CYB5R3 Research Articles

Oxidative Metabolism Genes in Ovarian Neoplasms

BACKGROUND: Reactive oxygen species play important and ambiguous role in carcinogenesis, and local oxidative metabolism may differ significantly from systemic metabolism and determine the processes occurring in tumor tissues.
 AIM: This study aimed to examine the expressions of key oxidative metabolism genes, particularly CYB5R, POR, NOX4, SOD1, NF-B, and NRF2, in ovarian neoplasm tissues, and determine cytochrome b5 reductase and cytochrome P450 reductase activity, blood neutrophil activity, and antioxidant indices in the blood plasma and peritoneal fluid.
 MATERIALS AND METHODS: The study included two groups of patients: a study group (n = 10) with ovarian adenocarcinoma and a comparison group (n = 6) with benign ovarian neoplasms. The expressions of CYB5R1, CYB5R2/R4, CYB5R3, POR, BIRC3, NOX4, NRF2, NF-B, SOD1, HMOX1, and BCL2 genes, cytochrome b5 reductase, and cytochrome P450 reductase activity, oxidative activity of blood neutrophils, and antioxidant potential of plasma and peritoneal fluid were evaluated in these two groups of women.
 RESULTS: The expression levels of CYB5R3 and BCL2 were significantly lower in adenocarcinoma tissues. The activities of cytochrome b5 reductase and cytochrome P450 reductase increased in the group with adenocarcinoma. On average, the activity of blood neutrophils corresponded to the reference values. For blood plasma, the antioxidant capacity were not different, whereas the antioxidant capacity in the peritoneal fluid increased approximately twofold in ovarian cancer.
 CONCLUSIONS: Significantly increased cytochrome b5 reductase activity in adenocarcinoma tissues may be a response to intracellular oxidative stress, whereas CYB5R3 gene expression may be reduced by a negative feedback mechanism. The activities of cytochrome P450 reductase and its gene change to a lesser extent in the presence of ovarian adenocarcinoma. The oxidative balance in the blood and peritoneal fluid correlated with the tissue expressions of NF-B and NRF2.

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.

Methemoglobinemia, Increased Deformability and Reduced Membrane Stability of Red Blood Cells in a Cat with a CYB5R3 Splice Defect

Methemoglobinemia is an acquired or inherited condition resulting from oxidative stress or dysfunction of the NADH-cytochrome b5 reductase or associated pathways. This study describes the clinical, pathophysiological, and molecular genetic features of a cat with hereditary methemoglobinemia. Whole genome sequencing and mRNA transcript analyses were performed in affected and control cats. Co-oximetry, ektacytometry, Ellman's assay for reduced glutathione concentrations, and CYB5R activity were assessed. A young adult European domestic shorthair cat decompensated at induction of anesthesia and was found to have persistent methemoglobinemia of 39 ± 8% (reference range < 3%) of total hemoglobin which could be reversed upon intravenous methylene blue injection. The erythrocytic CYB5R activity was 20 ± 6% of normal. Genetic analyses revealed a single homozygous base exchange at the beginning of intron 3 of the CYB5R3 gene, c.226+5G>A. Subsequent mRNA studies confirmed a splice defect and demonstrated expression of two mutant CYB5R3 transcripts. Erythrocytic glutathione levels were twice that of controls. Mild microcytosis, echinocytes, and multiple Ca2+-filled vesicles were found in the affected cat. Erythrocytes were unstable at high osmolarities although highly deformable as follows from the changes in elongation index and maximal-tolerated osmolarity. Clinicopathological presentation of this cat was similar to other cats with CYB5R3 deficiency. We found that methemoglobinemia is associated with an increase in red blood cell fragility and deformability, glutathione overload, and morphological alterations typical for stress erythropoiesis.

Molecular Dynamic Simulation Analysis of a Novel Missense Variant in CYB5R3 Gene in Patients with Methemoglobinemia.

Background and Objective: Mutations in the CYB5R3 gene cause reduced NADH-dependent cytochrome b5 reductase enzyme function and consequently lead to recessive congenital methemoglobinemia (RCM). RCM exists as RCM type I (RCM1) and RCM type II (RCM2). RCM1 leads to higher methemoglobin levels causing only cyanosis, while in RCM2, neurological complications are also present along with cyanosis. Materials and Methods: In the current study, a consanguineous Pakistani family with three individuals showing clinical manifestations of cyanosis, chest pain radiating to the left arm, dyspnea, orthopnea, and hemoptysis was studied. Following clinical assessment, a search for the causative gene was performed using whole exome sequencing (WES) and Sanger sequencing. Various variant effect prediction tools and ACMG criteria were applied to interpret the pathogenicity of the prioritized variants. Molecular dynamic simulation studies of wild and mutant systems were performed to determine the stability of the mutant CYB5R3 protein. Results: Data analysis of WES revealed a novel homozygous missense variant NM_001171660.2: c.670A > T: NP_001165131.1: p.(Ile224Phe) in exon 8 of the CYB5R3 gene located on chromosome 22q13.2. Sanger sequencing validated the segregation of the identified variant with the disease phenotype within the family. Bioinformatics prediction tools and ACMG guidelines predicted the identified variant p.(Ile224Phe) as disease-causing and likely pathogenic, respectively. Molecular dynamics study revealed that the variant p.(Ile224Phe) in the CYB5R3 resides in the NADH domain of the protein, the aberrant function of which is detrimental. Conclusions: The present study expanded the variant spectrum of the CYB5R3 gene. This will facilitate genetic counselling of the same and other similar families carrying mutations in the CYB5R3 gene.

Loss of Cyb5R3 Function Silences Fetal Hemoglobin and Hematocrit Responses to Hydroxyurea in Patients with Sickle Cell Anemia

BACKGROUND: Hydroxyurea (HU) is the first FDA-approved and most widely prescribed drug for sickle cell (SS) patients due to its beneficial effects (decreased Hb polymerization, pain crises and hospitalization) and relative safety profile. While the clinical benefits of HU have been found to associate with increased nitric oxide (NO) and fetal hemoglobin (HbF) in most SS patients, there has been considerable interpatient variability in HbF response to the drug. From this standpoint, HU is imperfectly understood mechanistically. Cytochrome b5 reductase 3 (Cyb5R3), also known as methemoglobin reductase, is an enzyme known to modulate redox signaling in erythrocytes and vascular cells by catalyzing one electron transfer reactions using reduced nicotinamide adenine dinucleotide (NADH) as an electron donor. Functionally, membrane-associated CYB5R3 exerts antioxidant properties by modulating heme iron reduction, plasma membrane coenzyme Q reduction, elongation and unsaturation of fatty acids, cholesterol biosynthesis and drug metabolism, while the soluble form of CYB5R3 reduces red blood cell methemoglobin. Unremitting oxidation of the soluble guanylate cyclase (sGC) heme iron in the absence of sufficient Cyb5R3 activity in vascular smooth muscle has been shown to explain some NO resistance that characterizes pulmonary arterial hypertension and other vasculopathy associated with SCD. More than 40 variants of Cyb5R3 have been identified. Of particular interest is a Cyb5R3 variant (T117S) that contains a threonine to serine substitution in the flavoprotein, which lessens the reductase's activity by roughly 50% compared to wild-type. This variant occurs with high frequency (0.23 minor allele) in persons of African ancestry but is rare (<1%) in all other races. Nothing is known about the role of Cyb5R3 in the anemia of sickle cell disease. We hypothesized that Cyb5R3 may act as a modifier of HbF induction by HU in sickle cell anemia. METHODS: Male and female SS patients (n=633, 12-70 years of age) from the Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (walk-PHaSST) trial were recruited for genetic analysis (University of Pittsburgh IRB Protocol Study 19060255). Genotyping of rs1800457 (T117S SNP) was performed using Taqman genotyping assay. One-way ANOVA and Holms Sidak were used for multiple comparisons of HbF levels by Cyb5R3 genotype and HU treatment status. Intrapatient correlation analyses of HbF and hematocrit (HCT) were conducted using Pearsons r. RESULTS: Comparisons of HbF levels by Cyb5R3 genotype (CC, wild-type; CG, heterozygous wild-type and T117S; GG, homozygous T117S) and HU treatment status (treated vs. untreated) at time of study enrollment (baseline) show that HU is a less potent HbF inducer in the GG group (4.32 mean diff, P=0.344) versus the CC and CG patient groups (CC: 6.027 mean diff, P<0.0001 and CG: 6.914 mean diff, P<0.0001). Correlation analyses showed that as HbF levels increased among HU-untreated patients, the trend was for HCT to decrease. This was true for all SS patients, irrespective of Cyb5R3 genotype (CC: r = -0.336, P<0.0001, n=320; CG: r = -0.437 P<0.0001, n=120; GG: r = -0.253, P=0.136, n=36). However, with HU therapy, the inverse relationship between HbF and HCT was less negative in SS patients carrying at least one copy of the wild-type Cyb5R3 gene (CC or CG). In these individuals, increasing levels of HbF (up to 10%) correlated with positive gains in HCT at HbF greater than 5-6% (CC: r = -0.161, P=0.097, n=107) and 6-7% (CG: r = -0.186, P=0.25, n=40). In patients homozygous for T117S, HCT continued trending downward as HbF increased (GG: r = -0.419, P=0.228, n=10). These data indicate that the positive relationship between HbF and HCT is subject to uncoupling by loss of Cyb5R3 function. In vitro studies with human erythroleukemic K562 cells reveal loss of gamma globin induction by HU treatment when wild-type Cyb5R3 is knocked down by siRNA, indicating a requirement for Cyb5R3 in HU-stimulated HbF induction. SUMMARY andCONCLUSION: Loss of Cyb5R3 activity negatively impacts HbF and HCT in HU treated SS patients. We conclude that the T117S variant may be a useful marker for predicting which SS patients will incur beneficial HbF and HCT responses to HU, thereby improving clinical decision-making about best drug choices for preventing HbS polymerization and treating anemia in SS patients.

Congenital methemoglobinemia masquerading as spastic diplegic cerebral palsy

Congenital methemoglobinemia is an autosomal recessive condition resulting from deficiency of methemoglobin reductase which is caused by homozygous or compound heterozygous mutation in the CYB5R3 gene on chromosome 22q13, manifesting clinically by decreased oxygen carrying capacity of the blood, with resultant cyanosis and hypoxia. The clinical features include microcephaly, developmental delay, spasticity and myelination defects, seizures, feeding difficulties. Methemoglobinemia is an abnormal increase of MetHb (&gt;3%) of total haemoglobin which can be either hereditary and acquired. The acquired methemoglobinemia is mostly due to anaesthetic drugs and genetic one is due to deficiency of CYB5R3 or cytochrome B5 systems. The type I congenital methemoglobinemia is more common and less severe and is caused by CYB5R functional deficiency in red blood cells compared to type II which is more severe and less frequent and CYB5R deficiency is present in all cells. Sometimes it might mimic as static insult sequalae in the form of spastic diplegia. Here we reported an 8-month-old female with spastic diplegia with recurrent episodes of bluish discoloration later found to have autosomal recessive congenital methemoglobinemia.

Three novel mutations in CYB5R3 gene causing NADH-cytochrome b5 reductase enzyme deficiency leads to recessive congenital methaemoglobinemia.

Methemoglobin is the reduced form of haemoglobin that is normally found in the blood in levels < 1%. Methemoglobinemia can occur as a congenital or acquired disease. Two types of recessive congenital methaemoglobinemia (RCM) are caused by the NADH-dependent cytochrome b5 reductase enzyme deficiency of the CYB5R3 gene. RCM-I is characterized by higher methaemoglobin levels (> 2g/dL), causing only cyanosis, whereas RCM-II is associated with cyanosis with neurological impairment. Routine haematological investigations were done by standard method. The methaemoglobin level was evaluated by the potassium ferricyanide assay. NADH-cytochrome b5 reductase (cytb5r) enzyme activities were measured by standard methods, and molecular analysiswas 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. 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 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 frameshift of termination codon are associated with neurological impairment. Molecular diagnosis ofRCM can help to conduct genetic counselling for novel mutations and, subsequently, prenatal diagnosis of high-risk genetic disorders.

Nadir bir Hipoksemi Sebebi; Herediter Methemoglobinemi

Hereditary methemoglobinemia is one of the rare causes of hypoxemia. Mutations in the CYB5R3 gene cause autosomal recessive hereditary methemoglobinemia. Mostly, symptoms such as shortness of breath, bruise and and fatique occur. It may not display any symptoms until adult ages. Our case was at the age of 18 and had sometimes recurring bruise in hands and lips, shortness of breath, palpitations and oxygen saturation (SaO2) was 85%. Methemoglobin (Methb) level was %20 (N;0-1.5) No cardiac or pulmonary cause could be detected, which could account for dyspnea and cyanosis, and due to low saturation and high Methb. levels, metheglobinemia was considered and high dose IV ascorbic acid was administered. In follow up period, saturation increased and Methb. level gradually decreased and with genetic tests, homozygous missense c.136C&amp;gt; T (p.R46W) mutation was detected with CYB5R3 gene sequence analysis. Patient was diagnosed with autosomal recessive hereditary methemoglobinemia type 1. This case is presented ın order to emphasize that hereditary methemoglobinemia should be kept in mind when shortness of breath, hypoxia and cyanosis, occur together and can not be attributed to pulmonary and cardiovascular causes.

Recommendations for diagnosis and treatment of methemoglobinemia.

Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro‐iron of hemoglobin (Hb) to ferri‐iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi‐like approach and the experts panel reached a final consensus >75% of agreement for all the questions.

Severe phenotype of an Iranian patient with methemoglobinemia type II due to a novel mutation in the CYB5R3 gene

Methemoglobinemia is a rare autosomal recessive genetic disease caused by disruptive mutations in the CYB5R3 gene (MIM: 250800). Herein, a novel mutation is reported in an Iranian patient affected with methemoglobinemia type II. In this case study, the patient is precisely described according to the thoroughly carried-out examinations and workups. In so doing, the peripheral blood sample was collected to evaluate the methemoglobin level and NADH-CYB5R3 activity test. Moreover, whole-exome sequencing (WES) was recruited to identify the mutation leading to this disorder. Subsequently, Sanger sequencing was employed to confirm the detected mutation. Magnetic Resonance Imaging was also performed to explore the structure of the brain. As identified by the blood test, the methemoglobin level increased up to 25%, and the NADH-CYB5R3 enzyme activity showed to be 13.8 IU/g of Hb. A novel homozygous mutation in CYB5R3 (NM_001171661: g.23435C&gt;T, c.181C&gt;T, p.R61X, rs1210302322) was identified as the cause of the Methemoglobinemia type II in the proband. This nonsense mutation alters arginine to the stop codon at position 61 of protein in the FAD-binding domain that results in a truncated protein. The MRI revealed brain atrophy and corpus calusom hypoplasticity. It was established that this variation can lead to Methemoglobinemia. The proband demonstrates Methemoglobinemia type II phenotype such as cyanosis, severe mental retardation, microcephaly, as well as developmental delay. The brain MRI revealed brain atrophy and corpus calusom hypoplasticity. The cyanosis symptom is managed by daily ascorbic acid uptake.

Mutation update: Variants of the CYB5R3 gene in recessive congenital methemoglobinemia

NADH-cytochrome b5 reductase 3 deficiency is an important genetic cause of recessive congenital methemoglobinemia (RCM) and occurs worldwide in autosomal recessive inheritance. In this Mutation Update, we provide a comprehensive review of all the pathogenic mutations and their molecular pathology in RCM along with the molecular basis of RCM in 21 new patients from the Indian population, including four novel variants: c.103A>C (p.Thr35Pro), c.190C>G (p.Leu64Val), c.310G>T (p.Gly104Cys), and c.352C>T (p.His118Tyr). In this update, over 78 different variants have been described for RCM globally. Molecular modeling of all the variants reported in CYB5R3 justifies association with the varying severity of the disease. The majority of the mutations associated with the severe form with a neurological disorder (RCM Type 2) were associated with the FAD-binding domain of the protein while the rest were located in another domain of the protein (RCM Type 1).

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&gt;C (p.Thr35Arg), c.192C&gt;G (p.Leu64Val), c.312G&gt;T (p.Gly104Cys), c.354C&gt;T (p.His118Tyr) and c.470T&gt;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

Two Novel C-Terminal Frame shift Mutations in the CYB5R3 Gene Lead to Global Growth and Developmental Delay Associated with Recessive Congenital Methemoglobinemia Type II

Two Novel C-Terminal Frame shift Mutations in the CYB5R3 Gene Lead to Global Growth and Developmental Delay Associated with Recessive Congenital Methemoglobinemia Type II

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.

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene.

BackgroundIn veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.ObjectivesTo analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.AnimalsThree Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.MethodsMethemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.ResultsMethemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).Conclusions and Clinical ImportanceThis dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.

MG-113 A case ‘out of the blue’

Objective To delineate a retrospective diagnosis in a case of severe fixed encephalopathy. Case description Propositus was a male child, first born to 3rd degree consanguineous South Asian parents. Pregnancy, delivery and clinical examination at birth were normal. During the first months of life, he had generalised hypotonia with spontaneous dystonic leg extension spasm and frequent vomiting. All the developmental milestones were delayed, he never sat alone or never attained speech. From his available photographs head size appeared to be small, and esotropia and adducted thumbs were noted. Magnetic resonance imaging of his brain showed delayed myelination. He also had grade II/III gastro oesophageal reflux evidenced by GER Scintigraphy. Other investigations including routine biochemical and haematological parameters, neurometabolic screening and electrophysiological analysis were within normal limits. He was treated as spastic cerebral palsy from another centre without much improvement to the symptoms. Child expired at the age of 2 years followed by an attack of pneumonia; clinical exome test was performed later from his DNA. Results Long read sequencing revealed a homozygous, pathogenic novel variation p. Val112ProFSTer8 resulting from the deletion of exons 5–8 of CYB5R3 gene, associated with recessive hereditary methemoglobinemia (RHM) type II, which could explain the clinical profile. This large deletion was validated by polymerase chain reaction and carrier status of parents was established. Conclusion Type II RHM is a rare condition, with less than 100 cases reported worldwide. 1. Cyanosis is an invariable presenting feature and important clinical clue to methemoglobinemia. There was no history or documentation of cyanosis in our case. In the absence/failure to elicit the sign in children with severe encephalopathy, possibility of RHM might get overlooked. 2. Majority of the reported mutations in RHM type II are point mutations with two prior documentations of single exonic deletion. Ours is the first family with the largest deletion of multiple exons of CYB5R3 gene, thus expanding the genotype spectrum.

NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer*

Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.

Congenital Recessive Methemoglobinemia Revealed in Adulthood: Description of a New Mutation in Cytochrome b5 Reductase Gene

Methemoglobinemia can be acquired (oxidizing drugs or chemicals products) or inherited either by mutations affecting globin chains [M hemoglobins (M Hbs)] or by defects in the enzymatic system involved in the reduction of spontaneous Hb oxidation: nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. It is encoded by the CYB5R3 gene: there are two phenotypes of autosomal recessive congenital methemoglobinemia, in type II CYB5R deficiency is generalized and affects all cells, leading to an early onset, whereas in type I, the enzyme deficiency is restricted to erythrocytes, usually discovered in infancy but not exclusively. We report a new case of methemoglobinemia discovered in a patient from Bahrain who exhibited an unknown dyspnea at the age of 37 years without trigger events or oxidizing products. We discovered a new mutation in the CYB5R3 gene: exon 9, codon 266 (delGAG) (GLU) (CYB5R3: c.726_729delGAG) in the homozygous state. Appearance of methemoglobinemia in an adult usually suggests an acquired cause but our case illustrated that it could also reveal a type I mutation of cytochrome b5 reductase.

Genetic variation in CYB5R3 is associated with methemoglobin levels in preterm infants receiving nitric oxide therapy.

In recent years, increasing numbers of preterm infants have been exposed to inhaled nitric oxide (iNO). This population has decreased methemoglobin (MetHb) reductase activity in their erythrocytes, which may increase the risk of MetHb toxicity. We sought to determine if genetic factors are associated with the observed variance in MetHb levels. A population of 127 preterm infants was genotyped for five single-nucleotide polymorphisms (SNPs) in the CYB5A and CYB5R3 genes. iNO dose and levels of MetHb were obtained by chart abstraction. ANOVA was performed to identify genetic associations with MetHb levels. An association was found between the heterozygous genotype (GA) of rs916321 in the CYB5R3 gene and the mean of the first recorded MetHb levels in Caucasian infants (P = 0.01). This result remained significant after adjustment for the iNO dose (P = 0.009), gender (P = 0.03), multiple gestation (P = 0.03), birth weight (P = 0.02), and gestational age (P = 0.02). No significant associations were found with the other SNPs. We demonstrate a novel genetic association with neonatal MetHb levels. Identification of genetic risk factors may be useful in determining which preterm infants are most at risk of developing MetHb toxicity with the use of iNO.

Population frequency and age of c.806 C&gt;T mutation in CYB5R3 gene as cause of recessive congenital methemoglobinemia in Yakutia

Population frequency and age of c.806 C&gt;T mutation in CYB5R3 gene as cause of recessive congenital methemoglobinemia in Yakutia