Transcript 2012_02_23_Tamara_Koopmann - Erasmus Observatory on Health …

How to find an association
between a disease and a gene?
Alex V. Postma
Department of Anatomy, Embryology & Phsyiology
Heart Failure Research Center
AMC
[email protected]
NHS
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Sudden cardiac death, the numbers
- 50% of cardiac mortality
- US: ~250.000 SCD / year
- NL: ~300 SCD / week, 15.000 / year
- VF most common underlying
arrhythmia
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Sudden cardiac death, the causes
> 40 y
1/1.000/year
> 85% coronary artery disease
< 40 y
0,8-1,6/100.000/year
~75% genetic disease
Cardiomyopathies, primary electrical
disease
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Monogenic Cardiac Arrhythmia
Syndromes
Long QT Syndrome
Brugada Syndrome
Catecholamine-induced PMVT/VF
Short QT Syndrome
Isolated Cardiac Conduction Disorders
Familial Atrial Fibrillation
Sinus Node Disease
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LQTS,
SQTS
LQTS, SQTS
Sinus
Node
Dysfunction
LQTS,
SQTS
LQTS
BrS
CD
AF
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GENE
KCNQ1
KCNH2
KCNE1
KCNE2
KCNJ2
KCNA5
SCN5A
SCN4B
RYR2
CASQ2
CACNA1C
HCN4
GJA5
CAV3
ANKB
GPD1L
DISORDER
LQTS, SQTS, AF
LQTS, SQTS
LQTS
LQTS
LQTS, SQTS
AF
LQTS, BrS, CD, SSS
LQTS
CPVT
CPVT
LQTS
SSS
AF
LQTS
LQTS
BrS
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From patient to genotype
• Patient goes to cardiologist (complaints, family
history,...)
• Genetic counseling
• DNA isolated
• Mutation screening (PCR, sequencing)
• Clinical geneticist and cardiologist discuss result
/ treatment with patient
• Family screening
OR
• Research
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Where to start?
• Linkage analysis
• Screen candidate genes
• Perform exome / genome sequencing
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Identifying genetic cause for a disease
Many human diseases/phenotypes are affected by
single genes. Examples include: Brown eyes
(dominant to blue eyes); Ability to curl or roll up
tongue (dominant to inability)
PEDIGREES of large families can help identify
whether a genetic disease is caused by a
dominant or a recessive allele, and whether the
disease is sex-linked or autosomal.
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Autosomal Dominant Transmission
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Autosomal Dominant Transmission
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Autosomal Recessive Transmission
Appears in both male and female
children of unaffected parents.
e.g. KCNQ1 mutations associated with
Jervell Lange Nielsen Syndrome
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Autosomal Recessive Transmission
Appears in both male and female
children of unaffected parents.
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Mutation Arising De Novo
e.g. Timothy Syndrome (CACNAIC)
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Mutation Arising De Novo
e.g. Timothy Syndrome (CACNAIC)
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Compound heterozygosity (multiple variants together)
Bezzina et al., Circ Res, 2003
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Linkage analysis
Technique to find causative regions / genes for a
genetic disease
Linkage is co-segregation between a disease
and a set of markers >> identify regions that
are consistently shared by affected relatives
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Genetic markers:
• Genetic markers are DNA sequences that show
variations in size or sequence in the population
• Occur random all over the genome off all species
• Probably errors of DNA copying
• Inherited to next generations
Markers
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Cross-over
Parents
A a
B
a
a

b
b
b
Affected
First generation
A a
B
a
 a
b
b
b
Affected
Second generation
A a
B
Crossover has taken place
b
Affected
A a
a
a
b
b
b
?
a
a
b
B
b
Affected
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Example linkage analysis
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Candidate gene approach
-Study literature, study disease, hypothesize
-Perform linkage analysis (on all candidate genes)
-Positive linkage -> look for interesting areas of
the gene
-Sequence
-Find mutation….
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Mutations
Mutations are heritable changes in genes or
chromosomes.
Most mutations are SINGLE-GENE MUTATIONS
that arise from errors in replication or from
unrepaired damage to DNA molecules.
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Types of mutations
Original DNA strand
Substitution
Deletion
Insertion
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Effect on the protein
Silent
Missense
Nonsense
Frameshift
Ser
Thr
Pro
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Why mutation screening?
• Diagnostic screening:
– Confirmation clinical diagnosis
– (Prenatal) counseling
• Research:
– Genotype-phenotype relationship
– Pathophysiological mechanisms
– Presymptomatic screening/therapy
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Mutation Interpretation
• Not every novel change leads to disease.
• Interpret in conjunction with:
– Inheritance pattern
– Mechanism of disease
– Amino acid conservation
– Cis vs. trans
– Population/Haplotype information
– Structural and functional domains
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How to find a mutation
• DNA isolation from:
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How to find a mutation
The Invention of PCR
Invented by Kary Mullis
in 1983
First published account
appeared in 1985
Awarded Nobel Prize for
Chemistry in 1993
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Intron-exon structure of a gene
Start (ATG)
5’
Stop (TAA, TAG or TGA)
AG
3’
GT
PCR fragments
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What is the Polymerase Chain Reaction?
• A means of selectively amplifying a particular segment of DNA
• The segment may represent a small part of a large complex
mixture of DNAs: e.g. a specific exon of a gene
• It can amplify a usable amount of DNA (visible by gel
electrophoresis) in ~ 2 hours
• It can amplify a single DNA molecule (e.g. from a single sperm)
• PCR product usually undergoes post-PCR analysis:
restriction digestion, sequencing, cloning etc
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Sanger sequencing
- DNA fragments are separated by size by gel
electrophoresis
- From the gel, the DNA sequence can be
determined
- Can produce DNA fragments 700-900bp long,
but low throughput
- The Human Genome Project
used Sanger sequencing,
completion took over 10 years
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wildtype
A I-1
ATC CAC ATA
I H I
ATC CRC ATA
I H/R I
?
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Next Generation Sequencing (NGS)
a.k.a. Whole Genome/Exome Sequencing
Mega Sequencing
2nd Generation Sequencing
Massive parallel Sequencing
High Throughput Sequencing
Deep Sequencing
General characteristics include:
- Amplification of genetic material by PCR
- Ligation of amplified material to a solid surface
- Short reads applications; sequence and then
use computers to assemble the small pieces
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Next Generation Sequencers
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However...
• 20K variants identified per exome
• ~45% missense, 150 nonsense
• 150-250 unique variants / individual
• ETHICAL ISSUES!!!
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Monogenic vs. polygenic
Monogenic disease
Polygenic (complex) disease
• Severe phenotype
• Mild phenotype
• Early onset
• Late onset
• Rare
• Common
• Mendelian inheritance
• Complex inheritance
Cause: Mutations (<1%)
Polymorphisms (>1%)
Many polymorphisms with each a small effect, combined with
environmental factors, explain inter-individual differences in
risk
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Genetic modifiers
• The ultimate clinical presentation not only
depends on the gene affected or the type /
location of the mutation but also on the
GENETIC BACKGROUND on which it occurs.
• Clinical presentation = effect of mutation +
genetic variation in genes (polymorphisms)
encoding other players in that particular
biological pathway
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Polymorphisms
~1/300 bp is polymorphic in a population
(3 million loci = 0.1% of the genome)
~1/1200 bp will differ between 2 random
subjects
Single Nucleotide Polymorphisms (SNPs) are
the most common variants
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What is the evidence for a role of polymorphisms in
susceptibility to sudden cardiac death ?
1.
Variability in clinical severity among patients with
primary electrical disease
2.
Heritability of various ECG parameters
3.
Family history
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Large family with SCN5A 1795insD mutation
Total family members:
Unexplained SCD:
Nocturnal :
SCD <40 yr :
232
26
17
20/26
Total:
HR (bpm):
PQ (s):
QRS (ms):
QTc (s):
ST-elev V1-V3:
ST-elev (mm):
Carriers
Non-carriers
114
65.4±16.8
118
72.0±12.1
0.20±0.02
115±15
0.48±0.09
21/43 (49%)
1.6±1.0
0.16±0.03 #
93±13 #
0.40±0.03 #
3/36 (8%) #
0.5±0.6 #
Bezzina et al., Circ Res 1999;85:1206
van den Berg et. al. JCE. 2001;12:630-6
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Reduced penetrance and variable disease
expressivity and severity in patients with
SCN5A 1795insD
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What is the evidence for a role of polymorphisms
in susceptibility to sudden cardiac death ?
1.
Variability in clinical severity among patients with
primary electrical disease
2.
Heritability of various ECG parameters
3.
Family history
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Common sodium channel promoter
haplotype in Asian subjects underlies
variability in cardiac conduction
T
T
T ---
G
C
75.5%
Haplotype B
C
C
G ins
C
T
24%
Haplotype C
C
T
T ---
C
C
0.5%
T-847G
-835insGC
G-354C
C287T
T-1418C
Haplotype A
T-1062C
Frequency
promoter
intron 1
exon 1
(non-coding)
-2000 bp
-1000
0
1000
Bezzina et al., Circulation 2006;113:338-44.
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Haplotype B is associated with slower
conduction; Haplotype pair effects on PR-interval
Brugada syndrome
baseline
PR (msec)
350
Non-Brugada
syndrome
controls
**
300
*
**
250
*
200
150
100
BB
(5)
AB
(20)
AA
(45)
BB
(8)
AB
(33)
AA
(60)
*P<0.05; **P≤0.001
Bonferroni-corrected significance level: 0.002
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Reporter activity is reduced for Haplotype B
CHO cells
Cardiomyocytes
n=13, p=0.04
n=9, p=0.006
Fold activity
18
15
12
9
6
3
0
Hap A
Hap B
Hap A
?
Hap B
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What is the evidence for a role of polymorphisms
in susceptibility to sudden cardiac death ?
1.
Variability in clinical severity among patients with
primary electrical disease
2.
Heritability of various ECG parameters
3.
Family history
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Family history of sudden death is an important predictor of
ventricular fibrillation risk in myocardial infarction
MI + VF (cases)
Family history of sudden death
Male gender
Smoking, at time of infarct
Hypertension
Diabetes
Hypercholesterolemia
BMI
Medication
Cardiac history (last 12 mo)
Anterior location infarct
Center
ST deviation
versus
MI - VF (controls)
Univariate
OR
95% CI
P
2.33
0.72
1.17
0.96
0.58
0.65
0.95
0.99
1.86
1.25
1.65–3.28
0.49–1.05
0.85–1.61
0.69–1.32
0.31–1.06
0.48–0.89
0.91–0.99
0.69–1.43
1.50–3.28
0.92–1.70
1.58
1.30–1.91
<0.0001
0.084
0.34
0.75
0.1
0.007
0.01
0.98
0.033
0.15
0.002
<0.0001
Multivariate
OR
95% CI
P
2.72
0.72
1.32
1.01
0.64
0.64
0.96
1.04
1.92
1.26
1.84–4.03
0.45–1.14
0.90–1.95
0.64–1.61
0.30–1.35
0.44–0.95
0.91–1.00
0.62–1.74
0.95–3.85
0.86–1.84
1.59
1.25–2.02
<0.0001
0.16
0.16
0.96
0.24
0.027
0.066
0.88
0.068
0.23
0.001
<0.0001
Dekker et al., Circulation 2006; 114: 1140 - 1145
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ASSOCIATION STUDIES
How to find the polymorphisms that confer susceptibility
to sudden cardiac death?
- Large sample sizes
- Strict definition of clinical phenotype > same
pathophysiological mechanism
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Identification of genetic determinants of
SCD risk
• Association studies in disease populations with the SCD phenotype
genetic
variation
SCD
phenotype
• Identification of genetic variation associated with specific
endophenotyes
genetic
variation
SCD
phenotype
intermediate
phenotypes
(endophenotypes)
conduction (QRS duration)
repolarization (QT interval)
- accurate measurement in large sample sizes
- quantitative traits; more powerful for
genetic analysis
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Prolonged QT interval and risk of sudden
death in the population
Straus et al.,JACC 2006
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Genome-wide association study
of QT-interval in the general population
[Nitric oxide synthase 1
adaptor protein]
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Key points
Several cardiac arrhythmia syndromes that for long were
considered idiopathic are now known to have a genetic basis
The genetic basis for most arrhythmia syndromes is
heterogeneous
Genetic testing is recommended to support the clinical diagnosis,
to identify silent carriers and to install gene-specific therapy
Considerable heterogeneity may exist in disease manifestation
among family members carrying the same mutation.
Future research in genetics of arrhythmias will likely focus on
identification of common genetic variation to risk of arrhythmia /
sudden cardiac death
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Opdrachten
• Duo 1: presenteer het artikel van Alders et al. (2009)
• Duo 2: presenteer het artikel van Bezzina et al. (2010)
• Duo 3: presenteer het artikel van Sotoodehnia et al.
(2010)
• PowerPoint-presentatie van maximaal 10 minuten
• PowerPoint slides in het Engels
• Mondelinge presentatie naar keuze in het Nederlands
of in het Engels
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