Novel hemoglobin alpha chain elongation resulting from a 15-residue insertion and tandem duplication of the F helix.
OBJECTIVE
To determine the cause of an unusual hemoglobin pattern with two novel components eluting after HbA(2) on cation exchange HPLC. This variant was detected during HbA1c measurement and was associated with a normal blood count and a positive isopropanol test.


METHODS
Whole 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.


RESULTS
ESI MS showed a mass increase of 1614Da in 9% of the alpha globin chains and sequence analysis of the alpha2 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 alpha79-93 and signature ions from the new peptide were clearly visible in peptide maps of the unstable hemoglobin.


CONCLUSIONS
The 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, (alpha(L) beta), 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 alpha(L) chain.
Molecular Pathology, Canterbury Health Laboratories, Christchurch, New Zealand. steve.brennan@chmeds.ac.nz
Stephen O
SO
Brennan
Maurice C
MC
Owen
Tim
T
Chan
Anna
A
Ruskova
D000328:Adult; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D020440:Gene Duplication; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D007474:Ion Exchange; D008297:Male; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010441:Peptide Chain Elongation, Translational; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics*; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000378:metabolism
D006454:Hemoglobins; D010446:Peptide Fragments; D010455:Peptides; C070077:alpha(A) globin; D014357:Trypsin