ABSTRACT
Background
The factors influencing the phenotypic heterogeneity of patients with β‐thalassemia have been receiving much attention in the field of hematology research. Activating the sustained expression of fetal hemoglobin (HbF) has proven to be one of the effective ways to alleviate the clinical symptoms of β‐thalassemia. Studies have reported that single nucleotide polymorphisms (SNP) in KLF1, BCL11A, and HBS1L‐MYB can increase the expression level of HbF in patients with β‐thalassemia and have an impact on the phenotype.
Methods
In this study, SNaPshot and Sanger sequencing were used to detect SNPs of BCL11A, HBS1L‐MYB, and KLF1 in patients with different types of β‐thalassemia collected in Hainan. Linkage disequilibrium and haplotype analysis were performed on mutant sites.
Results
As a result, 41 mutation types of the above genes were detected (high mutation frequency and wide distribution range), and there was strong linkage disequilibrium at multiple mutation sites, resulting in multiple haplotypes. However, there are no significant differences in the distribution of gene polymorphisms between different types of β‐thalassemia, suggesting that the modifications of KLF1, BCL11A, and HBS1L‐MYB may have little impact on the β‐thalassemia phenotype in this region.
Conclusion
Our study provides data support for assessing the impact of modified genes on the phenotype of patients with β‐thalassemia in Hainan, and also promotes the clinical accurate diagnosis and classification evaluation of β‐thalassemia.
Keywords: β‐thalassemia, BCL11A, HBS1L‐MYB, KLF1
In this article, SNaPshot and Sanger sequencing were used to detect SNPs of BCL11A, HBS1L‐MYB, and KLF1 in patients with different types of β‐thalassemia collected in Hainan. 41 mutation types of the above genes were detected, and there was strong linkage disequilibrium at multiple mutation sites, resulting in multiple haplotypes.
1. Introduction
β‐thalassemia is a blood system disease caused by changes in the structure of the β‐globin gene, which leads to the reduction or cessation of the synthesis of β‐globin chain in the hemoglobin structure. The disease generally occurs frequently in tropical and subtropical regions, including the Mediterranean Sea, the Indian subcontinent, Southeast Asia, Guangdong, Guangxi, and Hainan of China (Birgens and Ljung 2007; Weatherall 2018). Clinical phenotypes of patients with β‐thalassemia have wide heterogeneity, ranging from asymptomatic to severe anemia requiring blood transfusion treatment. The severity of their phenotypes is related to the imbalance in the synthesis ratio of globin α and β chains. In addition to β‐globin gene mutations, any factor that can change the degree of globin chain imbalance can be used as a modifier that affects the phenotype. At present, there are two main mechanisms for modifying the clinical symptoms of β‐thalassemia: one is the combination of α‐thalassemia, resulting in a simultaneous reduction of α‐globin expression and alleviating the imbalance (Mettananda 2021). The other is to reactivate the expression of the γ globin gene and combine with the free α chain to form HbF that can normally carry oxygen, reducing the imbalance between the α and β chains (Sankaran and Weiss 2015).
In recent years, reactivating the expression of HbF has been proven to be one of the effective ways to alleviate the clinical symptoms of β‐thalassemia. Genome‐Wide Association Studies (GWAS) have identified that the genetic factors affecting HbF are mainly concentrated in the BCL11A gene (OMIM: 606557), HBS1L‐MYB gene (OMIM: 189990) and the 11p15 β globin gene cluster. In addition, studies have reported that KLF1 gene (OMIM: 600599) mutations can significantly affect HbF expression (Thein 2018; Hariharan et al. 2021). For example, Emmanuelle Charpentier's team has developed the first CRISPR gene‐editing therapy of the world, which activates HbF expression by targeting the BCL11A and can be used to treat β‐thalassemia and sickle cell disease (Frangoul et al. 2021). Previously, we conducted an epidemiological survey on thalassemia in Hainan, which showed that the human carrier rate of β‐thalassemia was about 1.70%. Based on previous research, this study further tested the SNPs of KLF1, BCL11A, and HBS1L‐MYB in the local population and analyzed the association between mutation sites and the phenotype of patients with β‐thalassemia, providing data support for precise clinical diagnosis and efficacy evaluation.
2. Materials and Methods
2.1. Ethical Compliance
This study was approved by the Institutional Ethics Committee of Hainan Hospital Affiliated to Hainan Medical University (no. YYL [2022]‐164). Blood samples were obtained from the remaining samples after clinical testing. The studies were conducted in accordance with the local legislation and institutional requirements.
2.2. Sample Collection
A total of 513 subjects (Age: 2 months–40 years old) were included in our study. According to the results of gene detection and clinical diagnosis, the subjects were divided into 5 groups: group A, 58 patients with severe β thalassemia (β0/β0); group B, 18 patients with intermediate β thalassemia (β+/β0); group C, 255 patients with mild β thalassemia (β0/βN or β+/βN); group D, 132 patients with “HbF > 2%” (Most of the genotypes in group D were normal, only a few were β+/βN, severe and intermediate β thalassemia excluded, used to enrich modified gene mutations); group E, 50 patients with normal genotype (HbF < 1%). Hematology parameters were collected, including MCV, MCH, Hb, HbA, HbA2, and HbF.
2.3. Nucleic Acid Extraction and Quality Testing
Nucleic acid extraction was performed using a Genomic DNA Extraction Kit by Magnetic Beads (NanoMagBio) in conjunction with a KingFisherFlex automated nucleic acid extractor (ThermoFisher). Use NanoDROP8000 ultra‐micro spectrophotometer (ThermoFisher Company) to detect nucleic acid concentration and purity. DNA detection results should be: concentration (ng/μL) ≥ 30 ng/μL, A260/A280 ratio should be in the range of 1.8–2.0.
2.4. Detection of SNPs in BCL11A and HBSIL‐MYB by SNaPshot
2.4.1. Primer Synthesis
PrimerPlex2 was used to design PCR reaction primers, and primer3‐plus was used to design single‐base extension primers online. Primer synthesis was completed by Wuhan Tianyi Huayu Gene Technology Co. Ltd.
2.4.2. Classification Experiment
PCR amplification system consists of 9 μL PCR master mix and 1 μL template DNA (20 ng/μL). After alkaline phosphatase treatment, single base extension reaction was carried out. SNaPshot reaction products were amplified and analyzed by 3730xL DNA Analyzer (Applied Biosystems). BCL11A (GenBank ID: NM_001405735.1) detection sites include: rs10184550 (NC_000002.12:g.60502159G>A), rs10189857 (NC_000002.12:g.60486100A>G), rs6545816 (NC_000002.12:g.60487726A>C), rs1427407 (NC_000002.12:g.60490908T>G), rs766432 (NC_000002.12:g.60492835C>A), rs7599488 (NC_000002.12:g.60491212C>T), rs4671393 (NC_000002.12:g.60493816A>G), rs189984760 (NC_000002.12:g.60511373A>G), rs6706648 (NC_000002.12:g.60494905C>T), rs61742690 (NC_000002.12:g.60460564C>T), rs6732518 (NC_000002.12:g.60481462C>T), rs141538797 (NC_000002.12:g.60496880C>T). HBS1L‐MYB (GenBank ID: NM_001363686.2) detection sites include: rs9376090 (NC_000006.12:g.135090090T>C), rs7776054 (NC_000006.12:g.135097778A>G), rs9399137 (NC_000006.12:g.135097880T>C), rs9389268 (NC_000006.12:g.135098493A>G), rs4895440 (NC_000006.12:g.135105420A>T), rs4895441 (NC_000006.12:g.135105435A>G), rs9402686 (NC_000006.12:g.135106679G>A), rs9402685 (NC_000006.12:g.135098550A>G), rs11759553 (NC_000006.12:g.135101158A>T), rs35959442 (NC_000006.12:g.135103041C>G), rs9494142 (NC_000006.12:g.135110502T>C), rs6934903 (NC_000006.12:g.135130426T>A), rs6929404 (NC_000006.12:g.135132889C>A), rs6904897 (NC_000006.12:g.135061842T>G), rs76288258 (NC_000006.12:g.135146091C>G), rs28384513 (NC_000006.12:g.135055071T>G), rs9389269 (NC_000006.12:g.135106021T>C), rs9376092 (NC_000006.12:g.135106006C>A).
2.5. Genome Re‐Sequencing for KLF1
According to the structural characteristics of the KLF1 (GenBank ID: NM_006563.5), 4 segments (KLF1‐01–KLF1‐04) and specific primers are designed. Set up the amplification system and amplification program. The amplified products were amplified and analyzed by 3730xL DNA Analyzer (Applied Biosystems).
2.6. Statistical Analysis
Hardy–Weinberg equilibrium (HWE) was tested for all the SNPs, and X 2 was determined by SPSS ver.20 to evaluate allele association. Linkage disequilibrium (LD) test was carried out using HaploView 4.2 software to identify the non‐random association of these SNPs. Pairwise correlations are measured in r 2 and displayed in each diamond (×100). Haplotype blocks were constructed using the HaploView 4.2 program. Haplotypes associated with the study subjects were inferred based on the partition‐ligation approach through an EM algorithm. p‐value < 0.05 was considered significant for all statistical analyses.
3. Results
3.1. Phenotypic Data
From Table 1, the clinical diagnosis data showed that the subjects in group A had a severe thalassemia phenotype; the patients were generally younger in age and had a history of regular blood transfusion therapy. The average expression level of HbF was 51.73% ± 4.39%. The anemia symptoms of group B subjects were slightly lighter than those of group A; patients generally did not require regular blood transfusion therapy, and the average expression level of HbF was 16.87% ± 5.53%. Hematological parameters in groups C, D, and E reflected anemia in different degrees; the anemia degree of the subjects in groups C and D was the lightest, and group E was a normal population. The average HbF expression level in group C and group E was 0.16% ± 0.24% and 0.16% ± 0.39%, respectively. Group D was mainly used to enrich modified gene mutations, and the average HbF expression level was 4.99% ± 4.15%.
TABLE 1.
Distribution of relevant hematological parameters in different types of β‐thalassia and control groups (Mean ± SD).
| Group | N | M/F | Age | HbF (%) | HbA2 (%) | HbA (%) | Hb (g/L) | MCV (fL) | MCH (pg) |
|---|---|---|---|---|---|---|---|---|---|
| A | 58 | 30/28 | 8.01 ± 4.60 | 51.73 ± 4.39 | 2.22 ± 0.30 | 46.04 ± 4.42 | 71.37 ± 9.98 | 74.07 ± 5.46 | 22.20 ± 2.01 |
| B | 18 | 13/5 | 11.50 ± 7.94 | 16.87 ± 5.53 | 5.00 ± 1.01 | 78.13 ± 5.47 | 81.83 ± 8.99 | 67.67 ± 7.41 | 20.83 ± 1.65 |
| C | 255 | 107/148 | 36.89 ± 9.03 | 0.16 ± 0.24 | 5.40 ± 0.45 | 94.43 ± 0.56 | 115.88 ± 10.67 | 61.58 ± 2.42 | 19.72 ± 0.84 |
| D | 132 | 37/93 | 17.65 ± 2.98 | 4.99 ± 4.15 | 3.08 ± 1.45 | 92.86 ± 3.33 | 125.97 ± 11.43 | 74.73 ± 8.87 | 24.14 ± 3.04 |
| E | 50 | 17/33 | 16.26 ± 1.41 | 0.16 ± 0.39 | 2.55 ± 0.25 | 97.22 ± 0.50 | 134.54 ± 7.90 | 84.40 ± 4.10 | 28.64 ± 1.42 |
Abbreviations: F, female; M, man.
3.2. SNPs Detection
3.2.1. SNaPshot Typing Results of BCL11A and HBSIL‐MYB
The results showed that 11 SNPs in BCL11A and 18 SNPs in HBSIL‐MYB were detected in 513 subjects. Hardy–Weinberg equilibrium was tested for all the SNPs, and genotype frequency, allele frequency, and MAF were calculated.
In the BCL11A, rs6732518, rs6706648, and rs141538797 were excluded due to their low mutation frequencies. The genotype frequencies, allele frequencies, and MAF of the other 8 SNPs are shown in Table 2. The 8 SNPs were distributed in HWE equilibrium in 5 groups; rs10184550 and rs189984760 were heterozygous with mutation frequencies ranging from 0% to 41.4%; the other 6 SNPs were homozygous with mutation frequencies ranging from 83.3% to 94.9%. The mutation frequencies of these 8 SNPs were compared between groups (compared with group E), and the results showed no significant differences (p > 0.05).
TABLE 2.
Genotype frequency and allelic frequency distribution of SNP sites of BCL11A gene in different groups.
| SNP | Group | HWE (P) | Genotype (%) | Allele (%) | MAF (%) | ||
|---|---|---|---|---|---|---|---|
| rs10189857 | AA | GA/GG | A | G | |||
| A | 0.587 | 4 (6.9) | 54 (93.1) | 26 (22.4) | 90 (77.6) | 22.4 | |
| B | 0.500 | 3 (16.7) | 15 (83.3) | 12 (33.3) | 24 (66.7) | 33.3 | |
| C | 1.000 | 14 (5.5) | 241 (94.5) | 118 (23.1) | 392 (76.9) | 23.1 | |
| D | 0.708 | 10 (7.6) | 122 (92.4) | 78 (29.5) | 186 (70.5) | 29.5 | |
| E | 1.000 | 4 (8.0) | 46 (92.0) | 27 (27.0) | 73 (73.0) | 27.0 | |
| rs6545816 | AA | CA/CC | A | C | |||
| A | 0.587 | 4 (6.9) | 54 (93.1) | 26 (22.4) | 90 (77.6) | 22.4 | |
| B | 1.000 | 2 (11.1) | 16 (88.9) | 11 (30.6) | 25 (69.4) | 30.6 | |
| C | 1.000 | 13 (5.1) | 242 (94.9) | 115 (22.5) | 395 (77.5) | 22.5 | |
| D | 0.708 | 10 (7.6) | 122 (92.4) | 78 (29.5) | 186 (70.5) | 29.5 | |
| E | 1.000 | 4 (8.0) | 46 (92.0) | 27 (27.0) | 73 (73.0) | 27.0 | |
| rs1427407 | TT | GT/GG | T | G | |||
| A | 0.587 | 4 (6.9) | 54 (93.1) | 26 (22.4) | 90 (77.6) | 22.4 | |
| B | 1.000 | 2 (11.1) | 16 (88.9) | 11 (30.6) | 25 (69.4) | 30.6 | |
| C | 1.000 | 13 (5.1) | 242 (94.9) | 114 (22.4) | 396 (77.6) | 22.4 | |
| D | 0.801 | 10 (7.6) | 122 (92.4) | 77 (29.2) | 187 (70.8) | 29.2 | |
| E | 1.000 | 4 (8.0) | 46 (92.0) | 27 (27.0) | 73 (73.0) | 27.0 | |
| rs766432 | CC | AC/AA | C | A | |||
| A | 0.852 | 4 (6.9) | 54 (93.1) | 28 (24.1) | 88 (75.9) | 24.1 | |
| B | 1.000 | 2 (11.1) | 16 (88.9) | 12 (33.3) | 24 (66.7) | 33.3 | |
| C | 0.874 | 14 (5.5) | 241 (94.5) | 116 (22.7) | 394 (77.3) | 22.7 | |
| D | 0.708 | 10 (7.6) | 122 (92.4) | 78 (29.5) | 186 (70.5) | 29.5 | |
| E | 0.617 | 5 (10.0) | 45 (90.0) | 28 (28.0) | 72 (72.0) | 28.0 | |
| rs4671393 | AA | GA/GG | A | G | |||
| A | 0.852 | 4 (6.9) | 54 (93.1) | 28 (24.1) | 88 (75.9) | 24.1 | |
| B | 1.000 | 2 (11.1) | 16 (88.9) | 11 (30.6) | 25 (69.4) | 30.6 | |
| C | 0.747 | 14 (5.5) | 241 (94.5) | 114 (22.4) | 396 (77.6) | 22.4 | |
| D | 0.708 | 10 (7.6) | 122 (92.4) | 78 (29.5) | 186 (70.5) | 29.5 | |
| E | 0.849 | 4 (8.0) | 46 (92.0) | 26 (26.0) | 74 (74.0) | 26.0 | |
| rs10184550 | GG | GA/AA | G | A | |||
| A | 0.392 | 34 (58.6) | 24 (41.4) | 91 (78.4) | 25 (21.6) | 21.6 | |
| B | 0.313 | 12 (66.7) | 6 (33.3) | 29 (80.6) | 7 (19.4) | 19.4 | |
| C | 0.269 | 159 (62.4) | 96 (37.6) | 407 (79.8) | 103 (20.2) | 20.2 | |
| D | 0.785 | 90 (68.2) | 42 (31.8) | 217 (82.2) | 47 (17.8) | 17.8 | |
| E | 0.710 | 32 (64.0) | 18 (36.0) | 79 (79.0) | 21 (21.0) | 21.0 | |
| rs189984760 | AA | GA/GG | A | G | |||
| A | 0.172 | 54 (93.1) | 4 (6.9) | 111 (95.7) | 5 (4.3) | 4.3 | |
| B | 1.000 | 18 (100) | 0 (0) | 36 (100) | 0 (0) | 0.0 | |
| C | 0.922 | 231 (90.6) | 24 (9.4) | 485 (95.1) | 25 (4.9) | 4.9 | |
| D | 0.996 | 113 (85.6) | 19 (14.4) | 245 (92.8) | 19 (7.2) | 7.2 | |
| E | 0.785 | 41 (82.0) | 9 (18.0) | 90 (90.0) | 10 (10.0) | 10.0 | |
| rs7599488 | CC | CT/TT | C | T | |||
| A | 0.852 | 4 (6.9) | 54 (93.1) | 28 (24.1) | 88 (75.9) | 24.1 | |
| B | 0.769 | 3 (16.7) | 15 (83.3) | 13 (36.1) | 23 (63.9) | 36.1 | |
| C | 0.867 | 13 (5.1) | 242 (94.9) | 120 (23.5) | 390 (76.5) | 23.5 | |
| D | 1.000 | 11 (8.3) | 121 (91.7) | 78 (29.5) | 186 (70.5) | 29.5 | |
| E | 0.617 | 5 (10.0) | 45 (90.0) | 28 (28.0) | 72 (72.0) | 28.0 | |
The statistical results of HBSIL‐MYB are shown in Table 3. Among them, rs7776054 and rs9399137 are mostly homozygous mutations, with mutation frequencies of 2.4%–12%. rs6929404 and rs76288258 are mostly heterozygous mutations, with mutation frequencies of 62.4%–75.9%. Other SNPs are mostly heterozygous mutations, with mutation frequencies of 16.7%–50.8%. In rs9389269, the mutation frequency in group B (p = 0.039) and group C (p = 0.027) was lower than that in group E, and other results (compared with group E) were not significantly different (p > 0.05).
TABLE 3.
Genotype frequency and allelic frequency distribution of SNP sites of HBS1L‐MYB gene in different groups.
| SNP | Group | HWE (P) | Genotype (%) | Allele (%) | MAF (%) | ||
|---|---|---|---|---|---|---|---|
| rs28384513 | TT | TG/GG | T | G | |||
| A | 1.000 | 37 (63.8) | 21 (36.2) | 93 (80.2) | 23 (19.8) | 19.8 | |
| B | 0.766 | 10 (55.6) | 8 (44.4) | 26 (72.2) | 10 (27.8) | 27.8 | |
| C | 0.193 | 148 (58.0) | 107 (42.0) | 394 (77.3) | 116 (22.7) | 22.7 | |
| D | 0.528 | 70 (53.0) | 62 (47.0) | 195 (73.9) | 69 (26.1) | 26.1 | |
| E | 1.000 | 33 (66.0) | 17 (34.0) | 82 (82.0) | 18 (18.0) | 18.0 | |
| rs6904897 | TT | TG/GG | T | G | |||
| A | 1.000 | 37 (63.8) | 21 (36.2) | 93 (80.2) | 23 (19.8) | 19.8 | |
| B | 0.766 | 10 (55.6) | 8 (44.4) | 26 (72.2) | 10 (27.8) | 27.8 | |
| C | 0.284 | 151 (59.2) | 104 (40.8) | 397 (77.8) | 113 (22.2) | 22.2 | |
| D | 0.774 | 73 (55.3) | 59 (44.7) | 198 (75.0) | 66 (25.0) | 25.0 | |
| E | 1.000 | 33 (66.0) | 17 (34.0) | 82 (82.0) | 18 (18.0) | 18.0 | |
| rs9376090 | TT | CT/CC | T | C | |||
| A | 0.049 | 38 (65.5) | 20 (34.5) | 90 (77.6) | 26 (22.4) | 22.4 | |
| B | 0.780 | 13 (72.2) | 5 (27.8) | 30 (83.3) | 6 (16.7) | 16.7 | |
| C | 0.766 | 161 (63.1) | 94 (36.9) | 407 (79.8) | 103 (20.2) | 20.2 | |
| D | 1.000 | 67 (50.8) | 65 (49.2) | 188 (71.2) | 76 (28.8) | 28.8 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| rs7776054 | AA | GA/GG | A | G | |||
| A | 2.08E−05* | 55 (94.8) | 3 (5.2) | 110 (94.8) | 6 (5.2) | 5.2 | |
| B | 1.000 | 17 (94.4) | 1 (5.6) | 35 (97.2) | 1 (2.8) | 2.8 | |
| C | 1.65E−11* | 248 (97.3) | 7 (2.7) | 497 (97.5) | 13 (2.5) | 2.5 | |
| D | 3.74E−12* | 123 (93.2) | 9 (6.8) | 247 (93.6) | 17 (6.4) | 6.4 | |
| E | 1.00E−04* | 44 (88.0) | 6 (12.0) | 90 (90.0) | 10 (10.0) | 10.0 | |
| rs9389268 | AA | GA/GG | A | G | |||
| A | 0.764 | 38 (65.5) | 20 (34.5) | 93 (80.2) | 23 (19.8) | 19.8 | |
| B | 0.780 | 13 (72.2) | 5 (27.8) | 30 (83.3) | 6 (16.7) | 16.7 | |
| C | 0.486 | 160 (62.7) | 95 (37.3) | 407 (79.8) | 103 (20.2) | 20.2 | |
| D | 0.993 | 67 (50.8) | 65 (49.2) | 189 (71.6) | 75 (28.4) | 28.4 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| rs9376092 | CC | AC/AA | C | A | |||
| A | 1.000 | 36 (62.1) | 22 (37.9) | 91 (78.4) | 25 (21.6) | 21.6 | |
| B | 1.000 | 12 (66.7) | 6 (33.3) | 29 (80.6) | 7 (19.4) | 19.4 | |
| C | 0.809 | 154 (60.4) | 101 (39.6) | 398 (78.0) | 112 (22.0) | 22.0 | |
| D | 0.993 | 67 (50.8) | 65 (49.2) | 189 (71.6) | 75 (28.4) | 28.4 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| rs35959442 | CC | CG/GG | C | G | |||
| A | 0.238 | 43 (74.1) | 15 (25.9) | 98 (84.5) | 18 (15.5) | 15.5 | |
| B | 0.338 | 15 (83.3) | 3 (16.7) | 32 (88.9) | 4 (11.1) | 11.1 | |
| C | 0.003 | 198 (77.6) | 57 (22.4) | 442 (86.7) | 68 (13.3) | 13.3 | |
| D | 1.00E−04* | 101 (76.5) | 31 (23.5) | 223 (84.5) | 41 (15.5) | 15.5 | |
| E | 0.003 | 36 (72.0) | 14 (28.0) | 80 (80.0) | 20 (20.0) | 20.0 | |
| rs4895440 | AA | TA/AA | A | T | |||
| A | 1.000 | 36 (62.1) | 22 (37.9) | 91 (78.4) | 25 (21.6) | 21.6 | |
| B | 1.000 | 12 (66.7) | 6 (33.3) | 29 (80.6) | 7 (19.4) | 19.4 | |
| C | 0.809 | 154 (60.4) | 101 (39.6) | 398 (78.0) | 112 (22.0) | 22.0 | |
| D | 0.993 | 67 (50.8) | 65 (49.2) | 189 (71.6) | 75 (28.4) | 28.4 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| TT | TA/AA | T | A | ||||
| rs6934903 | A | 0.587 | 32 (55.2) | 26 (44.8) | 88 (75.9) | 28 (24.1) | 24.1 |
| B | 1.000 | 13 (72.2) | 5 (27.8) | 31 (86.1) | 5 (13.9) | 13.9 | |
| C | 0.541 | 162 (63.5) | 93 (36.5) | 404 (79.2) | 106 (20.8) | 20.8 | |
| D | 0.429 | 72 (54.5) | 60 (45.5) | 192 (72.7) | 72 (27.3) | 27.3 | |
| E | 1.000 | 28 (56.0) | 22 (44.0) | 75 (75.0) | 25 (25.0) | 25.0 | |
| rs9399137 | TT | CT/CC | T | C | |||
| A | 0.002 | 55 (94.8) | 3 (5.2) | 111 (95.7) | 5 (4.3) | 4.3 | |
| B | 0.057 | 17 (94.4) | 1 (5.6) | 34 (94.4) | 2 (5.6) | 5.6 | |
| C | 1.27E−12* | 249 (97.6) | 6 (2.4) | 498 (97.6) | 12 (2.4) | 2.4 | |
| D | 0.001 | 128 (97.0) | 4 (3.0) | 258 (97.7) | 6 (2.3) | 2.3 | |
| E | 2.48E−06* | 46 (92.0) | 4 (8.0) | 92 (92.0) | 8 (8.0) | 8.0 | |
| rs9402685 | AA | GA/GG | A | G | |||
| A | 0.764 | 38 (65.5) | 20 (34.5) | 93 (80.2) | 23 (19.8) | 19.8 | |
| B | 0.780 | 13 (72.2) | 5 (27.8) | 30 (83.3) | 6 (16.7) | 16.7 | |
| C | 0.339 | 161 (63.1) | 94 (36.9) | 409 (80.2) | 101 (19.8) | 19.8 | |
| D | 0.749 | 67 (50.8) | 65 (49.2) | 190 (72.0) | 74 (28.0) | 28.0 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| rs4895441 | AA | GA/GG | A | G | |||
| A | 1.000 | 36 (62.1) | 22 (37.9) | 91 (78.4) | 25 (21.6) | 21.6 | |
| B | 1.000 | 12 (66.7) | 6 (33.3) | 29 (80.6) | 7 (19.4) | 19.4 | |
| C | 0.809 | 154 (60.4) | 101 (39.6) | 398 (78.0) | 112 (22.0) | 22.0 | |
| D | 0.749 | 67 (50.8) | 65 (49.2) | 190 (72.0) | 74 (28.0) | 28.0 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| rs11759553 | AA | AT/TT | A | T | |||
| A | 1.000 | 36 (62.1) | 22 (37.9) | 91 (78.4) | 25 (21.6) | 21.6 | |
| B | 1.000 | 12 (66.7) | 6 (33.3) | 29 (80.6) | 7 (19.4) | 19.4 | |
| C | 0.871 | 155 (60.8) | 100 (39.2) | 399 (78.2) | 111 (21.8) | 21.8 | |
| D | 0.380 | 65 (49.2) | 67 (50.8) | 189 (71.6) | 75 (28.4) | 28.4 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| rs9389269 | TT | CT/CC | T | C | |||
| A | 0.637 | 39 (67.2) | 19 (32.8) | 94 (81.0) | 22 (19.0) | 19.0 | |
| B | 0.338 | 15 (83.3) | 3 (16.7)★ | 32 (88.9) | 4 (11.1) | 11.1 | |
| C | 0.088 | 183 (71.8) | 72 (28.2)★ | 427 (83.7) | 83 (16.3) | 16.3 | |
| D | 0.515 | 79 (59.8) | 53 (40.2) | 202 (76.5) | 62 (23.5) | 23.5 | |
| E | 0.236 | 28 (56.0) | 22 (44.0) | 72 (72.0) | 28 (28.0) | 28.0 | |
| rs9402686 | GG | GA/AA | G | A | |||
| A | 1.000 | 36 (62.1) | 22 (37.9) | 91 (78.4) | 25 (21.6) | 21.6 | |
| B | 1.000 | 12 (66.7) | 6 (33.3) | 29 (80.6) | 7 (19.4) | 19.4 | |
| C | 0.809 | 154 (60.4) | 101 (39.6) | 398 (78.0) | 112 (22.0) | 22.0 | |
| D | 0.749 | 67 (50.8) | 65 (49.2) | 190 (72.0) | 74 (28.0) | 28.0 | |
| E | 0.331 | 27 (54.0) | 23 (46.0) | 71 (71.0) | 29 (29.0) | 29.0 | |
| rs9494142 | TT | CT/CC | T | C | |||
| A | 1.000 | 35 (60.3) | 23 (39.7) | 90 (77.6) | 26 (22.4) | 22.4 | |
| B | 1.000 | 11 (61.1) | 7 (38.9) | 28 (77.8) | 8 (22.2) | 22.2 | |
| C | 1.000 | 154 (60.4) | 101 (39.6) | 396 (77.6) | 114 (22.4) | 22.4 | |
| D | 1.000 | 65 (49.2) | 67 (50.8) | 186 (70.5) | 78 (29.5) | 29.5 | |
| E | 0.156 | 27 (54.0) | 23 (46.0) | 70 (70.0) | 30 (30.0) | 30.0 | |
| rs6929404 | CC | CA/AA | C | A | |||
| A | 0.842 | 14 (24.1) | 44 (75.9) | 59 (50.9) | 57 (49.1) | 49.1 | |
| B | 0.557 | 5 (27.8) | 13 (72.2) | 17 (47.2) | 19 (52.8) | 47.2 | |
| C | 0.993 | 96 (37.6) | 159 (62.4) | 314 (61.6) | 196 (38.4) | 38.4 | |
| D | 0.847 | 47 (35.6) | 85 (64.3) | 156 (59.1) | 108 (40.9) | 40.9 | |
| E | 0.347 | 14 (28.0) | 36 (72.0) | 57 (57.0) | 43 (43.0) | 43.0 | |
| rs76288258 | CC | GC/GG | C | G | |||
| A | 0.828 | 15 (25.9) | 43 (74.1) | 61 (52.6) | 55 (47.4) | 47.4 | |
| B | 0.557 | 5 (27.8) | 13 (72.2) | 17 (47.2) | 19 (52.8) | 52.8 | |
| C | 1.000 | 93 (36.5) | 162 (63.5) | 309 (60.6) | 201 (39.4) | 39.4 | |
| D | 0.757 | 45 (34.1) | 87 (65.9) | 152 (57.6) | 112 (42.4) | 42.4 | |
| E | 0.389 | 13 (26.0) | 37 (74.0) | 55 (55.0) | 45 (45.0) | 45.0 | |
Note: The mark “*” indicates that it does not conform to HWE. The mark “★” indicates that there is a statistical difference in mutation frequency compared to group E.
3.2.2. Genome Re‐Sequencing Results for KLF1
Twelve SNPs were detected of KLF1 in 513 subjects. Except for rs117351327 (NC_000019.10:g.12885905G>A) and rs2072597 (NC_000019.10:g.12885926A>G), which had higher mutation frequency, other SNPs were accidental mutations. From Table 4, rs117351327 and rs2072597 were heterozygous mutants with mutation frequencies of 8.0%–33.3% and 73.5%–79.3%, respectively. In rs117351327, the mutation frequency in group A (p = 0.04) and group D (p = 0.001) was higher than that in group E, and other results (compared with group E) were not significantly different (p > 0.05). In rs2072597, there were no significant differences in mutation frequencies in all groups compared to group E (p > 0.05).
TABLE 4.
Genotype frequencies and allele frequency distribution of KLF1 gene in different groups.
| SNP | Group | HWE (P) | Genotype (%) | Allele (%) | MAF (%) | ||
|---|---|---|---|---|---|---|---|
| rs117351327 | GG | GA/AA | G | A | |||
| A | 1.000 | 45 (77.6) | 13 (22.4)★ | 102 (87.9) | 14 (12.1) | 12.1 | |
| B | 1.000 | 15 (83.3) | 3 (16.7) | 33 (91.6) | 3 (8.4) | 8.4 | |
| C | 0.008* | 224 (87.8) | 31 (12.2) | 474 (92.9) | 36 (7.1) | 7.1 | |
| D | 1.000 | 88 (66.7) | 44 (33.3)★ | 216 (81.8) | 48 (18.2) | 18.2 | |
| E | 1.000 | 46 (92.0) | 4 (8.0) | 96 (96.0) | 4 (4.0) | 4.0 | |
| rs2072597 | AA | AG/GG | A | G | |||
| A | 0.648 | 12 (20.7) | 46 (79.3) | 50 (43.1) | 66 (56.9) | 43.1 | |
| B | 1.000 | 4 (22.2) | 14 (77.8) | 17 (47.2) | 19 (52.8) | 47.2 | |
| C | 0.996 | 40 (15.7) | 215 (74.3) | 201 (39.4) | 309 (60.6) | 39.4 | |
| D | 0.970 | 35 (26.5) | 97 (73.5) | 135 (51.1) | 129 (48.9) | 48.9 | |
| E | 0.979 | 11 (22.0) | 39 (78.0) | 46 (46.0) | 54 (54.0) | 46.0 | |
Note: The mark “*” indicates that it does not conform to HWE. The mark “★” indicates that there is a statistical difference in mutation frequency compared to group E.
3.3. LD and Haplotype Analysis
3.3.1. BCL11A
In BCL11A, LD analysis showed that rs10189857, rs6545816, rs1427407, rs7599488, rs766432, and rs4671393 had strong linkage disequilibrium in all groups and formed a block (Figure 1). Haplotype analysis showed that there were 4 haplotypes in Group A and Group B, 2 haplotypes in Group C and Group D, and 5 haplotypes in Group E. The haplotype frequencies of “GCGTAG” and “AATCCA” were higher in each group (Table 5).
FIGURE 1.
Linkage disequilibrium analysis and haplotype classification of SNP in BCL11A、HBS1L‐MYB and KLF1. In groups A‐D from BCL11A, rs10189857, rs6545816, rs1427407, rs7599488, rs766432 and rs4671393 form a block. From HBS1L‐MYB, 4 blocks in group A and C, 2 blocks in group B, and 3 blocks in group D and E. From KLF1, rs117351327 and rs2072597 had strong linkage disequilibrium in group A and D.
TABLE 5.
Haplotype and frequency distribution.
| Gene | Block | Haplotype | Frequency | ||||
|---|---|---|---|---|---|---|---|
| KLF1 | rs117351327, rs2072597 | A | B | C | D | E | |
| GG | 0.489 | ||||||
| GA | 0.330 | ||||||
| AA | 0.182 | ||||||
| BCL11A | rs10189857, rs6545816, rs1427407, rs7599488, rs766432, rs4671393 | GCGTAG | 0.741 | 0.639 | 0.757 | 0.701 | 0.700 |
| AATCCA | 0.224 | 0.306 | 0.220 | 0.288 | 0.260 | ||
| GCGCAG | 0.017 | 0.010 | |||||
| GCGTCA | 0.017 | ||||||
| ACGCAG | 0.028 | ||||||
| GCGCCG | 0.028 | ||||||
| GCGTCG | 0.020 | ||||||
| AATCAG | 0.010 | ||||||
| HBS1L‐MYB | rs28384513, rs6904897 | TT | 0.802 | 0.773 | 0.739 | 0.820 | |
| GG | 0.198 | 0.222 | 0.250 | 0.180 | |||
| GT | 0.011 | ||||||
| rs9389268, rs9402685 | AA | 0.802 | |||||
| GG | 0.198 | ||||||
| rs11759553, rs35959442, rs4895440, rs4895441, rs9376092, rs9389269, rs9402686, rs9494142 | ACAACTGT | 0.776 | |||||
| TGTGACAC | 0.138 | ||||||
| TCTGACAC | 0.052 | ||||||
| TGTGATAC | 0.017 | ||||||
| rs6929404, rs76288258 | CC | 0.509 | 0.472 | 0.606 | 0.576 | 0.550 | |
| AG | 0.474 | 0.528 | 0.384 | 0.409 | 0.430 | ||
| AC | 0.017 | ||||||
| CG | 0.015 | 0.020 | |||||
| rs11759553, rs35959442, rs4895440, rs4895441, rs9376092, rs9389269, rs9402686 | ACAACTG | 0.806 | |||||
| TGTGACA | 0.083 | ||||||
| TCTGATA | 0.056 | ||||||
| TGTGATA | 0.028 | ||||||
| TCTGACA | 0.028 | ||||||
| rs9376090, rs9389268, rs9402685 | TAA | 0.794 | |||||
| CGG | 0.198 | ||||||
| rs11759553, rs4895440, rs4895441, rs9376092, rs9389269, rs9402686 | AAACTG | 0.778 | |||||
| TTGACA | 0.159 | ||||||
| TTGATA | 0.059 | ||||||
| rs9376090, rs9389268, rs9402685, rs11759553, rs4895440, rs4895441, rs9376092, rs9389269, rs9402686 | TAAAAACTG | 0.697 | 0.710 | ||||
| CGGTTGACA | 0.223 | 0.280 | |||||
| CGGTTGATA | 0.045 | 0.010 | |||||
3.3.2. HBS1L‐MYB
In HBS1L‐MYB, Except for group B, LD analysis showed that “rs28384513 and rs6904897”, “rs9376090, rs9389268, rs9402685, rs11759553, rs4895440, rs4895441, rs9376092, rs9389269, rs9402686, rs9494142 and rs6934903”, “rs6929404 and rs76288258” had strong linkage disequilibrium in all groups (Figure 1). Haplotype analysis showed that there were 4 blocks in group A and C, 2 blocks in group B, and 3 blocks in group D and E (Table 5).
3.3.3. KLF1
In KLF1, LD analysis showed that rs117351327 and rs2072597 had strong linkage disequilibrium in groups A and D (Figure 1). In addition, there is 1 block in group D, forming 3 haplotypes (GG, GA and AA). Group C was eliminated because the genotype frequency distribution of rs117351327 did not satisfy HWE (Table 5).
4. Discussion
β‐thalassemia is a monogenic disease dominated by genetic mutations. It has a typical Mendelian inheritance pattern, but different patients have significant phenotypic heterogeneity. Studies have shown that approximately 5%–10% of β‐thalassemia patients with the same mutation type exhibit different clinical phenotypes. For example, 60% of homozygous patients develop severe anemia, clinically defined as β‐thalassemia major, within the first year of life, and 9% of homozygous patients develop moderate anemia, clinically defined as intermediate beta thalassemia, after 2 years of age (Hariharan et al. 2021). Factors that cause genetic polymorphisms generally do not directly contribute to disease occurrence, but they may be major contributors to disease severity (Soomro et al. 2023). Persistent expression of HbF in adulthood has been shown to significantly influence disease severity in sickle cell disease and β‐thalassemia and is associated with genes such as HBG2‐XMN1(OMIM: 142250), BCL11A, HSB1L‐MYB, and KLF1 (El‐Ghamrawy et al. 2020; Zakaria et al. 2021). Therefore, analysis of the effects of these modifiers on phenotypic heterogeneity in β‐thalassemia can help to achieve accurate diagnosis and targeted treatment of β‐thalassemia.
Hematology data from 513 subjects showed that HbF expression levels were higher in severe β‐thalassemia patients in group A and intermediate β‐thalassemia patients in group B, consistent with reported phenotypic characteristics. However, due to the completeness of clinical data and the short study period, the number of people included in Group A and Group B is small, which may have a certain impact on subsequent statistical analysis. We further examined the polymorphisms of KLF1, BCL11A, and HBS1L‐MYB on the basis of previous studies and analyzed the mutation frequency of these three genes in the Hainan population.
BCL11A is a Krüppel‐like zinc‐finger transcription factor of the BCL family, which is mainly found in brain and hematopoietic tissues (Saiki et al. 2000; Luc et al. 2016). It not only participates in many biological processes such as lymphocyte generation and neural development, but also reactivates γ globin gene expression after mutation in adult erythroid progenitor cells and has an important effect on β‐thalassemia phenotype (Bauer et al. 2013; Markenscoff‐Papadimitriou et al. 2020; Liu et al. 2018). For example, GWAS analysis showed that rs11886868 of BCL11A was highly correlated with high HbF expression level, and the LOVD‐DASH database summarized 6 important SNPs in BCL11A associated with HbF expression level, including rs766432, rs1427407, rs4671393, rs101899857, rs7599488, and rs6545816 (Uda et al. 2008; Zhang et al. 2019). Studies in many foreign regions show that these SNPs have different effects on the heterogeneity of individuals with HbE/β thalassemia and moderate anemia. Domestic studies have also reported that rs766432 and rs4671393 have higher mutation frequencies in Guangxi and Guangdong (Yu 2015; Xun et al. 2022). In this study, 11 SNPs were found in the Hainan population, among which rs766432, rs1427407, rs4671393, rs101899857, rs7599488, and rs6545816 were mainly homozygous mutations; the mutation rate was more than 83%, and there was strong linkage disequilibrium, forming 8 haplotypes in total, which was different from the results reported in Indonesia, Northern Punjab, and Thailand (Rujito et al. 2016; Nasreen et al. 2023; Mohammad et al. 2022). In addition, we did not find any association between SNP and HbF expression level and patient phenotype in different β‐thalassemia populations, which is the first conclusion reported in Hainan.
HBSIL‐MYB intergenic region, mainly composed of HMIP‐1 and HMIP‐2. GWAS analysis showed that SNPs in these regions were associated with HbF expression levels, such as rs28385413, rs66650371, rs9399137, rs9389269, rs940268, rs9494145, and rs9483788 (So et al. 2008; Stadhouders et al. 2014; Menzel and Thein 2019). At present, many studies support that rs9399137 is most significantly associated with HbF expression level in China, Europe, and Africa (Farrell et al. 2011; Bashir et al. 2021; Yuxi Yang et al. 2022). In addition, the effect of rs35959442 and rs4895441 on β‐thalassemia phenotype has been reported in the Zhuang population in China, among which the high mutation rate of rs35959442 is considered to be caused by blood transfusion therapy (Lai et al. 2017; Timmer et al. 2019). Therefore, the mutation frequency of HBSIL‐MYB may vary greatly among different populations in different regions. In this study, 18 SNPs of HBSIL‐MYB were detected in the Hainan population, and the mutation frequencies of these SNPs showed great differences, among which rs9399137 and rs7776054 had low mutation frequencies. Regarding the effect of HBSIL‐MYB on β‐thalassemia phenotype, a cohort study from Saudi Arabia showed that rs9376090, rs9399137, rs4895441, rs9389269, rs9402686, and rs9494142 were mainly associated with HbF expression levels in intermediate β‐thalassemia (Cyrus et al. 2017). The results of this study showed that there was no significant difference in the distribution of mutation frequency of each SNP (except rs9389269) among different groups, but there were strong linkage disequilibrium and multiple haplotypes in many SNPs in each group, indicating that HBSIL‐MYB had extensive polymorphism distribution characteristics in the Hainan population.
KLF1 has a variety of natural mutations in different populations, with more than 130 mutations reported according to the Human Mutation Database, and the level of variation in populations varies with HbF expression levels. Borg et al. reported an association between mutations in KLF1 and HbF expression levels, and reducing KLF1 expression can improve disease severity in β‐thalassemia patients (Borg et al. 2010). Liu Dun et al. conducted mutation analysis of KLF1 in the southern China population, and the results showed that KLF1 mutation frequency was higher in thalassemia high incidence areas, and the modification effect on β‐thalassemia phenotype was most significant (Dun Liu 2015). Twelve SNPs were detected by genome resequencing in this study, and only two of them had high mutation frequency. Compared with the results of studies in other regions, for example, in Thailand, researchers sequenced the KLF1 exome of 130 β0/HbE patients and identified a total of 9 mutations, of which rs3817621 was thought to be associated with the expression level of HbF, but clinical evidence showed that it was insufficient to improve the clinical performance of patients, indicating that KLF1 mutations may not be effective in alleviating β0/HbE thalassemia (Khamphikham et al. 2018). In central India, rs3817621, rs2072597, and rs2072596 were found in 118 SCD patients, but in subsequent analysis, they were not significantly associated with HbF expression levels (Gambari et al. 2024). Therefore, the role of KLF1 may vary between different populations and diseases, and its modifying role needs to be further evaluated.
5. Conclusion
Foreign research institutions have proposed to score and standardize the anemia degree of patients by detecting pathogenic genes and modification sites. Researchers have emphasized the role of genetic modification factors in the diagnosis and screening of β‐thalassemia, and suggested that the modification effect should be quantified and used to guide clinical decision‐making. This study confirmed that there are many mutations of KLF1, BCL11A, and HBS1L‐MYB in the Hainan native population, and there is no obvious association between these modifiers and HbF expression level and phenotype. However, this does not rule out that there may be other factors related to HbF regulation, including the influence of other modifiers, DNA hypomethylation, and histone modifications in the β‐globin gene cluster, which deserve further study.
Author Contributions
Junjie Hu: conceptualization, formal analysis, methodology, visualization, writing – original draft. Huaye Chen: writing – review and editing. Wei Gong: writing – review and editing. Min Feng: writing – review and editing. Shidong Fu: writing – review and editing. Weihua Xu: writing – review and editing. Zhichao Ma: writing – review and editing. Shengmiao Fu: supervision, validation, writing – review and editing. Xinping Chen: supervision, validation, writing – review and editing.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgments
We would like to thank our laboratory colleagues for their assistance in the data and sample collection and laboratory analysis.
Hu, J. , Chen H., Gong W., et al. 2025. “Association Between KLF1, BCL11A and HBS1L‐MYB Polymorphisms and Phenotypes With β‐Thalassemia Patients in Hainan.” Molecular Genetics & Genomic Medicine 13, no. 9: e70142. 10.1002/mgg3.70142.
Funding: This work was supported by Hainan Province Science and Technology Special Fund (ZDYF2022SHFZ023) and Joint Program on Health Science and Technology Innovation of Hainan Province (WSJK2024QN103).
Junjie Hu and Huaye Chen are co‐first authors.
Data Availability Statement
The labeled dataset used to support the findings of this study is available from the corresponding author upon request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The labeled dataset used to support the findings of this study is available from the corresponding author upon request.