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Meta-Analysis
. 2023 Jul;66(7):1247-1259.
doi: 10.1007/s00125-023-05914-7. Epub 2023 May 19.

Investigating causality in the association between DNA methylation and type 2 diabetes using bidirectional two-sample Mendelian randomisation

Diana L Juvinao-Quintero  1   2   3 , Gemma C Sharp  4   5 , Eleanor C M Sanderson  4   5 , Caroline L Relton  4   5 , Hannah R Elliott  6   7
Affiliations
Meta-Analysis

Investigating causality in the association between DNA methylation and type 2 diabetes using bidirectional two-sample Mendelian randomisation

Diana L Juvinao-Quintero et al. Diabetologia. 2023 Jul.

Abstract

Aims/hypothesis: Several studies have identified associations between type 2 diabetes and DNA methylation (DNAm). However, the causal role of these associations remains unclear. This study aimed to provide evidence for a causal relationship between DNAm and type 2 diabetes.

Methods: We used bidirectional two-sample Mendelian randomisation (2SMR) to evaluate causality at 58 CpG sites previously detected in a meta-analysis of epigenome-wide association studies (meta-EWAS) of prevalent type 2 diabetes in European populations. We retrieved genetic proxies for type 2 diabetes and DNAm from the largest genome-wide association study (GWAS) available. We also used data from the Avon Longitudinal Study of Parents and Children (ALSPAC, UK) when associations of interest were not available in the larger datasets. We identified 62 independent SNPs as proxies for type 2 diabetes, and 39 methylation quantitative trait loci as proxies for 30 of the 58 type 2 diabetes-related CpGs. We applied the Bonferroni correction for multiple testing and inferred causality based on p<0.001 for the type 2 diabetes to DNAm direction and p<0.002 for the opposing DNAm to type 2 diabetes direction in the 2SMR analysis.

Results: We found strong evidence of a causal effect of DNAm at cg25536676 (DHCR24) on type 2 diabetes. An increase in transformed residuals of DNAm at this site was associated with a 43% (OR 1.43, 95% CI 1.15, 1.78, p=0.001) higher risk of type 2 diabetes. We inferred a likely causal direction for the remaining CpG sites assessed. In silico analyses showed that the CpGs analysed were enriched for expression quantitative trait methylation sites (eQTMs) and for specific traits, dependent on the direction of causality predicted by the 2SMR analysis.

Conclusions/interpretation: We identified one CpG mapping to a gene related to the metabolism of lipids (DHCR24) as a novel causal biomarker for risk of type 2 diabetes. CpGs within the same gene region have previously been associated with type 2 diabetes-related traits in observational studies (BMI, waist circumference, HDL-cholesterol, insulin) and in Mendelian randomisation analyses (LDL-cholesterol). Thus, we hypothesise that our candidate CpG in DHCR24 may be a causal mediator of the association between known modifiable risk factors and type 2 diabetes. Formal causal mediation analysis should be implemented to further validate this assumption.

Keywords: Causality; DNA methylation; Epigenetics; Mendelian randomisation; Type 2 diabetes.

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Figures

Fig. 1
Fig. 1
Study design of the forward 2SMR analysis investigating the causal effect of type 2 diabetes on differences in DNAm at 58 CpG sites. CpGs were previously identified in association with type 2 diabetes in a meta-EWAS. T2D, type 2 diabetes; WBC, white-blood cell
Fig. 2
Fig. 2
Study design of the reverse 2SMR analysis investigating the causal effect of differences in DNAm on type 2 diabetes risk. A total of 58 CpGs were previously identified in association with type 2 diabetes in a meta-EWAS, but only 30 of them were proxied by an mQTL SNP in GoDMC with available GWAS data for type 2 diabetes in MR-Base. IV, instrumental variable; T2D, type 2 diabetes; UKB, UK Biobank; WBC, white-blood cell
Fig. 3
Fig. 3
Forest plot showing causal effect estimates from the 2SMR analysis of the effect of prevalent type 2 diabetes on the difference in DNAm at five meta-EWAS CpGs with the strongest evidence of causality in the forward 2SMR analysis. The black diamond represents the mean causal effect for each CpG and MR method and the horizontal line shows the 95% CI. p, unadjusted p value. Associations were borderline significant at an unadjusted p<0.05 and significant at an adjusted p<0.001. T2D, type 2 diabetes
Fig. 4
Fig. 4
Summary of evidence from the reverse 2SMR analysis for the association between inverse normal transformed residuals of DNAm at five meta-EWAS CpGs and risk of prevalent type 2 diabetes. The CpGs illustrated showed the smallest p values in the reverse 2SMR analysis. (a) Volcano plot showing MR estimates using the Wald ratio and a single SNP as a proxy for each CpG analysed. The CpG highlighted in red (cg25536676 [DHCR24]) was identified as causally associated with type 2 diabetes (p=0.001). The green dots represent CpGs with an unadjusted p<0.05 or –log10 (p value) >1.3 in the 2SMR analysis. The vertical dashed line represents the line of null associations at OR=1.0. (b) Forest plot showing means and SEMs of the causal estimates of the association between DNAm and T2D (outcome) for the top five CpGs identified in the reverse 2SMR analysis. The results for each CpG are shown according to the MR method applied, which differed only for the CpG cg25536676 (DHCR24), proxied by three mQTL. T2D, type 2 diabetes
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