1. Genome-Wide Association (GWA) Studies National Human Genome Research Institute National Institutes of Health U.S. Department of Health and Human Services National Institutes of Health National Human Genome Research Institute Teri A. Manolio, M.D., Ph.D. Senior Advisor to the Director, NHGRI, for Population Genomics Director, Office of Population Genomics

2. There’s a revolution going on… Williard, AM. Spirit of ‘76

3. There’s a revolution going on… Williard, AM. Spirit of ‘76 Technologic advances now allow us to measure hundreds of thousands of variable points across human genome –Relatively low cost –Relatively little DNA Can be applied to unrelated individuals studied over years or decades Can identify multitude of subtle genetic effects increasing risk of “complex” disease

4. What is a GWA Study? Method for interrogating all 10 million variable points across human genome Variation inherited in groups, or blocks, so not all 10 million points have to be tested Blocks are shorter (so need to test more points) the less closely people are related Technology now allows studies in unrelated persons, assuming ~10,000 base pair lengths in common (300,000 - 500,000 markers)

5. Christensen and Murray, N Engl J Med 2007; 356:1094-1097. Mapping the Relationships Among SNPs

6. Christensen and Murray, N Engl J Med 2007; 356:1094-1097. One SNP May Serve as Proxy for Many

7. Progress in Genotyping Technology 11010 2 10 3 10 4 10 5 10 6 Nb of SNPs Cost per genotype (Cents, USD) 10 1 10 2 ABI TaqMan ABI SNPlex Illumina Golden Gate Illumina Infinium/Sentrix Affymetrix 100K/500K Perlegen Affymetrix MegAllele 20012005 Affymetrix 10K Courtesy S. Chanock, NCI

8. Affymetrix 500K Illumina 317K Illumina 550K Illumina 650Y Continued Progress in Genotyping Technology Courtesy S. Gabriel, Broad/MIT July 2005Oct 2006 Cost per person (USD)

9. Courtesy, K. Doheny, Johns Hopkins Intensity Data for Three Combinations of Two Alleles

10. GWA Genotyping Data, Chromosome 22, Parkinson’s Study Study ID Case/ Control Status rs5747620rs2236639 Allele 1Allele 2Allele 1Allele 2 14CaseTTGG 20CaseTCGG 41CaseTCGG 412ControlTCGG 592ControlCCGG 665ControlTCAG http://ccr.coriell.org/ninds/

11. Association of rs2236639 Alleles with Development of Parkinson Disease (Made Up!) Development of Disease Variant Allele (A) Develop Disease Do Not Develop Disease Total Present10 70 80 Absent40880 920 Total509501,000 Relative Risk = Risk in Exposed = 10/80 = 12.5% =2.9 Risk in Unexposed40/9204.3%

12. Measures of Association: The Odds Ratio Odds are related to probability: odds = p/(1-p) –If probability of horse winning race is 50%, odds are 1/1 –If probability of horse winning race is 25%, odds are 1/3 for win or 3 to 1 against win If probability of exposed person getting disease is 25%, odds = p/(1-p) = 25/75 = 1/3 When don’t have denominators for risk estimates, can calculate odds ratio = cross- product ratio (“ad/bc”); computationally easier If disease is rare, odds ratio approximates relative risk but always overestimates effect

13. Association of rsxxxx3207 Alleles with Occurrence of Myocardial Infarction Presence of Disease Variant Allele (G) PresentAbsentTotal Present 8133,061? Absent 7943,667? Total1,5076,728?? OR = Odds in Exposed = 813 / 3,061 = 813 x 3,667 =1.23 Odds in Unexposed794 / 3,667794 x 3,061 Helgadottir et al, Sciencexpress 3 May 2007.

14. Association of rsxxxx3207 Alleles with Occurrence of Myocardial Infarction Presence of Disease Variant Allele (G) PresentAbsentTotal Present 8133,061? Absent 7943,667? Total1,5076,728?? OR = Odds in Exposed = 813 / 3,061 = 813 x 3,667 =1.23 Odds in Unexposed794 / 3,667794 x 3,061 Helgadottir et al, Sciencexpress 3 May 2007.

15. This is a tsunami of data… Hokusai, K. The Great Wave

16. This is a tsunami of data… Hokusai, K. The Great Wave New approaches needed for accessing, manipulating, visualizing Requires entirely new perspective Recognize potential for differences to be observed by chance alone

17. A Few Epidemiologic Definitions P-Value Probability of finding result as extreme or more extreme by chance alone (0.0001 or 1 x 10 -4 ) Type I error (α) Probability of finding a difference when in fact none exists (also called “spurious association”) Type II error (β) Probability of failing to find a difference when in fact one does exist Power Probability of finding a difference when one in fact does exist, = 1 - β Effect Size Magnitude of risk associated with variant Sample Size P-value Effect size Allele frequency Variability of measure

18. Klein et al, Science 2005; 308:385-389. P Values of GWA Scan for Age-Related Macular Degeneration

19. http://www.broad.mit.edu/diabetes/scandinavs/type2.html Genome-Wide Scan for Type 2 Diabetes in a Scandinavian Cohort

20. P-values for 8q24 SNPs Most Strongly Associated with Prostate Cancer Haiman et al, Nat Genet 2007; 39:638-44.

21. P-values for Chromosome 11 SNPs Most Strongly Associated with Diabetes Scott et al, Sciencexpress 26 April 2007.

22. Of 600 Gene-Disease Associations, Only 6 Significant in > 75% of Identified Studies Disease/TraitGenePolymorphismFrequency DVTF5Arg506Gln0.015 Graves’ DiseaseCTLA4Thr17Ala0.62 Type 1 DMINS5’ VNTR0.67 HIV/AIDSCCR532 bp Ins/Del0.05-0.07 Alzheimer’sAPOEEpsilon 2/3/40.16-0.24 Creutzfeldt- Jakob Disease PRNPMet129Val0.37 Hirschhorn J et al, Genet Med 2002; 4:45-61.

23. Aspects of GWA Studies that Make Data Sharing Crucial Expensive, generate many “false positives” Replication held as sine qua non of valid association Large sample sizes and multiple studies needed to replicate findings Massive data sets, analysis requires huge and specialized effort Better analytic methods needed Once genome is measured can be related to just about anything

24. Larson, G. The Complete Far Side. 2003.

25. The revolution is here… Williard, AM. Spirit of ‘76

26. The revolution is here… Williard, AM. Spirit of ‘76 Extensive characterization of individual person’s genome now feasible Can be applied to unrelated individuals Many existing studies have carefully characterized thousands of persons New approaches to manipulating and interpreting data needed Responsible and widespread data sharing key to fully exploring GWA datasets Collaboration for replication and functional determination is crucial

28. Measures of Public Health Impact: Population Attributable Risk Measures the proportion of disease that would be eliminated if particular causal factor were eliminated Directly related to prevalence of risk factor and risk it conveys Almost always over-estimates proportion attributable to risk factor PAR = (Prevalence of exposure) x (Relative risk - 1) 1 + (Prevalence of exposure) x (Relative risk - 1)

29. GWA Genotyping Data, Chromosome 22, Parkinson’s Study Study ID Case/ Control Status rs5747620rs2236639 Allele 1Allele 2Allele 1Allele 2 14CaseTTGG 20CaseTCGG 41CaseTCGG 412ControlTCGG 592ControlCCGG 665ControlTCAG http://ccr.coriell.org/ninds/

30. GWA Genotyping Data, Chromosome 22, One Person 412 1 T C G G G G A A A A C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T …

31. Chromosome 22, One Person, Continued …G A A A A C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T TC C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T …

32. Chromosome 22, One Person, Continued… …A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T TC C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T T T C C A A A A T C A G C C T C T T T C T C T T A G C C C A A A A T C A G C C T C T T T C T C T T A G C C A G A A T C C A A A A T C A G C C T C …