Cytochrome B5 Reductase Deficiency Research Articles

Inflammatory phenotype, clinicopathologic variables, and effects of long-term methylene blue in dogs with hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency

To determine whether dogs with cytochrome b5 reductase (CYB5R) deficiency have a constitutive proinflammatory phenotype, characterize hematologic and serum chemistry results, and describe changes in methemoglobin (MetHb) levels and serum C-reactive protein (CRP) concentrations after long-term per os (PO) methylene blue (MB) therapy. 21 client-owned dogs (CYB5R deficient, n = 10; healthy controls, 11). In this prospective, case-control study, methemoglobin levels were measured using a blood gas analyzer with co-oximetry. Plasma tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations were measured using a canine-specific multiplex bead-based assay. Serum CRP concentrations were measured with a canine-specific commercial ELISA kit. Serum CRP concentration and MetHb levels were measured in 6 dogs with CYB5R deficiency after ≥ 60 days of PO MB therapy. As expected, MetHb levels were higher in dogs with CYB5R deficiency compared to controls (P < .001). Plasma TNF-α, IL-6, IL-10, and serum CRP concentrations were no different between CYB5R-deficient and control dogs. Dogs with CYB5R deficiency had lower absolute lymphocyte (P = .005) and eosinophil counts (P = .04) and higher alanine transaminase (P = .04) and alkaline phosphatase activity (P = .02) than controls, but these changes were not clinically relevant. Methemoglobin levels decreased after PO MB therapy (P = .03). These results suggest that otherwise healthy dogs with CYB5R deficiency do not have a constitutive proinflammatory phenotype and clinically relevant abnormalities in hematologic and serum chemistry panels are not expected. Dogs with decreased quality of life attributed to methemoglobinemia from CYB5R deficiency might benefit from PO MB therapy.

Methemoglobinemia.

The targeted audience for this simulation are emergency medicine providers, including residents as well as advanced practice providers, to properly educate on recognizing, diagnosing, and managing methemoglobinemia. Methemoglobinemia is a blood disorder characterized by the presence of ferric form of hemoglobin in the blood. This form of hemoglobin can carry oxygen but is unable to release it effectively causing a range of symptoms including headache, dizziness, nausea, and cyanosis. It is rarely congenital and mostly caused by the exposure to oxidizing agents, such as local anesthetics and quinolones.1 Normally, oxygen can bind to hemoglobin while it is in the ferrous state (Fe2+). In cases of methemoglobinemia, the heme iron configuration is converted from ferrous (Fe2+) to ferric (Fe3+), making it unable to bind to oxygen. As a result, normal ferrous hemes experience an increased affinity for oxygen causing a leftward shift in the oxygen dissociation curve. This in turn causes functional anemia due to reduced oxygen carrying capacity.1 Methemoglobinemia can result from exposure to different medications as well as environmental factors and presents like other disease processes including chronic obstructive pulmonary disease exacerbations. Congenital methemoglobinemia due to cytochrome b5 reductase deficiency is very rare, but the actual incidence is not known. Increased frequency of disease has been found in Siberian Yakuts, Athabaskans, Eskimos, and Navajo.2 Although it is also an unusual occurrence, acquired methemoglobinemia is much more frequently encountered than the congenital form.1In a 10-year retrospective study looking at the incidence rate of topical anesthetic-induced methemoglobinemia, it was found that the overall prevalence was 0.035%. A major risk factor was hospitalization at the time of a procedure being performed. An increased risk was also seen with benzocaine-based anesthetics.3. At the end of this simulation case, participants should be able to: 1) recognize shortness of breath, cyanosis and respiratory distress, and the difference between all of them based on the clinical presentation 2) identify the underlying cause of the condition by conducting a thorough history and physical 3) know how to identify and treat methemoglobinemia by ordering necessary labs and interventions and understand the pathophysiology leading to methemoglobinemia 4) recognize patient's response to treatment and continue to reassess. This is a high-fidelity simulation case that allows participants to evaluate and treat methemoglobinemia in a safe environment. The case is followed by a debriefing and small group discussion to review patient care skills, medical knowledge, interpersonal communication, practice-based learning, and improvement. The educational content and efficacy were evaluated by oral feedback and a debriefing session immediately after completion of the simulation. A 5-point Likert scale was sent out to participants pre-simulation and post-simulation. Questions on the survey included whether they felt confident in their ability to recognize methemoglobinemia, understood the physiology and causes of methemoglobinemia, and felt confident in their ability to treat methemoglobinemia. Sixteen learners responded to the survey, consisting of EM residents and medical students. Post simulation, approximately 92% of EM residents answered agree or strongly agree in their ability to recognize and treat methemoglobinemia compared to pre-sim survey of about 62.5%. Post-simulation feedback also resulted in positive reception, and learners found it useful to run through an uncommonly seen case in the hospital. Results showed overall improvement in recognition and treatment of methemoglobinemia among residents and medical students. This simulation improved recognition of methemoglobinemia including signs and symptoms associated with it. Proper management and treatment options were included such as administration of methylene blue. Overall, this simulation was helpful in teaching EM residents how to recognize, manage, and treat methemoglobinemia. In addition, post-simulation debriefing allowed further discussion among residents, which they found valuable. Methemoglobinemia, shortness of breath, cyanosis, respiratory distress, anemia, methemoglobin, oxygen dissociation curve, emergency medicine simulation.

Cyanosis in a Previously Well Child.

Cyanosis in a Previously Well Child.

Congenital Methemoglobinemia Under Diagnosed Condition with Variiable Therapeurtic Options

Methemoglobinemia is a rare overlooked differential diagnosis in patients with cyanosis and dyspnea unrelated to cardiopulmonary causes.Methemoglobin results from oxidation of ferrous iron to ferric iron within the heme moiety of hemoglobin . In healthy individuals, less than 1% of hemoglobin is present in the oxidized form (methemoglobin), which has limited ability to carry oxygen, however with increased oxygen affinity at the remaining binding sites .This results in decrease of oxygen delivery to tissue leading to hypoxaemia and lactic acidosis.Methemoglobin reductase enzyme deficiency is either type I or type II; type I: Cytochrome b5 reductase deficiency, demonstrable only in the erythrocytes, presents as uncomplicated, benign methemoglobinemia, and associated with a normal life expectancy with only fatigue and dyspnea being the most commonly reported symptoms.Hereditary type II methemoglobinemia, is global (affects both red and white blood cells) and is associated with severe neurologic dysfunction and reduced life expectancy . The enzyme's activity is less than 20% of normal.Type II congenital methemoglobinemia constitutes approximately 10% of all cases of congenital methemoglobinemia and usually causes death within the first few years of lifeThere is a significant delay in the onset of symptoms in a patient with type I hereditary methemoglobinemia, as most studies indicate that patients are asymptomatic in infancy and childhood.we report a 29 year old Indian non-smoker male, his story started 6 months prior to presentation when he had generalized fatigue and discoloration of hands. He presented with persistent polycythaemia with elevated hemoglobin level. The patient was misdiagnosed in another center as polycythaemia and treated with Imatinib. The diagnosis of PV was revisited and ruled out in view of negative JAK2, normal erythropoietin level and absence of features of panmyelosis.Clinical cyanosis and low oxygen saturation in the presence of normal arterial oxygen tension was highly suggestive of methemoglobinemia.Arterial blood gas (ABG) revealed a methemoglobin level of 38% (normal: 0-1.5%). Cytochrome B5 reductase (Methemoglobin reductase B) wasdeficient at level of <2.6 U/g Hb) (normal: 6.6-13.3), consistent with methemoglobin reductase (cytochrome b5) deficiency and hence the diagnosis of congenital methemoglobinemia was established.The role of Imatinib in provoking methemoglobinemia is questionable and association between Imatinib and methemoglobinemia never described before. There was no history of other drugs offended in aggravating the patients' symptoms and cyanosis.The patient started on Vitamin C 500 mg for which he responded well with less cyanosis and significant reduction of methemoglobin level.Upon extensive review of English literature for cases of congenital Methaemoglobinaemia due to deficiency of cytochrome b5 reductase, we found 23 cases diagnosed as type I (including the case reported here).17 cases (~74%) of type I and 6 cases (27%) of type II. There is male predominance (73%) versus 26% in females. Almost half of reported cases (12 cases; 52%) are indian, 2 Japanese, 3 English, 2 Arabic, one Spanish and one Italian.For type I, the median calculated age is 31 years with cyanosis and shortness of breath being the most common sign and symptom.For type II: Six cases were reported, all in pediatric age group with median calculated age at presentation is 6 years with neurologic manifestations and mental retardation are the most common type II associated symptoms.Congenital methemogobinemia is a rare underreported hemoglobin disease. Due to lack of systematic epidemiological studies it is usually under investigated and frequently clinically missed especially when presenting in adulthood. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

SEVERE METHEMOGLOBINEMIA WITH UNKNOWN CAUSE – CASE REPORT

The aim: Methemoglobina is an oxidized form of hemoglobin, which normally doesn`t exceed 1%. It is stated that the amount of methemoglobin over 70% is fatal. The etiology vary, it might for instance be idiopathic, congenital or caused by toxic compounds. Material and methods: We analyzed the case of patient who had exceeded the fatal level of methemoglobin in a blood. Case report: Medical Air Rescue Team was dispatched to an 80-year-old patient who suddenly lost consciousness at home. Methemoglobin level was measured using Cobas b221 system and amounted to 75%. Given the patient’s advanced age, congenital causes of methemoglobinemia, such as deficiency of cytochrome-b5 reductase or nicotinamide adenine dinucleotide reductase (NADPH-MHb reductase) were excluded a priori. It seems that the only reason for the observed methemoglobinemia in the case of the examined patient was the acquired methemoglobinemia. The patient was treated with methylene blue with good results, her state improved soon. Despite a detailed interview, it was impossible to establish the etiological factor of methemoglobinemia. None of the household members exhibited clinical symptoms of poisoning with methemoglobinogenic substance. Although the methemoglobin level was potentially deadly, the patients survived. Conclusion: It should be stressed that it is not always possible to determine the etiological factor of the discussed disorders. Knowledge of the clinical picture of poisoning, as well as of the changes characteristic for poisoning and observable in laboratory, is extremely important, especially when it is not possible to determine the etiological factor of poisoning.

Methemoglobinemia Secondary to Herbal Products.

Dear Editor, We read with interest the recent article by Al-Qahtani et al. regarding methemoglobinemia in a young boy.[1] We would like to discuss few other essential points that we believe to be important for Saudi J Med Med Sci readers to have a comprehensive understanding of this topic.[23] While there seems to be a good temporal correlation between Ferula asafoetida ingestion and the development of methemoglobinemia in the case reported, we still strongly recommend that in such cases, a comprehensive workup should be performed to rule out congenital methemoglobinemia. In young patients, we should be very cautious and always consider congenital methemoglobinemia before labeling them as a case of acquired methemoglobinemia. We recommend genetic testing for cytochrome b5 reductase deficiency and hemoglobin (Hb) M to ensure that in such cases, the child is free from any genetic perturbations that can put him/her at risk for the recurrence of methemoglobinemia in future.[4] Hb M is a special variant in which heme iron can undergo auto-oxidation, resulting in methemoglobinemia. The common Hb M variants are Hb M-Boston, Hb M-Iwate, Hb Auckland, Hb Chile, Hb M-Saskatoon and Hb M-Milwaukee. Food products such as frozen items may have high nitrate content, and there have been reports of clusters of cases with many family members developing methemoglobinemia because of frozen food consumption.[5] In addition, it should be noted that rarely used topical agents such as eutectic mixture of local anesthetic cream, e-cigarettes, contaminated water and other herbal medicines such as choy sum can lead to methemoglobinemia.[6] In the case reported by Al-Qahtani et al., it is unclear why the patient had anemia of 10.4 g/dl and leukocytosis of 24,000/mm. Did the authors look for hemolysis in this case and what was the likely explanation of the high white blood cell count? Getting a serum lactate dehydrogenase and other hemolytic panel such as serum haptoglobin, direct antiglobulin test and peripheral blood smear would be beneficial to rule out concomitant hemolysis that can be associated with methemoglobinemia.[7] The family history is very relevant in this case, as rightly explained by Al-Qahtani et al.[1] As per the history provided, both the brothers of the infant had glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, we recommend that the parents should also be checked for this deficiency. In case of a male boy, there is a 50% chance of having a G6PD deficiency if the mother is a carrier. Therefore, genetic testing of parents is mandatory to predict the likelihood of transmitting the condition to children. Methylene blue (MB) is the drug of choice in the management of methemoglobinemia. In the case reported by Al-Qahtani et al., MB was not given for the fear that he may be positive for G6PD deficiency given the strong family history. This patient was critically sick and mechanically ventilated. We are not sure about the logistic resources at the author’s medical center, but a rapid fluorescent spot test for G6PD deficiency is now readily available, which can confirm G6PD deficiency in as early as a few minutes.[8] This patient was not given either MB or vitamin C and instead was successfully treated with hyperoxic ventilation. This is a great learning case, but we wish to reinforce the fact to the readers that MB should be the first choice and all efforts should be made to check the G6PD status so that patients are not denied the standard of care. It should also be noted that in addition to MB and ascorbic acid, other drugs such as riboflavin and cimetidine can be used to treat methemoglobinemia with variable outcomes.[3] Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

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

The Acute Treatment of Methemoglobinemia in Pregnancy

BackgroundMethemoglobinemia can be a potentially lethal condition due to the hypoxic stress placed on the body. In pregnancy, the deleterious effects can be even more catastrophic. The benefits of treatment in all patients, especially in those who are pregnant, must outweigh the inherent risks of the therapies used to treat methemoglobinemia. Case ReportWe present a case of a 26-year-old Hispanic pregnant female at 30 weeks gestation presenting to the emergency department for chest pain, hypoxia, and cyanosis. She was subsequently diagnosed with methemoglobinemia, treated with methylene blue, and admitted to the intensive care unit with toxicology and obstetrics consultations. As an outpatient, the patient underwent genetic testing and was diagnosed with homozygous cytochrome b5 reductase deficiency as the etiology of the methemoglobinemia. Why Should an Emergency Physician Be Aware of This?Methemoglobinemia is a rare, potentially lethal, but treatable condition. In the setting of pregnancy, methemoglobinemia can pose a significant risk to the mother and fetus by causing acute hypoxia. Because methemoglobinemia can be acquired or congenital, treatments vary based on the etiology. Methylene blue is the mainstay treatment for symptomatic methemoglobinemia of levels > 20%. The teratogenic risks of methylene blue require risk−benefit analysis and discussion with the patient before utilization. Systemic maternal administration is theorized to be of lowest risk to the fetus. In this case, methylene blue was used safely as an emergent therapy for congenital methemoglobinemia during pregnancy.

Congenital methemoglobinemia misdiagnosed as polycythemia vera: Case report and review of literature.

Methemoglobinemia is a rare overlooked differential diagnosis in patients presented with cyanosis and dyspnea unrelated to cardiopulmonary causes. Our patient is 29 year old Indian non-smoker male, his story started 6 months prior to presentation to our center when he had generalized fatigue and discoloration of hands. He presented with persistent polycythemia with elevated hemoglobin level. The patient was misdiagnosed in another center as polycythemia and treated with Imatinib. The diagnosis of PV was revisited and ruled out in view of negative JAK2, normal erythropoietin level and absence of features of panmyelosis. Clinical cyanosis and lowoxygen saturation in the presence of normal arterial oxygen tension was highly suggestive of methemoglobinemia. Arterial blood gas revealed a methemoglobin level of 38% (normal: 0-1.5%). Cytochrome B5 reductase (Methemoglobin reductase B) was deficient at level of <2.6 U/g Hb) (normal: 6.6-13.3), consistent with methemoglobin reductase (cytochrome b5) deficiency and hence the diagnosis of congenital methemoglobinemia was established. The role of Imatinib in provoking methemoglobinemia is questionable and association between Imatinib and methemoglobinemia never described before. In our case, there were no other offending drugs in aggravating the patients’ symptoms and cyanosis. The patient started on Vitamin C 500 mg once daily for which he responded well with less cyanosis and significant reduction of methemoglobin level. Congenital methemoglobinemia is a rare underreported hemoglobin disease and often clinically missed. Upon extensive review of English literature for cases of congenital methemoglobinemia due to deficiency of cytochrome b5 reductase, we found 23 cases diagnosed as type I (including the case reported here). 17 cases (~74%) of type I and 6 cases (27%) of type II. There is male predominance 73% versus 26% in females. Almost half of reported cases 12 cases (52%) are Indian, 2 Japanese, 3 English, 2 Arabic, one case Spanish and one case Italian. For type I, the median calculated age is 31 years with cyanosis and shortness of breath being the most common sign and symptoms. For type II: Six cases were reported in English literature, all in pediatric age group with median calculated age at presentation is 6 years with neurologic manifestations and mental retardation are the most common type II associated symptoms. Due to lack of systematic epidemiological studies, congenital methemoglobinemia is under diagnosed as it is under investigated and usually overlooked especially when presenting in adulthood and in absence of obvious acquired agents.

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.

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.

Biochemical and anatomical basis of brain dysfunctions caused by cytochrome b5 reductase deficiency or dysregulation

Cytochrome b5 reductase (Cb5R) and cytochrome b5 (Cb5) are coupled redox systems with a high potential as biomarkers of health and disease in the brain because they regulate metabolic pathways that are essential to maintain normal neuronal function, like lipid biosynthesis, steroid and xenobiotics metabolism, neuronal bioenergetics and production of reactive oxygen species. Mutations of the Cb5R reported in humans produce recessive congenital methemoglobinemia of type II, a disease with severe clinical neurological dysfunctions. The isoform 3 of Cb5R (Cb5R3) and Cb5 are highly expressed in pyramidal neurons of the primary and secondary motor areas of frontoparietal cerebral cortex, hippocampus, vestibular, reticular and motor nuclei of the cerebellum and brain stem, and also in Purkinje and granule neurons of the cerebellum cortex. These brain areas are highly prone to undergo oxidative stress-induced neurodegeneration and their functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia.

A novel nine base deletion mutation in NADH-cytochrome b5 reductase gene in an Indian family with recessive congenital methemoglobinemia-type-II

Recessive hereditary methemoglobinemia (RCM) associated with severe neurological abnormalities is a very rare disorder caused by NADH- cytochrome b5 reductase (cb5r) deficiency (Type II). We report a case of 11month old male child who had severe mental retardation, microcephaly and gross global developmental delay with methemoglobin level of 61.1%. The diagnosis of NADH-CYB5R3 deficiency was made by the demonstration of significantly reduced NADH-CYB5R3 activity in the patient and intermediate enzyme activity in both the parents. Mutation analysis of the CYB5R gene revealed a novel nine nucleotide deletion in exon 6 leading to the elimination of 3 amino acid residues (Lys173, Ser174 and Val 175). To confirm that this mutation was not an artifact, we performed PCR-RFLP analysis using the restriction enzyme Drd I. As the normal sequence has a restriction recognition site for Drd I which was eliminated by the deletion, a single band of 603-bp was seen in the presence of the homozygous mutation. Molecular modeling analysis showed a significant effect of these 3 amino acids deletion on the protein structure and stability leading to a severe clinical presentation. A novel homozygous 9 nucleotide deletion (p.K173–p.V175del3) is shown to be segregated with the disease in this family. Knowing the profile of mutations would allow us to offer prenatal diagnosis in families with severe neurological disorders associated with RCM — Type II.

Methemoglobinemia after a Mediastinal Stab Wound

BackgroundMethemoglobinemia is a relatively common, potentially fatal syndrome resulting from oxidative stress. Of the numerous causes identified, toxins are the most common precipitating event. ObjectivesDescribe methemoglobinemia after a stab wound in a man with previously undiagnosed cytochrome b5 reductase deficiency. Case ReportIn 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. ConclusionThis case describes an uncommon presentation of a common toxicologic condition and presents a discussion regarding the evaluation, management, and pathophysiology of methemoglobinemia.

Transient Neonatal Cyanosis Associated With a New Hb F Variant

Neonatal cyanosis in healthy newborns can be associated either with methemoglobin due to cytochrome b5 reductase deficiency or to M-hemoglobin, a group of hemoglobin variants resulting from mutations in the globin chain genes. We report the clinical case of a neonate with cyanosis and normal cardiac and respiratory function. At birth the hematological parameters were normal; however, the methemoglobinemia was 16%. Spontaneously, the cyanosis gradually decreased and by the fifth month of age the methemoglobin level was normal. A heterozygous Gγ-globin gene (HBG2) missense mutation 87 C-A (Leu28Met) was identified. His father, with a history of transfusion in the neonatal period, is heterozygous for the same mutation. This hemoglobin variant, not previously described, was called Hb F Viseu and is the sixth Gγ-chain variant reported in association with neonatal cyanosis.

Methemoglobin Reductase Deficiency

Cytochrome b5 reductase (CB5R) deficiency is a recessively inherited autosomal disorder that is either benign (type I) or associated with severe neurological problems (type II). Specific mutations in the CYB5R gene are not exclusive to each type. Two cyanotic children with developmental delay but with slow progression were investigated for CB5R deficiency. A novel mutation, p.Arg58Pro, was independently detected in both cases. The clinical variability and severity of the disease reflect the combined effects of impaired function of the 2 mutant enzymes. As illustrated by these 2 cases, inheritance of p.Arg58Pro with either p.Gly76Ser or pLeu188del causes a clinical condition more severe than type I and less severe than the type II cases reported to date.

Index of Suspicion

Index of Suspicion

A Novel Mutation in Type II Methemoglobinemia

Type II methemoglobinemia is a somatic deficiency of cytochrome b5 reductase with severe global neurologic impairment. We report a novel mutation in exon 3 of the CYB5R3 gene on chromosome 22 consisting of homozygous 1-base pair (bp) deletion noted as c.215delG; p.Gly72AlafsX100. The patient had improvement of gross motor skills, chewing, and swallowing that may be due to the initiation of daily ascorbic acid therapy. We hypothesize that a possible response to ascorbic acid may be related to the effect of making additional ferrous iron available for its role as a cofactor in carnitine synthesis.

Identification of Two Novel Mutations in the CYB5R3 Gene in Five Cases of Type I Methemoglobinemia

Congenital methemoglobinemia due to cytochrome b5 reductase (CYB5R3) deficiency are sporadic worldwide, although clusters of this disorder have been identified in certain ethnic groups. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients representing three distinct ethnic backgrounds. Three subjects were of Indian ethnicity one being phenotypically normal and his two cousins diagnosed with type I methemoglobinemia. Causative mutations in the globin genes were excluded after sequencing. A fourth subject, a 12 year old Mexican child with Turner's syndrome and apparent congenital cyanosis was referred to us for evaluation. Her methemoglobin level was elevated and erythrocyte cytochrome b5 reductase was reduced (4% of the normal range). The fifth individual, a 36 year old woman of Greek ancestry, had a past history of childhood cyanosis and low O2 saturation (82%) post-surgery in 2007. In early 2008 she was diagnosed with methemoglobinemia due to dyspnea, disproportionate fatigue, and significant increase in methemoglobin levels (10%). Genomic DNA from all 5 subjects was isolated, and all nine exons of the CYB5R3 gene were PCR amplified and sequenced. A non-synonymous heterozygous mutation (G>A transversion) in exon 6 resulting in the change of amino acid 178 from alanine to threonine (A178T) was identified in both affected Indian siblings and not in their unaffected cousin. This mutation which is located in the NADH binding domain and in close proximity to the adenosine moiety, and may influence binding of the NADH coenzyme had been previously reported (Dekker et al., 2001). A novel homozygous mutation in an evolutionarily conserved position located in exon 3 (C>G transition) resulting in substitution of serine 53 for arginine (S53R) was found in the child of Mexican descent. This mutation, located within the FAD binding domain, is hypothesized to cause disruption of hydrogen bonding and steric hindrance due to the bulkier arginine side chain, consequently leading to protein instability. Five heterozygous mutations were detected in the subject with Greek ancestry. A synonymous G>A substitution (Pro44Pro) in exon 2, a previously reported type II mutation G>A (Val253Met) and two 3′-UTR single nucleotide polymorphisms (A>G at 29690 nt, and C>A at 29914 nt), all in exon 9, were found in a cis configuration. A novel heterozygous compound mutation in exon 6, a T>G transversion, resulting in substitution of phenylalanine 157 for cysteine (F>C) was identified in the same subject with Greek ancestry (Figure 1). PCR amplified CYB5R3 transcripts harboring either wild-type, S53R, or F157C sequences were cloned, in frame with the 10X histidine tag, into the pET16b expression vector. Sequence fidelity and in frame fusion of all constructs was verified by sequencing from both strands. Recombinant protein was purified using 1 mL niquel-affinity columns. Kinetic studies using recombinant CyB5R3 enzyme will follow. [Display omitted]