δ-Globin Chain Variants Associated with Decreased Hb A2 Levels: A National Reference Laboratory Experience.

High prevalence of hemoglobin (Hb) disorders mandates national programs for screening and genetic counseling in many countries. Increased Hb A2 levels are commonly associated with ß-thalassemias, however, various disorders including alteration of δ chains may result in decreased production of Hb A2, thus hindering the diagnosis of ß-thalassemias. The reported data reflect the experience of a large reference laboratory in the United States. In the current study, we have attempted to assess the prevalence and also tried to characterize the identified mutations in the HBD gene resulting in decreased Hb A2 levels. In our cohort, 1.6% of 6486 patients were found to have Hb A2 values of <1.9%. Bidirectional sequencing of the HBD gene demonstrated mutations in 20 cases (19.0% of the individuals with decreased Hb A2). In addition to the previously reported variants, one novel mutation (Hb A2-Utah or HBD: c.46T>C).

Thalassemia Major and Intermedia Patients in East Java do not Show Fetal Hemoglobin Level Difference in Relation to XMNI Polymorphism.

INTRODUCTION: Thalassemia is a genetic disorder, which shows, varies phenotype due to genetic modifier. XmnI is one of the genetic modifiers which affect clinical severity in thalassemia. XmnI polymorphism may increase HbF production beyond fetal life, thus ameliorating the clinical phenotype. AIM: this study aimed to investigate the difference in HbF level and the relation of HbF level and XmnI polymorphism in Thalassemia Major (TM) and Thalassemia Intermedia (TI) patients. METHODS: forty-eight beta thalassemia patients (28 males and 20 females), consists of 16 TM and 32 TI; mean age, 25.30 year old. Hemoglobin Fetal and HbA2 level were determined using High performance Liquid Chromatography (HPLC), and XmnI polymorphism was confirmed by PCR-RFLP. Statistical analysis was done using T-test, Mann-Whitney and Pearson Chi-square. RESULTS: The frequency of heterozygote (+/-) XmnI polymorphism in TM and TI patients was 56.25% vs 71.87%, while the frequency of homozygote (-/-) in TM and TI was 43.75% vs 28.13% with p value >0.05. The insignificant difference also found in HbF level between XmnI +/- and -/- in TM and TI patients. CONCLUSION: This study revealed that thalassemia major and thalassemia intermedia patients in East Java showed similar XmnI polymorphism. These phenomena also showed by HbF level in relation to XmnI polymorphism in the phenotype groups (TM and TI).

Hemoglobins F, A2 , and E levels in Laotian children aged 6-23 months with Hb E disorders: Effect of age, sex, and thalassemia types.

INTRODUCTION: Determination of hemoglobins (Hbs) F, A2, and E is crucial for diagnosis of thalassemia. This study determined the levels of Hbs F, A2, and E in children aged 6-23 months and investigated the effect of age, sex, and types of thalassemia on the expression of these Hbs. METHODS: A total of 698 blood samples of Laotian children including 272 non-Hb E, 271 Hb E heterozygotes, and 155 Hb E homozygotes were collected. Hb profiles were determined using the capillary zone electrophoresis. Coinheritance of α-thalassemia and the homozygosity for Hb E mutation were checked by PCR-based assay. RESULTS: Children heterozygous and homozygous for Hb E had significantly higher Hb F and A2 levels than non-Hb E children (median Hb F = 1.1% for non-Hb E group, 2.7% for Hb E heterozygotes, and 9.4% for Hb E homozygotes; median Hb A2  = 2.6% for non-Hb E group, 3.8% for Hb E heterozygotes, and 5.2% for Hb E homozygotes). The median Hb E levels were 21.9% for Hb E heterozygotes and 85.3% for Hb E homozygotes. Comparing within group, there was a statistically significant difference between children with and without an α-gene defect for Hb A2 and E, but not Hb F. Based on a multiple regression analysis, age and sex were significantly associated with the expression of Hb F and A2 but not Hb E. CONCLUSIONS: Our findings can guide the development of a diagnostic approach to thalassemia in children aged 6-23 months.

Dominant ß-thalassaemia with unusually high Hb A2 and Hb F caused by ßCD121(-G) (HBB:c.364delG) in exon 3 of ß-globin gene.

We describe a dominant ß-thalassaemia caused by a deletion of G at nucleotide position 364 in exon 3 of the ß-globin gene. The heterozygosity of this mutation was found in a 36-year-old Thai patient who had moderate hypochromic microcytic anaemia with haemolytic blood picture. Haemoglobin (Hb) analysis revealed relatively higher Hbs A2 (6.8%) and F (4.7%) as compared with those of ß0-thalassaemia (n=278) and ß+-thalassaemia (n=55) carriers in our series. Secondary structure prediction of the elongated ß-globin chain showed that the α-helix at the C-terminal is disrupted dramatically by the random coil and ß-sheet, which should result in a highly unstable ß-globin variant, undetectable in peripheral blood and a dominant clinical phenotypic feature.

[Hyperthyroidism: acquired cause of an increase in haemoglobin A2 level]. / Hyperthyroïdie : cause acquise d'augmentation de l'hémoglobine A2.

Hyperthyroidism may be associated with several haematological disorders, including an increase in haemoglobin A2 level. We report the case of haemoglobin study in a 29-years old man of Mediterranean origin presenting microcytosis. Results showed a slight increase in haemoglobin A2 level, suggestive of a beta-thalassemia trait, which was finally related to the existence of a recently diagnosed Grave's disease.

Evaluation of the suitability of the World Health Organization International Reference Reagent for Hb A2 quantitation (89/666) for continued use.

INTRODUCTION: The accurate determination of Hb A2 is a key marker when screening for a ß-thalassaemia carrier. Data from external quality assessment (EQA) exercises have shown a lack of alignment of Hb A2 quantitation both within and between methods. The only reference material available for Hb A2 quantitative assay at the time of writing is the World Health Organization International Reference Reagent (89/666; WHO IRR) prepared in the 1980s and not validated for all current methodologies. METHOD: The WHO IRR was analysed for Hb A2 concentration by 52 laboratories using a representative range of high-performance liquid chromatography and capillary electrophoresis analysers. The results of the analysis were compared to those of a whole blood EQA specimen of similar Hb A2 concentration, distributed in the same week. RESULTS: The mean Hb A2 value obtained for the WHO IRR was 5.17%, compared to the assigned value of 5.3%. The range of results returned was wide (4.0%-6.2%), with differences in the results observed by between and within analyser groups. A similar range of results was seen with the whole blood sample, although the bias observed between analyser types was different from that seen with the WHO IRR. CONCLUSION: The results may indicate a lack of commutability of the WHO IRR material, resulting from deterioration, matrix effects or changes in reagent formulation or calibration parameters. Further examination of the suitability of the WHO IRR (89/666) for continued use is required.

Reference intervals for HbA2 and HbF and cut-off value of HbA2 for ß-thalassemia carrier screening in a Guizhou population of reproductive age.

OBJECTIVE: The aims of this study were to establish the reference intervals for HbA2 and HbF in a Guizhou population of reproductive age, and to determine the cut-off value of HbA2 for ß-thalassemia carrier screening. METHODS: Hemoglobin analysis was performed on 832 individuals without hypochromic microcytic anemia to calculate the reference intervals for HbA2 and HbF. Three hundred and ninety one ß-thalassemia carriers and non ß-thalassemia individuals were analyzed for their HbA2 levels followed by detecting ß-globin gene mutations, then cut-off value of HbA2 for ß-thalassemia carrier screening was determined using ROC curve analysis. RESULTS: The reference interval for HbA2 in overall normal individuals was 2.3%-3.1%, and reference intervals for HbF in normal males and females (including normal females and pregnant women) were 0-0.5% and 0-1.0% respectively. The cut-off values of HbA2 for ß-thalassemia carrier screening in males, non-pregnant women, pregnant women and the overall set were 4.40%, 3.75%, 3.70% and 3.95% respectively. CONCLUSION: Gender and pregnancy status had no obvious influence on reference interval for HbA2. The HbF level was higher in females than in males, but pregnancy status had no obvious influence on HbF level. Cut-off value of HbA2 for ß-thalassemia carrier screening was obviously affected by gender but not by pregnancy status.

Causes of microcytic anaemia and evaluation of conventional laboratory parameters in the differentiation of erythrocytic microcytosis in blood donors candidates.

CONTEXT AND OBJECTIVE: Microcytic anaemia results from defective synthesis of haemoglobin in the erythroid precursors, causing a reduction in its mean corpuscular volume (MCV). The most common causes of microcytosis, without the increase in HbA2 levels, are iron deficiency anaemia (IDA) and α-thalassemia. The aim of this study was to identify the causes of microcytic anaemia and evaluate the haematological parameters from blood donors deemed ineligible (due to the low haematocrit level) that would differentiate the IDA and α-thal, whether isolated or in association. METHODS: Genomic DNA was submitted to the polymerase chain reaction multiplex for the diagnosis of the most common allele deletions of α-thal and erythrogram and in order to verify haematological parameters. Iron deficiency (ID) was determined through the measurement of serum ferritin. RESULTS: Of the 204 samples, 82 (40.2%) were identified with ID, 24 (17.8%) with α-thal and 10 (4.9%) with ID associated with α-thal. In the α-thal with ID group haemoglobin (Hb), MCV, mean corpuscular Hb concentration (MCHC) and mean corpuscular Hb (MCH) values were significantly lower compared to the isolated α-thal. In the group with ID Hb, MCV, MCHC and MCH values were significantly lower compared to those with isolated α-thal. The α-thal with ID group, showed Hb, MCV, MCHC and MCH significantly reduced when compared to those with IDA. CONCLUSIONS: This study showed that the values of haematological parameters, especially haematocrit, Hb, MCV, MCH, MCHC and red blood cell distribution width (RDW), are lower in patients with IDA, especially when associated with α-thal and therefore it may be useful to discriminate between the different types of microcytic anaemia.

Hemoglobin A2 (HbA2) has a measure of unreliability in diagnosing ß-thalassemia trait (ß-TT).

INTRODUCTION: Detection of ß-thalassemia trait or carriers (ß-TT) depends significantly on an increase in Hemoglobin A2 (HbA2) levels, which is found at low levels (<3%) in normal healthy individuals and elevated levels (≥3.5%) in ß-TT individuals. The study was designed to evaluate the reliability of the diagnostic parameter HbA2 in the differentiation of ß-TT and non-ß-TT in Saudis. METHODS: The widely used high performance liquid chromatography (Variant II Bio-Rad) was used to measure HbA2 levels in blood. Sanger sequencing was used to screen the variation in globin genes (HBB, HBD, HBA1, and HBA2). All the study subjects were divided into ßTT and non-ßTT (wild) categories based on the presence or absence of HBB variations and further sub-divided into false positive, true positive, false negative, and true negative, based on HbA2 values. RESULTS: Out of 288 samples, 96 had HBB gene mutations. Of the 96 ß-TT samples, sickle cell trait (SCT) samples (n = 58) were excluded, while the remaining (38 ß-TT) were included in the detailed analysis: seven subjects with the HBB mutation had normal HbA2 (<3%), and three were borderline (3.1-3.9%). The remainder (n = 28) had an elevated HbA2 level (>4%). Based on HbA2 analysis alone, both these groups would be incorrectly diagnosed as normal. Similarly, of the 189 non-ß-TT samples, 179 had normal HbA2, eight had borderline HbA2, and two had a HbA2 level above 4%. Based on HbA2 analysis alone, borderline and >4% HbA2 individuals, negative for ß-TT, can be incorrectly diagnosed as carriers. CONCLUSION: Given the percentage of samples falling in the HbA2 "borderline" and "normal" categories, it can be concluded that HbA2 has a measure of unreliability in the diagnosis of ß-thalassemia carriers.

The ­α3.7 deletion in α­globin genes increases the concentration of fetal hemoglobin and hemoglobin A2 in a Saudi Arabian population.

The regions of Al­Qatif and Al­Ahssa in the Eastern Province of Saudi Arabia are known for their high prevalence of hemoglobinopathies, including ß­thalassemia and sickle cell anemia. Previously, the α­gene deletion has been demonstrated as highly prevalent among populations residing in these two regions. The present study was conducted in order to investigate the implications of the α­globin gene deletion on fetal hemoglobin (HbF) and hemoglobin α2 (HbA2) concentrations in patients with transfusion­dependent ß­thalassemia. A total of 166 Saudi patients with transfusion­dependent ß­thalassemia and 337 healthy Saudi patients were included in the study. The ­α3.7, ­α4.2, -­FIL, -­SEA, -­MED and -­(20.5) gene deletions were identified using multiplex α­globin deletion polymerase chain reaction. The present study revealed that the ­α3.7 gene deletion is the most prevalent (43.5%) in the Saudi populations that were analyzed and is characterized by the deletion of 3,804 base pairs. Numerous genotypes, namely ­3.7α2/α1α2, ­3.7α2/α1α12, ­3.7α2/­3.7α2, ­3.7α2HphI/α1α2HphI, ­3.7α2/α1­4.2, ­3.7α2/α1polyA­1α2, ­3.7α12/α1α12, ­­FIL/­3.7α2 and ­3.7α2/­3.7α2Hb Villiers le Bel were also identified in the investigated population. Furthermore, a gradual increase in the concentration of HbF and HbA2 in patients with ß­thalassemia and the number of α­gene deletions was demonstrated; whereas in healthy patients the level of HbA2 was demonstrated to decrease as the number of α­gene deletions increased. Therefore, it can be concluded that the high HbF concentration in the present study is predominantly associated with other mutations associated with ß­thalassemia rather than α­globin deletions. Furthermore, the results of the present study also revealed novel α­gene deletion genotypes prevalent in the population studied, namely α1α2/α1α2HphI, α1α2HphI/α1α2HphI, α1α2/α1α2Hb Handsworth, ­3.7α2HphI/α1α2HphI, ­3.7α2/­3.7α2Hb Villiers le Bel and ­-MED/α1α2HphI.

Changes in HbA2 and HbF in alpha thalassemia carriers with KLF1 mutation.

α-thalassemia carriers are common in Mediterranean regions, particularly in the Sardinian population. Their haematological phenotype is characterized by reduced MCV and/or MCH with normal or slightly reduced HbA2 levels and normal HbF. Krüppel-like factor 1 (KLF1) is a pleiotropic erythroid transcription factor that is essential for haematopoiesis. Mutations in the KLF1 gene trigger a series of benign human red blood phenotypes, such as an increase in HbA2 and HBF. Recently, it has been found that KLF1 mutations were a frequent cause of borderline HbA2 levels in a group of Sardinian subjects. Here, we found that KLF1 mutations modulate the phenotype in a cohort of α-thalassemia carriers.

Haematological and electrophoretic characterisation of ß-thalassaemia in Yunnan province of Southwestern China.

OBJECTIVES: ß-Thalassaemia is widely found in Southwestern China. Characterisation of ß-thalassaemia can improve screening and prenatal diagnosis for at-risk populations. DESIGN: A retrospective study. METHODS: In this study, the levels of haemoglobin alpha 2 (HbA2) and haemoglobin alpha (HbA) were analysed by gender for a total of 15 067 subjects screened by capillary electrophoresis. The cut-off value with the highest accuracy was established to identify ß-thalassaemia in 723 patients suspected to have this disease. Haematological and electrophoretic characterisation of eight common types of ß-thalassaemia were analysed in 486 ß-thalassaemia subjects. RESULTS: HbA levels were significantly higher in men than in women, but there was no significant difference on HbA2 levels. A new cut-off value for the diagnosis of ß-thalassaemia (HbA2≥4.0%) with the highest accuracy was proposed for the studied populations. Haemoglobin (Hb) was significantly higher in men compared with women (p<0.05), whereas no statistically significant differences were found for mean cell volume (MCV), mean cell haemoglobin (MCH), HbA and HbA2. The haemoglobin E (HbE) group showed comparatively higher values for haematological indices (Hb, MCV and MCH) than the other genotypes in heterozygous ß-thalassaemia groups (p<0.05), and -28 (A>G) (HBB (ß-globin):c.-78A>C) had significantly higher HbA2 values compared with other ß-thalassaemia. CONCLUSIONS: Ethnic groups have diversified ß-globin gene mutations and considerable haematological variations. Our study will lay the foundation for screening programmes and clinical management of thalassaemia in Southwestern China.

Haemoglobin F, A2, and S levels in subjects with or without sickle cell trait in south-eastern Gabon.

BACKGROUND: Infant mortality due to sickle cell disease in sub-Saharan Africa is high, necessitating a better understanding of the modulating factors of the disease in this region. METHODS: We assessed the hereditary persistence of foetal haemoglobin and α-thalassemia. We diagnosed 787 subjects, with or without sickle cell trait, by capillary electrophoresis in the Medical Diagnostic Laboratory of the CIRMF (Franceville, Gabon). RESULTS: Heterocellular and pancellular forms of hereditary persistence of foetal haemoglobin occurred at low rates of 10.9 and 2.3%, respectively. The distribution of HbS levels in individuals with sickle cell trait was trimodal, showing a high percentage (52.4%) of heterozygous subjects with α-thalassemia. The distribution of HbA2 levels was bimodal in individuals without sickle cell trait, estimated to be comprised of 12 and 15% of α and ß-thalassemic heterozygous subjects, respectively. CONCLUSIONS: In sub-Saharan Africa, α-thalassemia is a far more prevalent modulating factor than hereditary persistence of foetal haemoglobin. Our study highlights the need for further investigation of thalassemia, haemoglobinopathies that are neglected in sub-Saharan Africa.

The long-term and extensive efficacy of low dose thalidomide in a case of an untransfusable patient with Non-Transfusion-Dependent Thalassemia.

Patients with Non-Transfusion-Dependent Thalassemia may require regular transfusion therapy. However, these patients are at risk of developing irregular antibodies, making them untransfusable. Second line treatment usually includes hydroxyurea, which however is not effective in all patients. Other treatment options include thalidomide, which has been reported to be safe and effective in selected patients. We report the case of a patient who experienced improvement of hemoglobin levels and of a part of NTDT related complications, following 36months of continuous therapy with low doses of thalidomide.

"Silent" ß-thalassemia mutation (promoter nt-101 C > T) with increased hemoglobin A2.

One of the most common silent ß-thalassemia mutations is the C > T substitution at position -101 within the distal CACCC box, which leads to a mild reduction in the expression level of the ß-globin gene. Carriers of this mutation have a normal hematologic picture without microcytosis and borderline hemoglobin A2 values, and may be missed during screening. Co-occurrence of this mutation with one of the classical ß-thalassemia mutations leads to ß-thalassemia intermedia, and this is important for Mediterranean populations where ß-thalassemia is frequent. Awareness of this mutation, which may have a heterogeneous clinical presentation, is required. We herein present the unusual hematologic findings of a Turkish family carrying this mutation.

Challenges in prenatal diagnosis of beta thalassaemia: couples with normal HbA2 in one partner.

OBJECTIVES: To undertake ß-genotyping in couples having normal/borderline HbA2 levels in one partner to offer the possibility of prenatal diagnosis of thalassaemia. METHODS: A total of 967 couples were screened for ß-thalassaemia. Haematological analysis was carried out on a Sysmex K-1000 analyser. The HbA2 and HbF levels were measured by High Performance Liquid Chromatography-Variant II analyser (Bio-Rad, USA). ß-globin gene analysis was done by reverse dot blot hybridization, amplification refractory mutation system or DNA sequencing. Alpha globin gene triplication was determined by Multiplex PCR. RESULTS: In 33 of 967 couples, one partner had a normal/borderline HbA2 level (1-3.5%); however a ß-thalassaemia mutation could be identified in 24 of these individuals. Molecular analysis of the ß-globin gene revealed the presence of the capsite +1 (A → C) [HBB: c.-50 A → C] mutation in 15 cases (60%), Poly A(T → C) [HBB: c.*110 T → C] mutation in two cases (8%), IVS 1-5 (G → C) [HBB: c. 92 + 5 (G → C)] mutation in four cases (17%) and the CD 15 (G → A) [HBB: c. 47 G → A] mutation, CD 16 (-C) [HBB: c. 51 del C] mutation and CD 30 (G → C) [HBB: c. 93 G → C] mutation in one case each (4%). Alpha gene triplication was found in five cases, while four cases remained uncharacterized. CONCLUSIONS: ß-genotyping should always be done in a couple if one partner is a ß-thalassaemia carrier irrespective of the RBC indices and HbA2 levels of the other partner.

Experimental Characterization of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) and Hb Boghé (HBA2: c.177 C > A, p.His > Gln) Reveals Contradictory HBA2 Expression and Translation Patterns Despite Identical Amino Acid Substitutions.

In this study, we describe the clinical features and provide experimental analyses of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) that contrasted with Hb Boghé (HBA2: c.177 C > A, p.His > Gln). Despite the identical amino acid substitution in both variants, Hb Flurlingen shows the phenotype of α-thalassemia (α-thal), whereas Hb Boghé has no impact on α2-globin (HBA2) production. For in vitro transcription analysis, HBA2 expression constructs carrying the HBA2-WT (wild type), Hb Flurlingen and Hb Boghé sequences were generated and expressed in human bladder carcinoma 5637 cells for downstream analyses by quantitative real time-polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC). In silico analysis of secondary folding structures of the HBA2-WT, Hb Flurlingen and Hb Boghé mRNA sequences was performed using Mfold software. The gene transcription and translation analyses revealed that cells transfected with the Hb Flurlingen construct had significantly lower HBA2 transcription (-55.4%, p ≤ 0.01) and reduced protein synthesis when compared to the wild type group. In contrast, cells transfected with the Hb Boghé construct showed no significant changes in HBA2 transcription or translation activities when compared to the wild type group. The in silico prediction of possible effects of these mutations on the folding structures of the HBA2 transcripts showed a change of secondary folding pattern in the Hb Flurlingen transcript when compared to those of HBA2-WT and Hb Boghé. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Boghé, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions.

Elevated Hb A2 Levels in a Patient with a Compound Heterozygosity for the (ß⁺) -31 (A > G) and (ß°) Codon 17 (A > T) Mutations Together with a Single α-Globin Gene.

We report the molecular and hematological feature of a Thai woman who had clinical diagnosis of ß-thalassemia intermedia (ß-TI). Hemoglobin (Hb) high performance liquid chromatography (HPLC) analysis identified Hb A (64.4%), Hb F (12.3%) and Hb A2/E (15.9%) with small peaks of Hb Bart's (γ4) and Hb H (ß4). She was initially diagnosed as EA Bart's disease, which occurs from combination of Hb H disease and Hb E (HBB: c.79G > A) trait. However, the Hb analysis using capillary electrophoresis (CE) demonstrated no Hb E, 68.5% Hb A, 15.5% Hb F and 16.0% Hb A2. DNA analysis showed a compound heterozygosity for (ß(+)) -31 (A > G) (HBB: c.-81A > G) and (ß(0)) codon 17 (A > T) (HBB: c.52A > T) mutations and deletional Hb H (- -(SEA)/-α(3.7)). Thus, she was finally diagnosed with a combination of Hb H disease and compound heterozygosity of ß(+)/ß(0)-thalassemia (ß(+)/ß(0)-thal). The ß-globin mutations could affect not only hematological parameters but also elevate the Hb A2 levels. These effects could not be ameliorated by the coinheritance of Hb H disease. Therefore, a better understanding of the effects of this combination on hematological analysis data will be useful for providing accurate diagnosis, genetic counseling, prevention and control programs of ß-thalassemia major (ß-TM).