Abstract 2661: Evaluation of genetic variability of cytochrome P-450 members in the canine preclinical model

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Abstract Dogs are used as pharmacokinetic and preclinical models, but no study has yet evaluated polymorphisms in a large dog population. We hypothesize that genetic variation exists in the coding region of the canine cytochrome P-450 homologue genes CYP1A1, CYP1A2, CYP2E1, and CYP2B6. The canine CYP1A1, CYP1A2, CYP2E1, and CYP2B11 (homologue to CYP2B6) genomic sequences were identified using the UCSC Genome Browser. Intronic primers, flanking individual exons were designed using Primer3. Amplification of all exons was carried out by PCR, products were electrophoresed in agarose gel, and visualized using ethidium bromide. Genomic DNA from 20 dogs (10 breeds, 2 dogs/breed) from our lab's genomic DNA bank was used as template. Direct sequencing of the PCR amplicons was performed and the results were analyzed using the DNASTAR Lasergene 7. PCR restriction fragment length polymorphism (RFLP) assay was designed for each polymorphism identified by sequencing. PCR-RFLP assays were used to evaluate identified polymorphic sites in dogs of 10 different breeds (10 dogs/breed). The potential effect of each polymorphism to the protein function was estimated using PolyPhen and SIFT software. In CYP1A1 a total of 6 SNPs were identified: three SNPs in exon 1, and three SNPs in exon 6. One SNP was a synonymous change (c.1362T>A), while five were nonsynonymous (c.53C>G → p.A18G; c.146G>T → p.W49L; c.345C>G → p.F115L; c.C1305C>A → p.F435L; and c.1561T>C → p.S521P). In CYP1A2, A total of 6 SNPs were identified. Four of these SNPs have been already described (c.1117C>T; c.1173C>G; c.1299C>T; and c.1303G>A), while 2 novel SNPs were identified (c.1165A>G; and c.1451A>G). Of the novel SNPs, one is a synonymous change (1451A>G), and one is nonsynonymous (c.1165A>G → p.N389D). A potential InDel was identified at the 6th exon in 7/13 Siberian huskies initially assayed. Sequencing of the cloned potential InDel - containing exon revealed a 10 base long duplication (1464_1473dupCCCCATCTAT). All 7 dogs harboring the duplication, appeared to be heterozygotes for it, hence we expanded our screening of the duplication to a set of a total of 274 Siberian husky genomic DNA. We identified 75 homozygotes for the wild type, and 199 dogs that appeared heterozygotes for the duplication. When we assayed 28 Alaskan malamutes for the duplication identified in the Siberian huskies, we failed to identify any dog positive for this duplication. In CYP2E1, a total of 2 SNPs in the coding region and 2 intronic SNPs were identified. The 2 exonic SNPs are nonsynonymous (c.85C>T → p.R29C; c.1453T>C → p.Y485H). The two intronic SNPs are located at the 5th (IVS5+5G>A), and the 6th intron, (IVS6+27C>T). In CYP2B11 a total of 3 SNPs were identified. Two SNPs were synonymous (c123G>A; c966G>A) and 1 was nonsynonymous (c220C>T → p.R74C). Genetic variation in the canine CYP1A1, CYP1A2, CYP2E1, and CYP2B11 genes exists, and may have significant implications in the use of dogs as preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2661.