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Genome-Wide Association Studies-Linking Genes to Disease


Computational Molecular Biology

Genome-Wide Association Studies: Linking Genes to Disease

A Primer of Genome Science Gibson 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
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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
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invests in preventive medicine one targets the cause of disease rather than symptoms

Penetrance and Environmental Factors
? Highly penetrant Mendelian single gene diseases
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Huntington’s Disease caused by excess CAG repeats in huntingtin’s protein gene Autosomal dominant, 100% penetrant, invariably lethal

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Reduced penetrance, some genes lead to a predisposition to a disease
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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
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)

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Complex diseases requiring alleles in multiple genes
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? ?

Some complex diseases have multiple causes
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Genetic vs. spontaneous vs. environment vs. behavior
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 lifestyle, smoking etc.).

Some complex diseases can be caused by multiple pathways
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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 PPARγ

Variant B DR3,4 E4 Leiden βS Δ32 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 Disease Population

WTCCC,

Nature 2007

Thanks to Daniel Newburger

A Quantitative Gene-Expression Association

Sample Population
SNP chip Expression

cDNA Levels 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. ? That’s $10 billion for each disease – completely out of the question

? Francis Collins, 2008

Progress in Genotype Technology

102 Cost per genotype (Cents, USD)

ABI TaqMan ABI SNPlex Illumina
Golden Gate

10

1

Affymetrix Affymetri MegAllele x Illumina
10K

Infinium/Sentr Perlegen Affymetrix ix 100K/500K

1

10 2001

102

103

104

105 2005

Nb of 106 SNPs

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, MI

? 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
TCF7L2 IGF2BP2 CDKN2A/B FTO

OR
1.34 1.18 1.20 1.11

p-value
1.3 x 10-8 2.1 x 10-4 .0022 0.016

OR
1.38 1.17 1.20 1.03

p-value
2.3 x 10-31 1.7 x 10-9 5.4 x 10-8 0.25

OR
1.37 1.11 1.19 1.23

p-value
6.7 x 10-13 1.6 x 10-4 4.9 x 10-7 7.3 x 10-14

OR
1.37 1.14 1.20 1.17

p-value
1.0 x 10-48 8.9 x 10-16 7.8 x 10-15 1.3 x 10-12

CDKAL1 KCNJ11
HHEX SLC30A8 Chr 11 PPARG

1.12 1.11
1.10 1.18 1.48 1.20

0.0095 0.013
0.026 7.0 x 10-5 5.7 x 10-8 0.0014

1.08 1.15
1.14 1.07 1.16 1.09

0.0024 1.0 x 10-7
1.7 x 10-4 0.047 0.12 0.019

1.16 1.15
1.13 1.12 1.13 1.23

1.3 x 10-8 0.0013
4.6 x 10-6 7.0 x 10-5 0.068 0.0013

1.12 1.14
1.13 1.12 1.23 1.14

4.1 x 10-11 6.7 x 10-11
5.7 x 10-10 5.3 x 10-8 4.3 x 10-7 1.7 x 10-6

? Francis Collins, 2008

Top 10 Results From Combined Analysis
FUSION DGI WTCCC/UKT2D All Samples

Gene
TCF7L2 IGF2BP2 CDKN2A/B FTO

OR
1.34 1.18 1.20 1.11

p-value
1.3 x 10-8 2.1 x 10-4 .0022 0.016

OR
1.38 1.17 1.20 1.03

p-value
2.3 x 10-31 1.7 x 10-9 5.4 x 10-8 0.25

OR
1.37 1.11 1.19 1.23

p-value
6.7 x 10-13 1.6 x 10-4 4.9 x 10-7 7.3 x 10-14

OR
1.37 1.14 1.20 1.17

p-value
1.0 x 10-48 8.9 x 10-16 7.8 x 10-15 1.3 x 10-12

CDKAL1 KCNJ11 HHEX
SLC30A8 Chr 11 PPARG

1.12 1.11 1.10
1.18 1.48 1.20

0.0095 0.013 0.026
7.0 x 10-5 5.7 x 10-8 0.0014

1.08 1.15 1.14
1.07 1.16 1.09

0.0024 1.0 x 10-7 1.7 x 10-4
0.047 0.12 0.019

1.16 1.15 1.13
1.12 1.13 1.23

1.3 x 10-8 0.0013 4.6 x 10-6
7.0 x 10-5 0.068 0.0013

1.12 1.14 1.13
1.12 1.23 1.14

4.1 x 10-11 6.7 x 10-11 5.7 x 10-10
5.3 x 10-8 4.3 x 10-7 1.7 x 10-6

? Francis Collins, 2008

K+

Glucose

Calcium Channel

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 controls
Nature 447, 661-678 (7 June 2007)

2007: The Year of GWA Studies?

Hokusai, K. The Great Wave

The Genomics Gold Rush
Disease
Prostate Cancer Acute Lymphoblastic Leukemia Obesity Multiple Solid Tumors Diabetes, Type II Myocardial Infarction, Coronary Artery Disease Breast Cancer Crohn’s Disease Diabetes, Type I Bipolar Disorder Rheumatoid Arthritis Celiac Disease

Gene or Loci
8q24 PAX 5 and others FTO CASP8 CDKAL1 and 6 others 9p21 FGFR2, TNCR9, MAP3K1, LSP and others IRGM 12q24 and others 16p12 6p21, 1p13 IL-2, IL-21

Date Reported
April 1, 2007 April 12, 2007 April 12, 2007 April 22, 2007 April 26,2007 May 3, 2007 May 27, 2007 June 7, 2007 June 7, 2007 June 7, 2007 June 7, 2007 June 10, 2007

Atrial Fibrillation

4q25

July 1, 2007

? Topol, Murrary & Frazer, JAMA 2007 218-221.

The Genomics Gold Rush
Disease
Diabetes, Type II Prostate Cancer

Gene or Loci
WFS1 TCF2; 17p

Date Reported
July 1, 2007 July 1, 2007

Asthma (childhood)
Colon, Prostate Cancer

ORMDL3
8q24

July 4, 2007
July 8, 2007

Diabetes, Type I
Gallstone Disease Restless Leg Syndrome Coronary Artery Disease

KIAA0350
ABCG8 MEIS1, BTBD9, MAP2K5 6q25, 2q36

July 15, 2007
July 15, 2007 July 18, 2007 July 18, 2007

Age-Related Macular Degeneration
HIV Host Control Multiple Sclerosis Amyotrophic Lateral Sclerosis Diabetes, Type I Glaucoma Rheumatoid Arthritis

CF3
HLA-B*5701 IL7Rα; IL2Rα FLJ10986 IL2Rα LOXL1 TRAF1-C5

July 18, 2007
July 19, 2007 July 28, 2007 August 1, 2007 August 5, 2007 August 9, 2007 August 31, 2007

? Topol, Murrary & Frazer, JAMA 2007 218-221.

The Genomics Gold Rush
Disease
Colorectal Cancer Ankylosing Spondylitis Autoimmune Thyroid Disease Rheumatoid Arthritis Psoriasis Systemic Lupus Erythematosus

Gene or Loci
SMAD7 ARTS1, IL23R TSHR, FCRL3 6q23 β-Defensin CNV TNFSF4

Date Reported
October 14, 2007 October 21, 2007 October 21, 2007 November 4, 2007 December 2, 2007 December 2, 2007

Amyotrophic Lateral Sclerosis
Colorectal Cancer Systemic Lupus Erythematosus Lipoprotein Disorders Hypercholesterolemia Prostate Cancer Gout Schizophrenia

DPP6
CRAC1 (HMPS) PXK, KIAA1542, BANK1, C8orf-BLK, ITGAM MLX1PL and Multiple Others CELSR2 2p15, Xp11.22 and Others SLC2A9 ERBB4, SLC1A3 and Others

December 16, 2007
December 16, 2007 January 20, 2008 January 13, 2008 February 9. 2008 February 10, 2008 March 9, 2008 March 27, 2008

? Topol, Murrary & Frazer, JAMA 2007 218-221.

The Genomics Gold Rush
Disease
Colorectal Cancer Diabetes, Type 2 Nicotine Add, Lung Ca, PAD Hypertension Crohn’s Disease and Ulcerative Colitis Breast Cancer (ER +) Osteoporosis Obesity Neuroblastoma Melanoma and Basal Cell Ca Gastric Cancer Macular Degeneration Alzheimer’s Disease Crohn’s Disease Obesity Knee Osteoarthritis Statin Myopathy

Gene or Loci
10p14,8q23.3,18q21,11q23 JA2F1 and others 15q25 SLC12A3, SLC12A1,KCNJ1 ECM1and others PTPN2, HERC2, STAT3 5p12 RANKL1,OPG, ESR MC4R 6p22 20q11.22, ASIP, TYR PSCA ARMS2 CALHM1 JAK2, CDKAL1, ITLN1, more PCSK1 DVWA SLCO1B1

Date
March 30, 2008 March 30, 2008 April 3, 2008 April 6, 2008 April 27, 2008 April 27, 2008 April 29, 2008 May 4, 2008 May 7, 2008 May 18, 2008 May 18, 2008 May 30, 2008 June 27, 2008 June 29, 2008 July 7, 2008 July 14, 2008 July 24, 2008

? Topol, Murrary & Frazer, JAMA 2007 218-221.

The Genomics Gold Rush
Disease
Restless Leg Syndrome Schizophrenia Systemic Lupus Erythematosus Sarcoidosis Bipolar Disorder Diabetes, Type II Crohn’s Disease Prostate Cancer CLL Pediatric Inflammatory Bowel Dz Rheumatoid Arthritis Bladder Cancer ESRD, Focal Glomerulosclerosis Narcolepsy Fatty Liver Disease (non-EtOH) Gout

Gene or Loci
PTPRD 1q21, 15q13 TNAIP3 ANXA11 ANK3, CACNA1C KCNQ1 IRGM HNF1B 2q13, 2q37, and others 20q13, 21q22 CD40, CD244, 10p15, 12q13, 22q13 8q24 MYH9 CPT1B, CHKB PNPLA3 SLC2A9, SLC17A3

Date
July 27, 2008 July 31, 2008 August 1, 2008 August 10, 2008 August 17, 2008 August 17, 2008 August 24, 2008 August 31, 2008 August 31, 2008 August 31, 2008 September 14, 2008 September 14, 2008 September 14, 2008 September 28, 2008 September 28, 2008 October 1, 2008

? Topol, Murrary & Frazer, JAMA 2007 218-221.

The Genomics Gold Rush
Disease
Male Pattern Baldness Basal Cell Carcinoma Asthma Lung Cancer Diabetes, Type 1 Multiple Sclerosis Intracranial Aneurysm Colon Cancer

Gene or Loci
20p11 1p36, 1q42 17q21 5p15, 6p21 4q27, BACH2, PRKCQ KIF1B SOX17, 2p33 BMP4, CDH1, RHPN2, 20p12

Date
October 12, 2008 October 12, 2008 October 15, 2008 November 2, 2008 November 2 ,2008 November 9, 2008 November 9, 2008 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

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?

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? ? ? ?

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 EuropeanHispanics Americans

Minor Allele Frequency

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 Fasting

PNPLA3 (Adiponutrin)

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 Finland–United 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) Huntington’s Disease Review. Lancet 2007; 369: 218–28. 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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