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Technologic Issues in GWAS and Follow-up
Studies
Stephen Chanock, M.D.Senior Investigator, POB,CCR &
Director, Core Genotyping Facility, DCEG NCIMay 22, 2007
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Types of Polymorphisms
Single nucleotide polymorphisms (SNPs)Common SNPs are defined as >1% in at
least one populationRare SNPs are hard to identify and validate
But, it is estimated that there are a large number per individual
MAF= minor allele frequency
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SNP in Unique Sequence
T/C
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SNPs & Function: We know so little..
• Majority are “silent”– No known functional change
• Alter gene expression/regulation– Promoter/enhancer/silencer– mRNA stability– Small RNAs
• Alter function of gene product– Change sequence of protein
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SNPs in Genes: Take one
Coding SNPsSynonymous- no change in amino acid
previously termed “silent” but…..Can alter mRNA stability
DRD2 (Duan et al 2002)Nonsynonymous- changes amino acid
conservative and radicalNonsense- insertion of stop codonIndel- Disrupts codon sequence
Rare but disruptive
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SNPs Outside Genes: Take many….
• Majority distributed throughout genome are “silent” (excellent as markers)
• Alter transcription– Promoter, enhancer, silencer
• Regulate expression– Locus control region, mRNA stability
• Most are assumed to be ‘silent hitchhikers’– No function by predictive models or analysis
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Linkage disequilibrium (LD)
• The non-random association of alleles in the population
• Alleles at neighboring loci tend to cosegregate
• Linkage disequilibrium implies population allelic association
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Strategy for SNP Selection
To test all SNPs is presently too costlyUtilize a strategy that capitalizes on linkage
disequilibrium between SNPs
Haplotype blocks defined by Gabriel et alBased on D’ values for linkage disequilibrium
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What can LD do for me?• Knowledge of patterns of linkage
disequilibrium can be quite useful in the design and analysis of genetic data
• Design:– Estimation of theoretical power to detect
associations– Evaluation of degree completeness of sampling
of genetic variants– Choice of most informative genetic variants to
genotype
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Genetic Association Testing: Finding Markers
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Large and Small Scale Polymorphisms• Copy Number of Polymorphisms
Regional “repeat” of sequence10s to 100s kb of sequence
Estimate of >10% of human genomeMulti-copy in many individuals
• Duplicons90-100% similarity for >1 kb5-10% of genome (5% exons elsewhere)Multi-copy (high N) in all individuals
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Germ-line DNA Copy Number Variation(CNV)
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Copy Number Variation Across the Genome
Redon et al Nature 2006
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Copy Number Variation
Across the GenomeFrequency of CNVs
Most are uncommon (<5%)Familial vs Unrelated Studies
Associated with DiseaseOPN1LW Red/green colorblindCCL3L1 Reduced HIV InfectionCYP2A6 Altered nicotine metabolismVKORC1 Warfarin metabolism
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Most SNPs Are In Unique Sequence !
T/C
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PSV (Paralogous Sequence Variant)
A G
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AA/C
SNP in Duplicon Sequence
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MSV: Multi-Site Variants
T G
GT G
T
G
T/G
T/G
T/G
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Progress in Genotyping Technology
1 10 102 103 104 105 106
# of SNPs
Cost per genotypeCents (USD)
10
1
102
ABITaqMan
ABISNPlex Illumina
Golden Gate
IlluminaInfinium/Sentrix
Affymetrix100K/500K
Perlegen
AffymetrixMegAllele
2001 2007
Affymetrix10K
Sequenom
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Genotype Technologies
• Dropping costs• Smaller amounts of DNA
< 1ug for > 1 Million SNPs• Economy of scale
Frequent Flier Paradigm• Increased density but with fixed products • Custom products bear high development cost• Challenge of mid-range (50 to 500 SNPs)
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from http://docs.appliedbiosystems.com/pebiodocs/00790910.pdf
TaqManTM (5’ Exonuclease)
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BRCA1-03
rs16941
SNP under both TM probes
Results in the wrong allele call
SNP Analysis- BEWARE
http://snp500cancer.nci.nih.gov
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Infinium™ Assay
Address 1 Address 2 Address 3
Array of capture probes (1 bead type/allele)Address 4
Address 1 Address 2 Address 3
Extension and uniform, single-color labelingAddress 4
Amplify genome uniformly
Fragment, Denature and Hybridize to immobilized 50- mers
Discriminate SNPs (enzyme-mediated)
Amplify signal and readout
1
3/4
“A/A” “A/B”
2
1
2
3
4
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Illumina HumanHap500
Good Cluster Poor Cluster
Read in BeadStudioTM
A/A A/G G/G ?/? ?/?* ?*/?*
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250 ng Genomic DNA
RE Digestion
Adaptor Ligation
Affymetrix GeneChip® Mapping Assay
Xba XbaXba
Fragmentationand Labeling
PCR: Universal Primer Amplification
Complexity Reduction
AA BB AB
Hyb & Wash
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Affymetrix 500K ChipsPoor Quality Good Quality
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Important Points
• Too many data points to review individually• Iterative algorithm for analysis
– Still undergoing improvements• Validation of notable SNPs with second
technology– “Neighboring SNP-land mines”
• Do not do this at home- Only for highly trained personnel
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Choice of Dense SNP Platforms
Basic PointsBased on ‘Spacing’100k, 500k, 1 MillionCNV AnalysisWGA compatible
IssuesLower price2 Enzyme ProblemCalling AlgorithmsRedundancy (useful)
Basic PointsBased on ‘tagging’317k, 550k, 1 MillionCNV AnalysisWGA not yet rec’d
IssuesHigher priceHapMap II Based
Affymetrix Illumina
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2007 What is Available for Whole Genome Scans
• Coverage analysis based on HapMap II Data• Build 20 MAF >5%, r2 > 0.8 (pair-wise)
• CEU YRI JPT/CHB
• Illumina HumanHap300 80% 35% 40%• Illumina HumanHap500 91% 58% 88%• Affymetrix* 500k Mapping 63*% 41% 63%
*77% (with 50k MegA)
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Quality control of genotype calls
& DNA handling
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Quality Control for Called Genotypes
PURPOSE:
Identification of unreliable SNPs and DNAs to be entirely removed from the analysis .
Evaluation of completion rate (DNAs)Evaluation of call rate (SNPs)
Evaluation of discordance rate (error rate)
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Attempted SNPs
CGEMS SNP DNA Success Rates
Threshold DNA completion rate > 94%
attempted failed Successrate
4696 66 0.986
CriteriumSNP call rate > 90%
attempted failedsuccess
rate
PLCO 561,494 1,490 0.973NHS 555,352 8,706 0.984
Number of individuals
Number of individuals
1.4%
2.1%
Atte
mpt
ed D
NA
s
SNPx
DNA y
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Discordance rate for CGEMS: HumanHap500 (Illumina)
CEPH-CGEMS74 duplicate pairs
Mean discordance
rate2 x 10-4
Participants142 duplicate pairs
Mean discordance
rateProstate 2.0 x 10-4
Breast 1.5 x 10-4
CEPH-HapMap
28 individuals(with 24 duplicates)
Mean discordance
rate1.4 x 10-3
Concordance rates >99.5%Subtle Differences in Quality of DNA
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Quality Control for Recruitment
DNA handlingChecking for:
Chromosome X PloidyIdentification of Familial RelationshipsEvaluation of Continental Admixture
Population StratificationPrincipal Component Analysis(Hardy Weinberg Statistics)
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Chromosome X1 copy 2 copies
Prostate 2279 3
Breast 0
Unexpected duplicates
1st & 2nd degree relatives
Other 3rd to 5th degree relatives
3 pairs 5 20*
*It was noted subsequently that both members of each pair had been recruited in the same center.
1 to be done3 pairs2299
Analysis of CGEMS Data Sets
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Admixture coefficient in PLCO samplesAsia
AfricaEurope
control
case
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-.15
-.1-.0
50
.05
V_Dim1
-.05 0 .05 .1V_Dim2
33 controls21 cases
69 controls91 cases
CGEMS Prostate Cases & ControlsPrincipal Component Analysis
Based on Price et al Nat Genet 2006
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0
log 1
0(p-
valu
e)
log10 (quantile)
corrected
uncorrected
-2
-4
-6
0-2-4-6
CGEMS Breast Cancer Scan
log quantile plot of p-values for the Entire Set of Markers
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Chromosomes1 2 3 4 5 6 7 8
-2
-4
-6
-4
-6
Log 1
0(p-
valu
e)p q p q
222120191817161514131211109 X-2
p q p q
incidence density sampling
8q24
CGEMS Prostate Cancer GWAS
P values < 0.01
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Replication Studies in CGEMS Prostate Cancer GWAS
PLCO
ACS
ATBC
FPCC
HPFS
ALL
Subjects
1157 1172
Predisposing allele frequency
0.55 0.49
1151 1150 0.55 0.50
896 894 0.57 0.51
459 455 0.56 0.51
636 625 0.57 0.51
4299 4296 0.56 0.50
P-value
2.4x10-05
3.2x10-03
1.9x10-03
1.2x10-01
1.0x10-02
9.4x10-13
Predisposing allele frequency
0.14 0.10
0.12 0.08
0.21 0.17
0.12 0.07
0.13 0.09
0.15 0.11
P-value
9.8x10-05
2.7x10-05
2.9x10-02
4.4x10-03
2.7x10-03
1.5x10-14
rs6983267 rs1447295
Cases Cont. Cases Cont.
Estimated Odds Ratios OverallHeterozygotes 1.26 1.43Homozygotes 1.58 2.23
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Meta-Analysis of 8q24 papers in Nature Genetics: J Witte
J Witte Nature Genetics 2007