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Recessive congenital methaemoglobinaemia: functional characterization of the novel D239G mutation in the NADH‐binding lobe of cytochrome b5 reductase

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (cytb5r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255‐ and G291D, have been identified in the NADH‐binding lobe of cytb5r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845–48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb5r mutations were also identified, including the R159‐ mutation, which causes loss of the entire NADH‐binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH‐binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb5r. In contrast to the E255‐ and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb5r deficient patients who are heterozygous for an NADH‐binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH‐binding lobe.