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Cis - regulatory SNPs altering transcription detected by allelic expression mapping

Cambridge, July 16, 2010. Cis - regulatory SNPs altering transcription detected by allelic expression mapping. Tomi Pastinen, MD, PhD Assistant Professor Departments of Human and Medical Genetics, McGill University McGill University and Genome Quebec Innovation Centre. Outline.

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Cis - regulatory SNPs altering transcription detected by allelic expression mapping

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  1. Cambridge, July 16, 2010 Cis-regulatory SNPs altering transcription detected by allelic expression mapping Tomi Pastinen, MD, PhD Assistant Professor Departments of Human and Medical Genetics, McGill University McGill University and Genome Quebec Innovation Centre

  2. Outline • Allelic expression: principle and methodology • Catalogs of cis-regulatory SNPs (cis-rSNPs) • Applications

  3. Regulatory SNPs & Phenotypes Coding variant Non-coding variant Coding variant Non-coding variant Type 1 Diabetes Asthma

  4. relative allele ratios can be used as quantitative trait for mapping local cis-regulatory variation in phased samples Allelic Expression (AE) mRNA (or pre-mRNA) T Expected equal expression of allelic transcripts T T C C 1 1 = C T T T T 2 1 Observed biased allelic expression C C =

  5. dECOUPLINGcisvs. trans Regulation

  6. Global expression/AE analysis by NGS

  7. Polymorphism targeted AE analysis

  8. Mapping cis-variation by AE test T T T Population panel of cells (CEU & YRI LCLs from HapMap) Illumina Human 1M Duo (currently 2.5 M quad) C T T Allelic Expression (AE) Measurement C C AE mapping in phased chromosomes A B A B A A AE association +ve cis-regulatory SNP (cis-rSNP) AE association -ve

  9. Variability of allele ratio [b = Y/(Y+X)] needs to be accounted for when comparing cDNA to gDNA Normalization of allele ratios cDNA cDNA gDNA gDNA

  10. Reproducibility of quantitative AE We use heterozygote ratio difference in phased gDNA and cDNA genotype data Dhet ratio = RNAc1/(c1+c2) - DNAc1/(c1+c2) R2 = 0.72 in biological replicates (CEU LCL) R2 = 0.83 in technical replicates (CEU LCL) R2 = 0.76 in biological replicates in same environment (osteoblast) R2 = 0.61 in individual but in different cell culture condition (osteoblast)

  11. USE of ae-phenotypes in mapping rSNPs • single point allelic expression is noisy • heterozygosity low using coding SNPs only Dhet ratio phased RNA (cDNA) and genomic DNA (gDNA) genotyping data from same individual are averaged across multiple sites in primary transcripts P = 2x10-9 BA BB/AA AB C1C2 genotype

  12. Full Transcript (AFF3 = ~600Kb), 5’ Association Heritable Allelic expression (1) A A   B B   AE measurements across large genes

  13. Differential 5’ Exon Usage (CUGBP2), 5’ Association Heritable Allelic expression (2) Allele-specific expression of long isoform

  14. Common SNPs govern cis-regulation • on average > 50% of population variance in cis-regulation can be explained by common SNPs in associated loci • 5-10x more fxn variation revealed as compared to cis-eQTL mapping • >90% of mapped cis-rSNPs behave as expected in the offspring (Mendelian inheritance)

  15. observe large effect sizes for associated variants common cis-variants affect >30% of measured RefSeq transcripts low-throughput methods show converging data for 75% of genome-wide significant AE mapping results, but diversity of mechanisms suggested Summary of AE Associations (CEU) Ge et al., Nature Genet. 2009

  16. Transcription Altering cis-rSNPs 1) Large effect size (> 1.2-fold difference between cis-rSNP heterozygotes) across full length transcripts 2) Most SNPs (>75%) of all available SNPs in primary transcript above signal cut-off 3) Consistent allelic effects across introns and exons of the primary transcript (for transcripts fulfilling criteria 1+2, the proportion with exon – intron r2 > 0.3 is >90%)

  17. Transcriptional Cis-rSNPs in 3 Panels • ~17% of genes

  18. of top cis-eQTLs up to 50% of AE-mapping data show converging cis-rSNP; but given the high discovery rate only ~10% of cis-rSNPs yield significant cis-eQTL (Ge et al. 2009) But comparison of AE mapping data in YRI LCLs vs. YRI RNA-seq. data shows converging effects for vast majority of transcriptional cis-rSNPs Comparison to cis-eQTLs

  19. 1000 Genomes & Catalogs of Cis-rSNPs 11 CEU SNP YRI SNP • simple scoring based on deviation from expected heterozygosity among samples showing unequal/equal AE Fine-map region of shared association to look for causal cis-rSNPs 6.8 CEU -log10(P-value) 6.3 YRI -log10(P-value) Instead of resequencing this region, use 1000 Genomes data CEU+YRI -log10(P-value) 96 14 94 % 1000G sites scored % HapMap 0-score sites captured 92 12 90 10 88 8 86 6 84 82 4 -5 -4 -3 -2 -1 0 1000G Score Cutoff

  20. Cis-rsnP localization (1)

  21. Cis-rSNPLocalization (2) Overlapping transcription altering cis-rSNP Pperm < 0.001 in FB Pperm > 0.001 in FB

  22. Cis-variation subtypes (1) 5’ proximal cis-rSNPs altering regulation of DISC1 in a cell type independent manner Most common type of cis-rSNPs

  23. Cis-variation subtypes (2) 5’ distal cis-rSNPs altering regulation of PTGER4 in a cell type dependent manner 3rd most common type of cis-rSNPs

  24. Cis-variation subtypes (3) 3’ distal cis-rSNPs altering regulation of EFNA5 in a cell type dependent manner least common type of cis-rSNPs 5’ distal cis-rSNPs altering regulation of EFNA5 in a cell type dependent manner

  25. Wg-ENCODE vs. 1000G fine-mapped AE

  26. Cis-rSNP validation rs17658686CG In vitro validation of intronic enhancer rSNP (rs909685) Input C*+non specificcompetitor G*+non specificcompetitor C*-nuclea extraction G*+G competitor C*+G competitor G*+C competitor C*+C competitor C*-competitor G*-competitor FAIRE In vitro validation of promoter rSNP (rs344071) MNase Allele-specific DNA-protein interactions

  27. 5’proximal cis-rSNPValiDation(D. Sinnett)

  28. Function of Disease Variants Genetic association Functional association Ge et al. Nature Genetics 2009

  29. Mechanisms of rSNP Action (1) Functional association Potential mechanism Verlaan et al. AJHG, 2009

  30. Mechanisms of rSNP Action (2)

  31. Dissection of GWAS associations Examples: Preliminary observations: Creutzfeld-Jacob’s disease: PRNP LDL cholesterol: HMGCR CRP levels: IL6R Crohn’s disease: IL23R Plasma homocysteine: CBS Tooth development: HOXB2 CIS-rSNPs POTENTIALLY EXPLAINING DISEASE ASSOCIATIONS ARE ENRICHED FOR TISSUE SPECIFIC VARIANTS

  32. common haplotypes harbor functional alleles altering cis-regulation in most human genes • cis-regulatory SNPs altering transcription can be characterized by: • specific assessment of population variation in cis-regulation (AE-mapping) • fine-mapping using sequenced genomes (1000G/imputation for common variants) • intersection with functional genomic data (ENCODE) • regulatory variation in complex genomic regions (overlapping transcripts), or causing post-transcriptional effects require other tools (strand-specific assays/RNA-seq.) • large-scale, orthogonal validation tools need to catch up with mapping Conclusions

  33. Acknowledgements McGill University and Génome Québec Innovation Centre • McGill University • Mathieu Blanchette • Brent Richards • Anna Naumova • SoizikBerlivet • SannyMoussette • Université de Montréal • Daniel Sinnett • Nina N’Diaye • Manon Ouimet • Vincent Gagné • Patrick Beaulieu • Robert Hamon • Illumina Inc. • Dmitry Pokholok • Jennie Le • Kevin Gunderson • GEFOS Consortium • 1000 Genomes Project • ENCODE Project • Southwest Foundation for Biomedical Research • Harald H.H. Göring • Harvard Medical School • Benjamin Raby • Scott Weiss • Jehyuk Lee • George M. Church Pastinen Lab Tony Kwan, Véronique Adoue, Lisanne Morcos, Dominique J Verlaan, Tomi Pastinen, Elin Grundberg, Vonda Koka, Kevin Lam, Bing Ge Alexandre Montpetit, Eef Harmsen, Joana Dias, Rose Hoberman, Ken Dewar

  34. Systematic annotation of cis-rSNPs Potential new transcript RLBP1L1 • top associated SNP from AE-mapping • highest scoring 1000 Genomes site

  35. Systematic annotation of cis-rSNPs Region of active chromatin Histone marks are tissue-specific Region for RNA polymerase 2 binding Highly conserved regions of regulatory potential

  36. REMINDER: not all AE is Mendelian But most comprehensive survey of imprinting to date suggests that <100 imprinted loci exist as compared to thousands of loci modulated by cis-rSNPs Morcos et al. manuscript in prep.

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