2 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 dinuc leotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). W e investigated the b5R gene of a patient with type II from a white Uni ted Kingdom (UK) family and found that the patient was a compound hete rozygote for two novel mutations, The first mutation was a C-to-A tran sversion changing codon 42 (TAC: Tyr) to a stop codon in the one allel e, 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 ch anged to His within the flavin adenine dinucleotide (FAD)-binding doma in of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fuse d b5R with the GST-fused mutant enzyme with the codon 95 missense muta tion (P95H) expressed in Escherichia coli, The mutant enzyme showed le ss catalytic activity, less thermostability, and a greater susceptibil ity to trypsin than did the normal counterpart, The absorption spectru m of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution inf luences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutatio ns apparently caused hereditary methemoglobinemia type II in this pati ent. (C) 1996 by The American Society of Hematology.