Pharmacogenomic studies and SNPS

1. THE USE OF SNPs IN
PHARMACOGENOMIC STUDEIS
CENTRE FOR INTERDISCIPLINARY BIOMEDICAL RESEARCH
Presented by:-
Rajveer Singh
Ph.D. scholar (Medical Pharmacology)

2. • Medicine is personal?
• Why does someone need twice the standard dose to be
effective?
• Why does this drug work for you but not me?
• Why do I have side-effects and you don’t?
• Why do some people get cancer and others don’t?
• Why is anecdotal information irrelevant to your own health
and treatment?

3. PERSONALISED MEDICINE

4. TRASTUZUMAB

5. The Goal of Personalized Medicine
• The Right Dose of
• The Right Drug for
• The Right Indication for
• The Right Patient at
• The Right Time

6. PHARMACOGENETICS & PHARMACOGENOMICS
• Pharmacogenetics The role of genetics/genetic factors in
drug responses.
o F. Vogel. 1959
• Pharmacogenomics is new science about how the systematic
identification of all the human genes, their products,
interindividual variation, intraindividual variation in
expression and function over time affects drug
response/metabolism

7. FUNCTIONAL GENOMICS
• Functional genomics is the study of the
relationships between particular genotypes
and specific phenotypes

8. POTENTIAL OF PHARMACOGENOMICS

9. THE FOUNDATION OF PHARMACOGENOMICS
MUTATION: difference in the DNA code that occurs in
less than 1% of population
• Often associated with rare diseases Cystic fibrosis, sickle cell
anemia, Huntington’s disease
POLYMORPHISM: difference in the DNA code
that occurs in more than 1% of the population
• A single polymorphism is less likely to be the main
cause of a disease
• Polymorphisms often have no visible clinical impact

10. • Single Nucleotide Polymorphism (SNP) are DNA
sequence variation that occurs when a single
nucleotide in the genome sequence is altered
• Occur in at least 1% of the population and make
up about 90% of all human genetic variation

11. OTHER SOURCES OF VARIATIONS

12. SNPs:-
• May result in a different amino acid or stop codon
• May result in a change in protein function
• No effect
• Genetic polymorphisms in drug-metabolizing enzymes,
transporters, receptors, and other drug targets
• Inter individual differences in the efficacy and toxicity of
many medications

13. IDENTIFYING SNPs
• rsID Reference Snip ID rs4680
• GENE COMT (Dopamine)
• CHANGE (nucleotide) G > A
rs4680 G VALINE
rs4680 A METHIONINE
“COMT VAL METH SNPs”
• Potential Impact of COMT-rs4680 G > A Gene Polymorphism in Coronary
Artery Disease, Depression, Parkinsonism and Psoriasis is known

14. METHODS FOR DETECTION OF SNPs

15. There are four commonly used methods for
the SNP (or mutation) detection:
1. Single strand conformation polymorphisms
2. Heteroduplex analysis
3. Direct DNA sequencing
4. DNA microarray technology

16. Single strand conformation polymorphisms

17. HETERODUPLEX ANALYSIS

18. DIRECT DNA SEQUENCING USING PCR

19. DNA MICROARRAY

20. SUCCES OF PHARMACOGENETICS IN
ONCOLOGY

21. Purine Analogs:
A Case Study in Pharmacogenetics
• 6-mercaptopurine, 6-thioguanine, azathioprine
• Used to treat lymphoblastic leukemia, autoimmune disease, inflammatory bowel
disease, after transplant
• Interferes with nucleic acid synthesis
• Therapeutic index limited by myelosuppression
(treatment limited by immune suppression side effect)
6-thioguanine azathioprine
6-mercaptopurine

22. Metabolism of 6-MP
TPMT: thiopurine methyltransferase
6-MMP: 6-methylmercaptopurine
HPRT: hypoxanthine-guanine phosphoribosyltransferase
TIMP: thioinosine monophosphate, thioinosinic acid
MeTIMP: methyl-thioinosine monophosphate

23. • In many ethnicities, TPMT polymorphisms that
result in decreased or absent TPMT activity
occur with a frequency of approximately 5%,
meaning that about 0.25% of patients
are homozygous for these variants
Relling MV et al. Clin Pharmacol Ther. 89 (3): 387–91

24. Pharmacogenetics: A Case Study

25. Pharmacogenetics: A Case Study

26. Pharmacogenetics: A Case Study

27. Evans Nature Reviews Cancer 2006

28. FDA APPROVED PHARMACOGENETIC TESTS FOR
ANTICANCER DRUGS
Gene Drug Consequence
TPMT 6MP Toxicity
CYP2D6 Tamoxifen Decreased efficacy
UGT1A1 Irinotecan Toxicity
CYP2D6 Codeine Ineffective analgesia
These genes all modulate Pharmokinetics

29. Second Example: Codeine and
Cytochrome P450 CYP2D6
• Codeine is a commonly used opioid
– Codeine is a prodrug
– It must be metabolized into morphine for activity
• Cytochrome P450 allele CYP2D6 is the metabolizing enzyme in
the liver
• 7% of Caucasians are missing one copy of the Cytochrome
P450 CYP2D6 gene
Leif Bertilsson et al. Br J Clin Pharmacol. 2002 Feb; 53(2): 111–122
Codeine does not work effectively in these individuals

30. • CYP2D6 converts codeine into morphine, which then
undergoes glucuronidation
• Life-threatening intoxication, including respiratory depression
requiring intubation, can develop over a matter of days in
patients who have multiple functional alleles of CYP2D6,
resulting in ultra-rapid metabolism of opioids such as codeine
into morphine
Lurcott G et al. Anesth Prog. 45 (4): 154–6

31. CODEINE AND MORPHINE METABOLISM

32. IRINOTECAN – IN COLON CANCER

33. • People with variants of the UGT1A1 called TA7, also known
as the "*28 variant", express fewer UGT1A1 enzymes in
their liver and often have Gilbert's syndrome
• During chemotherapy, they effectively receive a larger than
expected dose because their bodies are not able to clear
irinotecan as fast as others
• In studies this corresponds to higher incidences of severe
neutropenia and diarrhea
• In 2005, the FDA made changes to the labeling of irinotecan
to add pharmacogenomics recommendations, such that
irinotecan recipients with a homozygous (both of the two
gene copies) polymorphism in UGT1A1 gene, to be specific,
the *28 variant, should be considered for reduced drug
doses
• Irinotecan is one of the first widely used chemotherapy
agents that is dosed according to the recipient's genotype

34. Enzymes Example drugs
CYP2C9
(%) of drug
metabolism
10 Warfarin, Fluvastatin, Tolbutamide, ibuprofen,
mefenamic acid,, losartan, diclofenac
CYP2C19 25
CYP2D6 20-30
S-mephenytoin, amitriptyline, diazepam,
omeprazole, proguanil, hexobarbital, imipramine
Debrisoquine, metoprolol
propranolol, encainide, codeine,
dextromethorphan, clozapine, desipramine,
haloperidol, amitriptyline, imipramine
CYP3A4 40-45 Erythromycin, ethinylestradiol, nifedipine,
triazolam, cyclosporine, amitriptyline,
imipramine
CYP3A5 1-2 Erythromycin, ethinylestradiol, nifedipine,
triazolam, cyclosporine, amitriptyline,
aldosterone

35. • The Roche AmpliChip CYP450 Test is intended to identify a
patient's CYP2D6 and CYP2C19 genotype from genomic DNA
extracted from a whole blood sample
• An aid to clinicians in determining therapeutic strategy and
treatment dose for therapeutics
ROCHE AMPLICHIP P450 TEST

36. ROCHE
FDA APPROVED
AMPLICHIP
1. PCR amplification of the gene.
2. Fragmentation and labeling of the PCR
product
3. Hybridization and staining on the AmpliChip
DNA microarray.
4. Scanning the chip.
5. Data analysis.

37. SNP consortium (TSC)

38. SNP CONSORTIUM- BRIEF HISTORY
• The history of TSC goes back to early 1998
• The Consortium emerged from an initiative by Glaxo
Wellcome to establish a “private entity” to pursue the
following three important genomic activities:
a. Identify and map enough SNPs to create a high-quality,
genome-representative map
b. develop alone or in conjunction with other companies a cost-
effective technology for “scoring” (genotyping) high numbers
of SNPs in a reasonable time frame
c. develop “multi-point” software programs to facilitate the
analysis of pharmacogenomic studies
• The central concept was to establish the TSC as an entity that
would function in the PUBLIC GOOD

39. • The initial TSC funding ($53 million) was provided by the Wellcome Trust
($14 million) and top ten pharmaceutical companies
• In addition, Motorola, Amersham Pharmacia Biotech, and IBM became
members of the TSC, reflecting the commitment of the TSC mission to
building a membership beyond the pharmaceutical field
• The academic centers involved in the project include the Whitehead
Institute/MIT Center for Genome Research, Washington University, the
Sanger Center, Stanford University, and the Cold Spring Harbor Laboratory
• Stanford University and the Sanger Center are RH mapping the identified
SNPs.
• Cold Spring Harbor Laboratory’s bioinformatics group is organizing,
analyzing, and managing the resulting SNP database

40. OBJECTIVES OF SNP CONSORTIUM
1. Identify 3,00000 SNPs within two years of launch of the
scientific work plan
2. Map 150,000 of the SNPs over the two year term of the
program
3. Manage publication of the resulting SNP map in a manner
intended to maximize the number of SNPs that enter the
public domain (as that term is understood in the patent law)
4. Complete the project for less than $50 million .

43. • A high-quality, ordered SNP map of the human genome in the public
domain will hopefully facilitate pharmacogenetic/ genomic research by
accelerating the identification of specific markers involved in both
common and rare diseases
• It will facilitate the discovery of new ways to intervene in disease
processes, the development novel diagnostic tests, and the use of existing
and the development of new medicines to personalize therapies for more
effective clinical management of the patient
• The TSC initiative demonstrates the practicality and benefits of cross-
industry research collaboration
• This model will become increasing relevant to the life science industry as
the cost and risk of basic research continue to increase

44. THE USE OF SNP MAPS IN PHARMACOGENOMIC
STUDIES
Candidate Gene approach
Linkage Disequlibrium Mapping

45. CANDIDATE GENE APPROACH
Definition :
• Candidate gene approach is a technique that investigates the
associations between genetic variations within pre-specified
genes of interest and phenotypes or disease states
Testing: Genetic variation is analyzed within prespecified gene
of interest
Statistical power : High (Case control / Cohort studeis)
Prior knowledge of Genes: Needed
Selection of Genes : Yes
Example:
• Gene variants in a drug metabolizing enzyme ( TPMT) have been
linked to ADRs
• Variants in a disease susceptibility gene ( apolipoprotein E; APOE)
have been correalted with response to a cholinesterase inhibitor in
Alzhemier patients

46. • LINKAGE: two alleles are located on same
chromosome
• LINKAGE EQUILIBRIUM: The frequency of
alleles inherited from one parent in a given
population have the same value that they
would have if the alleles at each locus were
combined at random

47. • Two regions on the same chromosome are
sequenced using PCR. The frequency of
corresponding alleles are then analyzed in
population. The frequency of A allele is found
to be 0.5 and the frequency of allele B is found
to be 0.5. Assuming normal linkage
equilibrium, what is expected frequency of
AB?

48. LINKAGE EQUILIBRIUM

49. Linkage Disequilibrium
• LD is the tendency of alleles to be transmitted
together more or less often than expected by
chance alone
• Usually caused by close proximity of the genes
on the same chromosome

50. • Two regions of the chromosome are sequenced using
PCR. The frequency of the corresponding alleles are
analyzed in population. The frequency of allele A is
found to be 0.5 and frequency of allele B is found to
be 0.5. However the frequency of AB is found to be
0.49 in the same population. What genetic principle
most likely explains this finding?

51. LINKAGE DISEQUILIBRIUM

52. LINKAGE DISEQUILIBRIUM STUDIES
• Aim to identify which regions (or SNPs) in the genome are
associated with disease or certain phenotype
• Design:
– Identify population structure
– Select case subjects (those with disease)
– Select control subjects (healthy)
– Genotype a million SNPs for each subject
– Determine which SNP is associated

53. FACTORS AFFECTING LD
 The factors, which lead to an increase in LD, include
• Inbreeding,
• Small population size,
• Genetic isolation between lineages,
• Population subdivision,
• Low recombination rate,
• Population admixture,
• Natural and artificial selection,
• Balancing selection, etc.
 The factors, which lead to a decrease/disruption in LD, include
• Outcrossing,
• High recombination rate
• High mutation rate

54. ADVANTAGE OF LD MAPPING
• Linkage disequilibrium mapping has been employed
successfully in a limited fashion in genetic linkage
studies that take advantage of families with multiple
affected individuals to uncover genes for monogenic
diseases
• This mapping technique is now being considered in
the context of association studies

55. LIMITATION OF SNPs AS A TOOL
IN GENETIC ANALYSES

56. • DRUG RESPONSE is a trait whose expression
can only be determined after administration
of the therapeutic compound under study
• Ascertainment of responders cannot be made
from the non-exposed general population and
use of families, is generally avoided except in
the rare instance where multiple family
members are given drug

57. • CLINICAL TRIALS are the main source of patients for
pharmacogenomic studies and these are limited in
size, making estimation of linkage disequilibrium,
often impossible and usually imprecise
• Most of the clinical trials of new therapies are
performed on Caucasian Americans or Europeans
• Pharmacogenomic studies are therefore usually
limited to these genetically heterogeneous clinical
trial populations
• COST of genotyping SNPs, and the interpretation of
results are also problems

58. SAMPLE SIZE
• The number of patients required to find a
statistically significant association between an
SNP and an abnormal drug response depends
on a number of factors:
 including the frequency of the drug response
 the proportion of patients having the SNP allele, the
minimum detectable drug effect using existing diagnostic
criteria
 the level of statistical significance, and the power required.

59. INTERPRETATION OF RESULTS
Two questions should be asked:
1. Is any association detected real
2. and “are the results useful?
• Studies yielding statistically significant results are often considered
real even though the results are rarely replicated
• Consistency of results between studies is a major issue for both
candidate gene studies and studies employing genome-wide LD
mapping
• Furthermore, the use of low p values is recommended to allow for
the vast number of genetic hypotheses that will be tested
collectively in the field

60. FUTURE PROSPECTS
• The dropping genotyping costs
• All phenotype and genotype data will be made public and be
deposited in public databases
• Availability of large patient populations will be crucial for
discovering and validating SNPs
• Ultimately, as all human genes are discovered, the need for random
SNP markers diminishes and gene-based SNP approaches will
predominate
• Demonstrate convincing links between genetic variation and drug
responses and to translate that information into useful
pharmacogenomic tests.

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