Novel haemoglobin mutation (α127Lys→Glu) increases oxygen affinity and has a minor effect on haptoglobin binding

• Published in: Clinical biochemistry Vol. 45; no. 18; pp. 1587 - 1590
• Main Authors: Brennan, Stephen O., Pullon, Beverley, Owen, Maurice C., Brittain, Thomas
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01-12-2012; Elsevier
• Subjects: Aged; Amino Acid Substitution - genetics; Base Sequence; Biological and medical sciences; Chromatography; Chromatography, Reverse-Phase; DNA Mutational Analysis; DNA sequencing; Female; Haemoglobin mutation; Haptoglobin; Haptoglobin binding; Haptoglobins - metabolism; Hemoglobin; Hemoglobin A - genetics; Hemoglobins - chemistry; Hemoglobins - genetics; Humans; Investigative techniques, diagnostic techniques (general aspects); Ion exchange; Mass spectrometry; Medical sciences; Models, Molecular; Molecular modelling; Molecular Sequence Data; Mutation; Mutation - genetics; Oxygen; Oxygen - metabolism; Oxygen affinity; Peptide mapping; Phenotype; Polycythemia; Protein Binding; Spectrometry, Mass, Electrospray Ionization; RBC; Phenotype; MS; Hp; ESI; Hb; Oxygen affinity; Haptoglobin binding; Mass spectrometry; Haemoglobin mutation; Binding; Exploration; Haptoglobin; Clinical biology; Hemoglobin; Genetics; Minor; Mutation
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2012.08.021

To determine if a new haemoglobin (Hb) variant was the underlying cause of erythrocytosis in a subject with a high apparent HbA1c.
Haemolysate was analysed by ESI MS, and individual components purified by ion exchange and reverse phase chromatography. Peptide mapping was used to pinpoint the substitution and DNA sequencing to confirm the precise mutation. Oxygen affinity was measured and relative haptoglobin (Hp) binding estimated.
Intact protein analysis and peptide mapping suggested a mutation in peptide α13 and DNA sequencing confirmed a novel α127Lys→Glu substitution in the α 2 gene. The abnormal Hb had a significantly higher O2 affinity (5.8mmHg) than HbA (12.4mmHg). In addition the mutation caused a small but significant decrease in Hp binding.
Molecular models show that the side chain of α127Lys stabilises the T structure of deoxy Hb and that mutation to Glu would favour conversion to the high affinity R state. Notwithstanding this and the demonstrated high affinity, there was only a small increase in RBCs, Hb concentration and PCV in other female carriers of the mutation. The absence of a significant phenotype of erythrocytosis is most probably due to the low level (19%) of the variant.
► Novel Hb was fortuitously detected in carrier with erythrocytosis on A1c analysis. ► Molecular modelling showed critical functional location of mutation. ► Functional analysis showed increased O2 affinity and decreased haptoglobin binding. ► Family studies showed only a mild response to tissue anoxia. ► Substitution correlated with structure, function and phenotype changes.