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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.

Background One of the fundamental assumptions of DNA methylation in clinical epigenetics is that DNA methylation status can change over time with or without interplay with environmental and clinical conditions. However, little is known about how DNA methylation status changes over time under ordinary environmental and clinical conditions. In this study, we revisited the high frequency longitudinal DNA methylation data of two Japanese males (24 time-points within three months) and characterized the longitudinal dynamics. Results The results showed that the majority of CpGs on Illumina HumanMethylation450 BeadChip probe set were longitudinally stable over the time period of three months. Focusing on dynamic and stable CpGs extracted from datasets, dynamic CpGs were more likely to be reported as epigenome-wide association study (EWAS) markers of various traits, especially those of immune- and inflammatory-related traits; meanwhile, the stable CpGs were enriched in metabolism-related genes and were less likely to be EWAS markers, indicating that the stable CpGs are stable both in the short-term within individuals and under various environmental and clinical conditions. Conclusions This study indicates that CpGs with different stabilities are involved in different functions and traits, and thus, they are potential indicators that can be applied for clinical epigenetic studies to outline underlying mechanisms.

Babesia rossi infection has been reported to be associated with the high prevalence of pancreatitis in dogs. In this study, we retrospectively investigated whether pancreatitis occurs in B. gibsoni-infected dogs. The clinical manifestations, and hematological and serum biochemical examination results, including canine pancreatic-specific lipase (cPL), in 20 B. gibsoni-infected dogs were analyzed. The cPL concentration exceeded 400 µg/l in only 2 dogs, and they were suspected of having pancreatitis. Although the cPL concentration did not correlate with the degree of anemia or the level of parasitemia, it correlated with the band neutrophil count, platelet count, and blood urea nitrogen (BUN) level. Our study suggested that the prevalence of pancreatitis is lower among B. gibsoni-infected dogs than B. rossi-infected dogs.

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.

Babesia rossi infection has been reported to be associated with the high prevalence of pancreatitis in dogs. In this study, we retrospectively investigated whether pancreatitis occurs in B. gibsoni-infected dogs. The clinical manifestations, and hematological and serum biochemical examination results, including canine pancreatic-specific lipase (cPL), in 20 B. gibsoni-infected dogs were analyzed. The cPL concentration exceeded 400 µg/l in only 2 dogs, and they were suspected of having pancreatitis. Although the cPL concentration did not correlate with the degree of anemia or the level of parasitemia, it correlated with the band neutrophil count, platelet count, and blood urea nitrogen (BUN) level. Our study suggested that the prevalence of pancreatitis is lower among B. gibsoni-infected dogs than B. rossi-infected dogs.