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CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
CYP2C9 Haplotype Structure and Association with Clinical Outcomes
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CYP2C9 Haplotype Structure and Association with Clinical Outcomes

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  • 1. 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 O O OH CH3 OH CH3 CYP2C9 O O HO O O (S)-warfarin (S)-7-hydroxywarfarin• Crystal structure of CYP2C9 complexed with (S)-warfarin was solved recently
  • 2. CYP2C9 Crystal Structure
  • 3. CYP2C9 Crystal Structure + S- Warfarin
  • 4. CYP2C9 + Tienilic AcidIntact CYP2C9 (MW ~ 56,000 Da) covalent modification  2 species
  • 5. Human CYP2C9Allelic Variants3 alleles  6 genotypes: most studied to date *1/*1 (wild-type) *1/*2 *1/*3 *2/*2 *2/*3 *3/*3
  • 6. Population Distribution of CYP2C9 by Ethnicity Ethnicity Warfarin doses (mg/d) African higher (6.5) European intermed (5.0) Asian lower (3.0)
  • 7. *1*3*5
  • 8. 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
  • 9. May 2005Primary 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
  • 10. 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
  • 11. Genomic Sequence CYP2C9SNP = single nucleotide polymorphism – mutation in CYP2C9 gene may or may not lead to a point-mutation in the CYP2C9 proteinMAF = minor allele frequency Confidential 11
  • 12. CYP2C9 Sequence Analysis60.7 kb 2C9 genomic region 3’ exon and untranslated Upstream region (UTR) promoter CYP2C9 genomic sequence5’ intron 1 exon 1 intron 2 …….. exon 7 intron 8 exon 8 3’ 10 kb 1.7 kb 49 kb CYP2C9: ~ 500 amino acids  ~ 1.5 kb  ~ 47.5 kb intronic space Confidential 12
  • 13. Outline of Experiments 192 patients Warfarin dosing Daily WarfarinDNA resequencing Maintenance Dose 47 SNPs in promoter region correlation?132 SNPs 11 SNPs in coding region (exons) 74 SNPs in intronic sequence and 3’-UTR Haplotype Grouping (23 Haplotypes) MAFs 0.5 (60 SNPs) Haplotype Clustering Algorithm Confidential 13
  • 14. 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
  • 15. CYP2C9 Promoter Region SNPs 47 SNPs (10 kb).. . . . . . . . .. . . . . MAFs 0.05 are included in the haplotype analysis (21 SNPs)
  • 16. CYP2C9 Intron and 3’-UTR Region SNPs 74 SNPs (47.5 kb).. . . . . . . . .. . . . . MAFs 0.05 are included in the haplotype analysis (38 SNPs)
  • 17. 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
  • 18. 132 SNPs in CYP2C9 Haplotype Grouping MAF 0.560 CYP2C9 sequences computer alogorithms 384 individual CYP2C9 haplotypes inferred (192 patients)  23 different CYP2C9 haplotypes SNPs that correlated w/ each other - 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
  • 19. CYP2C9 Haplotypes
  • 20. CYP2C9 Haplotype Grouping and Evolutionary Analysis192 patients  384 individual haplotypes23 different haplotypes  6 groups 82.6% 10.9% 6.5%
  • 21. Combination of Haplotype Groups and Warfarin Dose (Primary Outcome)192 patients population mean (5.2 mg/d) 131 53 12 (68%) (28%) (6%)
  • 22. 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
  • 23. 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 not enough statistical power in this study) X warfarin maintenance dose (maybe – 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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