From DNA to Pharmacogenomics

Pharmacogenomics Course 3/13/2008

This patient should probably take the blue pill.

Yumi Kasai, PhD PGx Program Assoc. Director Dept. of Genetics and Genomic Sciences yumi.kasai@mssm.edu

From DNA to Pharmacogenomics

Goal to share a common language

Review of basic molecular biology Tools of molecular biology Genomic variation Pharmacogenomics

Many figures came from the

DNA Molecules
Cell Nucleus Chromosomes

Each cell has 46 molecules of double-stranded DNA. Each DNA molecule is made up of 50 to 250 million bases housed in a chromosome.

Human Chromosomes

10

11

12

13

14

15

16

17

18

19

20

21

22

X Y

The Genome Contains Genes

Gene 1 Coding region

Protein 1 Noncoding region

Gene 2 Coding region

Protein 2 Noncoding region

DNA to RNA to Protein

DNA Nucleus DNA bases Gene mRNA Cell membrane Chain of amino acids

Protein Ribosome

Genes to Proteins I

DNA

A U G C G U U A U A C G U A A

T A C G C A A T A T G C A T T

mRNA

Genes to Proteins II
Ribosome

tRNA

mRNA A U G C G U U A U A C G Methionine Tyrosine Threonine U A A

Arginine

Genes have multiple components

DNA
5 UTR cds 3 UTR

RNA

5 UTR

cds

3 UTR

spliced mRNA

5 UTR

cds

3 UTR

protein

A Few Basic Definitions

Two copies of each chromosome/gene Alleles - Any one of the alternative forms of a given gene Heterozygous - 2 different alleles for a trait Homozygous same alleles for a trait

Tools of Molecular Biology

Anneal Extend Denature Cycle 1 1 copy to 2

thermostable polymerase makes it happen

PCR

Anneal Extend Denature Cycle 2 2 copies to 4 Anneal Extend Denature Cycle 3 4 copies to 8

By cycle 30 ~1x109 copies

Sanger ddNTP chain termination sequencing

Sanger ddNTP chain termination sequencing

For

Genomic DNA 1
Rev

For

Genomic DNA 2
Rev

GCG > GAG Ala > Glu

the ability to query the whole genome

Microarrays

The Human Genome

Final sequence announced April 14, 2003
(National DNA Day is April 25!)

Genome size

Species Human Mouse C. elegans D. melanogaster

Size in bases 3x109 3x109 1x108 1.2x108

~ # of Genes 24,000 24,000 20,000 13,800

99.9% human DNA identical among ethnic groups and races. (98% identity with chimpanzee and gorilla)

Human Genome Variation

Large Scale

translocations

insertions, deletions

Medium scale - Structural variations

Human Genome Variation

Structural variation indels and rearrangements. ~1300 SVs identified in 2 individuals. 65% <10kb; 30% <5kb; 15% > 100 kb.
Korbel et al. Science 2008. 318; 420-426.

Medium scale Copy Number Variations

Human Genome Variation

270 people scanned 1477 CNV regions detected. 12% of the genome impacted.
Redon R. et al. 2006 Nature 444; 444-454.

Human Genome Variation

Repeat expansions
VNTRs variable number tandem repeats 14-100 bases long clusters of tandem repeats repeated in range of 4-40x/occurance STRs short tandem repeats 2 -10 bases long clusters of tandem repeats often occur in introns SSRs or microsatellites short simple repeats 1-4 bases long

Single Nucleotide Polymorphisms (SNPs)

SNP - 0.1% of genome Coding regions Missense Nonsense Silent Other regions Splice sites UTRs Promoters/enhancers Intergenic regions miRNAs

Human Genome Variation

SNP Databases
SNP discovery Population of people are sequenced Normal people Experimental data All chromosomes are sequenced. All SNPs recorded in databases.

Make (buy) a SNP chip

http://hapmap.org/

PGx data
http://www.ncbi.nlm.nih.gov/SNP/index.html

Origin of Haplotypes
Haplotype is a set of closely linked genetic markers present on one chromosome which tend to be inherited together (not easily separable by recombination).

http://hapmap.org/originhaplotype.html.en

Haplotype at a molecular level

SNPs

Samples

Blue - homozygous common allele Red - heterozygous Yellow homozygous rare allele http://pga.gs.washington.edu/

A Snapshot of SNPs for 1 Gene

http://pga.gs.washington.edu/

Variations Causing No Changes

= Variations in DNA that cause no changes

Variations Causing Harmless Changes

= Variations in DNA that cause harmless changes

Variations Causing Harmful Changes

No Disease Hemophilia

No Disease

= Variation in DNA that causes harmful change

Variations Causing Latent Changes

= Variations in DNA that cause latent effects

Many years later

or one drug later

Pharmacology terms
Pharmacogenetics/Pharmacogenomics is the study of patient variability in response to drugs due to genetic variation. Genetic variability can affect: Pharmacokinetics - Plasma clearance, delivery of drug or metabolite to target cells (what the body does to the drug) Pharmacodynamics - The relationship between the drug concentration and its therapeutic effect (what the drug does to the body) The likelihood of an adverse reaction

PGx simplified view

Where can trouble occur?

Cytochrome -metabolism

Target

Cytochrome -prodrug

Transporter Signaling, downstream pathway

HLA -hypersensitivity

FDA Relabeling of Drugs with PGx Information

Drug 6-MP Azathioprine Irinotecan Warfarin Enzyme TPMT TPMT UGT 2C9 and VKORC1 2D6 HLA-B*5701 Goal Safety Safety Safety Safety Safety Safety Year 2003 2003 2004 2007 2007 Pending? Status Complete Complete Complete Complete Complete Pending? Pending?

Carbamazepine HLA-B*1502 Tamoxifen Abacavir

Efficacy Pending?

Inclusion of genetic information in labels endorsed by FDA Advisory Committees comprised of experts in clinical pharmacology, medicine and pharmacogenetics.

Cost-benefit analysis does not enter into regulatory label decision.

SNP Profiles and Response to Drug Therapy

Responds to Standard Drug Treatment SNP profile A

Does Not Respond to Standard Drug Treatment SNP profile B SNP profile C

SNP profile E

SNP profile D

PGx and SNPs/CNVs/SVs in the future

PGx and SNPs for today

Genome-Wide Human SNP Array 6.0 1.8 million genetic markers 906,600 SNPs 946,000 probes for CNVs

Check out www.pharmgkb.org

Sources of Patient to Patient Variability Intrinsic Factors

Age Sex Concomitant disease Organ function Genetic variations

Extrinsic Factors
Medical practice Diet Concomitant drugs Adherence Smoking

3/13/08 Lecture Summary

The reference human genome sequence has been determined. Now studying the variation that exists amongst all of us. The molecular tools available impact the direction of PGx research and clinical emphasis. Drug effects vary from person to person & all drug effects are influenced by genes (and other factors). Like complex diseases, individual drug responses are multifactorial. PGx is a young field and has yet to be fully understood, accepted and utilized.

Selected Articles
Giacommi, KM et al. The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response. Clinical Pharmacology and Therapeutics. 2007; 81: 328-345. Ingelmann-Sundberg, M. Pharmacogenomic Biomarkers for Prediction of Severe Adverse Drug Reactions. N. Engl. J. Med. 2008; 358:637-639 Korbel, JO et al. Paired-End Mapping Reveals Extensive Structural Variation in the Human Genome. Science. 2007; 318: 420-426 Perry, GH et al. The Fine-Scale and Complex Architecture of Human Copy-Number Variation. AJHG. 2008; 82: 685-695. Redon, R. et al. Global variation in copy number in the human genome. Nature. 2006 444; 444-454. Wilke, RA et al. Identifying Genetic Risk Factors for Serious Adverse Drug Reactions: Current Progress and Challenges. Nature Reviews: Drug Discovery. 2007; 6: 904-916.

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