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Progress in Genotyping of Candidate Genes

This article discusses the progress made in genotyping candidate genes in the MESA Family Study, including the expanded candidate gene study, phase 1 and phase 2 genotyping, gene selection, and SNP selection.

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Progress in Genotyping of Candidate Genes

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  1. Progress in Genotyping of Candidate Genes Kent Taylor

  2. History Genetic Studies in MESA Family • MESA Family grant originally proposed to genotype 6-8 candidate genes with ~80 SNPs in MESA (parent study) AND • Genome scan in MESA Family subjects • Rapid technological developments allow a project of greater scope for the same budget

  3. Expanded Candidate Gene Study • Conduct an association study of 2,880 MESA participants (parent study) • 720 randomly selected from each ethnic group of Caucasian-, African-, Mexican-, and Chinese-Americans • Include the well-phenotyped “MESA 1000” • Genotype 1536 SNPs in candidate genes proposed by MESA Investigators, TWICE

  4. Candidate Genes • Phase 1 • 120 candidate genes • 1536 SNPs including 96 AIMs • 4 ethnic groups (~720 in each) • Phase 2 • Selection of another 1536 SNPs • Same study subjects as Phase 1 • Newly identified and untyped Phase 1 genes

  5. Phase 2 Genotyping • Additional SNPs to be genotyped using older technologies (being considered in the future) • ACE insertion/deletion, APOE • 1536 SNPs using Illumina run • DNA already at Illumina from Phase 1 • Some of same genes with additional SNPs • Additional genes proposed since Phase 1 was completed • Next 50 genes on the Phase 1 list • Additional AIMs

  6. Phase 2 Gene List

  7. Phase 1 Genotyping • Candidate genes and analyses • Focus of Mychaleckyj presentation (MESA Family Steering Committee meeting and MESA SC mtg) • High degree of performance • Few SNPs eliminated • High accuracy • Specific genes: focus of Bowden & Tsai presentations (MESA Family SC mtg) • Ancestry Informative Markers • 97 selected, 96 successfully genotyped • Focus of Arnett presentation (MESA Family SC mtg)

  8. Candidate Gene Selection • 267 candidate genes were initially proposed by MESA investigators, collated by DCC, and then ranked by MESA investigators • Further input by MESA laboratory and MESA Family Study Genetics Committee (294 genes) • Genes have continued to be added to list for the second 1536 genotyping run

  9. Selection of SNPs • SNPs proposed by MESA investigators • SNPs with feasibility of 80% or greater on Illumina platform • SNPs with minor allele frequency >0.05 • tagSNPs for Caucasian-American and African-American haplotype blocks as defined by Tagger or LDSelect for each candidate gene • List of SNPs sent to Illumina for evaluation • Candidate gene was re-analyzed for tagSNPs if necessary after feasibility scores were returned from Illumina • Ancestry informative SNPs added to panel (96)

  10. Data Overview • 1440/1536 SNPs were successfully genotyped • 119 genes represented • 96 ethnic-specific markers • Only 6 DNA samples did not genotype • These same samples are available for the second round (Group 2) genotyping

  11. Phase 1 Genotyping Results • Illumina report: • Data quality was very high • DNA success rate unprecedented (for such a large project) • Being pleased with the locus conversion rate (higher than predicted) • Excellent DNA quality (aided in achieving the high locus success rate) • Non-blinded & blinded sample pair genotyping concordance rates: >99.99%

  12. Phase 1 Genotyping ResultsAPOE and ACE #SNPs Gene/Marker Set Picked Succeed Fail APOE 7 2 5 ADORA2A 14 10 4 3 SNP FAIL (6 Genes) 3 2 SNP FAIL (17 Genes) 2 1 SNP FAIL (35 Genes) 1 TOTAL FAIL 96 # SUCCESSFUL SNPS 1440 (93.75%) What to do about APOE ?

  13. APOE Requires: “Classic” 2, 3, 4 determination (restriction enzyme or dotting procedure) Additional SNPs to divide 3

  14. ACE I/D polymorphism Requires agarose or capillary electrophoresis

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