The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology.

Central nervous system (CNS)-resident cells such as microglia, oligodendrocytes and astrocytes are gaining increasing attention in respect to their contribution to CNS pathologies including multiple sclerosis (MS). Several studies have demonstrated the involvement of pro-inflammatory glial subsets in the pathogenesis and propagation of inflammatory events in MS and its animal models. However, it has only recently become clear that the underlying heterogeneity of astrocytes and microglia can not only drive inflammation, but also lead to its resolution through direct and indirect mechanisms. Failure of these tissue-protective mechanisms may potentiate disease and increase the risk of conversion to progressive stages of MS, for which currently available therapies are limited. Using proteomic analyses of cerebrospinal fluid specimens from patients with MS in combination with experimental studies, we here identify Heparin-binding EGF-like growth factor (HB-EGF) as a central mediator of tissue-protective and anti-inflammatory effects important for the recovery from acute inflammatory lesions in CNS autoimmunity. Hypoxic conditions drive the rapid upregulation of HB-EGF by astrocytes during early CNS inflammation, while pro-inflammatory conditions suppress trophic HB-EGF signaling through epigenetic modifications. Finally, we demonstrate both anti-inflammatory and tissue-protective effects of HB-EGF in a broad variety of cell types in vitro and use intranasal administration of HB-EGF in acute and post-acute stages of autoimmune neuroinflammation to attenuate disease in a preclinical mouse model of MS. Altogether, we identify astrocyte-derived HB-EGF and its epigenetic regulation as a modulator of autoimmune CNS inflammation and potential therapeutic target in MS.

Simultaneous detection of DNA variation and methylation at HLA class II locus and immune gene promoters using targeted SureSelect Methyl-Sequencing.

The Human Leukocyte Antigen (HLA) locus associates with a variety of complex diseases, particularly autoimmune and inflammatory conditions. The HLA-DR15 haplotype, for example, confers the major risk for developing Multiple Sclerosis in Caucasians, pinpointing an important role in the etiology of this chronic inflammatory disease of the central nervous system. In addition to the protein-coding variants that shape the functional HLA-antigen-T cell interaction, recent studies suggest that the levels of HLA molecule expression, that are epigenetically controlled, also play a role in disease development. However, deciphering the exact molecular mechanisms of the HLA association has been hampered by the tremendous genetic complexity of the locus and a lack of robust approaches to investigate it. Here, we developed a method to specifically enrich the genomic DNA from the HLA class II locus (chr6:32,426,802-34,167,129) and proximal promoters of 2,157 immune-relevant genes, utilizing the Agilent RNA-based SureSelect Methyl-Seq Capture related method, followed by sequencing to detect genetic and epigenetic variation. We demonstrated successful simultaneous detection of the genetic variation and quantification of DNA methylation levels in HLA locus. Moreover, by the detection of differentially methylated positions in promoters of immune-related genes, we identified relevant pathways following stimulation of cells. Taken together, we present a method that can be utilized to study the interplay between genetic variance and epigenetic regulation in the HLA class II region, potentially, in a wide disease context.

Smoking affects epigenetic ageing of lung bronchoalveolar lavage cells in Multiple Sclerosis.

BACKGROUND: A compelling body of evidence implicates cigarette smoking and lung inflammation in Multiple Sclerosis (MS) susceptibility and progression. Previous studies have reported epigenetic age (DNAm age) acceleration in blood immune cells and in glial cells of people with MS (pwMS) compared to healthy controls (HC). OBJECTIVES: We aimed to examine biological ageing in lung immune cells in the context of MS and smoking. METHODS: We analyzed age acceleration residuals in lung bronchoalveolar lavage (BAL) cells, constituted of mainly alveolar macrophages, from 17 pwMS and 22 HC in relation to smoking using eight DNA methylation-based clocks, namely AltumAge, Horvath, GrimAge, PhenoAge, Zhang, SkinBlood, Hannum, Monocyte clock as well as two RNA-based clocks, which capture different aspects of biological ageing. RESULTS: After adjustment for covariates, five epigenetic clocks showed significant differences between the groups. Four of them, Horvath (Padj = 0.028), GrimAge (Padj = 4.28 × 10-7), SkinBlood (Padj = 0.001) and Zhang (Padj = 0.02), uncovered the sole effect of smoking on ageing estimates, irrespective of the clinical group. The Horvath, SkinBlood and Zhang clocks showed a negative impact of smoking while GrimAge detected smoking-associated age acceleration in BAL cells. On the contrary, the AltumAge clock revealed differences between pwMS and HC and indicated that, in the absence of smoking, BAL cells of pwMS were epigenetically 5.4 years older compared to HC (Padj = 0.028). Smoking further affected epigenetic ageing in BAL cells of pwMS specifically as non-smoking pwMS exhibited a 10.2-year AltumAge acceleration compared to pwMS smokers (Padj = 0.0049). Of note, blood-derived monocytes did not show any MS-specific or smoking-related AltumAge differences. The difference between BAL cells of pwMS smokers and non-smokers was attributable to the differential methylation of 114 AltumAge-CpGs (Padj < 0.05) affecting genes involved in innate immune processes such as cytokine production, defense response and cell motility. These changes functionally translated into transcriptional differences in BAL cells between pwMS smokers and non-smokers. CONCLUSIONS: BAL cells of pwMS display inflammation-related and smoking-dependent changes associated to epigenetic ageing captured by the AltumAge clock. Future studies examining potential confounders, such as the distribution of distinct BAL myeloid cell types in pwMS compared to control individuals in relation to smoking may clarify the varying performance and DNAm age estimations among epigenetic clocks.

Perturbed epigenetic transcriptional regulation in AML with IDH mutations causes increased susceptibility to NK cells.

Isocitrate dehydrogenase (IDH) mutations are found in 20% of acute myeloid leukemia (AML) patients. However, only 30-40% of the patients respond to IDH inhibitors (IDHi). We aimed to identify a molecular vulnerability to tailor novel therapies for AML patients with IDH mutations. We characterized the transcriptional and epigenetic landscape with the IDH2i AG-221, using an IDH2 mutated AML cell line model and AML patient cohorts, and discovered a perturbed transcriptional regulatory network involving myeloid transcription factors that were partly restored after AG-221 treatment. In addition, hypermethylation of the HLA cluster caused a down-regulation of HLA class I genes, triggering an enhanced natural killer (NK) cell activation and an increased susceptibility to NK cell-mediated responses. Finally, analyses of DNA methylation data from IDHi-treated patients showed that non-responders still harbored hypermethylation in HLA class I genes. In conclusion, this study provides new insights suggesting that IDH mutated AML is particularly sensitive to NK cell-based personalized immunotherapy.

Integration of small RNAs from plasma and cerebrospinal fluid for classification of multiple sclerosis.

Multiple Sclerosis (MS) is an autoimmune, neurological disease, commonly presenting with a relapsing-remitting form, that later converts to a secondary progressive stage, referred to as RRMS and SPMS, respectively. Early treatment slows disease progression, hence, accurate and early diagnosis is crucial. Recent advances in large-scale data processing and analysis have progressed molecular biomarker development. Here, we focus on small RNA data derived from cell-free cerebrospinal fluid (CSF), cerebrospinal fluid cells, plasma and peripheral blood mononuclear cells as well as CSF cell methylome data, from people with RRMS (n = 20), clinically/radiologically isolated syndrome (CIS/RIS, n = 2) and neurological disease controls (n = 14). We applied multiple co-inertia analysis (MCIA), an unsupervised and thereby unbiased, multivariate method for simultaneous data integration and found that the top latent variable classifies RRMS status with an Area Under the Receiver Operating Characteristics (AUROC) score of 0.82. Variable selection based on Lasso regression reduced features to 44, derived from the small RNAs from plasma (20), CSF cells (8) and cell-free CSF (16), with a marginal reduction in AUROC to 0.79. Samples from SPMS patients (n = 6) were subsequently projected on the latent space and differed significantly from RRMS and controls. On contrary, we found no differences between relapse and remission or between inflammatory and non-inflammatory disease controls, suggesting that the latent variable is not prone to inflammatory signals alone, but could be MS-specific. Hence, we here showcase that integration of small RNAs from plasma and CSF can be utilized to distinguish RRMS from SPMS and neurological disease controls.

Epigenetic clock indicates accelerated aging in glial cells of progressive multiple sclerosis patients.

Background: Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS) characterized by irreversible disability at later progressive stages. A growing body of evidence suggests that disease progression depends on age and inflammation within the CNS. We aimed to investigate epigenetic aging in bulk brain tissue and sorted nuclei from MS patients using DNA methylation-based epigenetic clocks. Methods: We applied Horvath's multi-tissue and Shireby's brain-specific Cortical clock on bulk brain tissue (n = 46), sorted neuronal (n = 54), and glial nuclei (n = 66) from post-mortem brain tissue of progressive MS patients and controls. Results: We found a significant increase in age acceleration residuals, corresponding to 3.6 years, in glial cells of MS patients compared to controls (P = 0.0024) using the Cortical clock, which held after adjustment for covariates (P adj = 0.0263). The 4.8-year age acceleration found in MS neurons (P = 0.0054) did not withstand adjustment for covariates and no significant difference in age acceleration residuals was observed in bulk brain tissue between MS patients and controls. Conclusion: While the findings warrant replication in larger cohorts, our study suggests that glial cells of progressive MS patients exhibit accelerated biological aging.

DNA methylation changes in glial cells of the normal-appearing white matter in Multiple Sclerosis patients.

Multiple Sclerosis (MS), the leading cause of non-traumatic neurological disability in young adults, is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). Due to the poor accessibility to the target organ, CNS-confined processes underpinning the later progressive form of MS remain elusive thereby limiting treatment options. We aimed to examine DNA methylation, a stable epigenetic mark of genome activity, in glial cells to capture relevant molecular changes underlying MS neuropathology. We profiled DNA methylation in nuclei of non-neuronal cells, isolated from 38 post-mortem normal-appearing white matter (NAWM) specimens of MS patients (n = 8) in comparison to white matter of control individuals (n = 14), using Infinium MethylationEPIC BeadChip. We identified 1,226 significant (genome-wide adjusted P-value < 0.05) differentially methylated positions (DMPs) between MS patients and controls. Functional annotation of the altered DMP-genes uncovered alterations of processes related to cellular motility, cytoskeleton dynamics, metabolic processes, synaptic support, neuroinflammation and signaling, such as Wnt and TGF-ß pathways. A fraction of the affected genes displayed transcriptional differences in the brain of MS patients, as reported by publically available transcriptomic data. Cell type-restricted annotation of DMP-genes attributed alterations of cytoskeleton rearrangement and extracellular matrix remodelling to all glial cell types, while some processes, including ion transport, Wnt/TGF-ß signaling and immune processes were more specifically linked to oligodendrocytes, astrocytes and microglial cells, respectively. Our findings strongly suggest that NAWM glial cells are highly altered, even in the absence of lesional insult, collectively exhibiting a multicellular reaction in response to diffuse inflammation.

Comparison of EM-seq and PBAT methylome library methods for low-input DNA.

DNA methylation is the most studied epigenetic mark involved in regulation of gene expression. For low input samples, a limited number of methods for quantifying DNA methylation genome-wide has been evaluated. Here, we compared a series of input DNA amounts (1-10ng) from two methylome library preparation protocols, enzymatic methyl-seq (EM-seq) and post-bisulfite adaptor tagging (PBAT) adapted from single-cell PBAT. EM-seq takes advantage of enzymatic activity while PBAT relies on conventional bisulfite conversion for detection of DNA methylation. We found that both methods accurately quantified DNA methylation genome-wide. They produced expected distribution patterns around genomic features, high C-T transition efficiency at non-CpG sites and high correlation between input amounts. However, EM-seq performed better in regard to library and sequencing quality, i.e. EM-seq produced larger insert sizes, higher alignment rates and higher library complexity with lower duplication rate compared to PBAT. Moreover, EM-seq demonstrated higher CpG coverage, better CpG site overlap and higher consistency between input series. In summary, our data suggests that EM-seq overall performed better than PBAT in whole-genome methylation quantification of low input samples.

TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis.

Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.

Small noncoding RNA profiling across cellular and biofluid compartments and their implications for multiple sclerosis immunopathology.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system (CNS). Small non-coding RNAs (sncRNAs) and, in particular, microRNAs (miRNAs) have frequently been associated with MS. Here, we performed a comprehensive analysis of all classes of sncRNAs in matching samples of peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells, and cell-free CSF from relapsing-remitting (RRMS, n = 12 in relapse and n = 11 in remission) patients, secondary progressive (SPMS, n = 6) MS patients, and noninflammatory and inflammatory neurological disease controls (NINDC, n = 11; INDC, n = 5). We show widespread changes in miRNAs and sncRNA-derived fragments of small nuclear, nucleolar, and transfer RNAs. In CSF cells, 133 out of 133 and 115 out of 117 differentially expressed sncRNAs were increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65 out of 67 differentially expressed PBMC sncRNAs were decreased in RRMS compared to NINDC. The striking contrast between the periphery and CNS suggests that sncRNA-mediated mechanisms, including alternative splicing, RNA degradation, and mRNA translation, regulate the transcriptome of pathogenic cells primarily in the CNS target organ.

Methylome and transcriptome signature of bronchoalveolar cells from multiple sclerosis patients in relation to smoking.

BACKGROUND: Despite compelling evidence that cigarette smoking impacts the risk of developing multiple sclerosis (MS), little is known about smoking-associated changes in the primary exposed lung cells of patients. OBJECTIVES: We aimed to examine molecular changes occurring in bronchoalveolar lavage (BAL) cells from MS patients in relation to smoking and in comparison to healthy controls (HCs). METHODS: We profiled DNA methylation in BAL cells from female MS (n = 17) and HC (n = 22) individuals, using Illumina Infinium EPIC and performed RNA-sequencing in non-smokers. RESULTS: The most prominent changes were found in relation to smoking, with 1376 CpG sites (adjusted P < 0.05) differing between MS smokers and non-smokers. Approximately 30% of the affected genes overlapped with smoking-associated changes in HC, leading to a strong common smoking signature in both MS and HC after gene ontology analysis. Smoking in MS patients resulted in additional discrete changes related to neuronal processes. Methylome and transcriptome analyses in non-smokers suggest that BAL cells from MS patients display very subtle (not reaching adjusted P < 0.05) but concordant changes in genes connected to reduced transcriptional/translational processes and enhanced cellular motility. CONCLUSIONS: Our study provides insights into the impact of smoking on lung inflammation and immunopathogenesis of MS.

Tobacco smoking induces changes in true DNA methylation, hydroxymethylation and gene expression in bronchoalveolar lavage cells.

BACKGROUND: While smoking is known to associate with development of multiple diseases, the underlying mechanisms are still poorly understood. Tobacco smoking can modify the chemical integrity of DNA leading to changes in transcriptional activity, partly through an altered epigenetic state. We aimed to investigate the impact of smoking on lung cells collected from bronchoalveolar lavage (BAL). METHODS: We profiled changes in DNA methylation (5mC) and its oxidised form hydroxymethylation (5hmC) using conventional bisulphite (BS) treatment and oxidative bisulphite treatment with Illumina Infinium MethylationEPIC BeadChip, and examined gene expression by RNA-seq in healthy smokers. FINDINGS: We identified 1667 total 5mC + 5hmC, 1756 5mC and 67 5hmC differentially methylated positions (DMPs) between smokers and non-smokers (FDR-adjusted P <.05, absolute Δß >0.15). Both 5mC DMPs and to a lesser extent 5mC + 5hmC were predominantly hypomethylated. In contrast, almost all 5hmC DMPs were hypermethylated, supporting the hypothesis that smoking-associated oxidative stress can lead to DNA demethylation, via the established sequential oxidation of which 5hmC is the first step. While we confirmed differential methylation of previously reported smoking-associated 5mC + 5hmC CpGs using former generations of BeadChips in alveolar macrophages, the large majority of identified DMPs, 5mC + 5hmC (1639/1667), 5mC (1738/1756), and 5hmC (67/67), have not been previously reported. Most of these novel smoking-associating sites are specific to the EPIC BeadChip and, interestingly, many of them are associated to FANTOM5 enhancers. Transcriptional changes affecting 633 transcripts were consistent with DNA methylation profiles and converged to alteration of genes involved in migration, signalling and inflammatory response of immune cells. INTERPRETATION: Collectively, these findings suggest that tobacco smoke exposure epigenetically modifies BAL cells, possibly involving a continuous active demethylation and subsequent increased activity of inflammatory processes in the lungs. FUND: The study was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, the Stockholm County Council (ALF), the King Gustav's and Queen Victoria's Freemasons' Foundation, Knut and Alice Wallenberg Foundation, Neuro Sweden, and the Swedish MS foundation.

Neuronal methylome reveals CREB-associated neuro-axonal impairment in multiple sclerosis.

BACKGROUND: Due to limited access to brain tissue, the precise mechanisms underlying neuro-axonal dysfunction in neurological disorders such as multiple sclerosis (MS) are largely unknown. In that context, profiling DNA methylation, which is a stable and cell type-specific regulatory epigenetic mark of genome activity, offers a unique opportunity to characterize the molecular mechanisms underpinning brain pathology in situ. We examined DNA methylation patterns of neuronal nuclei isolated from post-mortem brain tissue to infer processes that occur in neurons of MS patients. RESULTS: We isolated subcortical neuronal nuclei from post-mortem white matter tissue of MS patients and non-neurological controls using flow cytometry. We examined bulk DNA methylation changes (total n = 29) and further disentangled true DNA methylation (5mC) from neuron-specific DNA hydroxymethylation (5hmC) (n = 17), using Illumina Infinium 450K arrays. We performed neuronal sub-type deconvolution using glutamate and GABA methylation profiles to further reduce neuronal sample heterogeneity. In total, we identified 2811 and 1534 significant (genome-wide adjusted P value < 0.05) differentially methylated and hydroxymethylated positions between MS patients and controls. We found striking hypo-5mC and hyper-5hmC changes occurring mainly within gene bodies, which correlated with reduced transcriptional activity, assessed using published RNAseq data from bulk brain tissue of MS patients and controls. Pathway analyses of the two cohorts implicated dysregulation of genes involved in axonal guidance and synaptic plasticity, with meta-analysis confirming CREB signalling as the most highly enriched pathway underlying these processes. We functionally investigated DNA methylation changes of CREB signalling-related genes by immunohistofluoresence of phosphorylated CREB in neurons from brain sections of a subcohort of MS patients and controls (n = 15). Notably, DNA methylation changes associated with a reduction of CREB activity in white matter neurons of MS patients compared to controls. CONCLUSIONS: Our data demonstrate that investigating 5mC and 5hmC modifications separately allows the discovery of a substantial fraction of changes occurring in neurons, which can escape traditional bisulfite-based DNA methylation analysis. Collectively, our findings indicate that neurons of MS patients acquire sustained hypo-5mC and hyper-5hmC, which may impair CREB-mediated neuro-axonal integrity, in turn relating to clinical symptoms.

Competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells.

Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived replacements that are phenotypically indistinguishable from their embryonic counterparts. As the factors regulating this process are incompletely understood, we studied niche competition in the brain by depleting microglia with >95% efficiency using Cx3cr1CreER/+R26DTA/+ mice and monitored long-term repopulation. Here we show that the microglial niche is repopulated within weeks by a combination of local proliferation of CX3CR1+F4/80lowClec12a- microglia and infiltration of CX3CR1+F4/80hiClec12a+ macrophages that arise directly from Ly6Chi monocytes. This colonization is independent of blood brain barrier breakdown, paralleled by vascular activation, and regulated by type I interferon. Ly6Chi monocytes upregulate microglia gene expression and adopt microglia DNA methylation signatures, but retain a distinct gene signature from proliferating microglia, displaying altered surface marker expression, phagocytic capacity and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct.

Fatal demyelinating disease is induced by monocyte-derived macrophages in the absence of TGF-ß signaling.

The cytokine transforming growth factor-ß (TGF-ß) regulates the development and homeostasis of several tissue-resident macrophage populations, including microglia. TGF-ß is not critical for microglia survival but is required for the maintenance of the microglia-specific homeostatic gene signature1,2. Under defined host conditions, circulating monocytes can compete for the microglial niche and give rise to long-lived monocyte-derived macrophages residing in the central nervous system (CNS)3-5. Whether monocytes require TGF-ß for colonization of the microglial niche and maintenance of CNS integrity is unknown. We found that abrogation of TGF-ß signaling in CX3CR1+ monocyte-derived macrophages led to rapid onset of a progressive and fatal demyelinating motor disease characterized by myelin-laden giant macrophages throughout the spinal cord. Tgfbr2-deficient macrophages were characterized by high expression of genes encoding proteins involved in antigen presentation, inflammation and phagocytosis. TGF-ß is thus crucial for the functional integration of monocytes into the CNS microenvironment.

Usability of human Infinium MethylationEPIC BeadChip for mouse DNA methylation studies.

BACKGROUND: The advent of array-based genome-wide DNA methylation methods has enabled quantitative measurement of single CpG methylation status at relatively low cost and sample input. Whereas the use of Infinium Human Methylation BeadChips has shown great utility in clinical studies, no equivalent tool is available for rodent animal samples. We examined the feasibility of using the new Infinium MethylationEPIC BeadChip for studying DNA methylation in mouse. RESULTS: In silico, we identified 19,420 EPIC probes (referred as mEPIC probes), which align with a unique best alignment score to the bisulfite converted reference mouse genome mm10. Further annotation revealed that 85% of mEPIC probes overlapped with mm10.refSeq genes at different genomic features including promoters (TSS1500 and TSS200), 1st exons, 5'UTRs, 3'UTRs, CpG islands, shores, shelves, open seas and FANTOM5 enhancers. Hybridization of mouse samples to Infinium Human MethylationEPIC BeadChips showed successful measurement of mEPIC probes and reproducibility between inter-array biological replicates. Finally, we demonstrated the utility of mEPIC probes for data exploration such as hierarchical clustering. CONCLUSIONS: Given the absence of cost and labor convenient genome-wide technologies in the murine system, our findings show that the Infinium MethylationEPIC BeadChip platform is suitable for investigation of the mouse methylome. Furthermore, we provide the "mEPICmanifest" with genomic features, available to users of Infinium Human MethylationEPIC arrays for mouse samples.

Epigenetic research in multiple sclerosis: progress, challenges, and opportunities.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system. MS likely results from a complex interplay between predisposing causal gene variants (the strongest influence coming from HLA class II locus) and environmental risk factors such as smoking, infectious mononucleosis, and lack of sun exposure/vitamin D. However, little is known about the mechanisms underlying MS development and progression. Moreover, the clinical heterogeneity and variable response to treatment represent additional challenges to a comprehensive understanding and efficient treatment of disease. Epigenetic processes, such as DNA methylation and histone posttranslational modifications, integrate influences from the genes and the environment to regulate gene expression accordingly. Studying epigenetic modifications, which are stable and reversible, may provide an alternative approach to better understand and manage disease. We here aim to review findings from epigenetic studies in MS and further discuss the challenges and clinical opportunities arising from epigenetic research, many of which apply to other diseases with similar complex etiology. A growing body of evidence supports a role of epigenetic processes in the mechanisms underlying immune pathogenesis and nervous system dysfunction in MS. However, disparities between studies shed light on the need to consider possible confounders and methodological limitations for a better interpretation of the data. Nevertheless, translational use of epigenetics might offer new opportunities in epigenetic-based diagnostics and therapeutic tools for a personalized care of MS patients.

Contribution of Heritability and Epigenetic Factors to Skeletal Muscle Mass Variation in United Kingdom Twins.

CONTEXT: Skeletal muscle mass (SMM) is one of the major components of human body composition, with deviations from normal values often leading to sarcopenia. OBJECTIVE: Our major aim was to conduct a genome-wide DNA methylation study in an attempt to identify potential genomic regions associated with SMM. DESIGN: This was a mixed cross-sectional and longitudinal study. SETTING: Community-based study. PARTICIPANTS: A total of 1550 middle-aged United Kingdom twins (monozygotic [MZ] and dizygotic [DZ]), 297 of which were repeatedly measured participated in the study. MAIN OUTCOME MEASURE: Appendicular lean mass assessed using dual-energy X-ray absorptiometry technology, and methylated DNA immunoprecipitation sequencing DNA methylation profiling genome-wide were obtained from each individual. RESULTS: Heritability estimate of SMM, with simultaneous adjustment for covariates obtained using variance decomposition analysis, was h(2) = 0.809 ± 0.050. After quality control and analysis of longitudinal stability, the DNA methylation data comprised of 723 029 genomic sites, with positive correlations between repeated measurements (Rrepeated = 0.114-0.905). Correlations between MZ and DZ twins were 0.51 and 0.38 at a genome-wide average, respectively, and clearly increased with Rrepeated. Testing for DNA methylation association with SMM in 50 discordant MZ twins revealed 36 081 nominally significant results, of which the top-ranked 134 signals (P < .01 and Rrepeated > 0.40) were subjected to replication in the sample of 1196 individuals. Seven SMM methylation association signals replicated at a false discovery rate less than 0.1, and these were located in or near genes DNAH12, CAND1, CYP4F29P, and ZFP64, which have previously been highlighted in muscle-related studies. Adjusting for age, smoking, and blood cell heterogeneity did not alter significance of these associations. CONCLUSION: This epigenome-wide study, testing longitudinally stable methylation sites, discovered and replicated a number of associations between DNA methylation at CpG loci and SMM. Four replicated signals were related to genes with potential muscle functions, suggesting that the methylome of whole blood may be informative of SMM variation.

Regulation of Human PAX6 Expression by miR-7.

The paired box gene 6 (PAX6) is a powerful mediator of eye and brain organogenesis whose spatiotemporal expression is exquisitely controlled by multiple mechanisms, including post-transcriptional regulation by microRNAs (miRNAs). In the present study, we use bioinformatic predictions to identify three candidate microRNA-7 (miR-7) target sites in the human PAX6 3' untranslated region (3'-UTR) and demonstrate that two of them are functionally active in a human cell line. Furthermore, transient transfection of cells with synthetic miR-7 inhibits PAX6 protein expression but does not alter levels of PAX6 mRNA, suggesting that miR-7 induces translational repression of PAX6. Finally, a comparison of PAX6 3'-UTRs across species reveals that one of the functional miR-7 target sites is conserved, whereas the second functional target site is found only in primates. Thus, the interaction between PAX6 and miR-7 appears to be highly conserved; however, the precise number of sites through which this interaction occurs may have expanded throughout evolution.