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DNA chip Usage:
PharmacoGenomics –
personalized
medicine.
Alina Starovolsky
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.
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.
• 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
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 (‫)שעורה‬: 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.
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.
Pharmacogenomics
“Medicine tailored to the individual”
•The Study of how genetic differences
influence variability in patients’ responses
to drugs.
•Personalized drugs.
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.
The DNA Chip:
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.
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.
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.)
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.
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 drugmetabolizing 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.
About schizophrenia
• Does not mean split personality!
• Afflicts approximately 1% of the world’s
population.
• US spends 40 billion $ per year.
M=F for rate, onset: male(15-25), female(2535).
• 10% of the people with the disorder commit
suicide.
• Wide spectrum of illness Characterized by two
categories of symptoms:
- positive symptoms
- negative symptoms
Positive symptoms:
Negative symptoms:
(more responsive to drug treatment)
Thought disorders.
Delusions.
Hallucinations.
disorganized speech.
(e.g. frequent
incoherence)
 grossly disorganized
or catatonic behavior.




 Flattened emotional
response.
 Lack of initiative and
persistence.
 Anhedonia (inability to
experience pleasure).
 Social withdrawal.
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…
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.
What causes
schizophrenia?
 The Gray matter is the cortex of the brain
which contains nerve cells body.
parietal
lobe
logic
hearing
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, Ldopa, cocaine, amphetamine) – only effective
only for the positive symptoms.
Dopamine D2 receptor
•Found on
chromosome
11q22-23
•Binding site of
many psychoactive
drugs
Chlorpromazine
ANTIPSYCHOTIC DRUGS
TYPICAL
D2 Receptor
Treat mainly positive
symptom
Efficacy – 60%
ATYPICAL
Other dopamine receptors and
5HT2 receptor
Treat negative symptoms too,
Efficacy – 85%(less relapses)
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
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
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
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.
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 patient’s genotype).
Results 1 : the frequency of Dell
allele is higher in non
responders than in responders
141C Ins/Del Genotype
frequency
Ins/Ins
Ins/Del
Del/Del
Responders 61
53 (86.9)
6 (9.8)
2 (3.3)
Non responders 74
53 (71.6)
21 (28.4)
0(0)
P=0.01
Results 2 : no association
between A1 allele and the drug
response
TaqI A Genotype frequency
A2/A2
A1/A2
A1/A1
Responders 61
18 (29.5)
27 (44.3)
16
(26.2)
Non responders 74
22 (29.8)
32 (43.2)
20(27.)
NO SIGNIFICANT RESULTS!
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
Del allele
low therapeutic
response to
chlorpromazine
Other studies:
-
(Suzuki A, Kondo T, Mihara K, Yasui-Furukori N, Ishida M, Furukori H, Kaneko S, Inoue Y,
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.
Otani K.(2001).
-
Advantages
Disadvantages
• Diagnosissystematized
• investigators blinded
to the patient
genotype
• Prior medical
treatment
• Don’t separate
positive from negative
symptoms
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.
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?
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 350:2129-2139, 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 304:1497-500, 2004
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
Most of them were women, had never smoked, and had
bronchoalveolar tumors
(9 tumors available from 25 responders)
Overlap
AA 747-750
(25 untreated tumors evaluated)
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. (p<0.001)
Overlap
EGFR mutations in lung cancer: Correlation with
clinical response to gefitinib therapy. Science
304:1497, 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
EGFR mutations in lung cancer: Correlation
with clinical response to gefitinib therapy.
Science 304:1497, 2004
Pre-treatment tumors from treated patients: 6 responders, 4 non-responders
EGFR mutations in lung cancer: Correlation
with clinical response to gefitinib therapy.
Science 304:1497, 2004
exon 21
exon 18
exon 19
Sequence and substitutions alterations at kinase
active site.
EGFR mutations in lung cancer:
Correlation with clinical response to
gefitinib therapy.
Science 304:1497, 2004
EGFR mutations in lung cancer:
Correlation with clinical response to
gefitinib therapy.
Science 304:1497, 2004
Mutations may stabilize interaction of EGFR with both ATP
(enhancing phosphorylation) and with competitive inhibitor
geftinib -> both enhanced inhibition by drug.
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.
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.
Profiles
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.
Impact on
Bioinformatics
•
Genomics produces high-throughput, highquality 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.
Debate
Will it be economical to develop medications and
dosages for only a subset of the population?