Disease incidences increase with age, but the molecular characteristics of ageing that lead to increased disease susceptibility remain inadequately understood. Here we perform a whole-blood gene expression meta-analysis in 14,983 individuals of European ancestry (including replication) and identify 1,497 genes that are differentially expressed with chronological age. The age-associated genes do not harbor more age-associated CpG-methylation sites than other genes, but are instead enriched for the presence of potentially functional CpG-methylation sites in enhancer and insulator regions that associate with both chronological age and gene expression levels. We further used the gene expression profiles to calculate the ‘transcriptomic age' of an individual, and show that differences between transcriptomic age and chronological age are associated with biological features linked to ageing, such as blood pressure, cholesterol levels, fasting glucose, and body mass index. The transcriptomic prediction model adds biological relevance and complements existing epigenetic prediction models, and can be used by others to calculate transcriptomic age in external cohorts.

C-reactive protein (CRP) is a sensitive biomarker of chronic low-grade inflammation and is associated with multiple complex diseases. The genetic determinants of chronic inflammation remain largely unknown, and the causal role of CRP in several clinical outcomes is debated. We performed two genome-wide association studies (GWASs), on HapMap and 1000 Genomes imputed data, of circulating amounts of CRP by using data from 88 studies comprising 204,402 European individuals. Additionally, we performed in silico functional analyses and Mendelian randomization analyses with several clinical outcomes. The GWAS meta-analyses of CRP revealed 58 distinct genetic loci (p < 5 3 10 À8). After adjustment for body mass index in the regression analysis, the associations at all except three loci remained. The lead variants at the distinct loci explained up to 7.0% of the variance in circulating amounts of CRP. We identified 66 gene sets that were organized in two substantially correlated clusters, one mainly composed of immune pathways and the other characterized by metabolic pathways in the liver. Mendelian randomization analyses revealed a causal protective effect of CRP on schizophrenia and a risk-increasing effect on bipolar disorder. Our findings provide further insights into the biology of inflammation and could lead to interventions for treating inflammation and its clinical consequences.

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ObjectiveIdentify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.MethodsGenome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.ResultsAmong the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. TNFRSF11B and FRZB). Also several inflammatory genes such as CD55, PTGES and TNFAIP6, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of NGF in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex.ConclusionWe provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.

ObjectiveTo uncover the microRNA (miRNA) interactome of the osteoarthritis (OA) pathophysiological process in the cartilage.MethodsWe performed RNA sequencing in 130 samples (n=35 and n=30 pairs for messenger RNA (mRNA) and miRNA, respectively) on macroscopically preserved and lesioned OA cartilage from the same patient and performed differential expression (DE) analysis of miRNA and mRNAs. To build an OA-specific miRNA interactome, a prioritisation scheme was applied based on inverse Pearson’s correlations and inverse DE of miRNAs and mRNAs. Subsequently, these were filtered by those present in predicted (TargetScan/microT-CDS) and/or experimentally validated (miRTarBase/TarBase) public databases. Pathway enrichment analysis was applied to elucidate OA-related pathways likely mediated by miRNA regulatory mechanisms.ResultsWe found 142 miRNAs and 2387 mRNAs to be differentially expressed between lesioned and preserved OA articular cartilage. After applying prioritisation towards likely miRNA-mRNA targets, a regulatory network of 62 miRNAs targeting 238 mRNAs was created. Subsequent pathway enrichment analysis of these mRNAs (or genes) elucidated that genes within the ‘nervous system development’ are likely mediated by miRNA regulatory mechanisms (familywise error=8.4×10−5). Herein NTF3 encodes neurotrophin-3, which controls survival and differentiation of neurons and which is closely related to the nerve growth factor.ConclusionsBy an integrated approach of miRNA and mRNA sequencing data of OA cartilage, an OA miRNA interactome and related pathways were elucidated. Our functional data demonstrated interacting levels at which miRNA affects expression of genes in the cartilage and exemplified the complexity of functionally validating a network of genes that may be targeted by multiple miRNAs.

Thyroid dysfunction is an important public health problem, which affects 10% of the general population and increases the risk of cardiovascular morbidity and mortality. Many aspects of thyroid hormone regulation have only partly been elucidated, including its transport, metabolism, and genetic determinants. Here we report a large meta-analysis of genome-wide association studies for thyroid function and dysfunction, testing 8 million genetic variants in up to 72,167 individuals. One-hundred-and-nine independent genetic variants are associated with these traits. A genetic risk score, calculated to assess their combined effects on clinical end points, shows significant associations with increased risk of both overt (Graves’ disease) and subclinical thyroid disease, as well as clinical complications. By functional follow-up on selected signals, we identify a novel thyroid hormone transporter (SLC17A4) and a metabolizing enzyme (AADAT). Together, these results provide new knowledge about thyroid hormone physiology and disease, opening new possibilities for therapeutic targets.

MethylAid is implemented as an R/Bioconductor package (www.bioconductor.org/packages/3.0/bioc/html/MethylAid.html). A demo application is available from shiny.bioexp.nl/MethylAid.

ObjectivesTo investigate how the genetic susceptibility gene DIO2 confers risk to osteoarthritis (OA) onset in humans and to explore whether counteracting the deleterious effect could contribute to novel therapeutic approaches.MethodsEpigenetically regulated expression of DIO2 was explored by assessing methylation of positional CpG-dinucleotides and the respective DIO2 expression in OA-affected and macroscopically preserved articular cartilage from end-stage OA patients. In a human in vitro chondrogenesis model, we measured the effects when thyroid signalling during culturing was either enhanced (excess T3 or lentiviral induced DIO2 overexpression) or decreased (iopanoic acid).ResultsOA-related changes in methylation at a specific CpG dinucleotide upstream of DIO2 caused significant upregulation of its expression (β=4.96; p=0.0016). This effect was enhanced and appeared driven specifically by DIO2 rs225014 risk allele carriers (β=5.58, p=0.0006). During in vitro chondrogenesis, DIO2 overexpression resulted in a significant reduced capacity of chondrocytes to deposit extracellular matrix (ECM) components, concurrent with significant induction of ECM degrading enzymes (ADAMTS5, MMP13) and markers of mineralisation (ALPL, COL1A1). Given their concurrent and significant upregulation of expression, this process is likely mediated via HIF-2α/RUNX2 signalling. In contrast, we showed that inhibiting deiodinases during in vitro chondrogenesis contributed to prolonged cartilage homeostasis as reflected by significant increased deposition of ECM components and attenuated upregulation of matrix degrading enzymes.ConclusionsOur findings show how genetic variation at DIO2 could confer risk to OA and raised the possibility that counteracting thyroid signalling may be a novel therapeutic approach.