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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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Cambridge july 16 2010

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

Outline

  • Allelic expression: principle and methodology

  • Catalogs of cis-regulatory SNPs (cis-rSNPs)

  • Applications


Regulatory snps phenotypes

Regulatory SNPs & Phenotypes

Coding

variant

Non-coding

variant

Coding

variant

Non-coding

variant

Type 1

Diabetes

Asthma


Allelic expression ae

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

=


Decoupling cis vs trans regulation

dECOUPLINGcisvs. trans Regulation


Global expression ae analysis by ngs

Global expression/AE analysis by NGS


Polymorphism targeted ae analysis

Polymorphism targeted AE analysis


Mapping cis variation by ae test

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


Normalization of allele ratios

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


Reproducibility of quantitative ae

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)


Use of ae phenotypes in mapping rsnps

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


Heritable allelic expression 1

Full Transcript (AFF3 = ~600Kb), 5’ Association

Heritable Allelic expression (1)

A

A

B

B

AE measurements across large genes


Heritable allelic expression 2

Differential 5’ Exon Usage (CUGBP2), 5’ Association

Heritable Allelic expression (2)

Allele-specific expression of long isoform


Common snps govern cis regulation

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)


Summary of ae associations ceu

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


Transcription altering cis rsnps

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%)


Transcriptional cis rsnps in 3 panels

Transcriptional Cis-rSNPs in 3 Panels

  • ~17%

    of genes


Comparison to cis eqtls

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


1000 genomes catalogs of cis rsnps

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


Cis rsnp localization 1

Cis-rsnP localization (1)


Cis rsnp localization 2

Cis-rSNPLocalization (2)

Overlapping transcription altering cis-rSNP

Pperm < 0.001 in FB

Pperm > 0.001 in FB


Cis variation subtypes 1

Cis-variation subtypes (1)

5’ proximal cis-rSNPs

altering regulation

of DISC1 in a cell type

independent manner

Most common type of

cis-rSNPs


Cis variation subtypes 2

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


Cis variation subtypes 3

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


Wg encode vs 1000g fine mapped ae

Wg-ENCODE vs. 1000G fine-mapped AE


Cis rsnp validation

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


5 proximal cis rsnp validation d sinnett

5’proximal cis-rSNPValiDation(D. Sinnett)


Function of disease variants

Function of Disease Variants

Genetic

association

Functional

association

Ge et al. Nature Genetics 2009


Mechanisms of rsnp action 1

Mechanisms of rSNP Action (1)

Functional association

Potential mechanism

Verlaan et al. AJHG, 2009


Mechanisms of rsnp action 2

Mechanisms of rSNP Action (2)


Dissection of gwas associations

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


Conclusions

  • 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


Acknowledgements

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


    Systematic annotation of cis rsnps

    Systematic annotation of cis-rSNPs

    Potential new transcript

    RLBP1L1

    • top associated SNP from AE-mapping

    • highest scoring 1000 Genomes site


    Systematic annotation of cis rsnps1

    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


    Reminder not all ae is mendelian

    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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