Genetics for Epidemiologists Lecture 7: Replication and Functional Studies

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Genetics for EpidemiologistsLecture 7
Replication and Functional Studies
National Human Genome Research Institute
U.S. Department of Health and Human
Services National Institutes of Health National
Human Genome Research Institute
National Institutes of Health
Teri A. Manolio, M.D., Ph.D.Director, Office of
Population Genomics and Senior Advisor to the
Director, NHGRI, for Population Genomics
U.S. Department of Health and Human Services
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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

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Chanock S, Manolio T, et al, Nature 2007
447655-660.
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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

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Case in Point DTNBP1 and Schizophrenia

• First identified as putative schizophrenia-suscept
  ibility 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 79903-909.
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Phylogenetic Tree of Five Common Haplotypes of
DTNBP1
Mutsuddi et al, Am J Hum Genet 2006 79903-909.
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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 79903-909.
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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

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Case in Point Odds Ratio for Stroke Associated
with PDE4D in Three Studies
Rosand et al, Nat Genet 2006 381091-1092.
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• 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
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Lyon HN et al, PLoS Genet 2007 Apr 273(4)e61.
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• Nine large cohorts from eight populations across
  multiple ethnicities
• Family-based, population-based, case-control
  designs
• Association at p lt 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
  family-based (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 273(4)e61.
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• 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 lt 10-9), four others 0.003 lt p lt
  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 82849-58.
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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

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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?
• Dont change standards for definitiveness
• Dont 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

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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

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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

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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
447655-660.
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NCI-NHGRI Working Group Criteria for Positive
Replication (continued)

• Same inheritance model (dominant, co-dominant,
  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
447655-660.
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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
  winners curse)

Chanock S, Manolio T, et al, Nature 2007
447655-660.
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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

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Skol et al, Nat Genet 2006 38209-13.
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Narrowing the Association Region
Larson, G. The Complete Far Side. 2003.
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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
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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
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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
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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
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Linkage of Chromosome 13q12-13 and MI in 296
Icelandic Families
Helgadottir et al, Nat Genet 2004 36233-239.
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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 36233-239.
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Sequencing of ALOX5AP Gene
Helgadottir et al, Nat Genet 2004 36233-239.
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Sequencing for GWA 1,000 Genomes Project
http//www.1000genomes.org/
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LD (r2) among 8 TCF7L2 SNPs in Icelandic and West
African Population Samples
2906 9699 6992 0271 6744 8222 0833 1514
rs7752906 -- 0.55 0.66 0.56 0.56 0.67 0.66 0.65
rs1569699 -- 0.87 0.99 0.98 0.85 0.83 0.83
rs7756992 -- 0.86 0.86 0.99 0.97 0.96
rs9350271 -- 1.00 0.86 0.85 0.84
rs9356744 -- 0.87 0.86 0.85
rs9368222 -- 0.98 0.97
rs10440833 -- 0.99
rs6931514 --
Steinthorsdottir et al, Nat Genet 2007 39770-75.
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LD (r2) among 8 TCF7L2 SNPs in Icelandic and West
African Population Samples
2906 9699 6992 0271 6744 8222 0833 1514
rs7752906 -- 0.55 0.66 0.56 0.56 0.67 0.66 0.65
rs1569699 0.16 -- 0.87 0.99 0.98 0.85 0.83 0.83
rs7756992 0.32 0.61 -- 0.86 0.86 0.99 0.97 0.96
rs9350271 0.13 0.72 0.67 -- 1.00 0.86 0.85 0.84
rs9356744 0.14 0.72 0.67 0.99 -- 0.87 0.86 0.85
rs9368222 0.12 0.12 0.14 0.10 0.10 -- 0.98 0.97
rs10440833 0.07 0.07 0.08 0.04 0.04 0.86 -- 0.99
rs6931514 0.08 0.09 0.10 0.05 0.06 0.76 0.87 --
gt 0.9 0.80 0.89 0.60-0.79 0.30-0.59 lt 0.30
Steinthorsdottir et al, Nat Genet 2007 39770-75.
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LD (r2) among 8 TCF7L2 SNPs in Icelandic and West
African Population Samples
2906 9699 6992 0271 6744 8222 0833 1514
rs7752906 -- 0.55 0.66 0.56 0.56 0.67 0.66 0.65
rs1569699 0.16 -- 0.87 0.99 0.98 0.85 0.83 0.83
rs7756992 0.32 0.61 -- 0.86 0.86 0.99 0.97 0.96
rs9350271 0.13 0.72 0.67 -- 1.00 0.86 0.85 0.84
rs9356744 0.14 0.72 0.67 0.99 -- 0.87 0.86 0.85
rs9368222 0.12 0.12 0.14 0.10 0.10 -- 0.98 0.97
rs10440833 0.07 0.07 0.08 0.04 0.04 0.86 -- 0.99
rs6931514 0.08 0.09 0.10 0.05 0.06 0.76 0.87 --
gt 0.9 0.80 0.89 0.60-0.79 0.30-0.59 lt 0.30
Steinthorsdottir et al, Nat Genet 2007 39770-75.
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P-values for 8q24 SNPs Most Strongly Associated
with Prostate Cancer
Haiman et al, Nat Genet 2007 39638-44.
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CDKN2A/B and Coronary Disease
McPherson et al, Science 2007 3161488-91.
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CDKN2A/B and Type 2 Diabetes
Zeggini E et al, Science 2007 3161336-41.
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CDKN2A/B and Aortic and Intracranial Aneurysm
Helgadottir et al, Nat Genet 2008 40217-224.
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9p21 Region Associated with CAD
Genes () strand Genes () strand Conserved
regions
WTCCC, Nature 2007 447661-78.
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Functional Studies Correlation of SNPs with
Logical Intermediate Phenotypes

• rs7756992 on 6p22.3 strongly associated with type
  2 diabetes (OR 1.20, p lt 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 lt 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 39770-75.
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Relationship of Diabetes-Associated SNPs with
Insulin Secretion
(CDKAL1)
(SLC30A8)
Steinthorsdottir et al, Nat Genet 2007 39770-75.
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LTB4 Production of Ionomycin-Stimulated
Neutrophils in MI Cases and Controls
Helgadottir et al, Nat Genet 2004 36233-239.
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Co-Localization of Gene Product with
Histopathologic Changes

• CFH in retina and drusen
• (macular degeneration)
• GAB2 in dystrophic neurons
• (Alzheimers disease)

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Complement Deposition in Affected Retina
Complement deposition in Bruchs 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 Bruchs
membrane and choroidal vein
Klein et al, Science 2005 308385-89.
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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),
immuno-reactive structures resembling dendrites
(arrowheads)
Gab2 immunoreactive cell with flame-shaped
tangle-like inclusion
Reiman et al, Neuron 2007 54713-20.
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Conservation and Expression Studies Asthma and
ORMDL3
Moffatt et al, Nature 2007 448470-73.
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Conservation and Expression Studies Asthma and
ORMDL3
PSMD3 GSDM1 ZPBP2 IKZF3 GSDML ORMDL3
Moffatt et al, Nature 2007 448470-73.
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Conservation and Expression Studies Asthma and
ORMDL3
Moffatt et al, Nature 2007 448470-73.
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Conservation and Expression Studies Asthma and
ORMDL3
Moffatt et al, Nature 2007 448470-73.
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Knockdown and Knockout Studies

• Knockdown of ATG16L1
• Associated with Crohns disease
• Reduces phagocytosis of S. typhimurium in HeLa
  cells
• Knockdown of GAB2
• Associated with Azheimers disease
• Increases tau phosphorylation
• Knockout of MLXIPL
• Associated with lower triglyceride levels
• Knockout shows lower triglyceride levels
• Transgenic (knockin) shows higher levels

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Genome-Wide Associations in Crohns Disease
CARD15
IL23R
2q37.1 rs2241880 ATG16L1 exon 8 A197T
Rioux et al, Nat Genet 2007 39596-604.
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Identification of IBD1 Locus by Family-Based
Linkage and Fine Mapping
Hugot et al, Nature 2001 411599-603.
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Sequencing of IBD1 Region for Identification of
Potentially Causative SNPs

Hugot et al, Nature 2001 411599-603.
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CARD15 Sequence Variants and NF-?B Activation
NACHT
CARD2
LRRs
CARD1
1037
127
220
273
617
733
26
124
1040
1
A432V
P268S
R713C
R703C
N414S
R235C
V972I
V955I
N289S
D291N
A602V
W157R
R138Q
A725G
R684W
E441K
S431L
H352R
R311W
T294S
E843K
G978E
R702W
G908R
1007fs
L348V
A602T
R790Q
M863V
558DLG
V793M
E778K
Basal NF-kB Activation (vs WT)
100
10
1
SNP13
SNP8
SNP12
PGN induced NF-kB Activation (vs WT)
100

10
1
Chamaillard et al, PNAS 2003 1003455-3460.
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Gene Expression in Crohns Disease

• rs2241880 associated at p lt 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 39596-604.
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Expression of ATG16L1 in Human Primary Immune
Cells
Rioux et al, Nat Genet 2007 39596-604.
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Knockdown of Endogenous ATG16L1 by siRNA 2 in
HeLa Cells
Prevents encapsulation of S. typhimurium into
autophagosomes
Decreases transcripts
89?
89?
Rioux et al, Nat Genet 2007 39596-604.
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siRNA Knockdown of GAB2 Increases Tau
Phosphylation without Increasing Total Tau
Reiman et al, Neuron 2007 54713-20.
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Increased Triglyceride Levels in Mice Expressing
Transgenes of SREBP (Knockin)
Horton et al, J Clin Invest 1998 1012331-9.
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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

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