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

Abstract

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.

Keywords: CYB5R3 gene; Familial methemoglobinemia; Missense variant; NADH-cytochrome b5 reductase deficiency; Nonsynonymous SNP.

Figure 1

Clinical signs of cyanosis in the patient dog (Case 1). Cyanosis of the oral mucous membranes and tongue (A) and lower abdominal skin (B) as well as a spot test of venous blood from the patient (C; right) and a normal dog (C; left).

Figure 2

Family pedigree of the patient dog. *, Dogs were examined in the present study.

Figure 3

Detection by direct DNA sequencing of a homozygous single base substitution in exon 7 (c.580A>C) of the canine CYB5R3 gene in the affected dogs. This missense changes the ATC codon to a CTC codon, which results in the replacement of an isoleucine (Ile) by a leucine (Leu) at amino acid residue 194. The position of a base change is indicated by an arrow in the chromatograms. The codon with the A→C substitution is underlined.

Figure 4

A ribbon diagram of the 3‐dimensional model of the predicted canine soluble form b5R was created from the query sequence of the SWISS‐MODEL by the PyMOL program. The FAD‐binding domain is shown in cyan (Ile34 to Leu148), while the NADH‐binding domain is shown in yellow (Val172 to Phe301), and the hinge region is shown in green (Leu149 to Thr171). This structure was based on a previously reported porcine model.9 Ile194 is shown in pink and was replaced by leucine in the Pomeranian dogs with familial congenital methemoglobinemia. The FAD molecule is represented by a ball and stick model. X‐ray crystallography indicated that the FAD cofactor was able to noncovalently bind to the large, wide boundary cleft between the two major domains,8, 10 whereas the NADH molecule was able to fit into the interdomain cleft at the reside of the FAD isoalloxazine ring.10