Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 54: 1053-1059, 2008. First published April 17, 2008; 10.1373/clinchem.2007.097857
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
clinchem.2007.097857v1
54/6/1053    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zanella-Cleon, I.
Right arrow Articles by Francina, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zanella-Cleon, I.
Right arrow Articles by Francina, A.
Related Collections
Right arrow Hematology
(Clinical Chemistry. 2008;54:1053-1059.)
© 2008 American Association for Clinical Chemistry, Inc.

Hematology

Detection of a Thalassemic {alpha}-Chain Variant (Hemoglobin Groene Hart) by Reversed-Phase Liquid Chromatography

Isabelle Zanella-Cleon1, Michel Becchi1, Philippe Lacan2, Piero C. Giordano3, Henri Wajcman4,5 and Alain Francina2,a

1 Institut de Biologie et de Biochimie des Protéines, IFR128, Université Claude Bernard-Lyon I, Lyon, France; 2 Unité de Pathologie Moléculaire, Fédération de Biochimie et de Biologie Spécialisée, Hôpital Edouard Herriot, Hospices Civils, Université Claude Bernard-Lyon I, Lyon, France;3 Hemoglobinopathies Laboratory, Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands; 4 INSERM U 841, Créteil, France;5 Biochimie Génétique, Hôpital Henri Mondor, Créteil, France.

aAddress correspondence to this author at: Unité de Pathologie Moléculaire, Fédération de Biochimie et de Biologie Spécialisée, Hôpital Edouard Herriot, 69437 Lyon Cedex 03, France. Fax 33-0472110598; e-mail alain.francina{at}chu-lyon.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Hemoglobin (Hb) Groene Hart [{alpha}119 (H2)Pro->Ser ({alpha}1)], also known as Hb Bernalda, is a nondeletional {alpha}-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The {alpha}-thalassemic behavior of this variant has been explained as an impaired interaction between the {alpha}-globin chain and the {alpha}-Hb–stabilizing protein.

Methods: To separate globin chains, we developed a modified reversed-phase liquid chromatography (RPLC) procedure that uses acetonitrile–water solvents containing up to 3 mL/L trifluoroacetic acid. After RPLC, we characterized the isolated globin chains by electrospray ionization (ESI) mass spectrometry (MS) and analyzed their tryptic peptides with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nano-LC–ESI–MS/MS.

Results: RPLC detected an abnormal peak with a retention time substantially greater than that of the wild-type {alpha}A-globin chain. We identified this variant as Hb Groene Hart and found it in the hemolysates of 11 unrelated patients (1 homozygote, 9 heterozygotes, and 1 heterozygote associated with the –{alpha}3.7 deletion). These patients possessed abnormal hematologic features suggesting an {alpha}-thalassemia phenotype. Molecular modeling suggested that the increase in hydrophobicity was due to opening of the GH interhelical segment following replacement of amino acid residue 119 with a nonhelix breaker residue.

Conclusions: This method allows the detection of Hb variants at low concentrations, and adjusting the composition of the organic solvents enables the method to identify Hb variants with large changes in hydrophobicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Approximately 900 human hemoglobin (Hb)1 variants involving the {alpha} and β chains have been described(1). A few {alpha}-chain variants cause changes in the 3-dimensional structure of the Hb complex that impair the formation of the Hb {alpha}β dimer and/or tetramer(1)(2)(3), leading to the formation of highly unstable free {alpha}-Hb species. When present in low amounts, such species degrade rapidly; at higher concentrations, they precipitate within erythrocytes and erythrocyte precursors to form insoluble inclusions and cause substantial oxidative stress to erythrocyte membranes (Heinz bodies). Free β-globin is concurrently synthesized normally and accumulates within the erythrocyte, possibly forming Hb H (β4). This imbalance between {alpha}- and β-globins is typical of {alpha}-thalassemia. Assembly of the Hb {alpha}β dimer depends on the presence of a chaperone protein, the {alpha}-Hb–stabilizing protein (AHSP), which forms a heterodimer with {alpha}-Hb in erythroid precursors(4). Interactions between AHSP and {alpha}-Hb involve the last 2 {alpha}-helices (G and H) of the {alpha} chain(5)(6). Mutations in these helices impair the formation of the Hb {alpha}β dimer and thus decrease the availability of {alpha} chains for assembling the Hb complex, thereby increasing the pool of free {alpha} chains.

Mutations at position {alpha}119(H2)Pro have recently been demonstrated to impair the AHSP/{alpha}-Hb interaction(7). Under such conditions, the mutated {alpha}-globin chain is rapidly proteolyzed. In previous investigations of patients carrying the Groene Hart mutation [{alpha}119(H2)Pro->Ser ({alpha}1)], which is frequent in North Africa and known as Hb Bernalda in southern Italy(8)(9)(10), no abnormal Hb variant was detected with electrophoretic methods, liquid chromatography (LC), or electrospray ionization mass spectrometry (ESI MS). The mutation was identified by DNA analysis. This apparent absence of the protein was in agreement with the borderline {alpha}-thalassemic phenotype found in these carriers. Hb Groene Hart was thus hypothesized not to be present as an Hb species in vivo.

In the present study, we developed a reversed-phase LC (RPLC) method to investigate whether or not Hb Groene Hart occurs as an Hb species in vivo. We isolated the {alpha}Groene Hart-globin chain by preparative RPLC, confirmed its identity via DNA sequencing, and fully characterized this globin chain by MS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and hematologic procedures
The unrelated patients included in this study were 1 homozygous patient ({alpha}{alpha}Groene Hart/{alpha}{alpha}Groene Hart), 1 Hb Groene Hart heterozygote associated with a heterozygous {alpha}-thalassemia 3.7-kb deletion (–{alpha}3.7/{alpha}{alpha}Groene Hart), and 9 heterozygotes ({alpha}{alpha}/{alpha}{alpha}Groene Hart). Blood samples were drawn into collection tubes containing EDTA, and hematologic data were collected via standard methods. Hemolysates were analyzed by isoelectric focusing on polyacrylamide gels(11)(12), ion-exchange LC (Variant ITM, Beta-Thalassemia Short Program; Bio-Rad Laboratories)(11)(12), and RPLC of globin chains.

We used the hematologic results to check for the presence of a thalassemic phenotype and then investigated patients with borderline {alpha}-thalassemic erythrocyte data when we detected an abnormal peak that eluted at a high percentage of solvent B in the RPLC analysis (see below). DNA analysis was used to confirm the presence of the Hb Groene Hart mutation.

rplc of globin chains
RPLC analyses were performed on a C4 Uptisphere column (4.6 x 250 mm, 5-µm particle size, 30-nm mean pore size; Interchim). We eluted globin chains with a 2-solvent system [solvent A, 100 mL/L acetonitrile and 3 mL/L trifluoroacetic acid (TFA) in water; solvent B, 700 mL/L acetonitrile and 3 mL/L TFA in water] and a 3-step RPLC elution program consisting of a linear gradient of 50%–70% solvent B in 80 min, a linear gradient of 70%–90% solvent B in 10 min, and reequilibration with 50% solvent B for 10 min. Elution conditions were modified for some experiments, as is described later. The flow rate was 1 mL/min, and eluate absorbance was monitored at 220 nm. Samples were prepared from hemolysates adjusted to an Hb concentration of 40 g/L and were diluted further with water (75 µL hemolysate plus 925 µL water). We injected 20 µL of sample into the column for each assay. The same conditions were used in preparative RPLC runs, with the exception that the 20-µL injection volume was drawn from a sample dilution consisting of 225 µL hemolysate and 775 µL water.

mass spectrometry
sample preparation
Globin chain peaks eluting from the C4 RPLC column were collected (approximate solvent composition, 500 mL/L acetonitrile and 3 mL/L TFA) and dried in a vacuum concentrator. We oxidized 15 µg of the dried globin chain peak with 100 µL of 70 mL/L hydrogen peroxide for 15 min in the dark and dried the sample again in a vacuum concentrator. We dissolved the oxidized globin chain in 25 µL of 50 mmol/L ammonium bicarbonate buffer, pH 8.0, added 15 µL of a digestion solution [4.25 µmol/L trypsin (Promega) in 50 mmol/L ammonium bicarbonate buffer, pH 8.0)], and digested the globin chain for 2 h at 37°C. We stopped the digestion by adding 1 µL formic acid (98%–100% purity). From this final solution, 15 µL was used for MALDI-TOF-MS analysis after desalting and concentration of peptides on a Varian 10-µL OMIX® C18 pipette tip, following the manufacturer’s instructions. The remaining digested sample (25 µL) was used for LC–ESI–MS/MS experiments.

esi ms analysis of globin chains
We used a SCIEX API 165 instrument (Applied Biosystems) for MS analysis of the purified {alpha}-globin chain. We dissolved approximately 5 µg of the {alpha}-globin chain in 70 µL of a methanol/water mixture (1 part each) containing 1 mL/L formic acid and infused the globin solution directly into the instrument at a flow rate of 5 µL/min. Spectra were acquired at a 5000-V ion-spray voltage and a scan speed of 100 Da/s (scan range, 600–1800 Da).

maldi-tof ms analysis of tryptic peptides
We used an Applied Biosystems Voyager-DETM PRO MALDI-TOF mass spectrometer in the positive-ion reflector mode for mapping peptide masses. Spectra were acquired in the mass range of 700–5000 Da at a 20-kV accelerating voltage and with a 140-ns delay time. The matrix solution consisted of 5 g/L {alpha}-cyano-4-hydroxycinnamic acid (LaserBio Labs) dissolved in a water/acetonitrile mixture (1 part each) containing 1 mL/L TFA. We used the dried-droplet technique for depositing the sample on the target by mixing 2 µL of the eluate from the OMIX pipette tip with 1 µL of the matrix solution and allowing the mixture to dry. External calibration was carried out with the singly charged monoisotopic peaks produced with a peptide mixture from the SequazymeTM Peptide Mass Standards Kit (Applied Biosystems).

nano-lc–esi–ms/ms analysis of tryptic peptides
Experiments were performed on a Q-STAR® XL instrument (Applied Biosystems) equipped with a nanospray source equipped with a distal coated SilicaTipTM emitter (cat. no. FS150-20-10-D-20; New Objective) set at 2300 V. The instrument’s Information Dependent Acquisition (IDA) mode allowed peptide ions within a survey scan range of m/z 400–2000 to be analyzed for subsequent fragmentation. Tandem MS spectra were acquired in the m/z 65–2000 range for +2 to +4 charged ions. The collision energy was automatically set by the software (Analyst 1.1) and was related to the mass and charge of the precursor ion. The MS and MS/MS data were recalibrated with internal reference ions from a tryptic autolysis peptide at m/z 842.510 [M+H]+ and m/z 421.759 [M+2H]2+.

We injected 5 µL of the acidified solution and separated the tryptic peptides with a Dionex UltiMate nano LC system equipped with a Dionex C18 PepMap Micro Precolumn (300 µm x 5 mm, 5-µm particle size, 10-nm pore size) and a Dionex C18 PepMap nanocolumn (75 µm x 150 mm, 3-µm particle size, 10-nm pore size). We used a 2-solvent chromatography system (solvent A, 1 mL/L formic acid and 50 mL/L acetonitrile in water; solvent B, 0.8 mL/L formic acid and 800 mL/L acetonitrile in water) and separated the tryptic peptides with a 60-min linear gradient of 0%–50% solvent B at a flow rate of approximately 200 nL/min.

dna analysis
Genomic DNA was extracted from whole blood as previously described(11)(12). The sequence of the Hb Groene Hart mutation was identified via PCR of the HBA12 (hemoglobin, alpha 1) and HBA2 (hemoglobin, alpha 2) genes and direct DNA sequencing of the PCR products on a LI-COR 4200 sequencer (LI-COR Biosciences). The primers used for amplifying the {alpha}-globin genes have previously been described(13). In addition, we screened for common {alpha}-thalassemic deletions by gap multiplex PCR(14).

molecular modeling
The structure of the mutated Hb was drawn with the aid of Swiss-PdbViewer(15). The reference model was the deoxy-Hb structure [see file 3hhb at the RCSB Protein Data Bank Web site (http://www.rcsb.org/pdb/explore.do?structureId=3HHB)].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hB and dna analyses
Isoelectric focusing and ion-exchange LC did not detect the presence of an Hb variant; however, we did observe an abnormal peak with RPLC. DNA sequencing revealed the mutation CCT->TCT (Pro->Ser) in codon 119 of the third exon of the HBA1 gene. Gap multiplex PCR analysis of 1 patient revealed an association with a heterozygous –{alpha}3.7 deletion.

In the homozygous case involving a 26-year-old woman of Algerian origin, the {alpha}Groene Hart-globin chain represented 19% of the {alpha}-globin chains. With an RPLC gradient of 50%–68% solvent B over 80 min, the retention time for the {alpha}Groene Hart-globin chain was 83.53 min, compared with 53.38 min for the {alpha}A chain. This patient exhibited hypochromia and microcytosis as well as nonpathologic Hb A2 and F concentrations that strongly suggested {alpha}-thalassemia (erythrocyte count, 5.44 x 1012/L; Hb, 121 g/L; mean corpuscular hemoglobin, 22.3 pg; mean corpuscular volume, 72 fL; Hb A2, 3.3%; Hb F, 0.5%); however, the results of a search for a common {alpha}-thalassemic deletion were negative.

The compound heterozygote was a 4-year-old girl who showed values for hematologic variables that were similarly abnormal (erythrocyte count, 5.20 x 1012/L; Hb, 114 g/L; mean corpuscular hemoglobin, 21.9 pg; mean corpuscular volume, 67 fL; Hb A2, 2.5%; Hb F, 0.6%). Her parents were originally from Tunisia. With the elution conditions given in Materials and Methods (50%–70% solvent B), the {alpha}Groene Hart-globin chain had a retention time of 75.47 min, compared with 49.82 min for the {alpha}A chain (Fig. 1 ). The {alpha}Groene Hart-globin chain constituted 17.8% of the {alpha}-globin chains. Gap multiplex PCR analysis revealed a heterozygous –{alpha}3.7 deletion. The Hb Groene Hart mutation in this patient was located on the intact HBA1 gene (genotype, –{alpha}3.7/{alpha}{alpha}Groene Hart), as previously described(16). The hematologic and biochemical data for the 9 unrelated heterozygotes (3 from Algeria, 2 from Morocco, 3 from Tunisia, and 1 of undetermined origin) are summarized in Table 1 . We detected no Hb H (β4) in any of these patients.


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		Figure 1. RPLC elution pattern for a hemolysate from a compound heterozygote patient (–{alpha}3.7/{alpha}{alpha}Groene Hart).

mAU, milliabsorbance units.


View this table:
[in this window]
[in a new window]

  		Table 1. Hematologic and biochemical data for patients heterozygous for Hb Groene Hart.1

ms studies
esi ms analysis of globin chains
The ESI MS analysis of the globin chains showed a mean (SD) molecular mass of 15 116.8 (1.4) Da for the {alpha}Groene Hart chain and 15 126.3 Da for the wild-type {alpha} chain (Fig. 2 ). The experimentally obtained shift in mass was –9.5 Da. The expected theoretical mass shift was –10.0 Da.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Figure 2. ESI MS analysis of the {alpha}Groene Hart globin chain.

The mean (SD) experimental mass calculated after deconvolution is 15 116.8 (1.4) Da.

maldi-tof ms analysis of tryptic peptides
The results of MALDI-TOF MS analyses of hydrogen peroxide–treated tryptic digests for the {alpha}Groene Hart-globin and wild-type {alpha}-globin chains are shown in Fig. 1 of the Data Supplement that accompanies the online version of this article (http://www.clinchem.org/content/vol54/issue6). According to the ExPASy Molecular Biology Server(17), the theoretical masses for the protonated wild-type {alpha}T-12 peptide and the Hb Groene Hart {alpha}T-12 peptide are 2967.61 Da and 2957.59 Da, respectively. Under these experimental conditions, the cysteinyl residue of the {alpha}T-12 peptide was oxidized to a cysteic acid residue, yielding a mass difference of +47.985 Da (see Fig. 2 in the online Data Supplement); thus, the theoretical masses for the wild-type and mutated {alpha}T-12 protonated peptides are 3015.59 Da and 3005.57 Da, respectively.

nano-lc–esi–ms/ms analysis of tryptic peptides
The MS/MS experiments with the mutated {alpha}T-12 peptide (3004.60 Da; m/z = 752.17; charge = +4) showed a characteristic series of y daughter ions that allowed localization of the Pro->Ser substitution to position 119 (see Figs. 2Up and 3 and Tables 1 and 2 in the online Data Supplement). These experiments confirmed the oxidation of the cysteine residue at position 104.


Figure 3
View larger version (68K):
[in this window]
[in a new window]

  		Figure 3. Molecular modeling of the {alpha}-Hb molecule, showing Pro 119 (blue, Hb Groene Hart) and Pro 114 (green, Hb Nouakchott).

Residues with hydrophobic side chains located in helices G and H are shown in orange; heme is presented in red.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that Hb Groene Hart is actually produced at a low concentration in affected individuals. In the first description of Hb Groene Hart, Harteveld et al.(8) reported detecting no abnormal native or denatured fraction with electrophoresis, isoelectric focusing, ion-exchange HPLC, denaturing PAGE, and ESI MS. These investigators suggested that the mutated gene product was an abnormal monomer unsuitable for assembly into a normal Hb tetramer and that it was quickly catabolized by the ubiquitin-related proteolytic pathway(18). The mutation occurred in the heterozygous state in 2 Moroccan patients and was accompanied by typical {alpha}-thalassemic features with a balanced or slightly increased β/{alpha} ratio in globin chain synthesis. The mutation had also been found in a Tunisian child in association with the {alpha}-thalassemic –{alpha}3.7 deletion, and except for the expected presence of Hb Barts ({gamma}4) at birth, no abnormal Hb was detected with routine analytical techniques(9). More recently, Giordano et al.(10) were the first to describe a case of Hb Groene Hart in the homozygous state, in a Moroccan female. They confirmed that a thalassemia phenotype was associated with this abnormal Hb variant. In addition, this Hb variant has frequently been found in southern Italy, where it is known as Hb Bernalda(1).

Hb Groene Hart is a routine finding in reference laboratories during the screening of large populations of North African origin for suspected {alpha}-thalassemia. The hematologic indication is usually present, although this feature varies among individuals (see Results). After the common deletions have been excluded with gap multiplex PCR analysis, sequencing of the {alpha}-globin genes reveals the presence of point mutations, and multiplex ligation-dependent probe amplification may or may not show the presence of large deletions(19). When a common {alpha}-thalassemic deletion is found, the genes coding {alpha} chains are not routinely sequenced to search for additional point mutations. In such cases, the method we have described may represent a protein-level diagnostic alternative to molecular analysis.

The discovery of AHSP and its properties has altered our understanding of Hb assembly(4)(5)(6)(7). AHSP stabilizes {alpha}-Hb during Hb synthesis by acting as a chaperone to protect free {alpha}-Hb chains from oxidative stress and by facilitating their folding(20)(21). In vitro experiments with recombinant Hb proteins with mutations at amino acid residue 199(H2)Pro of the {alpha}-globin chain [Hb Groene Hart (->Ser) and Hb Diamant (->Leu)] have revealed impaired interactions between {alpha}-Hb and AHSP(22). The RPLC system used in our study clearly detected these 2 Hb variants: We observed a slight 3.17-min increase in the retention time for the {alpha}Diamant-globin chain relative to that of the {alpha}Groene Hart-globin chain (data not shown).

The integrity of helix H and the GH corner of the {alpha}-globin chain appears to be critical for the interaction between {alpha}-Hb and AHSP. With the exception of a few amphibians, a Pro residue occupies position H2 in the {alpha}-chain sequences of more than 280 animal species(23). The existence of a difference between the {alpha}Groene Hart- and {alpha}Diamant-globin chains in the AHSP-association constant could explain the observed differences in the clinical and biochemical phenotypes of these 2 Hbs(7). Molecular modeling with the Swiss-PdbViewer suggests that replacing the Pro residue at the GH corner accounts for the drastic increase in hydrophobicity, which is similar to that observed with Hb Nouakchott [{alpha}114(GH2)Pro->Leu](24). In these Hb variants, replacing the Pro residue with a nonhelix breaker residue may facilitate the opening of the GH corner, thereby exposing a series of residues with hydrophobic side chains (Fig. 3Up ). It is possible that replacing a Pro residue at position 114 or 119 with a Leu residue would retain some of these hydrophobic contacts in the cellular environment. This hypothesis would explain the absence of a thalassemic phenotype in individuals with Hb Diamant and Hb Nouakchott.

Our demonstration of the presence of Hb Groene Hart in the hemolysate ruled out the previously suggested possibility of rapid postsynthesis degradation(8), and we detected no posttranslational modifications. The failure to detect this Hb in earlier studies can be attributed to its considerably increased hydrophobicity, which makes it undetectable with conventional methods. The presence of the {alpha}Groene Hart-globin chain can be detected consistently with the modified RPLC method we have described.

A few technical points need to be emphasized. The highly increased hydrophobicity of the {alpha}Groene Hart-globin chain relative to that of the wild-type {alpha}-globin chain required the use of long gradients that reached higher organic solvent concentrations. Increased TFA concentrations in the elution solvents decrease the retention times of these very hydrophobic species and allow their recovery. TFA is a known anionic ion-pairing agent and is effective at higher concentrations (>1 mL/L) for separating peptides(25)(26)(27).

When a DNA study reveals a point mutation causing a single amino acid substitution in a patient with thalassemic hematologic features, we recommend that a careful Hb study be performed to estimate the relative concentration of the variant. Furthermore, when the mutant Hb cannot be isolated with conventional methods because of a low concentration or high hydrophobicity, we recommend RPLC analysis. Although one may choose among chromatographic techniques that have already been described(28)(29), the new LC method we have described appears to be highly efficient for isolating hydrophobic molecules.


   Acknowledgments
 
Grant/Funding Support: This work was supported by CNRS CNRS UMR 5086, IFR 128 (I.Z.C., M.B.), DHOS Maladies Rares and Hospices Civils de Lyon (P.L., A.F.), and INSERM Unité 841 and Assistance Publique-Hôpitaux de Paris (H.W.).

Financial Disclosures: None declared.

Acknowledgments: H.W. and P.C.G. acknowledge support by the EC action "Infrastructure for Thalassaemia Research Network" (ITHANET, RI-2004-026539; http://www.ithanet.eu). We thank Véronique Baudin-Creuza (INSERM, Unité 779, Le Kremlin-Bicêtre, France) for helpful discussion. We thank Martine Aubry for excellent technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: Hb, hemoglobin; AHSP, {alpha}-Hb stabilizing protein; RPLC, Reversed-phase liquid chromatography; LC, liquid chromatography; ESI MS, electrospray ionization mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; {alpha}Groene Hart, {alpha} chain encoded by the HBA1 allele carrying the C->T mutation in codon 119; TFA, trifluoroacetic acid.

2 Human genes: HBA1, hemoglobin, alpha 1; HBA2, hemoglobin, alpha 2.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Patrinos GP, Giardine B, Riemer C, Miller W, Chui DHK, Anagnou NP, et al. Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Nucleic Acids Res ;32(Database Issue)::D537-D541http://globin.cse.psu.edu/hbvar/menu.html (Accessed April 2008)..
  2. Hardison RC, Chui DHK, Giardine B, Riemer C, Patrinos GP, Anagnou N, et al. HbVar: a relational database of human hemoglobin variants and thalassemia mutations at the globin gene server. Hum Mutat 2002;19:225-233.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  3. Giardine B, van Baal S, Kaimakis P, Riemer C, Miller W, Samara M, et al. HbVar database of human hemoglobin variants and thalassemia mutations: 2007 update. Hum Mutat 2007;28:206.[Medline] [Order article via Infotrieve]
  4. Kihm AJ, Kong Y, Hong W, Russel JE, Rouda S, Adachi K, et al. An abundant erythroid protein that stabilizes free {alpha}-haemoglobin. Nature 2002;417:758-763.[CrossRef][Medline] [Order article via Infotrieve]
  5. Santiveri CM, Perez-Canadillas JM, Vadivelu MK, Allen MD, Rutherford TH, Watkins NA, Bycroft M. NMR structure of the {alpha}-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding. J Biol Chem 2004;279:34963-34970.[Abstract/Free Full Text]
  6. Feng L, Gell DA, Zhou S, Gu L, Kong Y, Li J, et al. Molecular mechanism of AHSP-mediated stabilization of {alpha}-hemoglobin. Cell 2004;119:629-640.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  7. Vasseur-Godbillon C, Marden MC, Giordano P, Wajcman H, Baudin-Creuza V. Impaired binding of AHSP to {alpha} chain variants: Hb Groene Hart illustrates a mechanism leading to unstable hemoglobins with {alpha} thalassemic like syndrome. Blood Cells Mol Dis 2006;37:173-179.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  8. Harteveld CL, Van Delft P, Plug R, Versteegh FGA, Hagen B, Van Rooijen I, Kok PJMJ, Wajcman H, Kister J, Giordano PC. Hb Groene Hart: a new Pro->Ser amino acid substitution at position 119 of the {alpha}1-globin chain is associated with a mild {alpha}-thalassemia phenotype. Hemoglobin 2002;26:255-260.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  9. Siala H, Ouali F, Messaoud T, Sfar R, Fattoum S. First description in Tunisia of a point mutation at codon 119 (CCT->TCT) in the {alpha}1-globin gene: Hb Groene Hart in association with the –{alpha}3.7 deletion. Hemoglobin 2005;29:263-268.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  10. Giordano PC, Zweegman S, Akkermans N, Arkesteijn SGJ, van Delft P, Versteegh FGA, et al. The first case of Hb Groene Hart [{alpha}119(H2)Pro->Ser, CCT->TCT ({alpha}1)] homozygosity confirms that a thalassemia phenotype is associated with this abnormal hemoglobin variant. Hemoglobin 2007;31:179-182.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  11. Lacan P, Kister J, Francina A, Souillet G, Galactéros F, Delaunay J, Wajcman H. Hemoglobin Debrousse [β 96(FG3)Leu->Pro]: a new unstable hemoglobin with twofold increased oxygen affinity. Am J Hematol 1996;51:276-281.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  12. Morlé F, Francina A, Ducrocq R, Wajcman H, Gonnet C, Philippe N, et al. A new {alpha} chain variant Hb Sallanches [{alpha}2 104(G11)Cys->Tyr] associated with Hb H disease in one homozygous patient. Br J Haematol 1995;91:608-611.[Web of Science][Medline] [Order article via Infotrieve]
  13. Dodé C, Rochette J, Krishnamoorthy R. Locus assignment of human {alpha} globin mutations by selective amplification and direct sequencing. Br J Haematol 1990;76:275-281.[Web of Science][Medline] [Order article via Infotrieve]
  14. Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-PCR screen for common deletional determinants of {alpha}-thalassemia. Blood 2000;95:360-362.[Abstract/Free Full Text]
  15. DeepView Swiss-PdbViewer. Accession no. P69905. http://www.expasy.org/spdbv/ (Accessed August 2007)..
  16. Lacan P, Aubry M, Couprie N, Francina A. Two new {alpha} chain variants: Hb Die [{alpha}93(FG5)Val -> Ala ({alpha}1)] and Hb Beziers [{alpha}99(G6)Lys -> Asn ({alpha}1)]. Hemoglobin 2004;28:59-63.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  17. The ExPASy Proteomics Server of the Swiss Institute of Bioinformatics. Accession no. P69905 (release of September 2007). http://www.expasy.org/tools/peptide-mass.html (Accessed April 2008)..
  18. Shaeffer JR. ATP-dependent proteolysis of hemoglobin {alpha} chains in β-thalassemic hemolysates is ubiquitin-dependent. J Biol Chem 1988;263:13663-13669.[Abstract/Free Full Text]
  19. Harteveld CL, Voskamp A, Phylipsen M, Akkermans N, den Dunnen JT, White SJ, Giordano PC. Nine unknown rearrangements in 16p13.3 and 11p15.4 causing {alpha}- and β-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet 2005;42:922-931.[Abstract/Free Full Text]
  20. Yu X, Kong Y, Dore LC, Abdulmalik O, Katein AM, Zhou S, et al. An erythroid chaperone that facilitates folding of {alpha}-globin subunits for hemoglobin synthesis. J Clin Invest 2007;117:1856-1865.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  21. Bank A. AHSP: a novel hemoglobin helper. J Clin Invest 2007;117:1746-1749.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  22. Préhu C, Riou J, Godart C, Bost M, Barro C, Promé D, et al. Hb Diamant [{alpha}119(H2)Pro->Leu]: a new variant with a modification at the {alpha}1β1 interface. Hemoglobin 2000;24:249-252.[Web of Science][Medline] [Order article via Infotrieve]
  23. Marden MC, Dewilde S, Kiger L, Hamdane D, Uzan J, Burmester T, et al. Exploiting a list of protein sequences. Gene 2007;398:35-41.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  24. Wajcman H, Delaunay J, Francina A, Rosa J, Galacteros F. Hemoglobin Nouakchott [{alpha}114(GH2)Pro->Leu]: a new hemoglobin variant displaying an unusual increase in hydrophobicity. Biochim Biophys Acta 1989;998:25-31.[CrossRef][Medline] [Order article via Infotrieve]
  25. Mant CT, Hodges RS. Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: implications for prediction of peptide retention behaviour. J Chromatogr A 2006;1125:211-219.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  26. Chen Y, Mehok AR, Mant CT, Hodges RS. Optimum concentration of trifluoroacetic acid for reversed-phase liquid chromatography of peptides revisited. J Chromatogr A 2004;1043:9-18.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  27. Guo DC, Mant CT, Hodges RS. Effects of ion-pairing reagents on the prediction of peptide retention in reversed-phase high-performance liquid chromatography. J Chromatogr 1987;386:205-222.[Web of Science][Medline] [Order article via Infotrieve]
  28. Leone L, Monteleone M, Gabutti V, Amione C. Reversed-phase high-performance liquid chromatography of human haemoglobin chains. J Chromatogr 1985;321:407-419.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. Wajcman H, Riou J, Yapo AP. Globin chain analysis by reversed phase high performance liquid chromatography: recent developments. Hemoglobin 2002;26:271-284.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
clinchem.2007.097857v1
54/6/1053    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zanella-Cleon, I.
Right arrow Articles by Francina, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zanella-Cleon, I.
Right arrow Articles by Francina, A.
Related Collections
Right arrow Hematology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS