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National Human Genome Research Institute. Genetics for Epidemiologists Lecture 7: Replication and Functional Studies. U.S. Department of Health and Human Services National Institutes of Health National Human Genome Research Institute. National Institutes of Health.

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genetics for epidemiologists lecture 7 replication and functional studies

National Human Genome Research Institute

Genetics for EpidemiologistsLecture 7: Replication and Functional Studies

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

topics to be covered
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
need for consensus on what constitutes replication circa november 2006
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
Case in Point: DTNBP1 and Schizophrenia
  • First identified as putative schizophrenia-susceptibility 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
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
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
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
Case in Point: Odds Ratio for Stroke Associated with PDE4D in Three Studies

Rosand et al, Nat Genet 2006; 38:1091-1092.

slide10

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, 12 Jan 2007

Science, 14 Apr 2006

slide12

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 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 27;3(4):e61.

slide13

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
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
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
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
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
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
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; 447:655-660.

criteria for true non replication or meaningful negativity
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
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
slide23

Narrowing the Association Region…

Larson, G. The Complete Far Side. 2003.

slide24

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

slide25

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

slide26

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

slide27

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
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
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
Sequencing of ALOX5AP Gene

Helgadottir et al, Nat Genet 2004; 36:233-239.

ld r 2 among 8 tcf7l2 snps in icelandic and west african population samples
LD (r2) among 8 TCF7L2 SNPs in Icelandic and West African Population Samples

Steinthorsdottir et al, Nat Genet 2007; 39:770-75.

ld r 2 among 8 tcf7l2 snps in icelandic and west african population samples1
LD (r2) among 8 TCF7L2 SNPs in Icelandic and West African Population Samples

Steinthorsdottir et al, Nat Genet 2007; 39:770-75.

ld r 2 among 8 tcf7l2 snps in icelandic and west african population samples2
LD (r2) among 8 TCF7L2 SNPs in Icelandic and West African Population Samples

Steinthorsdottir et al, Nat Genet 2007; 39:770-75.

slide35

P-values for 8q24 SNPs Most Strongly Associated with Prostate Cancer

Haiman et al, Nat Genet 2007; 39:638-44.

slide36

CDKN2A/B and Coronary Disease

McPherson et al, Science 2007; 316:1488-91.

slide37

CDKN2A/B and Type 2 Diabetes

Zeggini E et al, Science 2007; 316:1336-41.

slide38

CDKN2A/B and Aortic and Intracranial Aneurysm

Helgadottir et al, Nat Genet 2008; 40:217-224.

slide39

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
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.

slide41

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
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
Co-Localization of Gene Product with Histopathologic Changes
  • CFH in retina and drusen

(macular degeneration)

  • GAB2 in dystrophic neurons

(Alzheimers disease)

slide44

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.

slide45

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; 54:713-20.

conservation and expression studies asthma and ormdl3
Conservation and Expression Studies: Asthma and ORMDL3

Moffatt et al, Nature 2007; 448:470-73.

conservation and expression studies asthma and ormdl31
Conservation and Expression Studies: Asthma and ORMDL3

Moffatt et al, Nature 2007; 448:470-73.

knockdown and knockout studies
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
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
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
Sequencing of IBD1 Region for Identification of Potentially Causative SNPs

*

Hugot et al, Nature 2001; 411:599-603.

slide52

CARD15 Sequence Variants and NF-κB Activation

NACHT

CARD1

CARD2

LRRs

1037

26

124

127

220

273

617

733

1

1040

A432V

P268S

N289S

N414S

V972I

R235C

D291N

A602V

V955I

R713C

R703C

E441K

H352R

R790Q

R138Q

A725G

G908R

W157R

T294S

R311W

R684W

L348V

S431L

A602T

R702W

E843K

G978E

1007fs

M863V

558DLG

E778K

V793M

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; 100:3455-3460.

gene expression in crohn s disease
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.

slide54

Expression of ATG16L1 in Human Primary Immune Cells

Rioux et al, Nat Genet 2007; 39:596-604.

slide55

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; 39:596-604.

slide56

siRNA Knockdown of GAB2 Increases Tau Phosphylation without Increasing Total Tau

Reiman et al, Neuron 2007; 54:713-20.

slide57

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
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