1. Clinical Applications of Whole Genome/Whole Exome Sequencing Robert L. Nussbaum, MD, FACMG Division of Genomic Medicine, UCSF AMA – November 11, 2012

2. Chair of Genomic Medicine Advisory Board of Complete Genomics, Inc. Conflict of Interest Disclosures

3. Mythical Scenario A newborn blood spot undergoes whole genome sequencing. It is analyzed for Personal risk for a Mendelian disorder (BRCA1) Pharmacogenetic variants that predict efficacy, side-effects, adverse reactions (CYP2C19 and clopidogrel) Risk for carrying mutations that future children at risk (Ta- Sachs carrier) Tissue-type and Blood type (HLA, ABO) Variants (rare and common) that increase risk for common disorders (CFH and macular degeneration) All the results are recorded in an EMR, communicated to his health care providers, and used to guide health care over the lifespan

4. Outline Whole Genome and Whole Exome Sequencing Factors Impeding Implementation of WGS/WES sequencing – Limits of the Technology – Limits of Knowledge – Limits of Genetic Determinism

5. Evaluating A Genetic Test  Patient Sample  Right result from the right patient  Test has predictive value for patient care  Results have value for the patient and doctor  There is value to society in generalizing the testing Analytical Validity Clinical Validity Clinical Utility (“Actionability”) Social Utility

6. Whole Genome Sequencing (WGS) CLIA ’88 Test Performance Metrics Reportable Range: Portion of the genome from which sequence information can be reliably derived from WGS = ~96.5% Reference Range: Homopolymers, di- and tri-nucleotide repeats, microsatellites Deletions and duplications ~ 100-500 bp Single nucleotide variants sitting at the end of homopolymers Are outside the typical Reference Range of WGS

7. Whole Exome Sequencing (WES) by Exon Capture Elute Sequence

8. ~3-5% of Exons, Promoters, Untranslated Regions, and the Bulk of Intron Sequences are not Included in Exome Sequencing Start Stop What Do You Miss With Whole Exome Sequencing? 5’-UTR 3’-UTR

9. Why Do WES Rather Than WGS? Because you only sequence ~2% of the genome, what you do sequence is covered to tremendous depth You are sequencing the part of the genome we are better at interpreting Current cost of WES is ~$750-$1000 versus $4,000-$10,000 for WGS HOWEVER………

10. How Good are WES and WGS at Identifying Variants? Because of False Positives, neither approach provides stand-alone “clinical grade” sequencing at the present time and Variants need to be confirmed by conventional sequencing Increases the cost tremendously WES for research = $750 WES for Clinical Use = $8,000 -10,000

11. Variants in Whole Genome Sequence

12. “The” Human Genome There is no such thing – there are only Human Genomes There is a “Reference Genome” in databases but it is incomplete Variants are defined as differences from the Reference The more we learn, the more we realize that there are alternative Reference Genomes

13. Evaluating A Genetic Test  Patient Sample  Right result from the right patient  Test has predictive value for patient care  Results have value for the patient and doctor above and beyond current practice  There is value to society in generalizing the testing Analytical Validity Clinical Validity Clinical Utility (“Actionability”) Social Utility

14. Clinical Validity Positive Predictive Value Given a + test, how frequently does the patient have, or how frequently will he develop the disease? (“Penetrance”) Negative Predictive Value Given a – test, how frequently is the patient unaffected and will remain so?

15. The Reason for the Test Matters “Screening” a healthy executive for variants in her DNA that might be of interest Versus “Scanning” a child with a serious disorder for variants in her DNA that might explain the disease and suggest therapy

17. Genome-Wide Association Studies in Eight Common Diseases SNPS in a Region on Chr 9 are associated with CAD at P < 10 -15

18. Palomaki et al. Odds of Developing CAD Depending on 9p21 Genotype

19. 65 year old male No CAD risk factors 40 year old female No CAD risk factors 9p21Genotype 2 Risk 0 Risk Unknown Alleles Alleles 11% 13.2% 9.2% 2% 2.4% 1.7% Risk for Coronary Artery Disease Events over the Next 10 Years Palomaki et al. PPV for 9p21 Genotype for CAD

21. Combine 13 SNP Loci To Generate Genetic Risk Score for CAD Sipatti et al. A multilocus genetic risk score for coronary heart disease: case-control and prospective cohort analyses, The Lancet Volume 376, Issue 9750, Pages 1393-1400 (October 2010)Volume 376, Issue 9750, Pages 1393-1400

22. Fraction of the Population

23. Established Common Breast-Cancer Susceptibility Alleles. Pharoah PD et al. N Engl J Med 2008;358:2796-2803.

24. Pharoah P et al., N Engl J Med 2008; 358:2796-803. 56 of 10M UK women carry 14 low risk alleles (0.00056%) 7 of 10M UK women carry 14 high risk alleles (0.00007%) Distribution of Genetic Risk in the Population: Seven Breast Cancer Risk Alleles (Avg. risk allele freq. = ~0.35) 20,000 of 10M carry BRCA1/2 mutations Assuming a multiplicative model for interaction between these alleles

25. Evaluating A Genetic Test  Patient Sample  Right result from the right patient  Test has predictive value for patient care  Results have value for the patient and doctor above and beyond current practice  There is value to society in generalizing the testing Analytical Validity Clinical Validity Clinical Utility (“Actionability”) Social Utility

26. Clinical Utility of Genetic Testing Explain why a disease occurs Institute preventive measures Anticipate and prevent complications Affect choice of therapy Avoid adverse reactions Determine risk in other family members or in future offspring

27. Clinical Pharmacogenetics Implementation Consortium Gene-Drug Pairs

28. Clinical Validity ✔ Clinical Utility ? CYP2C19 genotype was not associated with modification of the effect of clopidogrel on CVD end points or bleeding…Overall there was no significant association of genotype with cardiovascular events Individuals with 1 or more CYP2C19 alleles associated with lower enzyme activity had lower levels of active clopidogrel metabolites less platelet inhibition lower risk of bleeding Individuals with 1 or more CYP2C19 alleles associated with lower enzyme activity had lower levels of active clopidogrel metabolites less platelet inhibition lower risk of bleeding

29. Actionability: In the Eye of the Beholder

30. What is “Actionable Information”? How does it differ from Clinical Utility? Information with high Clinical Validity Information that allows a medical decision to be made or therapeutic action to be taken (or not). Founded on evidence (A real problem in genetics where diseases are rare) Information that informs an individual and helps him/her make health decisions

31. Berg J. et al. Genetics IN Medicine Volume 13, Number 6, June 2011 “Actionability” Rating

32. Conclusions 1.Genetic Testing is often not straightforward and requires substantial interpretation 2.We do not know how to interpret a lot of genetic information 3.Genetic Testing is not static and what a result means can change over time. WES/WGS only magnify the problems enormously

33. Barriers to the adoption of pharmacogenetic tests in clinical practice Fragmentation of health-care systems that preclude linking a “lifetime” genetic test result with future medical care (exception: the VA) Limited use of electronic medical records vital to linking test results with medication prescribing/dispensing Health-care systems that do not reward the prevention of disease (or adverse drug effects),

34. Barriers to the adoption of pharmacogenetic tests in clinical practice Lack of sufficient awareness about genomics on the part of many clinicians, Little of such testing is done preemptively and therefore the results are not available when the prescribing decision is made. Some of these barriers will persist for many years to come.