Δ-globin Chain Research Articles

Wide spectrum of novel and rare hemoglobin variants in the multi-ethnic Indian population: A review.

The hemoglobin (Hb) variants are qualitative abnormalities due to production of structurally abnormal globin proteins. They are categorized based on the type of mutation present in the α1, α2, β, Gγ, Aγ and δ globin genes. So far, more than 1550 Hb variants are reported in the database. They could lead to Hb polymerization, Hb instability, altered oxygen affinity and decreased oxygen-carrying capacity of Hb or have no clinical manifestations. In India, ethnic diversity, consanguinity, regional variations and migration result in the presence of different Hb variants. We have compiled all the variants of α, β and δ globin chains in heterozygous, homozygous and in compound heterozygous forms reported from India in the last 52 years. Of the 63 rare and novel hemoglobin variants reported from India, 22 were α-globin chain variants, 37 were β-globin chain variants and 4 were δ-globin chain variants. Twelve novel Hb variants (Hb J Rajappan, Hb Koya Dora, Hb Rampa, Hb Godavari, Hb Chandigarh, Hb D Agri, Hb Lucknow, Hb Vellore, Hb Midnapore, Hb Bijnor, Hb A2Tianhe and Hb A2Saurashtra) were identified among persons of Indian origin. Majority of them were picked up on HPLC. Some of the variants like Hb Titusville, Hb Shimonoseki, Hb Chandigarh, Hb D Agri, Hb Yaizu and Hb Vellore eluted in the HbS window whereas variants like HbD Iran, Hb St. Louis, Hb G Coushata, HbM Saskatoon, Hb Lucknow, Hb Grange-Blanche and Hb Tianshui showed falsely elevated HbA2. Hence, careful and systematic investigations are required to identify them.

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.

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 β-thalassemias, however, various disorders including alteration of δ chains may result in decreased production of Hb A2, thus hindering the diagnosis of β-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).

Hematological and Molecular Characterization of a Novel Hb A2 Variant with Homozygous α-Thalassemia-2 in a Southern Thai Individual

We report here the hematological and molecular features of a novel δ-globin chain variant found in a Southern Thai woman. Her complete blood count was as follows: red blood cell (RBC) count 5.90 × 1012/L, hemoglobin concentration (Hb) 12.6 g/dL, packed cell volume (PCV) 0.41 L/L, mean corpuscular volume (MCV) 69.5 fL, mean corpuscular Hb (MCH) 21.4 pg, mean corpuscular Hb concentration (MCHC) 30.7 g/dL and RBC distribution width (RDW) 13.1%. The blood smear demonstrated microcytic hypochromic RBCs suggestive of thalassemia trait. Hemoglobin analysis identified Hb A2 + Hb A2-Kiriwong (2.4%) and Hb F (0.1%) on high performance liquid chromatography (HPLC). To characterize the α-thalassemia (α-thal) genotype, common α-thal-1 and α-thal-2 alleles were characterized by multiplex gap-polymerase chain reaction (gap-PCR). The results revealed homozygous α-thal-2 (–α3.7/–α3.7) in this case. DNA sequencing showed the presence of a novel δ-globin gene mutation [δ77(EF1)His→Arg; HBD: c.233A>G] that we named Hb A2-Kiriwong for the village from where the proband lived. In summary, the presence of microcytic hypochromic RBCs in this case was likely the result of the homozygous –α3.7 (rightward) deletion and was not affected by this Hb A2 variant.

Hb A2 Episkopi – a novel δ-globin chain variant [HBD:c.428C>T] in a family of mixed Cypriot–Lebanese descent

ABSTRACTObjectives: Thalassaemia is a potentially lethal inherited anaemia, caused by reduced or absent synthesis of globin chains. Measurement of the minor adult haemoglobin Hb A2, combining α- with δ-globin, is critical for the routine diagnosis of carrier status for α- or β-thalassaemia. Here, we aim to characterize a novel δ-globin variant, Hb A2 Episkopi, in a single family of mixed Lebanese and Cypriot ancestry with mild hypochromic anaemia and otherwise normal globin genotype, which also presents with a coincidental 0.78-Mb sequence duplication on chromosome 1 (1q44) and developmental abnormalities.Methods: Analyses included comprehensive haematological analyses, cation-exchange high-performance liquid chromatography (CE-HPLC), cellulose acetate electrophoresis (CAE), Sanger sequencing and structure-based stability predictions for Hb A2 Episkopi.Results: The GCT > GTT missense mutation, underlying Hb A2 Episkopi, HBD:c.428C > T, introduces a cd142 codon change in the mature protein, resulting in reduced normal Hb A2 amounts and a novel, less abundant Hb A2 variant (HGVS: HBD:p.A143V), detectable as a delayed peak by CE-HPLC. The latter was in line with structure-based stability predictions, which indicated that the substitution of a marginal, non-helical and non-interface residue, five amino acids from the δ-globin chain carboxy-terminus, was moderately destabilizing.Discussion: Detection of the new variant depends on the diagnostic set-up and had failed by CAE and on an independent CE-HPLC system, which, in unfavourable circumstances, may lead to misdiagnoses of β-thalassaemia as α-thalassaemia. Given the mixed background of the affected family, the ethnic origin of the mutation is unclear, and this study thus suggests awareness for possible detection of Hb A2 Episkopi in both the Cypriot and the Lebanese populations.

Hemoglobin Variants in Northern Thailand: Prevalence, Heterogeneity and Molecular Characteristics.

There are limited data on hemoglobin (Hb) variants among peoples of northern Thailand. Hence, we determined the prevalence of Hb variants among a large cohort from this region. A study was done on 23,914 subjects recruited from eight provinces during June 2012-January 2014. Hb was analyzed by high performance liquid chromatography (HPLC) and capillary electrophoresis, and corresponding mutations were identified by polymerase chain reaction. Among 23,914 subjects examined, 211 (0.88%) were found to carry 14 different Hb variants. Five α-globin chain variants were identified: Hb Q-Thailand (n = 40; 19.0%), Hb Hekinan (n = 8, 3.8%), Hb Siam (n = 2, 0.9%), Hb Beijing (n = 1, 0.5%), and Hb Kawachi (n = 1, 0.5%), not previously described in the Thai population. Seven β-globin variants, including Hb Hope, Hb Tak, Hb S, Hb J-Bangkok, Hb G-Makassar, Hb C, and Hb Korle-Bu, were found in 115 (54.5%), 30 (14.2%), 3 (1.4%), 3 (1.4%), 1 (0.5%), 1 (0.5%), and 1 (0.5%) subjects, respectively. The remaining five subjects (2.4%) were carriers of two different δ-globin chain variants. A different spectrum and frequencies of Hb variants were noted compared to other geographical areas. Haplotype analysis demonstrated multiple origins for Hbs Hope and Tak and confirmed a non-African origin of Hb C. Several genetic interactions between these variants with other hemoglobinopathies were encountered. Associated hematological phenotypes and novel Hb derivatives formed were presented. The prevalence and molecular heterogeneities of the Hb variants found in this large cohort of the northern Thai people's should prove useful in developing a screening program, and for the performance of additional population genetics studies of hemoglobinopathy in the region.

HbA2 : biology, clinical relevance and a possible target for ameliorating sickle cell disease.

HbA2 , a tetramer of α- and δ-globin chains, provides a diagnostic clue to the presence of β-thalassaemia trait. This minor haemoglobin, which forms about 2-3% of the total, has no known physiological role, but has the interesting property of preventing polymerization of deoxy-sickle haemoglobin. If it were possible to increase the level of HbA2 sufficiently it could have a benefit in sickle cell disease similar to that of foetal haemoglobin. Moreover, HbA2 is present in all erythrocytes, an advantage not found with foetal haemoglobin, which is heterocellularly expressed. The molecular basis of HbA2 gene (HBD) expression is partially understood, and with new molecular tools, it might be possible to induce levels of HbA2 that could be clinically important. However, high concentrations of this positively charged haemoglobin might damage the erythrocyte membrane; also, the reciprocal relationship of δ- and γ-globin gene (HBD and HBG1/2, respectively) expression might negate any benefit of increasing transcription of the former.

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

An Intriguing High Performance Liquid Chromatogram of a Double Heterozygosity for Hb Q-India/Hb D-Punjab

Cation exchange high performance liquid chromatography (HPLC) is commonly utilized as the first method of screening for thalassemias and hemoglobinopathies worldwide. This method of diagnosis requires knowledge of the clinical background and complete blood counts as well as skill and experience in interpreting the sometimes complex results produced. An asymptomatic 27-year-old pregnant North Indian woman was found to have a highly unusual chromatographic pattern with multiple unexpected peaks during routine antenatal screening. Most concerning was a C-window peak as Hb C (HBB: c.19G>A) is rare in ethnic Asian Indian populations. Cellulose acetate electrophoresis at alkaline pH (8.6) and parental screening were performed. These revealed the correct diagnosis to be a double heterozygosity for Hb Q-India (HBA1: c.193G>C) (an uncommon asymptomatic α-globin chain variant) plus Hb D-Punjab (HBB: c.364G>C) (a β-globin chain variant that is common in this region and is asymptomatic in the heterozygous state). The unexpected C-window peak was the hybrid of the abnormal α-Q-India and β-D-Punjab globin chains. Another small peak was explained as a variant Hb A2 formed by the combination of α-Q-India and δ-globin chains. Hematopathologists should be cognizant of the complex pattern resulting from coinheritance of both α- and β-globin structural variants. Second-line testing and parental testing are invaluable in resolving unknown peaks, especially if rare or unexpected variants are being considered. Although both Hb Q-India and Hb D-Punjab are relatively common in northwestern India, to the best of our knowledge, only two recent reports describe a total of three cases of such diagnostically puzzling coinheritance.

Known and new hemoglobin A2 variants in Thailand and implication for β-thalassemia screening

BackgroundWe reported molecular and hematological characteristics of δ-globin chain variants and addressed diagnostic consideration of complex hemoglobinopathies caused by their interactions with α- and β-thalassemias. MethodsStudy was done on four unrelated Thai subjects with second Hb A2 fractions. Hb analysis was carried out using automated HPLC and capillary electrophoresis. Mutations were identified by DNA analysis. Novel diagnostic methods based on PCR-RFLP and allele specific PCR were developed. ResultsHb analysis revealed Hb A2 variant in all cases. DNA analysis of δ-globin gene identified the Hb A2-Melbourne [δ43(CD2)Glu→Lys] in combination with α+-thalassemia, α0-thalassemia and β0-thalassemia in the first three cases, respectively. Analysis of the remaining case identified a novel δ-Hb variant namely the Hb A2-Lampang [δ47(CD6)GAT→AAT; Asp→Asn] found in association with Hb E and α+-thalassemia. These mutations could be identified using PCR-RFLP and allele specific PCR assays developed. ConclusionsIt is necessary to recognize the Hb A2 variant and to combine the amounts of Hb A2 and Hb A2-variant for a total Hb A2 value to make better diagnostic of these complex syndromes. Co-inheritance of these multiple globin gene defects could lead to complex hemoglobinopathies requiring comprehensive Hb and molecular assessments.

Hb Famagusta—analysis of a novel δ-globin chain variant [HBD:c.60C&gt;A] in four families with diverse globin genotypes

Table S1 HbA2 stability predictions for different δ-globin-variants. All δ-globin variants so far identified in Cyprus [1] carry amino-acid changes compared to the normal protein either in α-helices (T4I, G25D, A27S, R116C and L141P) or in a loop sequence at the interface of the α1δ1-α2δ2 heterodimers (V98M). Structure-based meta-analysis by iStable [2] was used to predict the stability of the novel δglobin N19K variant monomer for comparison with other Cypriot variants. According to the calculated ΔΔG value, the novel δ-globin variant is in Cyprus second in stability only to the δ-globin[T4I] mutation, which is positioned at the very start of the first δ-globin αhelix towards the flexible N terminus.

Evolutionary constraints in the β-globin cluster: the signature of purifying selection at the δ-globin (HBD) locus and its role in developmental gene regulation.

Human hemoglobins, the oxygen carriers in the blood, are composed by two α-like and two β-like globin monomers. The β-globin gene cluster located at 11p15.5 comprises one pseudogene and five genes whose expression undergoes two critical switches: the embryonic-to-fetal and fetal-to-adult transition. HBD encodes the δ-globin chain of the minor adult hemoglobin (HbA2), which is assumed to be physiologically irrelevant. Paradoxically, reduced diversity levels have been reported for this gene. In this study, we sought a detailed portrait of the genetic variation within the β-globin cluster in a large human population panel from different geographic backgrounds. We resequenced the coding and noncoding regions of the two adult β-globin genes (HBD and HBB) in European and African populations, and analyzed the data from the β-globin cluster (HBE, HBG2, HBG1, HBBP1, HBD, and HBB) in 1,092 individuals representing 14 populations sequenced as part of the 1000 Genomes Project. Additionally, we assessed the diversity levels in nonhuman primates using chimpanzee sequence data provided by the PanMap Project. Comprehensive analyses, based on classic neutrality tests, empirical and haplotype-based studies, revealed that HBD and its neighbor pseudogene HBBP1 have mainly evolved under purifying selection, suggesting that their roles are essential and nonredundant. Moreover, in the light of recent studies on the chromatin conformation of the β-globin cluster, we present evidence sustaining that the strong functional constraints underlying the decreased contemporary diversity at these two regions were not driven by protein function but instead are likely due to a regulatory role in ontogenic switches of gene expression.

Origin and mechanism of thermal insensitivity in mole hemoglobins: a test of the ‘additional’ chloride binding site hypothesis

The structural and evolutionary origins underlying the effect of temperature on the O(2) binding properties of mammalian hemoglobins (Hbs) are poorly understood, despite their potential physiological importance. Previous work has shown that the O(2) affinities of the blood of the coast mole (Scapanus orarius) and the eastern mole (Scalopus aquaticus) are significantly less sensitive to temperature changes than that of the star-nosed mole (Condylura cristata). It was suggested that this difference may arise from the binding of 'additional' chloride ions within a cationic pocket between residues 8His, 76Lys and 77Asn on the β-like δ-globin chains of coast and eastern mole Hbs. To test this hypothesis, we deduced the primary sequences of star-nosed mole and American shrew mole (Neurotrichus gibbsii) Hb, measured the sensitivity of these respiratory proteins to allosteric effector molecules and temperature, and calculated their overall oxygenation enthalpies (ΔH'). Here we show that the variability in ΔH' seen among mole Hbs cannot be attributed to differential Cl(-) binding at δ8, δ76 and δ77, as the Cl(-) sensitivity of mole Hbs is unaffected by amino acid changes at this site (i.e. the proposed 'additional' Cl- binding site is not operational in mole Hbs). Rather, we demonstrate that the numerically low ΔH' of coast and eastern mole Hbs results from heightened proton binding relative to other mole Hbs. Comparative sequence analysis and molecular modelling moreover suggest that this attribute evolved in a common ancestor of these two fossorial lineages and arises from the development of a salt bridge between a pair of amino acid residues (δ125His and α34Glu/Asp) that are not present in other mole Hbs.

Thalassemia-like Phenotype in a Novel Complex Hemoglobinopathy With α, β, δ Globin Chain Abnormalities

The occurrence of multiple abnormalities of α, β, δ, and γ globin genes may lead to unusual and complex phenotypes when they arise simultaneously in the same individual. Here, we report the findings of an African American boy who coinherited 3 heterozygous globin gene abnormalities: the unstable β-globin chain variant; hemoglobin (Hb) Showa-Yakushiji [β110(G12) Leu→Pro], the δ-globin chain variant; HbB2 [δ16(A13) Gly→Arg] and α-thalassemia (α-thal); (α-/αα). Hb Showa-Yakushiji had been previously described in Japanese, Indian, and European populations. We report its first occurrence in a child of African ancestry who presented with anemia not responsive to iron and an incomplete β-thalassemia minor phenotype. Although the clinical and laboratory features of Hb Showa-Yakushiji mimic those of a β-thalassemia, the coinheritance of the δ-globin chain variant Hb B2 suppressed the relative increase in Hb A2 usually observed in heterozygotes for the Hb Showa-Yakushiji mutation. Protein-based methods detected only a trace amount of HbB2 and failed to reveal presence of Hb Showa-Yakushiji and α-thal. The latter were only identified through DNA analyses. The diagnostic difficulties, molecular characteristics, and genotype/phenotype correlations of this novel complex hemoglobinopathy syndrome are reviewed.

Co-Inheritance of Delta Thalassemia Might Contribute to the High Fetal Hemoglobin in Sickle Cell Anemia Patients with the Saudi-Indian Haplotype

Abstract 1056Most sickle cell anemia patients (HBB glu6val homozygotes) indigenous to the Eastern Province of Saudi Arabia have a fetal hemoglobin (HbF) level of about 20% that is associated with a mild clinical phenotype. Their HbS gene is on the Saudi-Indian (SI) haplotype characterized by an Xmn1 restriction site at position −158 5’ to HBG2 (rs7482144), a Hinc2 site 5’ to HBE (rs3834466) and other polymorphisms in the HBB gene-like cluster. However, the functional elements within the HBB gene-like cluster and elsewhere in the genome causing high HbF are yet to be determined. In a previous study we found that Saudi HbS homozygotes with the SI haplotype had a common region of autozygosity that spanned about 126 kb and included the complete HBB cluster. We have sequenced 13.6 kb in the HBD-HBG1 intergenic region (chr11:5255683-5269326, HG19), the region of the Corfu deletion. We found a SNP at position −68 bp 5’ to HBD (c.-118 C>T) only in individuals with a SI haplotype. This SNP was not present in dbSNP build 132 or the 1000 Genomes databases. No other mutation in HBD was identified. This same SNP was recently associated with δ thalassemia (Phylipsen et al. Int. J. Lab. Hematol. 2011, 33: 85–91). Homozygotes for the −68 HBD SNP, who were not on hydroxyurea, had a mean HbF of 23%, range 12.1%-33.4% and mean HbA2 of 2.1%, range 1.2%-3%. We did not find the −68 HBD SNP in 15 African Americans with sickle cell anemia selected because of their unusually high HbF (mean HbF 17.2%, range 11%-28.9%). Parents and sickle cell trait carriers from the families of Saudi Eastern Province patients were heterozygous for the −68 HBD mutation (mean HbF 1.6%, range 0–4.2% and mean HbA2 2.7%, range 2.4%-3.2%) and one normal sibling did not carry this mutation (HbF 0 and HbA2 2.9%). Thirty patients with sickle cell disease indigenous to the Southwestern Province of Saudi Arabia, with the HbS gene on an African origin haplotype, (mean HbF 15.5%, range 4.5%-23% and mean HbA2 2.9%, range 2.1%-3.4% in HbS homozygotes) and 13 normal Saudi controls were examined and none carried the −68 HBD mutation. We also sequenced HBD and its promoter in 8 Southwestern Province HbS homozygotes with a Benin haplotype, 4 with HbF <5% and 4 with HbF >15%, and none had the −68 HBD SNP or any other mutation in HBD. Inverse relationships between percent HbF level and percent HbA2 was seen in 3 groups of HbS homozygotes:1) SI haplotype homozygotes; 2) Benin haplotype homozygotes from the Southwestern Province; 3) African Americans with diverse HbS haplotypes. The increased HbF in sickle cell anemia with δ thalassemia might involve both post-translational and transcriptional mechanisms. Increased HbF levels might in part be due to the preferential post-translational assembly of αγ dimers compared with αδ and αβS dimers. When δ thalassemia reduces available δ-globin chains, this might further favor preferential binding of less positively charged γ-globin subunits to positively charged α-globin compared with more positively charged δ-globin subunits. Perhaps more importantly, at the transcriptional level, the −68 HBD δ+-thalassemia promoter mutation might favor the interactions of transcription complexes with HBG promoters. The stimulus of hemolytic anemia and expanded erythropoiesis might be required before the favorable genetic milieu for increased HbF production can be fully utilized with the achievement of clinically significant increases in HbF that modulates the course of disease. For example, increased HbF levels of only 3.3%-4.7% have been reported in some hematologically normal individuals with homozygous δ0 thalassemia (Ohta et al. Hemoglobin 1980, 4: 417–425). High HbF levels in SI haplotype HbS homozygotes might involve the interactions of one or more HBG regulatory regions linked to the HBB gene-like cluster, like the −68 HBD SNP, perhaps with trans acting elements. Although the −68 HBD δ+-thalassemia mutation is associated with the SI haplotype and high HbF, functional studies are needed to establish causation. Disclosures:No relevant conflicts of interest to declare.

Hb A2′ (Hb B2) in the Omani Population and Diagnostic Significance

Hb A2′ [δ16(A13)Gly→Arg], also called Hb B2, is a δ-globin chain variant that has been identified in several populations of African origin or ancestry and is easily identifiable in alkaline acetate cellulose electrophoresis as doubling of the Hb A2 band. However, in high performance liquid chromatography (HPLC), commonly employed nowadays, it elutes in the S window. Over a period of 2 years at the Sultan Qaboos University Hospital, Muscat, Oman, we identified 25 Omanis with this variant. The quantity of Hb A2 ranged from 0.9 to 1.8% in heterozygotes and was undetectable in the single homozygous case. As both α- and β-thalassemia (α- and β-thal) as well as Hb S [β6(A3)Glu→Val] are common in the Omani population, it is important to be aware of the presence of Hb A2′ in this population to avoid misinterpretation of the HPLC data in terms of underdiagnosis of β-thal carriers and overestimation of α-thal based on Hb A2 levels in sickle cell carriers. The haplotype associated with Hb A2′ in Oman is identical to that described in African populations, suggesting a common origin for this mutation and its introduction into Oman by gene flow.

Sandwich ELISA for hemoglobin A2 quantification and identification of β-thalassemia carriers

Hemoglobin (Hb) A2 (α2δ2) is a minor hemoglobin in human red blood cells. An abnormal increase in the level of HbA2 is the most significant parameter in the diagnosis of β-thalassemia carriers. In this study, we produced two monoclonal antibodies (mAbs) that specifically react to the δ-globin chain of HbA2. A sandwich type ELISA was developed employing the produced anti-HbA2 mAbs. HbA2 levels quantified by the developed sandwich ELISA were highly correlated with those obtained from the standard HPLC method (r = 0.934, p < 0.001). HbA2 levels determined by the ELISA were 4.4 ± 1.9% in β-thalassemia heterozygotes compared to 1.4 ± 0.8, 1.9 ± 0.8, 1.5 ± 0.8 and 1.5 ± 0.6% in normal subjects, HbE heterozygotes, suspected α-thalassemia heterozygotes and HbE homozygotes, respectively. Using a cut-off value of 2.5%, β-thalassemia heterozygotes could be separated from non-β-thalassemia heterozygotes with the same accuracy as obtained using the standard HPLC method. More importantly, the developed ELISA was able to determine HbA2 levels in HbE-bearing individuals which could not be done by the HPLC method. Our results suggest that this sandwich ELISA can be applied for mass screening for β-thalassemia heterozygotes, especially in resource-limited countries, where β-thalassemia is highly prevalent.

Molecular basis of a novel adaptation to hypoxic-hypercapnia in a strictly fossorial mole

BackgroundElevated blood O2 affinity enhances survival at low O2 pressures, and is perhaps the best known and most broadly accepted evolutionary adjustment of terrestrial vertebrates to environmental hypoxia. This phenotype arises by increasing the intrinsic O2 affinity of the hemoglobin (Hb) molecule, by decreasing the intracellular concentration of allosteric effectors (e.g., 2,3-diphosphoglycerate; DPG), or by suppressing the sensitivity of Hb to these physiological cofactors.ResultsHere we report that strictly fossorial eastern moles (Scalopus aquaticus) have evolved a low O2 affinity, DPG-insensitive Hb - contrary to expectations for a mammalian species that is adapted to the chronic hypoxia and hypercapnia of subterranean burrow systems. Molecular modelling indicates that this functional shift is principally attributable to a single charge altering amino acid substitution in the β-type δ-globin chain (δ136Gly→Glu) of this species that perturbs electrostatic interactions between the dimer subunits via formation of an intra-chain salt-bridge with δ82Lys. However, this replacement also abolishes key binding sites for the red blood cell effectors Cl-, lactate and DPG (the latter of which is virtually absent from the red cells of this species) at δ82Lys, thereby markedly reducing competition for carbamate formation (CO2 binding) at the δ-chain N-termini.ConclusionsWe propose this Hb phenotype illustrates a novel mechanism for adaptively elevating the CO2 carrying capacity of eastern mole blood during burst tunnelling activities associated with subterranean habitation.

Generation and characterization of human δ-globin-specific monoclonal antibodies

The human δ-globin chain, unique to the hemoglobin A2 (HbA2) heterotetramer, is important for the evaluation of hemoglobinopathy. However, there are no well-defined antibodies specific for the δ-globin chain, a fact that is attributed a striking similarity (93%) in amino acid sequence between δ-globin and β-globin of the hemoglobin A (HbA). In this study, two monoclonal antibodies (mAbs) against the δ-globin chain were generated and designated as 2H4 and 1H11. These antibodies were specific to HbA2 and do not cross-react with HbA and HbF (fetal hemoglobin). Moreover, the expression of HbA2 in fetal liver and mature erythrocytes was determined using these two mAbs. In addition to being useful tools for research or diagnosis, these antibodies could be valuable for development of rapid and effective antibody-based immunoassays of HbA2 expression in erythroid cells and non-erythroid tissue.

Premature Neonate With Apnea and Trace Amounts of Hemoglobin S and A Detected by High-Performance Liquid Chromatography

Premature Neonate With Apnea and Trace Amounts of Hemoglobin S and A Detected by High-Performance Liquid Chromatography