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MESA FS SC Meeting

This report presents the results of genotyping for cardiovascular candidate genes using Illumina Marker Panels #1 and #2 in the MESA study. It includes information on marker sets, SNPs, genotyping quality, sex mismatches, duplicates, and initial data analysis.

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MESA FS SC Meeting

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  1. MESA FS SC Meeting Genetics Report

  2. Illumina Marker Panel #1: Design Marker Set # SNPs Cardiovascular Candidate Genes 1439 Ancestry Informative Markers (AIMs) 97 Mean Picked SNPs/gene 12.8 Max SNPs/gene 49 (VWF) Min SNPs/gene 1 (VEGFB, MRPL10) TOTAL SNPs ASSAYED BY 1536 ILLUMINA

  3. Illumina Marker Panel #2: Results of Genotyping #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%)

  4. DNA Samples Sent to Illumina Unique Samples 2880 Illumina non-Blinded Duplicates 2 per 32 plates = 64 Illumina Blinded Duplicates 4 (ethnicity) x 23 = 92 (plate 33) Total Duplicates 156 TOTAL SENT TO ILLUMINA 3036 TOTAL DNA PLATES 33

  5. The Illumina Data Set Samples SNPs Total Total Genotypes Attempted 3036 1536 4663296 *Specific Non-called Genotypes 184 Total Genotypes Returned 3030 1440 3030 x 1440 – 184 = 4,360,016 * Not due to sample failure

  6. Analysis of Sex Mismatches

  7. Analysis of Sex Mismatches • Compare sex of individual in MESA database with genetically determined sex • Illumina includes additional sex specific markers on X,Y chromosomes for each sample they assay • they do not share this data with customers • Illumina reported 12 discrepancies between MESA reported sex and genetic sex

  8. Other Evidence • X chromosome SNPs typed • 47 X chromosome SNPs • 31 in 4 candidate genes (AGTR2, CD40LG, IRAK1, TLR7) • 16 in AIMs

  9. Sex Mismatches • 47 X SNPs appear to confirm 11 / 12 Illumina genetic sex assignments • 7014627 (MESA Male, ILMN Female) has N(het) = 0 / 47 SNPs suggesting that the individual is Male. • Intriguingly, many of the low N(het) individuals are Male and CHN 47 / 57 individuals with N(het) = 1,2,3 are CHN • More work with CC required

  10. Analysis of Duplicates

  11. Analysis of DuplicatesGenotyping Quality • Spike each of 32 (96 well) DNA plates plates with 2 duplicate samples = 64 in total • Illumina notified of duplicates (non-blinded) • Add an extra plate (#33) of 4 ethnicities x 23 duplicates = 92 duplicates • Illumina not notified that these were duplicates (blinded) • Total: 64 + 92 = 156 duplicate pairs of DNAs

  12. Analysis of DuplicatesIllumina Non-Blinded I64 x 1440 – 45 – 31 = 92,084 genotype pairs (less missing 1 or 2 genotypes) Remove and do not count: 1 pair of clearly non-identical samples, mismatch at 628 / 1439 SNPs total typed (lab error ?). One other single pair of genotypes do not match Non-blinded pair concordance rate = (92084 -*1439-1) /(92084 – 1439) = >99.998% *1439 not 1440 – 1 missing SNP genotypes.

  13. Analysis of DuplicatesIllumina Blinded 92 x 1440 – 138 – 32 = 132,310 genotype pairs (less missing 1 or 2 genotypes) Remove and do not count: 2 pairs of clearly non-identical samples: 717 / 1439 and 717 / 1438 SNPs mismatch (lab error?). 4 other genotype pair mismatches Blinded pair concordance rate = (132310 -1438-1439-4) /(132310 –1438-1439) = >99.996% *1438 not 1440 – 2 missing SNP genotypes.

  14. Initial Processing & Development

  15. Initial Processing • Tests for Hardy-Weinberg Equilibrium • For each SNP; maintain all in analyses • Deviations from HWE • Genotyping errors • Deviations from underlying assumptions • No mutation, migration, selection • Small sample size (genetic drift) • True association with phenotype • Estimates of Linkage Disequilibrium • D’ and r2 • Examination of LD block structure

  16. Non-X SNPs p<0.05/1393 (Bonf Corrected) EUA rs3918232NOS3 AFA rs352162 TLR9 rs3918232NOS3 rs679620 MMP3 rs678815 MMP3 rs10507391 ALOX5AP HIS rs2001660 AIM-ETHNIC rs1022573 AIM-ETHNIC rs1534649 LPL rs879293 PLAT rs1243164 SERPINA1 rs6647 SERPINA1 rs225412 ABCG1 CHN rs3918232NOS3

  17. Links to Gene Pages • SNPs genotyped for the candidate • Basic information • SNP allele frequency • HWE • LD panel • D’ and r2 for SNPs in gene by ethnicity • Haploview representation • Link to permit data download • Link to initial data analyses

  18. Gene Pages • Establish a ‘portal of gene pages’ • Hosted by Coordinating Center & WFU & CSMC • Candidate gene on each ‘page’ • Gene symbol (e.g., OLR1) • Gene name (Low Density Lipoprotein, Oxidized, Receptor 1) • Genome location (12p13-p12) • Description of function (the OLR1 gene encodes a cell-surface endocytosis receptor for oxidized low density lipoprotein (OxLDL). LDL is oxidized in vascular endothelial cells to a highly injurious product that results in endothelial cell injury, which is implicated in the development of atherosclerosis. Vascular endothelial cells also internalize and degrade OxLDL though the OLR1 receptor)

  19. Actual Inter-SNP Distance (successful SNPs) Typed = Successful Click to access gene details MESA Candidate Gene List – Entry Page

  20. Links to Page Detail: • Map Summary • Genotype Data (download) • Genotype Stats • etc.. Gene-specific links open browser to gene view in each of these databases (chromosome range)

  21. MESA Gene Pages • Still in development • Investigating use of tabbed browsing (like amazon.com - for gene page detail) • Future enhancements: • Include a Gene Page section on association results • Suggestions ? Please contact us

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