Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia

Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the bSR gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. From this mutant allele, the product without the catalytic portion of the enzyme is generated. The second one was a missense mutation at codon 95 (CCC → CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coli. The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart. The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.