A New Hemoglobin Variant Found During Hb A_1c Measurement: Hb Hokusetsu [β52(D3)Asp→Gly]

Abstract

A new β chain variant was accidentally found through the assay of Hb A_1c in a diabetic patient. The variant was detected by polyacrylamide gel isoelectrofocusing and electrospray ionization mass spectrometry. For sequence determination, globin was cleaved with combination of trypsin and lysyl endopeptidase and analyzed by high performance liquid chromatography connected to electrospray ionization mass spectrometry. An abnormal βT-5 peptide was found by reconstructed selected ion monitoring. The collision-induced dissociation spectrum of an ion derived from the abnormal βT-5 peptide revealed a new substitution, [β52(D3)Asp→Gly], named Hb Hokusetsu. The sequence was confirmed with an automatic sequencer using peptides isolated by reversed phase high performance liquid chromatography. Amplification of the β-globin exon 2 and nucleotide sequencing revealed a GATG`T mutation in codon 52 corresponding to an Asp→Gly replacement. Electrospray ionization mass spectrometry analysis of the hemolysate showed a reasonable value of 10.4% for glycated globin. The variant migrated as Hb S on isoelectrofocusing. Hematological analysis revealed normal parameters. The patient's hemolysate showed normal stability in the isopropanol test. Oxygen equilibrium studies on the patient's red blood cells and hemolysate showed no significant change in oxygen affinity or cooperativity.