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Hemoglobinopathies are the most frequent genetic diseases in the world. Among them, the Hb S variant [beta6(A3)Glu-->Val], which, in the homozygous state, produces a severe disease known as sickle cell anemia with polymerization of Hb S inside red blood cells under hypoxic conditions. Additional mutations, in cis or in trans of the beta(S)-globin chain, may inhibit or enhance the polymerization process. We describe here a new hemoglobin (Hb) variant (Hb S-Clichy) which carries the beta(S)-globin chain and an additional mutation beta8(A5)Lys-->Thr. The variant was detected by routine electrophoretic techniques and cation exchange liquid chromatography (CE-LC). Globin chain separation by reversed phase LC (RP-LC) showed normal and abnormal beta chains, confirming that the additional abnormality was located in cis to Hb S. Electrospray ionization mass spectrometry (ESI-MS) gave a 57 Da mass decrease for the abnormal globin chain. The abnormal chain was isolated and submitted to trypsin digestion. Normal peptides betaT-1 and betaT-2 were not observed on the matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrum but a new peptide betaT-1,2 was detected. Nano LC-ESI-MS/MS of the new peptide showed that the glutamic acid at codon 6 was replaced by a valine residue, and the lysine at codon 8 was replaced by a threonine residue, as confirmed by DNA sequencing. This example demonstrates that in a population where Hb S is present, every unidentified Hb needs to be clearly characterized to prevent major sickle cell syndromes. In addition, the identification of these variants must be considered in newborn screening for sickle cell disease, using either classical biochemical methods or MS techniques.

We present here five new hemoglobin (Hb) variants which have been identified during routine Hb analysis before their genotypic characterization. Four of these result from a classical missense mutation: Hb Canuts [α85(F6)Asp→His (α1)], Hb Ambroise Pare [α117(GH5)Phe→Ile (α2)], Hb Beaujolais [β84(EF8)Thr→Asn] and HbA2-North Africa [δ59(E3)Lys→Met]. The last one, Hb Monplaisir [β147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)], results from a frameshift mutation at the stop codon of the β-globin gene which leads to a modified C-terminal sequence in the β-globin chain. None of these variants seem to have a particular clinical expression in the heterozygous state. The circumstances of the discovery of these five new Hb variants emphasize the fact that an association of techniques is necessary for a complete screening of Hb variants during routine Hb analysis. Globin chain separation by reversed phase liquid chromatography (RP‐LC) appears to be the most relevant method.

Background: Hemoglobin (Hb) Groene Hart [α119 (H2)Pro→Ser (α1)], also known as Hb Bernalda, is a nondeletional α-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 α-thalassemic behavior of this variant has been explained as an impaired interaction between the α-globin chain and the α-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 αA-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 −α3.7 deletion). These patients possessed abnormal hematologic features suggesting an α-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.

We used a 2D-electrophoresis (2-DE) proteomic approach to identify novel biomarkers in node-negative breast cancers. This retrospective study focused on a population of patients with ductal pN0M0 tumours. A subset of patients who developed metastases and in whose tumours were found high levels of uPA and PAI-1 (metastatic relapse, MR: n=20) were compared to another subset in whom no metastatic relapse occurred and whose tumours were found to have low levels of uPA and PAI-1 (no relapse, NR: n=21). We used a 2-DE coupled with MS approach to screen cytosol fractions using two pH-gradient scales, a broad scale (3.0-11.0) and a narrower scale focussing in on a protein rich region (5.0-8.0). This study was conducted on 41 cytosol specimens analyzed in duplicate on two platforms. The differential analysis of more than 2,000 spots in 2-DE gels, obtained on the two platforms, allowed the identification of 13 proteins which were confirmed by western blotting. Two proteins, GPDA and FABP4 were down-regulated in the MR subset whereas all the others were up-regulated. An in silico analysis revealed that GMPS (GUAA), GAPDH (G3P), CFL1 (COF1) and FTL (FRIL), the most informative genes, displayed a proliferation profile (high expression in basal-like, HER2+ and luminal B molecular subtypes). Inversely, similar to FABP4, GPD1 [GPDA] displayed a high expression in luminal A subtype, a profile characteristic of tumour suppressor genes. Despite the small size of our cohort, the 2-DE analysis gave interesting results which were confirmed by the in silico analysis showing that some of the corresponding genes had a strong prognostic impact in breast cancer, mostly because of their link with proliferation: GMPS, GAPDH, FTL and GPD1. A validation phase on a larger cohort is now needed before these biomarkers could be considered for use in clinical practice.

DNA analysis is currently the easiest way to identify a hemoglobin variant in most cases. Nevertheless, in case of complex gene rearrangements, mass spectrometry studies may be required to orientate the DNA diagnosis. The present report shows the use of mass spectrometry techniques prior to DNA analysis for the identification of the rare P-Nilotic fusion hemoglobin. Complete protein analysis is performed by liquid chromatography–tandem mass spectrometry on the abnormal globin chain isolated by reversed-phase liquid chromatography.

We present here five new hemoglobin (Hb) variants which have been identified during routine Hb analysis before their genotypic characterization. Four of these result from a classical missense mutation: Hb Canuts [alpha85(F6)Asp--His (alpha1)], Hb Ambroise Pare [alpha117(GH5)Phe--Ile (alpha2)], Hb Beaujolais [beta84(EF8)Thr--Asn] and HbA(2)-North Africa [delta59(E3)Lys--Met]. The last one, Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)], results from a frameshift mutation at the stop codon of the beta-globin gene which leads to a modified C-terminal sequence in the beta-globin chain. None of these variants seem to have a particular clinical expression in the heterozygous state. The circumstances of the discovery of these five new Hb variants emphasize the fact that an association of techniques is necessary for a complete screening of Hb variants during routine Hb analysis. Globin chain separation by reversed phase liquid chromatography (RP-LC) appears to be the most relevant method.

A new hemoglobin (Hb) variant in the heterozygous state, Hb Al-Hammadi Riyadh [codon 75 (GAC--GTC); alpha75(EF4)Asp--Val (alpha2)] corresponding to an A--T transversion on the second exon of the alpha2-globin gene, is described. The variant was characterized by DNA sequencing and mass spectrometry (MS). The variant was found during a routine Hb analysis for anemia in a 16-month-old boy who lived in Riyadh, Kingdom of Saudi Arabia.

A new G(gamma) hemoglobin (Hb) variant, Hb F-Bron [gamma20(B2)Val--Ala] on the first exon of the G(gamma)-globin gene is described. The variant was characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities included hypochromia and microcytosis and were probably caused by an interaction with an alpha-thalassemia (thal) (3.7 kb) deletion in the heterozygous state.

Two new hemoglobin (Hb) variants: Hb Brem-sur-Mer [codon 9 (TCT--TAT); beta9(A6)Ser--Tyr] on the first exon of the beta-globin gene and Hb Passy [codon 81 (TCC--CCC); alpha81(F2)Ser--Pro (alpha2)] on the second exon of the alpha2-globin gene, are described. The two variants were characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities: microcytosis and hypochromia were found only in the carrier of Hb Passy. In the absence of an association with an alpha-thalassemic deletion or mutation, the mutation 81(F2)Pro could induce a possible alpha-thalassemia (thal).

Two new beta-globin chain variants: Hb Tripoli: codon 26, GAG--GCG [beta26(B8)Glu--Ala] and Hb Tizi-Ouzou: codon 29, GGC--AGC [beta29(B11)Gly--Ser] are described on the first exon of the beta-globin gene. The two variants are characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities were found in the two carriers. The presence of microcytosis and hypochromia is explained by an additional homozygous 3.7 kb alpha(+) thalassemic deletion for the carrier of Hb Tizi-Ouzou. Hb Tizi-Ouzou showed a slight instability in vitro. The same hematological abnormalities associated with anemia are difficult to explain for Hb Tripoli's carrier in the absence of an alpha-globin genes abnormality and could suggest a possible abnormal splicing.