1. Prevalence of Cytochrome p450 CYP2C9*2 and CYP2C9*3 in the York Hospital Blood Bank. Andy Ngo Department of Biological Sciences, York College Introduction Cytochrome p450 is a family of drug metabolizing enzymes found primarily in the liver. Enzymes within the family are known as isoforms (Taube et al. 2000). A prevalent enzyme in the Cytochrome p450 family is the CYP2C9 isoform which metabolizes specific drugs like warfarin. CYP2C9*1 is the wild type and metabolizes warfarin normally. The enzyme, however, has two polymorphisms known as CYP2C9*2 and CYP2C9*3 which do not metabolize warfarin normally. Both forms have been known to increase sensitivity to warfarin, and if both polymorphisms are found in a person their sensitivity is exacerbated (Taube et al. 2000). Warfarin reduces the coagulation of blood. The presence of either one of the polymorphisms increases the anti-coagulation effect of warfarin increasing the chances of serious bleeding (Taube et al. 2000). The prevalence of the CYP2C9 polymorphisms in a human population varies depending on ethnicity (Xia et al 2002). Objective Find the prevalence of CYP2C9*2 and CYP2C9*3 in the York Hospital Blood Bank. Methods A purified blood sample from the York Hospital Blood Bank was taken and amplified by PCR. To amplify CYP2C9*2, n= 197, and CYP2C9*3, n=199, specific primers were used. After amplification, RFLP was performed. CYP2C9*2 was cut with the restriction enzyme AvaII, and CYP2C9*3 was cut with the restriction enzyme NisI. A gel was run to determine if polymorphisms exist. PCR-RFLP flowchart Investigation of the CYP2C9*2 and CYP2C9*3 polymorphisms are performed at different times. CYP2C9*2 PCR-RFLP is done independently of CYP2C9*3 PCR-RFLP, but the bands representing heterozygosity, homozygosity, and wild type are similar in appearance, so the gels can be interpreted in the same way. In the following picture, the ladder is used as a comparison. The ladder allows the base pair size, bps, of DNA fragments to be measured since the size of the ladder’s bands are known. The wild-type connotation means no polymorphism was found. A wild-type CYP2C9*1 has a single band and is relatively smaller in bps to the polymorphisms, and so moves further along in the gel. The heterozygote denotes a single polymorphism within a person which could have originated from the paternal or maternal allele. The origin is not detected during this process. One band is the same bps as the wild type and the other the same bps as the homozygote. Homozygote polymorphisms have been found on the paternal and maternal allele. It shows up in the gel as a single band and has a higher bps than the wild type band. 1 2 3 4 1- Ladder 2- Wild-Type 3- Heterozygote 4- Homozygote Results For CYP2C9*2, 25.38% were heterozygote, and 1.01% were homozygote. For CYP2C9*3, 19.1% were heterozygote, and 3.5% were homozygote. Only 189 samples were usable for comparison with a previously performed study in Sweden, n=430. To analyze the how similar the two population are, a contingency table was used which analyzes ratios. The two were significantly associated with one another, P=0.0280. Discussion The results indicate genotypic make up of the York Hospital Blood Bank are similar to the genotypic makeup of sample populations in Sweden. Possibly the populations are similar in their sensitivity to warfarin. This study’s importance is lies in its eventual use in a larger study which will involve additional analysis of the York Hospital Blood Bank blood samples. The analysis will involve further exploration of the prevalence of different soforms and their polymorphism and if any correlation between the polymorphisms exist. Literature Cited Taube, Janis., Halsall, D., and Baglin, T. 2000.“Influence of cytochrome P-450 CYP2C9 polymorphisms on warfarin sensitivity and risk of over-anticoagulation in patients on long-term treatment.” The American Society of Hermatology. 96: 1816-1819. Xie, H., Prasad, H.C., Kim, R.B., and Stein, C. 2002. CYP2C9 allelic variants: ethnic distribution and functional significance. Advanced Drug Delivery Reviews. 54: 1257-1270. Acknowledgements Thanks to Ted Bell in the Emig Research Center for helping me during this project and thanks to Dr. Rehnberg for helping me with my work. Molecular model of warfarin Coumadiin, brand name of warfarin. Blood sample from York Hospital Blood Bank Polymerase Chain Reaction (PCR) Restriction Fragment Length Polymorphism (RFLP) Run samples on 2% agarose gel