HBB Gene Mutations and Their Pathological Impacts on HbE/β-Thalassaemia in Kuala Terengganu, Malaysia

Background: β-thalassaemia is a disorder caused by mutations in the β-globin gene, leading to defective production of haemoglobins (Hb) and red blood cells (RBCs). It is characterised by anaemia, ineffective erythropoiesis, and iron overload. Patients with severe β-thalassaemia require lifelong blood transfusions. Haemoglobin E beta-thalassaemia (HbE/β-thalassaemia) is a severe form of β-thalassaemia in Asian countries. More than 200 alleles have been recognised in the β-globin region. Different geographical regions show different frequencies of allelic characteristics. In this study, the spectrum of β-thalassaemia (β-thal) alleles and their correlation with iron overload, in HbE/β-thalassaemia patients, β-thalassaemia trait, and HbE trait were studied. Methods: Blood samples (n = 260) were collected from 65 β-thalassaemia patients, 65 parents (fathers and/or mothers) and 130 healthy control individuals. Haematological analyses, iron profiles, and serum hepcidin levels were examined for all participants. DNA was extracted from patients’ and their parents’ blood samples, then subjected to PCR amplification. Multiplex amplification refractory mutation system PCR (MARMS-PCR) was conducted for eighteen primers to detect the mutations. Results: There was severe anaemia present in HbE/β-thalassaemia patients compared to their parents and healthy controls. The ferritin and iron levels were significantly increased in patients compared to their parents and healthy controls (p = 0.001). Two common mutations were detected among the patient group and three mutations were detected among their parents, in addition to seven novel mutations in HbE/β-thalassaemia patients (explained in results). Conclusion: Some mutations were associated with severe anaemia in β-thalassaemia patients. The detection of mutations is a prognostic marker, and could enhance the appropriate management protocols and improve the haematological and biochemical statuses of β-thalassaemia patients.


Introduction
Thalassaemia is a group of inherited haematologic disorders caused by defects in the synthesis of one or more of the haemoglobin chains. These are among the most common genetic diseases globally, occurring more frequently in the Mediterranean, Indian subcontinent, Southeast Asia, and West Africa [1,2].
Thalassaemia can be classified generally into alpha-thalassaemia (α-Thal) and β-Thal. Each is subdivided into different types according to the severity of the mutation. Alpha Multiplex ARSM PCR was used to detect 20 main expected point mutations in the HBB gene in HbE/β-thalassaemia patients and their parents in Kuala Terengganu, Malaysia. This allowed these point mutations to be characterised directly by the presence or absence of the amplification using specific primers. These point mutations were Codon 26, Codon 41/42, Codon 8/9, Cd 17, IVS 1-1 (G>T), -28, Cd 71/71, Cd 43, Poly A, Cd 19, CAP + 1, Cd 15, IVS 2-654, IVS 1-1 (G>A), Cd 30, Cd 16, -86, and IVS-1-5. This analysis was applied sequentially to the DNA samples extracted from the blood of 65 HbE/β-thalassaemia patients, 65 parents, and 130 healthy controls to reveal the presence of specific point mutations. For each reaction, a pair of allele-specific primers were used; one primer had 3' terminal nucleotides complementary to the point mutation, while the other primer had 3' terminal nucleotides complementary to the internal control DNA sequence. The internal control primer was used to ensure that the process of amplification was successful. The sequence of primer sets used for the detection of beta-globin gene mutations by MARMS is listed in Table 1.

PCR Mix Preparation
A 20 µL volume of PCR reaction mix was performed, containing 2 µL of DNA; 2X lyophilised Green master mix; and 0.1-1.0 µL of each of the internal control primers (A, B, C, D, E, and F common primer and mutation-specific primer). Nuclease-free water was added for a final volume of 20 µL. Then, the PCR amplification protocol was applied as follows: a denaturing step for 3 min at 95 • C for 1 cycle, followed by 30 cycles of denaturation for 30 s at 95 • C, annealing for 75 s at 60 • C for 30 cycles, and extension for 150 s at 72 • C by using a Dream Taq Green PCR Master Mix (2X) kit (cat. no. K9021; Thermo Scientific, Thermo Fisher Scientific Inc., Wilmington, DE, USA). The components of the PCR reaction mix are shown in Table 2.

PCR Protocol
Six separate reactions were performed for each sample to test each mutation primer. Primers were divided into six groups according to their PCR protocol, and the same conditions and number of cycles were used for each group [11,12].
Multiplex-ARSM A was applied to screen for the IVS 1-5 (G>C), Cd 41/42 (-TTCT), Cd 17 (A>T), and Cd 26 (G>A) HbE mutations. In MARMS-B, we screened for the IVS 1-1 (G>T), Cd 8/9 (+G), -28 (A>G), Cd 30 (G>C), and Cd 71/72 (+A) mutations. In MARMS-C, we screened for the IVS 1-1 (G>A), Cd 43 (G>T), Cd 16 (-C), and Poly A (A>G) mutations. In MARMS-D, we screened for the Cd 19 (A>G) and cap +1 (A>C) mutations. In MARMS-E, we screened for the Cd 15 (G>A) mutation. Another mutation of IVS 2-654 (C>T) was detected in a single ARMS, MARMS-F. Subsequently, gel electrophoresis was performed to confirm the successful amplification and correct size of the PCR products. In all successful ARMS-PCR reactions, an internal control product of 861 bp molecular weight was observed. It was considered a mandatory sign of a successful reaction upon gel electrophoresis, and its band was located between the 800 bp and 900 bp bands of a 100 bp ladder marker. Additionally, the ARMS-PCR products for normal, heterozygous, and/or homozygous samples for each type of studied mutation were observed. For normal samples, the ARMS-PCR products were found within the normal primer reactions, while in positive diagnosed patients and parents, ARMS-PCR products were found within both normal and mutant reactions in heterozygous samples, and only within mutant primer reactions in homozygous samples.

Sanger Sequencing Analysis
Sanger sequencing was performed for three HbE/β-thalassaemia patients' samples by the Institute for Medical Research (IMR), Kuala Lumpur, using a Big Dye cycle sequencing kit. The samples were then analysed on the ABI 3730XL DNA Analyser (Applied Biosystems, Singapore). This assay is able to detect the presence of β-thal mutations from −100 of the 5'untranslated region to +320 of the 3'untranslated region.

Statistical Analysis
The data obtained in the present study were statistically analysed using the Statistical Package for Social Sciences (SPSS Inc., version 20, IBM Corporation, Somers, NY, USA). Comparisons between the mean values of different parameters were performed using the independent t-test and one-way ANOVA statistical analysis. A p < 0.05 was considered statistically significant. The results were presented as frequency (n) and percentage (%). Normality and homogeneity of variance were tested for each group included in the study, and the assumptions were met. Therefore, we conducted parametric tests (independent t-test and one-way ANOVA).

Results
The participants in this study comprised 260 subjects divided into two groups: the patient group (n = 130), which included 65 HbE/β-thalassaemia patients and 65 parents of patients; and the control group, which involved 130 apparently thalassaemia-free participants. The percentages and frequencies for each group based on genders are provided in Table 3. No significant differences were observed in the mean gender of the patients, their parents, or the controls included in this study. In the current study, the mutational analysis revealed 18 types of β-thalassaemia mutations detected in 130 out of 260 participants (65 HbE/β-thalassaemia patients; 65 patients' parents, designated as fathers and/or mothers; and 130 healthy controls). Among the 65 HbE/β-thalassaemia patients, 61 showed heterozygous mutations and 4 showed homozygous mutations (Table 4). In addition, there were 59 parents with homozygous mutations and 6 with heterozygous mutations (Table 5). On the other hand, the mutational analysis showed that all 130 of the healthy control samples were negative for β-thal alleles. Table 4. Types of compounds and heterozygous and homozygous mutations in HbE/β-thalassaemia patients.

Mutations
Number

Haematological and Biochemical Characteristics According to the Genotypes of HbE/β-Thalasasemia Patients and Their Parents
To relate the severity of thalassaemia to the type of mutation, the respective haematological and iron profiling results of the patients and parents were also investigated. The results revealed that the codon 26 (GAG>AAG) HbE (βE) mutation was associated with severe hypochromic microcytic anaemia, with Hb concentrations between 55-95 g/dL in patients (Table 4) compared to those between 100-125 g/dL for their parents ( Table 5).
The results also revealed a significant difference in red cell indices of HbE/β-thalassaemia patients and their parents compared to healthy controls. In addition, morphological RBCs changes and poikilocytosis were seen in more than 85.7% of HbE/β-thalassaemia patients. The results also showed remarkable thrombocythemia and leukocytosis in patients compared to their parents and healthy controls.
The results from the iron profile revealed a significant increase in serum ferritin in HbE/βthalassaemia patients compared to their parents and healthy controls (p < 0.001). Serum iron levels were also significantly increased in HbE/β-thalassaemia patients (p = 0.001) compared with their parents and healthy controls. Serum hepcidin levels were reduced significantly (<0.5 ng/mL) both in patients and their parents compared to healthy controls.
Patients with mutations of Cd 41/42 (-TTCT) (β0), IVS 1-5 (G>C) (β+), and Cd 17 (A>T) (β0) showed mild anaemia (Hb 98 g/dL) with significantly lower serum ferritin and serum iron levels compared to patients with other mutations in the present study (Tables 6 and 7).  The haematological characteristics of patients with compound heterozygous mutation showed low Hb concentration and RBC indices compared to parents and healthy controls. No significant differences were found between the different genotypes regarding WBCs and PLT compared to other parents with homozygous mutations. The biochemical parameters showed significantly higher serum ferritin and serum iron levels and reduced hepcidin levels compared to other parents' mutations (Tables 8 and 9). Table 8. Haematological characteristics according to the mutation types of parents (mother/father). ANOVA, analysis of variance. Data represent mean ± s.d. of n = 65 parents. a hepcidin in heterozygous mutations of parents vs. homozygous mutation types (p < 0.001); b serum ferritin in heterozygous mutations of parents vs. homozygous mutation types (p = 0.001); c iron in novel mutations of HbE/β-thalassaemia patients vs. heterozygous mutations of parents (p < 0.001); d TIBC and UIBC heterozygous mutations of parents vs. homozygous mutation types (p = 0.001); TIBC: total iron binding capacity; UIBC: unsaturated iron binding capacity.
The haematological characteristics of parents with compound heterozygous mutations included low Hb concentration and RBC indices compared to parents with homozygous mutations and healthy controls. No significant differences were found between the different genotypes regarding WBCs and PLT compared to other parents with homozygous mutations. On the other hand, biochemical parameters showed significantly higher serum ferritin and serum iron levels, which led to reduced hepcidin levels compared to the genotypes of other parents and healthy controls (Tables 8 and 9).

Discussion
β-Thalassaemia (β-Thal) is caused by mutations in the β-globin gene (HBB), and is one of the most common genetically inherited haemoglobin disorders globally [12,13]. Identification of the spectrum of β-Thal mutations might support the prevention and community control of β-Thal [14]. β-Thal can be associated with an abnormal β-globin chain, such as haemoglobin E (HbE) disease exhibiting severe anaemia. Hb E is a haemoglobin (Hb) variant caused by a single base substitution of glutamic acid to lysine at position 26 of the globin chain, commonly found in Southeast Asian countries such as Malaysia. It can be classified into three types: heterozygous, homozygous, or compound heterozygous. When the HbE trait is coinherited with β-Thal, it is called compound heterozygous, a condition known as HbE/β-thalassaemia which clinically and haematologically resembles homozygous β0-Thalassaemia [15,16].
The presence of the Chinese and Indian races in the Malay Peninsula at different times throughout the centuries introduced various genetic mixtures into the Malaysian population [23]. Before British colonisation, the Chinese and Indian traders had established strong trading links with the Malay Peninsula, resulting in widespread intermarriage and integration between them and the Malaysians [24].
In the present study, compound heterozygous (β0)-thalassaemia Filipino~45 kb deletion with codon 26 (GAG>AAG) HbE (βE) were found in 1 (1.54%) patient. Filipino β0-deletion was reported for the first time in Australia by Motum et al. in a Filipino family, and it was found in 45.8% of the β-globin mutant alleles in Taiwan, Filipinos [25,26]. This mutation was also found in Indonesians from the eastern part of Indonesia due to its geographical location adjacent to the Philippines [27,28]. Filipino β0-deletion is present among Malay and Chinese populations in the indigenous communities of East Malaysia, such as Sabah (homozygosity: 219 (86.9%), and especially in Kadazandusun (137 (62.6%)) [29]. Therefore, in our study, Filipino β0-deletion was considered as a novel mutation among the participants of the study population. Common haemoglobin (Hb) variants were found to coexist with the Filipino β0-deletion. The Hb variant in this study was HbE, which is consistent with a previous report in which HbE was a common β+-Hb variant among Malaysians [30]. The coexistence of Hb E with Filipino β0-deletion has also been reported in Thai and Indonesian populations [28,31].
In the current study, compound heterozygous codon 26 (GAG>AAG) HbE (βE) with IVS I-2 (T→G; AGGTTGGT→AGGGTGGT) (NM_000518.4:c.92 + 2T > G) was found in 3 (4.6%) patients. IVS I-2 is commonly found in Tunisians. This rare mutation results in β0-thalassaemia with the transition of T to G at intron 1 in chromosome 11. The transition changes in the GT dinucleotide disturb the normal splicing event, and no normal mRNA is produced (Itha ID: 104; Hb Var ID: 821) [29]. The relativity of the genetic variations observed in our study might be attributable to the history of human migration and hybridisation, which resulted in large-scale intermarriage and integration between indigenous communities in Sabah and Terengganu, Malaysia.

Conclusions
In conclusion, molecular investigation of thalassaemia patients is recommended for accurate diagnosis. This process could contribute to appropriate management protocols and improve the haematological and biochemical statuses of patients, especially those with mutations associated with severe anaemia. However, further studies into the ethnicity background of this group would be required. There is molecular heterogeneity of the HBB mutation among HbE/β-thalassaemia patients in Terengganu. A deteriorated haematological and biochemical picture was seen in the patients, which would require a more appropriate management protocol, especially for those with severe mutations. Moreover, the role of genetic modifiers in disease severity needs to be addressed in future studies in order to better understand disease pathophysiology. In the end, a feasible regional preventive program is key to reducing the number of affected births in settings with a high prevalence of thalassaemia. Finally, it is noteworthy that the absence of evidence-based transfusional regimens and follow-up protocols are not the only gaps in managing patients with thalassaemia. Furthermore, although genetic testing of thalassaemia is becoming critical, especially for complex atypical thalassaemia [34], molecular diagnosis is rarely performed. This service is not provided in MOH facilities, which is another gap in managing patients with thalassaemia in Kuala Terengganu, Malaysia.