Transcript Genetics for EpidemiologistsLecture 7: Replication and Functional

Genetics for Epidemiologists
National Human
Genome Research
Institute
National
Institutes of
Health
U.S. Department
of Health and
Human Services
Lecture 7: Replication and Functional
Studies
U.S. Department of Health and Human Services
National Institutes of Health
National Human Genome Research Institute
Teri A. Manolio, M.D., Ph.D.
Director, Office of Population Genomics and
Senior Advisor to the Director, NHGRI,
for Population Genomics
Topics to be Covered
• Replication
– Past challenges
– Criteria
– Reasons for inability to replicate findings
• Finding the causal variant
– Neighboring regions: conservation, nearby
genes
– Sequencing
– Protein product
– Expression studies
– Experimental studies: Knockdown, knockout,
knockin
Chanock S, Manolio T, et al, Nature 2007; 447:655-660.
Need for Consensus on What Constitutes
Replication: circa November, 2006
• Avalanche of GWA and candidate gene studies
anticipated in near future
• Replication held as sine qua non
• Likelihood of single study establishing an
association is low until sample sizes increase
sufficiently and analytical methods improve
substantially
• Common problem of how to interpret confusing
and spurious findings
Case in Point: DTNBP1 and Schizophrenia
• First identified as putative schizophreniasusceptibility gene in Irish pedigrees
• Reported confirmation in several replication
studies in independent European samples but
reported risk alleles and haplotypes appeared
to differ between studies
• Comparison among studies difficult because
different marker sets used by each group
• HapMap data and all identified polymorphisms
typed in CEPH samples to produce high density
reference map
Mutsuddi et al, Am J Hum Genet 2006; 79:903-909.
Phylogenetic Tree of Five Common
Haplotypes of DTNBP1
Mutsuddi et al, Am J Hum Genet 2006; 79:903-909.
Positively Associated Haplotypes Differ in
All Six Studies
Each common DTNBP1 haplotype was tagged by association signal of at
least one study, implying there is not one common variant contributing to
schizophrenia risk at DTNBP1 locus
Mutsuddi et al, Am J Hum Genet 2006; 79:903-909.
How NOT To Do A Replication Study
• Use a different phenotype
• Use different markers
• Mix fine-mapping and replication
• Use different analytic methods (haplotype vs.
single marker)
• Use different populations
Case in Point: Odds Ratio for Stroke
Associated with PDE4D in Three Studies
Rosand et al, Nat Genet 2006; 38:1091-1092.
• Association between minor allele of rs7566605 near
INSIG2 and increased BMI and homozygosity in 923
related Framingham Heart Study (FHS) participants
• Association reproduced in four additional cohorts
• Not seen in fifth cohort
Science, 14 Apr 2006
Science, 12 Jan 2007
Lyon HN et al, PLoS Genet; 2007 Apr 27;3(4):e61.
• Nine large cohorts from eight populations across
multiple ethnicities
• Family-based, population-based, case-control
designs
• Association at p < 0.05 in five cohorts but none in
three cohorts
• Variability in strength of association over time
• Replication both in unrelated (p = 0.046) and familybased (p = 0.004) samples
• Suggests initial finding unlikely to be spurious but
effect likely to be heterogeneous
Lyon HN et al, PLoS Genet; 2007 Apr 27;3(4):e61.
• Second variant (rs1455832-C) intronic to ROBO1 with
age-varying association to BMI over time
• Minor allele homozygote associated with increased
BMI diminishing after age 45
• Replicated age-varying association in same direction
in five of eight other cohorts totaling 13,584 subjects
• One childhood cohort showed very strong interaction
(p < 10-9), four others 0.003 < p < 0.05; overall 10-9
• In all replication cohorts but one, association would not
have been detected if testing only for main genetic
effect and not for age-by-’5832 interaction
Lasky-Su J et al, Am J Hum Genet; 2008; 82:849-58.
Definition of Robust Initial Finding
• Sufficient statistical power to observe reported
effect, which will vary by magnitude of
observed effect
• Highly significant analysis using stable method
• Consistent findings using simple,
straightforward analytic approach
• Consistent findings in epidemiologically sound
study
• Consistent findings overall and within key
subgroups of initial study
• Consistent findings in same or highly similar
phenotypes
Value of Single/First Study
• Initial study rarely definitive by itself but often
represents important discovery tool
– If consortium of multiple studies, stronger
• What to do with studies not having option for
replication?
– Don’t change standards for definitiveness
• Don’t just rely on GWA-- have multiple tools for
identifying and understanding associations
• May need different standards for findings of
major clinical significance, particularly
pharmacogenomic demonstration of adverse
effect that would be unethical to try to replicate
Importance of Significance Level
• Should we promulgate a specific number– NO, but
in general, smaller is better
• General agreement: range is very broad, higher
threshold for difficult to measure phenotype
• Beware of the very smallest
• If significance depends on analytic method or
multiple comparison correction, BEWARE
• If significance or association depends on phenotype
definition, BEWARE
• Randomize the phenotypes and report number
significant at that level
• Biologic information may be useful A PRIORI but a
posteriori can come up with almost anything
Importance of Genotyping Quality
• Report results of known study sample duplicates,
HapMap or other standard duplicates
• Replicate small number of “significant” SNPs with
second technology at some late stage
• May not be needed if nearby SNPs in strong LD
show same results
• Strong caveats are needed regarding fallibility of
genotyping
- Results can change based on genotype
calling algorithm
- QC filters and consistency of results after
applying them must be described
NCI-NHGRI Working Group Criteria for
Positive Replication
• Sufficient sample size to distinguish proposed
effect from no effect convincingly
• Same or very similar trait (extension to related
trait may increase confidence in finding, such as
consistent finding for both dichotomized obesity
and continuous BMI)
• Same or very similar population (extension to
other populations may also increase confidence in
finding, such as consistent association in
populations of European, Asian, or even recent
African ancestry)
Chanock S, Manolio T, et al, Nature 2007; 447:655-660.
NCI-NHGRI Working Group Criteria for
Positive Replication (continued)
• Same inheritance model (dominant, codominant, recessive), though not necessarily
same analytic method
• Same gene, same SNP (or SNP in complete LD
with prior SNP, r2 = 1), same direction as
original finding
• Highly significant association
• N.B.: Initial study must adequately describe
these parameters
Chanock S, Manolio T, et al, Nature 2007; 447:655-660.
Criteria for True Non-Replication or
“Meaningful Negativity”
• Same as for positive replication (same trait,
same gene, same SNP, same direction, same
genetic model)
• Must be identical trait and population to claim
non-replication
• Powered to appropriate effect size (account for
“winner’s curse”)
Chanock S, Manolio T, et al, Nature 2007; 447:655-660.
Replication in Samples of Different
Ancestral Origin
• Shorter LD blocks may explain failure of
associations identified in European ancestry
populations to replicate in recent African
ancestry samples
• Shorter LD may permit better localization of risk
variants
• Allele frequency differences may also explain
lack of replication
Skol et al, Nat Genet 2006; 38:209-13.
Narrowing the Association Region…
Larson, G. The Complete Far Side. 2003.
Flow of Investigation: From Genome-Wide Association to
Clinical Translation
Initial Genome-Wide
Association (GWA) Studies
Replication/Fine Mapping
Sequencing/Genotyping
Functional Studies
Translational Studies
Flow of Investigation: From Genome-Wide Association to
Clinical Translation
Initial Genome-Wide
Association (GWA) Studies
Replication/Fine Mapping
Sequencing/Genotyping
Functional Studies
Translational Studies
Flow of Investigation: From Genome-Wide Association to
Clinical Translation
Initial Genome-Wide
Association (GWA) Studies
Replication/Fine Mapping
Sequencing/Genotyping
Functional Studies
Translational Studies
Flow of Investigation: From Genome-Wide Association to
Clinical Translation
Initial Genome-Wide
Association (GWA) Studies
Replication/Fine Mapping
Sequencing/Genotyping
Functional Studies
Translational Studies
Linkage of Chromosome 13q12-13 and MI
in 296 Icelandic Families
Helgadottir et al, Nat Genet 2004; 36:233-239.
Fine Mapping of 1-LOD Drop Region
Containing ALOX5AP
Most significant in males
Most significant in females
• Single marker
-- Two-marker haplotype
-- Three-marker haplotype
-- Four-marker haplotype
-- Five-marker haplotype
Helgadottir et al, Nat Genet 2004; 36:233-239.
Sequencing of ALOX5AP Gene
Helgadottir et al, Nat Genet 2004; 36:233-239.
Sequencing for GWA: 1,000 Genomes Project
http://www.1000genomes.org/
LD (r2) among 8 TCF7L2 SNPs in Icelandic
and West African Population Samples
2906
rs7752906
rs1569699
rs7756992
rs9350271
rs9356744
--
9699 6992 0271
6744 8222 0833
1514
0.55 0.66 0.56 0.56 0.67 0.66 0.65
--
0.87 0.99 0.98 0.85 0.83 0.83
--
0.86 0.86 0.99 0.97 0.96
--
1.00 0.86 0.85 0.84
--
rs9368222
rs10440833
rs6931514
Steinthorsdottir et al, Nat Genet 2007; 39:770-75.
0.87 0.86 0.85
--
0.98 0.97
--
0.99
--
LD (r2) among 8 TCF7L2 SNPs in Icelandic
and West African Population Samples
2906
9699 6992 0271
6744 8222 0833
rs7752906
--
rs1569699
0.16
rs7756992
0.32 0.61
rs9350271
0.13 0.72 0.67
rs9356744
0.14 0.72 0.67 0.99
rs9368222
0.12 0.12 0.14 0.10 0.10
0.55 0.66 0.56 0.56 0.67 0.66 0.65
--
0.87 0.99 0.98 0.85 0.83 0.83
--
0.86 0.86 0.99 0.97 0.96
--
1.00 0.86 0.85 0.84
--
0.87 0.86 0.85
--
rs10440833 0.07 0.07 0.08 0.04 0.04 0.86
rs6931514
> 0.9
0.98 0.97
--
0.08 0.09 0.10 0.05 0.06 0.76 0.87
0.80 – 0.89
1514
0.60-0.79
0.30-0.59
Steinthorsdottir et al, Nat Genet 2007; 39:770-75.
< 0.30
0.99
--
LD (r2) among 8 TCF7L2 SNPs in Icelandic
and West African Population Samples
2906
9699 6992 0271
6744 8222 0833
rs7752906
--
rs1569699
0.16
rs7756992
0.32 0.61
rs9350271
0.13 0.72 0.67
rs9356744
0.14 0.72 0.67 0.99
rs9368222
0.12 0.12 0.14 0.10 0.10
0.55 0.66 0.56 0.56 0.67 0.66 0.65
--
0.87 0.99 0.98 0.85 0.83 0.83
--
0.86 0.86 0.99 0.97 0.96
--
1.00 0.86 0.85 0.84
--
0.87 0.86 0.85
--
rs10440833 0.07 0.07 0.08 0.04 0.04 0.86
rs6931514
> 0.9
0.98 0.97
--
0.08 0.09 0.10 0.05 0.06 0.76 0.87
0.80 – 0.89
1514
0.60-0.79
0.30-0.59
Steinthorsdottir et al, Nat Genet 2007; 39:770-75.
< 0.30
0.99
--
P-values for 8q24 SNPs Most Strongly
Associated with Prostate Cancer
Haiman et al, Nat Genet 2007; 39:638-44.
CDKN2A/B and Coronary Disease
McPherson et al, Science 2007; 316:1488-91.
CDKN2A/B and Type 2 Diabetes
Zeggini E et al, Science 2007; 316:1336-41.
CDKN2A/B and Aortic and Intracranial Aneurysm
Helgadottir et al, Nat Genet 2008; 40:217-224.
9p21 Region Associated with CAD
Genes (+) strand
Genes (–) strand
Conserved regions
WTCCC, Nature 2007; 447:661-78.
Functional Studies: Correlation of SNPs with
Logical Intermediate Phenotypes
• rs7756992 on 6p22.3 strongly associated with
type 2 diabetes (OR 1.20, p < 8 x 10-8), resides
in intron 5 of CDK5 regulatory subunit
associated protein 1-like1 (CDKAL1)
• rs13244434 on 8q24 also associated with
T2DM: OR 1.15, p < 4 x 10-6
• Nonsynonymous arginine to tryptophan change
in last exon of solute carrier family 30 (zinc
transporter), member 8 (SLC30A8)
• Specific to pancreas and expressed in beta cells
Steinthorsdottir et al, Nat Genet 2007; 39:770-75.
Relationship of Diabetes-Associated
SNPs with Insulin Secretion
(CDKAL1)
(SLC30A8)
Steinthorsdottir et al, Nat Genet 2007; 39:770-75.
LTB4 Production of Ionomycin-Stimulated
Neutrophils in MI Cases and Controls
Helgadottir et al, Nat Genet 2004; 36:233-239.
Co-Localization of Gene Product with
Histopathologic Changes
• CFH in retina and drusen
(macular degeneration)
• GAB2 in dystrophic neurons
(Alzheimers disease)
Complement Deposition in Affected Retina
Complement
deposition in Bruch’s
membrane (thin
black arrows)
Deposition also in
choroidal artery
(double headed
arrow, pt C) and
choroidal vein (white
arrow, both)
Deposition in drusen
(*) as well as Bruch’s
membrane and
choroidal vein
Klein et al, Science 2005; 308:385-89.
Gab2 Colocalizes with Dystrophic Neurons
in LOAD Brain
Dystrophic neuron (arrow)
and neurites (arrowheads)
Tangle-containing neuron
(arrow), dystrophic neurites
(arrowheads)
Tangle-bearing neuron
(open arrow), immunoreactive structures
resembling dendrites
(arrowheads)
Gab2 immunoreactive cell
with flame-shaped tanglelike inclusion
Reiman et al, Neuron 2007; 54:713-20.
Conservation and Expression Studies:
Asthma and ORMDL3
Moffatt et al, Nature 2007; 448:470-73.
Conservation and Expression Studies:
Asthma and ORMDL3
Moffatt et al, Nature 2007; 448:470-73.
Knockdown and Knockout Studies
• Knockdown of ATG16L1
– Associated with Crohn’s disease
– Reduces phagocytosis of S. typhimurium in
HeLa cells
• Knockdown of GAB2
– Associated with Azheimer’s disease
– Increases tau phosphorylation
• Knockout of MLXIPL
– Associated with lower triglyceride levels
– Knockout shows lower triglyceride levels
– Transgenic (knockin) shows higher levels
Genome-Wide Associations in Crohn’s Disease
CARD15
IL23R
2q37.1; rs2241880
ATG16L1 exon 8 A197T
Rioux et al, Nat Genet 2007; 39:596-604.
Identification of IBD1 Locus by Family-Based
Linkage and Fine Mapping
Hugot et al, Nature 2001; 411:599-603.
Sequencing of IBD1 Region for Identification of
Potentially Causative SNPs
*
Hugot et al, Nature 2001; 411:599-603.
CARD15 Sequence Variants and NF-κB Activation
CARD1
26
NACHT
CARD2
124 127
220
LRRs
273
617
1037
733
A602T
A602V
R684W
R702W
R703C
R713C
A725G
E778K
R790Q
V793M
E843K
M863V
G908R
V955I
V972I
G978E
1007fs
558DLG
A432V
E441K
P268S
N289S
D291N
T294S
R311W
L348V
H352R
N414S
S431L
1040
R138Q
W157R
R235C
1
100%
Basal
NF-kB
Activation 10%
(vs WT)
1%
SNP8
PGN
100%
induced
NF-kB
Activation 10%
(vs WT)
1%
Chamaillard et al, PNAS 2003; 100:3455-3460.
SNP12
SNP13
Gene Expression in Crohn’s Disease
• rs2241880 associated at p < 10-8
• Nonsynonymous amino acid change in exon 8
of autophagy-related 16-like 1 (ATG16L1)
• Autophagy is biologic process involved in
protein degradation, antigen processing,
absorption of cellular organelles, initiation and
regulation of inflammatory response
Rioux et al, Nat Genet 2007; 39:596-604.
Expression of ATG16L1 in Human
Primary Immune Cells
Rioux et al, Nat Genet 2007; 39:596-604.
Knockdown of Endogenous ATG16L1 by
siRNA 2 in HeLa Cells
Decreases transcripts
Prevents encapsulation
of S. typhimurium
into autophagosomes
89%↓
Rioux et al, Nat Genet 2007; 39:596-604.
89%↓
siRNA Knockdown of GAB2 Increases Tau
Phosphylation without Increasing Total Tau
Reiman et al, Neuron 2007; 54:713-20.
Increased Triglyceride Levels in Mice
Expressing Transgenes of SREBP (Knockin)
Horton et al, J Clin Invest 1998; 101:2331-9.
Post GWA: Finding (Putative) Causal Variants
• Narrowing region with fine mapping,
sequencing
• Structure of association region: nearby genes,
conservation
• Association with levels of protein product
• Co-localization with histopathologic changes
• Association with expression levels
• Knockdown, knockout