Genotype-Phenotype Correlation of β-Thalassemia in Malaysian Population: Toward Effective Genetic Counseling.

Effective prevention of β-thalassemia (β-thal) requires strategies to detect at-risk couples. This is the first study attempting to assess the prevalence of silent β-thal carriers in the Malaysian population. Hematological and clinical parameters were evaluated in healthy blood donors and patients with β-thal trait, Hb E (HBB: c.79G>A)/β-thal and β-thal major (β-TM). β-Globin gene sequencing was carried out for 52 healthy blood donors, 48 patients with Hb E/β-thal, 34 patients with β-TM and 38 patients with β-thal trait. The prevalence of silent β-thal carrier phenotypes found in 25.0% of healthy Malaysian blood donors indicates the need for clinician's awareness of this type in evaluating β-thal in Malaysia. Patients with β-TM present at a significantly younger age at initial diagnosis and require more blood transfusions compared to those with Hb E/β-thal. The time at which genomic DNA was extracted after blood collection, particularly from patients with β-TM and Hb E/β-thal, was found to be an important determinant of the quality of the results of the β-globin sequencing. Public education and communication campaigns are recommended as apparently healthy individuals have few or no symptoms and normal or borderline hematological parameters. β-Globin gene mutation characterization and screening for silent β-thal carriers in regions prevalent with β-thal are recommended to develop more effective genetic counseling and management of β-thal.

Keywords: β-Thalassemia (β-thal); genetic counseling; genotype; Malaysia; phenotype; prevalence; silent carriers

The "Thalassemia Belt" describes the regions extending from sub-Saharan Africa, through the Mediterranean region and Middle East, to the Indian subcontinent and East and Southeast Asia, where people are affected with this inherited disease [[1]]. An estimated 1.0–5.0% of the global population are carriers for thalassemia [[1]]. In Malaysia, the estimated carrier rate for β-thalassemia (β-thal) is 3.5–4.0%. The cutoff value of Hb A2 for β-thal carrier detection is equal to or more than 4.0%. However, some mutations can result in Hb A2 levels between 3.2% and 3.9% [[3]]. The mean of the newly diagnosed and treated Malaysian patients with Hb E (HBB: c.79G>A)/β-thal and those with β-thal major (β-TM) from 2013 to 2017 is 2423.8/year and 2501.2/year, respectively [[4]]. Currently, 934 entries involving the β-globin gene have been reported on the HbVar database [[5]]. However, small deletions removing all or part of the β-globin gene are rare [[4]]. Moreover, some β-thal are caused by deletions or mutations affecting the regulatory regions upstream of the β-globin gene complex [collectively referred to as the β-globin locus control region (LCR)], leaving the β-globin genes intact [[5]]. β-Globin gene mutation are detected using polymerase chain reaction (PCR)-based procedures followed by sequence analysis. Automated DNA sequencing employing the Dye Terminator method is preferably used for complete sequencing of the whole β-globin gene [[6]]. Three β-globin gene mutations were reported in 73.1% of Malays with β-thal; these include codon 26 (or Hb E) (GAG>AAG) (HBB: c.79G>A), IVS-I-5 (G>C) (HBB: c.92+5G>C) and IVS-I-1 (G>T) (HBB: c.92+1G>T). The five reported β-globin mutations that account for about 90.0% of β-thal in the Chinese-Malaysians were codons 41/42 (–TCTT) (HBB: c.126_129delCTTT), IVS-II-654 (C>T) (HBB: c.316-197C>T), −28 (A>G) (HBB: c.-78A>G), codon 17 (A>T) (HBB: c.52A>T) and codons 71/72 (+A) (HBB: c.216-217insA). The –45 kb Filipino (NG_000007.3: g.66258_184734del118477) deletion was reported in over 90.0% of transfusion-dependent thalassemia patients in Sabah State [[3]].

The cause of the phenotypic diversity in β-thal is not known but may be related to the different chromosomal background or to the C>T polymorphism at position –158 upstream of the Gγ-globin gene, which is associated with increased Hb F production under conditions of erythropoietic stress [[9]]. However, phenotypic diversity of thalassemia depends on the level of imbalance between α- and non-α-globin chains [[10]]. The Hb E/β-thal are classified as mild, moderate, or severe, with the severe form being similar to β-TM, and the mild and moderate forms being similar to β-thal intermedia (β-TI) [[1]]. The Hb E variant is characterized by weak union between α- and β-globin genes [[11]]. The coexistence α-thalassemia (α-thal) in patients with β-thal leads to a milder phenotype because of the reduction in the excess of α-globin chains. Patients with severe β-thal who inherit more α genes than normal, also tend to have more severe phenotypes. Other patients have milder β-thal because of their genetically determined ability to produce more γ-globin chains, and thus Hb F, resulting in less globin chain imbalance [[12]]. Mutations associated with the Hb E variant lead to abnormal, inefficient splicing of the associated β-globin gene, resulting in the phenotype of Hb E/β-thal [[13]]. This study aimed to evaluate the phenotype-genotype correlation of β-thal and to assess the prevalence of silent β-thal carriers in the Malaysian population.

Approval of the ethics committees of the National Medical Research Register (NMRR) [NMRR-3-1471-15105], Medical Research and Ethics Committee (MREC) [(8) dlm.KKM/NIHSEC/P14-462] and Jawatankuasa Etika Penyelidikan (Manusia), Universiiti Sains Malaysia (USM) (JEPeM) [JEPeM Code: USM/JEPeM/15] and Universiti Sultan Zainal Abidin (UniSZA) [UniSZA/C/2/CRIM/431-2 (24)] were obtained at the initial stage of the study.

In this cross-sectional study, healthy blood donor selection was based on the criteria adopted by the National Blood Center in Malaysia, which is in accordance with that of the WHO guidelines. The diagnosis of the clinical phenotypes of β-thal, including β-TM, Hb E/β-thal and β-thal trait, was based of the international guidelines and the standard clinical and hematological criteria. Prior to enrollment of each respondent, patient information and written consent was obtained. The hematological profile and β-globin gene sequencing of genomic DNA (gDNA) were carried out for the 52 healthy blood donors enrolled, 65 patients with Hb E/β-thal, 45 patients with β-TM and 48 patients with β-thal trait (Table 1). Some clinical parameters of the enrolled patients with transfusion dependent β-thal were obtained including: age at disease presentation and at receiving first blood transfusion, spleen size, requirement for transfusion and growth and development. A validated clinical scoring for Asian populations was used to determine the category of each patient with Hb E/β-thal [[15]].

Table 1. Demographics of the studied groups.

1 Hb: hemoglobin; β-TM: β-thalassemia major. There were 210 recruited responders. The majority of β-TM and Hb E/β-thal patients were transfusion-dependent, and were enrolled in the regular tranfusion program at the Paediatric Department of Hospital Kuala Lumpur, Kuala Lumpur, Malaysia. Those with β-thal trait were included at the time of initial diagnosis.

Venous blood samples (5 mL) were collected from selected healthy blood donors who had normal clinical and hematological profile as well as from patients with different type of β-thal included in this study (β-thal trait, β-TM and Hb E/β-thal) (Table 2). The blood samples were processed for full blood cell (FBC) count using an automated hematology system (Sysmex Corporation, Chuo-ku, Kobe, Japan) at Hospital Kuala Lumpur (HKL), Jalan Pahang, Kuala Lumpur, Malaysia, and stored at 4 °C until high performance liquid chromatography (HPLC) analysis was performed, generally within 2 weeks. The HPLC analysis of hemoglobin (Hb) was done using VARIANT II™, β-Thalassemia Short Program (Bio-Rad Laboratories, Hercules, CA, USA) as recommended by the manufacturer's instructions for quantification of Hb variants for all blood samples. For patients on a regular blood transfusion regimen, the blood samples for the FBC and HPLC analysis were collected just before the start of the scheduled blood transfusion.

Table 2. Verified mutations on the β-globin gene of the studied groups.

2 ID: identity; gDNA: genomic DNA; Hb: hemoglobin; β-TM: β-thal major. Human β-globin gene mutations were detected in exon 1 and intron 1 of healthy blood donors and patient groups. No mutations were detected in exons 2 and 3. Mutations caused no amino acid property changes, except one mutation leading to a stop of amino acid property detected in exon 1 of patients with β-TM (codon 17) at position 102. Failed sequencing occurred in 17/65 (26.1%), 11/45 (24.4%) and 10/48 (20.8%) of samples from Hb E/β-thal, β-TM and β-thal trait patients, respectively. No failed sequencing occurred in samples from healthy blood donors.

Genomic DNA was isolated from peripheral blood mononuclear cells using the LaboPass Genomic DNA isolation kit (Qiagen Biotechnology Malaysia Sdn Bhd, Kuala Lumpur, Malaysia) according to the manufacturer's recommendations. Extracted gDNA was determined quantitatively by absorbance measurement at 260 nm using NanoDrop™ ND-100 micro volume spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) to assess the purity of extracted DNA, and the gDNA was stored at –20 °C for about 2–3 months. Extracted gDNA was amplified using two sets of primers specific for the β-globin gene as described previously [[6]]. Briefly, the PCR amplification was performed using a DNA thermal cycler in, a 25 µl reaction volume containing 10 ng/µl genomic DNA, 6.5 µl distilled water, 12.5 µl polymerase (AmpliTaq Gold 360 Master Mix; Applied Biosystems, Foster City, CA, USA), and 0.5 µl of primers. Amplification was done with an initial denaturation step of 5 min. at 95 °C, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s.

β-Globin gene sequencing was carried out using the fluorescence dye terminator chemistry method on ABI PRISM® 3700 DNA Analyzer (Applied Biosystems, 1st BASE, Selangor, Malaysia), and analyzed using the 310 Genetic Analyzer (Sanger sequencing; Applied Biosystems, 1st BASE, Selangor, Malaysia). Using the same primers with a BigDye® Terminator v3.1 cycle sequencing kit chemistry (Applied Biosystems, 1st BASE, Selangor, Malaysia), the amplified DNA product was sequenced. The reaction is completed by the addition of template and primer to the master mix [[16]].

The UGENE (version 1.25) bioinformatics tool [[18]] was used to interpret the alignments of multiple sequences. All sequencing amplicons were screened for quality prior to analysis. Regions with erroneous base-calling were disregarded in the downstream analyses. Samples with highly incongruous sequences despite acceptable base-calling quality were removed from the multiple alignments. As a reference, the Hb subunit β sequence was acquired from the National Center for Biotechnology Information (NCBI) database Homo sapiens chromosome 11, GRCh38 reference primary assembly, obtained using the accession number NC_059281.

We recruited 210 respondents: 52 healthy blood donors, 48 individuals with β-thal trait, 45 individuals with β-TM and 65 individuals with Hb E/β-thal. The majority of respondents with Hb E/β-thal and those with β-TM were transfusion dependent (Table 1).

Sequencing of the β-globin gene was carried out using two sets of primers specific for the β-globin gene. Amplification of exons 1 and 2 and intron 1 (Target PCR I) employed forward (5′-CGA TCT TCA ATA TGC TTA CCA A-3′) and reverse (5′-CAT TCG TCT GTT TCC CAT TCT A-3′) primers. Amplification of exon 3 (Target PCR II) was done using forward (5′-CAA TGT ATC ATG CCT CTT TGC A-3′) and reverse (5′-TGC AGC CTC ACC TTC TTT CAT-3′) primers. The PCR I and PCR II products size were 916 and 667 bp, respectively. The β-globin gene sequencing employed the same primers. Failed sequencing occurred in 17/65 (26.1%), 11/45 (24.4%) and 10/48 (20.8%) of samples from patients with Hb E/β-thal, β-TM and β-thal trait, respectively. No failed sequencing occurred in samples from the healthy blood donors.

Human β-globin gene mutations were detected in exon 1 and intron 1 of healthy donors and patient groups. No mutation was detected in exons 2 and 3. Mutations were identified in 13/52 (25.0%) healthy blood donors with mutations at position 59 of exon 1 found in 12/52 (23.1%) donors and the IVS-I-5 (G>C) (HBB: c.92+5G>C) of intron 1 found in one donor (1.9%). Mutations in each of exon 1 and intron 1 were identified in 27/48 (56.3%) and patients with Hb E/β-thal. Mutations in exon 1 and intron 1 were identified in 23/38 (60.5%) and 14/38 (36.8%) patients with β-thal trait, respectively. No mutation was detected in exons 2 and 3 of patients and healthy blood donors (Table 2). Mutations caused no amino acid property change, except one mutation leading to stop of amino acid property detected in exon 1 of patients with β-TM (codon 17).

Significantly higher Hb F levels were found in patients with Hb E/β-thal and β-TM (p values 0.001 and 0.003, respectively), while Hb A2 was significantly higher (p value 0.001) in the Hb E/β-thal group compared with the other two patient groups. The mean of the mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) values were significantly lower in the patient groups compared to their values in healthy blood donors (p = 0.001). The red blood cell (RBC) distribution width (RDW-CV) was significantly higher in patient groups compared to its value in the healthy blood donors (p = 0.001) (Table 3).

Table 3. Hematological parameters of the studied groups.

• 3 Results are expressed as mean ± SD (min-max). β-TM: β-thal major; Hb: hemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular Hb; RDW-CV: RBC distribution width. Hb F levels were significantly higher in Hb E/β-thal and β-TM patients (p 0.001 and p 0.003, respectively), while Hb A2 was significantly higher (p 0.001) in the Hb E/β-thal group compared to the other two patient groups. The mean MCV and MCH values were significantly lower in patient groups compared to their values in the healthy blood donors (p 0.001). The RDW-CV was significantly higher in patient groups compared to its value in the healthy blood donors (p 0.001).
• 4 aA p value of <0.05 was considered to be significant using the one-way analysis of variance (ANOVA) test.

Clinical parameters (age at receiving first blood transfusion, spleen size, required transfusion interval to maintain Hb at target level of 9.5 g/dL, and growth development) [[15]] in the patients with Hb E/β-thal were compared to those with β-TM. Patients with β-TM were significantly (p = 0.0002) younger at initial diagnosis. Significantly (p = 0.00001) more transfusion requirements, as indicated by shorter transfusion interval (3.97 weeks), was observed in patients with β-TM compared to those with Hb E/β-thal (4.63 weeks) (Table 4).

Table 4. Clinical characteristics in patients with Hb E/β-thalassemia and those with β-TM.

• 5 β-TM: β-thal major; 95% CI: 95% confidence interval; t Statistic (df): independent test degrees of freedom. Patients with β-TM were significantly younger (p = 0.0002) at initial diagnosis. Significantly more transfusions were required (p 0.00001), as indicated by the shorter transfusion interval (3.97 weeks), was observed in patients with β-TM compared to those with Hb E/β-TM (4.63 weeks).
• 6 aA p value of <0.05 was considered to be statistically significant.
• 7 bHemoglobin levels were obtained from the average Hb levels at steady state or before receiving a blood transfusion.

The screening programs for β-thal are based on prospective screening and prenatal diagnosis. The current screening method for β-thal carrier detection has its limitations as mutations can exist and be missed in the screening process when the RBC indices are normal [[19]]. Confirmation of β-thal diagnosis requires Hb analysis by HPLC or capillary electrophoresis (CE) and DNA analysis of the globin genes. Specific guidance for β-thal diagnosis is available from recent clinical management guidelines [[20]]. The typical clinical phenotypes of β-thal include β-TM, β-TI and β-thal trait (β-thal minor, carrier state) [[1]].

Otherwise healthy individuals who carry the β-globin gene mutation of β-thal with no identified hematological abnormalities are called silent β-thal carriers [[1], [21]]. The interaction of silent β-thal carriers with β-thal trait results in a silent β-thal variant of reduced severity [[21]]. Silent β-thal carriers are typically discovered during family genetic studies [[1]]. Awareness of silent β-thal is essential for effective detection of carriers and prevention of β-thal, and represents a diagnostic challenge to the clinician. Individuals with silent β-thal are asymptomatic and have nearly normal β/α chain ratio and no hematological abnormalities with normal levels of Hb A2. This is the first study attempting to assess the prevalence of silent β-thal carriers in the Malaysian population, who have β-globin gene mutations without any clinical or hematological abnormality. Haplotype studies of intragenic single nucleotide polymorphisms (SNPs) are powerful discriminators between cases and controls in disease association studies. Despite that, haplotype determinations done in this study, have limitations in terms of determining haplotypes without parental genotypes; it reduces the number of tests to be carried out in disease association studies.

β-Globin gene complex point mutations include deletions or insertions of nucleotides are the predominant genetic defects causing β-thal [[1], [5]]. The mutations identified in this study cause no amino acid property change, except one mutation (codon 17) detected at position 102 in exon 1 of a patient with β-TM (Table 2). These findings agree with the fact that mutation in the β-globin gene in β-thal lead to quantitative defects in the Hb production [[8], [11]] rather than the production of structurally abnormal Hb molecules (qualitative defects).

The mutation at position 59 of exon 1 of the β-globin gene was found in 13/52 (25.0%) of healthy blood donors and the IVS-I-5 (G>C) mutation on the β-globin gene was found in one donor (1.9%). The same mutation was identified in patients with all the studied β-thal types (Table 2). This prevalence (25.0%) of silent β-thal in healthy Malaysian blood donors indicates the need for the clinician's awareness of this type of β-thal in the evaluation of β-thal in Malaysia, and might explain the occurrence of new cases of β-thal trait in the children of couples with no family history of β-thal.

The only abnormal parameter in silent β-thal carrier with normal RBC indices and Hb A2 level is the mild imbalance of globin chain synthesis. The –101 (C>T) (HBB: c.-151C>T) (silent β+-thal) interacts with a variety of more severe β-thal mutations to produce milder forms of β-thal in the Mediterranean populations. However, other mutations have been reported with highly variable Hb A2 levels despite similar hematological parameters and globin chain synthesis ratios [[9]]. Thalassemia carrier screening programs employed for different populations vary in many aspects, including whether the programs are mandatory or voluntary, the education and counseling provided and whether screening is offered pre pregnancy or prenatally [[23]].

To date, there are 934 recognized, disease-causing, mutations of the β-globin gene that have been reported, ranging from silent β-thal carriers to mutations causing relative low (β+) to complete lack of β-globin chain synthesis (β0) [[5]]. However, β-globin gene deletions are rare [[1]]. Mutations in each of exon 1 and intron 1 were identified in 27/48 (56.3%) patients with Hb E/β-thal, in 19/34 (55.9%) and 14/34 (41.2%) patients with β-TM, and in 23/38 (60.5%) and 14/38 (36.8%) patients with β-thal trait, respectively. No mutations were detected in exons 2 and 3. These findings are in contrast to reported mutations in β-thal which are found in all the exons [[1], [6], [16]].

In the other β-thal phenotypes, Hb E/β-thal is the most common in South Asian and Southeast Asian populations [[2]]. Clinical characteristics in patients with Hb E/β-thal and those with β-TM were compared, aiming to evaluate these groups of patients in addition to those with silent β-thal carrier, to give a more clear view of the clinical spectrum of the disease, as this is a clinically-oriented study. The clinical forms of Hb E/β-thal are classified as mild, moderate, and severe, with the severe form being similar to β-TM, and the mild and moderate forms being similar to β-TI [[1]]. Phenotypic variability is directly affected by the level of globin chain imbalance [[19]]. The severity of globin chain imbalance attributes to the finding of significantly higher Hb F levels in patients with Hb E/β-thal and β-TM (Table 3). The higher level of Hb A2 found in patients with Hb E/β-thal, as compared with the other two patient groups, is consistent with other reported findings [[1], [4], [24]].

The significantly lower mean MCV and MCH values in the patient groups as compared with the values in the healthy blood donors (p = 0.001), indicating ineffective erythropoiesis in patients group compared with normal erythropoiesis in healthy blood donors. However, the values in patients with Hb E/β-thal and β-TM showed no significant difference (p = 0.837) (Table 3). This is due to the same level of suppression of endogenous erythropoiesis caused by the transfusion program in the management of these two types of thalassemia. The significantly higher RDW-CV values in patient groups compared to its value in the healthy blood donors (p = 0.001) indicate the more severe degree of anisopoikilocytosis in β-thal secondary to the effects of ineffective and dyserythropoiesis [[25]].

Patients with β-TM were a significantly (p = 0.0002) younger age at initial diagnosis, which might be attributed to the Hb switching from γ to β type in early infancy (Table 4). B-TM patients had significantly (p = 0.00001) more transfusion requirements to maintain their steady Hb at a target level of 9.5 g/dL (Table 4), which might reflect the more severe hemolysis due to decreased deformability of the red cells, inclusion body formation, or senescence antigen exposure of thalassemic red cells [[1], [25]].

The prevalence of silent β-thal carriers in 25.0% of Malaysian healthy blood donors indicates the need for clinician's awareness of this type of β-thal in the evaluation of β-thal in Malaysia, and might explain the occurrence of new cases of β-thal trait in the children of couples with no family history of β-thal. Incorporating molecular analysis in the β-thal screening programs to identify the type and frequency of β-globin gene mutation even in apparently healthy individuals in regions prevalent with β-thal is recommended as the majority of the population in these regions is unaware of their thalassemia status. Patients with β-TM were diagnosed at a significantly younger age, and required significantly more transfusions. Public education and communication campaign are recommended as apparently healthy individuals have fewer or no symptoms and normal or borderline hematological parameters. Mutation characterization is required for couples with consanguineous marriages at-risk of having a child with β-thal to develop more effective genetic counseling. Our findings recommend the need for implementing oriented prenatal diagnostic strategies even in apparently healthy individuals in family with a history of β-thal. To limit the future burden of β-thal disease, consanguineous marriages should be avoided even between apparently healthy individuals in families who have a history of patients with β-TM. This preventive approach will be more effective, particularly in regions endemic for this inherited blood disease.

The limitations of this study were (a) the technical limitation in the control of the time elapsed from the blood sample collection to time of the analysis (the storage period), which affects the success and quality of the gene sequencing; (b) sequencing of intron 2 of the β-globin gene was not performed; (c) multiple sequence alignment analysis was not done for the upstream and downstream regions of the three exons due to the poor quality of sequencing in these regions.

The authors are grateful to all the respondents who participated in this study. The authors provided intellectual input into all aspects of this study. The corresponding author had full access to data in the study and had final responsibility for the decision to submit the study for publication.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.