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

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.     

A single‐nucleotide polymorphism in the canine cytochrome b5 reductase (CYB5R3) gene is associated with sulfonamide hypersensitivity and is overrepresented in Doberman Pinschers

Canine sulfonamide hypersensitivity (HS) has been associated with a variant in the cytochrome b₅ reductase gene (CYB5R3 729A>G), which encodes a drug‐detoxifying enzyme. Study objectives were to determine variant allele frequency in Doberman Pinschers (DOBE), a breed which may be predisposed to sulfonamide HS, and to characterize the effects of CYB5R3 729G on gene expression and function. CYB5R3 729A>G allele frequencies were compared between DOBE (n = 24) vs. non‐Doberman (non‐DOBE; n = 60) dogs. CYB5R3mRNA expression, protein expression, and reduction of sulfamethoxazole hydroxylamine were compared between banked canine liver samples of 729AA vs. GG genotype. The 729G allele was overrepresented in DOBE (1.00) vs. non‐DOBE dogs (0.567, p < .0001). mRNA and protein expressions as well as cyt b₅ reductase activity were similar between livers of AA and GG genotype. All Doberman Pinschers in this study were homozygous for CYB5R3 729G, which could contribute to this breed's apparent predisposition to sulfonamide HS. However, CYB5R3 729G does not alter sulfamethoxazole detoxification capacity, so a direct role could not be demonstrated. It is possible that this marker is linked to another contributing variant.     

Precision Medicine in Cats: Novel Niemann‐Pick Type C1 Diagnosed by Whole‐Genome Sequencing

State‐of‐the‐art health care includes genome sequencing of the patient to identify genetic variants that contribute to either the cause of their malady or variants that can be targeted to improve treatment. The goal was to introduce state‐of‐the‐art health care to cats using genomics and a precision medicine approach. To test the feasibility of a precision medicine approach in domestic cats, a single cat that presented to the University of Missouri, Veterinary Health Center with an undiagnosed neurologic disease was whole‐genome sequenced. The DNA variants from the cat were compared to the DNA variant database produced by the 99 Lives Cat Genome Sequencing Consortium. Approximately 25× genomic coverage was produced for the cat. A predicted p.H441P missense mutation was identified in NPC1, the gene causing Niemann‐Pick type C1 on cat chromosome D3.47456793 caused by an adenine‐to‐cytosine transversion, c.1322A>C. The cat was homozygous for the variant. The variant was not identified in any other 73 domestic and 9 wild felids in the sequence database or 190 additionally genotyped cats of various breeds. The successful effort suggested precision medicine is feasible for cats and other undiagnosed cats may benefit from a genomic analysis approach. The 99 Lives DNA variant database was sufficient but would benefit from additional cat sequences. Other cats with the mutation may be identified and could be introduced as a new biomedical model for NPC1. A genetic test could eliminate the disease variant from the population.     

Whole‐genome resequencing to identify candidate genes for the QTL for oleic acid percentage in Japanese Black cattle

In our previous study, we detected a QTL for the oleic acid percentage (C18:1) on BTA9 in Japanese Black cattle through a genome‐wide association study (GWAS). In this study, we performed whole‐genome resequencing on eight animals with higher and lower C18:1 to identify candidate polymorphisms for the QTL. A total of 39,658 polymorphisms were detected in the candidate region, which were narrowed to 1993 polymorphisms within 23 genes based on allele differences between the high and low C18:1 groups. We subsequently selected three candidate genes, that is, CYB5R4, MED23, and VNN1, among the 23 genes based on their function in fatty acid metabolism. In each candidate gene, three SNPs, that is, CYB5R4 c.*349G > T, MED23 c.3700G > A, and VNN1 c.197C > T, were selected as candidate SNPs to verify their effect on C18:1 in a Japanese Black cattle population (n = 889). The statistical analysis showed that these SNPs were significantly associated with C18:1 (p < 0.05), suggesting that they were candidates for the QTL. In conclusion, we successfully narrowed the candidates for the QTL by detecting possible polymorphisms located within the candidate region. It is expected that the responsible polymorphism can be identified by demonstrating their effect on the gene's function.     

Long‐term Treatment with Methylene Blue in a Dog with Hereditary Methemoglobinemia Caused by Cytochrome b5 Reductase Deficiency

A juvenile male mixed breed dog was presented for lethargy, exercise intolerance, and aggression when touched on the head. Cyanosis, tachycardia, and tachypnea were observed and persisted during oxygen supplementation. Arterial blood gas analysis by co‐oximetry identified an increased methemoglobin concentration (27%; normal, <2%) with normal arterial oxygen tension. The methemoglobinemia and associated clinical signs resolved after administration of methylene blue (1 mg/kg) IV, and the dog was discharged. The affected dog's whole‐genome sequence contained 2 potentially causal heterozygous CYB5R3 missense mutations suggesting that cytochrome b5 reductase deficiency was responsible for the methemoglobinemia. This hypothesis was confirmed by enzyme analysis that identified cytochrome b5 reductase activity in the affected dog's erythrocytes to only approximately 6% of that in a control sample. Clinical signs recurred 11 days after discharge but normalized and the methemoglobin concentration decreased with methylene blue administration PO (1.5 mg/kg, initially daily and then every other day).     

COMPARISON OF LUNG ATTENUATION AND HETEROGENEITY BETWEEN CATS WITH EXPERIMENTALLY INDUCED ALLERGIC ASTHMA,NATURALLY OCCURRING ASTHMA AND NORMAL CATS

Airway remodeling is a prominent feature of feline allergic asthma but requires biopsy for characterization. Computed tomography (CT) has appeal as a minimally invasive diagnostic test. The purpose of this prospective case–control study was to compare indices of airway remodeling between cats with experimentally induced, spontaneous asthma and healthy unaffected cats using CT. We hypothesized that experimental and spontaneous feline asthma would have similar CT airway remodeling characteristics and that these would be significantly different in healthy cats. Experimentally induced asthmatic research cats (n = 5), spontaneously asthmatic pet cats (n = 6), and healthy research cats (n = 5) were scanned unrestrained using a 64‐detector row CT scanner. Inspiratory breath‐hold CT scans were also performed in experimentally induced asthmatic and healthy cats. Mean ± extent variation of lung attenuation for each cat was determined using an airway inspector software program and CT images were scored for lung heterogeneity by a board‐certified veterinary radiologist who was unaware of cat group status. Groups were compared using one‐way ANOVA (unrestrained scans) and the Student's t‐test (anesthetized scans) with significance defined as P < 0.10. Experimentally asthmatic and spontaneously asthmatic cats had significantly (P = 0.028 and P = 0.073, respectively) increased lung attenuation compared to healthy cats. Heterogeneity scores were higher in experimentally induced asthmatic cat than in healthy cats. Objective quantification of lung heterogeneity and lung volume did not differ among the three groups (P = 0.311, P = 0.181, respectively). Findings supported our hypothesis. Inspiratory breath‐hold anesthetized CT scans facilitated discrimination between asthmatic and healthy cats in comparison to unrestrained CT scans.     

Identification of a nonsense mutation in feline ABCB1

The aim of this study was to sequence all exons of the ABCB1 (MDR1) gene in cats that had experienced adverse reactions to P‐glycoprotein substrate drugs (phenotyped cats). Eight phenotyped cats were included in the study consisting of eight cats that experienced central nervous system toxicosis after receiving ivermectin (n = 2), a combination product containing moxidectin and imidacloprid (n = 3), a combination product containing praziquantel and emodepside (n = 1) or selamectin (n = 2), and 1 cat that received the product containing praziquantel and emodepside but did not experience toxicity (n = 1). Fifteen exons contained polymorphisms and twelve exons showed no variation from the reference sequence. The most significant finding was a nonsense mutation (ABCB11930_1931del TC) in one of the ivermectin‐treated cats. This cat was homozygous for the deletion mutation. All of the other phenotyped cats were homozygous for the wild‐type allele. However, 14 missense mutations were identified in one or more phenotyped cats. ABCB11930_1931del TC was also identified in four nonphenotyped cats (one homozygous and three heterozygous for the mutant allele). Cats affected by ABCB11930_1931del TC would be expected to have a similar phenotype as dogs with the previously characterized ABCB1‐1Δ mutation.     

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.     

Pneumothorax in cats with a clinical diagnosis of feline asthma: 5 cases (1990–2000)

Objective: This paper characterizes the clinical findings in 5 cats with feline asthma complicated by concurrent pneumothorax. Design: Retrospective study. Medical records of cats with concurrent diagnoses of asthma and pneumothorax that were presented to the Veterinary Hospital of the University of Pennsylvania from 1990 to 2000 were reviewed. Results: Of 421 cases of feline asthma, 5 cats fulfilled the inclusion criteria (1.2%). All 5 had respiratory distress at presentation. One cat was panting, and the other 4 cats had respiratory rates of 28, 52, 58 and 120 breaths per minute (bpm), respectively (mean RR 65±39 bpm). Historical findings included untreated chronic cough (n=3), previously treated asthma (n=1), and no previous illness (n=1). Thoracocentesis was performed in 4/5 cats, and 3 of those cats required thoracostomy tubes. Four cats required immediate oxygen supplementation, and 1 of those cats required ventilation. All 5 cats had evidence of pneumothorax on initial radiographs. Follow‐up radiographs revealed partial or complete resolution of pneumothorax in 4 cats which were discharged alive with total hospitalization of 2–7 days, but were then lost to follow‐up. One cat was euthanized because it could not be weaned off mechanical ventilation, and necropsy confirmed end‐stage feline asthma and emphysema. Conclusion: Small airway obstruction can predispose asthma patients to increased alveolar pressure, emphysema, and spontaneous pneumothorax, which can lead to dyspnea in affected cats. The short‐term outcome in these cats was good despite the severity of dyspnea at presentation.     

Effects of a novel missense polymorphism within the SIGLEC5 gene on fertility traits in Holstein‐Friesian cattle

The aim of the study was to find functional polymorphism within two exons of the SIGLEC5 (sialic acid‐binding Ig‐like lectin‐5) gene and to examine its effects on the production and fertility traits of cows and bulls. Two hundred seventytwo Holstein‐Friesian cows and 574 bulls were included in the study. Novel missense polymorphism (A > G) within exon 3 causing substitution of amino acid arginine by glutamate in position 260 of SIGLEC5 protein (R260Q) was identified by sequencing and digestion by restriction enzyme Msp I. Basic production and fertility traits of cows and estimated breeding values (EBV) of bulls were analysed. The study demonstrated a significant association of SIGLEC5 R260Q polymorphism with days open and calving interval in cows as well as with breeding value for calving interval in bulls. An opposite effect of SIGLEC5 alleles for production and fertility traits was observed: the allele G increased the breeding value for the protein yield, while the allele A increased the breeding value for the calving interval. The current study suggests the involvement of SIGLEC5 R260Q polymorphism in biological processes related to fertility traits. This finding can be applied as a biomarker for a genetic improvement programme in Holstein‐Friesian cattle.     

MC1R c.310G>‐ and c.871G > A determine the coat color of Kumamoto sub‐breed of Japanese Brown cattle

Coat color is one of the important factors characterizing breeds for domestic animals. Melanocortin 1 receptor (MC1R) is a representative responsible gene for this phenotype. Two single‐nucleotide polymorphisms (SNPs) in bovine MC1R gene, c.296T > C and c.310G>‐, have been well characterized, but these SNPs are not enough to explain cattle coat color. As far as we know, MC1R genotypes of Kumamoto sub‐breed of Japanese Brown cattle have not been analyzed. In the current study, genotyping for c.296T > C and c.310G>‐ was performed to elucidate the role of MC1R in determining the coat color of this sub‐breed. As a result, most animals were e/e genotype, suggesting the coat color of this sub‐breed is derived from the e allele of MC1R gene. However, we found six animals with E/e genotype, which coat color would be black theoretically. Subsequently, sequence comparison was performed with these animals to identify other polymorphisms affecting coat color, elucidating that these animals possessed the A allele of c.871G > A commonly. c.871G > A was a non‐synonymous mutation in the seventh transmembrane domain, suggesting alteration of the function and/or the structure of MC1R protein. Our data indicated that the A allele of c.871G > A might be a loss‐of‐function mutation.     

Prevalence of zoonotic parasites in feral cats of Central Virginia,USA

Felis catus, the domestic cat, is the definitive host for parasites that may result in adverse health outcomes in humans. Prevalence data of zoonotic parasites in feral cats, which are free‐roaming domestic cats that are born and live in the wild, are limited. The objective of this study was to assess seroprevalence of Toxoplasma gondii antibodies and copro‐prevalence of potentially zoonotic parasites in feral cats and to evaluate risk factors for seropositivity and faecal excretion of parasites. In this cross‐sectional survey, 275 feral cats at Trap‐Neuter‐Release clinics in Central Virginia were tested for parasites via faecal flotation, direct immunofluorescence assay (faeces) and modified agglutination testing (serum). Toxoplasma gondii seroprevalence was 22.35% (95% CI: 17.47–27.86). Faecal prevalence of T. gondii‐like oocysts was 1.04% (95% CI: 0.13–3.71), Toxocara cati 58.85% (95% CI: 51.54–65.89), Ancylostoma spp. 18.75% (95% CI: 13.49–25.00), Giardia duodenalis 5.73% (95% CI: 2.89–10.02) and Cryptosporidium spp. 3.33% (95% CI: 1.37–7.24). Female cats were more likely than males to excrete faecal Ancylostoma spp. eggs (OR 2.88; 95% CI 1.34–6.17). Adults were more likely than immature cats to be seropositive (OR 2.10; 95% CI: 1.11–3.97) and to excrete faecal Ancylostoma spp. eggs (OR 2.57; 95% CI: 1.10–5.99). However, immature cats were more likely than adults to excrete T. cati eggs (OR 6.79; 95% CI: 3.31–13.90) and to excrete one or more potentially zoonotic species (OR 4.67; 95% CI: 2.28–9.55) in faeces. Results of this study have implications for human and animal health and highlight the importance of collaboration between public health, medical and veterinary communities in preventive efforts.     

In vitro glucuronidation of the angiotensin II receptor antagonist telmisartan in the cat: a comparison with other species

Ebner, T., Schänzle, G., Weber, W., Sent, U., Elliott, J. In vitro glucuronidation of the angiotensin II receptor antagonist telmisartan in the cat: a comparison with other species. J. vet. Pharmacol. Therap.  36 , 154–160. Glucuronidation of telmisartan comprises nearly its entire metabolic clearance in several mammalian species including human. However, data were lacking for the cat, a species noted for its inability to glucuronidate some drugs. Therefore, the glucuronidation of telmisartan was investigated using feline liver microsomes and compared to liver microsomes of rats, dogs, and human, intestinal human microsomes and cell lines expressing human UDP‐glucuronosyltransferases (UGT). Incubation of telmisartan with cat liver microsomes readily yielded telmisartan glucuronide, and pooled (N = 3 for each gender) cat liver microsomes even showed the highest glucuronidation rate (cat > dog >> human > rat). Michaelis Menten kinetics were observed with K_m of 7.5 and 10 μm and V_max of 3.9 and 3.3 nmol/min/mg for male and female cats, respectively. Confirming the in vitro data, telmisartan glucuronide was detected as the major circulating metabolite in cat plasma. To elucidate which UGT enzymes are involved, telmisartan was incubated with cell lines expressing human UGTs. The highest glucuronidation activity was observed for UGT1A8, UGT1A7, and UGT1A9. In conclusion, telmisartan was effectively glucuronidated in cats. Defects of the UGT1A6 gene in cats do not affect the glucuronidation of telmisartan as it is not a substrate of human UGT1A6.     

Q Fever (Coxiella burnetii) Knowledge and Attitudes of Australian Cat Breeders and Their Husbandry Practices

A Q fever outbreak in a small animal veterinary hospital, associated with a cat caesarean section, initiated a cat seroprevalence study (n = 712) that found circulating antibodies to Coxiella burnetii was highest in cattery‐confined breeding cats (9.3%). These findings stimulated interest about potential sources of C. burnetii infection for cats and humans associated with cats. Cat breeders are potentially a group at increased risk of C. burnetii infection, and this study sought to identify potential risk factors. A cross‐sectional online survey was conducted targeting all domestic cat breeders registered with an affiliate member body in Australia in 2015. Responses from 177 cat breeders across Australia were analysed. Forty per cent of responding cat breeders had not heard of Q fever. Raw meat was fed as an integral constituent of the diet by 89% of respondents. Eighty per cent of respondents allowed queens access to the home for parturition, and assistance of queens and resuscitation of kittens at the time of birth were reported by 97% of respondents. Respondents who perceived some level of exposure to Q fever through their breeding activities were three times less likely to perform mouth‐to‐snout resuscitation (OR 0.3 95% CI 0.1–0.9; P = 0.034) than those who did not perceive a risk of exposure. Similarly, respondents who perceived Q fever as a risk through breeding activities were close to eight times more likely to use personal protective equipment during parturition (OR 7.7 95% CI 1.5–39.9; P = 0.015) than those who did not. Husbandry practices of cat breeders that may increase the risk of C. burnetii transmission require further targeted investigations to assess the contribution of these risk factors to the acquisition of disease. Concurrent education forums are recommended to inform Australian cat breeders of the aetiopathogenesis of Q fever.     

P‐70 Idiopathic facial dermatitis of the Persian cat: three cases controlled with cyclosporine

Facial dermatitis in cats is a poorly understood clinical problem observed in Persian and Himalayan cats. This report describes three cases of idiopathic facial dermatitis in the Persian cat controlled with cyclosporine. The syndrome was observed in a 5‐year‐old intact female, a 1.5‐year‐old intact male, and a 3‐year‐old neutered male Persian cat. The lesions developed over 2 years, 2 months and 18 months, respectively. Cutaneous lesions were mainly localized to the face. A black patchy waxy exudate matted the hair, especially on the chin. Mild crusts and black exudate were also noted on the vulvar folds in one case and on the ventral aspect of the neck in another case. Erythematous, ceruminous otitis was observed in one case. The histopathological findings were exactly the same for all three cases and compatible with idiopathic facial dermatitis of the Persian cat, or eventually an allergic reaction. All cases were managed with cyclosporine (Neoral^® 6–7 mg/kg/day). Lesions were completely controlled after 4–6 weeks. During a 6‐month follow‐up for two cases, the lesions seemed to be more resistant to therapy. For these two cats, secondary infections with cocci and Malassezia occasionally occurred. No adverse reactions were observed in our three treated cats. Funding: Self‐funded.     

Lack of the Associations of the Polymorphisms in IGF2, MC4R and GNAS Genes with Reproduction Traits in Pigs and Imprinting Analysis of IGF2 Gene in Ovary and Cornus Uteri

In recent years, intensive attention has been put on improving reproductive performance of pigs. Several experiments aimed to identify markers associated with prolificacy, but this issue still remains open. In our study, we investigated associations between polymorphisms in IGF2, GNAS and MC4R genes with reproductive traits of Polish Landrace and Large White pigs. We did not find any significant associations for g. GNAS314T > 324C, IGF2 intron3‐g.3072G > A or g. MC4R 1426G > A in Polish Landrace and Large White pigs. In the case of IGF2 intron3‐g.3072G > A, this information is of great importance, because this marker is widely implemented in pigs breeding and previous experiments suggested its role in prolificacy of pigs. We also investigated expression of IGF2 gene and showed that this gene is monoallelically expressed in reproductive organs (ovary and cornus uteri).     

Multiple marker effects of single nucleotide polymorphisms in three genes,AKIRIN2, EDG1 and RPL27A,for marbling development in Japanese Black cattle

Marbling in beef, measured by Beef Marbling Standard (BMS) number, is an economically important trait for beef cattle breeding and markets in Japan. We previously detected three single nucleotide polymorphisms (SNPs) associated with BMS number of Japanese Black in Oita prefecture: c.*188G>A in AKIRIN2, g.1471620G>T in EDG1 and g.3109537C>T in RPL27A. Here, we carried out single and multiple marker association analyses for the three SNPs in a different commercial Japanese Black population of 892 genotyped animals. The single marker analyses with the model including a single SNP showed significant associations of all SNPs with BMS number. The multiple marker analysis with the model including the main effects of the three SNPs and their interactions detected only significant main effects of g.1471620G>T and g.3109537C>T and a significant interaction between c.*188G>A and g.1471620G>T. These findings suggest the presence of inter‐allelic interactions among genes affecting the development of beef marbling. For effective marker‐assisted selection for BMS number, interactions among these markers need to be considered.     

Evaluation of Pax5 expression and comparison with BLA.36 and CD79αcy in feline non‐Hodgkin lymphoma

Paired box gene 5 (Pax5) is a widely used B‐cell marker for human and canine non‐Hodgkin's lymphoma (nHL); however, in the literature there is only one case report using Pax5 in a cat B‐cell lymphoma. The purposes of this study were to investigate the expression and detection of B‐cell specific activator protein (BSAP) using a monoclonal anti‐Pax5 antibody in feline nHL (FnHL) tissue samples to evaluate its diagnostic relevance as a B‐cell marker. A total of 45 FnHL samples in 45 cats were evaluated. B‐cell lymphoma was the most common immunophenotype (51.1%) for all the samples and T‐cell the most common immunophenotype (64.3%) for the gastrointestinal (GI) form. Pax5 stained 82.6% of all B‐cell lymphomas and no expression was found in any of the T‐cell lymphomas. Anti‐Pax5 antibody staining in FnHL is similar to that reported in human and canine counterparts and may offer an excellent B‐cell marker in cats.     

Development of genotyping method for functionally relevant variants of cytochromes P450 in cynomolgus macaques

In cynomolgus macaques (Macaca fascicularis), widely used in drug metabolism studies, CYP2C9, CYP2C76, CYP2D6, CYP3A4, and CYP3A5, important drug‐metabolizing enzymes, are abundantly expressed in liver and metabolize cytochrome P450 substrates. CYP2C9 (c.334A>C), CYP2C76 (c.449TG>A), CYP2D6 (c.891A>G), CYP3A4 (IVS3 + 1G>del), and CYP3A5 (c.625A>T) substantially influence metabolic activity of enzymes, and thus are important variants in drug metabolism studies. In this study, a real‐time PCR method was developed for genotyping these variants. The validity of the methods was verified by genotyping two wild type, two heterozygous, and two homozygous DNAs and was used to genotype 41 cynomolgus macaques (from Cambodia, Indonesia, the Philippines, or Vietnam) for the five variants, along with another important variant CYP2C19 (c.308C>T). The CYP2C9 and CYP2C19 variants were found only in Cambodian and Vietnamese animals, while the CYP2C76 and CYP2D6 variants were found only in Indonesian and Philippine animals. The CYP3A4 and CYP3A5 variants were not found in any of the animals analyzed. Mauritian animals, genotyped using next‐generation sequencing data for comparison, possessed the CYP2C19 and CYP2D6 variants, but not the other variants. These results indicated differences in prevalence of these important variants among animal groups. Therefore, the genotyping tool developed is useful for drug metabolism studies using cynomolgus macaques.