Congenital methaemoglobinaemia Type I in a Turkish infant due to a novel mutation, Pro144Ser, in NADH-cytochrome b5 reductase

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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A novel mutation found in the 3' domain of NADH-cytochrome B5 reductase in an African-American family with type I congenital methemoglobinemia

Blood ◽

10.1182/blood.v87.7.2993.bloodjournal8772993 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2993-2999 ◽

Cited By ~ 23

Author(s):

MM Jenkins ◽

JT Prchal

Keyword(s):

African American ◽

Novel Mutation ◽

Cytochrome B5 ◽

Alpha Helix ◽

Type I ◽

African American Family ◽

American Family ◽

Cytochrome B5 Reductase ◽

Specific Pcr ◽

Function Relationship

Congenital methemoglobinemia caused by an erythrocytic deficiency of cytochrome b5 reductase (b5R; type I) in African-American individuals was first reported by this laboratory. The rarity of this observation is possibly due to the difficulty detecting cyanosis that is masked by naturally occurring dark skin pigment. Since previous biochemical studies on the African-American family with variant enzyme b5R- Shreveport showed enzyme instability, we focused on molecular analysis of its transcript. The transcript size was the same as that of a normal control. The nucleotide sequence of both normal and variant transcripts were examined by directly sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) product. The propositus was found to be homozygous for a G to A transition at codon 212 in exon 8, changing a glutamate to a lysine (E212K). In addition, a C to G transversion was found at codon 116 in exon 5, changing a threonine to a serine (T116S). Using allele-specific PCR, we determined that E212K was found only in the propositus and her heterozygous mother. Furthermore, E212K is predicted to disrupt an alpha-helix peptide structure of b5R, suggesting that this is likely the disease-causing mutation. In contrast, T116S was found to be a high-frequency polymorphism specific for the African-American population. The E212K mutation is uniquely present in the 3′ end of the b5R gene (exon 8), which differs from those b5R mutations found among Japanese subjects (exons 3 and 5) and in an Italian subject (exon 4) and, thus, further contributes to our understanding of the structure/function relationship of this housekeeping enzyme.

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Three Novel Mutations in CYB5R3 Gene Causing NADH-cytochrome b5 Reductase Enzyme Deficiency Leads to Recessive Congenital Methaemoglobinemia

10.21203/rs.3.rs-732442/v1 ◽

2021 ◽

Author(s):

Anuradha Deorukhkar ◽

Anuja KULKARNI ◽

Prabhakar S Kedar

Keyword(s):

Splice Site ◽

Genetic Disorders ◽

Cytochrome B5 ◽

Neurological Impairment ◽

Enzyme Deficiency ◽

Type I ◽

Cytochrome B5 Reductase ◽

Pathogenic Variants ◽

Nucleotide Insertion ◽

Nadh Cytochrome

Abstract Two types of recessive congenital methaemoglobinemia (RCM) is caused by NADH-dependent cytochrome b5 reductase enzyme deficiency encoded by CYB5R3 gene. RCM-I is characterized by higher methaemoglobin levels (>2 g/dL), causing only cyanosis, whereas RCMR-II is associated with cyanosis with neurological impairment. The present study discovered three novel homozygous pathogenic variants of CYB5R3 causing RCM I and II in four unrelated Indian patients. In patient-1 and patient-2 of are of RCM type I caused due to novel c.175C>T (p.Arg59Cys) and other reported c.469T>C (p.Phe157Ser) missense pathogenic variants respectively, whereas patient-3 and patient-4 presented with the RCM type II are related to developmental delay with cyanosis since birth due to a novel homozygous (g.25679_25679delA) splice-site deletion and novel homozygous c.824_825insC (p.Pro278ThrfsTer367) single nucleotide insertion. The CYB5R3 transcript levels were estimated by qRT-PCR in the splice-site deletion, which was 0.33fold of normal healthy control. The insertion of nucleotide C resulted in a frame-shift of termination codon are associated with neurological impairment. This study can help to conduct genetic counselling and, subsequently, prenatal diagnosis of high-risk genetic disorders.

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Enzymatic instability of NADH-cytochrome b5 reductase as a cause of hereditary methemoglobinemia type I (red cell type).

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)88718-5 ◽

1992 ◽

Vol 267 (28) ◽

pp. 20416-20421

Author(s):

K Shirabe ◽

T Yubisui ◽

N Borgese ◽

C.Y. Tang ◽

D.E. Hultquist ◽

...

Keyword(s):

Cytochrome B5 ◽

Type I ◽

Red Cell ◽

Cell Type ◽

Cytochrome B5 Reductase ◽

Nadh Cytochrome

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A novel G143D mutation in the NADH-cytochrome b5 reductase gene in an Indian patient with type I recessive hereditary methemoglobinemia

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2007.09.006 ◽

2008 ◽

Vol 40 (3) ◽

pp. 323-327 ◽

Cited By ~ 5

Author(s):

Prabhakar S. Kedar ◽

Prashant Warang ◽

Anita H. Nadkarni ◽

Roshan B. Colah ◽

Kanjaksha Ghosh

Keyword(s):

Cytochrome B5 ◽

Type I ◽

Indian Patient ◽

Cytochrome B5 Reductase ◽

Reductase Gene ◽

Nadh Cytochrome

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Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase

Blood ◽

10.1182/blood.v81.3.808.808 ◽

1993 ◽

Vol 81 (3) ◽

pp. 808-814 ◽

Cited By ~ 1

Author(s):

T Nagai ◽

K Shirabe ◽

T Yubisui ◽

M Takeshita

Keyword(s):

Enzyme Activity ◽

Cytochrome B5 ◽

Base Change ◽

Mutant Enzyme ◽

Type I ◽

Wild Type ◽

Cytochrome B5 Reductase ◽

Type Iii ◽

Patient Reported ◽

Nadh Cytochrome

Abstract A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu- 148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

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NADH-cytochrome b5 reductase in a Turkish family with recessive congenital methaemoglobinaemia type I: Table 1

Journal of Clinical Pathology ◽

10.1136/jcp.2008.058701 ◽

2008 ◽

Vol 61 (10) ◽

pp. 1122-1123 ◽

Cited By ~ 7

Author(s):

M J Percy ◽

D Aslan

Keyword(s):

Cytochrome B5 ◽

Blood Gas Analysis ◽

Gas Analysis ◽

Type I ◽

Missense Mutations ◽

Arterial Blood Gas ◽

Cytochrome B5 Reductase ◽

Arterial Blood ◽

Turkish Family ◽

Nadh Cytochrome

The development of cyanosis at birth, the so-called blue baby syndrome, alerts paediatricians to the presence of congenital heart disease. In rare cases where the arterial blood gas analysis is normal the cyanosis is a consequence of methaemoglobinaemia. There are three distinct origins of methaemoglobinaemia; the presence of a haemoglobin variant, environmental toxicity and deficiency of cytochrome b5 reductase (cb5r). Two children born to two sets of first-degree related parents were cyanotic from birth. Differential diagnosis eliminated cardiac and pulmonary abnormalities. Measurement of methaemoglobin levels confirmed recessive congenital methaemoglobinaemia (RCM) and treatment with ascorbic acid was commenced. In the absence of neurological defects, type I disease was diagnosed. Sequence analysis of CYB5R3 revealed two different missense mutations (one which is novel, Ile85Ser) in the two families. Neither of the mutations was located in the FAD or the NADH binding sites of cb5r, thus supporting a diagnosis of type I disease.

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