Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH-cytochrome b5 reductase

In 1943, the first description of familial idiopathic methemoglobinemia in the United Kingdom was reported in 2 members of one family. Five years later, Quentin Gibson (then of Queen's University, Belfast, Ireland) correctly identified the pathway involved in the reduction of methemoglobin in the family, thereby describing the first hereditary trait involving a specific enzyme deficiency. Recessive congenital methemoglobinemia (RCM) is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)–cytochrome b5 reductase. One of the original propositi with the type 1 disorder has now been traced. He was found to be a compound heterozygote harboring 2 previously undescribed mutations in exon 9, a point mutation Gly873Ala predicting a Gly291Asp substitution, and a 3-bp in-frame deletion of codon 255 (GAG), predicting loss of glutamic acid. A brother and a surviving sister are heterozygous; each bears one of the mutations. Thirty-three different mutations have now been recorded for RCM. The original authors' optimism that RCM would provide material for future genetic studies has been amply justified.

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

Blood ◽

10.1182/blood.v95.10.3250 ◽

2000 ◽

Vol 95 (10) ◽

pp. 3250-3255 ◽

Cited By ~ 21

Author(s):

Yao Wang ◽

Yu-Shui Wu ◽

Pei-Zhen Zheng ◽

Wen-Xi Yang ◽

Guo-An Fang ◽

...

Keyword(s):

Clinical Symptoms ◽

Novel Mutation ◽

Cytochrome B5 ◽

Clinical Manifestations ◽

Mutant Enzyme ◽

Chinese Family ◽

Coding Region ◽

Cytochrome B5 Reductase ◽

Nadh Cytochrome

Abstract 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.

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Recessive hereditary methemoglobinemia: Two novel mutations in the NADH-cytochrome b5 reductase gene

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2008.02.002 ◽

2008 ◽

Vol 41 (1) ◽

pp. 50-55 ◽

Cited By ~ 20

Author(s):

Elisa Fermo ◽

Paola Bianchi ◽

Cristina Vercellati ◽

Anna Paola Marcello ◽

Massimo Garatti ◽

...

Keyword(s):

Cytochrome B5 ◽

Novel Mutations ◽

Cytochrome B5 Reductase ◽

Reductase Gene ◽

Nadh Cytochrome

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Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia

Blood ◽

10.1182/blood.v88.8.3208.bloodjournal8883208 ◽

1996 ◽

Vol 88 (8) ◽

pp. 3208-3215 ◽

Cited By ~ 27

Author(s):

J Manabe ◽

R Arya ◽

H Sumimoto ◽

T Yubisui ◽

AJ Bellingham ◽

...

Keyword(s):

Catalytic Activity ◽

Missense Mutation ◽

Nicotinamide Adenine Dinucleotide ◽

Cytochrome B5 ◽

Mutant Enzyme ◽

Type Ii ◽

Novel Mutations ◽

Nadh Cytochrome ◽

Reduced Nicotinamide Adenine Dinucleotide ◽

Generalized Type

Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. 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 changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll. The mutant enzyme showed less catalytic activity, less thermostability, and a greater 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 suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.

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

Blood ◽

10.1182/blood.v95.10.3250.010k23_3250_3255 ◽

2000 ◽

Vol 95 (10) ◽

pp. 3250-3255 ◽

Cited By ~ 1

Author(s):

Yao Wang ◽

Yu-Shui Wu ◽

Pei-Zhen Zheng ◽

Wen-Xi Yang ◽

Guo-An Fang ◽

...

Keyword(s):

Clinical Symptoms ◽

Novel Mutation ◽

Cytochrome B5 ◽

Clinical Manifestations ◽

Mutant Enzyme ◽

Chinese Family ◽

Coding Region ◽

Cytochrome B5 Reductase ◽

Nadh Cytochrome

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.

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