Search

Background: Delta-chain (δ-chain) variants are a group of rare hemoglobin (Hb) variants resulting from mutations within the δ-globin gene. Although quantification of Hb A2 levels is a useful screening tool for the beta-thalassemia trait, the coinheritance of a δ-globin gene mutation can lead to misinterpretation of diagnostic results. Objective: To identify an unreported Hb A2 variant in Thailand and to develop a high resolution melting (HRM) curve assay for the four δ-globin chain variants found in the Thai population. Materials and Methods: Allele-specific polymerase chain reaction (ASPCR) was used to analyze a total of 18 DNA samples for Hb variants comprising 10 wild-type controls, 4 Hb A2-Melbourne, 1 Hb A2-Lampang, 2 Hb A2-Kiriwong, and an unknown variant via HRM assays. Results: The unreported Hb A2 variant in Thailand was found to be Hb A2-Walsgrave resulting from δ-globin gene mutation at codon 52 (GAT>CAT). This was also confirmed using ASPCR. In addition, we demonstrated that the HRM curve profile for Hb A2-Melbourne, Hb A2-Lampang, Hb A2-Walsgrave, and Hb A2-Kiriwong could be identified so as to distinguish the mutant alleles from one another and from wild-type alleles. Conclusion: This HRM assay detected both known and unknown mutations with simultaneous differentiation between heterozygous and homozygous alleles on a polymerase chain reaction fragment spanning four of the δ-globin variants found in Thailand. This assay may help to support the prevention and control of thalassemias and hemoglobinopathies in Thailand.

We report a case of an extremely rare hemoglobin (Hb) variant-Hb Broomhill, which has been only reported once in the literature. Hemoglobin fractions were determined by capillary electrophoresis (Sebia Capillarys 2 Flex piercing) and high performance liquid chromatography (HPLC) (Bio-Rad Variant™ II Hemoglobin Testing System), respectively. Complete blood count and DNA sequencing were also performed. The capillary electrophoregram revealed a tiny shoulder peak before the HbA peak and a subtle abnormal HbA 2 peak (slightly wider and lower), even though the percentage of each hemoglobin fraction was within the reference range (HbA, 97.4%; HbA 2 , 2.6%). On HPLC, not only the percentage but also the peak shape of each hemoglobin fraction was normal (HbA 88.2%, HbA 2 2.5%, HbF 0.6%). Eventually, sequencing analysis of α genes confirmed a missense mutation (CCC>GCC at codon 114 in alpha1 gene) which caused Hb Broomhill variant. Our report suggest that capillary electrophoresis may be an accurate tool for screening and diagnosis of Hb disorders., (© 2017 by the Association of Clinical Scientists, Inc.)

Haemoglobinopathies may interfere with the haemoglobin A 1c (HbA 1c ) measurement, leading to incorrect diagnosis and inappropriate treatment. It is essential that HbA 1c assays are capable of identifying haemoglobinopathies. We report two cases of haemoglobin New York (HbNY) discovered through HbA 1c analysis using capillary electrophoresis (Capillarys 2 Flex Piercing [C2FP], Sebia). We used these samples to evaluate the ability of three other HbA 1c assays to identify this variant: ion-exchange high-performance liquid chromatography (Variant II Turbo [VII-T], Bio-Rad); boronate affinity high-performance liquid chromatography (Ultra 2 , Trinity Biotech) and immunoassay (Cobas c501 Tina-quant Generation 3, Roche Diagnostics). Each method was used for HbA 1c assay of in samples from two cases of heterozygous haemoglobinopathy: β 0 -thalassemia/HbNY (Case 1) and HbA/NY (Case 2). Only the C2FP system detected HbNY (an additional peak appeared between HbA 1c and HbA 0 ). Clinical laboratories should be aware of the limitations of their HbA 1c assay methods especially in geographic areas, where haemoglobinopathy prevalence is high.

A 65-year-old Japanese man presented with acute myocardial infarction (AMI) and polycythemia. Biochemical studies of the patient's hemoglobin (Hb) and the sequencing of his globin genes revealed that the polycythemia was secondary to a high oxygen affinity Hb variant, Hb Fuchu-II. Hb variants with high oxygen affinity can be an additional thrombotic risk factor in older patients and/or those with other risk factors. The patient was diagnosed with hemoglobinopathy after the development of AMI and exemplifies the importance of recognizing such conditions and of taking appropriate prophylactic interventions.

Thalassemia is an inherited autosomal recessive blood disorder characterized by the underproduction of globin chains as a consequence of globin gene defects, resulting in malfunctioning red blood cells and oxygen transport. Analysis of globin chains is an important aspect of thalassemia research. In this study we developed a capillary zone electrophoresis (CZE) method for human globin determination in the diagnosis of thalassemia and hemoglobin variants. To demonstrate the utility of this approach, α/β area ratios were determined for samples from 310 thalassemia patients and healthy controls. The separation was performed on uncoated capillary with simple preparation. Distinct globin peaks were resolved in 17 min, and coefficients of variation (CV) for migration time and areas ranged from 0.37%-1.69% and 0.46%-6.71%, respectively. Receiver operating characteristic (ROC) curve analysis of the α/β area ratios gave 100% sensitivity and specificity for indicating β-TI/TM, and 100% sensitivity and 97.4% specificity for Hb H disease. Hemoglobin G-Honolulu (Hb G-Honolulu) and Hb Westmead (Hb WS) were successfully detected using this CZE method. This automated methodology is simple, rapid and cost-effective for the fast determination of human globin chains, which could be an important diagnostic tool in the field of hemoglobinopathies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A 52-year-old man presented for an evaluation of worsening glycemic control secondary to glucocorticoid administration. The glycated hemoglobin (HbA1c) level was 8%, and oral glucose tolerance testing revealed impaired tolerance, whereas the plasma glucose and glycoalbumin levels were normal. The results of high-performance liquid chromatography (HPLC) for HbA1c, isoelectrofocusing of the hemolysate and precise HPLC measurements of the HbA1c level supported the presence of an Hb variant, and DNA sequencing of the β-globin gene revealed Hb Yahata [β112 TGT (Cys)→TAT (Tyr)] (heterozygote). In this case, the discrepancy between the plasma glucose and HbA1c levels raised suspicion of this rare Hb variant.

Hb Handsworth is a rare α-globin structural variant caused by a missense mutation either on the α2 or α1-globin gene (HBA2 or HBA1: c.55G>C, p.Gly18Arg). This variant might be erroneously diagnosed as HbS unless secondary confirmative tests are carried out. We encountered a child with a prominent peak eluting in the 'S' window on high-performance liquid chromatography (HPLC). Sickle solubility test, gel electrophoresis, and selective direct nucleotide sequencing of α1, α2, and β globin genes were performed on the patient's sample. In addition, previous HPLC results on a cord blood sample were retrieved. Sickle solubility test was negative. Gel electrophoresis revealed a band migrating at the S region with an extra faint band seen on acid gel electrophoresis. Molecular analysis of α2 globin gene revealed heterozygous state of Hb Handsworth. Hb Handsworth is a rare variant that can mimic HbS on HPLC. Failure to recognize this rare variant in regions where HbS is highly prevalent may result in serious misdiagnosis and subsequent incorrect genetic counseling., (© 2013 John Wiley & Sons Ltd.)

Context: Hemoglobin (Hb) Austin was defined in 1977, using amino acid sequencing of samples from 3 unrelated Mexican-Americans, as a substitution of serine for arginine at position 40 of the β-globin chain (Arg40Ser). Its electrophoretic migration on both cellulose acetate (pH 8.4) and citrate agar (pH 6.2) was reported between Hb F and Hb A, and this description persists in reference literature. OBJECTIVES.-To review the clinical features and redefine the diagnostic characteristics of Hb Austin., Design: Eight samples from 6 unrelated individuals and 2 siblings, all with Hispanic surnames, were submitted for abnormal Hb identification between June 2010 and September 2011. High-performance liquid chromatography, isoelectric focusing (IEF), citrate agar electrophoresis, and bidirectional DNA sequencing of the entire β-globin gene were performed., Results: DNA sequencing confirmed all 8 individuals to be heterozygous for Hb Austin (Arg40Ser). Retention time on high-performance liquid chromatography and migration on citrate agar electrophoresis were consistent with that identification. Migration on IEF, however, was not between Hb F and Hb A, as predicted from the report of cellulose acetate electrophoresis. By IEF, Hb Austin migrated anodal to ("faster than") Hb A., Conclusions: Hemoglobin Austin (Arg40Ser) appears on IEF as a "fast," anodally migrating, Hb variant, just as would be expected from its amino acid substitution. The cited historic report is, at best, not applicable to IEF and is probably erroneous. Our observation of 8 cases in 16 months suggests that this variant may be relatively common in some Hispanic populations, making its recognition important. Furthermore, gene sequencing is proving itself a powerful and reliable tool for definitive identification of Hb variants.

Background: The currently recommended technologies of HPLC and isoelectric focusing for newborn blood spot screening for sickle cell disease (SCD) identify both the disease and carrier states, resulting in large numbers of infants being followed up unnecessarily. Analysis of blood spot tryptic peptides performed by using tandem mass spectrometry (MS/MS) is an alternative technology to detect hemoglobin (Hb) variant disorders., Methods: We analyzed 2154 residual newborn blood spots and 675 newborn blood spots from infants with Hb variants by using MS/MS after trypsin digestion. Screening cutoffs were developed by using the ratio between the variant peptide-to-wild-type peptide abundance for HbS, C, D(Punjab), O(Arab), Lepore, and E peptides. A postanalytical data analysis protocol was developed using these cutoffs to detect only the disease states of SCD and not to identify carrier states. A parallel study of 13 249 newborn blood spots from a high-prevalence SCD area were analyzed by both MS/MS and HPLC., Results: Screening cutoffs developed distinguished the infants with the disease states of SCD, infants who were carriers of SCD, and infants with normal Hb. In the parallel study no false-negative results were identified, and all clinically relevant cases were correctly identified using the MS/MS protocol. Unblinding the data revealed a total of 328 carrier infants that were successfully excluded by the protocol., Conclusions: The screening protocol developed correctly identified infants with the disease states of SCD. Furthermore, large numbers of sickle cell carrier infants were successfully not identified, thereby avoiding unnecessary follow-up testing and referral for genetic counseling.

Introduction: Hb Hofu (HBB:c. 380T>A) is a rare inherited hemoglobin abnormality with few case reports in the world literature., Methods: Screening for the sickle cell gene mutation and other hemoglobinopathies was carried out using the sickle slide test, Hb electrophoresis, and HPLC under an ongoing central government project., Results: We detected twelve Hb Hofu heterozygotes and three sickle Hb Hofu compound heterozygotes. The heterozygotes were asymptomatic except for one individual who had chronic kidney disease and moderate anemia. Only one HbS-Hofu case was symptomatic and presented with intermittent attacks of painful crisis. In the carrier state, the Hb Hofu eluted as a hump at the beginning of the HbA(0) window. But in HbS-Hofu cases, Hb Hofu eluted as a single peak in the HbA(0) window, with the HbA(2) levels being >4% consistently., Conclusion: HbS-Hofu has a variable clinical presentation. The retention time of Hb Hofu on HPLC is very close to that of HbA(0) and often elutes in the A0 window. Thus, there is every possibility of the HbS-Hofu chromatogram to be misinterpreted as that of a sickle cell trait/transfused sickle cell-beta-thalassemia case. This is the first time where Hb Hofu has been detected by HPLC, which is the widely accepted screening technique for hemoglobinopathies around the world., (© 2013 John Wiley & Sons Ltd.)

Many abnormal α-chain hemoglobins (Hbs) are caused by single nucleotide mutations in α1- or α2-goblin genes. One of these Hbs is Hb Q-Iran which is resulted from a point mutation at codon 75 of the α1-globin gene (Asp→His). The identification of Hb Q-Iran was observed in two members of a family from the Central Province of Iran. In this study, Globin chain analysis on high performance liquid chromatography (HPLC) and DNA sequencing were applied. An unusual Hb variant, like HbS on alkaline pH electrophoresis was identified from samples of a father and his son from Arak city in the Central Province of Iran. The variant was further characterized by globin chain analysis and DNA sequencing methods. Globin chain analysis revealed an unknown globin chain peak after α-globin chain peak with a different retention time from βs-globin chain, as the control in both samples. Genetic analysis led to the identification of an unknown Hb variant, Hb Q-Iran. Globin chain analysis showed the presence of an unknown globin chain, and likewise DNA sequencing revealed HbQ-Iran. In other words, Globin chain analysis procedure could preliminarily detect an unknown globin chain.

Commonly used methods for assay of haemoglobin A(1c) (HbA(1c)) are susceptible to interference from the presence of haemoglobin variants. In many systems, the common variants can be identified but scientists and pathologists must remain vigilant for more subtle variants that may result in spuriously high or low HbA(1c) values. It is clearly important to recognize these events whether HbA(1c) is being used as a monitoring tool or, as is increasingly the case, for diagnostic purposes. We report a patient with a rare haemoglobin variant (Hb Sinai-Baltimore) that resulted in spuriously low values of HbA(1c) when assayed using ion exchange chromatography, and the steps taken to elucidate the nature of the variant.

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O₂ affinity compared with normal cells (P₅₀ = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O₂ affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO₂, decreasing the rate approximately twofold. These kinetic data help explain the high O₂ affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure., (Copyright © 2012 The Protein Society.)

Hb Acharnes or [β53(D4) Ala - Thr] is a newly discovered unstable hemoglobin variant. It has been reported in very few literatures across the world and no cases have been reported from India till date. Hb Acharnes is known to interact with β°-thalassemia to produce thalassemia intermedia and heterozygotes may present with borderline HbA2 levels. Here we report a rare case discovered during routine screening of thalassemia and hemoglobinopathies in a 19 year old pregnant lady. To emphasize the diagnostic difficulties and importance of detection of a rare hemoglobin variant Hb Acharnes [β53(D4) Ala - Thr] in an asymptomatic patient in West Bengal, India. The 19 year old asymptomatic pregnant lady P1+0, LMP - 21.01.2011 reported in antenatal OPD of Burdwan Medical College & Hospital, Burdwan, West Bengal, India for routine follow up. After proper antenatal check up her blood was collected as a routine screening for Thalassemia, EDTA blood was taken on 5th April 2011 and was subjected to Hemoglobin estimation by Cyanmethemoglobin method, Cell parameters in automated cell counter (SYSMEX KX21) and Hemoglobin analysis by HPLC in BIORAD VARIANT system. The patient was normal with respect to clinical examination and urinalysis. Routine blood counts revealed mild microcytic hypochromic anemia and on HPLC an unknown band (retention time 2.2 minutes, 21.2%, appearing as a shoulder of HbA0 band) of hemoglobin variant was discovered with normal level of HbF and HbA2. Hemoglobin analysis of her mother showed similar pattern while her father and her husband had normal Hb-HPLC pattern. The unknown hemoglobin variant was identified as Hemoglobin Acharnes or [β53 (D4) Ala - Thr] by the Biorad laboratories upon consulting the standard chromatogram patterns for this particular hemoglobin variant. Only Haemoglobin Electrophoresis by conventional gel technique may miss the case and the HPLC pattern may be used as a standard control for identification.

Hemoglobinopathies are the most common inherited disorders. Newborn blood screening for clinically significant hemoglobin variants, including sickle (HbS), HbC, and HbD, has been adopted in many countries as it is widely acknowledged that early detection improves the outcome. We present a method for determination of Hb variants by direct surface sampling of dried blood spots by use of an Advion Triversa Nanomate automated electrospray system coupled to a high-resolution mass spectrometer. The method involves no sample preparation. It is possible to unambiguously identify homozygous and heterozygous HbS, HbC, and HbD variants in <10 min without the need for additional confirmation. The method allows for repeated analysis of a single blood spot over a prolonged time period and is tolerant of blood spot storage conditions.

We report two new point mutations of the alpha1-globin gene found in a Greek and a Burmese patient, both living in Western Australia. The patients were initially selected for their microcytic hypochromic parameters as belonging to a group suspected for uncommon (deletion) defects. Gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA) technologies were applied, and in those cases not showing deletions, direct sequencing was performed. We have found 1) HBA1:c.86C>T, Hb Nedlands [alpha28(B9)Ala-->Val] which, based on the red cell indices and phenotype prediction scores, is presumed to be clinically silent, and 2) HBA1:c.98T>A, Hb Queens Park [alpha32(B13)Met-->Lys] which seems to be associated with a mild alpha-thalassemia (alpha-thal) phenotype. The phenotype/genotype correlation is briefly described.

We describe a novel hemoglobin (Hb) variant, caused by a CCC > TCC transition at codon 77 on the alpha gene. The mutation was found in two unrelated patients, in one patient on the alpha1 gene and in the other patient on the alpha2 gene. Both are anemic patients of African origin. Due to the neutral Pro-->Ser substitution, Hb Nile could not be separated from Hb A with common short-run screening methods for high performance liquid chromatography (HPLC) and capillary electrophoresis, but was evidently present after prolonged cation exchange HPLC or separation by isoelectric focusing (IEF). Reversed phase HPLC separation of the globin chains revealed the normal and abnormal alpha chains with an expression of about 20% for Hb Nile[A1], indicative of normal expression and stability of the mutant protein.

We report a case of compound heterozygous hemoglobins S [beta6(A3)Glu6Val] and Korle-Bu [beta73(E17)Asp73Asn] in a 2-year-old girl. This hemoglobin genotype is associated with a benign clinical course, much like the sickle cell trait; however, its laboratory characteristics are very similar to compound heterozygous hemoglobin S and hemoglobin D-Los Angeles [beta121(GH4)Glu121Gln], which produces severe sickling hemolytic anemia. We describe laboratory data used to resolve this important differential diagnosis and review the interactions between hemoglobin S and the variant hemoglobins that may account for the different clinical phenotypes in compound heterozygotes.

The Sebia Capillarys (capillary zone electrophoresis [CE]) and the Primus Resolution high-pressure liquid chromatography (HPLC) were used to prospectively evaluate 297 samples for hemoglobinopathies. Hemoglobin (Hb) A levels were similar on both techniques (mean, 96.2% and SD, 5.7% by CE; mean, 96.8% and SD, 5.5% by HPLC), but HbA2 levels were higher by CE (mean, 2.8%; SD, 0.8%) than by HPLC (mean, 2.3%; SD, 0.8%). HbS had higher values by CE (mean, 40.6%; SD, 18.9%) than by HPLC (mean, 38.4%; SD, 18.9%). In cases with Hg S, HbA2 levels were greater by HPLC (mean, 4.0%; SD, 1.0%) than by CE (mean, 3.1%; SD, 0.8%). HbA2 was occasionally not separated sufficiently from HbC for measurement by CE, but did separate from HbE by CE. Both methods identified HbS, HbC, HbE, HbS, and HbC together, HbA2&prime, HbD-Los Angeles, HbF variant, HbG-Philadelphia, HbS-G Philadelphia, and Hb Lepore.

This paper summarizes the results on the epidemiology and molecular basis of thalassemias and other hemoglobinopathies in the Republic of Macedonia. Over the past 40 years, population surveys of more than 22,000 participants (school children and workers) from all over the country, have shown that the average incidence of beta-thalassemia (thal) trait is 2.6%, ranging from less than 1% in the northeast to 10% in the south. The frequency of deltabeta-thal is 0.2%, while the frequency of the Swiss type of hereditary persistence of fetal hemoglobin (HPFH) is 0.3%. Screening of 9,619 newborns has shown that the frequency of alpha-thal trait is 1.5%, of which alpha-thal-2 is 1.45% and alpha-thal-1 is 0.05%. The molecular basis of the different forms of beta-thal and other hemoglobinopathies has been completely defined. Among the Macedonians, over 450 beta-thal chromosomes have been studied. Fifteen different beta-thal mutations have been detected, four of which [IVS-I-110 (G-->A), IVS-I-6 (T-->C), IVS-I-1 (G-->A), codon 39 (C-->T)] account for 85% of all beta-thal chromosomes. Among the Albanians, 48 beta-thal chromosomes have been studied. Eight different mutations have been detected, four of which [codon 39, -30 (T-->A), IVS-I-110, IVS-I-1] account for 85% of all beta-thal chromosomes. Four new mutations [-101 (C-->A), -87 (C-->G), -30, polyadenylation signal (poly A) (AATAAA-->AATGAA)] have been characterized. Molecular analyses of DNA from over 20 unrelated cases with deltabeta-thal have shown that this condition is caused by a 13 kb deletion (Sicilian type); in two families a deletion of 18 to 23 kb (Macedonian type of deltabeta-thal) was discovered. Molecular analyses of alpha-thal in the Republic of Macedonia have shown the following types of molecular defects: 20.5 kb deletion, 17.5 kb deletion, 3.7 kb deletion, poly A mutation (AATAAA-->AATGAA), and Hb Icaria [alpha142, Term-->Lys, TAA-->AAA (alpha2)]. The incidence of abnormal hemoglobins (Hbs) in the Republic of Macedonia is 0.4%. Three different alpha chain variants among 10 families, seven different beta chain variants among 33 families, two gamma chain variants in two newborns, one variant with an extended alpha chain, and Hb Lepore among 105 families, have been observed. Structural analysis of numerous cases with Hb Lepore showed that the variant was of the Washington-Boston type.

We describe a new delta-globin variant, Hb A2-Pasteur-Tunis [delta59(E3)Lys-->Asn, AAG-->AAC]. This hemoglobin (Hb) displayed an electrophoretic mobility faster than normal Hb A2 and was expressed at 2.2 %. The molecular defect was characterized by DNA sequencing and confirmed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-designed protocol. Hb A2-Pasteur-Tunis was found in a carrier of a codon 39 (C-->T) beta0-thalassemia (thal), presenting with a normal Hb A2 level. Phenotype and genotype investigations revealed that the total Hb A2 level of the patient was that expected for a minor beta-thal (4.8%).

We describe a new hyperunstable beta-chain variant due to a complex genomic rearrangement. The abnormal hemoglobin (Hb) was found as a de novo mutation in a 2-year-old Bulgarian girl with severe hemolytic anemia. The mutation was detected through RNA/DNA analysis. It represents a complex genomic rearrangement involving an insertion of 23 nts after IVS-II-535 (derived by triplication of the 12-nts adjacent sequence and subsequent deletion of 1 nt), a deletion of 310 nts extending from IVS-II-550 to the first nt of Cd 108 and an insertion of 28 nts at the deletion junctions (derived from the inverted sequence between nts +3,707 and +3,734 3' to the beta-globin gene termination codon). At the protein level this mutation leads to a deletion of 4 amino acid residues (Leu-Leu-Glu-Asn) at positions 105-108 and an insertion of 9 residues (Val-Pro-Ser-Val-Thr-Leu-Phe-Phe-Asp) at the same location, creating an abnormal elongated beta-chain of 151 amino acid residues. This highly unstable variant was named 'Hb Jambol' after the geographic location in which the patient resides., (2007 S. Karger AG, Basel)

In human deoxyhemoglobin (deoxyHb), the hydrogen bond between Aspbeta99(G1) and Tyralpha42(C7), located in the alpha1beta2 interface, is crucial for the stability of the T structure. All the variants that could arise from a single point mutation affecting codon beta99 have already been observed, leading always to erythrocytosis. Conversely, up to now, Hb Barika is the only example found in a patient in whom the alpha42 is mutated. From a biochemical point of view, for theoretical reasons, this substitution has already been extensively studied on recombinant hemoglobin (rHb). In the patient, Hb Barika is expressed at a level lower than expected for an alpha2 gene variant and leads to an alpha+-thalassemic-like syndrome.

Hb La Coruña [beta38(C4)Thr --> Ile] is a new hemoglobin (Hb) variant that has an increased oxygen affinity. Clinically, this Hb leads to erythrocytosis. Hb La Coruña is an electrophoretically silent variant that can be detected by reversed phase high performance liquid chromatography (HPLC) and characterized by DNA sequencing. The patient was a 22-year-old Spanish male whose family lived in La Coruña, in the northwest of Spain. His mother was also a carrier.

Hemoglobin (Hb) Bart's (gamma4) was isolated and purified from Hb Bart's hydrops fetalis syndrome blood by CM-Sephadex C-50 chromatography. The isolated Hb Bart's was analyzed for its purity by high performance liquid chromatography. Balb/c mice were immunized intraperitoneally with Hb Bart's. The immunized mouse splenic cells were hybridized with mouse myeloma, X63-Ag8.653, by polyethylene glycol. There were 12 hybridoma clones, out of several thousand culture wells, secreting antibody against purified Hb Bart's. However, when those 12 monoclonal antibodies (mAb) were tested with Hb Bart's (gamma4), HbF (alpha2gamma12), HbH, HbE, and HbA2, there was only 1 hybridoma clone secreting mAb highly reactive to Hb Bart's with very low reactivity to HbF. A rabbit polyclonal antibody with relative high reactivity to Hb Bart's compared to HbF with the ratio of 2.4:1 was also produced by affinity column chromatography for the purpose of developing an enzyme-linked immunosorbent assay (ELISA) base for qualitative and quantitative determination of Hb Bart's in adult hemolysates. Preliminary results in quantitative determination of Hb Bart's in Hb solution of 3 alpha thalassemia families having at least 1 child with HbH disease and 6 normal subjects indicated that it was possible to quantify Hb Bart's by our developed ELISA with appropriate sensitivity and specificity.

Hemoglobin (Hb) chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb chains also after sample extraction. We describe a CE method for analysis of Hb chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the hemoglobinopathies.

A boy presented at age 4 years with severe congenital hemolytic anemia characterized by highly elevated reticulocyte count (30-50%) and prominent basophilic stippling. Hb had been 4 g/dL at age 7 months. The patient was on a monthly transfusion regimen up to the age of 7 years, when he underwent splenectomy. After removal of the spleen, his Hb stabilized at 11 g/dL. No abnormal pattern was detected in hemoglobin electrophoresis at pH 9 and 6. In-vitro globin synthesis revealed the presence of an abnormal beta-chain in front of the gamma-chain. The beta(A)/beta(X) ratio was 0.77 at 30 min and 0.74 at 2 hr of incubation. Molecular analysis revealed that the patient had GCC-->GAC alteration at codon 27 (beta27(B9)Ala-->Asp) causing the abnormal hemoglobin Volga. The beta-cDNA derived from the beta-Hb Volga allele could be differentiated from HbA beta-cDNA on silver-stained gel. No imbalance in the mRNA of beta(A)/beta(Hb Volga) ratio was observed., (Copyright 2004 Wiley-Liss, Inc.)

Rare abnormal haemoglobin (Hb) often poses a problem in the diagnosis of relative electrophoresis mobility. The rare neutral Hbs interact with the Hb S and thus can increase the severity of sickle cell anaemia. In the present study, we investigated the use of reversed-phase high-performance liquid chromatography (RP-HPLC) as a method for the identification of neutral or unstable Hb, and compared it with the well-known techniques urea-triton electrophoresis (UT) and isoelectrofocusing (IEF). The results of our experiments showed that Bucuresti Hb was separated without ambiguity by RP-HPLC. To date, no other electrophoresis techniques have been able to isolate this Hb. We also examined the Hb Nouakchott, which was the most hydrophobic Hb. Moreover, the adult and foetal chains of globins (especially the Agamma(t) and Agamma(i) chains) were quantified on the same chromatogram., (Copyright 2004 Wiley-Liss, Inc.)

Hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, was crystallized by the hanging drop vapor diffusion technique with a precipitant solution containing 10% polyethylene glycol 3350 and 50 mM HEPES-Na, pH 7.5. The Hb D crystals of G. gigantea, which diffract to at least a 2.0 A resolution, belong to the monoclinic space group C2 with unit cell dimensions of a = 112.1 A, b = 62.4 A, c = 54.0 A, and beta = 110.3 degrees. One alphabeta dimer molecule of Hb D existed in an asymmetric unit, with a calculated value of Vm of 2.77 A(3)Da(-1).

We report a novel mutation, Hb A2-Monreale [delta146(HC3)His-->Arg], detected by cation exchange high performance liquid chromatography in a family from West Sicily. The mutation is due to a CAT-->CGT substitution at codon 146 of the delta-globin gene. The two carriers had reduced levels of normal Hb A2 (1.1%), but comparable levels (0.9%) of the Hb A2 variant. Most likely the new variant has the same characteristics as Hb Cochin-Port Royal [beta146(HC3)His-->Arg], that is stable but has a 75% reduction of the Bohr effect. The finding of the new variant increases the genotype heterogeneity of the delta-globin gene in the Mediterranean area, and is relevant to the study and prevention of Cooley's Anemia.

Hb O-Tibesti, carries in the same chain the substitution of Hb O-Arab [beta121(GH4)Glu-->Lys] and that of Hb Hamilton [beta11(A8)Val-->Ile]. Hb O-Tibesti may be distinguished from Hb O-Arab by polyacrylamide gel electrophoresis in the presence of urea and Triton-X100, and by reversed phase high performance liquid chromatography. It was found in a compound heterozygous condition with Hb S [beta6(A3)Glu-->Val] in a child of Chad-Sudanese descent, suffering from a sickle cell syndrome. Compared to the classical description of the Hb S/Hb O-Arab association, the additional Hb Hamilton mutation does not seem to modify the clinical presentation.

Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H2S to the bacterial endosymbiont. To further study HbI, expression studies of this protein were performed in Escherichia coli. This is the first time that the recombinant HbI was produced using a recombinant DNA expression system. Hemoglobin I cDNA was amplified and cloned into the TOPO-PBAD expression vector, which contains a fusion tag of six histidine residues (6XHis tag). Plasmid clone sequence analysis was carried out in order to ensure that the insert was in the correct reading frame for proper protein expression in E. coli. The expression of recombinant HbI was optimal when induced for 5 hr with 0.002% of L-arabinose as detected by Western blot analysis. The proto-porphyrin group was inserted into the recombinant HbI. Purification of the heme-bound recombinant protein was performed under native conditions by affinity chromatography using Ni-NTA and Probond resins. The sodium dithionite-reduced recombinant protein presented a shift from the Soret band at 413-435 nm, indicating the presence of the heme group in the adequate amino acid environment of HbI. These results indicate that recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding.

The Asn108 beta-->Lys mutation in hemoglobin (HbPresbyterian mutation) endows a low O(2) affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two alpha 99 lysine residues also induces a low O(2) affinity into HbA. We have now engineered an alpha alpha-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O(2) affinity-inducing structural perturbations. Despite the presence of the additional epsilon-amino group of Lys108(beta) within the central cavity, the epsilon-amino group of Lys99(alpha alpha) of deoxy Hb-Presbyterian retained high selectivity for alpha alpha-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The alpha alpha-fumaryl cross-linking of Hb-Presbyterian reduced its O(2) affinity much more significantly than that observed with HbA, indicating a synergy between the two low O(2) affinity-inducing structural perturbations. Apparently, the alpha alpha-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O(2) affinity-inducing potential of Lys108(beta) that is generally activated in the presence of chloride. The synergy between the Asn108(beta)-->Lys mutation and the alpha alpha-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, alpha alpha-fumaryl Hb-Presbyterian accessed a low O(2) affinity T-state that is accessed by HbA, alpha alpha-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the alpha alpha-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(alpha) and Lys108(beta) [presumably His103(alpha) and/or Glu101(beta)] to compensate for the extra positive charge of Lys108(beta). Molecular modeling studies identified two potential chloride binding sites per alpha beta dimer within the middle of the central cavity of alphaalpha-fumaryl HbA involving residues His103(alpha), Arg104(beta) and Asn108(beta). The affinity of these sites is increased in alpha alpha-fumaryl Hb-Presbyterian as a result of the Asn108(beta)-->Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the alpha alpha-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the alpha alpha- fumaryl-Hb Presbyterian to access a low O(2) affinity T-state structure much more readily than either Hb-Presbyterian or alpha alpha-fumaryl HbA.

Hb Mont Saint-Aignan [beta128(H6)Ala-->Pro] is a mildly unstable variant, associated with hemolytic anemia, marked microcytosis and increased alpha/beta biosynthetic ratio (1.55 versus 1.1 +/- 0.1 in the control). The abnormal chain was isolated by selective precipitation with isopropanol and the structural modification determined by protein chemistry methods (reversed phase high performance liquid chromatography and mass spectrometry). Possible mechanisms underlying the beta(+)-thalassemia-like expression of this variant are discussed.

Hb Sitia [beta128(H6)Ala-->Val] was found in a Greek female with slightly reduced red blood cell indices. The abnormal hemoglobin was indistinguishable from Hb A by electrophoresis but eluted after Hb A on cation exchange high performance liquid chromatography. DNA sequence analysis revealed a GCT-->GTT mutation at codon 128, which is predicted to encode an Ala-->Val substitution. This was confirmed by mass spectrometry analyses of the beta-globin chain. Since alanine at beta128(H6) interacts with several amino acids of the alpha1beta1 contact, its replacement by a larger residue results in a mild instability of the molecule and slight modifications of the oxygen binding properties.

Haemoglobin, the 'honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158-161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglobin I from the South American fish Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 A resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6(1)22 and preliminary structural analysis revealed the presence of one dimer (alphabeta) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques.

A new, simple and fast spectrophotometric method for the determination of the H(2)S concentration is reported. This method, based on the 1:1 reaction between H(2)S and the ferric derivative of hemoglobin I (HbI) from the bivalve mollusc Lucina pectinata, allows the quantitative determination of H(2)S dissolved in a given solution even at concentrations as low as 1 x 10(-6) M. Note that L. pectinata HbI is considered the physiological receptor of H(2)S.