MESA FS SC Meeting Genetics Report 1 Illumina

MESA FS SC Meeting Genetics Report 1

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, MRPL 10) TOTAL SNPs ASSAYED BY ILLUMINA 1536 2

Illumina Marker Panel #2: Results of Genotyping #SNPs Succeed Gene/Marker Set Picked Fail APOE 7 2 5 ADORA 2 A 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%) 3

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

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

Analysis of Sex Mismatches 6

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 7

Other Evidence • X chromosome SNPs typed • 47 X chromosome SNPs • 31 in 4 candidate genes (AGTR 2, CD 40 LG, IRAK 1, TLR 7) • 16 in AIMs 8

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 9

Analysis of Duplicates 10

Analysis of Duplicates Genotyping Quality • Spike each of 32 (96 well) DNA 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 notified that these were duplicates (blinded) • Total: 64 + 92 = 156 duplicate pairs of DNAs 11

Analysis of Duplicates Illumina Non-Blinded I 64 x 1440 – 45 – 31 = 92, 084 genotype pairs (less missing 1 or 2 genotypes) Remove and do not count: 1 pair of clearly nonidentical 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. 12

Analysis of Duplicates Illumina 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 nonidentical samples: 717 / 1439 and 717 / 1438 SNPs mismatch (lab error? ). 4 other genotype pair mismatches Blinded pair concordance rate = (132310 -14381439 -4) /(132310 – 1438 -1439) = >99. 996% *1438 not 1440 – 2 missing SNP genotypes. 13

Initial Processing & Development 14

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 r 2 – Examination of LD block structure 15

16

Non-X SNPs p<0. 05/1393 (Bonf Corrected) EUA rs 3918232 NOS 3 AFA rs 352162 rs 3918232 rs 679620 rs 678815 rs 10507391 TLR 9 NOS 3 MMP 3 ALOX 5 AP HIS rs 2001660 rs 1022573 rs 1534649 rs 879293 rs 1243164 rs 6647 rs 225412 AIM-ETHNIC LPL PLAT SERPINA 1 ABCG 1 CHN rs 3918232 NOS 3 17

Links to Gene Pages • SNPs genotyped for the candidate • Basic information – SNP allele frequency – HWE • LD panel – D’ and r 2 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. , OLR 1) Gene name (Low Density Lipoprotein, Oxidized, Receptor 1) Genome location (12 p 13 -p 12) Description of function (the OLR 1 gene encodes a cellsurface endocytosis receptor for oxidized low density lipoprotein (Ox. LDL). 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 Ox. LDL though the OLR 1 receptor) 19

MESA Candidate Gene List – Entry Page Actual Inter-SNP Distance (successful SNPs) Typed = Successful Click to access gene details 20

Links to Page Detail: 1. Map Summary 2. Genotype Data (download) 3. Genotype Stats 4. etc. . Gene-specific links open browser to gene view in each of these databases (chromosome range) 21

22

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 23