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Consideration for Planning a Candidate Gene Association Study With TagSNPs

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Consideration for Planning a Candidate Gene Association Study With TagSNPs Shehnaz K. Hussain, PhD, ScM skhussain_at_ucla.edu Epidemiology 243: Molecular Epidemiology – PowerPoint PPT presentation

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Title: Consideration for Planning a Candidate Gene Association Study With TagSNPs


1
Consideration for Planning a Candidate Gene
Association Study With TagSNPs
  • Shehnaz K. Hussain, PhD, ScM
  • skhussain_at_ucla.edu
  • Epidemiology 243 Molecular Epidemiology

2
Objectives
  • Molecular genetics primer
  • Databases and tools to conduct in silico analyses
    for tagSNP selection/prioritization
  • Factors influencing statistical power

3
Central dogma
A T C G
DNA
mRNA
Protein
4
What are SNPs?
  • More than 99 of all nucleotides are the same in
    all humans
  • 1 of nucleotides are polymorphic
  • SNPsgtgt insertions-deletions
  • Bi-nucleotide T (80) A (20)
  • Where do SNPs occur?
  • Exons
  • Introns
  • Flanking regions

5
What are haplotypes?
  • A haplotype is the pattern of nucleotides on a
    single chromosome
  • Two copies of each chromosome
  • The haplotype inference problem

? T ? G ? A
T T C G T A
TA TT CG GG TA AA
? T ? G ? A
A T G G A A
6
What is linkage disequilibrium?
  • Linkage disequilibrium (LD) describes the
    non-random association of nucleotides on the same
    chromosome in a population
  • One nucleotide at one position (locus) predicts
    the occurrence of another nucleotide at another
    locus

LD
No LD
7
What are markers?
Disease Phenotype
Test for genetic association between the
phenotype and the DSL
Test for association between phenotype and
marker loci
LD
Candidate gene
Marker loci (SNPs)
Disease Susceptibility Locus
8
What are tagSNPs?
  • TagSNPs are a subset of all SNPs in a gene that
    mark groups of SNPs in LD
  • Avoids redundant genotyping

LD
LD
Marker loci (SNPs)
Disease Susceptibility Locus
9
The joint effect of tagSNPs in cytokine genes and
cigarette smoking in cervical cancer risk
10
T-cell proliferation

IL
-
2
IL
-
2
IL
-
2
gene
IL
-
2
gene
Activated T
-
cell
Activated T
-
cell
11
Background
  • Cigarette smoking ? 1.5- to 3-fold cancer risk
  • Cigarette smoking ? levels of IL-2 and IFN?
    (cervical and circulating)
  • ? levels of IL-2 and IFN?
  • HPV persistence in the cervix
  • Cervical neoplasia
  • Decreased survival from invasive cervical cancer

12
Model
Cigarette smoking
HPV-associated squamous cell cervical cancer
SNPs in IL-2, IL-2R, and IFNG
13
Methods
  • Study design
  • Population-based case-only study
  • Subjects
  • 308 Caucasian squamous cell cervical cancer cases
    diagnosed 1986-2004
  • Residing in 3 western Washington counties
  • Data collection
  • Structured inperson interviews
  • DNA isolated from buffy coats

14
Objectives
  • Molecular genetics primer
  • Databases and tools to conduct in silico analyses
    for tagSNP selection/prioritization
  • Factors influencing statistical power

15
Multi-stage tagSNP design
Select reference panel
Re-sequence panel, identify SNPs (many markers,
few subjects)
Choose tagSNPs
Genotype tagSNPs in main study (few markers, many
subjects)
16
1. Select reference panel
  • Definition
  • A sample of your study population
  • Most representative
  • Samples from the Coriell Repository
  • Ability to integrate your data with other
    resources

Candidate gene SNPs
HapMap SNPs
17
2. Re-sequence reference panel
18
Alternatives to re-sequencing
  • Program for Genomic Applications (PGA)
  • SeattleSNPs inflammation
  • NIEHS SNPs environmental response
  • Innate Immunity
  • International HapMap Project
  • 5 million SNPs in four ethnically distinct
    populations

19
3. Choose tagSNPs (LD)
Option LDSelect (Carlson, 2002) Tagger (de Bakker, 2005)
r2 threshold (0.80) Yes Yes
SNP exclusions/inclusions No Yes
SNP design score No Yes
20
LDSelect output for IL-2 SeattleSNPs, r20.80,
MAF 0.05, Caucasians
Bin Total Number of Sites TagSNPs
1 2 rs2069763 rs2069772
2 2 rs2069776 rs2069778
3 2 rs2069777 rs2069779
4 1 rs2069762
21
Genomic context
  • Exons (cSNPs)
  • SIFT (Ng, 2002)
  • PolyPhen (Ramensky, 2002)
  • Upstream flanking region
  • Intron-exon junctions

22
Sequence conservation
  • UCSC Genome Browser, PhasCons (Siepel, 2005)

Score
Repeat region
Unique region
23
Objectives
  • Molecular genetics primer
  • Databases and tools to conduct in silico analyses
    for tagSNP selection/prioritization
  • Factors influencing statistical power

24
Minor allele frequency and genetic model
300 cases, 300 controls, alpha0.05
25
LD
SNPs genotyped SNPs not genotyped r2 Sample size requirement Sample size requirement
S1 and S2 - - 600 600
S1 S2 1.00 600 600
S1 S2 0.85 600 706
S1
S2
  • N/r2 (Pritchard, 2001)

26
Genotype error
  • Generally non-differential
  • Reduces your power
  • Every 1 increase in genotyping error rates
    requires sample size increased by 2-8 (Zou
    et al, 2004, Genetic Epidemiology)
  • Depends on error model

27
Power calculators
  • Quanto
  • G, E, G X E, G X G
  • Case-control, case-sibling, case-parent, and
    case-only designs
  • Quantitative or binary outcome
  • htPowercc
  • r2
  • Power for Association With Error (PAWE)
  • Genotyping errors

28
TagSNP summary
  • Efficient yet comprehensive coverage of the
    genetic variation in our candidate genes
  • Reduce costs
  • Preference should be given to putatively
    functional variants
  • Literature, gene context, sequence conservation
  • Influences of statistical power
  • MAF, genetic model, LD, and genotyping error

29
  • Programs for Genomic Applications
  • SeattleSNPs, http//pga.mbt.washington.edu
  • NIEHS, http//egp.gs.washington.edu/
  • Innate Immunity, http//innateimmunity.net/
  • International HapMap, http//www.hapmap.org/
  • Coriell cell repository, www.coriell.org
  • cSNP predictive analysis
  • SIFT, http//blocks.fhcrc.org/sift/SIFT.html
  • PolyPhen, http//coot.embl.de/PolyPhen
  • Vista, http//genome.lbl.gov/vista/index.shtml
  • The following programs can be found at the
    Rockefeller site, http//linkage.rockefeller.edu/s
    oft/
  • Tagger
  • LDSelect
  • PAWE
  • Quanto
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