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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 CYB5R3 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 CYB5R3 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 CYB5R3 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.

but few veterinary studies have explored an underlying genetic cause. 2 Three types of CM have been described. Type I CM is caused by CYB 5 R 3 enzyme activity deficiency in erythrocytes. Type II CM is caused by CYB 5 R 3 enzyme activity deficiency in all tissues, resulting from several different genetic changes that either affect the catalytic domains of the enzyme or truncate the enzyme. Type IV CM is rarely reported and is caused by a deficiency in cytochrome b5 (CYB 5 ). 3 Abnormal hemoglobins (Hbs) such as Hb-M and Hb-Hana also have been reported to cause CM. [4][5][6][7] Before our investigation, another report documented type IV CM in a Pit Bull mixed breed dog, in which a 37 bp deletion in the promoter region and a missense mutation in 1 of the exons of the CYB 5 gene were suspected to be the genetic causes. 8 Another case of familial type II CM in Pomeranian dogs was reported in which the genetic abnormalities included a single nucleotide substitution in the promoter region and a missense mutation in the coding sequence of the CYB 5 R 3 gene. 9 In this report, we document the diagnostic investigation and genetic analysis of a Pomeranian dog referred to the Companion Animal Health Centre (CAHC), University of Adelaide, for marked cyanosis under general anesthesia and subsequently diagnosed with CM.

| CASE SUMMARY
A 6-year-old spayed female Pomeranian dog was anesthetized for surgery to treat right medial patella luxation and suspected cranial cruciate ligament rupture. During anesthesia, marked cyanosis of the tongue Abbreviations: CM, congenital methemoglobinemia; CYB 5 , cytochrome b 5 ; CYB 5 R 3 , cytochrome b 5 reductase; Hb, hemoglobin; MetHb, methemoglobin; NADH, nicotinamide adenine dinucleotide; SNP, single nucleotide polymorphism. and sublingual vessels was noted; the surgery was abandoned; and after recovery from anesthesia, the dog was referred to the CAHC.
The owner reported that the dog had a long history of exercise intolerance and its tongue had always been blue. On presentation at the CAHC, the dog's vital signs were within reference ranges, but its tongue appeared dark blue, and SpO 2 was 97% when the dog was breathing room air. Orthopedic examination confirmed cranial cruciate ligament rupture and medial patella luxation. The dog was admitted to hospital with surgery tentatively planned for the next day. However, observation of marked darkening of the cyanotic tongue and gingiva when the dog was walked or handled, together with the history and physical examination findings, prompted further investigation.
Cyanosis can be classified clinically as either central or peripheral.
Central cyanosis is a result of global deoxygenation of arterial blood as a consequence of cardiopulmonary diseases or decreased oxygen carrying capacity of circulating blood as is the case with methemoglobinemia. Peripheral cyanosis is caused by local reduction of oxygenated Hb. Central cyanosis is usually first observed in oral mucous membranes, especially the tongue, and peripheral cyanosis usually is limited to the affected region. 10 In our case, marked cyanosis of sublingual vessels was indicative of central cyanosis. However, no functional or structural cardiac abnormalities were detected during clinical examination or on echocardiography, and abdominal ultrasonography was unremarkable. 11 Airway and pulmonary diseases were unlikely given the dog's history, physical examination results, and unremarkable thoracic radiographs. 12 No abnormalities were observed on a serum biochemistry profile. Hematology abnormalities included moderate absolute erythrocytosis (red blood cells [RBC], 10.8 × 10 12 /L; reference range, 4.9-8.2 × 10 12 /L), marginal thrombocytopenia (platelet count, 183 × 10 9 /L; reference range, 200-500 × 10 9 /L), mild to moderate neutropenia (neutrophils, 1.5 × 10 9 /L; reference range, 3.5-12 × 10 9 /L), and lymphopenia (lymphocytes, 0.7 × 10 9 /L; reference range, 0.9-3.5 × 10 9 /L; Veterinary Diagnostic Laboratory, School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy, South Australia). Absolute erythrocytosis in the presence of cyanosis suggested that the erythrocytosis was a physiologic response to tissue hypoxemia, most likely as a result of methemoglobinemia. 13 Initial pulse oximetry and blood gas analysis using the epoc Blood Analysis System (Siemens Healthcare GMBH, Germany) indicated adequate oxygenation. However, because neither modality can detect methemoglobin (MetHb), venous and arterial blood were collected for MetHb assay using an ABL90 Flex blood gas machine (Radiometer) for co-oximetry. Published reference intervals for MetHb are ≤1.2%. 8 The dog's percentage MetHb was markedly increased, 26.3% in arterial blood and 26.0% in venous blood.

| MATERIALS AND METHODS
Reads were aligned to the reference genome Canis familiaris 3.1 using bwa. 14 Variants were called using GATK 4.0. 15 Protein altering variants of the CYB 5 R 3 gene were identified, and the likely pathogenicity of each was assessed using snpEff and Release 105. 16

| RESULTS
In CYB 5 R 3 genomic DNA, 1 potentially pathogenic variant was identi- To clarify the exact location of the mutation described both here and in another report, 9 we aligned our data with the stable, annotated transcript NCBI Reference Sequence for CYB5R3, NM_001048084.1. 17 The mutation site corresponds to the same c.580AàC in NM_001048084.1 as previously described. 9 Our analysis also showed that the dog's genotype was heterozygous T/G at position 22 819 977 bp in the putative promoter region upstream of the CYB 5 R 3 gene (Figure 2), whereas a previous report observed this site was homozygous for the G allele. 9 We found multiple other single nucleotide polymorphisms (SNPs) in the canine CYB 5 R 3 genome sequence, but based on snpEff analysis, these were deemed unlikely to be of functional significance.
To investigate whether isoleucine is conserved at the same site in other species, we used Gapped BLAST to compare the sequence of CYB 5 R 3 from 6 different mammalian species. 18 We found that pIle194 and the flanking sequence (particularly the upstream sequence) is highly conserved in 5 species, with 1 species encoding a conservative variant, valine, at position 194 (Table 1).

| DISCUSSION
Here we report a case of CM in a mature Pomeranian dog, discovered incidentally during investigation of abnormal mucous membrane color before surgical treatment for lameness. Systemic administration of methylene blue is appropriate for temporary relief of the clinical signs in situations such as advanced methemoglobinemia or when an animal undergoes surgery. 8 In our case, the canine lameness improved with medical management and surgery was not performed. Combined with the history of the canine ability to cope with its CM since birth, specific treatment for CM was deemed unnecessary. Genetic investigation identified a homozygous missense mutation in chr10: 22836951, within an exon common to the 2 splice variant CYB 5 R 3 gene transcripts, resulting in p.Ile194Leu substitution, a strong candidate for causing CM in this dog.
In humans, several distinctly different membrane-associated or soluble forms of the CYB 5 R 3 enzyme are encoded by the CYB 5 R 3 gene, generated from splice variant transcripts that differ only in the combination of the first exons that are used. 19 Genome and protein annotations from NCBI and Ensembl support the existence in dogs of at least 2 enzyme isoforms generated in the same way. The missense variant at chr10: 22836951 (cDNA580AàC in NM_001048084.1) would be predicted to impact both forms of CYB 5 R 3 enzymes, potentially producing a severe clinical phenotype. 19 In dogs and humans, the membranebound form of the CYB 5 R 3 enzyme (Membrane-CYB 5 R 3 ) is present in many tissue types and is involved in lipid metabolism among other functions. 19,20 As in humans, the soluble form of the CYB 5 R 3 enzyme (Soluble-CYB 5 R 3 ) is thought to be restricted to erythrocytes in dogs and is responsible for reducing MetHb. 21 This observation presents a puzzle because the clinical phenotype of our dog was not typical of Type II CM in humans who have severe clinical signs involving cyanosis, neurological dysfunction, and early death associated with deficiency of the membrane CYB5R3 isozyme. Instead, the phenotype of our dog was more typical of Type I CM associated with erythrocyte CYB 5 R 3 enzyme deficiency. In another study, 9 it was speculated that the mutation may contribute to decreased protein half-life, decreased catalytic efficiency, or both. Therefore, a possible explanation for the mild phenotype could be related to different protein synthesis capacities of erythrocytes compared with other tissue cells. Mature erythrocytes rapidly lose the ability to synthesize proteins after loss of the nucleus  as functionally conservative, although instances of marked functional alteration of proteins hosting this mutation have been reported. [23][24][25] When we compared the sequence of CYB 5 R 3 from 6 different mammalian species, we found the amino acid isoleucine is conserved at p194 in 5 species, with 1 species having a different conservative substitution with valine (Table 1). Together, our data and that of published reports provide further support for the likely functional significance of the p.Ile194Leu missense mutation.
Our study has several limitations the most important of which is that it remains possible that the mutation reported here and in another report 9 has no pathological impact, and that other genomic alterations might affect, for example, expression or degradation of soluble-CYB 5 R 3 . 26 As previously reported, 9 we agree that to confirm the pathological significance of the p.Ile194Leu missense variant, further investigations should be considered, including expression analysis in erythrocytes and other tissues. In our case, it would be useful to directly measure this canine CYB 5 R 3 enzymatic activity, in comparison with that of healthy dogs. 27 To probe enzyme properties, such as stability against heat and protease and affinity for NADH, in vitro protein expression systems could be devised to obtain purified reference and p.Ile194Leu mutated soluble and membrane CYB 5 R 3 enzymes. We did not assess the clinical history or laboratory abnormalities of Pomeranian dogs related to this dog as this information was unknown. However, given that the canine genotype is homozygous, it is reasonable to suspect that close or more distant family members related to both dam and sire must be carrying the pIle194Leu mutation. Because the identical mutation was found in a family of Japanese Pomeranian dogs, it is possible that, barring the highly unlikely event that identical de novo mutations have arisen independently in different lines of Pomeranian dogs, the mutation may be present within the breed internationally. Genetic testing may help to detect carriers of the mutation so as to estimate its prevalence and enable it to be eliminated from the breed.