The first Chinese with Hb Chile leading to chronic anemia and methemoglobinemia: a case report.

BACKGROUND: Hemoglobin (Hb) Chile [ß28(B10) Leu > Met; HBB: c.85 C > A] is a rare hemoglobin variant caused by a missense mutation in the HBB gene. Only one case of Hb Chile has been reported worldwide so far. It is an unstable hemoglobin, characterized by cyanosis associated with chronic methemoglobinemia and hemolytic anemia induced by sulfonamides or methylene blue. CASE PRESENTATION: A 9-year-3-month-old girl had mild anemia of unknown etiology for more than 6 years. She had a slight pallor without other symptoms or signs. The complete blood count revealed normocytic normochromic anemia with a sometimes-elevated reticulocyte count, and the bone marrow cytology showed marked erythroid hyperplasia, but the tests related to hemolysis were normal. Therefore, the whole exome sequencing was performed and showed a heterozygous mutation for HBB: c.85 C > A. With asymptomatic methemoglobinemia confirmed later, she was eventually diagnosed with Hb Chile. CONCLUSIONS: This is the first report of Hb Chile in China and the second worldwide. This case shows that Hb Chile is clinically heterogeneous and difficult to diagnose and expands our understanding on the clinical and hematological traits of the disease.

Whole-blood ribonucleic acid sequencing analysis in methemoglobinemia: a case report.

INTRODUCTION: Methemoglobinemia is a condition in which methemoglobin is increased and the oxygen carrying capacity of tissues is decreased, causing a lack of oxygen to the whole body. RNA (ribonucleic acid) sequencing technologies have made it possible to systematically examine how the human transcriptome responds to invasive pathologies. To our knowledge, no previous studies have reported the results of RNA sequencing in a patient with methemoglobinemia. We describe the analysis of RNAs from the whole blood of a patient with methemoglobinemia. CASE PRESENTATION: A 31-year-old Japanese man was brought to our hospital with symptoms of dyspnea due to inhalation of gas from an acetic acid phosphonitrate storage tank at a factory. The nitrogen oxide concentration measured around the storage tank was over 2500 ppm, and he witnessed orange-brown smoke at that time. After entering the area and taking a few breaths, he suddenly became unwell, with dyspnea and numbness in his extremities. He was evacuated from the area within a few minutes, at which time he was suffering from whole-body cyanosis and was still aware of the above symptoms. On arrival at the hospital, his respiration rate was 18 breaths/minute, and his SpO2 ranged from 80% to 85% on 15 L/minute of oxygen by mask (2.5 hours postexposure). Arterial blood gas testing revealed a methemoglobin level of 23.1%. After the administration of methylene blue, the patient's methemoglobin level normalized and his symptoms improved. Chest X-ray and chest computed tomography showed no evidence of pulmonary edema or interstitial pneumonia, and no other abnormal findings were observed. RNA sequencing was performed on the blood samples obtained at the time of the visit, with the blood sample collected on day 5 used as a control. To our knowledge, the present study is the first to describe the analysis of RNAs from the whole blood of a patient with methemoglobinemia. The RNA sequencing analysis showed that an activated "hydrogen peroxide catabolic process" may be associated with the pathogenesis of methemoglobinemia. CONCLUSION: The results reported in the present study may explain the pathogenesis of methemoglobinemia.

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.

Hereditary Congenital Methemoglobinemia Diagnosed at the Age of 79 Years: A Case Report.

Background: Cardiopulmonary disorders are the most common cause of central cyanosis, and methemoglobinemia is often overlooked in the differential diagnosis of patients with central cyanosis. In most cases, methemoglobinemia is acquired and hereditary congenital methemoglobinemia is rare. Only a few case reports of congenital methemoglobinemia can be found in PubMed. To date, only four cases of congenital methemoglobinemia diagnosed after the age of 50 years have been reported. Case Presentation: A 79-year-old Japanese woman presented at our hospital with the chief complaints of dyspnea and cyanosis. She exhibited cyanosis of the lips and extremities, and her SpO2 was 80%, with oxygen administration at 5 L/min. Blood gas analysis revealed a PaO2 of 325.4 mmHg and methemoglobin level of 36.9%. The SpO2 and PaO2 values were dissociated, and methemoglobin levels were markedly elevated. Genetic analysis revealed a nonsynonymous variant in the gene encoding nicotinamide adenine dinucleotide cytochrome (NADH) B5 reductase 3 (CYB5R3), and the patient was diagnosed with congenital methemoglobinemia. Conclusions: It is important to consider methemoglobinemia in the differential diagnosis of patients with central cyanosis. At 79 years of age, our patient represents the oldest patient with this diagnosis. This report indicates that it is crucial to consider the possibility of methemoglobinemia regardless of the patient's age.

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.

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

OBJECTIVE: 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. ANIMALS: 21 client-owned dogs (CYB5R deficient, n = 10; healthy controls, 11). PROCEDURES: 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. RESULTS: 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). CLINICAL RELEVANCE: 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.

Cytochrome b5 reductases: Redox regulators of cell homeostasis.

The cytochrome-b5 reductase (CYB5R) family of flavoproteins is known to regulate reduction-oxidation (redox) balance in cells. The five enzyme members are highly compartmentalized at the subcellular level and function as "redox switches" enabling the reduction of several substrates, such as heme and coenzyme Q. Critical insight into the physiological and pathophysiological significance of CYB5R enzymes has been gleaned from several human genetic variants that cause congenital disease and a broad spectrum of chronic human diseases. Among the CYB5R genetic variants, CYB5R3 is well-characterized and deficiency in expression and activity is associated with type II methemoglobinemia, cancer, neurodegenerative disorders, diabetes, and cardiovascular disease. Importantly, pharmacological and genetic-based strategies are underway to target CYB5R3 to circumvent disease onset and mitigate severity. Despite our knowledge of CYB5R3 in human health and disease, the other reductases in the CYB5R family have been understudied, providing an opportunity to unravel critical function(s) for these enzymes in physiology and disease. In this review, we aim to provide the broad scientific community an up-to-date overview of the molecular, cellular, physiological, and pathophysiological roles of CYB5R proteins.

CYB5R3 homozygous pathogenic variant as a rare cause of cyanosis in the newborn.

Detailed below is a very illustrative case of a rare pathology of recessive congenital methemoglobinemia. The patient, a newborn female, was homozygous for c.535G > A, p.(Ala179Thr) a pathogenic variant in the CYB5R3 gene. The reported population frequency of the allele is 0.853%, demonstrating why it is remarkable to find both parents are heterozygous carriers without consanguinity. A brief review of previously published cases is also presented.

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.

Interpreting sulfhemoglobin and methemoglobin in patients with cyanosis: An overview of patients with M-hemoglobin variants.

INTRODUCTION: Methemoglobin (MetHb) and sulfhemoglobin (SHb) measurements are useful in the evaluation of cyanosis. When one or both values are elevated, additional analysis is important to establish the etiology of the disorder. Methemoglobinemia occurs from acquired or hereditary causes with diverse treatment considerations, while true sulfhemoglobinemia is only acquired and treatment is restricted to toxin removal. Some toxic exposures can result in a dual increase in MetHb and SHb. Hereditary conditions, such as M-Hemoglobin variants (M-Hbs), can result in increased MetHb and/or SHb values but are clinically compensated and do not require treatment if they are cyanotic but otherwise clinically well. METHODS: Herein, we report 53 hemoglobin variant cases that have associated MetHb and SHb levels measured by an adapted Evelyn-Malloy laboratory assay method. RESULTS: Our data indicate M-Hbs cause variable patterns of MetHb and SHb elevation in a fairly reproducible pattern for the particular variant. In particular, α globin chain M-Hbs can mimic acquired sulfhemoglobinemia due to an isolated increased SHb value. CONCLUSION: If the patient appears clinically well other than cyanosis, M-Hbs should be considered early in the evaluation process to differentiate from acquired conditions to avoid unnecessary testing and treatment regimens and prompt genetic counseling.

Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia.

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.

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.

The First Korean Family with Hemoglobin-M Milwaukee-2 Leading to Hereditary Methemoglobinemia.

Hemoglobin M (HbM) is a group of abnormal hemoglobin variants that form methemoglobin, which leads to cyanosis and hemolytic anemia. HbM-Milwaukee-2 is a rare variant caused by the point mutation CAC>TAC on codon 93 of the hemoglobin subunit beta (HBB) gene, resulting in the replacement of histidine by tyrosine. We here report the first Korean family with HbM-Milwaukee-2, whose diagnosis was confirmed by gene sequencing. A high index of suspicion for this rare Hb variant is necessary in a patient presenting with cyanosis since childhood, along with methemoglobinemia and a family history of cyanosis.

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).

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

Two non-pedigreed male castrated cats had persistent cyanosis over a 3-year observation period. Clinical cardiopulmonary evaluations did not reveal abnormalities, but the blood remained dark after exposure to air. Erythrocytic methemoglobin concentrations were high (~40% of hemoglobin) and cytochrome b5 reductase (CYB5R) activities in erythrocytes were low (≤15% of control). One cat remained intolerant of exertion, and the other cat developed anemia and died due to an unidentified comorbidity. Whole-genome sequencing revealed a homozygous c.625G>A missense variant (B4:137967506) and a c.232-1G>C splice acceptor variant (B4:137970815) in CYB5R3, respectively, which were absent in 193 unaffected additional cats. The p.Gly209Ser missense variant likely disrupts a nicotinamide adenine dinucleotide (NADH)-binding domain, while the splicing error occurs at the acceptor site for exon 4, which likely affects downstream translation of the protein. The 2 novel CYB5R3 variants were associated with methemoglobinemia using clinical, biochemical, genomics, and in silico protein studies. The variant prevalence is unknown in the cat population.

Exon sequencing of the alpha-2-globin gene for the differential diagnosis of central cyanosis in newborns: a case report.

BACKGROUND: Cyanosis is usually associated with serious conditions requiring urgent treatment in the neonatal intensive care unit (NICU). Hemoglobin M (Hb M) disease is one type of congenital methemoglobinemia characterized by cyanosis. Among these variants, α-globin chain mutations such as Hb M Boston present cyanosis from birth while other variants usually manifest later in life. CASE PRESENTATION: We report a case of a male newborn with cyanosis apparent since birth. Surprisingly, his respiratory and hemodynamic status including normal arterial blood oxygen saturation was stable, but oxygen saturation on pulse oximetry did not increase after 100% supplemental oxygen was started. In addition to routine pulmonary and cardiologic evaluation, further evaluation for dyshemoglobin was conducted; α2-globin gene sequencing showed a single-point variant causing Hb M Boston. Methemoglobin (MetHb) level estimated by co-oximetry was normal. After a 14-day stay in the NICU, the patient remained respiratory and hemodynamically stable without supplemental oxygen except for cyanosis. CONCLUSIONS: Hb M disease is a benign disease and does not require any treatment whereas acquired methemoglobinemia is a potentially fatal condition. Neonatologists should be aware that low oxygenation status on pulse oximetry in the face of normal arterial blood saturation values might indicate the possibility of Hb M disease in early neonatal cyanosis, irrespective of MetHb value.

Hereditary methemoglobinemia caused by Hb M-Hyde Park (Hb M-Akita) (HBB:c.277C > T; p.His93Tyr).

Healthy human blood contains only a trace amount of methemoglobin (Hb M), less than 1%. In Hb M iron is present in the oxidized ferric state (Fe3+) not in the reduced ferrous form (Fe2+) and this reduces the ability of hemoglobin to bind oxygen. The described rare hemoglobin variant Hb M­Hyde Park (also known as Hb M-Akita) results from the substitution of amino acid tyrosine by histidine at position 93 of the beta-globin chain of hemoglobin. The rare Hb variant Hb M­Hyde Park (Hb M­Akita) is mainly inherited autosomal dominant and causes methemoglobinemia. Due to the low frequency of inherited Hb M variants, the diagnosis is challenging. Here, we here report on a family with Hb M­Hyde Park (Hb M­Akita) whose members demonstrated Hb M > 10%, but were, asymptomatic except for chronic cyanosis. Due to human mobility and migration other hemogobin variants, such as beta-thalassemia minor have spread to Austria . A genetic combination of two different hemoglobin variants may result in severe anemia. Genetic counseling for patients with hemoglobin variants, including Hb M­Hyde Park (Hb M­Akita) and beta-thalassemia minor, is essential.

Genetic cause for congenital methemoglobinemia in an Australian Pomeranian dog.

Little is known about genetic causes of congenital methemoglobinemia in dogs. Here, we report a CYB5 R3 mutation in a Pomeranian dog with congenital methemoglobinemia. A 6-year-old neutered female Pomeranian dog was investigated for cyanosis noticed during anesthesia for an orthopedic procedure. The history included lifelong mild exercise intolerance and bluish tongue. Methemoglobinemia was diagnosed using co-oximetry. The CYB5 R3 gene was analyzed by comparing the patient's genomic DNA with the reference canine sequence. Mutation functional significance was investigated using snpEff and multispecies protein homology analyses. A homozygous missense single nucleotide CYB5 R3 mutation (ATC ➔ CTC at codon 194) caused a p.Ile194Leu substitution. The pIle194 residue is highly conserved in other mammals, supporting the likely pathogenicity of the substitution. The mutation described here is identical to that associated with familial methemoglobinemia in a family of Japanese Pomeranian dogs. This observation, together with the homozygous mutation found in our case, indicates that the mutant allele may be widespread within the Pomeranian breed internationally.