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Strategies for investigating the
genetics of psychiatric disorders
Margit Burmeister, Ph.D.
Molecular & Behavioral Neuroscience Institute
(formerly Mental Health Research Institute)
Department of Psychiatry
Department of Human Genetics
Neuroscience Program
Bioinformatics Program
University of Michigan, USA
Overview
• Heritability
• Terminology
• Linkage analysis
• Association studies – candidate genes
• Whole genome association studies
• Phenotypes
• Gene x environment interaction
Familiality is not heritability
• Bipolar Disorder, Depression, Schizophrenia
and Alcoholism run in families
–But: Going to Medical School, being Catholic
and speaking Mandarin also run in families.
• Heritable due to genes?
• Familial due to environment?
–Adoption: risk of child more influenced by
biological parents (genetic) or adoptive
(environment) parents?
Twins: Monozygotic (MZ, identical) twins share
100% of genes, dizygotic (DZ, fraternal) twins
share 50% of genes.
–
Twin concordance rates
(From: Plomin et al. 1994, Science 264: 1733-9)
Locus (plural: Loci)
From Latin: Place
The position on a chromosome
of a gene or a genetic marker or a SNP
A locus that comes in several forms is called
polymorphic
Allele
one of several forms of a locus
We inherit one allele from each parent
Genetic marker
• DNA segment with known location on a
chromosome whose inheritance can be followed
• DNA segments near each other on a chromosome
tend to be inherited together
–so markers are an indirect way to track inheritance of an
unknown gene
• Eye color and bristle length are used as easily
recognizable genetic marker in Drosophila crosses
• Molecular markers are now used in human genetics
–Restriction fragment length polymorphisms (RFLPs).
–minisatellites, often also called VNTR markers
–microsatellite or STRP markers, e.g. CA/GT repeats
–SNPs: Single nucleotide polymorphism
Linkage analysis
Hypothetical marker with alleles A, B, C and D is is linked to the disease.
In the first pedigree, allele A segregates with the disease, but in the second
pedigree, allele B segregates with the disease. The indicated recombination
event leads to allele C segregating with the disorder in the right branch.
What is linkage?
• Linkage is caused by loci (e.g. the risk gene
•
•
•
and a genetic marker) being close to each
other on a chromosome.
The recombination fraction θ is the
probability that, in any meiosis, there will
be a recombination between them.
θ=0.5 Mendelian segregation – no linkage
θ=0.0 very tight linkage – no recombination.
How sure are we of linkage?
LOD score!
Logarithm of Odds
(Probability of a particular family constellation
if the marker and disease locus are linked,
with 10% recombination between them)
LOD (q=0.1) = log
----------------------------------------------(Probability of the same family constellation
of markers and disease if the
marker is unlinked to the disease locus)
When is linkage significant?
How many families are needed?
• LOD score of 3.0 - odds of 1000:1 are
equivalent to p= 0.05
• With pedigree at hand, the best possible and
a likely LOD score can be calculated.
• For a Mendelian disease, only 10 informative
meioses are needed to get a LOD score of 3.03
– single families can be enough to find linkage
Family with recessive ataxia: SCASI
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+*
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*
*
*
* *
*
In this single family with a rare recessive disorder, we found
linkage to 1p36 with a LOD score of 3.28
Unaffected;
Affected; * DNA available
Complex Disorders
• Psychiatric disorders are complex - unlike
Mendelian disorders, where a single mutation is
both necessary and sufficient to bring about the
disorder. Complexity can be due to
– genetic heterogeneity: many different risk genes
– small effect of each genetic factor: risk allele only
increases risk by a small fraction
– diagnostic uncertainty: is a depressed or alcoholic
subject in a Bipolar pedigree affected or not? What
about a BP II subject?
interaction with the environment
–
LOD score for complex traits –
in multipoint analysis
(Probability of this particular data constellation
if a locus that increases risk for the
disorder is in a particular chromosomal region)
LOD = log
----------------------------------------------(Probability of this particular data constellation
if there is no locus that increases disease risk
in this particular chromosomal region)
COGA Genome Linkage Scan (Max. Drinks)
Saccone et al. (2000) Am J Med Genet 96:632-7.
Summary linkage/LOD score
• looks at chromosomal location, with the help of
informative genetic markers
• powerful for simple Mendelian inheritance – no
need for large samples
• assumes you know the genetic model
• no assumptions about the biology involved
• Assumes one or just a few genes are involved
• Families easily combined – add LOD scores
• More powerful if less heterogeneous subtypes
can be defined (e.g. Bipolar Disorder with
psychosis, antisocial alcoholism; early onset
MDD)
• How do I follow up my positive linkage finding
•
that showed 3 great POSITIONAL candidate
genes in the region linked to the disease?
How do I test whether a new BIOLOGICAL
candidate gene is involved in alcoholism?
Association
Cases with Alcoholism
ALDH2: 1/1: 283 (83%)
1/2: 57 (17%)
2/2: 0
Control sample
ALDH2: 1/1: 304 (56%)
1/2: 218 (40%)
2/2: 23 ( 4%)
ALDH2*2 allele significantly protects against alcoholism
in this sample from China (Chen et al., Am J Hum Genet. 1999,795-807).
FLUSHING SYMPTOMS
IN RESPONSE TO DRINKING ALCOHOL
• Blood Flow to Face
• Blood Flow to Body
• Feel Itchy
• Feel Dizzy
• Feel Tired
• Feel Anxious
• Pounding Head
• Have Sweats
• Increased Heart Rate
• Feel Nauseous
These symptoms are experienced by those heterozygote for
the ALDH2*2 allele, and are severe in homozygotes
Pharmacogenetic association
• Within a disease population,
pharmacogenetic studies ask about
drug side effects and/or response:
–drop outs versus drug continuation
–“responders” versus non-responders
Greer and Schatzberg, 2003
Am J Psychiatry 160:1830-5.
Zanardi et al., 2001
Biol. Psych. 50: 323-30.
Confounding of association by
ethnicity (stratification)
cases
controls
Example:
alcoholics in San Francisco.
Asians (stripes) will be underrepresented among cases.
ANY genetic marker more common among Asians will also
be overrepresented in the controls – e.g. HLA.
Linkage disequilibrium (LD)
Linkage disequilibrium shows the history of the mutation.
starting chromosomes that exist in population at one point
A,a B,b C,c and D,d are different alleles at four different nearby loci:
A
b
c
D
A
b
C
d
a
b
C
d
New mutation arises on one particular chromosome:
a
B
C
d
Haplotype
Combination of specific SNPs on chromosome
I: A,c,D
A
b
c
D
II: A,C,d
A
b
C
d
III: a,C,d
a
b
C
d
a
B
C
d
We might find association of a disorder with haplotype III
because of linkage disequilibrium with the (untyped) allele B!
Applying the data:
• There are >10 Million SNPs in the human
genome.
• Because of LD (linkage disequilibrium), typing
300,000-500,000 SNPs may give us some
information about all the >10 Million SNPs
• Single SNPs as well as haplotypes can be
tested for association with disease
COGA Genome Linkage Scan (Max. Drinks)
Saccone et al. (2000) Am J Med Genet 96:632-7.
0
20
40
60
80
100
120
140
160
180
200
220
cM/Mb
45800000 46000000 46200000 46400000 46600000 46800000 47000000 47200000 47400000
1
84
141
GABRG1
GABRA2
68
GABRA4
bp
395
GABRB1
1,400 kb
0.1
0.01
0.001
SNPs
haplotypes
0.0001
Drawn by Jeffrey Long from Edenberg HJ, et al. (2004) Am J Hum Genet 74:705-14
Linkage - versus - Association
• Needs
families (e.g.
sibpairs) in which the
• Needs
unrelated cases
and unrelated controls
disease segregates
•
Assumes that only one or
• powerful when there are just few common variants in
•
•
•
a few predisposing genes
Works well even when many
different mutations or alleles
in those few genes increase
risk for the disease
Requires no hypothesis
about the nature of the
defect in the disorder
usually requires a model of
the pattern of inheritance
•
•
each gene are risk factors
Powerful even when there
are many different genes,
each with a small effect,
as long as the variant in
each gene is common
Until recently, required
having a limited number of
genes in mind
2005 and beyond:
Whole Genome Association Studies
•
•
•
•
500-1000 cases
500-1000 controls
Extract DNA – hybridize to whole genome chip
Construct haplotypes
Calculate which of 300-500,000 SNPs and ??? haplotypes
is more frequent in cases than controls
Currently: $1,000 per individual – planned: $100
Evidence from Twin Studies
for involvement of genes in depression
Risk of Major Depression Onset by Genotype and Severe Life Event
Probability of Onset of Major Depression
20
MZ Co-Twin Aff
MZ Co-twin Unaff
DZ Co-Twin Aff
DZ Co-twin Unaff
15
10
5
0
Absence
of severe life event
Presence
Redrawn from
Kendler et al., 1995
Am. J. Psychiatry
152: 833
Genes, Stress and Depression
Genetic Vulnerabilities
(Personality, Physiology, …?)
Early experiences
(stress, rearing,..?)
Vulnerable Phenotype
Daily stress
Traumatic event
Life event
Exercise,
antidepressants
psychotherapy
Depression
High Neuroticism score is
a risk factor for depression
• Endophenotypes or intermediate traits mark the genetic
•
predisposition to a disorder, less dependent on the
additional external triggers or vulnerabilities.
Neuroticism is a psychological domain, measured with
the established NEO-PI. Sample questions:
– I often feel tense and jittery (anxiety facet)
– Sometimes I feel completely worthless (depression)
–When I’m under a great deal of stress, sometimes I feel like I’m
going to pieces (vulnerability)
• Twin and high risk studies suggest that a high
Neuroticism score is a trait marker for depression
• At least in women, about 55% of the genetic liability to
depression is shared with Neuroticism
Serotonin Transporter Promoter
Variant 5-HTTLPR
• 5-HTT target of SSRIs
• S-short and L- long form of promoter:
• The L form has higher promoter activity
• Both are quite common in Caucasians
5-HTTLPR is associated
with Neuroticism
92
90
88
p=0.015
86
84
Total variance
explained: 1.8%
82
80
78
76
N=
83
183
149
s/s
l/s
l/l
5 HTTLPR G enoty pe
From: Sen et al. 2004, Biol Psychiatry. 55:244-9.
Genes, Stress and Depression
Genetic Vulnerabilities
(Personality, Physiology, …?)
Early experiences
(stress, rearing,..?)
Vulnerable Phenotype
Daily stress
Traumatic event
Life event
Exercise,
antidepressants
psychotherapy
Depression
Is there an effect of childhood
maltreatment on depression risk?
Caspi et al., Science 2003
Genotype x life events interact with respect
to 5-HTTLPR genotype and depression
(Caspi et al., 2003: Science 301:386-9)
symptoms
MDD episode
suicidality
MDD per informant
Conclusions
• Twin and adoption studies suggest genes are involved in
nearly all psychiatric disorders
• Psychiatric disorders are complex
• Linkage studies are a powerful means to find where
genes are located
• Subtypes of psychiatric disorders that decrease
heterogeneity will increase power of linkage analysis
• Association studies can help identify common genetic
risk factors even with very small effect size
• Association studies are now feasible for the whole
genome, not just candidate genes
• Research into intermediate traits/endophenotypes can
help identify genetic risk variants
• Genes and environment interact