Oral Methylene Blue Treatment in A Dog with Cytochrome B5 Reductase Deficiency And 78, XX Testicular Disorder of Sex Development.

A 6-month-old mixed breed dog was referred for evaluation of a potential disorder of sex development (DSD) and lower than expected energy level. Genitourinary examination revealed ambiguous external genitalia, hypospadias, and a subtle pouch of skin that resembled an empty scrotum. Corrective surgery was planned and subsequently aborted after cyanosis was identified preoperatively and an arterial blood gas analysis by co-oximetry identified increased methemoglobin (MetHb) concentration (35%, normal <2%) with normal arterial oxygen tension. Ensuing investigations confirmed hereditary methemoglobinemia caused by cytochrome b5 reductase (CYB5R) deficiency via molecular genetic (Arg219Pro homozygous variant in CYB5R3 gene) and biochemical (cytochrome b5 reductase enzyme activity of 8% [normal, 100% activity] testing. Karyotyping and molecular analysis of sex chromosomes revealed the dog was genetically female with a normal female karyotype (78,XX), and was negative for the Y-linked SRY gene and positive for the X-linked androgen receptor gene. Methylene blue (MB, 3.3 mg/kg per os [PO] q24 h) was administered and the MetHb concentration decreased to 9% within 14 days. Urogenital revision surgery proceeded without complication and the dog was maintained on MB (3-4 mg/kg PO q24 h) long-term without adverse effects. This is the first report to describe the use of PO MB to decrease MetHb concentrations in a dog with CYB5R deficiency in preparation for anesthesia and highlights its potential as a viable alternative to the intravenous formulation for elective procedures. In addition, this report describes the clinical, molecular, imaging, surgical, and macroscopic and microscopic pathological features of a dog with SRY-negative, 78,XX testicular DSD.

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

BACKGROUND: 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 (> 2 g/dL), causing only cyanosis, whereas RCM-II is associated with cyanosis with neurological impairment. METHODS: 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 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: 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. CONCLUSIONS: Molecular diagnosis of RCM can help to conduct genetic counselling for novel mutations and, subsequently, prenatal diagnosis of high-risk genetic disorders.

Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1.

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.

Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with ß-cell failure.

OBJECTIVE: Diabetes is characterized by pancreatic ß-cell dedifferentiation. Dedifferentiating ß cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the ß-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear. METHODS: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to ß-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios. RESULTS: The expression of Cyb5r3 is decreased in FoxO1-deficient ß cells. Mice with ß-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient ß cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient ß cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability. CONCLUSIONS: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate ß-cell failure.

An Updated View on the Molecular Pathomechanisms of Human Dihydrolipoamide Dehydrogenase Deficiency in Light of Novel Crystallographic Evidence.

Dihydrolipoamide dehydrogenase (LADH, E3) deficiency is a rare (autosomal, recessive) genetic disorder generally presenting with an onset in the neonatal age and early death; the highest carrier rate has been found among Ashkenazi Jews. Acute clinical episodes usually involve severe metabolic decompensation and lactate acidosis that result in neurological, cardiological, and/or hepatological manifestations. Clinical severity is due to the fact that LADH is a common E3 subunit to the alpha-ketoglutarate, pyruvate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes, and is also a constituent in the glycine cleavage system, thus a loss in LADH function adversely affects multiple key metabolic routes. However, the severe clinical pictures frequently still do not parallel the LADH activity loss, which implies the involvement of auxiliary biochemical mechanisms; enhanced reactive oxygen species generation as well as affinity loss for multienzyme complexes proved to be key auxiliary exacerbating pathomechanisms. This review provides an overview and an up-to-date molecular insight into the pathomechanisms of this disease in light of the structural conclusions drawn from the first crystal structure of a disease-causing hE3 variant determined recently in our laboratory.

Application of hepatic cytochrome b5/P450 reductase null (HBRN) mice to study the role of cytochrome b5 in the cytochrome P450-mediated bioactivation of the anticancer drug ellipticine.

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.

Congenital methaemoglobinaemia in a 61-year-old patient with normal haemoglobin levels.

A 61-year-old Ghanaian woman presented with dizziness and low oxygen saturations whereupon a methaemoglobin level of 24.9% was obtained. Initially it was thought to be caused by an unknown toxin. However, failure to normalise spontaneously and a short recurrence following administration of methylene blue suggested a congenital cause. Subsequently a novel variant in the CYB5R3 gene, coding for Cytochrome b5 reductase, was demonstrated. Absence of polycythaemia prompted additional analysis for a concomitant haemoglobinopathy.

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

BACKGROUND: In 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. OBJECTIVES: To analyze the NADH-cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency. ANIMALS: Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls. METHODS: Methemoglobin 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. RESULTS: Methemoglobin 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 IMPORTANCE: This 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.

NCB5OR Deficiency in the Cerebellum and Midbrain Leads to Dehydration and Alterations in Thirst Response, Fasted Feeding Behavior, and Voluntary Exercise in Mice.

Cytosolic NADH-cytochrome-b5-oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues. We have previously reported that global ablation of NCB5OR in mice results in early-onset lean diabetes with decreased serum leptin levels and increased metabolic and feeding activities. The conditional deletion of NCB5OR in the mouse cerebellum and midbrain (conditional knock out, CKO mice) results in local iron dyshomeostasis and altered locomotor activity. It has been established that lesion to or removal of the cerebellum leads to changes in nutrient organization, visceral response, feeding behavior, and body weight. This study assessed whether loss of NCB5OR in the cerebellum and midbrain altered feeding or metabolic activity and had an effect on serum T3, cortisol, prolactin, and leptin levels. Metabolic cage data revealed that 16 week old male CKO mice had elevated respiratory quotients and decreased respiratory water expulsion, decreased voluntary exercise, and altered feeding and drinking behavior compared to wild-type littermate controls. Most notably, male CKO mice displayed higher consumption of food during refeeding after a 48-h fast. Echo MRI revealed normal body composition but decreased total water content and hydration ratios in CKO mice. Increased serum osmolality measurements confirmed the dehydration status of male CKO mice. Serum leptin levels were significantly elevated in male CKO mice while prolactin, T3, and cortisol levels remain unchanged relative to wild-type controls, consistent with elevated transcript levels for leptin receptors (short form) in the male CKO mouse cerebellum. Taken together, these findings suggest altered feeding response post starvation as a result of NCB5OR deficiency in the cerebellum.

Long-term Treatment with Methylene Blue in a Dog with Hereditary Methemoglobinemia Caused by Cytochrome b5 Reductase Deficiency.

A juvenile male mixed breed dog was presented for lethargy, exercise intolerance, and aggression when touched on the head. Cyanosis, tachycardia, and tachypnea were observed and persisted during oxygen supplementation. Arterial blood gas analysis by co-oximetry identified an increased methemoglobin concentration (27%; normal, <2%) with normal arterial oxygen tension. The methemoglobinemia and associated clinical signs resolved after administration of methylene blue (1 mg/kg) IV, and the dog was discharged. The affected dog's whole-genome sequence contained 2 potentially causal heterozygous CYB5R3 missense mutations suggesting that cytochrome b5 reductase deficiency was responsible for the methemoglobinemia. This hypothesis was confirmed by enzyme analysis that identified cytochrome b5 reductase activity in the affected dog's erythrocytes to only approximately 6% of that in a control sample. Clinical signs recurred 11 days after discharge but normalized and the methemoglobin concentration decreased with methylene blue administration PO (1.5 mg/kg, initially daily and then every other day).

Enzymopenic Congenital Methemoglobinemia in Children of the Republic of Sakha (Yakutia).

Type I congenital methemoglobinemia is an autosomal recessive disorder. A high frequency of congenital methemoglobinemia has been reported among Native Americans inhabiting the Yukon-Kuskokwim Delta. Other rare cases of congenital methemoglobinemia of types I and II have been reported in Japan and other countries. In Russia-namely, in Yakutia-a high frequency of type I congenital methemoglobinemia has been reported. In 2009, the Consultation Polyclinic of the Pediatric Center in Yakutsk city established a registry of children with congenital methemoglobinemia. In total, 43 patients were registered between 2005 and 2009. The median methemoglobin level was 13.5% (ranging between 4.2% and 33.9%) and physical examination revealed cyanosis of the skin and mucus membranes. There were significant positive relationships between percentage of methemoglobin and erythrocyte count, hemoglobin concentration, and hematocrit among male patients, consistent with an upregulation of the hypoxic response. The prevalence per 100,000 children ranged from 12.7 to 47.0 in 3 geographic regions of Yakutia. Further research is needed to clarify the clinical consequences of congenital methemoglobinemia in the children of Yakutia and the reasons for the high variability in the prevalence of the condition.

Congenital Methemoglobinemia Type II-Clinical Improvement with Short-Term Methylene Blue Treatment.

We report a case of prophylactic management with methylene blue (MB) in an almost 4-year-old male with congenital methemoglobinemia type II. He has a CYB5R3 compound heterozygote mutation, causing a cytochrome-b(5) reductase deficiency. Since the MB treatment regimen has commenced, his methemoglobin level has been significantly lower. He has shown modest behavioral improvements (as assessed on the Achenbach behavior report scales). There have been no iatrogenic side effects. These findings are encouraging for symptomatic improvement with regular prophylactic MB treatment but represent a single case report, which must be interpreted with caution.

HbM methaemoglobinaemia as a rare case of early neonatal benign cyanosis.

Early neonatal central cyanosis that is unrelated to cardiopulmonary causes, alerts clinicians to possibility of methaemoglobinaemia. Congenital methaemoglobinaemia due to haemoglobin M is an autosomal dominant disorder characterised by lifelong cyanosis. We report a case presentation and review of diagnostic pitfalls of a newborn who presented with central cyanosis; investigations revealed a low methaemoglobin reductase (2.2 IU/g Hb), with normal maternal levels (9.1 IU/g Hb). Therefore, haemoglobinopathy investigations were completed on the mother and her baby, which showed an α-globin variant in both. The maternal α2 globin gene sequencing showed heterozygosity for haemoglobin M Boston (α58 His → Tyr).

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.

Congenital methaemoglobinaemia: a rare cause of cyanosis in an adult patient.

Cyanosis is a physical finding that can occur at any age but presents a great challenge as the causes are multiple and varied. When patients present with cyanosis and dyspnoea that are unrelated to cardiopulmonary causes, methaemoglobinaemia should be considered as a possible diagnosis although rare. Methaemoglobinaemia can be asymptomatic even when methaemoglobin (metHb) levels are as high as 40% of the total haemoglobin values. Although acquired methaemoglobinaemia caused by environmental oxidizing agents is common; congenital deficiency of the innate reducing enzymes is so rare that very few cases have been documented. We report this case of type I congenital methaemoglobinaemia.

Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2.

AIMS: Membrane-bound CYB5R3 deficiency in humans causes recessive hereditary methaemoglobinaemia (RHM), an incurable disease that is characterized by severe neurological disorders. CYB5R3 encodes for NADH-dependent redox enzyme that contributes to metabolic homeostasis and stress protection; however, how it is involved in the neurological pathology of RHM remains unknown. Here, the role and transcriptional regulation of CYB5R3 was studied under nutritional and oxidative stress. RESULTS: CYB5R3-deficient cells exhibited a decrease of the NAD(+)/NADH ratio, mitochondrial respiration rate, ATP production, and mitochondrial electron transport chain activities, which were associated with higher sensitivity to oxidative stress, and an increase in senescence-associated ß-galactosidase activity. Overexpression of either forkhead box class O 3a (FOXO3a) or nuclear factor (erythroid-derived 2)-like2 (Nrf2) was associated with increased CYB5R3 levels, and genetic ablation of Nrf2 resulted in lower CYB5R3 expression. The presence of two antioxidant response element sequences in the CYB5R3 promoter led to chromatin immunoprecipitation studies, which showed that cellular stressors enhanced the binding of Nrf2 and FOXO3a to the CYB5R3 promoter. INNOVATION: Our findings demonstrate that CYB5R3 contributes to regulate redox homeostasis, aerobic metabolism, and cellular senescence, suggesting that CYB5R3 might be a key effector of oxidative and nutritional stress pathways. The expression of CYB5R3 is regulated by the cooperation of Nrf2 and FOXO3a. CONCLUSION: CYB5R3 is an essential gene that appears as a final effector for both nutritional and oxidative stress responses through FOXO3a and Nrf2, respectively, and their interaction promotes CYB5R3 expression. These results unveil a potential mechanism of action by which CYB5R3 deficiency contributes to the pathophysiological underpinnings of neurological disorders in RHM patients.

Methemoglobinemia after a mediastinal stab wound.

BACKGROUND: Methemoglobinemia is a relatively common, potentially fatal syndrome resulting from oxidative stress. Of the numerous causes identified, toxins are the most common precipitating event. OBJECTIVES: Describe methemoglobinemia after a stab wound in a man with previously undiagnosed cytochrome b5 reductase deficiency. CASE REPORT: In this case report, we describe a 27-year-old man with no past medical history who developed clinically significant methemoglobinemia after a mediastinal stab wound. After an extensive toxicologic work-up failed to reveal the etiology of the symptoms, genetic testing was performed, which revealed the individual to have a previously undiagnosed cytochrome b5 reductase deficiency. It is hypothesized that the physiologic stress from the expanding mediastinal stab wound resulted in enough oxidative stress to cause methemoglobinemia in this predisposed individual. A discussion of methemoglobinemia ensues. CONCLUSION: This case describes an uncommon presentation of a common toxicologic condition and presents a discussion regarding the evaluation, management, and pathophysiology of methemoglobinemia.