A novel mutation in the NADH-cytochrome b5 reductase gene of a Chinese patient with recessive congenital methemoglobinemia

Recessive congenital methemoglobinemia due to nicotinamide adenine dinucleo tide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into 2 c linical types: type 1 (erythrocyte type) and type 2 (generalized type). We found a Chinese family with type 1 recessive congenital methemoglobinemia, the patients from which were diagnosed according to clinical symptoms and b 5R enzyme activity in the blood cells. To learn the molecular basis of type 1 recessive congenital methemoglobinemia in this Chinese family, we isolat ed total RNA from the peripheral leukocytes of the propositus and b5R compl ementary DNA (cDNA) by reverse transcription-polymerase chain reaction (RT- PCR). The coding region of the b5R cDNA was analyzed by sequencing the clon ed PCR products. The results showed that the propositus was homozygous for a G-A transition at codon 203 in exon 7, changing a cysteine to a tyrosine (Cys203Tyr), To characterize the mutant enzyme, both glutathione S-transfer ase (GST)-fused wild-type b5R and GST-fused mutant Cys203Tyr b5R were expre ssed in Escherichia coli and affinity purified, The results showed that the catalytic activity of the enzyme was not much affected by this amino acid substitution, but the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin, These properties of the mutant enzyme would account for the restricted b5R deficiency and mild clinical manifesta tions of these type 1 patients. The finding of this novel mutation makes co don 203 the only position within the b5R gene at which more than 1 mutation has been found. (C) 2000 by The American Society of Hematology.