Molecular analysis of hereditary methemoglobinemia

Hereditary methemoglobinemla is an autosomal recessive disorder characterized by NADH-cytochrome bo reductase (b5R) deficiency. This disorder is classified Into two types: an erythrocyte (type I) and a generalized (type II) types. We analyzed the bSRgenes of a patient with type Üfrom a white U. K. family using PCR-related techniques, and found that the patient was a compound hétérozygote for two novel mutations, a nonsense mutation at codon 42 (TAC-TAA) and a mlssense mutation at codon 95 (CCC; Pro-CAC; His). From the allele with the nonsense mutation, the product without the catalytic portion of the enzyme Is generated. To characterize the effect of the missense mutation on the enzyme function, we compared GST-fused b5R with the GST-fused mutant enzyme expressed in Escherichia. call The mutant enzyme exhibited less catalytic activity, less thermostability and more 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 suggested that this amlno acid substitution within the FAD-blnding domain of the enzyme Influences both a secondary structure and a catalytic activity of the enzyme. One Japanese case of type n was also Investigated. A novel nonesense mutation (GAG;Glu-TAG;Stop) was detected In the codon 255 of one allele, however no mutation is observed in another allele. A Greek-Cyprus case with typell, in which no obvious mutation Is associated with the b5R gene, was presented. Possible molecular basis of such cases was discussed.