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Selecting TagSNPs in Candidate
Genes for Genetic Association Studies
Shehnaz K. Hussain, PhD, ScMAssistant ProfessorDepartment of Epidemiology, [email protected]
Epidemiology 244: Cancer Epidemiology Methods
Objectives
Molecular genetics primer
Databases and tools to conduct in silico analyses for tagSNP selection/prioritization
Central dogma
DNA
Protein
mRNA
A T C G
What are SNPs?
More than 99% of all nucleotides are the same in all humans
1% of nucleotides are polymorphic SNPs>> insertions-deletions
Bi-nucleotide – T (80%) A (20%)
Where do SNPs occur? Exons
Introns
Flanking regions
? T ? G ? A
? T ? G ? A
A T G G A A
T T C G T A
What are haplotypes?
A haplotype is the pattern of nucleotides on a single chromosome
Two “copies” of each chromosome
The haplotype inference problem
TA TT CG GG TA AA
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
No LD LD
Disease Susceptibility
Locus
Disease Phenotype
Test for genetic association between the phenotype and the DSL
Marker loci (SNPs)
LD
Test for association between phenotype and
marker loci
What are markers?
Candidate gene
What are tagSNPs?
TagSNPs are a subset of all SNPs in a gene that mark groups of SNPs in LD
Avoids redundant genotyping
Disease Susceptibility
Locus
LD LD
Marker loci (SNPs)
The joint effect of tagSNPs in cytokine genes and cigarette
smoking in cervical cancer risk
T-cell proliferation
IL-2 gene
IFNγ gene
Activated T-cell
Proliferation of TH1-cells
IL-2
IFNγ
IL-2 receptor
IL-2 gene
Activated T-cell
IL-2
IL-2 gene
Activated T-cell
Proliferation of TH1-cellsProliferation of TH1-cells
IL-2
IL-2 receptorIL-2 receptor
IFNγ gene
IFNγ
IFNγ gene
IFNγ
Background
Cigarette smoking ↑ 1.5- to 3-fold cancer risk
Cigarette smoking ↓ levels of IL-2 and IFNγ (cervical and circulating)
↓ levels of IL-2 and IFNγ HPV persistence in the cervix
Cervical neoplasia
Decreased survival from invasive cervical cancer
Cigarette smoking
HPV-associated squamous cell cervical cancer
Model
SNPs in IL-2, IL-2R, and IFNG
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 in–person interviews DNA isolated from buffy coats
Methods
Multi-stage tagSNP design
Re-sequence panel, identify SNPs (many markers, few subjects)
Choose tagSNPs
Genotype tagSNPs in main study(few markers, many subjects)
Select reference panel
A sample of your study population Most representative
Samples from the Coriell Repository Ability to integrate your data with other
resources
1. Select reference panel
= Candidate gene SNPs = HapMap SNPs
2. Re-sequence reference panel
PolyPhred
Phred Phrap
(Nickerson, 1997)
(Ewing, 1998) (Ewing, 1998)
Amplify and Sequence DNA
Gene
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
3. Choose tagSNPs
Option LDSelect(Carlson, 2002)
Tagger(de Bakker, 2005)
r2 threshold Yes Yes
SNP exclusions/inclusions No Yes
SNP design score No Yes
LDSelect output for IL-2
SeattleSNPs, r2≥0.80, MAF ≥0.05, Caucasian
BinTotal Number
of SitesTagSNPs
1 2rs2069763 rs2069772
2 2rs2069776 rs2069778
3 2rs2069777 rs2069779
4 1 rs2069762
Exons (cSNPs) SIFT (Ng, 2002) PolyPhen (Ramensky, 2002)
Upstream flanking region Intron-exon junctions
Genomic context
Sequence conservation
Repeat region Unique region
UCSC Genome Browser, PhasCons (Siepel, 2005)
Sco
re
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
Thanks for your attention!
Questions?