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Selecting TagSNPs in Candidate Genes for Genetic Association Studies

Selecting TagSNPs in Candidate Genes for Genetic Association Studies. Shehnaz K. Hussain, PhD, ScM Assistant Professor Department of Epidemiology, UCLA skhussain@ucla.edu Epidemiology 244: Cancer Epidemiology Methods. Objectives. Molecular genetics primer

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Selecting TagSNPs in Candidate Genes for Genetic Association Studies

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  1. Selecting TagSNPs in Candidate Genes for Genetic Association Studies Shehnaz K. Hussain, PhD, ScM Assistant Professor Department of Epidemiology, UCLA skhussain@ucla.edu Epidemiology 244: Cancer Epidemiology Methods

  2. Objectives • Molecular genetics primer • Databases and tools to conduct in silico analyses for tagSNP selection/prioritization

  3. A T C G DNA mRNA Protein Central dogma

  4. 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

  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 TTCG TA TATTCGGGTAAA ?T?G?A A TGGAA

  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. IL - 2 IL - 2 gene IFN IFN IFN γ γ γ gene gene gene IL IL IL - - - 2 2 2 receptor receptor receptor Proliferation Proliferation Proliferation of TH1 of TH1 of TH1 - - - cells cells cells IFN IFN IFN γ γ γ Activated T - cell T-cell proliferation IL IL - - 2 2 IL IL - - 2 2 gene gene Activated T Activated T - - cell cell

  11. 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

  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 in–person interviews • DNA isolated from buffy coats

  14. 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)

  15. 1. Select reference panel • 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

  16. Amplify and Sequence DNA Gene Phred Phrap (Ewing, 1998) (Ewing, 1998) PolyPhred (Nickerson, 1997) 2. Re-sequence reference panel

  17. 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

  18. 3. Choose tagSNPs

  19. LDSelect output for IL-2SeattleSNPs, r2≥0.80, MAF ≥0.05, Caucasian

  20. Genomic context • Exons (cSNPs) • SIFT (Ng, 2002) • PolyPhen (Ramensky, 2002) • Upstream flanking region • Intron-exon junctions

  21. Sequence conservation • UCSC Genome Browser, PhasCons (Siepel, 2005) Score Repeat region Unique region

  22. 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

  23. Thanks for your attention! Questions?

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