The role of one-carbon metabolism in health and disease: A genetic epidemiology perspective

The overarching aim of this thesis is to provide mechanistic and aetiological insight into the existing evidence that links one-carbon metabolism (OCM) variability with healthy ageing and disease. There is increased urgency in the field of research to understand the distribution and determinants of human common diseases, as healthcare costs and the burden of complex diseases are on the rise. This thesis supports the view that understanding the inter-individual variability in levels of key OCM vitamins is a fundamental building block in redressing malnutrition/ nutrient deficiency in population groups, and in preventing disease.At the crossroads of genetics and epidemiology is genetic epidemiology, which examines the role of genetic factors in disease aetiology. Throughout the decades, the advancement of human health has been highlighted by the ability to move from a molecular perspective of understanding pathways of disease aetiology to extrapolating the implications of the latter to broader public health ventures. The thesis itself is a smaller-scale visual of how this works, as it builds up from a molecular-scale stance to a more public health viewpoint that may potentially lead to improved understanding of the genetic architecture of disease, improved diagnosis of specific markers, and targeted disease treatments.More specifically, the significance of this work is that it informs on (1) the genetic determinants of variability in key OCM metabolites and cofactors, which in turn informs on (2) the development of novel diagnostic and prognostic tools that predict vitamin B12 deficiency in healthy adults and in pregnancy; (3) a mechanism of gene-nutrient interaction leading to site-specific changes in DNA methylation patterns in humans – a molecular outcome of OCM-, that have also been associated with disease risk; (4) the effects of OCM- associated genetic variants on risk of ischaemic stroke, and finally; (5) how can we deploy/ translate findings from genetic research in the field of nutrition to aid in the development of public health recommendations

Genetic heterogeneity in cardiovascular disease across ancestries: Insights for mechanisms and therapeutic intervention

Cardiovascular diseases (CVDs) are complex in their aetiology, arising due to a combination of genetics, lifestyle and environmental factors. By nature of this complexity, different CVDs vary in their molecular mechanisms, clinical presentation and progression. Although extensive efforts are being made to develop novel therapeutics for CVDs, genetic heterogeneity is often overlooked in the development process. By considering molecular mechanisms at an individual and ancestral level, a richer understanding of the influence of environmental and lifestyle factors can be gained and more refined therapeutic interventions can be developed. It is therefore expedient to understand the molecular and clinical heterogeneity in CVDs that exists across different populations. In this review, we highlight how the mechanisms underlying CVDs vary across diverse population ancestry groups due to genetic heterogeneity. We then discuss how such genetic heterogeneity is being leveraged to inform therapeutic interventions and personalised medicine, highlighting examples across the CVD spectrum. Finally, we present an overview of how polygenic risk scores and Mendelian randomisation can foster more robust insight into disease mechanisms and therapeutic intervention in diverse populations. Fulfilment of the vision of precision medicine requires more exhaustive leveraging of the genetic variability across diverse ancestry populations to improve our understanding of disease onset, progression and response to therapeutic intervention

LDL-c Lowering, Ischemic Stroke, and Small Vessel Disease Brain Imaging Biomarkers: A Mendelian Randomization Study.

BACKGROUND: The effects of lipid-lowering drug targets on different ischemic stroke subtypes are not fully understood. We aimed to explore the mechanisms by which lipid-lowering drug targets differentially affect the risk of ischemic stroke subtypes and their underlying pathophysiology. METHODS: Using a 2-sample Mendelian randomization approach, we assessed the effects of genetically proxied low-density lipoprotein cholesterol (LDL-c) and 3 clinically approved LDL-lowering drugs (HMGCR [3-hydroxy-3-methylglutaryl-CoA reductase], PCSK9 [proprotein convertase subtilisin/kexin type 9], and NPC1L1 [Niemann-Pick C1-Like 1]) on stroke subtypes and brain imaging biomarkers associated with small vessel stroke (SVS), including white matter hyperintensity volume and perivascular spaces. RESULTS: In genome-wide Mendelian randomization analyses, lower genetically predicted LDL-c was significantly associated with a reduced risk of any stroke, ischemic stroke, and large artery stroke, supporting previous findings. Significant associations between genetically predicted LDL-c and cardioembolic stroke, SVS, and biomarkers, perivascular space and white matter hyperintensity volume, were not identified in this study. In drug-target Mendelian randomization analysis, genetically proxied reduced LDL-c through NPC1L1 inhibition was associated with lower odds of perivascular space (odds ratio per 1-mg/dL decrease, 0.79 [95% CI, 0.67-0.93]) and with lower odds of SVS (odds ratio, 0.29 [95% CI, 0.10-0.85]). CONCLUSIONS: This study provides supporting evidence of a potentially protective effect of LDL-c lowering through NPC1L1 inhibition on perivascular space and SVS risk, highlighting novel therapeutic targets for SVS

Associations of Genetically Predicted Vitamin B12 Status across the Phenome

Variation in vitamin B12 levels has been associated with a range of diseases across the life-course, the causal nature of which remains elusive. We aimed to interrogate genetically predicted vitamin B12 status in relation to a plethora of clinical outcomes available in the UK Biobank. Genome-wide association study (GWAS) summary data obtained from a Danish and Icelandic cohort of 45,576 individuals were used to identify 8 genetic variants associated with vitamin B12 levels, serving as genetic instruments for vitamin B12 status in subsequent analyses. We conducted a Mendelian randomisation (MR)-phenome-wide association study (PheWAS) of vitamin B12 status with 945 distinct phenotypes in 439,738 individuals from the UK Biobank using these 8 genetic instruments to proxy alterations in vitamin B12 status. We used external GWAS summary statistics for replication of significant findings. Correction for multiple testing was taken into consideration using a 5% false discovery rate (FDR) threshold. MR analysis identified an association between higher genetically predicted vitamin B12 status and lower risk of vitamin B deficiency (including all B vitamin deficiencies), serving as a positive control outcome. We further identified associations between higher genetically predicted vitamin B12 status and a reduced risk of megaloblastic anaemia (OR = 0.35, 95% CI: 0.20–0.50) and pernicious anaemia (0.29, 0.19–0.45), which was supported in replication analyses. Our study highlights that higher genetically predicted vitamin B12 status is potentially protective of risk of vitamin B12 deficiency associated with pernicious anaemia diagnosis, and reduces risk of megaloblastic anaemia. The potential use of genetically predicted vitamin B12 status in disease diagnosis, progression and management remains to be investigated

Interaction between plasma homocysteine and the MTHFR c.677C > T polymorphism is associated with site-specific changes in DNA methylation in humans

One-carbon metabolism provides a direct link between dietary folate/vitamin B12 exposure, the activity of the enzyme methylenetetrahydrofolate reductase (MTHFR) and epigenetic regulation of the genome via DNA methylation. Previously, Friso et al. (2002) showed that the common c.677C>T polymorphism in MTHFR influences global DNA methylation status through a direct interaction with folate status and (indirectly) with homocysteine (tHcy) levels. To build on this and other more recent observations that have further highlighted associations between MTHFR c.677C>T, tHcy and aberrations in DNA methylation, we investigated whether interaction between mildly elevated plasma tHcy and the c.677C>T polymorphism is associated with site-specific changes in DNA methylation in humans. We used data on plasma tHcy levels, c.677C>T polymorphism, and site-specific DNA methylation levels for a total of 915 Caucasian females and 335 males from the TwinsUK (n=610) and Rotterdam study (n=670). We carried out methylome-wide association analyses in each cohort to model the interaction between levels of tHcy and c.677C>T genotypes on DNA methylation beta values. Our meta-analysis identified a total of 13 probes significantly associated with [rs1801133 x tHcy] levels (FDR<0.05). The most significant associations were with a cluster of probes at the AGTRAP-MTHFR-NPPA/B gene locus on chromosome 1 (FDR=1.3E-04), with additional probes on chromosomes 2, 3, 4, 7, 12, 16 and 19. Our top 2 hits on chromosome 1 were functionally associated with variability in expression of the TNFRSF8 gene/locus on this chromosome. This is the first study to provide a direct link between perturbations in one-carbon metabolism, through an interaction of tHcy and the activity of MTHFR enzyme on epigenetic regulation of the genome via DNA methylation

Associations of genetically predicted fatty acid levels across the phenome: A mendelian randomisation study.

BACKGROUND: Fatty acids are important dietary factors that have been extensively studied for their implication in health and disease. Evidence from epidemiological studies and randomised controlled trials on their role in cardiovascular, inflammatory, and other diseases remains inconsistent. The objective of this study was to assess whether genetically predicted fatty acid concentrations affect the risk of disease across a wide variety of clinical health outcomes. METHODS AND FINDINGS: The UK Biobank (UKB) is a large study involving over 500,000 participants aged 40 to 69 years at recruitment from 2006 to 2010. We used summary-level data for 117,143 UKB samples (base dataset), to extract genetic associations of fatty acids, and individual-level data for 322,232 UKB participants (target dataset) to conduct our discovery analysis. We studied potentially causal relationships of circulating fatty acids with 845 clinical diagnoses, using mendelian randomisation (MR) approach, within a phenome-wide association study (PheWAS) framework. Regression models in PheWAS were adjusted for sex, age, and the first 10 genetic principal components. External summary statistics were used for replication. When several fatty acids were associated with a health outcome, multivariable MR and MR-Bayesian method averaging (MR-BMA) was applied to disentangle their causal role. Genetic predisposition to higher docosahexaenoic acid (DHA) was associated with cholelithiasis and cholecystitis (odds ratio per mmol/L: 0.76, 95% confidence interval: 0.66 to 0.87). This was supported in replication analysis (FinnGen study) and by the genetically predicted omega-3 fatty acids analyses. Genetically predicted linoleic acid (LA), omega-6, polyunsaturated fatty acids (PUFAs), and total fatty acids (total FAs) showed positive associations with cardiovascular outcomes with support from replication analysis. Finally, higher genetically predicted levels of DHA (0.83, 0.73 to 0.95) and omega-3 (0.83, 0.75 to 0.92) were found to have a protective effect on obesity, which was supported using body mass index (BMI) in the GIANT consortium as replication analysis. Multivariable MR analysis suggested a direct detrimental effect of LA (1.64, 1.07 to 2.50) and omega-6 fatty acids (1.81, 1.06 to 3.09) on coronary heart disease (CHD). MR-BMA prioritised LA and omega-6 fatty acids as the top risk factors for CHD. Although we present a range of sensitivity analyses to the address MR assumptions, horizontal pleiotropy may still bias the reported associations and further evaluation in clinical trials is needed. CONCLUSIONS: Our study suggests potentially protective effects of circulating DHA and omega-3 concentrations on cholelithiasis and cholecystitis and on obesity, highlighting the need to further assess them as prevention treatments in clinical trials. Moreover, our findings do not support the supplementation of unsaturated fatty acids for cardiovascular disease prevention

Associations of genetically predicted fatty acid levels across the phenome: A mendelian randomisation study

Background Fatty acids are important dietary factors that have been extensively studied for their implication in health and disease. Evidence from epidemiological studies and randomised controlled trials on their role in cardiovascular, inflammatory, and other diseases remains inconsistent. The objective of this study was to assess whether genetically predicted fatty acid concentrations affect the risk of disease across a wide variety of clinical health outcomes. Methods and findings The UK Biobank (UKB) is a large study involving over 500,000 participants aged 40 to 69 years at recruitment from 2006 to 2010. We used summary-level data for 117,143 UKB samples (base dataset), to extract genetic associations of fatty acids, and individual-level data for 322,232 UKB participants (target dataset) to conduct our discovery analysis. We studied potentially causal relationships of circulating fatty acids with 845 clinical diagnoses, using mendelian randomisation (MR) approach, within a phenome-wide association study (PheWAS) framework. Regression models in PheWAS were adjusted for sex, age, and the first 10 genetic principal components. External summary statistics were used for replication. When several fatty acids were associated with a health outcome, multivariable MR and MR-Bayesian method averaging (MR-BMA) was applied to disentangle their causal role. Genetic predisposition to higher docosahexaenoic acid (DHA) was associated with cholelithiasis and cholecystitis (odds ratio per mmol/L: 0.76, 95% confidence interval: 0.66 to 0.87). This was supported in replication analysis (FinnGen study) and by the genetically predicted omega-3 fatty acids analyses. Genetically predicted linoleic acid (LA), omega-6, polyunsaturated fatty acids (PUFAs), and total fatty acids (total FAs) showed positive associations with cardiovascular outcomes with support from replication analysis. Finally, higher genetically predicted levels of DHA (0.83, 0.73 to 0.95) and omega-3 (0.83, 0.75 to 0.92) were found to have a protective effect on obesity, which was supported using body mass index (BMI) in the GIANT consortium as replication analysis. Multivariable MR analysis suggested a direct detrimental effect of LA (1.64, 1.07 to 2.50) and omega-6 fatty acids (1.81, 1.06 to 3.09) on coronary heart disease (CHD). MR-BMA prioritised LA and omega-6 fatty acids as the top risk factors for CHD. Although we present a range of sensitivity analyses to the address MR assumptions, horizontal pleiotropy may still bias the reported associations and further evaluation in clinical trials is needed. Conclusions Our study suggests potentially protective effects of circulating DHA and omega-3 concentrations on cholelithiasis and cholecystitis and on obesity, highlighting the need to further assess them as prevention treatments in clinical trials. Moreover, our findings do not support the supplementation of unsaturated fatty acids for cardiovascular disease prevention. Loukas Zagkos and colleagues conduct Mendelian randomisation analysis to investigate potential causal associations of circulating fatty acids and 845 disease outcomes in the UK Biobank. Author summary Why was this study done? Epidemiological studies have investigated the influence of dietary fatty acids on several chronic diseases including cardiovascular disease (CVD), inflammatory diseases, cancers, and neurodegenerative conditions. The evidence is conflicting and inconclusive and is not supported by randomised controlled trials (RCTs) on fatty acids supplementation. The fact that the human fatty acid metabolome partly reflects the fatty acid intake was leveraged in this work. What did the researchers do and find? Mendelian randomisation analysis was conducted to investigate potential causal associations of circulating fatty acids and 845 disease outcomes in UK Biobank. We found that 1 mmol/L increase in genetically predicted docosahexaenoic acid (DHA) was associated with 24% reduced odds of cholelithiasis and cholecystitis, as well as 17% reduced odds of obesity. One mmol/L higher genetically predicted linoleic acid (LA) was associated with 27% increased odds of ischemic heart disease. When assessed with other fatty acid classes simultaneously, 1 mmol/L higher genetically predicted LA and omega-6 fatty acids were associated with higher odds of coronary heart disease (64% and 81% increase, respectively). What do these findings mean? Our results support the prioritisation of RCTs examining the role of primary and secondary prevention of cholelithiasis via dietary modification or supplementation of DHA and other omega-3 fatty acids. Our principal findings also provide evidence against the supplemental use of fatty acids for CVD prevention. Triangulation of evidence for causal inference of fatty acid levels through experimental studies susceptible to different assumptions is needed to strengthen the evidence