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

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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Compound heterozygosity of two missense mutations in the NADH-cytochrome b5 reductase gene of a Polish patient with type I recessive congenital methaemoglobinaemia

European Journal Of Haematology ◽

10.1034/j.1600-0609.2003.00070.x ◽

2003 ◽

Vol 70 (6) ◽

pp. 404-409 ◽

Cited By ~ 12

Author(s):

Dorota Grabowska ◽

Danuta Plochocka ◽

Ewa Jablonska-Skwiecinska ◽

Anna Chelstowska ◽

Irmina Lewandowska ◽

...

Keyword(s):

Cytochrome B5 ◽

Type I ◽

Missense Mutations ◽

Compound Heterozygosity ◽

Cytochrome B5 Reductase ◽

Reductase Gene ◽

Nadh Cytochrome ◽

Polish Patient

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A novel Pro92His, and previously described, Glu255‐, double mutation of NADH‐cytochrome b5 reductase associated with apparent type I RCM

The FASEB Journal ◽

10.1096/fasebj.21.6.a1044 ◽

2007 ◽

Vol 21 (6) ◽

Author(s):

Melanie J Percy ◽

Louis J Crowley ◽

Mark Layton ◽

Terry Lappin ◽

Michael Barber

Keyword(s):

Cytochrome B5 ◽

Type I ◽

Double Mutation ◽

Cytochrome B5 Reductase ◽

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.bloodjournal813808 ◽

1993 ◽

Vol 81 (3) ◽

pp. 808-814 ◽

Cited By ~ 11

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

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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