A compound heterozygote in the NADH-cytochrome b5 reductase gene from a Chinese patient with hereditary methemoglobinemia type I.

To elucidate the mutation in the nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) gene from a Chinese patient with hereditary methemoglobinemia type I, we analyzed the coding sequences of b5R cDNA from the patient and from normal subjects by direct sequencing the reverse transcriptase-polymerase chain reaction (RT-PCR) products. The PCR-amplified genomic DNA fragments of the b5R gene from the patient, his mother, and normal controls were analyzed by restriction enzymes MspI and RsaI. A compound heterozygote Arg57Gln (CGG-->CAG)/Cys203Tyr (TGC-->TAC) was found in the b5R gene from the patient, and a CGG-->CAG mutant allele occurred in a chromosome inherited from his mother, while TGC-->TAC occurred in a chromosome inherited from his father. In this report, we discuss a compound heterozygote first observed in the b5R gene from a patient with hereditary methemoglobinemia type I.

Antibody-based spot test for NADH-cytochrome b5 reductase activity for the laboratory diagnosis of congenital methemoglobinemia.

NADH (reduced Coenzyme I)-cytochrome b5 reductase (b5R) is a multifunctional redox enzyme, whose deficiency causes recessive congenital methemoglobinemia. A novel procedure for the detection of b5R activity in human hemolysates was developed, in which b5R monoclonal antibodies dot-blotted on nitrocellulose membrane was used to capture and enrich b5R from hemolysates, and the captured b5R activity was subsequently visualized with the substrate 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Application of this simple method to the detection of b5R activity in the hemolysates from different subjects demonstrated that it was both sensitive and reliable. Our method would be useful for the laboratory diagnosis of congenital methemoglobinemia.

Detection of Shigella and enteroinvasive Escherichia coli using polymerase chain reaction.

Two oligonucleotide primers were used in a polymerase chain reaction (PCR) procedure to amplify a region of the invasive-associated locus (ial) of Shigella and enteroinvasive Escherichia coli (EIEC). Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of these two pathogens. PCR is done using DNA extracted directly from faeces. The procedure can be completed in 7 h. These findings demonstrate a novel method for rapid, sensitive, specific, and simple diagnosis of diarrhoea caused by Shigella and EIEC.

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 dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into 2 clinical 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 b5R enzyme activity in the blood cells. To learn the molecular basis of type 1 recessive congenital methemoglobinemia in this Chinese family, we isolated total RNA from the peripheral leukocytes of the propositus and b5R complementary DNA (cDNA) by reverse transcription- polymerase chain reaction (RT-PCR). The coding region of the b5R cDNA was analyzed by sequencing the cloned 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-transferase (GST)-fused wild-type b5R and GST-fused mutant Cys203Tyr b5R were expressed 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 manifestations of these type 1 patients. The finding of this novel mutation makes codon 203 the only position within the b5R gene at which more than 1 mutation has been found.