Genome-Wide Association Studies: Linking Genes to Disease

Genome-Wide Association Studies: Linking Genes to Disease

Computational Molecular Biology Biochem 218 BioMedical Informatics 231 http://biochem218.stanford.edu/ Genome-Wide Association Studies: Linking Genes to Disease Doug Brutlag Professor Emeritus Biochemistry & Medicine (by courtesy) A Primer of Genome ScienceGibson and Muse Gibson and Muse 2009 Preventive Medicine Preventive Medicine Prevent disease from occurring Identify the cause of the disease Treat the cause of the disease rather than the

symptoms o o Example peptic ulcers Pyrogens Genomics identifies the cause of disease All medicine may become pediatrics Paul Wise, Professor of Pediatrics, Stanford Medical School, 2008 Effects of environment, accidents, aging, penetrance Health care costs can be greatly reduced if o o invests in preventive medicine one targets the cause of disease rather than symptoms

Penetrance and Environmental Factors Highly penetrant Mendelian single gene diseases o o Reduced penetrance, some genes lead to a predisposition to a disease o o o o Many cancers (solid tumors) require somatic mutations that induce cell proliferation, mutations that inhibit apoptosis, mutations that induce angiogenesis, and mutations that cause metastasis

Cancers are also influenced by environment (smoking, carcinogens, exposure to UV) Atherosclerosis (obesity, genetic and nutritional cholesterol) Some complex diseases have multiple causes o BRCA1 & BRCA2 genes can lead to a familial breast or ovarian cancer Disease alleles lead to 80% overall lifetime chance of a cancer, but 20% of patients with the rare defective genes show no cancers Complex diseases requiring alleles in multiple genes o Huntingtons Disease caused by excess CAG repeats in huntingtins protein gene Autosomal dominant, 100% penetrant, invariably lethal

Genetic vs. spontaneous vs. environment vs. behavior Some complex diseases can be caused by multiple pathways o Type 2 Diabetes can be caused by reduced beta-cells in pancreas, reduced production of insulin, reduced sensitivity to insulin (insulin resistance) as well as environmental conditions (obesity, sedentary Genes & Disease http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/gnd/tocstatic.html OMIM Home Page http://www.ncbi.nlm.nih.gov/omim/ Genetics Home Reference http://ghr.nlm.nih.gov/ Medline Plus http://medlineplus.gov/ Common Gene Variation in Complex

Disease Case-control studies, comparing the frequencies of common gene variants can identify susceptibility and protective alleles. Many have multiple identified genes (*) Phenotype Peptic ulcer IDDM* Alzheimer dementia Deep venous thrombosis* Falciparum malaria* AIDS* Colorectal cancer* NIDDM* Gene ABO HLA APOE F5 HBB CCR5 APC

PPAR Variant B DR3,4 E4 Leiden S 32 3920A 12A Gibson & Muse, A Primer of Genome Science Francis Collins, 2008 2007 Scientific Breakthrough of the Year International HapMap Project http://www.hapmap.org/

International HapMap Project http://www.hapmap.org/ Using SNPs to Track Predisposition to Disease and other Genetic Traits Gibson & Muse, A Primer of Genome Science GWAS: Genome-Wide Association Study A Brief Primer SNP chip Control Population WTCCC, Nature Disease Population

Thanks to Daniel Newburger A Quantitative Gene-Expression Association Sample Population cDNA Levels SNP chip Expression and Expression Quantitative Trait Loci (eQTLs) Modified from WTCCC, Nature 2007 Thanks to Daniel Newburger Genome-Wide Association Approach to Common and Complex Diseases

Identify all 10 million common SNPs Collect 1,000 cases and 1,000 controls Genotype all DNAs for all SNPs That adds up to 20 billion genotypes In 2002, this approach cost 50 cents a genotype. Thats $10 billion for each disease completely out of the question Francis Collins, 2008 Progress in Genotype Technology Cost per genotype (Cents, USD)

102 ABI TaqMan ABI SNPlex 10 Illumin a Affymetr Golden Affymet ix Gate rix MegAllele 10K 1 Illumina

Perleg Infinium/ Affymet en Sentrix rix 1 10 2001 102 103 104 100K/ 500K 5

10 2005 Nb of 106SNPs Courtesy S. Chanock, NCI Francis Collins, 2008 Genome-Wide Association Approach to Common and Complex Diseases Identify an optimum set of 300,000 tag SNPs Collect 1,000 cases and 1,000 controls Genotype all DNAs for all SNPs That adds up to 600 million genotypes In 2008, genotyping dropped to $0.0010, amounting to $600,000 for each disease Francis Collins, 2008

Francis Francis Collins, Collins, 2008 2008 The FUSION Study Finland-United States Investigation of NIDDM n = 10,068 Subject Recruitment and Clinical Testing National Public Health Institute Helsinki, Finland Molecular Genetics National Human Genome Research Institute, Bethesda, MD University of North Carolina, Chapel Hill, NC Biochemical Measurements USC Keck School of Medicine, Los Angeles, CA

Statistical Analysis University of Michigan School of Public Health, Ann Arbor, M Francis Collins, 2008 Results of Genome-Wide Association of Type 2 Diabetes with 317,503 SNPs Stage 1: FUSION only (1161 cases + 1174 controls) Francis Collins, 2008 Results of Genome-Wide Association of Type 2 Diabetes with 317,503 SNPs Stage 2 FUSION + DGI + WTCCC (4549 cases + 5579 controls) Francis Collins, 2008 Genome-Wide Scan for Type 2 Diabetes in a Scandinavian Cohort

http://www.broad.mit.edu/diabetes/scandinavs/type2.html Francis Collins, 2008 Top 10 Results From Combined Analysis FUSION DGI WTCCC/UKT2D All Samples Gene OR p-value OR

p-value OR p-value OR p-value TCF7L2 1.34 1.3 x 10-8 1.38 2.3 x 10-31 1.37

6.7 x 10-13 1.37 1.0 x 10-48 IGF2BP2 1.18 2.1 x 10-4 1.17 1.7 x 10-9 1.11 1.6 x 10-4 1.14

8.9 x 10-16 CDKN2A/B 1.20 .0022 1.20 5.4 x 10-8 1.19 4.9 x 10-7 1.20 7.8 x 10-15 FTO

1.11 0.016 1.03 0.25 1.23 7.3 x 10-14 1.17 1.3 x 10-12 CDKAL1 1.12 0.0095

1.08 0.0024 1.16 1.3 x 10-8 1.12 4.1 x 10-11 KCNJ11 1.11 0.013 1.15 1.0 x 10-7

1.15 0.0013 1.14 6.7 x 10-11 HHEX 1.10 0.026 1.14 1.7 x 10-4 1.13 4.6 x 10-6

1.13 5.7 x 10-10 SLC30A8 1.18 7.0 x 10-5 1.07 0.047 1.12 7.0 x 10-5 1.12 5.3 x 10-8

Chr 11 1.48 5.7 x 10-8 1.16 0.12 1.13 0.068 1.23 4.3 x 10-7 PPARG 1.20

0.0014 1.09 0.019 1.23 0.0013 1.14 1.7 x 10-6 Francis Collins, 2008 Top 10 Results From Combined Analysis FUSION DGI WTCCC/UKT2D

All Samples Gene OR p-value OR p-value OR p-value OR p-value TCF7L2

1.34 1.3 x 10-8 1.38 2.3 x 10-31 1.37 6.7 x 10-13 1.37 1.0 x 10-48 IGF2BP2 1.18 2.1 x 10-4

1.17 1.7 x 10-9 1.11 1.6 x 10-4 1.14 8.9 x 10-16 CDKN2A/B 1.20 .0022 1.20 5.4 x 10-8

1.19 4.9 x 10-7 1.20 7.8 x 10-15 FTO 1.11 0.016 1.03 0.25 1.23 7.3 x 10-14

1.17 1.3 x 10-12 CDKAL1 1.12 0.0095 1.08 0.0024 1.16 1.3 x 10-8 1.12 4.1 x 10-11

KCNJ11 1.11 0.013 1.15 1.0 x 10-7 1.15 0.0013 1.14 6.7 x 10-11 HHEX 1.10

0.026 1.14 1.7 x 10-4 1.13 4.6 x 10-6 1.13 5.7 x 10-10 SLC30A8 1.18 7.0 x 10-5 1.07

0.047 1.12 7.0 x 10-5 1.12 5.3 x 10-8 Chr 11 1.48 5.7 x 10-8 1.16 0.12 1.13

0.068 1.23 4.3 x 10-7 PPARG 1.20 0.0014 1.09 0.019 1.23 0.0013 1.14

1.7 x 10-6 Francis Collins, 2008 K+ Calcium Channel Glucose Insulin Zn2+ Ca2+ ATP ADP SLC30A8 Zn2+ SLC30A8 A Beta Cell Zinc

Transporter Francis Collins, 2008 The Wellcome Trust Case Control Consortium Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared Nature 447, 661-678 (7 June 2007) controls 2007: The Year of GWA Studies? Hokusai, K. The Great Wave The Genomics Gold Rush Disease Gene or Loci

Date Reported Prostate Cancer 8q24 April 1, 2007 Acute Lymphoblastic Leukemia PAX 5 and others April 12, 2007 Obesity FTO April 12, 2007 Multiple Solid Tumors

CASP8 April 22, 2007 Diabetes, Type II CDKAL1 and 6 others April 26,2007 Myocardial Infarction, Coronary Artery Disease 9p21 May 3, 2007 Breast Cancer FGFR2, TNCR9, MAP3K1, LSP and others

May 27, 2007 Crohns Disease IRGM June 7, 2007 Diabetes, Type I 12q24 and others June 7, 2007 Bipolar Disorder 16p12 June 7, 2007 Rheumatoid Arthritis

6p21, 1p13 June 7, 2007 Celiac Disease IL-2, IL-21 June 10, 2007 Atrial Fibrillation 4q25 July 1, 2007 Topol, Murrary & Frazer, JAMA 2007 218-221. The Genomics Gold Rush Disease

Gene or Loci Date Reported Diabetes, Type II WFS1 July 1, 2007 Prostate Cancer TCF2; 17p July 1, 2007 Asthma (childhood) ORMDL3 July 4, 2007

Colon, Prostate Cancer 8q24 July 8, 2007 Diabetes, Type I KIAA0350 July 15, 2007 Gallstone Disease ABCG8 July 15, 2007 Restless Leg Syndrome MEIS1, BTBD9, MAP2K5

July 18, 2007 Coronary Artery Disease 6q25, 2q36 July 18, 2007 Age-Related Macular Degeneration CF3 July 18, 2007 HIV Host Control HLA-B*5701 July 19, 2007 Multiple Sclerosis

IL7R; IL2R July 28, 2007 Amyotrophic Lateral Sclerosis FLJ10986 August 1, 2007 Diabetes, Type I IL2R August 5, 2007 Glaucoma LOXL1

August 9, 2007 Rheumatoid Arthritis TRAF1-C5 August 31, 2007 Topol, Murrary & Frazer, JAMA 2007 218-221. The Genomics Gold Rush Disease Gene or Loci Date Reported Colorectal Cancer SMAD7 October 14, 2007

Ankylosing Spondylitis ARTS1, IL23R October 21, 2007 Autoimmune Thyroid Disease TSHR, FCRL3 October 21, 2007 Rheumatoid Arthritis 6q23 November 4, 2007 Psoriasis -Defensin CNV

December 2, 2007 Systemic Lupus Erythematosus TNFSF4 December 2, 2007 Amyotrophic Lateral Sclerosis DPP6 December 16, 2007 Colorectal Cancer CRAC1 (HMPS) December 16, 2007 Systemic Lupus Erythematosus

PXK, KIAA1542, BANK1, C8orf-BLK, ITGAM January 20, 2008 Lipoprotein Disorders MLX1PL and Multiple Others January 13, 2008 Hypercholesterolemia CELSR2 February 9. 2008 Prostate Cancer 2p15, Xp11.22 and Others

February 10, 2008 Gout SLC2A9 March 9, 2008 Schizophrenia ERBB4, SLC1A3 and Others March 27, 2008 Topol, Murrary & Frazer, JAMA 2007 218-221. The Genomics Gold Rush Disease Gene or Loci

Date Colorectal Cancer 10p14,8q23.3,18q21,11q23 March 30, 2008 Diabetes, Type 2 JA2F1 and others March 30, 2008 Nicotine Add, Lung Ca, PAD 15q25 April 3, 2008 Hypertension

SLC12A3, SLC12A1,KCNJ1 April 6, 2008 Crohns Disease and Ulcerative Colitis ECM1and others PTPN2, HERC2, STAT3 April 27, 2008 Breast Cancer (ER +) 5p12 April 27, 2008 Osteoporosis RANKL1,OPG, ESR

April 29, 2008 Obesity MC4R May 4, 2008 Neuroblastoma 6p22 May 7, 2008 Melanoma and Basal Cell Ca 20q11.22, ASIP, TYR May 18, 2008 Gastric Cancer

PSCA May 18, 2008 Macular Degeneration ARMS2 May 30, 2008 Alzheimers Disease CALHM1 June 27, 2008 Crohns Disease JAK2, CDKAL1, ITLN1, more June 29, 2008

Obesity PCSK1 July 7, 2008 Knee Osteoarthritis DVWA July 14, 2008 Statin Myopathy SLCO1B1 July 24, 2008 Topol, Murrary & Frazer, JAMA 2007 218-221. The Genomics Gold Rush Disease

Gene or Loci Date Restless Leg Syndrome PTPRD July 27, 2008 Schizophrenia 1q21, 15q13 July 31, 2008 Systemic Lupus Erythematosus TNAIP3 August 1, 2008

Sarcoidosis ANXA11 August 10, 2008 Bipolar Disorder ANK3, CACNA1C August 17, 2008 Diabetes, Type II KCNQ1 August 17, 2008 Crohns Disease IRGM

August 24, 2008 Prostate Cancer HNF1B August 31, 2008 CLL 2q13, 2q37, and others August 31, 2008 Pediatric Inflammatory Bowel Dz 20q13, 21q22 August 31, 2008 Rheumatoid Arthritis

CD40, CD244, 10p15, 12q13, 22q13 September 14, 2008 Bladder Cancer 8q24 September 14, 2008 ESRD, Focal Glomerulosclerosis MYH9 September 14, 2008 Narcolepsy CPT1B, CHKB

September 28, 2008 Fatty Liver Disease (nonEtOH) PNPLA3 September 28, 2008 Gout SLC2A9, SLC17A3 October 1, 2008 Topol, Murrary & Frazer, JAMA 2007 218-221. The Genomics Gold Rush Disease Gene or Loci Date

Male Pattern Baldness 20p11 October 12, 2008 Basal Cell Carcinoma 1p36, 1q42 October 12, 2008 Asthma 17q21 October 15, 2008 Lung Cancer 5p15, 6p21

November 2, 2008 Diabetes, Type 1 4q27, BACH2, PRKCQ November 2 ,2008 Multiple Sclerosis KIF1B November 9, 2008 Intracranial Aneurysm SOX17, 2p33 November 9, 2008 Colon Cancer

BMP4, CDH1, RHPN2, 20p12 November 16, 2008 Topol, Murrary & Frazer, JAMA 2007 218-221. Catalog of GWAS Studies http://www.genome.gov/26525384 Catalog of GWAS Studies http://www.genome.gov/26525384 Catalog of GWAS Studies http://www.genome.gov/26525384 Published Genome-Wide Associations through 12/2009, 658 published GWA at p<5x10-8 NHGRI GWA Catalog www.genome.gov/GWAStudies Study Designs Used in Genome-wide

Association Studies . Copyright restrictions may apply. Pearson, T. A. et al. JAMA 2008;299:1335-1344 Replication A Must Replication Replication Replication Hirschhorn & Daly Nat. Genet. Rev. 6: 95, 2005 NCI-NHGRI Working Group on Replication Nature 447: 655, 2007 Examples of Multistage Designs in Genome-wide Association Studies

Copyright restrictions may apply. Pearson, T. A. et al. JAMA 2008;299:1335-1344 Hypothetical Quantile-Quantile Plots in Genome-wide Association Studies Copyright restrictions may apply. Pearson, T. A. et al. JAMA 2008;299:1335-1344 Interleukin 23R & Inflammatory Bowel Disease Pearson, T. A. et al. JAMA 2008;299:1335-1344 Genome-Wide Associations in Rheumatoid Arthritis Pearson, T. A. et al. JAMA 2008;299:1335-1344 Association of Alleles & Genotypes

Pearson, T. A. et al. JAMA 2008;299:1335-1344 Ten Basic Questions to Ask About a Genome-wide Association Study Report 1. Are the cases defined clearly and reliably so that they can be compared

with patients typically seen in clinical practice? 2. Are case and control participants demonstrated to be comparable to each other on important characteristics that might also be related to genetic variation and to the disease? 3. Was the study of sufficient size to detect modest odds ratios or relative risks (1.3-1.5)? 4. Was the genotyping platform of sufficient density to capture a large proportion of the variation in the population studied? 5. Were appropriate quality control measures applied to genotyping assays, including visual inspection of cluster plots and replication on an independent genotyping platform? 6. Did the study reliably detect associations with previously reported and replicated variants (known positives)? 7. Were stringent corrections applied for the many thousands of statistical tests performed in defining the P value for significant associations? 8. Were the results replicated in independent population samples? 9. Were the replication samples comparable in geographic origin and phenotype definition, and if not, did the differences extend the applicability of the findings? 10. Was evidence provided for a functional role for the gene polymorphism identified?

Pearson, T. A. et al. JAMA 2008;299:1335-1344 Helen H. Hobbs Chief Clinical Genetics, Internal Medicine Helen Hobbs 2009 Do genetic differences between ethnic groups contribute to differences in fatty liver disease? Normal Steatosis Steatohepatitis Cirrhosis 10-20% 1-2% Hispanics

European-Americans African-Americans First Hit Obesity Type 2 diabetes Ethanol Hepatitis C Second Hit Oxidative Stress Lipid Peroxidation Anti-virals Cytokines Helen Hobbs 2009 Hepatic Steatosis Normal Hepatic Steatosis

Obesity Type 2 diabetes Ethanol Hepatitis C Helen Hobbs, Nature Genetics V40, pp 1461, 2008 Genome-Wide Association Study for Hepatic Triglyceride Content in the Dallas Heart Study Restricted to nonsynonymous SNPs Chip-based oligonucleotide hybridization (Perlegen) Quality filter: n = 12,138 9,229 Association with hepatic fat, adjusted for ancestry (2,270 ancestry informative SNPs)

1,032 African-Americans 696 European-Americans 383 Hispanics n = 2,111 Romeo, et al.(2008) Genetic Variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nature Genetics 40, 1461-1465 Helen Hobbs 2009 Helen Hobbs 2009 Genome-wide Association Study in DHS Non-synonymous SNPs (n = 9,229) P=5.9 X 10-10 5.4 x 10-6 Chromosome

Helen Hobbs, Nature Genetics V40, pp 1461, 2008 Helen Hobbs 2009 PNPLA3: A Member of the Patatin-like Phospholipase Family Resembles patatin: major potato protein Nonspecific lipid acyl hydrolase activity (TG>PL) Expressed high level in fat & liver Increased with feeding (especially carbohydrates) Helen Hobbs, Nature Genetics V40, pp 1461, 2008 Helen Hobbs 2009 Ethnic Differences in the Frequency of PNPLA3-I148M AfricanAmericans Minor Allele Frequency

EuropeanHispanics Americans 0.17 0.23 0.49 Prevalence of Hepatic Steatosis (%) 0 Helen Hobbs, Nature Genetics V40, pp 1461, 2008 Helen Hobbs 2009 PNPLA3: I148M and Hepatic TG Content Helen Hobbs, Nature Genetics V40, pp 1461, 2008 Helen Hobbs 2009

I148M & Catalytic Dyad of PNPLA3 Ile148 Met148 Asp166 Asp166 Ser47 Ser47 Helen Hobbs 2009 PNPLA3 & Hepatic Triglyceride Metabolism Liver Acetyl CoA Mito

Remnants Adipose Tissue + PNPLA2 (ATGL) VLDL PNPLA3 (Adiponutrin) Fasting Feeding Translation of Genetic Discoveries TRAIT GENE PUBLIC HEALTH

Therapeutic target PNPLA3: TG metabolism Prevention strategy Risk stratification Helen Hobbs 2009 DNAdirect: Clinical Genetic Testing Direct to Consumer DNADirect 2009 Navigenics Navigenics 2009 23andMe

23andMe 2009 23andMe Kit 23andMe Spittoon 23andMe Sample Tube 23andMe Tube in Envelope 23andMe Fedex Mailer 23andMe Login 23andME Opt-In Statement 23andMe Clinical Reports 23andMe Clinical Reports 23andMe Research Reports

23andMe Carrier Status 23andMe Traits 23andMe Maternal Inheritance 23andMe Paternal Inheritance 23andMe Ancestry Genome-Wide Association Study References How to Use an Article About Genetic Association: A: Background Concepts John Attia; John P. A. Ioannidis; Ammarin Thakkinstian; et al. JAMA. 2009;301(1):74-81 How to Interpret a Genome-wide Association Study Thomas A. Pearson; Teri A. Manolio JAMA. 2008;299(11):1335-1344 The Genomics Gold Rush Eric J. Topol; Sarah S. Murray; Kelly A. Frazer JAMA. 2007;298(2):218-221 The Genome Gets Personal: Almost W. Gregory Feero; Alan E. Guttmacher; Francis S. Collins JAMA. 2008;299(11):1351-1352 Mapping Genes for NIDDM: Design of the FinlandUnited States Investigation of NIDDM Genetics(FUSION) Study

Valle et al. DIABETES CARE, VOLUME 21, NUMBER 6, JUNE 1998 Romeo, et al.(2008) Genetic Variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nature Genetics 40, 1461-1465, Francis O. Walker (2007) Huntingtons Disease Review. Lancet 2007; 369: 21828. The Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447, 661-678 (7 June 2007) The HapMap and Genome-Wide Association Studies in Diagnosis and Therapy Manolio T. and Collins, F. Annual Review of Medicine (2009) 60: 443-456. Finding the missing heritability of complex diseases. Manolio TA et al. Nature 2009 461: 747-753

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