Screening For Hemoglobinopathies Research Articles

An Integration of Statistical and Anthropometric Measurement Approach Towards Improving Ergonomic Design for Production Workbench

Capillary zone electrophoresis (CZE) is a method of haemoglobin (Hb) analysis for the screening of haemoglobinopathies or variant haemoglobins. In our centre, few samples sent for Hb analysis showed Hb peak at zone 10 requiring further evaluation. This pattern was observed in patients with history of diabetes mellitus (DM); hence it was postulated the peak observed is glycated Hb (HbA1c). The objective of this study was to determine whether the expression of Hb peak at zone 10 among blood samples from DM patients correlate with HbA1c values measured using high performance liquid chromatography (HPLC). Methods: This was a cross-sectional study involving blood samples from DM patients. Samples with HbA1c values ≥ 6.5% by HPLC were selected and subsequently analysed by CZE method. Presence of Hb peak in zone 10 were correlated and analysed with the HbA1c levels measured by HPLC method. Results: A total of 131 samples were analysed. Hb peak was detected at zone 10 in 50/131(38.2%). Out of 50 samples, 47 (94%) were from patients with HbA1c level > 10%. Correlation study showed it was 18 times more likely for Hb peak to appear in zone 10 in patients with HbA1c > 10%. Cut off point for HbA1c to appear in CZE is 10.5% with AUC of 0.965. Conclusion: Hb peak detected at zone 10 of CZE was most likely to be HbA1c. However, it is recommended that for every primary method of Hb analysis used be confirmed by secondary method. Therefore, both zone 10 in CZE and P2 peak in HPLC must be correlated together to achieve final diagnosis

Hemoglobinopathies by HPLC: A 3 Year Study of 106,277 Cases

Introduction: Hemoglobinopathies and thalassemias are the most common single gene disorders in the world. World Health Organization figures estimate that 5% of the world populations are carriers of a potentially pathological hemoglobin (Hb) gene. The general incidence of thalassemia trait and sickle cell anaemia in India varies between 3-17% and 1-44% respectively3 but because of consanguinity, caste and area endogamy, some communities show a very high incidence, making the disease a major public health problem in our country. Cation exchange high-performance liquid chromatography (CE-HPLC) is one of the best methods for screening, detection, and identification of various hemoglobinopathies.
 Material &Method: A retrospective study was carried out from period of 2017 to 2019 with 106277 cases evaluated with an aim to identify various hemoglobinopathies seen in Indian population by high-performance liquid chromatography. Cases outside Indian geographical location were excluded from the study
 Result: A total of 18,936(17.82%) cases with abnormal haemoglobin variants was reported in the study with 35 difference variants across India. Northeast India reported maximum abnormal hemoglobinopathies (50.16%). Beta Thalassemia Trait was the most common abnormal variant found.Such a high incidence emphasises premarital and prenatal screening for prevention of dangerous effects of hemoglobinopathies in the population.
 Conclusion: CE-HPLC should be used for early detection and proper management of these haemoglobinopathies. The most common hemoglobinopathy observed in our study was Beta thalassemia trait followed by Sickle cell trait and then sickle cell disease. It was also observed that Northeast India had maximum abnormal hemoglobinopathies.

THE INCIDENCE OF BETA-THALASSEMIA MINOR IN PREGNANT FEMALES BY MEASURING HBA2 THROUGH HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Objectives: The aim of the study was to study the incidence of thalassemia minor by determining HbA2 levels in pregnant females attending Obstetrics and Gynaecology OPD and HbA2 levels of husbands of positive females for beta-thalassemia trait. Methods: The prospective study spanning over 1½ years was conducted in the Department of Pathology in 1020 pregnant females who attended the Gynaecology and Obstetrics OPD of Government Medical College, Patiala for antenatal check-up. The pregnant females of any trimester without any specific sign and symptoms whose Hb level was <10 g/dL were screened in the study. Levels of HbA2 and HbF were determined by high performance liquid chromatography (HPLC) and the cases with raised HbA2 value above the cutoff limit (>3.5%) were labeled as BTT. Husbands of BTT positive females were also screened for the trait. Incidence of all these cases was calculated and analyzed statistically. Results: The majority of the females were in the age group of 21–30 years. In present study, we found that total 134 (13.1%) patients were having beta thalassemia trait. Husbands of all these positive patients were also screened for BTT and only 2 (1.49%) of them were found to be positive. Conclusion: HPLC has the advantage for screening and detection of various hemoglobinopathies by providing rapid and accurate results. HPLC can detect and measure HbF and HbA2 in a single system. Early diagnosis and management of thalassemia can help in reduction of burden on society as well as government.

Dual hemoglobinopathy presenting as lymphadenopathy: A diagnostic challenge

Abstract: Hemoglobinopathies, including sickle cell disease and beta-thalassemia, are genetic disorders that can manifest with diverse clinical presentations. Recognizing variations in hematological profiles is crucial for accurate diagnosis and management, particularly in regions where certain hemoglobinopathies are not so prevalent such as in regions of North India. Such hematological profiles can present simply as nonspecific lymphadenopathy. Here, we present a case of a 16-year-old boy with a complex presentation involving widespread lymphadenopathy, initially misdiagnosed as tuberculosis, ultimately revealing a dual hemoglobinopathy thus implying the need for family screening and consideration of late-onset hemoglobinopathies in cases with atypical presentations.

Screening and diagnosis of hemoglobinopathies in Germany: Current state and future perspectives

This monocentric study conducted at the Pediatric and Adult Hemoglobinopathy Outpatient Units of the University Hospital of Essen summarizes the results of hemoglobinopathies diagnosed between August 2018 and September 2021, prior to the introduction of a general newborn screening (NBS) for SCD in Germany (October 2021). In total, 339 patients (pts.), 182 pediatric [50.5% males (92/182)] and 157 adult pts. [75.8% females (119/157)] were diagnosed by molecular analysis. The most common (parental) descent among affected pts. were the Middle Eastern and North African/Turkey (Turkey: 19.8%, Syria: 11.8%, and Iraq: 5.9%), and the sub-Saharan African region (21.3%). Median age at diagnosis in pediatric carriers [N = 157; 54.1% males (85/157)] was 6.2 yrs. (range 1 (months) mos.–17.8 yrs.) and 31 yrs. (range 18–65 yrs.) in adults [N = 53; 75.2% females (115/153)]. Median age at diagnosis of homozygous or compound-heterozygous disease in pediatric pts. (72% (18/25) females) was 3.7 yrs., range 4 mos.–17 yrs. (HbSS (N = 13): 2.5 yrs., range 5 mos.–7.8 yrs.; HbS/C disease (N = 5): 8 yrs., range 1–8 yrs.; homozygous/compound heterozygous β-thalassemia (N = 5): 8 yrs., range 3–13 yrs.), in contrast to HbH disease (N = 5): 18 yrs. (median), range 12–40 yrs. Hemoglobinopathies represent a relevant health problem in Germany due to immigration and late diagnosis of second/third generation migrants. SCD-NBS will accelerate diagnosis and might result in reduction of disease-associated morbidity. However, diagnosis of carriers and/or disease-states (i.e. thalassemic syndromes) in newly immigrated and undiagnosed patients will further be delayed. A first major step has been taken, but further steps are required.

Electrophoretic and molecular profile of the hemoglobinopathies in newborns at a reference hospital in Mozambique

Hemoglobinopathies are monogenic diseases with a significant impact on public health. It is estimated that 5.2% of the world population has at least one genetic alteration in the genes that code for globins and in Africa, 18.2% of the population have hemoglobinopathies of clinical relevance. Using hemoglobin capillary electrophoresis, we screen newborns from the Central Maputo Hospital in Mozambique for hemoglobinopathies and then used various PCR to genotype-affected newborns. Screening of newborns revealed that 35.8% had hemoglobinopathy traits, with variants running in the position of Hb Bart (33.2%), Hb C (1.7%), Hb D (0.8%), and Hb S (sickle cell) (0.4%). The molecular test identified that 9.9% of the newborns were homozygous for 3.7 kb and/or 4.2 kb deletion genotype and that there may be an uncharacterized deletion of MCS-R located on chromosome 16. This study also confirmed the presence of hemoglobin variants, Hb Maputo and Hb S, through the identification of their defining mutations. No mutations for Hb C were found; thus, we believe that it was a result of a screening error or contamination. This study successfully identified hemoglobinopathies of clinical significance within the Mozambican population, α-thalassemia and hemoglobin S, as well as their associated genotypes. Future research should include samples from other regions of the country and the use of a more comprehensive molecular diagnostic tool. With the confirmation of clinically significant genotypes, the local health system should promote the importance of screening of hemoglobinopathies in Mozambican newborns and adults.

T225 The development of the laboratory algorithm for screening and diagnosis of hemoglobinopathies

T225 The development of the laboratory algorithm for screening and diagnosis of hemoglobinopathies

Hb Kirikiriroa [α57(E6)Gly→Cys; HBA1: c.172G>T]: A Novel Unstable α-Globin Variant with Oxidized Derivatives Interfering with Hb A1c

We report the identification of a novel hemoglobin (Hb) variant [α57(E6)Gly→Cys; HBA1: c.172G>T], to be referred to as Hb Kirikiriroa. The variant was detected in five subjects from two families, with familial relationship established between the families following diagnosis. A persistently elevated Hb A1c over a 1-year period prompted hemoglobinopathy screening in an adolescent male of New Zealand (NZ) European descent (case 1). Capillary electrophoresis (CE) revealed the variant was negatively charged and susceptible to oxidation, with multiple abnormal peaks detected (0.4–5.1% total Hb). Hb A1c analysis by cation exchange high performance liquid chromatography (HPLC) was the first indication of the variant in a pregnant female of NZ European descent (case 2). Cases 1 and 2 had normal complete blood counts. Isopropanol stability testing provided evidence the variant was unstable. We herein describe the characterization of Hb Kirikiriroa and clinical significance of the variant for interference with Hb A1c analysis by CE and cation exchange HPLC.

The design and post-implementation review of an algorithm for antenatal haemoglobinopathy screening

Background: Haemoglobinopathies are autosomal recessive disorders caused by quantitative (thalassaemia) or qualitative (variants) changes in alpha or beta-globin chains due to variants in HBA1, HBA2 and HBB. An effective carrier screening program requires identification of couples at risk of offspring affected by clinically severe disease.

Arab-Indian −530 β-distal promoter haplotype and sickle/Hb D heterozygosis in Badagas of Nilgiris: is it suggestive of Harappan origin?

Origin and ancestry of the Badagas of Nilgiris, an indigenous community of Nilgiris, Tamil Nadu in India, is a controversial topic. During our massive screening for haemoglobinopathies among Badagas, we identified a rare south Indian haemoglobin variant, the haemoglobin D (Hb D) Punjab (HBB: c.364G>C; rs33946267). The observed Hb D cases were double heterozygous with sickle cell trait (HbSD-Punjab) and manifested symptoms. The beta-globin distal promoter motif (-530 cap site BP1U) sequencing revealed a characteristic heterozygous combination of Arab-Indian sickle haplotype ((AT)9 (T)5) with universally common beta gene haplotype ((AT)7 (T)7). The report is a pioneer one informing the presence of Hb-D Punjab from an indigenous or aboriginal group of southern India. By the above information, we support the theory of unicentric tribal origin and distribution during Harappan civilization.

Prevalence and aetiologies of anaemia among first trimester pregnant women in Sri Lanka; the need for revisiting the current control strategies

BackgroundThe Sustainable development goals, which focus strongly on equity, aim to end all forms of malnutrition by 2030. However, a significant cause of intergenerational transfer of malnutrition, anaemia in pregnancy, is still a challenge. It is especially so in the low- and middle-income settings where possible context-specific aetiologies leading to anaemia have been poorly explored. This study explores the prevalence of etiological factors significantly contributing to anaemia in pregnancy in Sri Lanka, a lower-middle-income country with a high prevalence of malnutrition albeit robust public health infrastructure.MethodsAll first-trimester pregnant women registered in the public maternal care programme in the Anuradhapura district from July to September 2019 were invited to participate in Rajarata Pregnancy Cohort (RaPCo). After a full blood count analysis, high-performance liquid chromatography, peripheral blood film examination, serum B12 and folate levels were performed in anaemic participants, guided by an algorithm based on the red cell indices in the full blood count. In addition, serum ferritin was tested in a random subsample of 213 participants. Anaemic women in this subsample underwent B12 and folate testing.ResultsAmong 3127 participants, 14.4% (95%CI 13.2–15.7, n = 451) were anaemic. Haemoglobin ranged between 7.4 to 19.6 g/dl. 331(10.6%) had mild anaemia. Haemoglobin ≥13 g/dl was observed in 39(12.7%). Microcytic, normochromic-normocytic, hypochromic-normocytic and macrocytic anaemia was observed in 243(54%), 114(25.3%), 80(17.8%) and two (0.4%) of full blood counts in anaemic women, respectively. Microcytic anaemia with a red cell count ≥5 * 106 /μl demonstrated a 100% positive predictive value for minor haemoglobinopathies. Minor hemoglobinopathies were present in at least 23.3%(n = 105) of anaemic pregnant women. Prevalence of iron deficiency, B12 deficiency and Southeast Asian ovalocytosis among the anaemic was 41.9% (95%CI 26.4–59.2), 23.8% (95%CI 10.6–45.1) and 0.9% (95%CI 0.3–2.3%), respectively. Folate deficiency was not observed.ConclusionEven though iron deficiency remains the primary cause, minor hemoglobinopathies, B 12 deficiency and other aetiologies substantially contribute to anaemia in pregnancy in this study population. Public health interventions, including screening for minor hemoglobinopathies and multiple micronutrient supplementation in pregnancy, should be considered in the national programme for areas where these problems have been identified.

Synthetization, Distortion, and Geometric Correction of Isoelectric Focusing Gels for Newborn Screening

Hemoglobinopathies are inherited red blood cell disorders. They are commonly screened from newborns early after birth, and if not detected early and treated properly, some of them can be fatal. One of the most common method for screening hemoglobinopathies in newborns is isoelectric focusing (IEF). IEF separates different hemoglobins to distinct bands based on their isoelectric point. Different hemoglobinopathies can be detected from IEF as additional bands from normal hemoglobins and also from quantitative issues in normal hemoglobins. However, distortions are commonly present in IEF gels that hinder the interpretation of the gel. In this study, we showcase a method for straightening distorted images of IEF gels. Since no dataset containing images of IEF gels was present, we created a novel Sig2Img synthetic sample generator. This allowed us to create gel sides with any desired bands and intensities. Artificial distortions were also applied with basic image processing functions, to represent different kinds of distortions commonly found in IEF gels. Fifteen experiments were created to evaluate the method. The experiments showed that the correction method managed to straighten gel sides even with added difficulties, such as within-sample and sample-to-sample distortions, and added Gaussian noise. Experiments also showed that the method retained the relative quantities of the bands accurately. The straightening enables easier visual interpretation of the gel when screening for hemoglobinopathies. It also allows accurate quantitation of bands which was not originally possible from distorted samples. This gives the method clinical significance, as quantitation can be added to the clinical workflow.

The First Case of Hemoglobin Görwihl [α2β25(A2) Pro→Ala] Identified in Türkiye

We present the first individual with hemoglobin (Hb) Görwihl in Türkiye and to our knowledge, this is the 3rd case reported worldwide. During premarital thalassemia screening an abnormal unidentified Hb peak [Retention Time (RT): 4.950 min., 42.4%] was determined by cation-exchange high-performance liquid chromatography (CE-HPLC) method at Premier Resolution (Trinity-Biotech) system. Variant Hb was eluted with HbA (RT: 2.47 min., 82%) at Variant II Turbo, Bio-Rad system. He was 22 years old and didn't show any clinical symptoms or haematological abnormality. HbA1c measured with Boronate affinity method at (Premier Hb9210, Trinity-Biotech) system was 5.2% and consistent with his blood glucose value (5.5 mmol/L) while it was 4.6% with CE-HPLC method at Variant II Turbo system. β-globulin gene sequencing revealed a heterozygote codon c16C>C variant which was identical to Hb Görwihl. Although clinically insignificant, Hb Görwihl may be important in hemoglobinopathy screening and HbA1c measurements.

Acceptance, Knowledge, and Experiences of Pediatric Hematologists in the Philippines on Newborn Screening for Hemoglobinopathies.

Hemoglobinopathies as a group is one of the most common conditions confirmed through the newborn screening (NBS) program of the Philippines. This led to the increased participation of pediatric hematologists in the NBS program. The aim of the study was to assess newborn screening acceptance and knowledge of pediatric hematologists using an online questionnaire. Members of the Philippine Society of Pediatric Hematology (PSPH), who are practicing pediatric hematologists in the Philippines, were invited to answer an online questionnaire. Sixty members of the PSPH (65.2%) answered the survey. All the respondents are familiar with the newborn screening program. Fifty-seven respondents (95 %) have already managed a case of hemoglobinopathy identified through the newborn screening program. Differences in the approach to management and level of confidence with diagnostic test result interpretation have been noted. General themes of their concerns include being unaware of the protocol, concerns on delays in confirmatory tests, request for guidelines on follow-up, and incongruence of results with clinical picture. The information collected may be used to develop strategies to better equip our pediatric hematologists and assist the PSPH standardize management protocols for hemoglobinopathies.

Prevalence of anemia among reproductive-age females in the Tharu tribe of the Indo-Nepal border region.

ABSTRACTBackground:India shares the largest number of anemia patients globally. Anemia in reproductive-age female, affects not only the maternal mortality and morbidity but inversely affect the pregnancy outcome also. Tribals in India are geographically isolated and most of them belong to very low socio-economic status. Regarding Tharu tribes, to date, there is no study to find the prevalence of anemia in this community.Aim:We aim to find the prevalence of anemia in the reproductive-age female of the Tharu community. Additionally, we also accessed the severity and possible causes of anemia.Materials and Methods:A total of 440 reproductive age females from the Tharu community were recruited in this study from Tharu predominant Tulsipur tehsil of Balrampur district on 8 December 2019. CBC of each sample was performed by a 3-part basic hematology analyzer.Results:The mean (SD) hemoglobin level of the study group observed was 11.11 (±1.96) gm/dl with a median value of 11.3 gm/dl. Prevalence of anemia was noted in 283 (64.32%) participants. Amongst the anemic subjects, mild, moderate, and severe anemia was noted in 33.57%, 57.24%, and 9.19%, respectively. Microcytic, normocytic, and macrocytic anemia was noted in 53.00%, 44.88%, and 2.12%, respectively.Conclusion:The high prevalence of mild and moderate anemia in reproductive-age women is a matter of concern and should be dealt with priority. Community-based screening for hemoglobinopathies is also needed to evaluate the actual cause of anemia.

Thalassaemia

Thalassaemias are among the most common genetic disorders worldwide; an estimated 1.5% of the global population carries a thalassaemia mutation. Originally associated with tropical and sub-tropical regions, migration has facilitated wider dispersal, concentrated in larger multi-ethnic conurbations. Thalassaemias are heterogeneous, ranging from asymptomatic to causing severe haemolytic anaemia with pan-corporal sequelae and complications. Carriers benefit from access to pre-conception and antenatal advice and screening. The National Health Service Antenatal Haemoglobinopathy Screening Programme has operated since 2001, but carrier diagnoses may still emerge incidentally. This article provides an overview of the genetics, presentation and management of thalassaemia.

Screening for hemoglobinopathy with capillary electrophoresis in adult patients

Abstract Hemoglobinopathy screening is frequently needed in adult patients, including prenatal carrier screen, workup of unexplained anemia, and bone marrow donor and recipient screening. However, the preferred test method for screening of hemoglobinopathy is not well established due to limited guidance from professional societies. American College of Obstetricians and Gynecologists’ Committee on Genetics recommended hemoglobin electrophoresis as the screening method of hemoglobinopathy in pregnancy; nevertheless, electrophoresis employs various methodologies, including acid gel electrophoresis, alkaline gel electrophoresis, and capillary electrophoresis (CE) with alkaline buffer. For other adult patient populations who need hemoglobinopathy screening, no clear guidelines dictate the method of choice. A previous study has shown that CE captures major hemoglobinopathies with comparable performance to high-performance liquid chromatography (HPLC) in pediatric patients but no study has investigated using CE alone in adult patient screening. In this retrospective study, we evaluated the utility of CE as a screening method to rule out clinically significant hemoglobin variants. During eight months, 312 adult patients without previously identified hemoglobin variants had hemoglobinopathy screening performed using a comprehensive testing algorithm. This cascade algorithm screens for hemoglobinopathy using both CE (Capillarys, Sebia, Paris, France) and HPLC (laboratory-developed test) with reflex to more advanced variant identification such as mass spectrometry and genetic analyses. Categories of abnormal findings were reviewed to determine if hemoglobinopathy can be identified by using CE only. The patient population mainly consists of pregnant women and anemic patients with hematologic malignancies with an average age of 42. Out of the 312 screened patients, 47 had abnormal results. The most frequent condition was elevated hemoglobin F (N=25) ranging 2-5% seen in leukemia patients on chemotherapy attributed to bone marrow stress. Eight cases of beta plus thalassemia (featuring hemoglobin A2 >4%) and 3 cases of hemoglobin C trait were identified in patients with little to mild clinical manifestations (red blood cell indices suggesting anemia). Decreased hemoglobin A2 fraction was observed in 7 patients, and potential causes were alpha thalassemia or iron deficiency. Other less common hemoglobinopathies included heterozygote A2 prime (N=3, a benign delta chain variant that migrates separately from hemoglobin A2 on CE) and hemoglobin G-Philadelphia (N=1). All of the abnormal results are identifiable by CE alone, although HPLC and more advanced methods help confirm the diagnosis. Our study shows that CE as the first line of screening method would rule out major hemoglobinopathies in adults. There have been reports that rare but clinically significant hemoglobin variants like hemoglobin Malmo may not be detected by CE, and therefore, certain pre-test probability factors need to be considered when testing for hemoglobinopathies, such as race/ethnicity background, family history, red blood cell indices, and iron deficiency status.

A Different Look at Premarital Hemoglobinopathy Screening in Primary Care

Objective: Despite the high prevalence of hemoglobinopathies (HBP), the most common single-gene disorders in Turkey, data in some regions are lacking. 
 We aimed to evaluate the effectiveness of the hemoglobinopathy premarital screening program (PMS) and to investigate the contribution of efficient use of complete blood count (CBC) parameters on cost-effectivity.
 Method: Hemoglobinopathy diagnosed 49171 subjects in 4 years and CBC of subjects with hemoglobinopathy in a year were evaluated retrospectively.
 Result: The total incidence rate of HBP was 3.41%, β-thalassemia trait (β-TT) was 1.98%, Hemoglobinopathy incidence in the PMS group was 2.43%, β-TT was 1.08%. Moreover, HbF, HbD, HbC, HbS, HbE and HbJ were detected with the incidences of 0.49%, 0.14%, 0.05%, 0.04%, 0.007% and 0.009%, respectively. RDW/MCH ratio compared to other indices was the most successful for both sexes in diagnostic efficiency of HBP (AUC: male:0.922 - female:0.961) and β-TT (AUC: male;0.928 - female:0.961). 
 Conclusion: PMS was found to be an effective application program in hemoglobinopathy screening. RDW/MCH ratio was the most useful and easy parameter in detecting HBP and β-TT in PMS and in terms of reducing unnecessary test requests and cost-effectiveness in public health screenings.

The Evolving Role of Next-Generation Sequencing in Screening and Diagnosis of Hemoglobinopathies

During the last few years, next-generation sequencing (NGS) has undergone a rapid transition from a research setting to a clinical application, becoming the method of choice in many clinical genetics laboratories for the detection of disease-causing variants in a variety of genetic diseases involving multiple genes. The hemoglobinopathies are the most frequently found Mendelian inherited monogenic disease worldwide and are composed of a complex group of disorders frequently involving the inheritance of more than one abnormal gene. This review aims to present the role of NGS in both screening and pre- and post-natal diagnostics of the hemoglobinopathies, and the added value of NGS is discussed based on the results described in the literature. Overall, NGS has an added value in large-scale high throughput carrier screening and in the complex cases for which common molecular techniques have some inadequacies. It is proven that the majority of thalassemia cases and Hb variants can be diagnosed using routine analysis involving a combined approach of hematology, hemoglobin separation, and classical DNA methods; however, we conclude that NGS can be a useful addition to the existing methods in the diagnosis of these disorders.

Haemoglobin switching modulator SNPs rs5006884 is associated with increased HbA2 in β-thalassaemia carriers.

IntroductionHaemoglobin A2 (HbA2), the tetramer of α- and δ-globin chains, is used as a diagnostic biomarker for β-thalassaemia carriers. The HbA2 levels are regulated by the presence of HPFH, δ-thalassaemia, HbA1/2 gene triplication, and variants of KLF1, β-globin gene, and HbF regulating QTLs. Saudi Arabia has a high incidence of borderline HbA2 levels, thereby making it difficult to classify the haemoglobinopathies. This study aims to investigate the association of known HbF enhancer QTL gene SNPs with HbA2 levelsMaterial and methods14 Specific SNPs in BCL11A, HMIP, OR51B6, HBBP1, and HBG2 loci were genotyped in 164 Saudi β-thalassaemia carriers by TaqMan assay to validate their role as regulators of HbA2 levels. HbA2 levels were determined using the Variant II β-Thalassemia Short Program Recorder kit. The non-random association of these SNPs was tested using HaploView software. Protein interaction was assessed using 3D structure modelling for OR51B6 (rs5006884), comparative energy minimisation, and root-mean-square deviation (RMSD) prediction.ResultsElevated HbA2 levels were associated with SNPs in HBBP1, OR51B6, and TCT haplotype from HBG2 promoter region. The bioinformatics modelling and prediction revealed that the exonic rs5006884 had RMSD value deviations and significantly varied binding energy minimisation. α-globin variations were found in 57.92% of individuals but were not associated with elevated HbA2.ConclusionsThe haemoglobin switching modulators rs2071348, rs7482144, and rs5006884 are involved in regulation of HbA2 level with rs5006884 influencing the tetramer formation. Screening for haemoglobinopathies should take these SNPs into consideration, specifically in borderline HbA2 cases. Assiduous analysis of rs5006884 as HbA2 modulator for amelioration of disease severity is recommended.