Hereditary methemoglobinemia is an autosomal recessive disorder characterized by NADH-cytochrome b5 reductase (b5R) deficiency. In an attempt to clarify the molecular mechanisms involved in the enzyme deficiency, we isolated the b5R gene from a patient homozygous for hereditary methemoglobinemia, generalized type, and compared its nucleotide sequence with that of the normal NADH-cytochrome b5R gene. Only one difference was observed; a thymidine at the first position of codon 127 (TCT) was altered to a cytidine in the b5R gene of the patient, resulting in replacement of serine with proline. Dot blot hybridization of the amplified DNA samples with allele-specific oligonucleotide probes showed that the proband and her brothers were homozygous for this mutation and that their father was heterozygous. Although the activity of b5R in lymphoblastoid cells from homozygotes was reduced to 10% of the normal level, RNA blot and protein blot analyses of the lymphoblastoid cells showed that synthesis of b5R messenger RNA and the b5R polypeptide were normal. Serine at residue 127 is presumed to be in an alpha-helix structure that is part of a nucleotide-binding domain. These observations suggest that replacement of Pro-127 causes a significant conformation change in the nucleotide- binding domain that affects electron transport from NADH to cytochrome b5. Functional enzyme deficiency results in a generalized type of hereditary methemoglobinemia.

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