A hemoglobin variant associated with neonatal cyanosis and anemia

Abstract

Globin-gene mutations are a rare but important cause of cyanosis. We identified a missense mutation in the fetal Gγ-globin gene (HBG2) in a father and daughter with transient neonatal cyanosis and anemia. This new mutation modifies the ligand-binding pocket of fetal hemoglobin by means of two mechanisms. First, the relatively large side chain of methionine decreases both the affinity of oxygen for binding to the mutant hemoglobin subunit and the rate at which it does so. Second, the mutant methionine is converted to aspartic acid post-translationally, probably through oxidative mechanisms. The presence of this polar amino acid in the heme pocket is predicted to enhance hemoglobin denaturation, causing anemia.

Figure 1. The Hemoglobin Toms River Mutation

Panel A depicts the human β-globin gene cluster on chromosome 11. Affected persons are heterozygous for a transition from guanine to adenine (G→A) at nucleotide position 202 of the G γ-globin gene. This switch substitutes methionine (Met) for valine (Val) at codon 68, or amino acid 67, after the post-translational cleavage of methionine at position 1. The mutant methionine residue is converted to aspartic acid (Asp) post-translationally. Valine is at position 11 in α-helix E of γ-globin. Panel B depicts the hemoglobin Toms River pedigree, with squares representing male members and circles female members and shading indicating members with neonatal cyanosis and anemia. The individual G γ-globin genotypes for each person are indicated. Panel C shows the structure of the oxygen-binding pockets of the γ-globin subunits in persons with wild-type (left image) and mutant (right image) V67M deoxygenated hemoglobin F. The wild-type subunit shows valine at position 67; the other subunit shows the predicted structure of the Toms River mutation, in which methionine has been inserted into the wild-type structure at position 67. In both images, the γ subunits are depicted as yellow ribbons. The planar heme group and its associated proximal histidine are depicted with red and yellow stick models, respectively. The iron atom is shown as an orange sphere. Oxygen enters the heme pocket in the wild-type subunit (indicated by the blue arrow) and binds iron on the upper face of the heme ring next to the valine 67 side chain. In the mutant subunit, the residues at position 67 are depicted as spheres, with blue indicating the additional methionine atoms that prevent oxygen from having access to the iron atom in the center of the heme ring. The insets show wild-type valine and mutant methionine in stick and dot format to indicate the difference in size between the amino acids.

Figure 2. Rates of Ferricyanide-Induced Methemoglobin Formation in the Patient and a Control

Blood samples from the patient with the hemoglobin (Hb) Toms River mutation were analyzed when she was 6 days old and again when she was 6 months old. Erythrocyte lysates were diluted to a final heme concentration of 25 μM in 10 mM HEPES buffer (pH 7.4). The change in heme spectra was monitored after the addition of 25 μM of potassium ferricyanide, which oxidizes ferrous (Fe²⁺) Hb to ferric (Fe³⁺) methemoglobin. The initial rate of oxidation was more rapid when the patient was 6 days old. Similar results were obtained at a range of ferricyanide concentrations. HbA indicates data derived from purified hemoglobin A, which was used as an additional control.