# Presentation: 090523

## Genetics for EpidemiologistsLecture 7: Replication and Functional Studies

- National Human Genome Research Institute

- National  Institutes of Health

- U.S. Department of Health and Human Services

**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.

- 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 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

- Mutsuddi et al, *Am J Hum Genet  *2006; 79:903-909.

## Positively Associated Haplotypes Differ in All Six Studies

- Mutsuddi et al, *Am J Hum Genet  *2006; 79:903-909.

- 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

## 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 

*Science, *14 Apr 2006

***Science, *****12 Jan 2007**

- 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

## 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 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.

## Second variant (rs1455832-C) intronic to ROBO1 with age-varying association to BMI over time

- 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

## 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, 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”

- 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.

- Skol et al, *Nat Genet*  2006; 38:209-13.

## Narrowing the Association Region...

**Narrowing the Association Region...**

## Flow of Investigation: From Genome-Wide Association to Clinical Translation

**Sequencing/Genotyping**

**Functional Studies**

**Translational Studies**

**Initial Genome-Wide**
**Association (GWA) Studies**

**Replication/Fine Mapping**

## Flow of Investigation: From Genome-Wide Association to Clinical Translation

**Sequencing/Genotyping**

**Functional Studies**

**Translational Studies**

**Initial Genome-Wide**
**Association (GWA) Studies**

**Replication/Fine Mapping**

## Flow of Investigation: From Genome-Wide Association to Clinical Translation

**Sequencing/Genotyping**

**Functional Studies**

**Translational Studies**

**Initial Genome-Wide**
**Association (GWA) Studies**

**Replication/Fine Mapping**

## Flow of Investigation: From Genome-Wide Association to Clinical Translation

**Sequencing/Genotyping**

**Functional Studies**

**Translational Studies**

**Initial Genome-Wide**
**Association (GWA) Studies**

**Replication/Fine Mapping**

## Linkage of Chromosome 13q12-13 and MI in 296 Icelandic Families

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

**Notes:**
7.6Mb 1-lod drop region contains 40 known genes
Typed 120 microsatellites

## Fine Mapping of 1-LOD Drop Region Containing ALOX5AP

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

- Most significant in males

- Most significant in females

-   Single marker
- -- Two-marker haplotype
- -- Three-marker haplotype

- -- Four-marker haplotype
- -- Five-marker haplotype

**Notes:**
b) Microsatellite association for all individuals with myocardial infarction: single-marker association (black dots) and two-, three-, four- and five-marker haplotype association (black, blue, green and red horizontal lines, respectively). The blue and red arrows indicate the location of the most significant haplotype association across ALOX5AP in males and females, respectively. 

## Sequencing of ALOX5AP Gene

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

**Notes:**
144 SNPs identified in 93 cases and 93 controls, excluded 96 from further analysis due to low MAF or complete correlation with other SNPs
48 SNPs chose for genotyping, only one in coding region (exon 2) and was synonymous
Several haplotypes were significantly associated with the disease at an adjusted significance level of P < 0.05 (Supplementary Table 5 online). We observed the most significant association with a four-SNP haplotype spanning 33 kb, including the first four exons of ALOX5AP (Fig. 1c), with a nominal P value of 0.0000023 and an adjusted P value of 0.005. This haplotype, called HapA, has a haplotype frequency of 15.8% (carrier frequency 29.1%) in affected individuals versus 9.5% (carrier frequency 18.1%) in controls (Table 1). 

## Sequencing for GWA: 1,000 Genomes Project

- http://www.1000genomes.org/

**Notes:**
144 SNPs identified in 93 cases and 93 controls, excluded 96 from further analysis due to low MAF or complete correlation with other SNPs
48 SNPs chose for genotyping, only one in coding region (exon 2) and was synonymous
Several haplotypes were significantly associated with the disease at an adjusted significance level of P < 0.05 (Supplementary Table 5 online). We observed the most significant association with a four-SNP haplotype spanning 33 kb, including the first four exons of ALOX5AP (Fig. 1c), with a nominal P value of 0.0000023 and an adjusted P value of 0.005. This haplotype, called HapA, has a haplotype frequency of 15.8% (carrier frequency 29.1%) in affected individuals versus 9.5% (carrier frequency 18.1%) in controls (Table 1). 

## 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; 39:770-75.

## 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 | -- |

| > 0.9 |  | 0.80 – 0.89 |  | 0.60-0.79 |  | 0.30-0.59 |  | < 0.30 |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

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

## 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 | -- |

| > 0.9 |  | 0.80 – 0.89 |  | 0.60-0.79 |  | 0.30-0.59 |  | < 0.30 |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

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

**Notes:**
Icelandic above diagonal
West African below diagonal

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

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

## CDKN2A/B and Coronary Disease

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

***CDKN2A/B *****and Coronary Disease**

## CDKN2A/B and Type 2 Diabetes

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

***CDKN2A/B *****and Type 2 Diabetes**

## CDKN2A/B and Aortic and Intracranial Aneurysm

***CDKN2A/B *****and Aortic and Intracranial Aneurysm**

## 9p21 Region Associated with CAD

- Genes (+) strand
- Genes (–) strand
- Conserved regions

**9p21 Region Associated with CAD**

**Notes:**
Red bars are finescale recombination rates in cM per Mb
Purple lines are genetic distance in cM
Gray dots are imputed SNPs

## 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

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

- (*CDKAL1*)

- (*SLC30A8*)

**Notes:**
CIR = Corrected insulin response

## LTB4 Production of Ionomycin-Stimulated Neutrophils in MI Cases and Controls

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

**Notes:**
Protein product of ALOX5AP pathway associated with MI and with at-risk haplotype

## 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

- Klein et al, *Science* 2005; 308:385-89.

**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**

## Gab2 Colocalizes with Dystrophic Neurons in LOAD Brain

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

**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**

## 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.

***PSMD3***
***GSDM1***
***ZPBP2***
***IKZF3***
***GSDML***
***ORMDL3***

## 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

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

***CARD15***

***IL23R***

**2q37.1; rs2241880**
***ATG16L1 *****exon 8 A197T**

**Notes:**
Protein product of ALOX5AP pathway associated with MI and with at-risk haplotype

## Identification of IBD1 Locus by Family-Based Linkage and Fine Mapping

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

**Notes:**
Numerous examples of the use of fine mapping strategies are available in the cardiovascular field but I have chosen this one from inflammatory bowel disease because it is from my colleague Gilles Thomas and it nicely demonstrates the subsequent steps of sequencing and functional studies.
An approximately 9-cm linkage peak localized the IBD1 locus to the pericentromeric region of chromosome 1 with a maximum non-parametric LOD score of 3.49, as shown in panel a.  Fine mapping was concentrated on the area between markers D16S541 and D16S2623.  26 additional microsatellite  markers were tested in this region, the location of 9 of which are shown in panel B above.
The third microsatellite from the left, D16S3136, was associated with the disease in 108 trios at p < 0.05, and the association was replicated in another 76 families at p < 0.01, though with a different allele.
This difference in allelic associations at the same locus could be due to chance or could reflect a true association in two sets of families drawn from genetically distinct populations.  This would suggest that D16S3136 is in linkage disequilibrium with the disease-causing variant, though the microsatellites could have diverged after the disease-related variant arose.  We’ll come back to this example.

## Sequencing of IBD1 Region for Identification of Potentially Causative SNPs

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

- *

**Notes:**
To return to the IBD1 example from my colleague, the BAC clone containing the marker with two different alleles associated with the disease was sequenced completely, along with several neighboring clones.  Expressed sequence tags suggested the presence of a candidate gene in this region, which was named “IBD1.” Thirteen SNPs were identified in this region and typed in 235 available families.

## CARD15 Sequence Variants and NF-κB Activation

**1%**

**10%**

**100****%**

**1%**

**10****%**

**100****%**

**Basal **
**NF-********B **
**Activation**
**(vs WT)**

**PGN**
**induced**
**NF-********B**
**Activation**
**(vs WT)**

**SNP8**

**SNP12**

**SNP13**

**W157R**

**R138Q**

**V793M**

**E778K**

**A725G**

**R713C**

**R703C**

**R684W**

**E441K**

**A432V   **

**S431L**

**N414S**

**H352R**

**R311W**

**T294S**

**R235C**

**E843K**

**M863V**

**NACHT**

**CARD2**

**LRRs**

**127**

**220**

**273**

**617**

**733**

**1037**

**1040**

**26**

**124**

**CARD1**

**V972I**

**G978E**

**R702W**

**P268S**

**G908R**

**V955I**

**1007fs**

**1**

**L348V**

**N289S**

**D291N**

**A602T**

**A602V**

**558DLG**

**R790Q**

***CARD15 *****Sequence Variants and NF-****κ****B Activation**

**Chamaillard et al, *****PNAS***** 2003; 100:3455-3460.**

**Notes:**
The candidate IBD1 gene has high expression in leukocytes and encodes a 1,013-aa protein identical to NOD2, an apoptosis regulator (of the CED4/A_AF1 superfamily).  The C-terminal leucine-rich region has binding activity for  bacterial lipopolysaccharides and its deletion stimulates NF-kB activation.  NF-kB is activated by inflammatory stimuli such as cytokines and bacterial or viral products.  Inappropriate activation has been linked to inflammatory events associated with numerous autoimmune and inflammatory diseases, while inhibition of NF-kB has been linked to apoptosis.   
Three independent associations were demonstrated with SNPs 8, 12, and 13; their rare alleles were never found on the same haplotype and associated with different alleles of D16S3136, explaining the original conflicting associations with this marker. The IBD1/NOD2 gene, which was renamed CARD15 due to the two caspase-recruitment domains at its N-terminal, was demonstrated through functional assays to activate the NF-kB pathway in vitro in response to various baterial components.  This provided a functional assay which would permit testing of bacterial peptidoglycan response in cells transfected with CARD15 variants.  
As seen in panel A, SNPs 8 and 12 had impaired basal NF-kB activation compared to wild type, as did several other SNPs carried by Crohn’s disease patients.  SNPs 8, 12, and 13 had impaired induced activation as shown in Panel B. Quantitative parametrization of this response, estimated from the patients' CARD15 genotypes, was associated with several clinical manifestations of Crohn’s disease. 
Prototype I: variants indistinguishable from wildtype (white/white)
Prototype 2: 2-fold reduction in both basal activity and PGN-induced response, indicating a constitutive defect of CARD15 function (gray/pink)
Prototype 3: almost unchanged basal activity (at least 80% residual activity), but a major impairment of the PGN-response (20% that of WT), thus demonstrating a specific defect in PGNsensing caused by alteration in the LRR domain. (black/dark blue)
Cross-hatched=cyan= intermediate 2 and 3
The availability of this functional assay provided strong experimental evidence for a role of these three variants, and the protein produced by CARD15, 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.

## 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

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

**89%****↓**

**Decreases transcripts**

**Prevents encapsulation of *****S. typhimurium***** **
**into autophagosomes**

**89%****↓**

**Notes:**
(b) Endogenous ATG16L1 mRNA knockdown (mean relative abundance   s.d.) by siRNA 2 in HeLa cells within 48 h of transfection, as assessed by real-time quantitative RT-PCR normalized to GAPDH. Compared with control duplex, siRNA 2 yielded an 89% reduction in transcripts. RT-PCR was performed in triplicate; results represent two independent experiments. 
(c) Knockdown of ATG16L1 prevented effective autophagy of S. typhimurium in HeLa cells. Forty-eight hours after cotransfection with control siRNA or duplex 2 and LC3-GFP plasmid, HeLa cells were infected for 1 h with Salmonella typhimurium SL1344, fixed and examined microscopically. Mean percentages (  s.e.m.) of bacteria per cell encapsulated by LC3+ membranes (autophagosomes) are shown. Bacterial counts were pooled from two separate experiments; each counted a minimum of 100 infected cells. Significance was assessed by two-tailed Students t-test, assuming unequal variances. 

## 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

## Slide 61