Post on 14-Feb-2017
transcript
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