Transcript SUSCEPTIBILITY LOCI FOR BIPOLAR DISORDER

Genetics of Addiction
Wade Berrettini, MD, PhD
Karl E. Rickels Professor of Psychiatry
Center for Neurobiology and Behavior,
University of Pennsylvania
Philadelphia, Pennsylvania, USA
[email protected]; 215-8980092
Anais Nin: “He does not need opium.
He has the gift of reverie.”
Tallulah Bankhead: “Cocaine isn’t
habit-forming. I should know---I’ve
been using it for years.”
George Carlin:
“Just because you got the monkey
off your back doesn’t mean the
circus has left town.”
101st Anniversary of Origin of Species


We celebrate the 101st anniversary
of the publication of Darwin’s
Origin of Species, which first
appeared in print (1909) only after
his death. Among other subjects,
Darwin investigated the
evolutionary adaptation of beak
length among finches.
But today our subject is the
genetics of addiction. It is unclear
whether Darwin completely
understood the advantages of a
longer beak among finches…
Courtesy of Nils D’Aulaire
OUTLINE
 Epidemiology
of addictions
 Genetics
and Genome-wide
Association Study (GWAS)
 Molecular
study of nicotine
addiction (NA)
 Molecular
study of alcohol
addiction (AA)
A View from Inside the Addicted Brain
 “My
coke (sic) use hasn’t harmed
me all that much, other than my
brain going out the window, and I’m
unproductive.”
 Of 100 pack-a-day smokers
admitted to hospital for myocardial
infarction, 50% had returned to their
former smoking practices within 3
weeks after discharge.
Public Health Perspectives
 If
current trends continue, the annual
number of deaths (world-wide) from
tobacco-related diseases will double
from 5 million in the year 2000 to 10
million in 2020 (WHO, http://www.who.int/whosis).
 13% of US adults are nicotine
addicted, smoking a pack or more
daily.
 Smoking is the largest preventable
cause of morbidity and mortality in
the US, with obesity being the second
largest.
Public Health Perspectives
 Alcohol
addiction (AA) is an
adolescent-onset disease, with peak
age-at-onset in the 3rd decade of life.
 In the US, ~ 8% of adults have AA.
 Men are at somewhat higher risk than
women for AA.
 ~ 80% of AA individuals are also
nicotine addicted (NA).
Drug addiction* is > 3 of These

1. Tolerance (e.g., the need to take more drug to achieve the desired effect)
 2. Withdrawal: Daily use for at least several weeks; for nicotine,
following abrupt cessation, four or more of the following signs:
•
•
•
•
•
•
•
•
(A) dysphoric or depressed mood
(B) insomnia
(C) irritability, difficulty managing anger
(D) anxiety
(E) difficulty in concentration
(F) restlessness
(G) decreased heart rate
(H) increased appetite or weight gain

3. Repeated unsuccessful attempts to quit or reduce using
 4. Continued drug use despite medical or psychological harm
 5. Drug use is often greater than intended or longer than intended.
 6. Reduction or elimination of social, recreational or occupational activities
because of drug use
 7. Much time is spent obtaining and using the drug, & recovering from use.
For nicotine addiction (NA) #6 & #7 are not very relevant. Thus, DSM criteria for drug
addiction are not ideal for NA.
*From the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, 1994
Two Questions to Diagnose Nicotine
Addiction
 How
many cigarettes per day did
you smoke when your smoking was
at its worst?
 Answer: > 15
 During that time, how soon after
arising did you have your first
cigarette of the day?
 Answer: within 30 minutes
GENES FOR POLYDRUG ADDICTION
Polydrug addiction is the rule:
1. ~25% of cocaine dependent persons are
alcohol addicted (AA);
2. ~ 80% of AA persons are NA.
3. ~ 30% of opioid addicted persons are AA
Thus, it is reasonable to theorize that some alleles
predispose to drug addiction without regard to the
pharmacologic class of drug (eg, stimulant or
opioid). These alleles might influence a common
brain reward pathway activated by drugs of abuse.
But,…
Opioid Addiction Aggregates
in Families


Merikangas (Arch Gen Psych 55: 973,
1998) studied 87 opioid addict (OA)
probands & 332 1o relatives
10% of OA proband 1o relatives had OA,
a significant increase compared to 1o
relatives of cocaine addicted probands,
AA probands or control probands
(p<0.001), suggesting specificity in
familial aggregation of the predominant
drug
Separated at birth, the Mallifert twins meet accidentally
Opioid Addiction (OA) in Twins



Tsuang et al (AGP 55:967, 1998) used
the Vietnam Era Twin Registry, 3372
twin pairs
54% of the variance in risk for (OA) was
genetic
~70% of genetic variance in OA risk was
specific to opioids, suggesting that most
of the genetic risk for OA is due to
variation in endogenous opioid system
genes
GENES FOR SPECIFIC DRUG ADDICTION
Given that opioid addiction aggregates in
families, it is expected that some alleles
predispose to drug addiction which is
specific to a pharmacologic class of drug.
(eg, alleles in genes which code for
proteins involved in a mechanism of action
of the drugs (eg, an opioid receptor gene
allele as a risk factor for opioid addiction).
It is expected that some risk alleles will
decrease aversive effects of a drug, while
other risk alleles will increase the
rewarding effects.
Hall et al, Tob Control 11:119, 2002
NICOTINE
Nicotine (an alkaloid) is an agonist at
nicotinic cholingeric receptors. It is
the only compound in tobacco which
mammals will self-administer.
An average cigarette may contain ~ 10
mg of nicotine, which is rapidly
absorbed through lung membranes
into the blood, as smoke is inhaled.
10-20 seconds are required from inhalation of tobacco smoke for
nicotine to reach the brain. After brief nicotine exposure, receptors
assume a desensitized state.
The T1/2 of nicotine is ~1.5 hours, with metabolism primarily in liver,
through CYP2A6 and other pathways.
NEURONAL NICOTINIC RECEPTORS

Neuronal nicotinic receptors (NachRs) are
heterogeneous pentomeric cell membrane
receptors which act as ligand-gated ion channels,
mediating conductance of calcium and sodium.

They are found both on cell bodies and dendrites of
CNS neurons, where they increase release of multiple
neurotransmitters, including dopamine and 5HT.
 NachRs are diverse, composed of pentamers, alpha 2
through alpha 9 and beta 2 through beta 4 subunits.

Nicotine activates NachRs, opening the ion channel, but, after
brief exposure to nicotine, NachRs can assume a desensitized
state, with the channel closed.

The endogenous neurotransmitter is acetylcholine.
Prominent Neuronal NachR Subtypes
Alpha 5 subunits cannot form
functioning receptors alone or
in combination with only one other
Subunit type. Alpha 5 subunits
do not participate in forming
the acetylcholine binding site.
courtesy of Jon Linstrom, PhD
19
Dopamine neurons, with cell bodies in the
VTA project to the
n. Acc. & the
medial
prefrontal
cortex.
(n. Acc)
(ventral
tegmental area)
Activation of these neurons is
a key brain signal of reward.
A MAJOR CNS REWARD SYSTEM
NachRs Influence Transmitter Release
NachR
dopamine molecules
NachRs modulate
neurotransmitter release
via pre-synaptic receptors.
Nicotine’s ability to
modulate release of
dopamine may be key to
its addictive quality.
dopamine
receptors
Post-synaptic NachRs
modulate the response of
the post-synaptic neuron.
Nicotine Patch Efficacy
*
For someone smoking > 20 cigarettes per day, start
with the 21 mg patch. After ~2 weeks, reduce to
14 mg patch, after ~2 weeks, reduce to 7 mg. Few
data exist to guide the duration of therapy.
*Note the poor overall success rate.
Bupropion for Smoking Cessation
300mg used for 9weeks
Mechanism may involve blockade of DA/NE reuptake
~ 30% of participants abstinent after a 9 week trial!
Does not alleviate nicotine withdrawal symptoms.
May be combined with nicotine replacement therapy
Effectiveness of Bupropion (vs. Placebo)
No. of studies
21
No. of participants
7171
Odds Ratio (95% CI)
1.99 [1.73, 2.30]
Fiore et al., 2000; Hughes et al., 2005
Varenicline
 a4b2 nicotinic receptor partial agonist
 Eliminates reward from smoking and craving and
withdrawal
 AEs are comparable to placebo (nausea is biggest
Onckene et al., 2005
concern)
60
48
50
37.3
40
4-week
Continuous 30
Abstinence
20
%
33
28.6
17.1
10
0
Placebo
Bupropion
0.3mg
Once/day
1mg
Once/day
1mg
Twice/day
OUTLINE
 Epidemiology
of addictions
 Genetics
and Genome-wide
Association Study (GWAS)
 Molecular
study of nicotine
addiction (NA)
 Molecular
study of alcohol
addiction (AA)
The Human Genome
The 4 DNA bases (A, G, C, T,
shown in light green, dark
green, orange, purple),are
arranged in pairs as a linear
sequence along two sugar
backbones. G pairs with C and
A with T. 3 billion bases
comprise the human genome.
Variation in the linear
sequence of bases makes each
of us unique.
As usual, the problem begins in the
gonads (no surprise here).
DNA is inherited in huge chunks, millions of
bases long, through recombination, which
occurs during meiosis in germ cells
MEIOSIS
STARTS IN
A DEVELOPING
SPERM
OR EGG CELL
Locus 1
*Locus 2
Mat Pat
+
*
+
+
*
*
4 recombined chromosomes
in 4 new eggs or sperm cells
Single Nucleotide Polymorphisms (SNPs)
The most common variation in DNA is the
SNP. An example of a SNP is shown below. At
a hypothetical point in the human genome
(the fifth base pair in the sequence), humans
can have either a T:A base pair or a C:G base
pair .
ALLELE 1
ALLELE 2
CGATTGCACC
CGATCGCACC
GCTAACGTGG
GCTAGCGTGG
SNPs are the variants which increase risk for common
diseases. A common SNP occurs every 1000 base pairs
across the 3 billion bases of DNA
THE HUMAN GENOME PROJECT
Although we have completed the linear
sequence of the 3 billion bases in the human
genome, this accomplishment is only the
beginning.
Knowing the sequence of the human genome,
without understanding the functional
significance of the variation, is similar to
knowing the alphabet of a language without
comprehending any of the words.
This state of ignorance has given rise to the
expected cartoons….
The Human Genome Project
Among the ~3 million
common SNPs, which
are functional?
WHAT IS A HAPLOTYPE?
A haplotype refers to a combination of two
or more alleles that are found on the same
chromosome. Haplotypes in this example
are TC and AG.
There are four possible
T
A
haplotypes: TC, TG, AC and AG.
If alleles at these two loci are
found together more often than
C
G randomly among unrelated
individuals, the two loci are in
linkage disequilibrium (LD) or
they are associated.

LINKAGE DISEQUILIBRIUM
(GENETIC ASSOCIATION)
1. For uncertain reasons, recombination tends not to
occur at many sites in our genome, giving rise to
blocks of DNA which remain relatively intact over
human evolutionary time.
2. When an illness-causing variant first appears in a
population, it is transmitted with large flanking DNA
sequences to the next generation.
3. With each subsequent generation the flanking
sequence transmitted always with the variant becomes
smaller & smaller due to recombination.
4. Hundreds of generations later, “unrelated” persons
(with the variant) share a small block of DNA, flanking
the variant.
LINKAGE DISEQUILIBRIUM (LD)
Abraham’s ancestral chromosome
Isaac
= disease allele
2 x 107 bp
shared
Ismael
Present day Middle East population
5000 generations later, ~4,000 bp shared
LINKAGE DISEQUILIBRIUM (LD)
Abraham’s ancestral chromosome
Isaac
^
^
=disease allele
^=neutral allele
Ismael
^
Present day Middle East Population
^
5000 generations later, ~4,000 bp shared
LINKAGE DISEQUILIBRIUM (LD)
Abraham’s ancestral chromosome
Isaac
^
^
Bethlehem
=disease allele
^=neutral allele
Ismael
^
Beirut
^
^
~5000 generations later, ~4000 bp shared
ADVANCES in SNP TECHNOLOGY
~1,000,000 SNP CHIPs provide the ability
to obtain a genotype at 1 SNP every ~
4000 base pairs across the genome,
allowing study of most haplotype blocks.
Allele-specific fluorescently-tagged DNA
fragments (known as oligonucleotides)
are mounted on the slide. The
oligonucleotides are sequence-specific
for one of the two alleles of a given SNP.
The fluorescent tag will emit energy at a
specific wavelengths (color), only in the
presence of the specific SNP allele for
which they have been designed.
The fluorescent signal is detected by a
CCD camera.
The fluorescent signals are read and
translated into genotypes by computer
scripts.
DNA SNP Chip Image
Homozygous for allele 1 = red
Homozygous for allele 2 = green
Heterozygous = blue
Successes in GWAS
*
Cichon et al, AJP 166:5, 2009
*1 million SNPs have been tested for association with
disease, creating a small multiple testing problem, so
the p = 5 x 10-8 is the equivalent of p = 0.05.
OUTLINE
 Epidemiology
of addictions
 Genetics
and Genome-wide
Association Study (GWAS)
 Molecular
study of nicotine
addiction (NA)
 Molecular
study of alcohol
addiction (AA)
Genome-wide association study of 1 million
-15
L
O
G
P
SNPs for cigarettes per day in 41,000 persons
CHRNA3-
of European origin revealed only a single
CHRNA5
cluster
region of the genome (chromosome 15q25)
conveying a small amount (odds ratio = 1.3)
of risk for nicotine addiction (P = 10-16)
Liu et al, Nature Genetics, in press
-5
1
2
3
CHROMOSOME
15
CHRNA5
CHRNB4
CHRNA3
Bierut et al, 2008, P = 0.007;
Chen et al, in press
P=0.003 and 0.007;
Weiss et al, 2008
p = 0.0009
Saccone, et al, 2007, P=0.0003
Thorgeirsson et al, 2008, P=10-20
Saccone et al, 2007
Berrettini et al, 2008 (P=0.000003
P=0.003
Berrettini et al, 2008 (p = 0.00007)
Risk alleles are boxed; all are
on a common European
haplotype (frequency = 38%)
CHRNA3/5 Alleles Increase Lung CA Risk (Hung et al, Nature, 2008)
This was found even though cases and controls were
closely matched for smoking history!
CHRNA3/5 Alleles Increase Risk for Lung CA # 2
This was found even though cases and controls were
closely matched for smoking history!
Amos et al,
Nat Genetics, 2008
CHRNA5
CHRNA3
PNAS, 2006
Lung CA cell lines
Model of Carcinogenesis in Smoking and Lung
Cancer
 Carcinogens in tobacco smoke cause a lung cell to become pre-malignant.
 The pre-malignant cell begins to overexpress CHRNA5 and underexpress
CHRNA3, so that nicotinic signalling is markedly amplified.
 Immune surveillance cells detect this and send a pro-apoptotic signal to the
pre-malignant lung cell.
 Because of the presence of nicotine, the cell is able to escape the apoptotic
signal.
 The lung cell becomes fully malignant and is able to ignore further messages.
 If true, this model has implications for the treatment of nicotine addiction (with
nicotine replacement therapies) in individuals with lung cancer, especially when
such persons are undergoing chemotherapy.
rs1051730
odds ratio = 1.3, Cases (n = ~2000) & controls were carefully
matched for smoking history, suggesting
p = 5.7 x 10-10
that the NA risk haplotype increases risk
for COPD by a mechanism independent
of smoking.
Phenotypes Associated with the NA Risk Haplotype
 1. Cigarettes per day (CPD): Berrettini et al, 2008; Thorgeirsson et al,
2008; Caporoso et al, 2009; Stevens et al, 2009; Chen et al, 2009;
Freathy et al, 2009; Spitz et al, 2008, Liu et al, in press, 2010.
 2. Fagerstrom test for Nicotine Dependence: Beirut et al, 2007; Weiss et
al, 2008; Thorgeirsson et al, 2008; Chen et al, 2009
 3. Lung cancer: Amos et al, 2008; Thorgeirsson et al 2008; Hung et al,
2008;
Spitz et al, 2008; Liu et al, 2008;
 4. Inability to quit smoking: Freathy et al, 2009; Baker et al, 2009;
 5. Increased risk to relapse to smokingafter quitting: Baker et al, 2009;
 6. Early onset of smoking: Weiss et al, 2008; Schlaepfer et al, 2008
 7. COPD: Pillai et al, 2009
Prominent Neuronal NachR Subtypes
Alpha 5 subunits cannot
form functioning
receptors alone or in
combination with only
one other Subunit type.
Alpha 5 subunits do not
participate in forming the
acetylcholine binding
site.
courtesy of Jon Linstrom, PhD
48
OUTLINE
 Epidemiology
of addictions
 Genetics and Genome-wide
Association Study (GWAS)
 Molecular study of nicotine
addiction (NA)
 Molecular study of alcohol
addiction (AA)
Naltexone Treatment of Alcohol Addiction
Naltrexone (NTX), a mu opioid receptor antagonist, is
efficacious (at 50-100 mg daily) in the treatment of alcohol
addiction.
It reduces risk for relapse to heavy drinking (> 4 drinks daily
for a woman, > 5 drinks daily for a man), but does not
influence abstinence.
It reduces the euphoria from ethanol, and thereby reduces
the drive to drink excessively.
Because alcohol addiction is so common in our country,
widespread use of naltrexone could improve public health
considerably.
Randomized Placebo Controlled NTX Trials
Studies supporting efficacy
Study
Studies not supporting efficacy
# Ss
Notes
Volpicelli et al 1992
70
None
O’Malley et al 1992
97
Volpicelli et al 1997
Study
# Ss
Notes
Oslin et al 1997
44
Older
None
Kranzler et al 2000
183
None
97
None
Krystal et al 2001
627
VA only
Kranzler et al 1998
20
Depot
Lee et al 2001 (Singapore)
53
None
Anton et al 1999
131
None
Gastpar et al 2002 (Germ.)
171
None
Chick et al 2000 (UK)
169
Adherence
Kranzler et al 2004
315
Depot
Monterosso et al 2001
183
None
Killeen et al 2004
145
None
Morris et al 2001 (Australia)
111
None
Oslin et al in press
240
None
Heinala et al 2001 (Finland)
121
Nonabst.
Latt et al 2002 (Australia)
107
None
Ahmadi and Ahmadi 2002 (Iran)
116
None
Guardia et al 2002 (Spain)
202
None
Balldin 2003
118
None
Kiefer et al 2003 (Germany)
160
None
Kranzler et al 2003
153
None
Kranzler et al 2004
315
For drinking not
relapse
Anton et al 2004
270
None
Mu Opioid Receptor Amino Acid Sequence
Extracellular Space
N40D (A118G) SNP; N is a glycosylation site
cytoplasm
Alcohol Effects by A118G Genotype
Ray and Hutchinson, 2004
50
45
40
35
30
25
20
15
10
5
0
AA allele
AG allele
0.02
0.04
Breath Alcohol Concentration
0.06
Naltrexone (NTX) in Clinical
Trials for Alcoholism
Patients were selected from 3 NTX multiple double-blind, placebocontrolled trials. These trials were 12 weeks, and include selfreport measures of drinking along with biochemical verification
(liver enzymes and carbohydrate deficient transferrin). Primary
endpoint was relapse to heavy drinking.
Medication was given in blister packs and compliance was
determined by pill counts. Participants had twice weekly
abstinence counseling sessions.
Only those persons completing the trial, with outcome data,
were contacted later to provide a blood sample for DNA studies.
Cumulative Survival (time to relapse)
OPRM1 VARIANTS IN NALTREXONE Rx
FOR ALCOHOL DEPENDENCE
Naltrexone / Asp40 Allele
(A/G, G/G), N = 23*
Naltrexone Asn40
Allele (A/A), N = 48
Placebo / Asp40 Allele
(A/G, G/G), N = 41
*p = 0.04 vs NTX Asn40
odds ratio = 3.4
Days
Placebo / Asn40
Allele (A/A), N = 18
Oslin et al 2003
Fig 6: 118G Allele Predicts Good NTX Response
%
G
O
O
D
P = 0.005, genotype
x medication
interaction
odds ratio = 5.8,
A/A vs. G/*
PLA
NTX
NTX
PLA
O
U
T
C
O
M
E
A/A
G/A, G/G
The COMBINE
study
was a multi-site
trial of NTX,
acamprosate or
both drugs, +/addiction
counseling, for
alcohol addiction.
The NTX & placebo
groups were
compared by
A118G genotype for
outcome after 14
weeks of treatment.
SUMMARY
Genetic influences in drug addiction are
varied, with some being specific to a
pharmacologic given class of agents, while
others cross these boundaries.
 A haplotype at the CHRNA3/5 locus
increases risk for nicotine addiction, COPD
and lung cancer, through independent
mechanisms.
 The A118G SNP in the mu opioid receptor
may predict response to naltrexone for
alcoholism.

I trust your genetic code is in order, Mr. Jones?