NADH-cytochrome B5 Reductase Deficiency Research Articles

Identification of High-Risk Single Nucleotide Polymorphisms in the Human CYB5R3 Gene Responsible for Recessive Congenital Methemoglobinemia: A Computational Approach

Introduction: NADH-cytochrome b5 reductase deficiency due to pathogenic variants in the CYB5R3 gene causes recessive congenital methemoglobinemia (RCM) type I or type II. In type I, cyanosis from birth is the only major symptom, and the enzyme deficiency is restricted only to erythrocytes. Whereas in type II, cyanosis is associated with severe neurological manifestations, and the enzyme deficiency is generalized to all tissues. Methods: In this study, several computational methods (SIFT, Polyphen-2, PROVEAN, Mutation Assessor, Panther, Phd-SNP, SNPs&GO, SNAP2, Align, GVGD, MutPred2, I-Mutant 2.0, MUpro, Duet, ConSurf and Netsurf-2.0 tools) were used to find the most deleterious nsSNPs in the CYB5R3 gene. Furthermore, structural analysis by Swiss-PDB viewer, protein-ligand docking using FTSite, and protein-protein interaction using STRING were carried out to evaluate the impact of these nsSNPs on the protein structure and function. Results: Our in silico analysis suggested that out of 339 nsSNPs of the CYB5R3 gene, 17 (L47H, L47P, R61P, L73R G76D, G76C, P96H, G104C, S128P, G144D, P145S, L149P, Y151H, M177T, I178T, I216N, and G251V), are the most deleterious. Among them, two (P96H and S128P) were reported to be associated with the severe form RCM type II, six are related to RCM type I (G104C, G144D, P145S, L149P, M177T, and I178T), and the remaining nine high-risk nsSNPs have not yet been reported in RCM patients. Discussion: This study highlighted the potential pathogenic nsSNPs of the CYB5R3 gene. To comprehend how these most harmful nsSNPs contribute to disease, it is crucial to experimentally validate their functional effects.

PS1201 : MOLECULAR STUDY OF RED CELL NADH‐CYTOCHROME B5 REDUCTASE DEFICIENCY IN 21 INDIAN PATIENTS WITH RECESSIVE HEREDITARY METHEMOGLOBINAEMIA

Background:Recessive congenital methemoglobinemia (RCM) due to NADH–cytochrome b5 reductase 3 (cyb5r3) deficiencies is an autosomal recessive disorder that occurs sporadically worldwide. Over seventy six different mutations have been described for RCM.Aims:The aim of this study molecular basis of RCM in 21 patients and prenatal diagnosis in 4 cases with severe mental retardation and neurological disabilities along with a comprehensive review of all the pathogenic mutations in cyb5r3 and their molecular pathology in RCM.Methods:Twenty‐five cases were analyzed amongst which 21 cases were diagnosed with NADH‐CYB5R deficiency based on significantly reduced NADH‐CYB5R activity while 4 were referred for prenatal screening. The study has been approved by the National Institute of Immunohematology Ethical committee and as per protocol, written informed consents were obtained from all the patients or their parents. Complete blood counts were measured by automated hematology analyzer XN (Sysmex, Kobe, Japan). Methemoglobin estimation, G6PD, and NADH‐Cyb5R enzyme assay were performed as described by Beutler et al. method. DNA was extracted from the Blood/CVS using Qiagen DNA extraction kit according to the manufacturer's instructions. PCR exon‐specific primers were utilized and PCR and Sanger's sequencing was performed by a standard method. Molecular modeling of cyb5r3 was done using the PYMOL and SWISS‐PDB viewer tool.Results:In the 21 patients analyzed in this study, all index cases from twenty unrelated families were referred for the cause of cyanosis. All patients showed mild to moderate cyanosis and few patients had mental retardation or any neurologic abnormalities. Methemoglobin levels were increased, varying from 8.5% to 65%. NADH‐Cyb5r3 activity was seen to be in the range of 6 to 19%. In the molecular analysis, eighteen mutations (fourteen missense, one deletion, one‐stop codon, and 1 splice‐site mutation) were observed (Figure 1) in this study. Amongst fourteen missense mutations 5 were novel mutations [c.105A>C (p.Thr35Arg), c.192C>G (p.Leu64Val), c.312G>T (p.Gly104Cys), c.354C>T (p.His118Tyr) and c.470T>G (p.Phe157Cys)]. Over seventy‐six different mutations have been described for RCM globally (Figure 1). Same mutations are associated with the severe form with a neurological disorder (RCM type 2) were associated with the FAD‐binding domain of the protein while rests were located in the other domains of the protein (RCM Type 1). One of the prenatal analyses showed splice‐site mutation p.Gly76Ser at nucleotides 226 in exon 3 of the CYB5R3 gene in the homozygous state while in another one it was found in the heterozygous state. In the third case stop codon mutation p.Gln77X at nucleotide position 229 in exon 4 was seen in the heterozygous condition. In the fourth case, normal wild type genotype for the CYB5R3 gene was found.Summary/Conclusion:This study presents the results of the first molecular study of a large number of cases of NADH cytochrome b5 reductase deficiency in India Molecular modeling of all the mutations reported in CYB5R3 justifies the association of the mutations with the varying severity of the disease. Knowing the profile of mutations in families with severe neurological disorders allowed us to offer a prenatal diagnosis of RCM Type 2 for the first time in the Indian population. Thus severity and recurrence risk of type II RCM merit could be prevented by prenatal diagnosis in affected families.image

Novel mutation (R192C) in CYB5R3 gene causing NADH-cytochrome b5 reductase deficiency in eight Indian patients associated with autosomal recessive congenital methemoglobinemia type-I

ABSTRACTObjective: To investigate the cause of recessive congenital methemoglobinemia (RCM) in Indian families and to identify molecular defect associated with RCM.Methods: Eight cases of RCM have been addressed to our laboratory in order to investigate the cause of cyanosis associated with genetic disorders. NADH-cytochrome b5 reductase (cytb5r) enzyme activities were measured by standard methods, and molecular analysis was performed by polymerase chain reaction (PCR) followed by DNA sequencing. The interpretation of mutation effect and the molecular modeling were performed by using specific software DEEP VIEW SWISS-PDB VIEWER and Pymol molecular graphics program.Results and Discussion: Eight index cases from four unrelated families were referred for the cause of cyanosis. All patients showed mild to moderate cyanosis without mental retardation or any neurologic abnormalities. The methemoglobin levels were in the range of 11.5–22.41% with 50–70% reduction in CYTB5R activity. Spectroscopic analysis of the hemolysate showed normal peaks suggesting the absence of Hb-M. Molecular characterization showed a novel homozygous mutation p.Arg192Cys in CYB5R3 gene is an evolutionarily conserved position located in exon 7 in all eight index cases. The substitution of Cys is located on the interface of two domains of NADH-binding domain and is close proximity to the adenosine moiety would preclude the reciprocal ionic interaction (salt bridge) between Arg192 and Ile97 and may influence binding of the NADH coenzyme is hypothesized to cause disruption of hydrogen bonding and instability. Our study indicated that novel homozygous mutation p.Arg192Cys in CYB5R3 gene present in eight cases and the possibility of high prevalence of heterozygous in Indian population causing Type I RCM.

Association of Heme Oxygenase-1 (Number of GT Repeats) with The Methemoglobin Levels in Recessive Congenital Methemoglobinemia in Indian Population

Heme oxygenase-1 (HO-1) is important in the defense against oxidative stress. Length polymorphisms in this GT repeat region correlate with levels of HO-1 expression and associates with several diseases. The aim of this study was to test for possible association of HO-1 (number of GT repeats) with methemoglobin levels in recessive congenital methemoglobinemia in Indian population. Genotyping of DNA isolated from whole blood of 25 RCM patients due to NADH cytochrome b5 reductase deficiency and 50 healthy controls was performed. Fragment size analysis by sequencing was used for genotype/allele definition by ABI 3130 genetic analyzer and size of PCR product was determined by Gene Mapper software. Significance of findings was tested using χ(2) test. The HO-1 (number of GT repeats) polymorphisms was significantly associated with RCM. Results of genotyping analysis indicated that a genotype carrying short alleles (<24 (GT)n repeats was preferentially associated with RCM patients than in control (P<0.001). The short allele (<24 GT repeats) genotype were present in 82% of the methemoglobinemia patients group and long allele (≥31 GT repeats) observed in the normal control group. This study is supporting the association of HO-1 (number of GT repeats) polymorphism and RCM. . Allele and genotype frequencies for HO-1 polymorphisms show significant association with disease severity. This data could also be valuable to the clinicians, mainly hematologists, when attempting a definitive diagnosis for the cause of methemoglobinemia that will help clinical decisions for treatment.

Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India

We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH-cytochrome b5 reductase (NADH-CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice-site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three-dimensional (3D) structure of human erythrocyte NADH-CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype-phenotype correlation in NADH-CYB5R deficiency.

Recessive congenital methemoglobinemia due to NADH-cytochrome b5 reductase deficiency associated with recurrent early pregnancy loss (REPL) in an Indian family

Recessive congenital methemoglobinemia due to NADH-cytochrome b5 reductase deficiency associated with recurrent early pregnancy loss (REPL) in an Indian family

Cytochrome b5 Reductase T116S Mutation and Hemolysis in Sickle Cell Disease.

Abstract 903Abstract Background:Deficiency of NADH-cytochrome b5 reductase (cytb5r, EC 1.6.2.2) is responsible for congenital methemoglobinemia. This enzyme exists in soluble and membrane-bound forms. The soluble erythrocytic cytb5r isoenzyme is involved in cytochrome b5 reduction and in erythrocyte methemoglobin reduction; the membrane-bound microsomal enzyme participates in a fatty acid desaturation complex and in drug metabolism. The cytb5r isoforms are the product of a single gene locus, DIA1 (or CYB5R3), on chromosome 22. More then 40 mutations which cause methemoglobinemia have been reported to date; the majority are missense mutations and are associated with mild type I methemoglobinemia. The CYBR5 T116S mutation is the most common genetic polymorphism among African Americans known (gene frequency as high as 20%) and it has not yet been detected in other ethnic and racial groups. This polymorphism is not associated with methemoglobinemia and its functional significance is not yet known. We studied the relationship of CYBR5 T116S with the degree of hemolysis and the tricuspid regurgitation velocity (which correlates with systolic pulmonary artery pressure) in patients with sickle cell disease. Methods:Two hundred sixty one children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Patients with other sickle genotypes were excluded from this analysis of CYBR5 T116S. Principal component analysis was used to develop a hemolytic component from reticulocyte count and concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of the CYBR5 T116S mutation. Multivariate models were employed to determine the independent effects of this genotype on degree of hemolysis and tricuspid regurgitation velocity. Results:Ninety-eight of the patients (38%) were CYBR5 T116S heterozygotes and 26 (10%) were homozygotes, consistent with Hardy-Weinberg equilibrium. Both heterozygosity (beta = -0.4) and homozygosity (beta = -0.5) were associated with reduction in the hemolytic component (N = 261; P for trend = 0.002) (Figure 1). This relationship persisted after adjusting for α-thalassemia, hemoglobin F percent and hydroxyurea treatment in a subset of 113 patients with all of this information available (P for trend = 0.037) and it also persisted in a subset of 87 patients with no α-globin gene deletion who were not being treated with hydroxyurea (P for trend = 0.029). In none of these analyses did G6PD-202/-376 have an effect on hemolysis. Both heterozygosity (beta = -0.04) and homozygosity (beta = -0.14) for the CYBR5 T116S mutation were also associated with lower tricuspid regurgitation velocity (P for trend = 0.024). Conclusions:CYBR5 T116S is a common polymorphism among patients with sickle cell disease that appears to be associated with less hemolysis and lower tricuspid regurgitation velocity. We speculate that this polymorphism may be related to a previously reported subpopulation of African Americans with increased cytochrome b5 reductase activity, and that increased anti-oxidant activity may explain the polymorphism's hemolysis-reducing effect. Functional studies to investigate this possibility are planned. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

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

Recessive congenital methemoglobinemia is characterised by a deficiency of NADH-cytochrome b5 reductase and exhibits two phenotypes. Both types are associated with cyanosis, type I is benign while ...

A Novel Heterologous Expression System for Characterization of Individual NADH-Cytochrome b5 Reductase Variants in Recessive Congenital Methemoglobinemia.

Deficiency of NADH-cytochrome b5 reductase (cb5r) causes two clinically distinct phenotypes of recessive congenital methemoglobinemia (RCM). Type I patients often manifest cyanosis from birth, and in type II patients the cyanosis is accompanied by severe neurological impairment. The mechanisms responsible for the phenotypic differences between the two subgroups remain to be defined. The majority of patients harbor two different mutant alleles. To date 39 mutant variants of cb5r have been identified, 2 of which are common to both types of RCM. In order to characterize the individual cb5r variant proteins we have developed a novel heterologous expression system based on the structures of the rat and human proteins derived by X-ray crystallography. The system permits the investigation of the catalytic efficiencies, protein thermostability, FAD cofactor properties and substrate (NADH/NAD+) affinities of the variants. We have investigated four patients with type I RCM, one of whom was homozygous for the D239G mutation. The other three were compound heterozygous: R159-/D239G; G75S/V252M; and P275L/G291D, and one mutation, P275L, was novel. All patients showed reduced enzyme activity, in the range 0.5 to 5.8 IU/g Hb compared to normal values of 7.2 to 26.9 IU/g Hb. Individual variant proteins were prepared and the analytical data are summarised in the Table below.VariantCatalytic Efficiency (% of normal)Thermal Stability (T50°C)NADH affinity (Km)NAD+ affinity (Ks)G75S1148Normal9-fold ↑R159-0NDNDNDD239G25640-fold ↓NDV252M9539-fold ↓18-fold ↑E255-0.451100-fold ↓NDP275L0.253437-fold ↓NDG291D43491.3-fold ↓1.1-fold ↑Wild type10057normalnormalND - not determinedAs expected all of the variants generated had decreased enzyme activity compared to wild type heterologous protein, supporting the validity of this approach. Thermal stability was decreased in the G75S, V252M and G291D variants. G75 is present in a highly conserved region in the FAD-binding lobe. Although it does not interact directly with the FAD prosthetic group it is important for association with cytochrome b5. Substitution of glycine at residue 75 by serine resulted in decreased enzyme activity and stability, with a marginal decrease in NADH affinity. The R159- variant protein was unstable and could not be isolated. Both the D239G and P275L mutations significantly reduced the affinity of cb5r for NADH, by 40-fold and 437-fold respectively. The rat cb5r model suggests that residue D239 is key for selecting between the NADPH and NADH pyridine nucleotides. This was confirmed by the 40-fold decrease in affinity for NADH and a 125-fold increase in affinity for NADPH. Residue P275 is located in a highly conserved region, which is important for the correct positioning and binding of NADH. Consequently, substitution of proline at 275 would affect the affinity of cb5r towards NADH, which was confirmed by the affinity constant measurements. These studies provide important information about the structure-function relationships of the variant cb5r proteins which may impart useful insights into the pathophysiological differences between type I and type II RCM.

A case of methemoglobinemia type II due to NADH-cytochrome b5 reductase deficiency: Determination of the molecular basis

Clinical, biochemical and molecular findings in a patient with methemoglobinemia type II are described. Furthermore, a comparison between methemoglobinemia type I and type II, both caused by a deficiency of NADH-cytochrome b5 reductase (b5R), is made. Although the clinical pictures of type I and II are strikingly different, mutations in the diaphorase (DIA1) gene located on chromosome 22 have been described in both types. In the present patient, two newly identified mutations, both leading to a stop codon in exon 4 (Gln77Ter) and in exon 6 (Arg160Ter), were found. Identification of different mutations at different positions in the DIA1 gene might shed light on the clinical and biochemical differences between methemoglobinemia type I and type II. Hum Mutat 16:18–22, 2000. © 2000 Wiley-Liss, Inc.

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH-cytochrome b5 reductase gene.

Hereditary methaemoglobinaemia, caused by deficiency of NADH-cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C-to-T substitution in codon 8 3 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C-to-T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life-span of erythrocytes.

Analysis of Mutant NADH-Cytochrome bs Reductase: Apparent “Type III” Methemoglobinemiα Can Be Explained as Type I With an Unstable Reductase

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.

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

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--&gt;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 &lt; 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.

NADH cytochrome b5 reductase activity in lymphoid cell lines. Expression of the defect in epstein Barr virus transformed lymphoblastoid cell lines from patients with recessive congenital methemoglobinemia.

Recessive congenital methemoglobinemia (RCM) is due to the homozygous deficiency of NADH-cytochrome b5 reductase (EC 1.6.2.2.). In type I disease, in which the patients are only methemoglobinemic, the enzyme defect is fully expressed in the erythrocytes, whereas the leukocytes are much less affected. In type II disease, in which the patients are, in addition, mentally retarded, the defect is generalized to all the tissues including cultured fibroblasts. In the present study we have investigated Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) derived from patients with both types of cytochrome b5 reductase deficiency and from nondeficient individuals. The total cytochrome b5 reductase activity of the control LCL was found to be similar whatever the LCL origin, except for one lymphoma line (Daudi). The enzyme from the control LCL (c 252/B 95) was found to be immunologically related to the human soluble erythrocyte cytochrome b5 reductase, indicating that it is the product of the same gene: the DIA1 (diaphorase) locus. The LCL derived from one patient with the type I disease and two patients with the type II disease were investigated.l In the former the defect was expressed to a lesser degree than in the cases with mental retardation in which the defect was much pronounced, and involved both the mitochondrial and the microsomal fraction. This indicated that all the subcellular forms of the cytochrome b5 reductase are under the same genetic control. Altogether, these data show that the LCL are a favorable material for studying both types of cytochrome b5 reductase deficiency and for investigating in depth the molecular aspects of this metabolic disease.

Analyis of met-form haemoglobins in human erythrocytes of normal adults and of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase.

Isoelectric-focusing analysis on an Ampholine/polyacrylamide-gel plate revealed that met-form haemoglobins are present as half-oxidized haemoglobins such as the (alpha 2+ beta 3+)2 and (alpha 3+ beta2+)2 forms rather than as methaemoglobin in the erythrocytes of normal human adults and also of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase.

Acceleration of methaemoglobin reduction by riboflavin in human erythrocytes.

The effect of riboflavin on nitrite treated erythrocytes from normal subjects and patients with hereditary methaemoglobinaemia due to the deficiency of NADH-cytochrome b5 reductase was studied in the presence of glucose, 2-deoxy-D-glucose or lactate. When glucose or 2-deoxy-D-glucose was used as a substrate for these erythrocytes, the rate of methaemoglobin reduction in these cells was accelerated more than two-fold in the presence of riboflavin. The acceleration was dependent on the concentration of riboflavin and was suppressed by the addition of atebrin. The stimulative effect of riboflavin was, however, not observed when lactate was used in place of glucose or 2-deoxy-D-glucose. On the basis of these results, the acceleration of methaemoglobin reduction by riboflavin was considered to be due to the activation of NADPH-flavin reductase (Yubisui et al, 1977) in erythrocytes by the reagent. The availability of riboflavin for patients with methaemoglobinaemia due to the deficiency of NADH-cytochrome b5 reductase and for those with toxic methaemoglobinaemia is discussed in relation to methaemoglobin reducing systems in erythrocytes.