Association study of common KLF1 variants with Hb F and Hb A2 levels in ß-thalassaemia carriers of Portuguese ancestry.

Kruppel-like factor 1 (KLF1) is an essential erythroid-specific transcription factor. Several reports have shown that KLF1 gene mutations are associated with increased levels of Hb F and Hb A2. However, scarce population studies have analysed common KLF1 variations. This study examines the potential association with Hb F and Hb A2 levels in ß-thalassemia (ß-thal) carriers of Portugueseancestry of the four common KLF1 gene variants: -251C>G (rs3817621) and -148G>A (rs79334031), in the promoter region; and c.115A>C (p.Met39Leu) (rs112631212) and c.304T>C (p.Ser102Pro) (rs2072597), in exon 2. Ninety-two Portuguese ß-thal carriers (43 males and 49 females) aged 2 to 77 years old (mean 32.55 years) were engaged in the study. Hb F levels range from 0.2 to 12.5% and Hb A2 was above the normal level, ranging from 3.6 to 6%. The Hb A2 and Hb F levels were determined by high-performance liquid chromatography. Single-nucleotide polymorphisms were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Minor allele frequencies for SNPs rs3817621 (G), rs79334031 (A), rs112631212 (C) and rs2072597 (C) were 0.196, 0.016, 0.011 and 0.169, respectively. Basic simple linear regression in the total population showed no significant associations with the levels of Hb F (P>0.05). For the low-frequency variant -148A, a statistically significant association was found with increased levels of Hb A2 (ß = 0.855; P = 0.017). In conclusion, an association signal with Hb A2 levels was observed for the variant -148A>G (rs79334031). The complex pattern of SNP interactions related to their influence on the KLF1 transcriptional activity mayexplain the absence of association with Hb F levels.

Hb A2-Guangxi [δ79 (EF3) Asp→Asn, HBD: C.238G > A] and polyA + 70 (HBD: C.*200G > A): Two Novel δ-Globin Gene Mutations Identified in a Chinese Family.

We report the molecular and hematological identifications of two novel δ-globin gene mutations found in Guangxi Zhuang Autonomous Region, China. Capillary electrophoresis of the proband showed 1.3% Hb A2, accompanied by a minor unknown peak (0.7%) within the Z1 zone. High-performance liquid chromatography also revealed the presence of 1.5% Hb A2 and a 0.6% unknown peak. Routine genetic testing (Gap-PCR and reverse dot-blot hybridization) for common α-thalassemia was performed, and no mutations were observed. Sanger sequencing identified a heterozygous mutation for GAC > AAC at codon 79 (HBD:c.238G > A) and G > A at polyA + 70 (HBD:c.*200G > A) of the δ-globin gene. This variant was named Hb A2-Guangxi [δ79 (EF3) Asp→Asn, HBD:c.238G > A] after the geographic origin of the proband.

Post-GWAS Validation of Target Genes Associated with HbF and HbA2 Levels.

Genome-Wide Association Studies (GWASs) have identified a huge number of variants associated with different traits. However, their validation through in vitro and in vivo studies often lags well behind their identification. For variants associated with traits or diseases of biomedical interest, this gap delays the development of possible therapies. This issue also impacts beta-hemoglobinopathies, such as beta-thalassemia and sickle cell disease (SCD). The definitive cures for these diseases are currently bone marrow transplantation and gene therapy. However, limitations regarding their effective use restrict their worldwide application. Great efforts have been made to identify whether modulators of fetal hemoglobin (HbF) and, to a lesser extent, hemoglobin A2 (HbA2) are possible therapeutic targets. Herein, we performed the post-GWAS in vivo validation of two genes, cyclin D3 (CCND3) and nuclear factor I X (NFIX), previously associated with HbF and HbA2 levels. The absence of Ccnd3 expression in vivo significantly increased g (HbF) and d (HbA2) globin gene expression. Our data suggest that CCND3 is a possible therapeutic target in sickle cell disease. We also confirmed the association of Nfix with γ-globin gene expression and present data suggesting a possible role for Nfix in regulating Kruppel-like transcription factor 1 (Klf1), a master regulator of hemoglobin switching. This study contributes to filling the gap between GWAS variant identification and target validation for beta-hemoglobinopathies.

[Thalassemia Genotypes and HbA2 levels of Children in Wuzhou, Guangxi].

OBJECTIVE: To analyze thalassemia genotypes and distribution of children in Wuzhou Guangxi, and evaluate the diagnostic value of HbA2 in children's thalassemia screening, so as to provide scientific evidence for the prevention and control strategies of thalassemia. METHODS: Four hundred and fifty-eight children suspected with thalassemia in Wuzhou were enrolled from March 2017 to June 2022. The level of HbA2 was detected using Bio-Rad VARIANT II Hb analysis system. The deletion of α-thalassemia was measured with gap-PCR assay, and the point mutation of α- and ß-thalassemia was tested with DNA reverse dot blot hybridization assay. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of HbA2 for children's thalassemia. RESULTS: A total of 304 thalassemia carriers were detected in 458 children, accounting for 66.38%. One hundred and seventy-five cases were defined to be α-thalassemia, with the main type of --SEA/αα (54.86%). Thirty-six cases were defined to be intermediate α-thalassemia, with the main type of -α3.7/--SEA (9.72%). In 108 cases with ß-thalassemia, ßCD41-42/ßN was the main type, accounting for 49.07%, followed by ßIVS-Ⅱ-654 /ßN (14.81%). Seven cases were moderate/severe ß-thalassemia (predominantly ß-28/ß-28 and ßCD41-42/ßCD17/). Twenty-one genotypes of α- and ß-thalassemia were found in the children. There was significant difference of HbA2 level between the children with different types of thalassemia and healthy controls (all P < 0.001). ROC curve analysis showed that the sensitivities of HbA2 for α-thalassemia, ß-thalassemia and αß-thalassemia were 74.3%, 82.4% and 85.7%, with the optimal cut-off values of 2.60%, 3.60% and 3.70%, respectively, the specificities were 64.3%, 96.1% and 96.8%, and the area under the curve were 0.690, 0.887 and 0.916, respectively. CONCLUSION: The thalassemia genotypes of children in Wuzhou are diverse. It is necessary to further strengthen the prevention and control measure of thalassemia to reduce birth defects and improve birth quality.

[Molecular Diagnosis and Pedigree Analysis of Rare Mutations in Non-coding Region of HBA2 Gene].

OBJECTIVE: To perform molecular diagnosis and pedigree analysis for one case with α-thalassemia who does not conform to the genetic laws, and explore the effects of a newly discovered rare mutation (HBA2:c.*12G>A) on clinical phenotypes. METHODS: Blood samples of the proband and her family members were collected for blood routine analysis, and the hemoglobin components were analyzed by capillary electrophoresis. The common α- and ß-globin gene loci in Chinese population were detected by conventional techniques (Gap-PCR, RDB-PCR). The α-globin gene sequences (HBA1, HBA2) were analyzed by Sanger sequencing. RESULTS: By analyzing the test results of proband and her family members, the genotype of the proband was -α3.7/HBA2:c.*12G>A, her father was HBA2:c.*12G>A heterozygous mutation carrier. CONCLUSION: This study identifies a rare α-globin gene mutation (HBA2:c.*12G>A) that has not been reported before. It is found that heterozygous mutation carriers present with static α-thalassemia.

Graves' Disease: Acquired Cause of a Moderate Increase in Hemoglobin A2 Level.

BACKGROUND: Hyperthyroidism can lead to diverse hematological disorders, such as microcytosis and a mild increase in hemoglobin A2 fraction. METHODS: This study reported a 31-year-old woman of Moroccan origin recently diagnosed with Graves' disease. Her blood tests revealed microcytosis, hypochromia, and a normal ferritin level. A phenotypic analysis of hemo-globin was performed using two techniques: capillary electrophoresis and reversed-phase high performance liquid chromatography. RESULTS: Both techniques indicated a slight increase in hemoglobin A2 level. These results initially suggested het-erozygous beta-thalassemia, eventually correlating with the concurrent presence of Graves' disease, as evidenced by the normalization of hemoglobin A2 level following treatment. CONCLUSIONS: This case highlights the importance of having clinical, biological, and therapeutic data for a relevant interpretation of a phenotypic hemoglobin study.

[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 Novel Frameshift Mutation(HBA2:C.337delC) Associated With α-Thalassemia Trait Detected by Next-Generation Sequencing in Southern China.

Here, we report a novel frameshift mutation caused by a single base deletion in exon 3 of the HBA2 gene (HBA2:c.337delC) detected by next-generation sequencing. The proband was a 26-year-old Chinese pregnant woman who originates from Hunan Province. Her mean corpuscular volume(MCV) and mean corpuscular hemoglobin (MCH) had a mild decrease. Capillary electrophoresis (CE) showed that both Hb A (97.8%) and Hb F (0.0%) values were within normal range, while the Hb A2 (2.2%) value was below normal. Sequence analysis of the α and ß-globin genes revealed a novel single base deletion at codon 112 (HBA2:c.337delC) in the heterozygous state, which resulted in a mild phenotype of α-thalassemia.

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.

A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F.

OBJECTIVES: Hemoglobinopathies, including thalassemia and hemoglobin (Hb) variants, are common hematological disorders in tropical countries. Accurate and precise separation of hemoglobin types and reliable quantitation are necessary for differential diagnosis of these disorders. METHODS: We have evaluated the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC; Trinity Biotech, Co. Wicklow, Ireland) to assist in the presumptive diagnosis of thalassemia and Hb variants commonly found in Southeast Asian countries. HbA0, HbA2, HbE, and HbF levels were separated and quantified in 120 blood samples from unrelated adult subjects and compared with those analyzed by capillary zone electrophoresis (CZE; CAPILLARYS™ 2, Sebia, Norcross, GA, US). The Hb analysis patterns of Hb variants obtained from the PR-HPLC system were also compared to those obtained from HPLC (VARIANT II, ß-thalassemia Short Program, Bio-Rad, Laboratories, Hercules, CA, US) and CZE systems. RESULTS: The PR-HPLC had excellent precision with a coefficient of variation (CV) for HbA2 quantitation of 3.8 % within-run and 5.2 % between-run. The levels of HbA2/E quantified by the PR-HPLC system correlated well with those of the CZE system (r=0.997). In addition, thalassemia interpretation results obtained from the PR-HPLC and the CZE showed 100 % agreement. Moreover, chromatograms of the PR-HPLC were also comparable to those of VII-HPLC and CAP2-CZE electropherograms. CONCLUSIONS: The PR-HPLC system would be applicable to diagnose common forms of thalassemia and Hb variants in Southeast Asia.

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.