Two step epigenetic Mendelian randomization:

a strategy for establishing the causal role of epigenetic processes in pathways to disease

Caroline L Relton & George Davey Smith

Int J Epidemiol 2012;41:161-176

Summary

• Epigenetic marks, like any other molecular biomarker are vulnerable to confounding and reverse causation

• Mendelian randomization (MR) provides a strategy for distinguishing causal associations from non-causal

• MR has been used extensively in the context of assessing the causal relationship between environmental exposures and disease risk

• Here we describe how it can be extended and applied to epigenetic data

The principle of Mendelian randomization

E Y

G

U

(A)

Body mass

index

Blood

pressure

FTO

SNP

U

(B)

The principle of mediation

E X Y

U1

U2

A two-step approach

Exposure Phenotype

SNP 1

CpG

(A) Step 1

Exposure Phenotype

SNP 2

CpG

(B) Step 2

Smoking and cardiovascular disease

Smoking

CVD

F2RL3

SNP

U

F2RL3

CpG E Y

G

U

X

(B) Step 2

E Y

G

U

X

(A) Step 1

Smoking

CVD

CHRN3/5

SNP

U

F2RL3

CpG

Identifying the proxy SNP for step 1

Smoking and cardiovascular disease: step 1

Smoking

CVD

CHRN3/5

SNP

U

F2RL3

CpG

Identifying the proxy SNP for step 2

1. Confirm location of CpG site

• CpG is located in exon 2 of F2RL3

Identifying the proxy SNP for step 2

2. Identify SNPs in vicinity of CpG site

• 29 SNPs within 100 Kb either side of CpG site

Identifying the proxy SNP for step 2

3. Construct LD plot of region of interest

• Data from 205 individuals of European ancestry)

• Plot of region 100 kb upstream and downstream of cg03636183. Values in diamonds are r2 values. Diamond colours represent LOD and D’ (white: D’ <1; shades of grey: D’=1, LOD <2; black: D’= 1, LOD≥ 2.

• SNPs included are on the Illumina 550 SNP array.

Identifying the proxy SNP for step 2

4. Identify tag SNPs and assess correlation with CpG site methylation

• Use existing genotype data if available

• Impute additional genotypes across region if appropriate and assess correlation with CpG site methylation

• Undertake custom genotyping if genotype data is unavailable or imputation is not possible

• Establish per allele effect upon CpG site methylation level

Smoking and cardiovascular disease: step 2

Smoking

CVD

F2RL3

SNP

U

F2RL3

CpG

Applying the two-step approach to in utero exposures and offspring outcomes

Em Yo

Gm

U

Xo

(A) Step 1

Alcohol in

pregnancy

Offspring

cognition

ADH1B

SNP

U

CpG

Em Yo

Go

U

Xo

(B) Step 2

Alcohol in

pregnancy Offspring

cognition

cis

SNP

U

CpG

Using multiple instruments

Exposure Phenotype

Ga, Gb, Gc

CpG

(B) Step 2

Exposure Phenotype

G1, G2, G3

CpG

(A) Step 1

Gene functions of smoking-sensitive loci

Wan ES et al. Hum Mol Genet Apr 2012

F2RL3 Platelet activation, cell signalling

GPR15 Shares homology with angiotensin AT1 and AT2 receptors

LRRN3 Neurodevelopment

LIM2 Lens development and cataract formation

MYLK Endothelial and epithelial barrier function, asthma

ADHFE1 Alcohol metabolism

CNTNAP2 Neurodevelopment

SLAMF1 Bi-directional T-cell to B-cell stimulation

APBA2* Neuronal adaptor protein that stabilises amyloid precursor protein

CEBPE Transcription factor

TIPARP Adaptive response to chemical exposure?

TM4SF19 Function unknown; putative bipolar disorder susceptibility region

ARHGAP25 GTPase activator, signal transduction

FASLG Cell signalling, apoptosis

* 2 CpG sites associated

Smoking and bipolar disorder

Smoking

Bipolar

disorder

TM4SF19

SNP

U

TM4SF19

CpG E Y

G

U

X

(B) Step 2

E Y

G

U

X

(A) Step 1

Smoking

Bipolar

disorder

CHRN3/5

SNP

U

TM4SF19

CpG

Gene functions of smoking-sensitive loci

Wan ES et al. Hum Mol Genet Apr 2012

F2RL3 Platelet activation, cell signalling

GPR15 Shares homology with angiotensin AT1 and AT2 receptors

LRRN3 Neurodevelopment

LIM2 Lens development and cataract formation

MYLK Endothelial and epithelial barrier function, asthma

ADHFE1 Alcohol metabolism

CNTNAP2 Neurodevelopment

SLAMF1 Bi-directional T-cell to B-cell stimulation

APBA2* Neuronal adaptor protein that stabilises amyloid precursor protein

CEBPE Transcription factor

TIPARP Adaptive response to chemical exposure?

TM4SF19 Function unknown; putative bipolar disorder susceptibility region

ARHGAP25 GTPase activator, signal transduction

FASLG Cell signalling, apoptosis

* 2 CpG sites associated

Smoking in pregnancy and offspring neurodevelopment

Em Yo

Gm

U

Xo

(A) Step 1

Smoking in

pregnancy

Offspring

Neurodevelopment

CHRN3/5

SNP

U

LRRN3 CpG

CTNAP2 CpG

LRRN3

SNP

U

CNTNAP2

SNP

Smoking in

pregnancy

Offspring

Neurodevelopment LRRN3 CpG

CTNAP2 CpG Em Yo

Go

U

Xo

(B) Step 2

Go

Strengths and limitations

Strengths

• MR very well-established

• Two steps can be used independently

• Many exposure-methylation associations have been reported

• Cis variation in methylation studies suggests SNP for use as instruments will be located near to CpG sites

Limitations

• No concrete published examples as yet

• Essential to define robust instruments for 2nd step

• Relationship between methylation and underlying genetic sequence is not well characterised

• Tissue specificity (Numata et al)

• Power?

• Pleiotropy