Strategy for Identification by Mass Spectrometry of a New Human Hemoglobin Variant with Two Mutations inCisin the β-Globin Chain: Hb S-Clichy [β6(A3)Glu→Val; β8(A5)Lys→Thr]

Abstract

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.

Hemoglobin Variant Analysis of Whole Blood and Dried Blood Spots by MS

“…TWIMS separates ions on the basis of both shape and m/z. Williams et al were able to demon strate that TWIMS could separate the future science group doubly charged tryptic peptide (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) containing the low-abundance (4%) Hb Fort Worth variant (a27 Glu→Gly, Dm -72.0211 Da), and the singly charged normal tryptic peptide (1)(2)(3)(4)(5)(6)(7) at m/z 729. They also demonstrated separation of the singly charged peptide (12)(13)(14)(15)(16) containing variant Hb J-Paris-I (a12 Ala→Asp, Dm +43.9898 Da) at m/z 576 and the third isotope of the doubly charged b-globin (133-144) peptide.…”

“…The compound variant HbSE (HbE is the result of substitution of a glutamic acid for a lysine at position 26 on the b-globin chain) is also uncommon and is generally asymptomatic, however, sickling disorders may develop that require medical intervention [16]. In addition, double mutations, such as HbS-Clichy, are possible, whereby the sickle variant is accompanied by another amino acid substitution in the same globin chain [17]. The additional mutations can enhance Hb polymerization.…”

A New Sickling Variant ‘Hb S-Wake β[(Glu6Val-Asn139 Ser)]’ Found in a Compound Heterozygote with Hb S β(Glu6Val) Coinherited with Homozygous α-Thalassemia-2: Phenotype and Molecular Characteristics

“…Hb SC-Harlem β[(Glu6Val)(Glu6Val-Asp73Asn)] and Hb SC-Ndjamena β[(Glu6Val)(Glu6Val-Trp37Gly)] are the other 2 cases with clinical symptoms that are similar to those with sickle cell anemia. Neither one of these 3 cases simultaneously inherited other globin gene abnormalities [3,13]. …”

“…Twelve similar Hb variants have been reported so far and 3 of them were found in a compound heterozygous state with Hb S that lead to sickling disorders. The names, the location of second amino acid substitutions, and related references are listed in a recently published article [3]. …”

Syndromes drépanocytaires atypiques : à propos de deux cas

“…L'électrophorèse de l'Hb de contrôle effectuée à l'âge de 12 ans a montré un profil électrophorétique proche de celui d'un porteur du trait drépanocytaire ( Quelques cas très rares de SDM, décrits dans la litté-rature, sont dus à des doubles mutants Hb comportant la mutation ␤ 6 val et une autre mutation en cis dans la chaîne ␤-globine. Plusieurs ont été décrits dans la littérature : HbS-Antilles [23], HbS-Oman [24], HbS-Cameroon [9], HbSProvidence [16], HbS-Travis [17], HbS-Clichy [18], HbS-South End [19] HbC-Ziguinchor [20], HbC-Harlem [8], Hb-Jamaïca Plain [21]. Ces doubles mutants ont un comportement élec-trophorétique à pH alcalin similaire à celui de l'HbS ou de l'HbC, plus rarement à celui de l'HbA (HbS-Providence, HbSSouth End).…”