Cocaine and Amphetamine Addiction Genetics Dr. Gerome Breen and Amphetamine ... Recently Italian, German or Japanese and Korean Not yet interbred One solution for this is to only use

Cocaine and Amphetamine

Addiction Genetics

Dr. Gerome Breen

The screen versions of these slides have full details of copyright and acknowledgements 1

1


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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)


79% (Kendler et al., 2000)


53% (Carmelli et al., 1990)

79% (Kendler & Prescott, 1998)




Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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%!


Addiction Genetics

Dr. Gerome Breen


37

Ice

38

Action

� Re-uptake inhibitor

� Release from synaptic vesicles

� Inhibition of MAO

39


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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


Addiction Genetics

Dr. Gerome Breen


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

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Cocaine and Amphetamine Addiction Genetics Dr. Gerome Breen and Amphetamine ... Recently Italian, German or Japanese and Korean Not yet interbred One solution for this is to only use