CYP2C9• Member of CYP2C family

– ~ 18% CYP protein content in HLMs– Catalyzes ~ 20% of CYP-mediated metabolic rxns of drugs on the

market– CYP2C9 is important for the metabolism of (S)-warfarin (5X more

potent than (R))• As well as phenytoin, tolbutamie, and various NSAIDs

• Crystal structure of CYP2C9 complexed with (S)-warfarin was solved recently

O O

OH

O

CH3

O O

OH

O

CH3

HO

(S)-warfarin (S)-7-hydroxywarfarin

CYP2C9

CYP2C9 Crystal Structure

CYP2C9 Crystal Structure + S-Warfarin

CYP2C9 + Tienilic Acid

covalent modification 2 species

Intact CYP2C9 (MW ~ 56,000 Da)

Human CYP2C9 Allelic Variants

3 alleles 6 genotypes:most studied to date*1/*1 (wild-type)

*1/*2*1/*3*2/*2*2/*3*3/*3

Population Distribution of CYP2C9 by Ethnicity

Ethnicity Warfarin doses (mg/d)African higher (6.5)European intermed (5.0)Asian lower (3.0)

*1

*3*5

Association Between CYP2C9 Genotype and Warfarin Dose

• Previous study – April 2002

• *2 and *3 associated with lower doses of warfarin• Lots of variability in *1/*1 and *1/*2• Why?

– More in-depth genetic analysis of CYP2C9

May 2005

Primary Goals: - establish haplotype structure in a European-American population - evaluate associations between CYP2C9 haplotype and clinical outcomes esp. is there a genetic variability in *1/*1 genotype that can explain

wide variability in dmaintenance dose of warfarin required

Outline of Methods• 192 patients enrolled– Ave age = 60– 64% male

• Primary outcome– Determine daily maintenance warfarin dose

• 3 consecutive clinic visits with INRs within therapeutic range at same mean daily dose

• INR = international normalized ratio used to describe clotting

• Secondary outcomes– Time to:

• First INR within therapeutic range (2-3 or 2.5-3.5)• First above-range INR (> 4 or 4.5)• Stable warfarin dosing (3 consecutive clinic visits…)• Serious (requiring treatment) or life-threatening bleeding event

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Genomic Sequence

CYP2C9

SNP = single nucleotide polymorphism – mutation in CYP2C9 genemay or may not lead to a point-mutation in the CYP2C9 protein

MAF = minor allele frequency

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CYP2C9 Sequence Analysis

5’ 3’10 kb

Upstream promoter

3’ exon anduntranslatedregion (UTR)

1.7 kb

60.7 kb 2C9 genomic region

intron 1 exon 1 intron 2 …….. exon 7 intron 8 exon 8

CYP2C9 genomic sequence

49 kb

CYP2C9: ~ 500 amino acids ~ 1.5 kb ~ 47.5 kb intronic space

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Outline of Experiments192 patients

Warfarin dosing

DNA resequencing

132 SNPs47 SNPs in promoter region

74 SNPs in intronic sequence and 3’-UTR

11 SNPs in coding region (exons)

MAFs 0.5 (60 SNPs)

Haplotype Clustering Algorithm

Haplotype Grouping(23 Haplotypes)

Daily WarfarinMaintenance Dose

correlation?

CYP2C9 Coding Region SNPs11 SNPs (1.5 kb)

MAFs 0.05 are included in the haplotype analysis (3 SNPs)but SNPs *2 and *3 were excluded from the analysis

CYP2C9 Promoter Region SNPs

... ... ...... ...

MAFs 0.05 are included in the haplotype analysis (21 SNPs)

47 SNPs (10 kb)

CYP2C9 Intron and 3’-UTR Region SNPs

... ... ...... ...

MAFs 0.05 are included in the haplotype analysis (38 SNPs)

74 SNPs (47.5 kb)

Genealogic Trees and Haplotype Clustering

• MEGA– Molecular Evolutionary Genetic Analysis– Tool for automatic and manual sequence alignment– Used to explore and analyze DNA and protein sequence variability from

an evolutionary perspective– Comparative sequence analysis estimation of evolutionary distances– Free

• UPGMA– Unweighted Pair Group Method with Arithmetic mean – clustering method used in bioinformatics for the creation of

phylogenetic trees

Haplotype Grouping132 SNPs in CYP2C9

MAF 0.5

60 CYP2C9 sequences

computer alogorithms

- Group 1 (17): A (6) contains most common or “wild type” haplotype (28%)

other 5 differ by one SNP

B (5) differ by 3-10 SNPs from wild-type C (2) most occur in intronic region D (4) none contain *2 or *3 allele

- Group 2 (2): both contain *2 allele- Group 3 (4): most contain *3 allele

384 individual CYP2C9 haplotypes inferred (192 patients) 23 different CYP2C9 haplotypes

SNPs that correlated w/ each other

CYP2C9 Haplotypes

CYP2C9 Haplotype Grouping and Evolutionary Analysis

192 patients 384 individual haplotypes23 different haplotypes 6 groups

10.9%

6.5%

82.6%

Combination of Haplotype Groups and Warfarin Dose(Primary Outcome)

131(68%)

53(28%)

12(6%)

192 patients

populationmean

(5.2 mg/d)

Secondary Outcomes• Time to:

– Therapeutic INR– Above-range INR– Stable dosing– Bleeding event

• Only 2 associations were statistically significant– Patients with a group 2 or 3 haplotype

• Took 2X as long to achieve stable dosing• Were at 2X greater risk of bleeding event

Conclusions• 1st whole-gene high-resolution haplotype-based analysis of CYP2C9 variants

– Although CYP2C9 gene is 50 kb, it is not complex

• 23 CYP2C9 haplotypes observed

– 8 of which were at 5% MAF

• Genetic variability in Group 1 haplotypes warfarin maintenance dose (maybe not enough statistical power in this study)

– Genotyping CYP2C9 at common polymorphic sites (other than *2 and *3) is not likely to provide clinicians with useful information about managing warfarin patients

• Lack of a correlation may be due to the genetic variability of other enzymes

– e.g. VKORC1 gene product vitamin K epoxide reductase

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