Association of dietary folate and vitamin B-12 intake with genome-wide DNA methylation in blood: a large-scale epigenome-wide association analysis in 5841 individuals.

BACKGROUND: Folate and vitamin B-12 are essential micronutrients involved in the donation of methyl groups in cellular metabolism. However, associations between intake of these nutrients and genome-wide DNA methylation levels have not been studied comprehensively in humans. OBJECTIVE: The aim of this study was to assess whether folate and/or vitamin B-12 intake are asssociated with genome-wide changes in DNA methylation in leukocytes. METHODS: A large-scale epigenome-wide association study of folate and vitamin B-12 intake was performed on DNA from 5841 participants from 10 cohorts using Illumina 450k arrays. Folate and vitamin B-12 intakes were calculated from food-frequency questionnaires (FFQs). Continuous and categorical (low compared with high intake) linear regression mixed models were applied per cohort, controlling for confounders. A meta-analysis was performed to identify significant differentially methylated positions (DMPs) and regions (DMRs), and a pathway analysis was performed on the DMR annotated genes. RESULTS: The categorical model resulted in 6 DMPs, which are all negatively associated with folate intake, annotated to FAM64A, WRAP73, FRMD8, CUX1, and LCN8 genes, which have a role in cellular processes including centrosome localization, cell proliferation, and tumorigenesis. Regional analysis showed 74 folate-associated DMRs, of which 73 were negatively associated with folate intake. The most significant folate-associated DMR was a 400-base pair (bp) spanning region annotated to the LGALS3BP gene. In the categorical model, vitamin B-12 intake was associated with 29 DMRs annotated to 48 genes, of which the most significant was a 1100-bp spanning region annotated to the calcium-binding tyrosine phosphorylation-regulated gene (CABYR). Vitamin B-12 intake was not associated with DMPs. CONCLUSIONS: We identified novel epigenetic loci that are associated with folate and vitamin B-12 intake. Interestingly, we found a negative association between folate and DNA methylation. Replication of these methylation loci is necessary in future studies.

Novel Genetic Variants for Cartilage Thickness and Hip Osteoarthritis.

Osteoarthritis is one of the most frequent and disabling diseases of the elderly. Only few genetic variants have been identified for osteoarthritis, which is partly due to large phenotype heterogeneity. To reduce heterogeneity, we here examined cartilage thickness, one of the structural components of joint health. We conducted a genome-wide association study of minimal joint space width (mJSW), a proxy for cartilage thickness, in a discovery set of 13,013 participants from five different cohorts and replication in 8,227 individuals from seven independent cohorts. We identified five genome-wide significant (GWS, P≤5·0×10-8) SNPs annotated to four distinct loci. In addition, we found two additional loci that were significantly replicated, but results of combined meta-analysis fell just below the genome wide significance threshold. The four novel associated genetic loci were located in/near TGFA (rs2862851), PIK3R1 (rs10471753), SLBP/FGFR3 (rs2236995), and TREH/DDX6 (rs496547), while the other two (DOT1L and SUPT3H/RUNX2) were previously identified. A systematic prioritization for underlying causal genes was performed using diverse lines of evidence. Exome sequencing data (n = 2,050 individuals) indicated that there were no rare exonic variants that could explain the identified associations. In addition, TGFA, FGFR3 and PIK3R1 were differentially expressed in OA cartilage lesions versus non-lesioned cartilage in the same individuals. In conclusion, we identified four novel loci (TGFA, PIK3R1, FGFR3 and TREH) and confirmed two loci known to be associated with cartilage thickness.The identified associations were not caused by rare exonic variants. This is the first report linking TGFA to human OA, which may serve as a new target for future therapies.

Latent infection with cytomegalovirus is associated with poor memory CD4 responses to influenza A core proteins in the elderly.

Influenza remains a major pathogen in older people. Infection with CMV and the accumulation of late-differentiated T cells associated with it have been implicated in poor Ab responsiveness to influenza vaccination in the elderly, most of whom are CMV positive. However, whether CMV infection also affects memory T cell responses to influenza remains unknown. To investigate this, we assessed T cell responses to influenza A matrix protein and nucleoprotein ex vivo in 166 Dutch individuals (mean age 62.2 y, range 42-82) and validated the results in a second cohort from North America (mean age 73.1 y, range 65-81, n = 28). We found that less than half of the CMV-infected older subjects mounted a CD4 T cell response to influenza Ags, whereas ∼80% of uninfected elderly did so. A similar proportion of younger subjects possessed influenza A virus-responsive CD4 T cells, and, interestingly, this was the case whether they were CMV-infected. Thus, the effect of CMV was only seen in the older donors, who may have been exposed to the virus for decades. The percentage of donors with CD8 responses to influenza A virus was lower than those with CD4; this was not influenced by whether the subjects were CMV seropositive or seronegative. CMV-seropositive responders had significantly higher frequencies of late-differentiated CD4 T-cells (CD45RA(+/-)CCR7(-)CD27(-)CD28(-)) compared with CMV-infected nonresponders. These data add to the accumulating evidence that infection with CMV has profound but heterogeneous effects on responses to the products of other viruses and have implications for the design of influenza vaccines, especially in the elderly.

Infection with cytomegalovirus but not herpes simplex virus induces the accumulation of late-differentiated CD4+ and CD8+ T-cells in humans.

Human cytomegalovirus (CMV) establishes persistent, usually asymptomatic, infection in healthy people. Because CMV infection is associated with the presence of lower proportions of peripheral naïve CD8+ T-cells and a higher fraction of late-differentiated CD8+ cells, commonly taken as biomarkers of age-associated compromised adaptive immunity ('immunosenescence'), we asked whether chronic exposure to any persistent virus mediates these effects. Herpes simplex virus (HSV) is also a widespread herpesvirus that establishes lifelong persistence, but, unlike CMV, its impact on the distribution of T-cell subsets has not been established. Here, we analysed T-cell subsets in 93 healthy people aged 42-81 years infected or not infected with CMV and/or HSV. Individuals harbouring CMV were confirmed to possess lower frequencies of naïve CD8+ T-cells (defined as CD45RA+CCR7+CD27+CD28+) and greater proportions of late-differentiated effector memory (CD45RA-CCR7-CD27-CD28-) and so-called TEMRA (CD45RA+CCR7-CD27-CD28-) CD4 and CD8 subsets, independent of HSV seropositivity. In CMV-seronegative donors, HSV did not affect T-cell subset distribution significantly. We conclude that these hallmarks of age-associated alterations to immune signatures are indeed observed in the general population in people infected with CMV and not those infected with a different persistent herpesvirus.

Common genetic variation in the Estrogen Receptor Beta (ESR2) gene and osteoarthritis: results of a meta-analysis.

BACKGROUND: The objective of this study was to examine the relationship between common genetic variation of the ESR2 gene and osteoarthritis. METHODS: In the discovery study, the Rotterdam Study-I, 7 single nucleotide polymorphisms (SNPs) were genotyped and tested for association with hip (284 cases, 2772 controls), knee (665 cases, 2075 controls), and hand OA (874 cases, 2184 controls) using an additive model. In the replication stage one SNP (rs1256031) was tested in an additional 2080 hip, 1318 knee and 557 hand OA cases and 4001, 2631 and 1699 controls respectively. Fixed- and random-effects meta-analyses were performed over the complete dataset including 2364 hip, 1983 knee and 1431 hand OA cases and approximately 6000 controls. RESULTS: The C allele of rs1256031 was associated with a 36% increased odds of hip OA in women of the Rotterdam Study-I (OR 1.36, 95% CI 1.08-1.70, p = 0.009). Haplotype analysis and analysis of knee- and hand OA did not give additional information. With the replication studies, the meta-analysis did not show a significant effect of this SNP on hip OA in the total population (OR 1.06, 95% CI 0.99-1.15, p = 0.10). Stratification according to gender did not change the results. In this study, we had 80% power to detect an odds ratio of at least 1.14 for hip OA (α = 0.05). CONCLUSION: This study showed that common genetic variation in the ESR2 gene is not likely to influence the risk of osteoarthritis with effects smaller than a 13% increase.

Amino acid profiling in urine by capillary zone electrophoresis - mass spectrometry.

Analysis of amino acid profiles in urine and plasma is an essential part of modern clinical diagnostic routine. Here we present an approach for the analysis of amino acids in urine by capillary electrophoresis/time-of-flight (TOF) mass spectrometry. At first a method combining improved separation, high dynamic range, and high sensitivity is presented. Detection limits in the mid nM-range are achieved through the use of pH-mediated stacking injection in combination with modern TOF detection technology. The method can be easily applied to detect differences in the amino acid profile in urine in a clinical context. Moreover, beside amino acids low molecular weight amines, peptides and related metabolites can be profiled. As a proof of concept, urine samples from patients suffering from osteoarthritis have been analyzed. Finally, the introduction of multivariate data analysis in the work flow was evaluated on spiked urine samples and real clinical material.