PharmacoGenomics

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DNA chip Usage
PharmacoGenomics personalized medicine.
Alina Starovolsky
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SNP snip Single Nucleotide Polymorphisms
One-letter variations in the DNA sequence. SNPs
contribute to differences among individuals. The
majority have no effect, others cause subtle
differences in countless characteristics,
including risk for certain diseases.
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Human genome diversity

• 28 of the human genome are coding genes. (all
  the rest is junk DNA).
• 1.4 are the exons.
• 30,000 genes.
• 40 of then have alterative splicing and thus
  there are more genes.

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• A multi-country effort (Japan, the United
  Kingdom, Canada, China, Nigeria, and the United
  States) to identify and catalog genetic
  similarities and differences in human beings.
• Analyzing DNA from populations with African,
  Asian, and European ancestry. Together, these DNA
  samples should enable HapMap researchers to
  identify most of the common haplotypes that exist
  in populations worldwide

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Polymorphism vs. mutation

• Polymorphism is defined as a variation in more
  than 1 of the population.
• Mutations Rare differences which occur in less
  than 1 of the population (usually much less than
  1).
• Typically, mutations have been discovered in
  coding sequences of genes causing rare inherited
  diseases.
• In Barley (?????) 1 out of 131 nucleotides is
  different between individuals (was calculated on
  75 different genes).
• In 4 types of chickens in comparison to their
  ancestor it was found that every 200 nucleotides
  there is an SNP.

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Polymorphism in humans

• Two random humans are expected to differ at
  approximately 1 in 1000 nucleotide pairs, whereas
  two random chimpanzees differ at 1 in 500
  nucleotide pairs.
• This is interpreted to mean that the human
  species is relatively young, perhaps too young to
  evolve subspecies.
• However, with a geonome of approximate 3 billion
  nucleotides, on average two humans differ at
  approximately 3 million nucleotides.
• Most of these SNPs are neutral, but some are
  functional and influence the phenotypic
  differences between humans. It is estimated that
  about 10 million SNPs exist in human populations.
• Amino acid-altering non-synonymous coding-region
  SNPs would be rare and harder to be found because
  of expected selection against them in human
  evolution.

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Pharmacogenomics Medicine tailored to the
individual

• The Study of how genetic differences influence
  variability in patients responses to drugs.
• Personalized drugs.

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SNPs rool

• Genetic polymorphisms in drug-metabolizing
  enzymes, transporters, receptors, and other drug
  targets have been linked to inter-individual
  differences in the efficacy and toxicity of many
  medications.
• Pharmacogenomic studies explain the inherited
  nature of these differences in drug disposition
  and effects.

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The DNA Chip
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SNP Genotyping

• Using DNA chips, it is possible to measure many
  thousands of SNPs simultaneously in a small
  sample from a patient.
• Can compare genotypes for SNP markers linked to
  virtually any trait.

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Examples traits complex and non complex
diseases.

• There are a number of classic genetic diseases
  caused by mutations of a single gene.
• There are also many diseases that are the result
• of the interactions of many genes
• Athsma, heart disease, cancer.
• Each of these genes may be considered to be a
  risk factor for the disease.
• Groups of SNP markers may be associated with a
  disease without determining mechanism.
• Pharmacogenomics
• personalized drugs.

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The Future
Soon it will be able to profile variations
between individuals DNA to predict responses to
a particular medicine. It will provide
information on the likelihood of efficacy and
safety of a drug for an individual patient It
Will change the practice and economics of
medicine (Faster clinical trials. Less drug side
effects.)
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The roots of pharmacogenetics
Clinical observations of inherited differences in
drug effects first documented in the 1950s. e.g.
In African American population it was found that
in response to the anti-malarial drug primaquine,
they developed hemolyitic anemia due to
polymorphic alleles of Glucose-6-phosphate
dehydrogenase. D-glucose 6-phosphate NADP
D-glucono-1,5-lactone 6-phosphate NADPH
(energy). Without enough normal G6PD to help red
blood cells get rid of harmful oxidative
substances, they can be damaged or destroyed,
leading to a condition known as hemolytic anemia.
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Cytochrome P450
The molecular genetic basis for the inherited
traits began to be revealed in the late 1980s,
with the initial cloning and characterization of
a polymorphic human gene encoding the
drug-metabolizing enzyme debrisoquin hydroxylase
(CYP2D6).

• Homozygousity for alleles of the Cytochrome P450
  gene CYP2D6 (in 10 of the Caucasian
  population) lead to dangerous vacular
  hypotension when receiving the hypertension drug
  debrisoquine.

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About schizophrenia

• Does not mean split personality!
• Afflicts approximately 1 of the worlds
  population.
• US spends 40 billion per year.
• MF for rate, onset male(15-25),
  female(25-35).
• 10 of the people with the disorder commit
  suicide.
• Wide spectrum of illness Characterized by two
  categories of symptoms
• - positive symptoms
• - negative symptoms

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• Negative symptoms
• Flattened emotional response.
• Lack of initiative and persistence.
• Anhedonia (inability to experience pleasure).
• Social withdrawal.

• Positive symptoms
• (more responsive to drug treatment)
• Thought disorders.
• Delusions.
• Hallucinations.
• disorganized speech. (e.g. frequent incoherence)
  
• grossly disorganized or catatonic behavior.

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What causes schizophrenia?

• The Genetic Risk known to run in the family

Each of the genetically identical girls was to
become schizophrenic before the age of 28
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What causes schizophrenia?

• Viral infection in the 2nd trimester of pregnancy
• Brain abnormality (enlarged lateral ventricles,
  low metabolic rate of the prefrontal cortex,
  abnormal cell arrange in the hippocampus).
  Usually correlated to negative symptoms
• Social influence highest in poor socioeconomic
  groups, stressful live events.

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What causes schizophrenia?

• The Gray matter is the cortex of the brain which
  contains nerve cells body.

parietal lobe logic
hearing
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What causes schizophrenia?

• Biochemistry - dopamine hypothesis - dopamine
  levels increase in the brain. (Dopamine is a
  neurotransmitter that transports signals between
  nerve endings in the brain).
• (antipsychotic drugs dopamine antagonists,
  L-dopa, cocaine, amphetamine) only effective
  only for the positive symptoms.

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Dopamine D2 receptor

• Found on chromosome 11q22-23
• Binding site of many psychoactive drugs
• Chlorpromazine

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ANTIPSYCHOTIC DRUGS
TYPICAL
ATYPICAL
D2 Receptor
Other dopamine receptors and 5HT2 receptor
Treat mainly positive symptom Efficacy 60
Treat negative symptoms too, Efficacy 85(less
relapses)
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THE PHARMACOGENOMIC HYPOTHESIS DRUG EFFICACY
RELEATE TO GENETIC REASONS

• Drug mechanism- identify how drug works
  block dopamine receptors
• Target identify those gene products implicated
  in the mechanism of the drug Dopamine
  receptor
• Candidate gene identify the gene that have been
  found to be associated with the disease
  DRD2 receptor (dopamine receptor D2 ).
• Gene variants 141 C Del/Ins, TaqI A

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141C Del/Ins polymorphism

• deletion of cytosine 141 in the promoter region
  upstream from the transcription start site
• Associated with schizophrenia in Japanese,
  Swedish and Portuguese population
• In vitro del allele is directly
• related to DRD2 expression
• Individuals with no del allele
• had lower striatal density of
• dopamine receptor

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TaqI polymorphism

• localized 9.5 kb downstream from the DRD2 gene
• restriction fragment length polymorphism creating
  A1 and A2 allels
• A1 allele -lower density of DRD2 in the caudate
  nuclei and striaum
• A2 allele - decrease in the binding potential of
  the D2 receptor
• Controversy about the linkage to schizophrenia

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Wu S,. Xing Q,. Gao R,. Li X, Gu N,. Feng G,. He
L. (2005).Response to chlorpromazine treatment
may be associated with polymorphisms of the DRD2
gene in Chinese schizophrenic patients. Neurosci
Lett. 376(1)1-4.
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Purpose of the study examine whether the DRD2
gene contribute to the therapeutic effect of
chlorpromazine in schizophrenia by investigating
the potential genetic role of the 141C Ins/Del
and TaqIA polymorphism in the DRD2 gene

• Patients - Chinese population
• - mean age 27.3
• - 2 or more characteristic
  symptoms
• according to the DSM 3R
  (Diagnostic and Statistical manual of Mental
  Disorder ).
• - first time to be treated with
• chlorpromazine
• - 8 weeks of treatment
• Assessment clinical symptoms were evaluated
• by BPRS (brief psychiatric
  rating scale) by two psychiatrics (given no
  information about the patients genotype).

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Results 1 the frequency of Dell allele is
higher in non responders than in responders
P0.01 
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Results 2 no association between A1 allele and
the drug response
 NO SIGNIFICANT RESULTS!
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conclusion

• Del allele of the 141C Ins/Del polymorphism
  might predict therapeutic response to
  chlorpromazine in schizophrenia probably due to
  alteration of the D2 receptor density but that
  the A1 allele of the TaqI A polymorphism have no
  such effect

Higher density of the D2 receptor
low therapeutic response to chlorpromazine
Del allele
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• Other studies
• (Suzuki A, Kondo T, Mihara K, Yasui-Furukori N,
  Ishida M, Furukori H, Kaneko S, Inoue Y, Otani
  K.(2001).The -141C Ins/Del polymorphism in the
  dopamine D2 receptor gene promoter region is
  associated with anxiolytic and antidepressive
  effects during treatment with dopamine
  antagonists in schizophrenic patients.
  Pharmacogenetics. 11(6)545-50)
• Arranz, M.J., Li, T., Liu, X., Murray, R.
  Collier, D.A. Kerwin, R.W.(1998). Lack of
  association between a polymorphism in the
  promoter region of the dopamine-2 receptor gene
  and clozapine response. Pharmacogenetics.
  8(6)481-4.

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Advantages Disadvantages

• Diagnosis-systematized
• investigators blinded to the patient genotype

• Prior medical treatment
• Dont separate positive from negative symptoms

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Non small cell lung cancer - NSCLC
Lung carcinoma is the Leading cause of cancer
deths in the USA and worldwide for both men and
women.
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Multi-center trial of EGFR inhibitor to treat
advanced lung cancer (NSCLC)

• Rationale
• EGFR (epidermal growth factor receptor)
  over-expressed in lung cancers (and other).
• EGFR inhibitors block signal transduction and
  cell proliferation
• Gefitinib A drug that targets the ATP cleft
  within the EGFR.
• Design
• 210 patients from Europe, Australia, South
  Africa, Japan
• Objective tumor response in 19 of patients -
  mean survival 8 months
• Response better among Japanese vs non-Japanese
  pts
• (27.5 vs. 10.4 response P 0.002)
• Response also better among female pts,
  adenocarcinoma pts, pts with prior
  hormonal/immuno treatment, pts with less
  morbidity
• What is molecular basis of the differential
  response?

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Lung cancer - EGFR inhibitors EGFR somatic
mutation
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S,
Okimoto RA, Brannigan BW, Harris PL, Haserlat SM,
Supko JG, Haluska FG, Louis DN, Christiani DC,
Settleman J, Haber DA. Activating mutations in
the epidermal growth factor receptor underlying
responsiveness of non-small-cell lung cancer to
gefitinib. N Engl J Med 3502129-2139, 2004
Paez JG, Janne PA, Lee JC, Tracy S, Greulich H,
Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon
TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers
WR, Johnson BE, Meyerson M. EGFR mutations in
lung cancer correlation with clinical response
to gefitinib therapy. Science 3041497-500, 2004
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Activating mutations in EGFR underlying
responsiveness of lung cancer to gefitinib

• EGFR sequenced in pre-treatment tumor tissue
  from
• 9 responders (tumors that were available), 7
  non-responders, 25 untreated patients

Example of improvement after 6 weeks treatment
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Most of them were women, had never smoked, and
had bronchoalveolar tumors
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Overlap AA 747-750
8 out of the 9 patients that were checked for
mutations in the tumors and responded to
gefitinib had deletions in the tumor cells. And
in 7 patients with no response no mutations were
observed. (plt0.001)
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Overlap
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EGFR mutations in lung cancer Correlation with
clinical response to gefitinib therapy. Science
3041497, 2004
119 primary lung tumors (58 Japan, 61 US), none
treated before, EGFR somatic mutations in 15/58
(26) of Japanese pts vs 1/61 (2) of US pts.
Among adenocarcinomas only, mutations in 14/41
(32) of Japanese pts vs. 1/29 (3) of US pts
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EGFR mutations in lung cancer Correlation with
clinical response to gefitinib therapy. Science
3041497, 2004
Pre-treatment tumors from treated patients 6
responders, 4 non-responders
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EGFR mutations in lung cancer Correlation with
clinical response to gefitinib therapy. Science
3041497, 2004
exon 21
exon 18
exon 19
Sequence and substitutions alterations at kinase
active site.
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EGFR mutations in lung cancer Correlation with
clinical response to gefitinib therapy. Science
3041497, 2004
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EGFR mutations in lung cancer Correlation with
clinical response to gefitinib therapy. Science
3041497, 2004
Mutations may stabilize interaction of EGFR with
both ATP (enhancing phosphorylation) and with
competitive inhibitor geftinib -gt both enhanced
inhibition by drug.
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In general Collect Drug Response Data

• These drug response phenotypes are associated
  with a set of specific gene alleles.
• Identify populations of people who show specific
  responses to a drug.
• In early clinical trials, it is possible to
  identify people who react well and react poorly.

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Make Genetic Profiles

• Scan these populations with a large number of SNP
  markers.
• Find markers linked to drug response phenotypes.
• It is interesting, but not necessary, to identify
  the exact genes involved.

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Profiles
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Use the Profiles - Summary

• Genetic profiles of new patients can then be used
  to prescribe drugs more effectively avoid
  adverse reactions.
• Can also speed clinical trials by testing on
  those who are likely to respond well.

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Impact on Bioinformatics

• Genomics produces high-throughput, high-quality
  data, and bioinformatics provides the analysis
  and interpretation of these massive data sets.
• It is impossible to separate genomics laboratory
  technologies from the computational tools
  required for data analysis.

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Debate
Will it be economical to develop medications and
dosages for only a subset of the population?