Novel hemoglobin α chain elongation resulting from a 15-residue insertion and tandem duplication of the F helix

ObjectivesTo determine the cause of an unusual hemoglobin pattern with two novel components eluting after HbA2 on cation exchange HPLC. This variant was detected during HbA1c measurement and was associated with a normal blood count and a positive isopropanol test.Design and methodWhole hemolysate and isopropanol precipitates were analysed by ESI MS, and individual components were purified by reverse phase and cation exchange HPLC. Tryptic peptide mapping of isopropanol precipitates was used to detect the molecular lesion and DNA sequencing was used to characterise the precise rearrangement.ResultsESI MS showed a mass increase of 1614Da in 9% of the α globin chains and sequence analysis of the α2 gene revealed the heterozygous insertion of 45 nucleotides after codon 93. The predicted in phase incretion of ALSALSDLHAHKLRV (+ 1613Da) is a direct repeat of residues α79–93 and signature ions from the new peptide were clearly visible in peptide maps of the unstable hemoglobin.ConclusionThe insertion probably results from replication slippage during DNA synthesis and the 15-residue repeat results in full repetition of the heme-linked F helix. The nature of the inserted sequence explains the molecular instability and the electrophoretic mobility, but not the twin peaks observed on cation exchange HPLC. These components had the same chain composition, (αL β), the same number of heme groups per chain, were not in rapid equilibrium with each other, and probably represent hemoglobin species with different conformers of the elongated αL chain.