Strategy for Identification by Mass Spectrometry of a New Human Hemoglobin Variant with Two Mutations in Cis in 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 [β6(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 β^S-globin chain, may inhibit or enhance the polymerization process. We describe here a new hemoglobin (Hb) variant (Hb S-Clichy) which carries the β^S‐globin chain and an additional mutation β8(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 β 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 βT-1 and βT-2 were not observed on the matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrum but a new peptide βT-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.

Keywords:

• Electrospray ionization mass spectrometry (ESI-MS)
• Matrix-assisted laser desorption-time of flight MS (MALDI-TOF MS)
• Tandem MS/MS
• Hb S-Clichy