1. Nathaniel Gustafson Dr. Garry Larson (City of Hope)

2. Cancer Studies Can we tie genetic variation (eg. SNPs) to Cancer Risk? Myriad Genetics found BrCa1 and BrCa2 Mutations in BrCa1/2 tied to 800% increase in breast cancer risk Most research is on exonic regions Changes protein composition http://members.cox.net/amgough/Fanconi-genetics-genetics-primer.htm

3. Our approach What about regulatory regions? Motif: Recurring sequence, usu. 6-20 bp. Generally functional Hypothesis: Regulatory motifs upstream of the transcriptional start site (TSS) may play some role in breast cancer stopATG 3` 5` 5` - upstream YGCGYRCGC ATCMNTCCGY TGAYRTCA GCTNWTTGK...

4. Disease Mutations in Phylogenetically Conserved Motifs Sequence phylogeny G A C C T A C T A C A Orthologous bases: identical by descent Nonorthologous Bases in red G A G C T A C T A C T ~5 myr G A C T T A A T T C A ~70 myr G A G C T A C - A G A ~300 myr G A G T T A A T G G T ~475 myr G A C C T T C T A C A BrCa Pt. Mutation

5. Background Meta-Analysis pools several brca ER+/-* studies Statistics used to find genes that have consistent differences in expression levels in ER+ vs ER- cell lines *ER = Estrogen Receptor – a common way of classifying breast cancer cells

6. GCCATnTT x 50 x 50 GCCATnTT x 9 x 9

7. Aims Investigate regulatory motifs for these genes Compare occurrences of each motif across gene sets Hypothesis: genes overexpressed in the same tumor type share motifs

8. Weak Results

9. Are we missing the signal? Old counting method 1510 -2000 -1500 -1000 -500 TSS ER+ < ER- gene set motif occurrences P-val:.30 ER+ > ER- gene set motif occurrences -2000 -1500 -1000 -500 TSS NOT significant

10. Are we missing the signal? New counting method: use position bias 123 -2000 -1500 -1000 -500 TSS ER+ < ER- gene set motif occurrences P-val:.03 ER+ > ER- gene set motif occurrences -2000 -1500 -1000 -500 TSS significant

11. Tools Perl Handy scripting language Great for parsing textual data mySQL Storage and retrieval of structured data www.yusoft.net/yu-graph/main/logo-mysql.jpg

12. Problems Lack of data specificity What do Xie’s pos. biases mean? Insufficient data Needed position of motif relative to TSS Improperly annotated data Position shown to be inconsistent Collaboration Norway is about 10 time zones away

13. Results Motif 1down count 1up cnt 5down count 5up cnt pos. bias Pval top1 Pval top5 SCGGAAGY583671-240.401160.00019... Motif 1down count 1up cnt 5down count 5up cnt pos. bias Pval Top1 Pval top5 SCGGAAGY 3141168206-240.102990.00719... Reading 100 bp from positional bias No window (Previous results)

14. Any SNPs in this motif? One SNP was found from HapMap in this motif But it was at a degenerate position (eg. Y = C or G) = still satisfied the motif Might still affect expression

15. Biological Significance SCGGAAGY found more in ER+ overexpressed genes Known as a binding site for ELK-1 Might provide some insight into ER+/ER- cell differentiation Verification in vivo remains to be done

16. 3’UTR Motif List -6/7mer miRNA seeds -Phylogenetic conser. motifs 3’UTR Motif List -6/7mer miRNA seeds -Phylogenetic conser. motifs HapMap BrCa GWAS Datasets BrCa GWAS Datasets Hunter, et al (CGEMS) Hunter, et al (CGEMS) Gold, et al. (MSKCC) Gold, et al. (MSKCC) Easton, et al.(UK) (unavailable) Easton, et al.(UK) (unavailable) Stacey (deCode) (unavailable) Stacey (deCode) (unavailable) SNP_list SNPs Rank & Biological Testing SNPs Rank & Biological Testing BrCa Somatic Mutations (Sjöblom) BrCa Somatic Mutations (Sjöblom) Linkage Studies in BrCa (Smith, et al.) Linkage Studies in BrCa (Smith, et al.) LOH (aCGH) in BrCa LOH (aCGH) in BrCa Thermodynamic Profiling (STarMir, PITA) Thermodynamic Profiling (STarMir, PITA) In-House Independent Association Studies In-House Independent Association Studies 3’UTR-luc Fusion Assay Reciprocal Allelic Testing-Effect Evolutionary Conser- vation (miRNA seeds) Evolutionary Conser- vation (miRNA seeds) LD Mapping Proxy SNPs Allele frequency in HapMap Population(s) Allele frequency in HapMap Population(s) Reciprocal Allelic testing-no effect Additional Biological Testing

17. GWAS “Genome Wide Association Study” Genotypes cases and controls at thousands of loci Intended to be an unbiased approach Potentially identifies pertinent mutations http://www2.bioinformatics.tll.org.sg/img /species/karyotype_Homo_sapiens.png

18. StudyAssay Platform Cases/ Controls Comment_1Comment_2Public Dataset Hunter, et al. (Nat Genet 39, 2007) Illumina Hap 550 (keep 528K) 1,145 / 1,142Prospective, post- menopausal women Logistic Regression YES (CGEMS) Easton, et al. (Nature 447, 2007) Affy, 266k SNPs (keep 227k) Stage I - 380 / 364 Stage 2-3,990 /3,916 ctrls Stage 3- 21,860/22,578 ctrls Stage 1-Cases (  2 first-degree relatives with Fam Hx) 3 stage association Stage 2-top 5% of stage 1 Stage 3-Top 30 SNPs from Stage 2 TNRC9 high score NO Stacey, et al (Nat Genet 39, 2007) deCode Dataset Illumina Hap300 (keep 311k) 1,600 Icelandic cases/ 11,563 ctrls Top 10 SNPs GTP’d in 2 nd Icelandic sample and 2-3 ind. European cohorts 1 SNP strong LD with 999  5 BRCA2- removed from study Found SNP near TNRC9 NO Gold, et al. (PNAS 105 March, 2008) Affy GTP 435K SNPs (keep 150k) 249 AJ Fam Hx (  3 cases, BRCA1 & 2 neg) vs. 299 Ca-free AJ ctrls 3 stage designReproduced FGFR2 region MAYBE? BrCa GWAS Datasets

19. StudyAssay Platform Cases/ Controls Comment_1Comment_2Public Dataset Hunter, et al. (Nat Genet 39, 2007) Illumina Hap 550 (keep 528K) 1,145 / 1,142Prospective, post- menopausal women Logistic Regression YES (CGEMS) Easton, et al. (Nature 447, 2007) Affy, 266k SNPs (keep 227k) Stage I - 380 / 364 Stage 2-3,990 /3,916 ctrls Stage 3- 21,860/22,578 ctrls Stage 1-Cases (  2 first-degree relatives with Fam Hx) 3 stage association Stage 2-top 5% of stage 1 Stage 3-Top 30 SNPs from Stage 2 TNRC9 high score NO Stacey, et al (Nat Genet 39, 2007) deCode Dataset Illumina Hap300 (keep 311k) 1,600 Icelandic cases/ 11,563 ctrls Top 10 SNPs GTP’d in 2 nd Icelandic sample and 2-3 ind. European cohorts 1 SNP strong LD with 999  5 BRCA2- removed from study Found SNP near TNRC9 NO Gold, et al. (PNAS 105 March, 2008) Affy GTP 435K SNPs (keep 150k) 249 AJ Fam Hx (  3 cases, BRCA1 & 2 neg) vs. 299 Ca-free AJ ctrls 3 stage designReproduced FGFR2 region MAYBE? BrCa GWAS Datasets YES

20. Bring this... SM70 SG74 LF52 SM17 SH14 L5721 SM56 SF63 L5957 L5349 L5420 L5713 SH5 LF48 SJG4 L6029 SG21 L5352 L6121 SG69 L5952 SM78 SM113 SF23 L5573 SN6 SF1 SM91 L5895 L5518 L5501 L5328 L5772 SG08 SG28 SM52 SM106 SM67 L5463 L5494 SA17 L5796 L6014 SN15 rs2180341 chr6 127642323 + ncbi_b35 MSKCCOffit AffyEAv3 PhaseIGold_et_al TT TT TT CT CT CC TT CT TT TT TT TT CT TT CT TT CT TT TT CT TT TT TT CT CT CT CT CT CT CT TT CT CT TT TT CT CC TT TT CT TT TT CT TT CT CT CT CT TT TT TT CT CT CT TT TT CT TT TT TT TT TT TT CT TT TT TT TT CT TT TT CT TT TT TT CT CT TT CT CT TT CT TT... rs6569480 chr6 127663441 + ncbi_b35 MSKCCOffit AffyEAv3 PhaseIGold_et_al GG GG GG GG AG AA GG AG GG GG GG GG AG GG GG GG AG GG GG AG GG GG GG AG AG AG AG AG AG AG GG AG AG GG GG AG AA GG GG AG GG GG AG GG AG AG AG AG GG GG GG AG AG AG GG GG AG GG GG NN GG GG GG AG GG GG GG GG AG GG GG AG GG GG GG AG GG GG AG AG GG AG GG... rs_num chrpos analysis_name p_valueOR_hetOR_hombuild rs10510126 chr10124992475chi square - genotype 4e-060.59180.6387 ncbi_b36 rs10510126 chr10124992475chi square - allele 2e-060.59180.6387 ncbi_b36 To this...

21. Future Work We’ve digested the Gold data set Employ this in the triage for producing a gene list Combine with other triage methods to find the most interesting genes Test these in vivo

22. Special Thanks Dr. Garry Larson SoCalBSI program SoCalBSI mentors City of Hope Funding Komen for the Cure National Science Foundation National Institute of Health Employment and Workforce Development

23. References Xie X, Mikkelsen TS, Gnirke A, Lindblad-Toh K, Kellis M, Lander ES. Systematic discovery of regulatory motifs in conserved regions of the human genome, including thousands of CTCF insulator sites. Proc Natl Acad Sci U S A. 2007 Apr 24;104(17):7145-50. D. Smith, P. Sætrom, O. Snøve Jr, C. Lundberg, G. Rivas, C. Glackin and G. Larson. Meta-analysis of breast cancer microarray studies in conjunction with conserved cis-elements suggest patterns for coordinate regulation. BMC Bioinformatics 2008, 9:63