[Cases Analysis of Hemoglobin H Disease Caused by HBA2:c.2T>C and HBA2:c.2delT Mutations].

OBJECTIVE: To investigate two cases of rare pathogenic genes, initiation codon mutations in HBA2 gene, combined with Southeast Asian deletion and their family members to understand the relationship of HBA2:c.2T>C and HBA2:c.2delT mutations with clinical phenotype. METHODS: The peripheral blood of family members was obtained for blood cell analysis and capillary electrophoresis hemoglobin analysis. Gap-PCR and reverse dot blotting (RDB) were used to detect common types of mutations in ɑ-thalassaemia gene. Sanger sequencing was used to analyze HBA1 and HBA2 gene sequence. RESULTS: Two proband genotypes were identified as --SEA/αα with HBA2:c.2T>C and --SEA/αα with HBA2:c.2delT. HBA2:c.2T>C/WT and HBA2:c.2delT/WT was detected in family members. They all presented with microcytic hypochromic anemia. CONCLUSION: When HBA2:c.2T>C and HBA2:c.2delT are heterozygous that can lead to static α-thalassemia phenotype, and when combined with mild α-thalassemia, they can lead to the clinical manifestations of hemoglobin H disease. This study provides a basis for genetic counseling.

A New δ-Globin Gene Variant: Hb A2-Yulin [δ46(CD5)Gly→Arg,HBD: C.139G > A].

We report a new δ-chain hemoglobin (Hb) variant observed in a 5-year-old female living in Yulin, Guangxi, China. Capillary electrophoresis revealed splitting of the Hb A2 peak into two fractions (Hb A2 and Hb A2 variant), and the Hb A2 variant was also detected by high-performance liquid chromatography. However, it could not be detected using matrix-assisted laser desorption lonization-time of flight mass spectrometry. CD41-42 (-TCTT) heterozygosity was observed on the HBB gene by PCR and reverse dot-blot hybridization. Sanger sequencing showed a new transition (G > A) at codon 46 of the HBD gene, resulting in glycine changing to arginine. Based on the patient's place of residence, the new variant was named Hb A2-Yulin [δ46(CD5)Gly→Arg,HBD:c.139G > A].

SUPT5H mutations associated with elevation of Hb A2 level: Identification of two novel variants and literature review.

ß-thalassemia is one of the most common monogenic disorders in areas of the tropics and subtropics, which represents a major familial and social burden to local people. The elevated Hb A2 level, generally specified as greater than 3.5 %, is commonly used as a high efficiency index for screening of ß-thalassemia carriers. However, mutations in other genes such as GATA1 and KLF1, could also result in increased Hb A2 level. In this study, we identified two novel variants in the SUPT5H gene: a frameshift mutation (SUPT5H: c.3032_3033delTG, p.M1011Mfs*9) and a nonsense mutation (SUPT5H: c.397C > T, p.Arg133*) in two Chinese individuals. Utilizing a combination of phenotype analysis, bioinformatics analysis, and functional analysis, we deduced that these two variants modified the SUPT5H protein's structure, thereby impacting its function and consequently leading to the heightened Hb A2 level phenotype found in the carriers. Furthermore, through a comprehensive literature review, a mutation spectrum was consolidated for SUPT5H, an investigation into the genotype-phenotype correlation was conducted, and factors known to influence Hb A2 levels were identified. Based on this in-depth understanding, clinicians are better equipped to carry out large scale screenings in regions with high prevalence of ß-thalassemia.

Reliability of hemoglobin A2 value as measured by the Premier Resolution system for screening of ß-thalassemia carriers.

OBJECTIVES: Accurate quantification of hemoglobin (Hb) A2 is vital for diagnosing ß-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE). METHODS: We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA2 cutoff value for ß-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the ß- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing. RESULTS: HbA2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias, -0.33; r, 0.991), ß-thalassemia (bias, -0.27; r, 0.927), and non-ß-thalassemia carriers (bias, -0.36; r, 0.928). An HbA2 cutoff value of ≥4.0 % for ß-thalassemia screening achieved 100 % sensitivity and 99.6 % specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3 , 99.8, 97.3, 99.8, and 99.7 %, respectively. We also identified a rare ß-Hb variant, Hb La Desirade [HBB:c.389C>T], associated with ß-thalassemia and co-inherited with a single α-globin gene. CONCLUSIONS: The Premier Resolution HPLC is a reliable and accurate method for routine ß-thalassemia carrier screening, aligning with existing CE methods.

Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

Molecular Characterization of Haemoglobin E.

OBJECTIVE: To detect mutation in cases having haemoglobin A2 level >7% on high performance liquid chromatography. METHODS: The cross-sectional, descriptive study was conducted from July 2017 to December 2018 at the Department of Haematology and Human Genetics and Molecular Biology, University of Health Sciences, Lahore, Pakistan, and comprised patients of either gender with haemoglobin A2 ≥7%. The samples were collected from different cities of Punjab in collaboration with the Punjab Thalassemia Prevention Programme, Lahore. The samples were subjected to complete blood count and high performance liquid chromatography using automated haematology analysers and variant-II beta thalassemia short programme, respectively. To analyse haemoglobin E mutations at the molecular level, polymerase chain reaction-restriction fragment length polymorphism (PCR_RFLP) was performed using a type IIS restriction endonuclease known as Mnl1 (derived from Moraxella nonliquefaciens) to cleave DNA at specific sites and the results were further confirmed on randomly selected samples using Sanger sequencing. Data was analysed using SPSS 25. RESULTS: Of the 39 patients, 15(38.5%) were males and 24(61.5%) were females. The overall median age was 14 (23) years. There were 29 (74.4%) patients with thalassemia family history, and 22(56.4%) had a positive family history of transfusion related to thalassemia, while no patient had a family history of iron therapy. The median haemoglobin A, haemoglobin A2 and haemoglobin F levels were 72.2 (65.2-79.1) %, 26.6 (19.1-34.0) % and 0.9 (-0.8-2.6) %, respectively. After molecular investigation, HbAE mutation was found in 23(59%) patients, while wild type HbAA genotype was found in 16(41%). The heterozygous HbE mutation was present in 23(59%) patients. CONCLUSIONS: Frequently missed/undiagnosed cases of haemoglobin E that co-elute with haemoglobin A2 in the same high performance liquid chromatography window were detected among those with haemoglobin A2 ≥7%.

Comparison of Capillary Zone Electrophoresis with High-pressure Liquid Chromatography in the Evaluation of Hemoglobinopathies

Objective: The capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) methods were compared in terms of HbA2 measurement for the assessment of hemoglobinopathies. Materials and Methods: CZE was compared with HPLC for the evaluation of patients without hemoglobinopathy (n=321), with ß-thalassemia trait (n=113), and with common (HbD-Punjab, E, C, S/A, and S/S) and rare (HbS/D, O-Arap, Lepore, G-Coushata, Setif, Hamadan, Q-Iran, and H) variants (n=21). The reference range for HbA2 was determined by CZE. Results: Among patients without hemoglobinopathy, the median (2.5th-97.5th percentiles) values were 97.4% (97.0-98.0%) and 97.5% (96.6-98.4%) for HbA (p=0.060) and 2.4% (1.6-3.0%) and 2.5% (1.6-3.1%) for HbA2 (p<0.001) by HPLC and CZE, respectively. The reference range for HbA2 was 1.6-3.1% by CZE. In the comparison of methods for HbA2, there was a constant error of 0.255 (confidence interval: 0.062-0.448) and bias of 0.10% (limit of agreement: 0.33-0.53), and higher values were obtained with CZE. A strong correlation was observed between the methods (r=0.782). Interrater agreement was almost perfect for clinical diagnosis (Ï°=0.911). The two methods detected and identified the common variants similarly. All rare variants, except HbH by HPLC and HbS/D by CZE, were detected as separate peaks by both methods. Conclusion: The two methods were in agreement regarding the preliminary identification of ß-thalassemia patients. Different Hb variants were detected by both methods but with possible methodological interference for HbA2 measurements. CZE is a reliable and simple alternative for the evaluation of hemoglobinopathies. The standardization of HbA2 measurements should be prioritized as more techniques become available in routine laboratory practice.

Mutational spectrum of HBD gene in the Chinese population: Description of 36 mutations including 11 novel variants.

INTRODUCTION: Mutations in the hemoglobin subunit delta (HBD) gene (MIM#142000) are associated with decreased levels of the Hemoglobin A2 (Hb A2 ) fraction. We aimed to examine the prevalence of HBD gene mutations and summarize their characteristics in the Chinese population. METHODS: Individuals who exhibited Hb A2 levels below 1.8%, with or without Hb A2 variant peaks, were chosen for further investigation. Hemoglobin analysis was conducted using capillary electrophoresis. Common α and ß-thalassemia in China were detected using gap-PCR and reverse dot blot hybridization. The presence of HBD gene mutations was confirmed by DNA sequencing. RESULTS: A total of 188 patients were identified as carriers of the HBD gene mutation, with a prevalence of approximately 0.46%. We discovered 36 types of mutations, 30 of which resulted in δ-globin variants, while the remaining 6 resulted in δ-thalassemia. The most common mutation was HBD:c.-127 T > C, accounting for 87.2% of δ-thalassemia cases. In addition, we identified 11 novel HBD gene mutations and found 10 cases compounded with other common thalassemias. CONCLUSION: We observed a high prevalence of HBD gene mutations in southern China. Our findings provide a genetic basis for screening for δ-thalassemia and enrich the spectrum of HBD gene mutations.

Prospective screening for δ-hemoglobinopathies associated with decreased hemoglobin A2 levels or hemoglobin A2 variants: A single center experience.

BACKGROUND: δ-hemoglobinopathies may lead to misdiagnosis of several thalassemia syndromes especially ß-thalassaemia carrier, it is important to evaluate the δ-globin gene defects in areas with high prevalence of globin gene disorders. We describe a prospective screening for δ-hemoglobinopathies in a routine setting in Thailand. METHODS: Study was done on a cohort of 8,471 subjects referred for thalassemia screening, 317 (3.7%) were suspected of having δ-globin gene defects due to reduced hemoglobin (Hb) A2 levels and/or appearance of Hb A2-variants on hemoglobin analysis. Hematologic and DNA analysis by PCR and related assays were carried out. RESULTS: DNA analysis of δ-globin gene identified seven different δ-globin mutations in 24 of 317 subjects (7.6%). Both known mutations; δ-77(T>C) (n = 3), δ-68(C>T) (n = 1), δ-44(G>A) (n = 8), Hb A2-Melbourne (n = 5), δIVSII-897(A>C) (n = 5), and Hb A2-Troodos (n = 1) and a novel mutation; the Hb A2-Roi-Et (n = 1) were identified. This Hb A2-Roi-Et, results from a double mutations in-cis, δCD82(AAG>AAT) and δCD133(GTG>ATG), was interestingly found in combination with an in trans, 12.6 kb deletional δß0-thalassemia in an adult Thai woman who had no Hb A2 and elevated Hb F. A multiplex-allele-specific PCR was developed to detect these novel δ-globin gene defects. CONCLUSIONS: The result confirms a diverse heterogeneity of δ-hemoglobinopathies in Thailand which should prove useful in a prevention and control program of thalassemia in the region.

[Causes of Abnormal Hemoglobin Electrophoresis].

OBJECTIVE: To investigate the possible causes of abnormal hemoglobin electrophoresis results. METHODS: The hemoglobin electrophoresis results of 5 696 patients in the First Affiliated Hospital of Chengdu Medical College from September 2018 to July 2021 were collected, and the abnormal results and clinical significance were analyzed. RESULTS: The results of 486 patients (accounting for 8.53%) were abnormal, of which 300 cases had increased HbA2, 135 cases had decreased HbA2, 44 cases had increased F alone, and 7 cases had abnormal hemoglobin bands. Among the 486 patients, 246 patients were thalassemia gene positive (the positive rate was 50.62%), including 29 cases of α thalassemia, 208 cases of ß thalassemia and 9 cases of αß thalassemia. Among the patients with elevated HbA2, 68.67% were detected ß thalassemia, 3.00% αß thalassemia, 9.33% were suspected to be caused by macrocytosis, 6.33% by thyroid dysfunction, and 12.67% by uncertainty of the method. Among the patients with reduced HbA2, 21.48% were detected α thalassemia, 60.00% iron deficiency anemia, 8.15% were suspected to be caused by thyroid dysfunction, and 10.37% by uncertainty of the method. Among the patients with elevated F alone, the results of thalassemia gene detection were negative, 40.91% of them were suspected to be caused by macrocytosis, 27.27% by hereditary persistence of fetal hemoglobin, 29.55% by special physiological condition of pregnant women, and 2.27% by hyperthyroidism. Abnormal hemoglobin bands were detected in 7 patients, including 4 cases of hemoglobin D, 2 cases of hemoglobin E, and 1 case of hemoglobin J. CONCLUSION: Thalassemia, iron deficiency anemia, macrocytosis such as megaloblastic anemia and non-severe aplastic anemia, thyroid dysfunction, hereditary persistence of fetal hemoglobin, abnormal hemoglobin diseases, the uncertainty of the method are all important causes of abnormal hemoglobin electrophoresis results. In clinical work, the patient's indicators should be comprehensively analyzed to determine the possible cause.

Molecular basis of a high Hb A2/Hb Fß-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction with α-globin gene triplication.

Background: ß 0-thalassemia deletion removing 5´ß-globin promoter usually presents phenotype with high hemoglobin (Hb) A2 and Hb F levels. We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the ß 0-thalassemia with 3.4 kb deletion. Methods: A total of 148 subjects, including 127 heterozygotes, 20 Hb E-ß-thalassemia patients, and a double heterozygote with α-globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at A γ-globin promoter, rs5006884 on OR51B6 gene, -158 G γ-XmnI, BCL11A binding motifs (TGGTCA) between 3´A γ-globin gene and 5´Î´-globin gene. Results: It was found that heterozygous ß 0-thalassemia and Hb E-ß 0-thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous ß 0-thalassemia with 3.4 kb deletion and α-thalassemia was associated with even higher MCV and MCH values. The Hb E-ß 0-thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous ß 0-thalassemia with 3.4 kb deletion and α-globin gene triplication presented as a plain ß-thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´ß-globin promoter may likely be responsible for this unusual phenotype. Conclusions: The results indicate that ß 0-thalassemia with 3.4 kb deletion is a mild ß-thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis.

Hemoglobin variant in disguise.

BACKGROUND: Hemoglobinopathies include thalassemia syndromes, where production of one or more globin subunits of hemoglobin (Hb) is reduced, and structural Hb variants. Over 1000 disorders of Hb synthesis and/or structure have been identified and characterized, with phenotypes ranging from having severe clinical manifestations to clinically silent. Various analytical methods are used to phenotypically detect Hb variants. However, molecular genetic analysis is a more definitive method for Hb variant identification. CASE REPORT: Here, we report a case of a 23-month-old male with results from capillary electrophoresis, gel electrophoresis (acid and alkaline), and high-performance liquid chromatography most consistent with HbS trait. Specifically, capillary electrophoresis showed slightly elevated HbF and HbA2, HbA of 39.4% and HbS of 48.5%. The HbS percentage was consistently higher than expected (typically 30-40%) for HbS trait with no concurrent thalassemic indices. The patient has not experienced any clinical complications due to the hemoglobinopathy and he is thriving. CONCLUSION: Molecular genetic analysis revealed the presence of compound heterozygosity for HbS and Hb Olupona. Hb Olupona is an extremely rare beta-chain variant that appears as HbA on all three common methods used for phenotypic Hb analysis. When the fractional concentration of Hb variants is unusual, more definitive methods should be used, such as mass spectrometry or molecular genetic testing. In this case, incorrectly reporting this result as HbS trait is unlikely to have a significant clinical impact, as current evidence suggests Hb Olupona is not a clinically significant variant.

Factors affecting the evaluation and use of a hemoglobin A2 method - Lot-to-lot variation, long-term deviation and carry-over.

BACKGROUND: We compared two Hb A2 methods, high pressure liquid chromatography and capillary zone electrophoresis, at two occasions. In 2014 the methods showed good agreement, while in 2020 HPLC results were clearly higher than CE results. This finding prompted us to investigate the external quality assessment (EQA) outcome and our total patient results obtained by high pressure liquid chromatography over several years. METHODS: The methods compared were Bio-Rad Variant II Beta-Thal Short Program (HPLC) and Sebia Capilllarys Flex Piercing (CE). RESULTS: Our annual patient results obtained by HPLC increased significantly from 2014 to 2020. A similar trend was also seen in our EQA results. When patient results were grouped according to different reagent lots it became evident that method comparisons might be severely affected by lot-to-lot variation. We also noticed that samples analyzed with the HPLC method following a sample containing a high proportion of Hb E where prone to give falsely increased Hb A2 results. CONCLUSION: Standardization of the measurement of Hb A2 is urgently needed. Furthermore, the lot-to-lot variation must be minimized. While waiting for these improvements each laboratory ought to repeatedly evaluate their distribution of patient Hb A2 results.

Effect of megaloblastic anemia on hemoglobin A2 and diagnosis of ß-thalassemia trait.

Context: ß-thalassemia trait is usually diagnosed by raised hemoglobin A2 (HbA2). The presence of megaloblastic anemia can cause an increase in HbA2 and create a diagnostic dilemma. Here, we have analyzed the effect of vitamin B12 and folic acid supplementation on HbA2 and diagnosis of ß-thalassemia trait in cases of megaloblastic anemia with raised HbA2. Materials and Methods: Cases of megaloblastic anemia with raised HbA2 on high-performance liquid chromatography (HPLC) were supplemented with vitamin B12 and folic acid. Post-treatment evaluation was done after 2 months. Cases showing adequate hematological response were subjected to statistical analysis. Based on post-treatment HbA2 value, the cases were diagnosed as normal, borderline raised HbA2, or ß-thalassemia trait. Pre- and post-treatment values of red cell parameters and HbA2 were analyzed. Results: There was a significant decrease in HbA2 value after vitamin B12 and folic acid supplementation. The diagnosis was changed in 70.97% of the cases after treatment. The chance of inconclusive diagnosis was decreased from more than 50% to less than 10%. Pre-treatment mean corpuscular volume (MCV) and HbA2% showed a significant difference between the thalassemic and normal groups. Conclusions: Megaloblastic anemia can lead to false-positive diagnosis of ß-thalassemia trait on HPLC. Repeat HPLC should be done after adequate supplementation of vitamin B12 and folic acid in cases of megaloblastic anemia with raised HbA2. Red cell parameters are not helpful to suspect ß-thalassemia trait in presence of megaloblastic anemia. However, HbA2% on HPLC can be a useful parameter to suspect or exclude ß-thalassemia trait in cases of megaloblastic anemia.

Work-up of Patients with Decreased Hemoglobin A2 Identified by Capillary Zone Electrophoresis: A North American Institutional Experience.

OBJECTIVE: Isolated low hemoglobin A2 (HbA2) is rarely encountered in our clinical practice using capillary zone electrophoresis. The study goal was to characterize the work-up at our institution of patients with low HbA2. METHODS: Patients with low HbA2 and a control cohort with normal capillary zone electrophoresis were identified and relevant information extracted from the medical record. RESULTS: Of 44 patients with isolated decreased HbA2, 28 (64%) had corresponding complete blood count/ferritin values. Compared to control patients, patients with low HbA2 were more likely to have iron deficiency and demonstrated a more microcytic, hypochromic blood picture. However, 46% (13/28) of patients with low HbA2 and ferritin for evaluation did not have iron deficiency. Only 2 patients had genetic testing. CONCLUSION: This study redemonstrates the association between low HbA2 and iron deficiency and reinforces the need for iron indices to interpret capillary zone electrophoresis results. Our study population showed incomplete or absent iron studies in most cases.

A stepwise haematological screening and whole-exome sequencing reveal multiple mutations from SUPT5H causing an elevation of Hb A2 from a cohort of 47336 individuals.

INTRODUCTION: Though an increase in Hb A2 is one of the most key markers of ß-thal carriers, a few independent cases are reported to show elevated Hb A2 levels caused by mutations in other genes beyond ß-globin gene. METHODS: We reviewed the haematological indices of 47336 individuals to analyse the phenotype-genotype correlation and identified 1439 individuals (3.04%) positive in the elevation of Hb A2 . Globin and KLF1 genes analysis was performed, and further whole-exome sequencing was carried to dissect the genetic causes of those positive samples without ß-thalassemic or KLF1 mutations. RESULTS: Of these 1439 individuals with elevated Hb A2 , 1381 had a molecular defect in globin genes, and most were ß-thalassemic mutation; 10 had a molecular defect in KLF1 gene. Finally, among the 38 individuals without ß-thalassemic or KLF1 mutations, 7 were identified to carried a loss-of-function mutation in SUPT5H. CONCLUSION: This study has provided a mutation spectrum of SUPT5H in a cohort screening leading to the elevation of Hb A2 . According to the previous observations that individuals with a combination of ß-thal mutation and a SUPT5H variant might present moderate ß-thaelassemia, these findings emphasized the importance of comprehensive molecular diagnosis to prevent birth defects of ß-thaelassemia caused by rare mutations from modifier genes.

Causes of Abnormal Hemoglobin Electrophoresis / 中国实验血液学杂志

OBJECTIVE@#To investigate the possible causes of abnormal hemoglobin electrophoresis results.@*METHODS@#The hemoglobin electrophoresis results of 5 696 patients in the First Affiliated Hospital of Chengdu Medical College from September 2018 to July 2021 were collected, and the abnormal results and clinical significance were analyzed.@*RESULTS@#The results of 486 patients (accounting for 8.53%) were abnormal, of which 300 cases had increased HbA2, 135 cases had decreased HbA2, 44 cases had increased F alone, and 7 cases had abnormal hemoglobin bands. Among the 486 patients, 246 patients were thalassemia gene positive (the positive rate was 50.62%), including 29 cases of α thalassemia, 208 cases of β thalassemia and 9 cases of αβ thalassemia. Among the patients with elevated HbA2, 68.67% were detected β thalassemia, 3.00% αβ thalassemia, 9.33% were suspected to be caused by macrocytosis, 6.33% by thyroid dysfunction, and 12.67% by uncertainty of the method. Among the patients with reduced HbA2, 21.48% were detected α thalassemia, 60.00% iron deficiency anemia, 8.15% were suspected to be caused by thyroid dysfunction, and 10.37% by uncertainty of the method. Among the patients with elevated F alone, the results of thalassemia gene detection were negative, 40.91% of them were suspected to be caused by macrocytosis, 27.27% by hereditary persistence of fetal hemoglobin, 29.55% by special physiological condition of pregnant women, and 2.27% by hyperthyroidism. Abnormal hemoglobin bands were detected in 7 patients, including 4 cases of hemoglobin D, 2 cases of hemoglobin E, and 1 case of hemoglobin J.@*CONCLUSION@#Thalassemia, iron deficiency anemia, macrocytosis such as megaloblastic anemia and non-severe aplastic anemia, thyroid dysfunction, hereditary persistence of fetal hemoglobin, abnormal hemoglobin diseases, the uncertainty of the method are all important causes of abnormal hemoglobin electrophoresis results. In clinical work, the patient's indicators should be comprehensively analyzed to determine the possible cause.

RS12574989 and haplotype associated with α/ß-chain imbalance and population HbA2 reduction.

Determining the associated relationship of genotype and phenomenon would benefit the understanding of disease and renew disease intervention means. 14,518 patients who underwent haemoglobin electrophoresis from June 2020 to December 2020 were enrolled in our study, and additional data including sex, age and routine blood examination results were collected. We focused on individuals with normal red blood cell indices and no common thalassemia pathogenic mutation and selected three groups for the following study: the control group (2.5% ≤ HbA2 ≤ 3.5%), the HbA2 under 2.5 group (HbA2 < 2.5%) and the HbA2 under 2.4 group (HbA2 < 2.4%). Four regions of ß-globin regulation were sequenced. Statistical analysis was conducted to compare the collected information of the three groups and the genotype distributions in the control group and sequenced group. The HbA2 under 2.5 group was characterized by a majority of females and lower red blood cell counts and haemoglobin compared with the control group. There were genotypes associated with the grouping as the T of rs12574989 and TTTAGC of the haplotype were significantly increased in the HbA2 under 2.4 group and CTTAGC was significantly decreased in the HbA2 under 2.4 group. This study demonstrated that the genotypes of the population associated with HbA2 were reduced in southern China.

First study to describe a novel HbA2: c.400A > C mutation and Hb Dongguan heterozygote in two unrelated Chinese families.

OBJECTIVES: Here we report two rare α-globin chain variants in two unrelated families: Hb Val de Marne [α133(H16) Ser > Arg (AGC > CGC); HBA2: c.400A > C] and Hb Dongguan [α52(E6) Ser > Cys (TCT > TGT); HBA1: c.158C > G]. Notably, HBA2: c.400A > C is an unreported new variant in the third exon of the α2 gene, and simple heterozygous unstable Hb Dongguan haematological characteristics are proposed for the first time. METHODS: Hb analysis was performed by using capillary electrophoresis (CE). Twenty-three common mutations were detected using a suspension array system. Mutations were identified by DNA sequencing. RESULTS: The CE results showed an abnormal peak with incomplete separation from Hb A at zone 8 in two members of Family 1. DNA sequencing confirmed the presence of Hb Val de Marne [α133(H16) Ser > Arg (AGC > CGC); HBA2: c.400A > C]. Five members of Family 2 exhibited an abnormal peak at zone 11, and DNA sequencing confirmed the presence of Hb Dongguan [α52(E6) Ser > Cys (TCT > TGT); HBA1: c.158C > G]. CONCLUSIONS: The discovery of HBA2: C.400A > C expands the existing spectrum of α-globin variants. The carriers of simple heterozygous Hb Dongguan generally do not have obvious clinical symptoms. The information in this study will help clinicians understand the screening, molecular diagnosis and clinical significance of Hb variants.

A New Mutation, Hb A2-Canakkale [δ10(A7)Ala→Val; HBD: c.32C>T], and Other Well-Known δ Variants Identified in a Selected Cohort with Low Hb A2 Levels.

Hemoglobinopathies are the most common single-gene disorders, and ß-thalassemia (ß-thal) imposes a tremendous health burden on Turkey. Thus, premarital carrier screening is obligatory in Turkey, as it is in some other countries. As a result of this mandatory procedure, at routine clinical checkups, many individuals who had undergone premarital screening but did not have any clinical symptoms and/or hematological findings, have compulsorily been required to undergo further evaluation due to abnormal levels of hemoglobin (Hb) fractions (Hb A, Hb A2 and Hb F). Many consequences, such as mutations in unrelated gene(s) or someone's nutritional status, have been reported to affect the Hb fractions levels. In the present study, we aimed to determine whether HBD has a molecular causative role in patients with low Hb A2 levels (below 1.8%). The study was conducted with 20 individuals with low Hb A2 levels who had applied to our outpatient clinic. All DNA samples were analyzed for the HBD gene. Nineteen of the 20 subjects were diagnosed to carry a mutation with one of four different δ-globin variants. Three of them had been described previously [Hb A2-Yialousa (HBD: c.82G>T), Hb A2-Bornova (HBD: c.350G>C) and Hb A2-Yokoshima (HBD: c.77G>A)]. The novel [δ10(A7)Ala→Val, HBD: c.32C>T] mutation was defined as a new δ variant and reported to the HbVar database as Hb A2-Canakkale. In conclusion, the molecular characterization of Hb A2 low levels has been suggested to be significant for a definite diagnosis and counseling.