Development of a genotyping tool for a functionally relevant CYP2C19 allele (Phe100Asn,Ala103Val and Ile112Leu) in cynomolgus macaques

In cynomolgus macaques, which are widely used in drug metabolism studies, CYP2C19 (formerly known as CYP2C75) is abundantly expressed in liver, metabolizes human CYP2C substrates and is thus an important drug-metabolizing enzyme. One of the cynomolgus CYP2C19 alleles (p.Phe100Asn, p.Ala103Val and p.Ile112Leu) results in substantially reduced metabolic activity and thus is an important allele in drug metabolism studies. For this allele, a genotyping tool was developed using allele-specific TaqMan probe. Genotyping 40 Cambodian cynomolgus macaques using this tool found 1 homozygote, 17 heterozygotes and 22 wild type animals, and the result was confirmed by direct-sequencing. Interestingly, this allele frequency was similar to that of Chinese cynomolgus macaques. The genotyping tool established is useful for drug metabolism studies using cynomolgus macaques.     

CYP1B1 is polymorphic in cynomolgus and rhesus macaques

Cytochrome P450 (CYP) 1B1 is involved in the metabolic activation of various procarcinogens, and some CYP1B1 genetic variants alter CYP1B1-dependent procarcinogen metabolism. Cynomolgus and rhesus macaques are frequently used in toxicity tests due to their evolutionary closeness to humans. In this study, we attempted to identify CYP1B1 genetic variants in 13 cynomolgus and 4 rhesus macaques. A total of 17 genetic variants were identified, including 8 non-synonymous genetic variants, indicating that, similar to humans, CYP1B1 is polymorphic in macaques. These CYP1B1 genetic variants could be the basis for understanding potential inter-animal differences in macaque CYP1B1-dependent metabolism of promutagens.     

Identification and Analysis of CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 in Cynomolgus Macaques

Cytochromes P450 (P450) are important for not only drug metabolism and toxicity, but also biosynthesis and metabolism of cholesterol and bile acids, and steroid synthesis. In cynomolgus macaques, widely used in biomedical research, we have characterized P450 cDNAs, which were isolated as expressed sequence tags of cynomolgus macaque liver. In this study, cynomolgus CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 cDNAs were characterized by sequence analysis, phylogenetic analysis and tissue expression pattern. By sequence analysis, these five cynomolgus P450s had high sequence identities (94–99%) to the human orthologs in amino acids. By phylogenetic analysis, each cynomolgus P450 was more closely related to the human ortholog as compared with the dog or rat ortholog. By quantitative polymerase chain reaction, among the 10 tissue types, CYP7A1 and CYP17A1 mRNAs were preferentially expressed in liver and adrenal gland, respectively. Cynomolgus CYP27A1 and CYP51A1 mRNAs were most abundantly expressed in liver and testis, respectively. Cynomolgus CYP20A1 mRNA was expressed in all the tissues, including brain and liver. Tissue expression patterns of each cynomolgus P450 were generally similar to that of the human ortholog. These results suggest the molecular similarities of CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 between cynomolgus macaques and humans.     

Isolation and characterization of arylacetamide deacetylase in cynomolgus macaques

Arylacetamide deacetylase (AADAC), a microsomal serine esterase, hydrolyzes drugs, such as flutamide, phenacetin and rifampicin. Because AADAC has not been fully investigated at molecular levels in cynomolgus macaques, the non-human primate species widely used in drug metabolism studies, cynomolgus AADAC cDNA was isolated and characterized. The deduced amino acid sequence, highly homologous (92%) to human AADAC, was more closely clustered with human AADAC than the dog, rat or mouse ortholog in a phylogenetic tree. AADAC was flanked by AADACL2 and SUCNR1 in the cynomolgus and human genomes. Moreover, relatively abundant expression of AADAC mRNA was found in liver and jejunum, the drug-metabolizing organs, in cynomolgus macaques, similar to humans. The results suggest molecular similarities of AADAC between cynomolgus macaques and humans.     

Variation in the chicken LPIN2 gene and association with performance traits

1. The objective of the study was to investigate the distribution of LPIN2 variants and haplotypes among breeds and perform an association analysis of the variants and haplotypes with the broiler traits in chickens.

2. Six breeds were used to study the variation and distribution of chicken LPIN2, and an F₂ resource population was used to measure growth traits, carcass traits, meat quality traits and serum biochemistry parameters.

3. A c.-599G>A variant was located in the promoter region of LPIN2 and c.444G>A and c.1730A>T (E577D) coding variant mutations were detected. Linkage disequilibrium tests showed that these three variants were under moderate linkage disequilibrium in the 6 breeds and 7 haplotypes were constructed. The distribution of variation/haplotypes presented clear differences among breeds.

4. Association analysis showed that c.-599G>A was associated with leg muscle weight, jejunum length, ileum length, leg muscle fibre density and leg muscle fibre diameter; c.444G>A was associated with spleen weight, ileum length, body weight at hatch and metatarsus length at 8 weeks; c.1730T>A had significant effects on chicken liver weight, heart weight, body weight at 10 weeks, serum albumin and glucose.

5. Diplotypes were significantly associated with body weight at hatch, heart weight, pancreas weight, duodenum length, leg muscle fibre density and lactate dehydrogenase.

     

Reaction phenotyping of vinblastine metabolism in dogs

Vinblastine is a vinca alkaloid used either as a single agent or in combination therapy for the treatment of canine mast cell tumours and lymphomas. The objective of this study was to determine which isoform of cytochrome P450 enzyme is responsible for the majority of vinblastine metabolism in dogs. A panel of eight recombinant canine cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A12, CYP3A26, CYP2B11, CYP2C41, CYP2C21 and CYP2D15) were incubated in vitro with vinblastine. Findings were confirmed by the use of canine polyclonal antibodies of cytochrome P450 enzymes (CYP1A1, CYP3A12, CYP2B11 and CYP2C21) that were pre‐incubated with individual and pooled hepatic microsomes that were purified from canine liver. Substrate depletion was observed in the presence of recombinant CYP3A12, whereas depletion did not substantially occur when microsomes were pre‐incubated with polyclonal antibodies against CYP3A12. These findings confirmed that CYP3A12 is the major cytochrome P450 isoform responsible for the metabolism of vinblastine in dogs.     

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.     

Analysis of polymorphisms of canine Cytochrome P 450-CYP2D15

Cytochrome P450 (CYP) proteins constitute a large ancient family of oxidative enzymes essential for the efficient elimination of a wide variety of clinically used drugs. Polymorphic variants of human CYP2D6 are associated with the conversion rate and efficacy of several drugs such as antidepressants. Polymorphisms of the canine orthologue CYP2D15 are of interest because these antidepressants are also used in dogs with behavioral problems and the outcome of the treatment is variable. However, the annotated CYP2D15 gene is incomplete and inaccurately assembled in CanFam3.1, hampering DNA sequence analysis of the gene in individual dogs. We elucidated the complete exon–intron structure of CYP2D15 to enable comprehensive genotyping of the gene using genomic DNA. We surveyed variations of the gene in four diverse dog breeds and identified novel polymorphisms in exon 2 in border collies. Further investigation to establish the impact of these canine CYP2D15 polymorphisms on interindividual variability in expression and function of this metabolizing enzyme is now feasible. Further knowledge of CYP pharmacogenetics will help individualize therapy and thereby increase therapeutic efficacy, especially in the use of antidepressants in veterinary behavioral medicine.     

Cynomolgus macaque CYP4 isoforms are functional, metabolizing arachidonic acid

Cytochrome P450 (CYP) is important for metabolism of not only xenobiotics such as drugs, but also endogenous compounds including arachidonic acids. CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 have been identified in cynomolgus macaque, an animal species widely used for investigation of drug metabolism due to its evolutionary closeness to human. However, their metabolic functions have not been investigated. In this study, proteins were heterologously expressed in Escherichia coli and characterized by metabolic assays using arachidonic acids as substrates that are metabolized by CYP4 isoforms in human. The results showed that all four CYPs metabolized arachidonic acids. Therefore, cynomolgus macaque CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 are functional enzymes.     

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p‐hydroxydiazepam [p‐OH‐DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p‐OH‐DZP was significantly less in the horse. Inhibition assays with a CYP‐specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos‐7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.     

Three SNPs within exons of INHA and ACVR2B genes are significantly associated with litter size in Dazu black goats

The aim of this study was to analyse the association between single-nucleotide polymorphisms within INHA and ACVR2B and litter size in Dazu black goats. In total, twenty-two SNPs were genotyped in 190 individuals by SNaPshot and resequencing. The results showed that three SNPs (SNP_1, SNP_12 and SNP_13 in this study) were detected to have significant additive genetic effect on the recorded goat litter size (p < .05). The SNP_1 (NC_030809.1), a non-synonymous substitution of G for T at chr2-g. 28314990 in the exon 2 of INHA gene (NM_001285606.1), resulted in homozygote 2 (HOM2) contributed 0.25 and heterozygote (HET) contributed 0.12 larger litter than homozygote 1 (HOM1). Meanwhile, SNP_12 (Chr22-g. 11721225 A > T) and SNP_13 (Chr22-g. 11721227 A > C) (NC_030829.1) simultaneously mutated at the first and third position of a triplet AAA (lysine, K) in the exon 4 of ACVR2B gene (XM_018066623.1) had estimated genetic effects of HOM1 (0.00) and HOM2 (0.03) larger than HET (−0.12). In conclusion, one SNPs (chr2-g. 28314990 T > G) within the exon 2 of INHA and two SNPs (Chr22-g. 11721225 A > T and Chr22-g. 11721227 A > C) i n the exon 4 of ACVR2B gene were highly recommended as candidate markers of litter size in Dazu black goats. A large-scale association study to assess the impact of these variants on litter size is still necessary.     

Polymorphisms in the canine glucocorticoid receptor alpha gene (NR3C1α)

Corticosteroids are one of the most extensively used class of therapeutic agents in dogs. In human patients, response to corticosteroid therapy has been correlated with the presence of certain polymorphisms of the glucocorticoid receptor gene (NR3C1). Depending on the polymorphism present, patients may show either increased sensitivity to glucocorticoid‐induced adverse effects or resistance to their therapeutic effects. Because response to corticosteroid therapy in dogs can also be variable and unpredictable, we hypothesized that genetic variability exists in the canine NR3C1 gene. The aim of this study was to sequence the coding regions of the canine NR3C1 gene in a representative sample of dogs. Samples from 97 dogs from four previously identified genetic groupings of domestic breeds (Asian/Ancient, Herding, Hunting, and Mastiff) were sequenced and evaluated. Four exons contained polymorphisms and four exons showed no variation from the reference sequence. A total of six single nucleotide polymorphisms (SNPs) were identified including four synonymous SNPs and two nonsynonymous SNPs (c.811A>T and c.2111T>C). No dogs were homozygous for either variant allele, while 23 dogs were heterozygous for the c.811A>T allele and 2 were heterozygous for c.2111T>C allele. The amino acid changes caused by c.811A>T (serine to cysteine) and c.2111T>C (isoleucine to threonine) were both predicted by in silico analysis to be ‘probably damaging’ to structure and function of the resulting protein. We conclude that NR3C1 polymorphisms occur in dogs and may cause individual variation in response to corticosteroid therapy.     

Evaluation of tizanidine as a marker of canine CYP1A2 activity

The dog CYP1A2 enzyme is likely an important contributor to the metabolism of veterinary drugs. Dog CYP1A2 is expressed in liver, plus it is inducible and polymorphic, creating the potential for intersubject differences in pharmacokinetics. Hence, the ability to probe dog CYP1A2 activity and inhibition is relevant toward veterinary drug development and drug–drug interaction assessment. Previous studies have relied on human probes with questionable specificity for CYP1A2, so it was hypothesized that recombinant CYP1A2 could be used to find a specific CYP1A2 substrate. Intrinsic clearance experiments demonstrated that tizanidine was a substrate of CYP1A2. Profiling of tizanidine metabolites generated by CYP1A2 identified the imidazole metabolite that was detectable in dog plasma. The imidazole metabolite was subsequently used to evaluate tizanidine as a CYP1A2 probe. Co‐administration of the CYP1A inhibitor enrofloxacin with tizanidine significantly decreased (30%; n = 3) the formation of the imidazole metabolite vs. control experiments. As enrofloxacin is a weak inhibitor, further studies are required to confirm the sensitivity of tizanidine as an in vivo probe. However, tizanidine may be a more selective CYP1A2 probe than phenacetin when conducting in vitro studies due to the presence of other phenacetin‐metabolizing enzymes in dog liver microsomes.     

Evidence for polymorphism in the cytochrome P450 2D50 gene in horses

Metabolism is an essential factor in the clearance of many drugs and as such plays a major role in the establishment of dosage regimens and withdrawal times. CYP2D6, the human orthologue to equine CYP2D50, is a drug‐metabolizing enzyme that is highly polymorphic in humans leading to widely differing levels of metabolic activity. As CYP2D6 is highly polymorphic, in this study it was hypothesized that the gene coding for the equine orthologue, CYP2D50, may also be prone to polymorphism. Blood samples were collected from 150 horses, the CYP2D50 gene was cloned and sequenced; and full‐length sequences were analyzed for single nucleotide polymorphisms (SNPs), deletions, or insertions. Pharmacokinetic data were collected from a subset of horses following the administration of a single oral dose of tramadol and probit analysis used to calculate metabolic ratios. Prior to drug administration, the ability of recombinant CYP2D50 to metabolize tramadol to O‐desmethyltramadol was confirmed. Sequencing of CYP2D50 identified 126 exonic SNPs, with 31 of those appearing in multiple horses. Oral administration of tramadol to a subset of these horses revealed variable metabolic ratios (tramadol: O‐desmethyltramadol) in individual horses and separation into three metabolic groups. While a limited number of horses of primarily a single breed were studied, the variability in tramadol metabolism to O‐desmethyltramadol between horses and preliminary evidence of what appears to be poor, extensive, and ultra‐rapid metabolizers supports further study of the potential for genetic polymorphisms in the CYP2D50 gene in horses.     

Effects of dietary sodium butyrate on hepatic biotransformation and pharmacokinetics of erythromycin in chickens

Butyrate, a commonly applied feed additive in poultry nutrition, can modify the expression of certain genes, including those encoding cytochrome P450 (CYP) enzymes. In comparative in vitro and in vivo experiments, the effect of butyrate on hepatic CYP genes was examined in primary cultures of chicken hepatocytes and in liver samples of chickens collected from animals that had been given butyrate as a feed additive. Moreover, the effect of butyrate on the biotransformation of erythromycin, a marker substance for the activity of enzymes of the CYP3A family, was investigated in vitro and in vivo. Butyrate increased the expression of the avian‐specific CYP2H1 both in vitro and in vivo. In contrast, the avian CYP3A37 expression was decreased in hepatocytes following butyrate exposure, but not in the in vivo model. CYP1A was suppressed by butyrate in the in vitro experiments, and overexpressed in vivo in butyrate‐fed animals. The concomitant incubation of hepatocytes with butyrate and erythromycin led to an increased CYP2H1 expression and a less pronounced inhibition of CYP3A37. In in vivo pharmacokinetic experiments, butyrate‐fed animals given a single i.m. injection of erythromycin, a slower absorption phase (longer T_half‐abs and delayed T_max) but a rapid elimination phase of this marker substrate was observed. Although these measurable differences were detected in the pharmacokinetics of erythromycin, it is unlikely that a concomitant application of sodium butyrate with erythromycin or other CYP substrates will cause clinically significant feed‐drug interaction in chickens.     

High interindividual variability in plasma clopidogrel active metabolite concentrations in healthy cats is associated with sex and cytochrome P450 2C genetic polymorphism

Clopidogrel response variability has been identified in cats. In humans, evidence suggests that variable clopidogrel active metabolite (CAM) generation is the primary explanation for clopidogrel response variability with differences in body weight, sex, and variable metabolism of clopidogrel primarily due to polymorphisms of the gene encoding cytochrome P450 (CYP) 2C19 as some proposed mechanisms. The aim of this study was to evaluate whether variation in CAM concentrations exists in healthy cats and what the cause of such variation might be. Nineteen healthy cats were given 18.75 mg clopidogrel by mouth. Blood was collected 2 hr later. Plasma CAM concentrations were measured using high performance liquid chromatography and tandem mass spectrometry. Clopidogrel metabolism was estimated by calculating CAM metabolic ratio. DNA was collected, and feline CYP2C genotyping was performed. The cats demonstrated high interindividual variation of plasma CAM concentrations. Approximately 69% of this interindividual variation was primarily explained by differences in clopidogrel metabolism as measured by CAM metabolic ratio with some influence by sex but not by weight. A single nucleotide polymorphism was identified in the feline CYP2C gene that explained in part individual differences in CAM metabolic ratio and CAM plasma concentrations.     

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.