Phenome-wide association study of genetically predicted B vitamins and homocysteine biomarkers with multiple health and disease outcomes: analysis of the UK Biobank

Abstract

Background: Although a number of health outcomes such as CVDs, metabolic-related outcomes, neurological disorders, pregnancy outcomes, and cancers have been identified in relation to B vitamins, evidence is of uneven quality and volume, and there is uncertainty about putative causal relationships.

Objectives: To explore the effects of B vitamins and homocysteine on a wide range of health outcomes based on a large biorepository linking biological samples and electronic medical records.

Methods: First, we performed a phenome-wide association study (PheWAS) to investigate the associations of genetically predicted plasma concentrations (genetic component of the circulating concentrations) of folate, vitamin B6, vitamin B12, and their metabolite homocysteine with a wide range of disease outcomes (including both prevalent and incident events) among 385,917 individuals in the UK Biobank. Second, 2-sample Mendelian randomization (MR) analysis was used to replicate any observed associations and detect causality. We considered MR P <0.05 as significant for replication. Third, dose-response, mediation, and bioinformatics analyses were carried out to examine any nonlinear trends and to disentangle the underlying mediating biological mechanisms for the identified associations.

Results: In total, 1117 phenotypes were tested in each PheWAS analysis. After multiple corrections, 32 phenotypic associations of B vitamins and homocysteine were identified. Two-sample MR analysis supported that 3 of them were causal, including associations of higher plasma vitamin B6 with lower risk of calculus of kidney (OR: 0.64; 95% CI: 0.42, 0.97; P = 0.033), higher homocysteine concentration with higher risk of hypercholesterolemia (OR: 1.28, 95% CI: 1.04, 1.56; P = 0.018), and chronic kidney disease (OR: 1.32, 95% CI: 1.06, 1.63; P = 0.012). Significant nonlinear dose-response relationships were observed for the associations of folate with anemia, vitamin B12 with vitamin B-complex deficiencies, anemia and cholelithiasis, and homocysteine with cerebrovascular disease.

Conclusions: This study provides strong evidence for the associations of B vitamins and homocysteine with endocrine/metabolic and genitourinary disorders.

Keywords: B vitamins; Mendelian randomization; homocysteine; phenome-wide association study.

Figure 1. Interrelation between folate, vitamin B6, vitamin B12 and homocysteine metabolism.

Folate is reduced to THF. A methyl group is then transferred to THF, forming 5,10-methylene-THF. 5,10-methylene-THF can be reduced by MTHFR to 5-methyl-THF. The methyl group of 5-methyl-THF is transferred to homocysteine by MS, generating methionine and regenerating THF. Vitamin B12 is a cofactor for MS. Methionine can also be generated independently of folate and B12, by the action of BHMT, which transfers a methyl group from betaine to homocysteine. Methionine is then activated to form SAM, which serves as a universal methyl donor for numerous reactions. SAH is one of the products of these methylation reactions and is subsequently hydrolyzed to generate homocysteine. Homocysteine is used either to regenerate methionine, or it is converted to cystathionine and then cysteine. BHMT, betaine homocysteine methyltransferase; DMG, dimethylglycine; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; THF, tetrahydrofolate.

Figure 2. Flowchart for the study design.

Step 1, PheWAS analysis was performed for each biomarker to investigate associations with a wide range of disease outcomes; Step 2, the observed significant associations were replicated using several MR approaches (IVW, weighted median, MR-Egger and MR-PRESSO); Step 3, dose-response, mediation and bioinformatics analyses were conducted to quantify and characterize the associations between the biomarkers and the associated outcomes.

Figure 3. Schematic diagram illustrating the rationale of mediation analysis.

Dotted lines (αβ₂) refer to the indirect effect of the exposure on the identified outcome through the mediator. Solid line (β₁) is the direct effect of the exposure on the identified outcome. The assumption under mediation analysis is that 1) the relationship between the exposure and the outcome is entirely mediated by the mediator; 2) the exposure is truly associated with the mediator; and 3) the exposure is not associated with any confounder of the relationship between the mediator and the outcome.

Figure 4. Plots for PheWAS associations of folate, vitamin B6, vitamin B12 and homocysteine (n=385917).

The x axis represents distinct phenotypic groups by using different colors, the y axis represents the p value for the phenotypic associations.

Figure 5. Dose-response relationships between genetically predicted biomarker concentrations and the risk of outcomes identified by PheWAS (n=385917).

(A) Non-linear relationship between genetically predicted vitamin B12 concentration and vitamin-B complex deficiencies risk; (B) Non-linear relationship between genetically predicted vitamin B12 concentration and megaloblastic anemia risk; (C) Non-linear relationship between genetically predicted homocysteine concentration and cerebrovascular disease risk; (D) Non-linear relationship between genetically predicted folate concentration and megaloblastic anemia risk; (E) Non-linear relationship between genetically predicted vitamin B12 concentration and cholelithiasis and cholecystitis risk.

Figure 6. Bioinformatics analysis for homocysteine-associated instrumental variants.

(A) eQTL violin plots of the associations between homocysteine-associated SNPs and expression of located genes; (B) Diagram of the interactions between homocysteine-associated genes and hyperlipidemia-associated genes; (C) Diagram of the interactions between homocysteine-associated genes and chronic renal failure-associated genes; (D) Diagram of the interactions between homocysteine-associated genes and chronic kidney disease-associated genes.