Transcript Prevalence of Cytochrome p450 CYP2C9*2 and *3 in York, Pa

Prevalence of Cytochrome p450 CYP2C9*2 and
CYP2C9*3 in the York Hospital Blood Bank.
Andy Ngo
Department of Biological Sciences, York College
Molecular model of warfarin
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.
1
2
3
4
PCR-RFLP flowchart
Coumadiin, brand name of warfarin.
Blood sample from York Hospital
Blood Bank
Discussion
Polymerase Chain Reaction (PCR)
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.
Restriction Fragment Length
Polymorphism (RFLP)
Run samples on 2% agarose gel
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.
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.
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.
York Hospital Blood Bank
Sweden
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.
Wild Type (*1*1)
103/189
287/430
2C9*2 Heterozygote (*1*2)
42/189
80/430
Acknowledgements
2C9*2 Homozygote(*2*2)
1/189
2/430
2C9*3 Heterozygote(*1*3)
30/189
50/430
2C9*3 Homozygote(*3*3)
5/189
3/430
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.
2C9*2*3 compound
heterozygote(*2*3)
8/189
8/430