cocaine and amphetamine addiction genetics dr. gerome breen and amphetamine ... recently italian,...
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Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 1
1
Cocaine and Amphetamine
Addiction Genetics
Gerome Breen, PhD
MRC SGDP Centre
and Psychological Medicine
Institute of Psychiatry
Kings College, University of London
2
� Third most commonly used illegal drug with 13.5 million
users worldwide (UNODC, 2004)
� Thus far, America (North and South) but in Europe
the problem/epidemic is growing
� For example, in the UK, powdered cocaine use during
previous year increased 5x in people 18-30
between 1996-2003
Cocaine
3
Coca-leaf
� Native to the Americas
� Used by native peoples
to increase stamina
� Probably evolved
as an insect defence -
potently inhibits
reuptake of the insect
neurotransmitter
octopamine
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 2
4
� Crop spraying in Columbia
has succeeded in greatly
reducing the cocaine crop
� Howev er, f armers
and cartels hav e now
bred a herbicide resistant,
>50% larger coca plant
which produces 4-8x cocaine
per kg of leav es
5
Late 19th and early 20th centur y
commercial products
often used cocaine
The most famous of these was
“Coca-Cola”. In fact coca leaves are still
used in its production but are now treated to remove the cocaine molecules
Now it is illegal in almost every country
but has become a widely used drug
of abuse. Powdered cocaine and ‘crack’ cocaine are the major forms
6
� Increased energy: “It sustains and refreshes both body and brain..... in the same space of time more than double the amount of work could be undergone...” Sears, Roebuck,
and Co. Consumers Guide, 1900
� Euphoria: “.....exhilarating and lasting
euphoria.... You perceive increase in self-control and possess more vitality and capacity for work.” Sigmund Freud, 1884
� Replace natural reward: "A coke shot...it's like... injectable sex, an orgasm in every cell.”
� Crav ing: “I found I was taking money meant to buy presents for my children.”
� Paranoia: “He thought he was being forcibly electrocuted and could see electric wires leading to his body.”
Transition to addiction
toGood Bad
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 3
7
� Unlike many psychiatric disorders we have a clear idea
of the biological bases of addiction
� Can follow cocaine’s binding in the brain
� Increase neurotransmitter levels in the synapse,
principally blocking reuptake at monoamine
transporters:
• Dopamine transporter (DAT)
• Serotonin transporter (SERT)
• Noradrenalin transporter (NET)
Cocaine action
9
4 drugs: 1 final destination
Cocaine
Amphetamine
THC
Morphine
Dopamine
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 4
10
The nucleus accumbens (NA) has been implicated
as an especially important structure of the brain reward
pathway because drugs of abuse target it. Other
structures important in brain reward include
the amygdala and the ventral tegmental area (VTA)
11
Cocaine addiction is genetic
� Twin studies demonstrate that cocaine
and other addictions are highly heritable (>70%)
� It is an adverse drug reaction, leading
in some individuals to addiction
� Environmental factors are perhaps most important
in mediating exposure(s) to drug
� Genetic factors are perhaps most important
in the transition from initiation to dependence
� But these interact: a gene-environment interaction
12
Twin studies
Drug Males Females
Sedatives
Marijuana
Cocaine
Hallucinogens
Nicotine
87% (Kendler, et al., 2000)
58% (Kendler, et al., 2000)
79% (Kendler et al., 2000)
79% (Kendler, et al., 2000)
53% (Carmelli et al., 1990)
79% (Kendler & Prescott, 1998)
81% (Kendler et al., 1999)
72% (Kendler et al., 1999)
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 5
13
The dopamine transporter gene (SLC6A3)
� 15 exons across 60kb of DNA
� Hundreds of SNPs
� Estimated 15 repeat type polymorphisms
� Most studies have focussed on a 40bp variable number
tandem repeat in the 3’ untranslated region
� Only one study has looked at DAT genotype
in cocaine addiction
14
10kb
Ex1
83bp
Ex2
331bp
Ex3
132bp
Ex4
235bp
Ex5
139bp
Ex6
135bp
Ex7
104bp
Ex8
125bpEx9113bp
Ex10129bp
Ex11100bp
Ex12101bp
Ex13168bp
Ex1472bp
Ex15
1965bp
In1
2.1kb
In2
1.4kb
In3
8.7kb
In4
10.3kb
In5
1.2kb
In6
3.7kb
In7
1.3kb
In8
3.3kb
In9
1.4kb
In10
0.6kbIn11
2.7kb
In12
2.9kb
In13
1.9kb
In14
6.2kb
Details of the exon/intron structure of DAT1
ATG at 130 TAG at 1990
1 2 3 4 5 6 7 8 9 10 1112 13 14 15
1
84
782
547
415
1056
1160
921
1285
1527
1627
1398
1728
1896
1968
3928
Genomic DNAapprox. 60 kb
Processed transcript 3928 bp
Coding sequence 1863 bp
Protein sequence
621 amino acids
15
21, I10+117
P+215
I1+1036
I2+48
P+2459
I6+9
6
E9+59,I9+102
I9-
I5+448
I7+922
I8+2086
E15+274, E15+352
E15 VNTR
E15+1811
I11+2478
I12+267
I13+1457
I14+4217
P+215
I2+48I1+1735, I1+1859,
I1+1860P+2459I1+1036
I6+96
E9+59, I9+102
I9- 21, I10+117
I5+448
I7+922
I8+2086
E15+274, E15+352E15 VNTR
E15+1811
I11+2478
I12+267
I13+145
7
I14+4217D’
I1+1735, I1+1859,
I1+1860
Structure
of LD
across
DAT1
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 6
16
The dopamine transporter gene
15 exons, highly polymor phic – rich in both single nucleotide
and repeat polymor phisms
17
Variable number tandem
repeats/microsatellites
� Multiallelic markers
• Many motifs: (CA)n, (CAG)n, (TGYCC)n, etc
• ‘Microsatellites’ have 1-6bp unit repeats
• VNTRs are larger - >14bp unit repeats
� Huge chemical/physical difference between alleles
and much greater effect on flanking regions than a SNP
� Perhaps 1 million VNTR/microsatellites
� >>Polymorphic than SNPs
18
Brazilian cocaine association sample
� Case-control cohort from Sao Paulo, Brazil
� Cases:
• 702 ICD 10 cocaine dependent abusers:
(95% male; 5% f emale)
• 35.8 + 10.9 years
� Controls:
• 866 healthy indiv iduals: (68% male; 32% f emale)
• 26.1 + 7.7 years
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 7
19
Study design
� Not a lot of money available thus:
• We conducted a detailed in silico (bioinf ormatic)
examination of the gene to select some polymorphisms
based on dif f erent criteria
� The most interesting polymorphisms are genotyped
to assess any association between the variants
and cocaine addiction
� Further gene expression studies are designed
to verify the functional importance
of any associated polymorphism
� Selected about 10, but the second one genotyped
was positive so we stopped for a while
20
DAT intron 8 VNTR
� 6 perfect copies of a 30bp VNTR
� In between the two major blocks of LD in the gene
� Originally found as a restriction/Southern blot VNTR
in 1993
� Had been genotyped in diverse human populations
by Ken Kidd
� But not diseases
21
30bp VNTR
� CACATACCATGCAACATACACACTCAGACA
� 3’-5’
� 2 Major alleles >90% of observed
� Allele 3 – 6 perfect copies of the VNTR
� Allele 2 – 5 perfect copies of the VNTR
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 8
22
0%
10%
20%
30%
40%
50%
60%
70%
80%
1 2 3 4 5
Controls
Cases
Χ2=12.4, DF=4p=0.008(Confirmed by simulation).
Intron 8 alleles in cocaine cases
and controls
23
0%
10%
20%
30%
40%
50%
60%
12 13 22 23 24 25 33 34 35
Control
Case
p=0.0002by 10,000 simulations
Intron 8 genotypes in cocaine cases
and controls
24
Is the association due to another
variation in the gene?
� 10 further markers were analysed in the gene to determine
if the association was due to the intron 8 VNTR’s relationshi p
with another variation in the gene
� We conducted haplotype analyses, which confirmed that the intron 8
VNTR is the sole contributor to the observed association
1 2 3 4 5 8 9 10 11 12 13 14 156 7
rs2963238
rs11564752
rs27048
rs6347
rs6876225
rs11564773
rs1042098
Indel_14
37bp_VNTR
Int8_VNTR
3’UTR_VNTR
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 9
25
The most obvious confounder
� However, this study could be confounded
by different ethnic, and thus genetic, patterns
in the case and control groups – a problem referred
to as “population stratification”
� If study does not correct for the ethnic differences
then they will find multiple false positives and,
more worryingly, false negatives
26
The Brazilian population is genetically
heterogeneous but this can be corrected for
� The Brazilian populati on consists of multiple populati on groups
� Ancestral populations - interbred
� Recently Italian, German or Japanese and Korean
� Not yet interbred
� One solution for this is to only use cases and controls derived
from relativel y homogenous and stable populati ons
� But it is possible to correct for stratification in a relativel y simple way
� Genotype polymor phisms with different allele frequencies
in different ethnic groups
� Use cluster or other statistical analysis methods to divide cases
and controls into multiple genetically similar subgroups
27
Stratification analysis
� Stratification analysis completed with about 23 markers
(using LPOP)
� Stratification analysis groups samples
into genetically similar subgroups
� Results in brief: no or little effect of stratification
on results’ positivity, in fact p-values improve
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 10
28
4.6%
27.3%
61.4%
81.0%
53.6%
27.8%
14.4%
19.1%
10.8%
Subpopulation 1
n=124
Subpopulation 2
n=590
Subpopulation 3
n=681
Indian
European
African
29
So it is associated? Now what?
� Is the VNTR functional?
� We also cloned the intron 8 VNTR into a special reporter
construct and transfect SN4741 cells with the vectors
� This vector is modified to have an MCS in an intron
� We then looked at different expression levels
of the alleles
� Also, used this system to test the response of the alleles
to stimulus/challenge
30
Reporter gene construct
Simply an easily monitored marker gene (producing a product
whose amount can easily be measured because of its properties,
e.g. a f luorescent protein)
PROMOTER MARKERVNTR
Intron with MCS where elements
can be inserted
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 11
31
The expression experiment
� Take the circular vectors and put them into cells
� Cell line chosen was derived from mouse
substantia nigra
� Immortalised via a retrovirus
� More importantly it expressed most
of the dopaminergic machinery
� We then exposed the cells to different stimuli
� Calculated expression of vector correcting for expression
of non-inserted vector under same conditions
32
Differential expression of intron 8 VNTR alleles
when exposed to cocaine, at baseline and chemical stimuli
0.1
1
10
Cocaine 10µM Cocaine 1µM Baseline KCl KCl + Forskolin
Treatment
Lo
g o
f re
lati
ve e
xpre
ss
ion
(vs
. ve
cto
r c
onta
inin
g n
o ins
ert
)
Int8-VNTR-2
Int8-VNTR-3
33
Summary
� Allele and genotype association
between DAT-intron 8 VNTR and cocaine
� Supported by reporter-gene assays indicating
that the VNTR is a differential response element
� Stratification analysis indicated no confounding
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 12
34
� Amphetamine was first synthesized
in Germany in 1887
� Methamphetamine first synthesised in Japan in 1919
35
Amphetamine
Amphetamine powder tends to be quite cheap -
about £8 to £12 a gram. Following a drop in purity
during the eighties, a rise in the late nineties,
it appears to be falling again at around 8 percent
pure (half what it was five years ago)
36
Methamphetamine
� Methamphetamine use common in America and Asia.
In crystal form (ice) it can be very strong,
resulting a quick, hard hit when smoked.
This can often lead to intense paranoia
and a very unpleasant comedown.
It has not (yet) become common in the UK
but there have been reports of it being sold as speed
in places like Glasgow and in some gay clubs.
Ice tends to be sold at £25 for a large rock
� In the Far East the purity is 99%!
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 13
37
Ice
38
Action
� Re-uptake inhibitor
� Release from synaptic vesicles
� Inhibition of MAO
39
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 14
40
Subjects for case control study (n= 800)
� Taipei City Psychiatric Center (TCPC) and Taipei
Detention Center (TDC)
� 433 healthy controls
� 233 no experience of delusions or hallucinations
(divided in occasional and regular users)
� 135 lifetime diagnosis of MAMP induced psychosis
(divided in brief and prolonged)
41
Marker selection
� 3’UTR VNTR: 40-bp repeat in 3’ untranslated region
(Vandenberg h et al., 1992)
� Int8 VNTR: 30-bp repeat in the intron 8
� Indel_14: 15-bp insertion/deletion polymorphism
in the intron 14 (Greenwood et al., 2003)
42
Genotype frequencies in cases and controls
13.00 22.00 23.00 24.00 33.00 34.00
9 2 54 1 315 11 392
2.3% 0.5% 13.8% 0.3% 80.4% 2.8% 100.0%
11 1 46 4 271 12 3453.2% 0.3% 13.3% 1.2% 78.6% 3.5% 100.0%
20 3 100 5 586 23 737
2.7% 0.4% 13.6% 0.7% 79.5% 3.1% 100.0%
11.0 12.0
413 18 43195.8% 4.2% 100.0%
355 11 366
97.0% 3.0% 100.0%768 29 797
96.4% 3.6% 100.0%
11.0 12.0 22.0
8 107 240 355
2.3% 30.1% 67.6% 100.0%
10 100 214 324
3.1% 30.9% 66.0% 100.0%
18 207 454 679
2.7% 30.5% 66.9% 100.0%
Control
Case
Total
DAT_Intron_8
Total
Control
Case
Total
Total
DAT_InDel_14
Total
Total
Control
Case
DAT3UTR
Genotyping is not complete:
92% UTR85% Intron 8 VNTR
99% Intron 14 Insertion Deletion
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 15
43
Association analyses
� Alleles and genotypes at each locus were not associated
with methamphetami ne abuse (p>0.05)
� We then performed haplotype analyses
� This in effect looks at the influence of combinati ons of genotypes
and alleles at the three locus
� Thus haplotype analysis programs calculate the number
of chromosomes carrying each combinati on of marker alleles
in cases and controls
� An example of a haplotype that could exist on one chromosome
would be:
• Allele 3 of intron 8 VNTR + allele 1 of intron 14 Indel + allele 10
of 3’UTR VNTR
44
Int8 VNTR Indel_14 3'UTR VNTR
3% 6% 2.28 0.044 2 1 9
4% 2% -2.64 0.023 3 1 9
12% 10% -1.25 0.099 2 1 10
77% 79% 0.84 0.467 3 1 10
HaplotypeControls Cases z score Emp p value
Haplotype analyses
The global significance of the difference in haplotype frequencies between
cases and controls was p=0.008
This was amplified in regular users (consumpti on 4 times or more per week
and at least 6 months continuous use)
Global χ2 Overall p value
Controls x Cases 26.9 0.008
Controls x Regular Users 27.6 0.00001
Controls x Occasional Users 15.6 >0.05
Occasional x Regular Users 21.9 0.01
45
The expression experiment
� Take the circular vectors and put them into cells
� Cell line chosen was derived from mouse
substantia nigra
� Immortalised via a retrovirus
� More importantly it expressed most
of the dopaminergic machinery
� We then exposed the cells to different stimuli
� Calculated expression of vector correcting for expression
of non-inserted vector under same conditions
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 16
46
Reporter gene construct
Simply an easily monitored marker gene (producing a product whose amount
can easily be measured because of its properties, e.g. a fluorescent protein)
PROMOTER MARKERVNTR
Intron with MCS where elements
can be inserted
This is a circular DNA molecule
that can exist as many copies
in a cell and be used to produce
marker proteins
Another
cloning site
We can use this reporter construct to investigate potenti al functional
interactions between alleles at different polymorphic sequence motifs
47
Preliminary functional work shows intron 8 is necessary
for expression and interacts with the 3’UTR VNTR
0% 0%
9%
22%
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
9 10 2-9 2-10 phRL
Vector
Exp
res
sio
n re
lativ
e to
un
alte
red
ph
RL
48
Conclusions
� Reasonably strong haplotypic association of functional
VNTRs in the DAT gene with metamphetamine abuse
� More work needs to be done
� Markers are functional and effects consistent across
subgroups – arguing against multiple testing problems
� Still possible there is an unknown functional variant
elsewhere in the gene causing the association
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 17
49
Intron 8 is also associated with ADHD (along with the 3’ UTR VNTR)
� A common haplotype of the dopamine transporter gene
is associated with attention deficit hyperactivity disorder
and interacts with prenatal exposure to alcohol
� Keeley-Joanne Brookes BSc1, Jon Mill PhD1, Camilla Guindalini BSc1,
Sarah Curran MRCPsych PhD1, Xiaohui Xu MD1, Jo Knight PhD1,
Chih-Ken Chen MD PhD2, Yu-Shu Huang MD2, Vaheshta Sethna
BSc1 Eric Taylor FRCP, FRCPsych PhD1, Wai Chen MRCPsych1,
Gerome Breen PhD1, *Philip Asherson MRCPsych PhD1
50
Figure: Transmissi on/
non-transmi ssi on ratios
from heterozygote parents
to their ADHD offspring
for the four common haplotypes
of the DAT1 VNTR markers
in the 3’UTR and intron 8,
by maternal drinking status
during pregnancy.
There is a significant interaction
between exposure to alcohol
during pregnancy
and the transmissi on ratios
for the observed haplotypes
(p=.04)0.1
1
10
Alcohol - Alcohol +
In ut ero exposure to alcohol
Odds ratio
9/2 Hap 9/3 Hap
10/2 Hap 10/3 Hap
51
Observations
� Addiction – you have a substrate
� May be complex but not as much
as schizophrenia or depression
� Any effects observed are likely
to be underestimated
Cocaine and Amphetamine
Addiction Genetics
Dr. Gerome Breen
The screen versions of these slides have full details of copyright and acknowledgements 18
52
Acknowledgements
IOP
� Camila Guindalini
� Phil Asherson
� Colin O’Gara
� Keeley Brookes
� Diana Prata
� Ursula D’Souza
� Dav id Ball
� Philip Asherson
� Dav id Collier
� Robin Murray
Brazil
� Homero Vallada
� Ronaldo Laranjeira
� Adalto Castelo
Taiwan
� Chih-Ken Chen
Liv erpool
� John Quinn
� Mark Howard
UCSD
� John Kelsoe
� Tif f any Greenwood
53