia variants were detected, which could also pose a problem for prenatal counseling if ignored. The main β-gene variants detected by Lu et al. were β^CD17 (37.12%), β^CD41/42 (34.52%), β^IVS-I−1 (6.65%), β^CD26 (6.16%), β^CD71/72 (5.51%), and β^–28 (3.73%). The detection of rare β-thalassemia variants was not reported. Compared with the present study, phenotypically milder variants, such as β^–28, were relatively underrepresented in the study by Lu et al. and the rare β-thalassemia variant, which accounted for 6.09% here, was not detected. The difference in the percentage of β^CD17 and β^CD41/42 might have arisen from differences in the individuals included in the research. In contrast to the above studies, relying solely on a hotspot thalassemia genetic variant detection protocol after a positive primary screening test can result in a proportion of less phenotypically significant thalassemia variants being missed.

He et al. surveyed 47,500 individuals from Baise, using a model of hematological screening and screening positive individuals before implementing the detection of hotspot thalassemia gene variants, adding some rare thalassemia gene variants[19]. They detected 15.35% carriers of α-thalassemia gene variants, 6.64% carriers of β-thalassemia gene variants, and 2.08% carriers of the α/β heterozygous thalassemia gene variants, for an overall prevalence of 24.07%. Although the total prevalence of thalassemia gene variants detected in the population of Baise in the study by He et al. was similar to the results of the present study (24.19%), there were some differences in the variants detected in the two study groups. Among the proportion of α-thalassemia gene variants, the composition of ααα^anti4.2, ααα^anti3.7 and –α^2.4 detected here amounted to 3.04%, while the proportion of –^SEA was not so high. For β-thalassemia gene variants, the large proportion of β⁻⁵⁰ detected here (4.43%) was not reported by He et al. The differences between the two studies could be due to differences in the detection technology (e.g. how the α triplets were detected) and the population selected, but the omission of β⁻⁵⁰ might be because that this variant was not included in the assays used. Therefore, a testing model based on primary screening of positive individuals followed by implementation of hotspot genetic variants might miss a proportion of thalassemia gene carriers.

Among the rare thalassemia genetic variants, detection of α-gene copy number variants is the most taxing. When an individual carries only the variant with increased copy number of the α-gene and is not heterozygous for the β-thalassemia variant, the individual's hematological phenotype does not change significantly[20]. However, when an individual carries both the variant with increased copy number of the α-gene and the β- gene variant, the increased expression of the α-gene leads to a further imbalance between the α and normal β chains in the individual, which can lead to moderate to severe anemia. In our previous study, we also detected several cases of individuals with similar causes of anemia[12]. This type of testing is difficult to perform in many secondary testing laboratories because most of the commercial thalassemia gene hotspot variant testing kits do not cover α-gene copy numbers. However, the consequences of such variants are serious, especially in regions of the population with a high incidence of the β-thalassemia variant. The clarification of α-gene copy number is of great importance to give more precise genetic counseling to the tested individuals. Therefore, it is necessary to test for such increases in priority individuals when implementing the genetic analysis of thalassemia in individuals from the Baise region.

Our present results are closer to the actual thalassemia gene prevalence in the Baise population because this study used an undifferentiated sample population, which was not filtered by primary screening. In addition, abnormal hemoglobin variants caused by point mutations and the rarer thalassemia gene variants were also not included here because of methodological limitations, and further data could be revealed by second- or third-generation sequencing.

Our results suggest that further improvement of the three-level prevention and control strategy for thalassemia is needed in regions with high thalassemia carriage rates to provide more accurate genetic counseling for subjects. At present, the strategy is still the mainstream approach for thalassemia prevention and control. Because the increased number of α-gene variants (mainly anti3.7 and anti4.2) are currently the ‘blind area’ of thalassemia genetic screening, under the premise of controlled testing cost and fully informed information, when implementing prenatal genetic testing, if one spouse is clearly a carrier of the β-thalassemia variant, both partners can be recommended for quantitative α-gene testing. The cost of using our quantitative testing protocol is about US$2 in 2022, which is relatively inexpensive. In addition, when implementing tertiary prevention and control, testing facilities need to further implement rare thalassemia genetic testing to prevent missed tests when the genotype and phenotype of an individual do not match. Currently, thalassemia analysis protocols based on next-generation sequencing (NGS) or third-generation sequencing (TGS) technologies are also being improved gradually, and if the price of the test drops to a level similar to that of traditional testing technology, testing institutions could consider adopting NGS or TGS testing technology.

5. Conclusions

The prevalence of thalassemia variants in the population of Baise, Guangxi region, was 24.19%. Among them, 16.69% of individuals carried α-thalassemia gene variant alone, 5.62% carried a β-thalassemia gene variant alone, and 1.88% were heterozygous for the α- and β-thalassemia gene variants. In the secondary prevention and control, if only the hotspot thalassemia gene variant kits are used to complete the test, some of the gene variants will be missed. In the prevention and control of thalassemia in Baise population, the testing organization should pay attention to the rare thalassemia gene variants such as ααα^anti4.2, ααα^anti3.7, –α^2.4, –α^21.9, β^–50, β^–90, and β^IVS-II−5, to provide more accurate genetic counseling.

Statement of Ethics

This study was reviewed and approved by the ethics committee of the Baise People’s Hospital.

Author Contributions

Wei Bixiao: Experiment

Zhou Weijie and Peng Mingkui: Methodology

Long Ju: Conceptualization, Methodology, Experiment and Writing-Original draft preparation

Wen Wangrong: Investigation, Methodology, Writing- Reviewing and Editing

Acknowledgements

We are grateful to all volunteers who provided the samples used in this study.

Disclosure statement

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

Data Availability Statement

The data and materials are available on request by contacting the corresponding author Long Ju.

Additional information

Funding

This work was supported by Guangxi Natural Science Foundation: [Grant Number 2017GXNSFDA198017]; "139" Training Program for High Level Medical Talents in Guangxi: [Grant Number G201903017]; Baise Scientific Research and Technology Development Projects: [Grant Number Baike-202118]; Qinzhou Scientific Research and Technology Development Projects: [Grant Number 201514924].