Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH‐cytochrome b₅ reductase gene

Abstract

Hereditary methaemoglobinaemia, caused by deficiency of NADH‐cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C‐to‐T substitution in codon 83 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C‐to‐T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life‐span of erythrocytes.

Seven new mutations in the nicotinamide adenine dinucleotide reduced–cytochrome b5 reductase gene leading to methemoglobinemia type I

“…Some of these mutant enzymes have been expressed in bacterial systems, purified and studied for kinetic properties, heat stability, and trypsin sensitivity. 33,34,38,39 In general, the recombinant type I mutant enzymes were found to have retained about 60% to 70% of the catalytic activity expressed by the recombinant wild-type enzyme, but to be more heat labile and trypsin sensitive. Similar experiments have been carried out with mutant enzymes from patients with type II methemoglobinemia.…”

“…In the literature, 7 additional amino acid substitutions have been reported in patients with type I methemoglobinemia [33][34][35][36][37][38][39] (Table 3). Some of these mutant enzymes have been expressed in bacterial systems, purified and studied for kinetic properties, heat stability, and trypsin sensitivity.…”

“…8 Until now, 7 different mutations in this gene have been described to lead to type I b5R deficiency, [33][34][35][36][37][38][39] and 11 different mutations to the type II disease. 31,32,34,[40][41][42][43][44] In general, type I mutations were found in "marginal portions" of the enzyme, giving rise to enzymatically active, but unstable proteins.…”

Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH-cytochrome b5 reductase

“…Previously, 15 mutations had been associated with the type 1 disorder 13-20 and 16 with type 2 RCM. 17,[20][21][22][23][24][25][26][27] Mutations have been found in all exons but 1 and 1S. Mutations that cause exon skipping and those predicted to give truncated proteins with severe impairment of function have been described only in type 2, except in one child in whom neurologic abnormalities were not observed at the age of 1 year.…”

Methemoglobinemia and Eccentrocytosis in Equine Erythrocyte Flavin Adenine Dinucleotide Deficiency

“…1). 25 Methemoglobinemia usually results from either increased production of methemoglobin by oxidants or decreased reduction of methemoglobin because of a deficiency in erythrocyte Cb 5 R. 19 Methemoglobinemia also has been reported in people with various hemoglobinopathies (referred to as hemoglobin M) that result in methemoglobin formation that is not amenable to reduction. 34 No source of exogenous oxidants was found in the mustang, and the rapid reduction of the methemoglobinemia with intravenous methylene blue excluded a similar hemoglobinopathy.…”