Genomic approach for drug target discovery and validation

Transcription

1 Genomic approach for drug target discovery and validation Hong-Hee Won, Ph.D. SAIHST, SKKU

2 Well-known example of genetic findings triggering drug development Genetics Mendelian randomisation Drug interventions Randomized Clinical Trials Humans with Normal PCSK9 Humans with Mutant PCSK9 Control Alirocumab LDL 140 mg/dl 28% LDL 100 mg/dl LDL 140 mg/dl 57% LDL 60 mg/dl ~40% Heart Attack TBD Odyssey Outcomes Cohen et al., NEJM 2006 Stein et al, NEJM 2012 Robinson et al, NEJM 2015 Ref: Andy Plump, Sanofi

3 From Bedside to Bench to Bedside in a decade Only ~10 years from genetic evidence in humans to FDA approaval of PCSK9 inhibitors Natarajan and Kathiresan, Cell, 165, 2016

4 PCSK9 inhibitors are expected to achieve annual sales of $11.5 billion by 2021

5 Questions to be addressed LDL cholestrol 을낮추는것이심혈관질환의예방에도움을준다? HDL cholesterol 을높이는것이심혈관질환의예방에도움을준다? LDL cholesterol 을낮추는약물의부작용이있을까? 심혈관질환위험을낮추는약물의효과를예측할수있을까? 지질농도와심혈관질환위험을낮추는새로운약물타겟을찾을수있을까? Do we have genetic evidence?

6 By frequency and effect size / Common complex / Common complex / Common complex

7 Increase in # of GWAS loci as a function of sample size

8 Large-scale meta-analysis consortia Nature Reviews Genetics 14, (2013)

9 GLGC cohort 188,577 individuals 157 genetic loci 10-15% of lipid

10 CARDIoGRAMplusC4D 200,000 CAD cases and controls from 46 genetic loci 10.6% of CAD heritability

11 GWA loci by 2015 Sept at p<5e- 8

12 Challenges in GWAS GWAS is a popular and successful approach, but Common SNPs (MAF>5%) >500 independent strong SNP associations (p<10-8 ) Very small effect sizes (increase in risk by a given causal variant) Less than modest heritability Only few associated with gene expression 90% lie in intergenic or intronic regions Causal gene? Nearest gene? Biological

13 Possible hypotheses for missing heritability Genetic interactions Genes work together Polygenic effects Many (common) variants with small effects Low-frequency and rare variants Large effects Zuk et al, PNAS Zuk et al, PNAS 2014.

14 Heritability of GWAS hits and all GWAS Height 100% SNPs 80% 45% Nat Genet 2010 GCTA 27% Nature 2017 Missing heritability 10% Nature 2010 Total variance Heritability (based on twin or family study) All SNP-heritability (variance explained by all SNPs) Variance explained by GW significant SNPs

15 Common polygenic variation contributes to the risk of SCZ, BD and MI/CAD Int. Schizophrenia Consortium data MIGen WTCCC CAD ISC, Nature 2009 Won et al, PLoS Genetics 2015

16 Vincent van Gogh Starry Night Over the Rhone, Musée d'orsay, Paris

17 Genetic risk and a healthy lifestyle (NEJM ) NYT Reuters ABC news CNS news

18 Study methods (NEJM ) Four cohorts (N=55,685 participants) Three prospective cohorts (N=7814, 21222, 22389) One cross-sectional study (N=4260) Genetic risk score 50 GWAS SNPs from CARDIoGRAM, CARDIoGRAMplusC4D studies Healthy lifestyle (4 factors) No current smoking, no obesity, regular physical activity, and a healthy diet End points Coronary artery events in prospective cohorts Coronary artery calcification in cross-sectional study

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20 Across four studies involving 55,685 participants, genetic and lifestyle factors were independently associated with susceptibility to coronary artery disease.

21 Among participants at high genetic risk, a favorable lifestyle was associated with a nearly 50% lower relative risk of coronary artery disease than was an unfavorable lifestyle.

22 Mendealian randomization approach Tada et al, J Hum Genet 2017

23 Mendelian randomization: HDL, LDL and MI risk Lancet 2012 HDL : 14 common variants LDL : 13 common variants myocardial infarction cases, controls

24 RCTs of CETP inhibitors (HDL increasing drugs) Torcetrapib Darcetrapib Evacetrapib Anacetrapib HDL-C LDL-C TG CHD +72% -25% -9% Fail +35% 0-7% Fail +130% -37% N/A Fail +139% -36% -5% Ongoing Schaefer EJ. Curr Op Lipidol Yamashita S, et al. Curr Op Lipidol 2016.

25 Mendelian randomization: low LDL and high T2D JAMA NEJM Lancet

26 PCSK9 variants

27 LDL-lowering drugs (genetic target) PCSK9 inhibitor (PCSK9) Statins (HMGCR) Ezetimibe (NPC1L1) Variants Lowering LDL-C 10mg/dL Reduced Risk of MI/CHD 19 percent Increased Risk of T2D 12 percent

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30 Possible hypotheses for missing heritability Genetic interactions Genes work together Polygenic effects Many (common) variants with small effects Low-frequency and rare variants Large effects Zuk et al, PNAS Zuk et al, PNAS 2014.

31 Statistical imputation : more variants

32 1000 Genomes based imputation 1000 Genomes HapMap MAF<0.05 MAF> Number of variants (million) 1000 Genomes Reference HapMap Project Reference

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34 Imputation reference

35 Exome chip Designed to include low-freq. coding variants Based on existing genomes and exomes sequenced (N=12,000) Nonsynonymous seen 3+ times across 2+ datasets Splice variants seen 1+ Stop gain or loss seen 1+ GWAS Common variants, etc.

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38 Effects of rare and low-frequency variants on height 458,927 individuals 697 known loci explained 23.3% of height heritability New loci explained additional 4.1% Rare variants give an increase of 1-2 cm per allele Nature 2017 Feb

39 Illumina NovaSeq

40 Precision Medicine Initiative (1M genomes) 2015

41 100K Genomes Projects Announced in 2012 (started in 2015) Whole genome sequencing of 100K by ,540 genomes sequenced (Nov 6, 2017) 100+ rare disease and 7 common cancers When the project ends Genomics as part of routine care

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43 32 projects Nearly 98,000 genomes have been sequenced Mean depth = 38x (Last modified on )

44 1000 Genomes results: Lots of rare variants that are private to a population Variants found to be rare (<0.5%) within the global sample but common within a population F ST -based population branch statistic (PBS) Common variants Skin pigmentation Rare variants Lactose tolerance Eye color A Auton et al. Nature 526, (2015) doi: /nature15393

45 Population specific rare mutations associated with disease TBC1D4 Arg684Ter 17% allele frequency in Inuit ~0% in other populations Homozygous carriers at OR=10.3 HNF1A E508K 2.1% in Latino cases ~0% in other populations Heterozygous carriers at OR=5.5

46 Estimated progress for research in PMI

47 Human gene knockouts

48 Human gene knockouts Variant in Exon ATGCTTCATGGTT ATGCTTTATGGTT Synonymous Missense Nonsense Splice site Indel frameshift Loss-of-function Protein-disruptive Protein-inactivatin Null Genetic variations predicted to completely disrupt the function of protein-coding genes

49 Normal: 2 working copies of a gene

50 With null mutations: one or two copies of a gene broken Gene Protein Gene Heterozygous null Homozygous null

51 Homozygotes of LoF variants are too rare to be seen

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53 Predicted efficacy of Lp-PLA2 inhibitor (GSK) Lack of human genetic evidence (Nature 2017) - Previous meta-analysis : high expression of Lp-PLA2 and risk of CAD (Lancet 2010) Failure of Darapladib (GSK) (NEJM 2014)

54 Large family with APOC3 knockouts

55 Crosby et al. NEJM, 201 Rare mutations in APOC3 and TG and CHD

56 CHD Hypothesis Genetics LDL Are there other genes than PCSK9 whose mutations lower LDL and protect against CHD?

57 Pharmacological treatments to reduce LDL-C Statins are most commonly prescribed treatment to reduce LDL-C Ezetimibe is a non-statin LDL-C lowering agent

58 NPC1L1 is the protein target of ezetimibe Ezetimibe inhibits the protein product of the Niemann-Pick C1-Like 1 (NPC1L1) gene NPC1L1 is expressed in the liver and small intestine NPC1L1 mediates intestinal cholesterol absorption Jia et al. Annu. Rev. Physio

59 ENHANCE randomized controlled trial Designed to test whether ezetimibe plus simvastatin (compared with simvastatin alone) will have beneficial effects on LDL-C and carotid intima-media thickness (CIMT), a surrogate measure of atherosclerosis Kastelein et al. NEJM, 20

60 Ezetimibe treatment lowers LDL-C by 17% 720 patients with familial hypercholesterolemia Treatment with simvastatin plus ezetimibe lowers LDL-C more than simvastatin alone ENHANCE trial 17% decrease Kastelein et al. NEJM, 20

61 Ezetimibe treatment did not alter a surrogate measure of atherosclerosis thickness of carotid artery Measured carotid intima-media thickness (CIMT), surrogate marker for atherosclerosis ENHANCE trial Combined therapy of ezetimibe and simvastatin did not result in change in CIMT, despite decrease in LDL-C Kastelein et al. NEJM, 20

62 It is uncertain if inhibiting NPC1L1 reduces risk for CHD Can human genetics yield insight into potential effects of NPC1L1 inhibition on CHD risk?

63 Rare variant association with CHD Focus on LoF (protein-inactivating) mutations Nonsense / Splice-site / Indel frameshift Sequencing of protein-coding region of NPC1L1 in seven case-control studies 7,364 individuals with CHD 14,728 individuals without CHD Genotyping of NPC1L1 p.arg406x in nine sample sets 22,590 individuals with CHD 68,412 individuals without CHD

64 Rare variant association study Three different scenarios Common variants 1. Collective frequency 2. Synergistic fashion 3. Specific regions

65 Rare inactivating mutations in NPC1L1 Discovered 15 protein-inactivating mutations from sequencing 10 nonsense 3 splice-site 2 frameshifts Among controls, 1 in 500 carried an inactivating mutation

66 Association of NPC1L1 inactivating mutations with LDL-C Cohort CHD free controls Total N (N carriers) 4586 (11) Difference [95% CI] [-49.51, 2.4 ARIC EA WGHS European subgroup (10) (11) [-29.58, [-32.97, [-26.50, 0.6 CHD free controls/jhs2287 (7) [-52.64, 9.5 ARIC AA AA subgroup 2671 (8) [-28.60, [-30.52, 10.0 All [-23.38, Mean difference in carriers (mg/dl)

67 Association of NPC1L1 inactivating mutations with LDL-C Cohort CHD free controls Total N (N carriers) 4586 (11) Difference [95% CI] [-49.51, 2.4 ARIC EA WGHS European subgroup (10) (11) [-29.58, [-32.97, [-26.50, 0.6 CHD free controls/jhs2287 (7) [-52.64, 9.5 ARIC AA African subgroup 2671 (8) [-28.60, [-30.52, 10.0 All [-23.38, Mean difference in carriers (mg/dl)

68 Association of NPC1L1 inactivating mutations with LDL-C Cohort Total N (N carriers) P value Difference [95% CI] CHD free controls 4586 (11) [-49.51, 2.4 ARIC EA WGHS European subgroup (10) (11) [-29.58, [-32.97, [-26.50, 0.6 CHD free controls/jhs2287 (7) [-52.64, 9.5 ARIC AA African subgroup 2671 (8) [-28.60, [-30.52, 10.0 All [-23.38, Mean difference in carriers (mg/dl)

69 Carrier frequency Rare alleles at NPC1L1 are enriched in controls free of CHD 0.09% 0.08% 0.07% 0.06% 0.05% 0.04% 0.03% 0.02% 0.01% 0.00% 0.09% 0.04% Controls 71/83, % CHD cases 11/29,954

70 Carrier frequency Rare alleles at NPC1L1 are enriched in controls free of CHD 0.09% 0.08% 0.07% 0.06% 0.05% 0.04% 0.03% 0.02% 0.01% 0.00% 0.09% 0.04% Controls 71/83, % CHD cases 11/29,954

71 Carrier frequency Rare alleles at NPC1L1 are enriched in controls free of CHD 0.09% 0.08% 0.07% 0.06% 0.05% 0.04% 0.03% 0.02% 0.01% 0.00% 0.09% 0.04% Controls 71/83,140 Odds ratio=0.47 (P=0.003) 0.04% CHD cases 11/29,954

72 Carrier frequency 0.09% 0.08% Individuals 0.07% with an inactivating 0.06% mutation 0.05% in NPC1L1 have 53% 0.04% 0.03% 0.02% 0.01% 0.00% 0.09% reduced 0.04% Controls 71/83,140 Odds ratio=0.47 (P=0.003) risk for CHD 0.04% CHD cases 11/29,954

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75 LDL reduction and vascular disease Pharmacological reduction Genetically mediated reducti Khera et al. EJPC, 2015.

76 Rare mutations in LPL and TG and CHD Khera*, Won*, et al. JAMA, 2017.

77 Khera and Kathiresan, Nat Rev Genet, 2017

78 CHD Risk Nature 2015 Carriers of LoF mutations at LDLR Had LDL-C of 279 mg/dl on aver Were at 13-fold increased risk fo LDLR PCSK9, NPC1L1 Plasma LDL Level

79 emerge (Electronic Medical Records and Genomics)

80 One Million Genomes by M genomes

81 MyCode Partnership with Regeneron (PCSK9 inhibitor) PCSK9 market could be worth $12 billion a year 5-10 year genome (WES) sequencing study 250,000 participants + EMR Health System 250 K genomes Covers 2.6 million people in 36 county 49.5% GHS patients for 20+ years 85% receive majority of care at GHS

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84 Health System 250 K genomes 2 M genomes 20,000 genes Combinations 100-1,000s human phenotypes

85 Summary Common variants GWAS successful and missing heritability Polygenic risk score and Mendelian randomization Cohorts (UK Biobank) and PheWAS Low-frequency / rare variants Statistical imputation (HRC) Exome chip, Korean chip Sequencing (human gene knockout and LoF) Drug target / efficacy / side effect Rare variants with large effects 5-20 odds ratio (compared to ORs of common variants)

86 Questions addressed LDL cholestrol 을낮추는것이심혈관질환의예방에도움을준다? Multiple lines of genetic evidence HDL cholesterol 을높이는것이심혈관질환의예방에도움을준다? Lack of genetic evidence, and failure of RCTs LDL cholesterol 을낮추는약물의부작용이있을까? T2D risk 심혈관질환위험을낮추는약물의효과를예측할수있을까? NPC1L1 (ezetimibe), Lp-PLA2 (darapladib) 지질농도와심혈관질환위험을낮추는새로운약물타겟을찾을수있을까? ANGPTL4, APOC3, LPL, etc. > Triglycerides & CAD

87 Thank you Hong-Hee Won, Ph.D. SAIHST, SKKU