Diagnostic Challenges of Borderline A2 Hemoglobin in β-Thalassemia Carriers: Insights from a Bangladeshi Cohort

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Abdul Aziz, Rowshon Zahan Luna, Salma Sadiya, Waqar Ahmed Khan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7223977/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The identification of beta-thalassemia (β-thal) carriers during prenatal screening relies on elevated hemoglobin A2 (HbA2) levels. However, β-thal minor may present as silent carriers, showing no hematologic abnormalities despite carrying a mutation. Borderline HbA2 levels pose a diagnostic challenge. This study aimed to characterize 120 individuals—selected from a pool of 1,650—with borderline HbA₂ levels, to distinguish true carriers from non-carriers. Methods Blood samples were collected in EDTA tubes from Bangladeshi subjects with borderline Hb A2 levels (3.0–3.9%) for this study. The samples were obtained from the Thalassemia Center at Bangladesh Shishu Hospital and Institute. Hematological parameters were measured using an automated blood counter (ADVIA 2120i hematology analyzer; Siemens Healthcare Diagnostics, Deerfield, IL, USA). HBB genotypes were identified using the Sanger sequencing method. Results Among the subjects, pathogenic mutations of the β-globin gene were identified in 3 cases (15%) with HbA₂ levels of 3.3–3.4%, and in 17 cases (85%) with levels between 3.5–3.9%. A total of seven pathogenic mutations were identified, with IVS1-5 (G > C) being the most common (55%, n = 11), followed by FS 41/42 (− CTTT), Codon 30 (G < C), and Codon 30 (G A), and FS 16 (− C) were each found in 5% of cases (n = 1). Conclusions This study supports improved identification of β-thal carriers with borderline HbA₂ levels, helping to reduce the risk of misdiagnosis. β-thalassemia prenatal screening borderline HbA2 levels mutation analysis Figures Figure 1 Introduction The inheritance of a single mutated β-thalassemia (β-thal) allele causes the β-thal trait, which is characterized by hypochromic, microcytic red blood cells and elevated HbA₂ levels. (Colaco et al., 2022 ). In some cases, the impact of this allele or its interaction with other genetic modifiers may be negligible or silent, leading to normal or borderline HbA₂ levels. Borderline HbA2 values for β-thal carriers typically range from 3.0–3.9% (Moradi et al., 2022 ; A. Noor et al., 2024 ). When both parents are carriers of β-thalassemia, each pregnancy has a one-in-four (25%) likelihood of resulting in a child affected by the condition. This chronic condition may result in severe health problems. There is a 50% probability that the child will be a carrier and a 25% chance that the child will inherit neither the mutation nor the disease (Mitro et al., 2024 ). The diagnosis of β-thal or carrier status is typically based on the detection of increased HbA₂ (α₂β₂) levels, with values exceeding 4.0% generally indicative of β-thal trait. (Giordano, 2015 ; Suresh et al., 2023 ). However, no studies have yet reported β-globin gene mutations in individuals with HbA2 levels below 4.0% in various populations of Bangladesh. As a result, individuals with borderline HbA2 levels (3.5–3.9%) are difficult to definitively classify as normal or β-thal carriers. Molecular analysis is necessary to detect β-globin gene mutations in these individuals, especially when one partner is a known β-thal carrier. To date, numerous β-globin gene mutations associated with β-thal have been identified. According to the HbVar database ( https://globin.bx.psu.edu/cgi-bin/hbvar/counter ), a total of 958 β-thalassemia alleles have been documented. However, population studies indicate that 20 mutations account for 80% or more of β-thalassemia cases worldwide (Rao et al., 2024 ). In Bangladesh, some studies have reported β-globin gene mutations, although none have specifically focused on individuals with borderline HbA2 levels. Furthermore, the reported mutations in the Bangladeshi population are not highly diverse (Banu et al., 2018 ; F. A. Noor et al., 2020 ). In this study, seven pathogenic mutations were found in individuals with borderline HbA2 levels. Borderline HbA2 values are commonly observed in regions with a high prevalence of β-thalassemia, such as South Asian countries—particularly Bangladesh and India (Rangan et al., 2011 ; Singh et al., 2023 ; Yadav et al., 2022 ). Approximately 4.1% of the Bangladeshi population are β-thalassemia carriers, while around 6.1% possess the hemoglobin E (HbE) trait. (W. Khan et al., 2005 ). It is estimated that over 14,000 children are born annually with thalassemia, primarily with HbE β-thal, followed by β-thalassemia major (W. A. Khan et al., 2021 ). In developing countries like Bangladesh, most affected infants are neither diagnosed nor treated, often dying in early infancy due to complications from anemia (Hossain et al., 2017 ). Given the high prevalence and the significant cost of treatment, prevention and control of new thalassemia cases are critical. As the number of β-thalassemia carriers continues to rise in developing countries such as Bangladesh, accurate detection of HbA2 levels is essential for effective screening and prevention on a global scale. Methods Selection of patients. This study was designed for patients with borderline HbA2 levels who visited the hospital for hemoglobin electrophoresis. Between January 2022 and December 2022, a total of 1,650 individuals were referred to our institute for hemoglobinopathy screening. Among them, 120 individuals (66.7% male [n = 80] and 33.3% female [n = 40]) were identified with HbA2 levels between 3.0% and 3.9%. The participants ranged in age from 2 to 65 years. Subjects were randomly selected for the study after obtaining written informed consent. Individuals who visited Bangladesh Shishu Hospital and Institute for treatment were included as study participants. Peripheral blood (2 ml) was collected in EDTA-containing tubes and stored at 4°C for further analysis. Hematology analysis Hematology analysis was done by SYSMEX Automated Hematology Analyzer (Model: XN1000). It measured the red blood cell (RBC) count, white blood cell (WBC) count, platelets, Hb, and hematocrit levels, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW). In addition, Hb electrophoresis was performed by Varant II (HPLC) for identification of Hb fraction. DNA extraction DNA was isolated from peripheral blood using the PureLink® Genomic DNA Purification Mini Kit (Invitrogen, USA), which utilizes phase separation by centrifugation of the aqueous sample. The procedure was carried out according to the manufacturer’s instructions. The purity and concentration of the extracted DNA (per microliter) were assessed using the Qubit® 2.0 Fluorometer. The extracted DNA samples were stored at -20°C for long-term preservation. Molecular analysis Mutation analysis was performed using the Sanger sequencing method. The analysis targeted the entire coding region (exons 1, 2, and 3), portions of introns 1 and 2, the promoter region, and the splice junctions of the β-globin gene (locus: NG_000007.3). Genomic DNA was first amplified using exon-specific primers for exons 1, 2, and 3 via the PCR method. Sequencing was conducted using an automated capillary electrophoresis (CE) system on the 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The resulting sequences were aligned with the reference sequence of the β-globin gene [NCBI RefSeq: NG_000007.3] using SeqScape Software version 2.5 (Applied Biosystems). Genetic variants and their clinical significance were identified using the HbVar and ClinVar databases ( http://globin.bx.psu.edu/hbvar and https://www.ncbi.nlm.nih.gov/clinvar/ ), which catalog hemoglobin variants and thalassemia mutations [13]. Results Demographic characteristics: According to this study, a total of 120 blood samples were characterized by various clinical features. Among them, the male was 66.7% (n=80) and female was 33.3% (n=40) (Figure 1a). The majority of participants were between 2 and 7 years old, accounting for 50% (n=60) of the sample. This was followed by participants aged 8–13 years (25%, n=30), 26–31 years (10.8%, n=13), 20–25 years (7%, n=7), over 31 years (6%, n=6), and 14–19 years (3.3%, n=4) (Figure 1b). β‑globin gene mutations Among the 120 individuals with borderline HbA2 levels, seven distinct β-thalassemia mutations were detected (Table 1). The IVS1-5 G>C (β⁺) mutation was the most frequently observed, present in 55% of cases (n=11). This was followed by FS 41/42 -CTTT (β⁰), Codon 30 G>A (β⁰), and Codon 30 G>C (β⁰), each found in 10% of individuals (n=2). Additionally, FS 8/9 +G (β⁰), Codon 15 G>A (β⁰), and FS 16 -C (β⁰) were each identified in 5% of cases (n=1). Overall, β⁰ mutations constituted 40% of all genetic variants detected in individuals with borderline HbA2 levels. Among the 25 individuals with HbA2 levels ranging from 3.3% to 3.4%, β-thalassemia alleles were detected in 3 cases (15%). Among them, two cases involved β⁺ mutations (IVS1-5 G>C), while one case was identified as a β⁰ mutation (Codon 30 G>A). Out of 18 individuals with HbA2 levels between 3.5% and 3.9%, β-thalassemia alleles were found in 17 cases, accounting for 85%. IVS1-5 G>C was the most frequently observed mutation among them, present in 45% of cases (n=9), followed by FS 41/42 -CTTT (β⁰) and Codon 30 G>C (β⁰), each found in 10% (n=2). Additionally, FS 8/9 +G (β⁰), Codon 15 G>A (β⁰), and FS 16 -C (β⁰) were each detected in 5% of individuals (n=1). Table 1. Mutation patterns observed in β-thalassemia carriers with borderline HbA2 levels. β-thalassemia mutation n (%) HbA2 3.0–3.2% HbA2 3.3–3.4% HbA2 3.5–3.9% IVS1-5 G>C [β+] 11 (55%) 0 2 9 FS 8/9+G [β0 ] 1 (5%) 0 0 1 FS 41/42CTTT [β0 ] 2 (10%) 0 0 2 codon 30 G>A [β0 ] 2 (10%) 0 1 1 codon 30 G>C [β0 ] 2 (10%) 0 0 2 codon 15 G>A [β0 ] 1 (5%) 0 0 1 FS 16 –C [β0 ] 1 (5%) 0 0 1 Total 20 (100%) 0 3 17 Relation between MCV and HbA2 levels Since carrier detection of β-thalassemia typically depends on MCV and MCH values in conjunction with HbA2 levels, we analyzed these parameters in the 20 β-thalassemia heterozygotes identified in this study (Table 2). All three β-thalassemia heterozygote mutations with HbA2 levels ranging from 3.3% to 3.4% had MCV values greater than 80 fL. In contrast, among the 17 heterozygous individuals with HbA2 levels between 3.5% and 3.9%, 70.6% (n=12) had MCV values under 80 fL, whereas 29.4% (n=5) had MCV values exceeding 80 fL. In terms of MCH values, two out of three β-thalassemia heterozygotes with HbA2 levels from 3.3% to 3.4% had MCH below 27 pg, while one exhibited MCH above 27 pg. Likewise, among the 17 individuals with HbA2 levels between 3.5% and 3.9%, 76.5% (n=13) had MCH values under 27 pg, and 23.5% (n=4) had values exceeding 27 pg. Among individuals with a normal β-globin genotype and MCV less than 80 fL, 30 had HbA2 levels in the 3.0–3.2% range, 7 had levels between 3.3–3.4%, and none exhibited HbA2 values between 3.5–3.9%. In contrast, among those with MCV greater than 80 fL, 47 individuals fell within the 3.0–3.2% HbA2 range, 15 were in the 3.3–3.4% range, and only one had HbA2 between 3.5–3.9%. With respect to MCH values, 55 individuals having MCH less than 27 pg exhibited HbA2 levels between 3.0–3.2%, while 12 individuals fell within the 3.3–3.4% range, and no cases were observed in the 3.5–3.9% range. Similarly, among 52 individuals with MCH values above 27 pg, 33 had HbA2 levels between 3.0–3.2%, 10 were between 3.3–3.4%, and just one had a value in the 3.5–3.9% range. Table 2. Distribution of study individuals according to HbA2, MCV and MCH. Parameters HbA2 values (%) Total 3.0-3.2 3.3-3.4 3.5-3.9 Total individuals n=77 n=25 n=18 n=120 βTT MCV 80 fL 0 3 5 8 Total βTT 0 3 17 20 MCH27 pg 0 1 4 5 Total βTT 0 3 17 20 Normal β-genotype MCV 80 fL 47 15 1 63 Total Normal β genotype 77 22 1 100 MCH27 pg 22 10 1 33 Total Normal β genotype 77 22 1 100 Of the 120 individuals investigated in this study, 49 individuals showed MCV 80fL (Table 3). Among 49 subjects, 12 individuals were identified as thalassemia heterozygotes mutations with MCV <80 fL. The average hematological parameters for these individuals were as follows: RBC count of 5.2 × 10⁶/µL (SD = 0.6), hemoglobin level of 10.5 g/dL (SD = 1.8), MCV of 66.85 fL (SD = 3.0), MCH of 19.92 pg (SD = 3.3), RDW at 18.12% (SD = 4.5), HbA2 at 3.6% (SD = 0.3), and HbF at 1.2% (SD = 2.1). Among the 8 thalassemia heterozygotes with MCV >80 fL, the hematological parameters were as follows: RBC = 3.83 × 10⁶/µL (SD = 0.5), Hb = 10.6 g/dL (SD = 2.1), MCV = 86.76 fL (SD = 4.2), MCH = 27.8 pg (SD = 2.0), RDW = 18.0% (SD = 6.47), HbA2 = 3.7% (SD = 0.2), and HbF = 0.9% (SD = 1.2). Table 3. Hematological analysis of the individuals with MCV≤80 fL and MCV≥80 fL. MCV80 fL n= 71 RBC × 106 /µl Hb g/dl MCV fL MCH pg RDW % HbA2 % HbF % No Defects identified 63 Defects identified 8 3.83 ± 0.5 10.65 ± 2.1 86.76 ± 4.2 27.8 ± 2.0 18 ± 6.47 3.7 ± 0.2 0.9 ± 1.2 Discussion Thalassemia is the most prevalent inherited recessive blood disorder globally. (Tesio & Bauer, 2023 ). An estimated 1.5% of the world’s population, equivalent to around 80–90 million individuals, are carriers of β-thalassemia. (Rao et al., 2024 ). It is important to emphasize that this complex disease is largely preventable through simple, cost-effective measures such as carrier screening and genetic counseling (Aziz et al., 2020 ; Pinto & Forni, 2020 ). The characterization of β-thal carriers show mild, persistent anemia and distinctly elevated levels of HbA2 which is basis for screening programs worldwide (Colaco & Nadkarni, 2021 ). In this context, while elevated HbA₂ levels are a key indicator for diagnosing β-thalassemia, the presence of borderline HbA₂ levels in some individuals poses a diagnostic challenge. In this study, the IVS1-5 (G > C) mutation was the most prevalent (55%) among the Bangladeshi population, aligning with other studies conducted in Bangladesh. Among the 11 IVS1-5 (G > C) mutations identified, 9 were associated with HbA2 values between 3.5% and 3.9%, while 2 were found in individuals with HbA2 values between 3.3% and 3.4%. Another study in Bangladesh found that the frequency of IVS1-5 (G > C) mutation was 42.68% (Mahzabin et al., 2021 ). According to Colaco et al. ( 2022 ), the IVS1-5 (G > C) mutation was identified in 4% of individuals with HbA₂ levels ranging from 3.0 to 3.2%, 24% of those with levels between 3.3 and 3.4%, and 72% of individuals with HbA₂ levels between 3.5% and 3.9%. Mutations including FS 8/9 (+ G), FS 41/42 (− CTTT), Codon 30 (G > A and G > C), Codon 15 (G > A), and FS 16 (− C) were detected in individuals with HbA₂ levels of 3.5–3.9%, while Codon 30 (G > C) was observed in only one case with HbA₂ between 3.3% and 3.4%. Among 20 individuals with heterozygous β-thalassemia, 12 had mutations with MCV 80 fL with HbA₂ ranging from 3.3–3.9%. Additionally, 15 had MCH 27 pg (1 with 3.3–3.4% and 4 with 3.5–3.9%). Among individuals with a normal β-globin genotype and MCV 80 fL, 47 had HbA₂ of 3.0–3.2%, 15 had 3.3–3.4%, and 1 had 3.5–3.9%. Among 67 individuals with normal β-genotype and MCH 27 pg, 22 had HbA₂ of 3.0–3.2%, 10 had 3.3–3.4%, and 1 had 3.5–3.9%. Among 49 individuals with MCV < 80 fL, 12 had detectable defects and 37 had none. In the group with detected mutations, the average hematological values were as follows: RBC count of 5.2×10⁶/µL (SD = 0.6), hemoglobin concentration of 10.5 g/dL (SD = 1.8), MCV at 66.85 fL (SD = 3.0), MCH at 19.92 pg (SD = 3.3), RDW at 18.12% (SD = 4.5), HbA₂ at 3.6% (SD = 0.3), and HbF at 1.2% (SD = 2.1). Among 71 individuals with MCV ≥ 80 fL, 8 had defects and 63 did not. Their mean values were: RBC = 3.83×10⁶/µL (SD = 0.5), Hb = 10.65 g/dL (SD = 2.1), MCV = 86.76 fL (SD = 4.2), MCH = 27.8 pg (SD = 2.0), RDW = 18% (SD = 6.47), HbA₂ = 3.7% (SD = 0.2), and HbF = 0.9% (SD = 1.2). We identified instances where HbA₂, MCV, and MCH values failed to reliably indicate β-thalassemia heterozygosity, posing a risk of misdiagnosis. Therefore, we strongly recommend molecular analysis of the β-globin gene in cases with borderline HbA₂ and normal or mildly reduced red cell indices—especially during partner screening when one individual is a known hemoglobinopathy carrier—to minimize false negatives and reduce the future burden of thalassemia. Conclusion Borderline HbA₂ levels may lead to missed diagnoses of β-thalassemia carriers. Our findings show that HbA₂, MCV, and MCH alone are sometimes insufficient to predict β-thalassemia heterozygosity, particularly in the Bangladeshi population. We strongly recommend molecular analysis of the β-globin gene when borderline HbA₂ levels are accompanied by normal or slightly reduced red cell indices, especially during routine screenings where one partner is a known hemoglobinopathy carrier. This approach will help reduce false negatives and mitigate the future burden of thalassemia. Declarations Ethics approval and consent to participate Ethical approval was obtained, and all procedures involving human participants were in compliance with the Declaration of Helsinki and local regulations. The Institutional Ethics Committee of Bangladesh Shishu Hospital and Institute, Dhaka, Bangladesh approved the study. Data were collected directly from the patients or their guardians. Before participation, a written consent form outlining the benefits of the research was provided. Signed consent was obtained from all participants. For participants under the age of 18, written informed consent was obtained from their parents or legal guardians, and assent was obtained from the minors themselves where appropriate. Patients or guardians who declined to participate were excluded from the study. Consent for publication Not applicable Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This research was supported by Bangladesh Medical Research Council (BMRC). Authors' contributions Md. Abdul Aziz, PhD analyzed and interpreted the patient data and Sanger sequencing data. Rowshon Zahan Luna and Salma Sadiya interpreted hemoglobin electrophoresis results. Waqar Ahmed Khan read and approved the final manuscript. Acknowledgement The authors would like to acknowledge Bangladesh Medical Research Council (BMRC)for financial support. We are grateful to Bangladesh Shishu Hospital and Institute (BSHI) for providing necessary laboratory and technical support. Special thanks to Prof. Dr. Belayet Hossain for their valuable guidance and assistance during the course of this study. References Aziz, Md. 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Abdul Aziz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYLCCBIYDDAzsDUCWgQUpWngOgLRIEG0PULlEAohBhBbd9sOPPzz4c8euX/L51Q0/CiQY+Nu7E/BqMTuTZmCQwPMseebsnLKbPUCHSZw5uwG/lgMJBgkJEoeTDW7npN3gAWoxkMgloOX88w9AXYeT7W+eSbv5hygtN3IMGxISDtsZSLAfu02cLTfeFDMkHHiWIHEmh+22jIEED2G/nE/f/PHHnzv2/O3Hn91888dGjr+9F78WGEhsYOAxADF4iFIOAvbAFPOAaNWjYBSMglEwsgAAVA5PoxgxpPEAAAAASUVORK5CYII=","orcid":"","institution":"Bangladesh Shishu Hospital and Institute","correspondingAuthor":true,"prefix":"","firstName":"Md.","middleName":"Abdul","lastName":"Aziz","suffix":""},{"id":511282823,"identity":"4c7764fb-d6eb-48e8-8c41-42ee04cc770f","order_by":1,"name":"Rowshon Zahan Luna","email":"","orcid":"","institution":"Bangladesh Shishu Hospital and Institute","correspondingAuthor":false,"prefix":"","firstName":"Rowshon","middleName":"Zahan","lastName":"Luna","suffix":""},{"id":511282824,"identity":"61d79018-6d8a-416b-b9d7-c7f468baef29","order_by":2,"name":"Salma Sadiya","email":"","orcid":"","institution":"Bangladesh Shishu Hospital and Institute","correspondingAuthor":false,"prefix":"","firstName":"Salma","middleName":"","lastName":"Sadiya","suffix":""},{"id":511282825,"identity":"e6cc0e89-e075-4bce-8cc3-eaa9611aa351","order_by":3,"name":"Waqar Ahmed Khan","email":"","orcid":"","institution":"Bangladesh Shishu Hospital and Institute","correspondingAuthor":false,"prefix":"","firstName":"Waqar","middleName":"Ahmed","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2025-07-27 04:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7223977/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7223977/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90984226,"identity":"d3c3bd11-418e-4cd8-8bed-9826b3f9dcad","added_by":"auto","created_at":"2025-09-10 09:40:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34589,"visible":true,"origin":"","legend":"\u003cp\u003eDemographic characteristics of the study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Schematic of gender among participants (N=120). Male and female participants were 67% (n=80) and 33% (n=40) respectively. \u003cstrong\u003eb\u003c/strong\u003e, Schematic of age groups.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7223977/v1/b89b6c7b04378a3833e877d1.png"},{"id":99798443,"identity":"13601eb4-a446-4bc2-8d66-7759ca34200a","added_by":"auto","created_at":"2026-01-08 13:48:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":544086,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7223977/v1/921eff54-9980-4d87-a5af-2a4e4f142f7c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diagnostic Challenges of Borderline A2 Hemoglobin in β-Thalassemia Carriers: Insights from a Bangladeshi Cohort","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe inheritance of a single mutated β-thalassemia (β-thal) allele causes the β-thal trait, which is characterized by hypochromic, microcytic red blood cells and elevated HbA₂ levels. (Colaco et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In some cases, the impact of this allele or its interaction with other genetic modifiers may be negligible or silent, leading to normal or borderline HbA₂ levels. Borderline HbA2 values for β-thal carriers typically range from 3.0\u0026ndash;3.9% (Moradi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; A. Noor et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). When both parents are carriers of β-thalassemia, each pregnancy has a one-in-four (25%) likelihood of resulting in a child affected by the condition. This chronic condition may result in severe health problems. There is a 50% probability that the child will be a carrier and a 25% chance that the child will inherit neither the mutation nor the disease (Mitro et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe diagnosis of β-thal or carrier status is typically based on the detection of increased HbA₂ (α₂β₂) levels, with values exceeding 4.0% generally indicative of β-thal trait. (Giordano, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Suresh et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, no studies have yet reported β-globin gene mutations in individuals with HbA2 levels below 4.0% in various populations of Bangladesh. As a result, individuals with borderline HbA2 levels (3.5\u0026ndash;3.9%) are difficult to definitively classify as normal or β-thal carriers. Molecular analysis is necessary to detect β-globin gene mutations in these individuals, especially when one partner is a known β-thal carrier.\u003c/p\u003e\u003cp\u003eTo date, numerous β-globin gene mutations associated with β-thal have been identified. According to the HbVar database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://globin.bx.psu.edu/cgi-bin/hbvar/counter\u003c/span\u003e\u003cspan address=\"https://globin.bx.psu.edu/cgi-bin/hbvar/counter\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a total of 958 β-thalassemia alleles have been documented. However, population studies indicate that 20 mutations account for 80% or more of β-thalassemia cases worldwide (Rao et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn Bangladesh, some studies have reported β-globin gene mutations, although none have specifically focused on individuals with borderline HbA2 levels. Furthermore, the reported mutations in the Bangladeshi population are not highly diverse (Banu et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; F. A. Noor et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, seven pathogenic mutations were found in individuals with borderline HbA2 levels. Borderline HbA2 values are commonly observed in regions with a high prevalence of β-thalassemia, such as South Asian countries\u0026mdash;particularly Bangladesh and India (Rangan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Approximately 4.1% of the Bangladeshi population are β-thalassemia carriers, while around 6.1% possess the hemoglobin E (HbE) trait. (W. Khan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). It is estimated that over 14,000 children are born annually with thalassemia, primarily with HbE β-thal, followed by β-thalassemia major (W. A. Khan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In developing countries like Bangladesh, most affected infants are neither diagnosed nor treated, often dying in early infancy due to complications from anemia (Hossain et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven the high prevalence and the significant cost of treatment, prevention and control of new thalassemia cases are critical. As the number of β-thalassemia carriers continues to rise in developing countries such as Bangladesh, accurate detection of HbA2 levels is essential for effective screening and prevention on a global scale.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eSelection of patients.\u003c/p\u003e\u003cp\u003eThis study was designed for patients with borderline HbA2 levels who visited the hospital for hemoglobin electrophoresis. Between January 2022 and December 2022, a total of 1,650 individuals were referred to our institute for hemoglobinopathy screening. Among them, 120 individuals (66.7% male [n\u0026thinsp;=\u0026thinsp;80] and 33.3% female [n\u0026thinsp;=\u0026thinsp;40]) were identified with HbA2 levels between 3.0% and 3.9%. The participants ranged in age from 2 to 65 years. Subjects were randomly selected for the study after obtaining written informed consent.\u003c/p\u003e\u003cp\u003eIndividuals who visited Bangladesh Shishu Hospital and Institute for treatment were included as study participants. Peripheral blood (2 ml) was collected in EDTA-containing tubes and stored at 4\u0026deg;C for further analysis.\u003c/p\u003e\u003cp\u003eHematology analysis\u003c/p\u003e\u003cp\u003eHematology analysis was done by SYSMEX Automated Hematology Analyzer (Model: XN1000). It measured the red blood cell (RBC) count, white blood cell (WBC) count, platelets, Hb, and hematocrit levels, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW). In addition, Hb electrophoresis was performed by Varant II (HPLC) for identification of Hb fraction.\u003c/p\u003e\u003cp\u003eDNA extraction\u003c/p\u003e\u003cp\u003eDNA was isolated from peripheral blood using the PureLink\u0026reg; Genomic DNA Purification Mini Kit (Invitrogen, USA), which utilizes phase separation by centrifugation of the aqueous sample. The procedure was carried out according to the manufacturer\u0026rsquo;s instructions. The purity and concentration of the extracted DNA (per microliter) were assessed using the Qubit\u0026reg; 2.0 Fluorometer. The extracted DNA samples were stored at -20\u0026deg;C for long-term preservation.\u003c/p\u003e\u003cp\u003eMolecular analysis\u003c/p\u003e\u003cp\u003eMutation analysis was performed using the Sanger sequencing method. The analysis targeted the entire coding region (exons 1, 2, and 3), portions of introns 1 and 2, the promoter region, and the splice junctions of the β-globin gene (locus: NG_000007.3). Genomic DNA was first amplified using exon-specific primers for exons 1, 2, and 3 via the PCR method. Sequencing was conducted using an automated capillary electrophoresis (CE) system on the 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The resulting sequences were aligned with the reference sequence of the β-globin gene [NCBI RefSeq: NG_000007.3] using SeqScape Software version 2.5 (Applied Biosystems). Genetic variants and their clinical significance were identified using the HbVar and ClinVar databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://globin.bx.psu.edu/hbvar\u003c/span\u003e\u003cspan address=\"http://globin.bx.psu.edu/hbvar\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/clinvar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which catalog hemoglobin variants and thalassemia mutations [13].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDemographic characteristics:\u003c/p\u003e\n\u003cp\u003eAccording to this study, a total of 120 blood samples were characterized by various clinical features. Among them, the male was 66.7% (n=80) and female was 33.3% (n=40) (Figure 1a). The majority of participants were between 2 and 7 years old, accounting for 50% (n=60) of the sample. This was followed by participants aged 8\u0026ndash;13 years (25%, n=30), 26\u0026ndash;31 years (10.8%, n=13), 20\u0026ndash;25 years (7%, n=7), over 31 years (6%, n=6), and 14\u0026ndash;19 years (3.3%, n=4) (Figure 1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026beta;‑globin gene mutations\u003c/p\u003e\n\u003cp\u003eAmong the 120 individuals with borderline HbA2 levels, seven distinct \u0026beta;-thalassemia mutations were detected (Table 1). The IVS1-5 G\u0026gt;C (\u0026beta;⁺) mutation was the most frequently observed, present in 55% of cases (n=11). This was followed by FS 41/42 -CTTT (\u0026beta;⁰), Codon 30 G\u0026gt;A (\u0026beta;⁰), and Codon 30 G\u0026gt;C (\u0026beta;⁰), each found in 10% of individuals (n=2). Additionally, FS 8/9 +G (\u0026beta;⁰), Codon 15 G\u0026gt;A (\u0026beta;⁰), and FS 16 -C (\u0026beta;⁰) were each identified in 5% of cases (n=1). Overall, \u0026beta;⁰ mutations constituted 40% of all genetic variants detected in individuals with borderline HbA2 levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the 25 individuals with HbA2 levels ranging from 3.3% to 3.4%, \u0026beta;-thalassemia alleles were detected in 3 cases (15%). Among them, two cases involved \u0026beta;⁺ mutations (IVS1-5 G\u0026gt;C), while one case was identified as a \u0026beta;⁰ mutation (Codon 30 G\u0026gt;A). Out of 18 individuals with HbA2 levels between 3.5% and 3.9%, \u0026beta;-thalassemia alleles were found in 17 cases, accounting for 85%. IVS1-5 G\u0026gt;C was the most frequently observed mutation among them, present in 45% of cases (n=9), followed by FS 41/42 -CTTT (\u0026beta;⁰) and Codon 30 G\u0026gt;C (\u0026beta;⁰), each found in 10% (n=2). Additionally, FS 8/9 +G (\u0026beta;⁰), Codon 15 G\u0026gt;A (\u0026beta;⁰), and FS 16 -C (\u0026beta;⁰) were each detected in 5% of individuals (n=1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Mutation patterns observed in \u0026beta;-thalassemia carriers with borderline HbA2 levels.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u0026beta;-thalassemia mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003en (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eHbA2 3.0\u0026ndash;3.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eHbA2 3.3\u0026ndash;3.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003eHbA2 3.5\u0026ndash;3.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eIVS1-5 G\u0026gt;C [\u0026beta;+]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e11 (55%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eFS 8/9+G [\u0026beta;0 ]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e1 (5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eFS 41/42CTTT [\u0026beta;0 ]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e2 (10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003ecodon 30 G\u0026gt;A [\u0026beta;0 ]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e2 (10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003ecodon 30 G\u0026gt;C [\u0026beta;0 ]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e2 (10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003ecodon 15 G\u0026gt;A [\u0026beta;0 ]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e1 (5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eFS 16 \u0026ndash;C [\u0026beta;0 ]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e1 (5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e20 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRelation between MCV and HbA2 levels\u003c/p\u003e\n\u003cp\u003eSince carrier detection of \u0026beta;-thalassemia typically depends on MCV and MCH values in conjunction with HbA2 levels, we analyzed these parameters in the 20 \u0026beta;-thalassemia heterozygotes identified in this study (Table 2).\u0026nbsp;All three \u0026beta;-thalassemia heterozygote mutations with HbA2 levels ranging from 3.3% to 3.4% had MCV values greater than 80 fL.\u0026nbsp;In contrast, among the 17 heterozygous individuals with HbA2 levels between 3.5% and 3.9%, 70.6% (n=12) had MCV values under 80 fL, whereas 29.4% (n=5) had MCV values exceeding 80 fL.\u0026nbsp;In terms of MCH values, two out of three \u0026beta;-thalassemia heterozygotes with HbA2 levels from 3.3% to 3.4% had MCH below 27 pg, while one exhibited MCH above 27 pg.\u0026nbsp;Likewise, among the 17 individuals with HbA2 levels between 3.5% and 3.9%, 76.5% (n=13) had MCH values under 27 pg, and 23.5% (n=4) had values exceeding 27 pg.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong individuals with a normal \u0026beta;-globin genotype and MCV less than 80 fL, 30 had HbA2 levels in the 3.0\u0026ndash;3.2% range, 7 had levels between 3.3\u0026ndash;3.4%, and none exhibited HbA2\u0026nbsp;values between 3.5\u0026ndash;3.9%.\u0026nbsp;In contrast, among those with MCV greater than 80 fL, 47 individuals fell within the 3.0\u0026ndash;3.2% HbA2 range, 15 were in the 3.3\u0026ndash;3.4% range, and only one had HbA2 between 3.5\u0026ndash;3.9%.\u0026nbsp;With respect to MCH values, 55 individuals having MCH less than 27 pg exhibited HbA2 levels between 3.0\u0026ndash;3.2%, while 12 individuals fell within the 3.3\u0026ndash;3.4% range, and no cases were\u0026nbsp;observed in the 3.5\u0026ndash;3.9% range.\u0026nbsp;Similarly, among 52 individuals with MCH values above 27 pg, 33 had HbA2 levels between 3.0\u0026ndash;3.2%, 10 were between 3.3\u0026ndash;3.4%, and just one had a value in the 3.5\u0026ndash;3.9% range.\u003c/p\u003e\n\u003cp\u003eTable 2. Distribution of study individuals according to HbA2, MCV and MCH.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eParameters\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 374px;\"\u003e\n \u003cp\u003eHbA2 values (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e3.0-3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e3.3-3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e3.5-3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eTotal individuals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003en=77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003en=25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003en=18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003en=120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 623px;\"\u003e\n \u003cp\u003e\u0026beta;TT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCV \u0026lt;80 fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCV \u0026gt;80 fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eTotal \u0026beta;TT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCH\u0026lt;27\u0026nbsp;pg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCH\u0026gt;27\u0026nbsp;pg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eTotal \u0026beta;TT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 623px;\"\u003e\n \u003cp\u003eNormal \u0026beta;-genotype\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCV \u0026lt;80 fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCV \u0026gt;80 fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eTotal Normal \u0026beta; genotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCH\u0026lt;27\u0026nbsp;pg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eMCH\u0026gt;27\u0026nbsp;pg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003eTotal Normal \u0026beta; genotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eOf the 120 individuals investigated in this study, 49 individuals showed MCV \u0026lt; 80fL and 71 individuals showed MCV \u0026gt; 80fL (Table 3). Among 49 subjects, 12 individuals were identified as thalassemia heterozygotes mutations with MCV \u0026lt;80 fL.\u0026lt;/mark\u0026gt; The average hematological parameters for these individuals were as follows: RBC count of 5.2 \u0026times; 10⁶/\u0026micro;L (SD = 0.6), hemoglobin level of 10.5 g/dL (SD = 1.8), MCV of 66.85 fL (SD = 3.0), MCH of 19.92 pg (SD = 3.3), RDW at 18.12% (SD = 4.5), HbA2 at 3.6% (SD = 0.3), and HbF at 1.2% (SD = 2.1). Among the 8 thalassemia heterozygotes with MCV \u0026gt;80 fL, the hematological parameters were as follows: RBC = 3.83 \u0026times; 10⁶/\u0026micro;L (SD = 0.5), Hb = 10.6 g/dL (SD = 2.1), MCV = 86.76 fL (SD = 4.2), MCH = 27.8 pg (SD = 2.0), RDW = 18.0% (SD = 6.47), HbA2 = 3.7% (SD = 0.2), and HbF = 0.9% (SD = 1.2).\u003c/p\u003e\n\u003cp\u003eTable 3. Hematological analysis of the individuals with MCV\u0026le;80 fL and MCV\u0026ge;80 fL.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"630\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eMCV\u0026lt;80 fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003en=\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eRBC \u0026times; 106 /\u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eHb g/dl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eMCV fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eMCH pg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eRDW %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eHbA2 %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eHbF %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eNo Defects identified\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 494px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eDefects identified\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e5.2 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e10.5\u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e66.85 \u0026plusmn; 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e19.92 \u0026plusmn; 3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e18.12 \u0026plusmn; 4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e3.6 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1.2 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eMCV \u0026gt;80 fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003en=\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eRBC \u0026times; 106 /\u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eHb g/dl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eMCV fL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eMCH pg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eRDW %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eHbA2 %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eHbF %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eNo Defects identified\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 494px;\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003eDefects identified\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e3.83 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e10.65 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e86.76 \u0026plusmn; 4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e27.8 \u0026plusmn; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e18 \u0026plusmn; 6.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e3.7 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.9 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eThalassemia is the most prevalent inherited recessive blood disorder globally. (Tesio \u0026amp; Bauer, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). An estimated 1.5% of the world\u0026rsquo;s population, equivalent to around 80\u0026ndash;90\u0026nbsp;million individuals, are carriers of β-thalassemia. (Rao et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It is important to emphasize that this complex disease is largely preventable through simple, cost-effective measures such as carrier screening and genetic counseling (Aziz et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pinto \u0026amp; Forni, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The characterization of β-thal carriers show mild, persistent anemia and distinctly elevated levels of HbA2 which is basis for screening programs worldwide (Colaco \u0026amp; Nadkarni, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this context, while elevated HbA₂ levels are a key indicator for diagnosing β-thalassemia, the presence of borderline HbA₂ levels in some individuals poses a diagnostic challenge.\u003c/p\u003e\u003cp\u003eIn this study, the IVS1-5 (G\u0026thinsp;\u0026gt;\u0026thinsp;C) mutation was the most prevalent (55%) among the Bangladeshi population, aligning with other studies conducted in Bangladesh. Among the 11 IVS1-5 (G\u0026thinsp;\u0026gt;\u0026thinsp;C) mutations identified, 9 were associated with HbA2 values between 3.5% and 3.9%, while 2 were found in individuals with HbA2 values between 3.3% and 3.4%. Another study in Bangladesh found that the frequency of IVS1-5 (G\u0026thinsp;\u0026gt;\u0026thinsp;C) mutation was 42.68% (Mahzabin et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to Colaco et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the IVS1-5 (G\u0026thinsp;\u0026gt;\u0026thinsp;C) mutation was identified in 4% of individuals with HbA₂ levels ranging from 3.0 to 3.2%, 24% of those with levels between 3.3 and 3.4%, and 72% of individuals with HbA₂ levels between 3.5% and 3.9%. Mutations including FS 8/9 (+\u0026thinsp;G), FS 41/42 (\u0026minus;\u0026thinsp;CTTT), Codon 30 (G\u0026thinsp;\u0026gt;\u0026thinsp;A and G\u0026thinsp;\u0026gt;\u0026thinsp;C), Codon 15 (G\u0026thinsp;\u0026gt;\u0026thinsp;A), and FS 16 (\u0026minus;\u0026thinsp;C) were detected in individuals with HbA₂ levels of 3.5\u0026ndash;3.9%, while Codon 30 (G\u0026thinsp;\u0026gt;\u0026thinsp;C) was observed in only one case with HbA₂ between 3.3% and 3.4%.\u003c/p\u003e\u003cp\u003eAmong 20 individuals with heterozygous β-thalassemia, 12 had mutations with MCV\u0026thinsp;\u0026lt;\u0026thinsp;80 fL and HbA₂ levels of 3.5\u0026ndash;3.9%, while 8 had MCV\u0026thinsp;\u0026gt;\u0026thinsp;80 fL with HbA₂ ranging from 3.3\u0026ndash;3.9%. Additionally, 15 had MCH\u0026thinsp;\u0026lt;\u0026thinsp;27 pg (2 with HbA₂ of 3.3\u0026ndash;3.4% and 13 with 3.5\u0026ndash;3.9%), whereas 5 had MCH\u0026thinsp;\u0026gt;\u0026thinsp;27 pg (1 with 3.3\u0026ndash;3.4% and 4 with 3.5\u0026ndash;3.9%). Among individuals with a normal β-globin genotype and MCV\u0026thinsp;\u0026lt;\u0026thinsp;80 fL (n\u0026thinsp;=\u0026thinsp;37), 30 had HbA₂ levels of 3.0\u0026ndash;3.2%, 7 had 3.3\u0026ndash;3.4%, and none had 3.5\u0026ndash;3.9%. In contrast, of 63 individuals with MCV\u0026thinsp;\u0026gt;\u0026thinsp;80 fL, 47 had HbA₂ of 3.0\u0026ndash;3.2%, 15 had 3.3\u0026ndash;3.4%, and 1 had 3.5\u0026ndash;3.9%. Among 67 individuals with normal β-genotype and MCH\u0026thinsp;\u0026lt;\u0026thinsp;27 pg, 55 had HbA₂ levels of 3.0\u0026ndash;3.2%, 12 had 3.3\u0026ndash;3.4%, and none had 3.5\u0026ndash;3.9%. Of 33 individuals with MCH\u0026thinsp;\u0026gt;\u0026thinsp;27 pg, 22 had HbA₂ of 3.0\u0026ndash;3.2%, 10 had 3.3\u0026ndash;3.4%, and 1 had 3.5\u0026ndash;3.9%.\u003c/p\u003e\u003cp\u003eAmong 49 individuals with MCV\u0026thinsp;\u0026lt;\u0026thinsp;80 fL, 12 had detectable defects and 37 had none. In the group with detected mutations, the average hematological values were as follows: RBC count of 5.2\u0026times;10⁶/\u0026micro;L (SD\u0026thinsp;=\u0026thinsp;0.6), hemoglobin concentration of 10.5 g/dL (SD\u0026thinsp;=\u0026thinsp;1.8), MCV at 66.85 fL (SD\u0026thinsp;=\u0026thinsp;3.0), MCH at 19.92 pg (SD\u0026thinsp;=\u0026thinsp;3.3), RDW at 18.12% (SD\u0026thinsp;=\u0026thinsp;4.5), HbA₂ at 3.6% (SD\u0026thinsp;=\u0026thinsp;0.3), and HbF at 1.2% (SD\u0026thinsp;=\u0026thinsp;2.1). Among 71 individuals with MCV\u0026thinsp;\u0026ge;\u0026thinsp;80 fL, 8 had defects and 63 did not. Their mean values were: RBC\u0026thinsp;=\u0026thinsp;3.83\u0026times;10⁶/\u0026micro;L (SD\u0026thinsp;=\u0026thinsp;0.5), Hb\u0026thinsp;=\u0026thinsp;10.65 g/dL (SD\u0026thinsp;=\u0026thinsp;2.1), MCV\u0026thinsp;=\u0026thinsp;86.76 fL (SD\u0026thinsp;=\u0026thinsp;4.2), MCH\u0026thinsp;=\u0026thinsp;27.8 pg (SD\u0026thinsp;=\u0026thinsp;2.0), RDW\u0026thinsp;=\u0026thinsp;18% (SD\u0026thinsp;=\u0026thinsp;6.47), HbA₂ = 3.7% (SD\u0026thinsp;=\u0026thinsp;0.2), and HbF\u0026thinsp;=\u0026thinsp;0.9% (SD\u0026thinsp;=\u0026thinsp;1.2).\u003c/p\u003e\u003cp\u003eWe identified instances where HbA₂, MCV, and MCH values failed to reliably indicate β-thalassemia heterozygosity, posing a risk of misdiagnosis. Therefore, we strongly recommend molecular analysis of the β-globin gene in cases with borderline HbA₂ and normal or mildly reduced red cell indices\u0026mdash;especially during partner screening when one individual is a known hemoglobinopathy carrier\u0026mdash;to minimize false negatives and reduce the future burden of thalassemia.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBorderline HbA₂ levels may lead to missed diagnoses of β-thalassemia carriers. Our findings show that HbA₂, MCV, and MCH alone are sometimes insufficient to predict β-thalassemia heterozygosity, particularly in the Bangladeshi population. We strongly recommend molecular analysis of the β-globin gene when borderline HbA₂ levels are accompanied by normal or slightly reduced red cell indices, especially during routine screenings where one partner is a known hemoglobinopathy carrier. This approach will help reduce false negatives and mitigate the future burden of thalassemia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained, and all procedures involving human participants were in compliance with the \u003cstrong\u003eDeclaration of Helsinki\u003c/strong\u003e and local regulations. The Institutional Ethics Committee of Bangladesh Shishu Hospital and Institute, Dhaka, Bangladesh approved the study. Data were collected directly from the patients or their guardians. Before participation, a written consent form outlining the benefits of the research was provided. Signed consent was obtained from all participants. For participants under the age of 18, written informed consent was obtained from their parents or legal guardians, and assent was obtained from the minors themselves where appropriate. Patients or guardians who declined to participate were excluded from the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Bangladesh Medical Research Council (BMRC).\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003eAuthors\u0026apos; contributions\u003c/h4\u003e\n\u003cp\u003eMd. Abdul Aziz, PhD analyzed and interpreted the patient data and Sanger sequencing data. Rowshon Zahan Luna\u0026nbsp;and Salma Sadiya interpreted hemoglobin electrophoresis results.\u0026nbsp;Waqar Ahmed Khan read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge Bangladesh Medical Research Council (BMRC)for financial support. We are grateful to Bangladesh Shishu Hospital and Institute (BSHI) for providing necessary laboratory and technical support. Special thanks to Prof. Dr. Belayet Hossain for their valuable guidance and assistance during the course of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAziz, Md. A., Khan, W. A., Banu, B., Das, S. A., Sadiya, S., \u0026amp; Begum, S. (2020). Prenatal Diagnosis and Screening of Thalassemia Mutations in Bangladesh: Presence of Rare Mutations. \u003cem\u003eHemoglobin\u003c/em\u003e, \u003cem\u003e44\u003c/em\u003e(6), 397\u0026ndash;401. https://doi.org/10.1080/03630269.2020.1830797\u003c/li\u003e\n\u003cli\u003eBanu, B., Khan, W. A., Selimuzzaman, Sarwardi, G., \u0026amp; Sadiya, S. (2018). Mutation pattern in beta thalassaemia trait population: A basis for prenatal diagnosis. \u003cem\u003eBangladesh Medical Research Council Bulletin\u003c/em\u003e, \u003cem\u003e44\u003c/em\u003e(2), 65\u0026ndash;70. https://doi.org/10.3329/bmrcb.v44i2.38688\u003c/li\u003e\n\u003cli\u003eColaco, S., Colah, R., \u0026amp; Nadkarni, A. (2022). Significance of borderline HbA2 levels in \u0026beta; thalassemia carrier screening. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1). https://doi.org/10.1038/s41598-022-09250-5\u003c/li\u003e\n\u003cli\u003eColaco, S., \u0026amp; Nadkarni, A. (2021). Borderline HbA2 levels: Dilemma in diagnosis of beta-thalassemia carriers. In \u003cem\u003eMutation Research - Reviews in Mutation Research\u003c/em\u003e (Vol. 788). https://doi.org/10.1016/j.mrrev.2021.108387\u003c/li\u003e\n\u003cli\u003eGiordano, P. C. (2015). Universal screening for hemoglobinopathies in today\u0026rsquo;s multi-ethnic societies: How and when. \u003cem\u003eWorld Journal of Obstetrics and Gynecology\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(4), 86. https://doi.org/10.5317/wjog.v4.i4.86\u003c/li\u003e\n\u003cli\u003eHossain, M. S., Raheem, E., Sultana, T. A., Ferdous, S., Nahar, N., Islam, S., Arifuzzaman, M., Razzaque, M. A., Alam, R., Aziz, S., Khatun, H., Rahim, A., \u0026amp; Morshed, M. (2017). Thalassemias in South Asia: clinical lessons learnt from Bangladesh. \u003cem\u003eOrphanet Journal of Rare Diseases\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 1\u0026ndash;9. https://doi.org/10.1186/s13023-017-0643-z\u003c/li\u003e\n\u003cli\u003eKhan, W. A., Banu, B., Aziz, M. A., Sadiya, S., Hossain, M. B., \u0026amp; Selimuzzaman, M. (2021). Frequency of G-Globin Promoter -158(C\u0026gt;T)Xmnl Polymorphism and its Correlation with Beta Thalassaemia Mutations in a Sample of Bangladeshi Population. \u003cem\u003eBangladesh Medical Research Council Bulletin\u003c/em\u003e, \u003cem\u003e47\u003c/em\u003e(2), 219\u0026ndash;224. https://doi.org/10.3329/bmrcb.v47i2.57783\u003c/li\u003e\n\u003cli\u003eKhan, W., Sadiya, S., Khan, W. A., Banu, B., Amin, S. K., Selimuzzaman, M., Rahman, M., Hossain, B., Sarwardi, G., Iqbal, A., Rahman, Y., \u0026amp; Razzaque, M. A. (2005). Prevalence of Beta thalassaemia trait and Hb E trait in Bangladeshi school children and health burden of thalassaemia in our population. Prevalence of Beta thalassemia trait and Hb E trait in Bangladeshi school children and health burden of thalassemia in our population. https://www.researchgate.net/publication/200176529\u003c/li\u003e\n\u003cli\u003eMahzabin, N., Islam, Md. A.-U.-, Islam, K. M. K., Islam, K. A., Rahman, Md. A.-U.-, Jahan, N., \u0026amp; Kabir, A. L. (2021). Spectrum of Genetic Mutation in Beta Globin Gene in Various Type of Thalassaemia in Bangladesh. \u003cem\u003eHaematology Journal of Bangladesh\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(02), 57\u0026ndash;60. https://doi.org/10.37545/haematoljbd202177\u003c/li\u003e\n\u003cli\u003eMitro, A., Hossain, D., Rahman, M. M., Dam, B., \u0026amp; Hosen, M. J. (2024). \u0026beta;-Thalassemia in Bangladesh: Current Status and Future Perspectives. \u003cem\u003eThalassemia Reports\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(3), 49\u0026ndash;59. https://doi.org/10.3390/thalassrep14030007\u003c/li\u003e\n\u003cli\u003eMoradi, K., Alibakhshi, R., Shafieenia, S., \u0026amp; Azimi, A. (2022). Problem of borderline hemoglobin A2 levels in an Iranian population with a high prevalence of \u0026alpha;- and \u0026beta;-thalassemia carriers. \u003cem\u003eEgyptian Journal of Medical Human Genetics\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(1). https://doi.org/10.1186/s43042-022-00279-9\u003c/li\u003e\n\u003cli\u003eNoor, A., Bozdar, M., Malik, H. S., Riaz, M. O., Mahmood, R., \u0026amp; Seema, N. (2024). PROGNOSTIC IMPACT OF CD34 EXPRESSION IN PAEDIATRIC PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKAEMIA. \u003cem\u003eJournal of Ayub Medical College\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e(4), 963\u0026ndash;969. https://doi.org/10.55519/JAMC-S4-14073\u003c/li\u003e\n\u003cli\u003eNoor, F. A., Sultana, N., Bhuyan, G. S., Islam, M. T., Hossain, M., Sarker, S. K., Islam, K., Khan, W. A., Rahman, M., Qadri, S. K., Shekhar, H. U., Qadri, F., Qadri, S. S., \u0026amp; Mannoor, K. (2020). Nationwide carrier detection and molecular characterization of \u0026beta;-thalassemia and hemoglobin e variants in Bangladeshi population. \u003cem\u003eOrphanet Journal of Rare Diseases\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(1), 1\u0026ndash;12. https://doi.org/10.1186/s13023-020-1294-z\u003c/li\u003e\n\u003cli\u003ePinto, V. M., \u0026amp; Forni, G. L. (2020). Management of iron overload in beta-thalassemia patients: Clinical practice update based on case series. 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In \u003cem\u003eHemoglobin\u003c/em\u003e (Vol. 46, Issue 1). https://doi.org/10.1080/03630269.2021.2001346\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"β-thalassemia, prenatal screening, borderline HbA2 levels, mutation analysis","lastPublishedDoi":"10.21203/rs.3.rs-7223977/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7223977/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe identification of beta-thalassemia (β-thal) carriers during prenatal screening relies on elevated hemoglobin A2 (HbA2) levels. However, β-thal minor may present as silent carriers, showing no hematologic abnormalities despite carrying a mutation. Borderline HbA2 levels pose a diagnostic challenge. This study aimed to characterize 120 individuals\u0026mdash;selected from a pool of 1,650\u0026mdash;with borderline HbA₂ levels, to distinguish true carriers from non-carriers.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eBlood samples were collected in EDTA tubes from Bangladeshi subjects with borderline Hb A2 levels (3.0\u0026ndash;3.9%) for this study. The samples were obtained from the Thalassemia Center at Bangladesh Shishu Hospital and Institute. Hematological parameters were measured using an automated blood counter (ADVIA 2120i hematology analyzer; Siemens Healthcare Diagnostics, Deerfield, IL, USA). HBB genotypes were identified using the Sanger sequencing method.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAmong the subjects, pathogenic mutations of the β-globin gene were identified in 3 cases (15%) with HbA₂ levels of 3.3\u0026ndash;3.4%, and in 17 cases (85%) with levels between 3.5\u0026ndash;3.9%. A total of seven pathogenic mutations were identified, with IVS1-5 (G\u0026thinsp;\u0026gt;\u0026thinsp;C) being the most common (55%, n\u0026thinsp;=\u0026thinsp;11), followed by FS 41/42 (\u0026minus;\u0026thinsp;CTTT), Codon 30 (G\u0026thinsp;\u0026lt;\u0026thinsp;C), and Codon 30 (G\u0026thinsp;\u0026lt;\u0026thinsp;A), each at 10% (n\u0026thinsp;=\u0026thinsp;2). FS 8/9 (+\u0026thinsp;G), Codon 15 (G\u0026thinsp;\u0026gt;\u0026thinsp;A), and FS 16 (\u0026minus;\u0026thinsp;C) were each found in 5% of cases (n\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study supports improved identification of β-thal carriers with borderline HbA₂ levels, helping to reduce the risk of misdiagnosis.\u003c/p\u003e","manuscriptTitle":"Diagnostic Challenges of Borderline A2 Hemoglobin in β-Thalassemia Carriers: Insights from a Bangladeshi Cohort","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 09:40:40","doi":"10.21203/rs.3.rs-7223977/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8cdb370-ed4d-4205-bb2e-6d55bbb66011","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-08T08:10:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 09:40:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7223977","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7223977","identity":"rs-7223977","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}