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SIRT1 is a NAD(+)-dependent deacetylase that governs a number of genetic programs to cope with changes in the nutritional status of cells and organisms. Behavioral responses to food abundance are important for the survival of higher animals. Here we used mice with increased or decreased brain SIRT1 to show that this sirtuin regulates anxiety and exploratory drive by activating transcription of the gene encoding the monoamine oxidase A (MAO-A) to reduce serotonin levels in the brain. Indeed, treating animals with MAO-A inhibitors or selective serotonin reuptake inhibitors (SSRIs) normalized anxiety differences between wild-type and mutant animals. SIRT1 deacetylates the brain-specific helix-loop-helix transcription factor NHLH2 on lysine 49 to increase its activation of the MAO-A promoter. Both common and rare variations in the SIRT1 gene were shown to be associated with risk of anxiety in human population samples. Together these data indicate that SIRT1 mediates levels of anxiety, and this regulation may be adaptive in a changing environment of food availability.
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Ansiedade/genética , Encéfalo/metabolismo , Comportamento Exploratório , Monoaminoxidase/genética , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sequência de Aminoácidos , Animais , Comportamento Animal , Impulso (Psicologia) , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Monoaminoxidase/química , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
Schizophrenia is a disabling disorder involving genetic predisposition in combination with environmental influences that likely act via dynamic alterations of the epigenome and the transcriptome but its detailed pathophysiology is largely unknown. We performed cell-type specific methylome-wide association study of neonatal blood (N = 333) from individuals who later in life developed schizophrenia and controls. Suggestively significant associations (P < 1.0 × 10-6) were detected in all cell-types and in whole blood with methylome-wide significant associations in monocytes (P = 2.85 × 10-9-4.87 × 10-9), natural killer cells (P = 1.72 × 10-9-7.82 × 10-9) and B cells (P = 3.8 × 10-9). Validation of methylation findings in post-mortem brains (N = 596) from independent schizophrenia cases and controls showed significant enrichment of transcriptional differences (enrichment ratio = 1.98-3.23, P = 2.3 × 10-3-1.0 × 10-5), with specific highly significant differential expression for, for example, BDNF (t = -6.11, P = 1.90 × 10-9). In addition, expression difference in brain significantly predicted schizophrenia (multiple correlation = 0.15-0.22, P = 3.6 × 10-4-4.5 × 10-8). In summary, using a unique design combining pre-disease onset (neonatal) blood methylomic data and post-disease onset (post-mortem) brain transcriptional data, we have identified genes of likely functional relevance that are associated with schizophrenia susceptibility, rather than confounding disease associated artifacts. The identified loci may be of clinical value as a methylation-based biomarker for early detection of increased schizophrenia susceptibility.
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Anxiety Disorders (ANX) such as panic disorder, generalized anxiety disorder, and phobias, are highly prevalent conditions that are moderately heritable. Evidence suggests that DNA methylation may play a role, as it is involved in critical adaptations to changing environments. Applying an enrichment-based sequencing approach covering nearly 28 million autosomal CpG sites, we conducted a methylome-wide association study (MWAS) of lifetime ANX in 1132 participants (618 cases/514 controls) from the Netherlands Study of Depression and Anxiety. Using epigenomic deconvolution, we performed MWAS for the main cell types in blood: granulocytes, T-cells, B-cells and monocytes. Cell-type specific analyses identified 280 and 82 methylome-wide significant associations (q-value < 0.1) in monocytes and granulocytes, respectively. Our top finding in monocytes was located in ZNF823 on chromosome 19 (p = 1.38 × 10-10) previously associated with schizophrenia. We observed significant overlap (p < 1 × 10-06) with the same direction of effect in monocytes (210 sites), T-cells (135 sites), and B-cells (727 sites) between this Discovery MWAS signal and a comparable replication dataset from the Great Smoky Mountains Study (N = 433). Overlapping Discovery-Replication MWAS signal was enriched for findings from published GWAS of ANX, major depression, and post-traumatic stress disorder. In monocytes, two specific sites in the FZR1 gene showed significant replication after Bonferroni correction with an additional 15 nominally replicated sites in monocytes and 4 in T-cells. FZR1 regulates neurogenesis in the hippocampus, and its knockout leads to impairments in associative fear memory and long-term potentiation in mice. In the largest and most extensive methylome-wide study of ANX, we identified replicable methylation sites located in genes of potential relevance for brain mechanisms of psychiatric conditions.
Assuntos
Epigenoma , Esquizofrenia , Humanos , Animais , Camundongos , Epigenoma/genética , Estudo de Associação Genômica Ampla , Esquizofrenia/genética , Metilação de DNA/genética , Transtornos de Ansiedade/genética , Ilhas de CpG/genéticaRESUMO
Postpartum depression (PPD) affects 1 in 7 women and has negative mental health consequences for both mother and child. However, the precise biological mechanisms behind the disorder are unknown. Therefore, we performed the largest transcriptome-wide association study (TWAS) for PPD (482 cases, 859 controls) to date using RNA-sequencing in whole blood and deconvoluted cell types. No transcriptional changes were observed in whole blood. B-cells showed a majority of transcriptome-wide significant results (891 transcripts representing 789 genes) with pathway analyses implicating altered B-cell activation and insulin resistance. Integration of other data types revealed cell type-specific DNA methylation loci and disease-associated eQTLs (deQTLs), but not hormones/neuropeptides (estradiol, progesterone, oxytocin, BDNF), serve as regulators for part of the transcriptional differences between cases and controls. Further, deQTLs were enriched for several brain region-specific eQTLs, but no overlap with MDD risk loci was observed. Altogether, our results constitute a convergence of evidence for pathways most affected in PPD with data across different biological mechanisms.
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Depressão Pós-Parto , Estudo de Associação Genômica Ampla , Resistência à Insulina , Depressão Pós-Parto/genética , Depressão Pós-Parto/metabolismo , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Resistência à Insulina/genética , Transcriptoma/genéticaRESUMO
Childhood trauma is robustly linked to a broad range of adverse outcomes with consequences persisting far into adulthood. We conducted a prospective longitudinal study to predict psychiatric disorders and other adverse outcomes from trauma-related methylation changes 16.9 years after trauma exposure in childhood. Methylation was assayed using a sequencing-based approach that provides near-complete coverage of all 28 million sites in the blood methylome. Methylation data involved 673 assays from 489 participants aged 13.6 years (SD = 1.9) with outcomes measures collected at age 30.4 (SD = 2.26). For a subset of 303 participants we also generated methylation data in adulthood. Trauma-related methylation risk scores (MRSs) significantly predicted adult depression, externalizing problems, nicotine dependence, alcohol use disorder, serious medical problems, social problems and poverty. The predictive power of the MRSs was higher than that of reported trauma and could not be explained by the reported trauma, correlations with demographic variables, or a continuity of the predicted health problems from childhood to adulthood. Rather than measuring the occurrence of traumatic events, the MRSs seemed to capture the subject-specific impact of trauma. The majority of predictive sites did not remain associated with the outcomes suggesting the signatures of trauma do not become biologically embedded in the blood methylome. Instead, the long-term effects of trauma therefore seemed more consistent with a developmental mechanism where the initial subject-specific impacts of trauma are magnified over time. The MRSs have the potential to be a novel clinical biomarker for the assessment of trauma-related health risks.
Assuntos
Experiências Adversas da Infância , Transtornos Mentais , Adulto , Humanos , Criança , Adolescente , Adulto Jovem , Estudos Prospectivos , Estudos Longitudinais , Metilação de DNA/genética , Transtornos Mentais/epidemiologiaRESUMO
Gene expression studies offer promising opportunities to better understand the processes underlying alcohol use disorder (AUD). As cell types differ in their function, gene expression profiles will typically vary across cell types. When studying bulk tissue, failure to account for this cellular diversity has a detrimental impact on the ability to detect disease associations. We therefore assayed the transcriptomes of 32,531 individual nuclei extracted from the nucleus accumbens (NAc) of nine donors with AUD and nine controls (72% male). Our study identified 17 clearly delineated cell types. We detected 26 transcriptome-wide significant differentially expressed genes (DEGs) that mainly involved medium spiny neurons with both D1-type and D2-type dopamine receptors, microglia (MGL) and oligodendrocytes. A higher than expected number of DEGs replicated in an existing single nucleus gene expression study of alcohol dependence in the prefrontal cortex (enrichment ratio 1.91, p value 0.019) with two genes remaining significant after a Bonferroni correction. Our most compelling result involved CD53 in MGL that replicated in the same cell type in the prefrontal cortex and was previously implicated in studies of DNA methylation, bulk gene expression and genetic variants. Several DEGs were previously reported to be associated with AUD (e.g., PER1 and MGAT5). The DEGs for MSN.3 seemed involved in neurodegeneration, disruption of circadian rhythms, alterations in glucose metabolism and changes in synaptic plasticity. For MGL, the DEGs implicated neuroinflammation and immune-related processes and for OLI, disruptions in myelination. This identification of the specific cell-types from which the association signals originate will be key for designing proper follow-up experiments and, eventually, novel clinical interventions.
Assuntos
Alcoolismo , Núcleo Accumbens , Masculino , Feminino , Animais , Camundongos , Núcleo Accumbens/metabolismo , Alcoolismo/genética , Alcoolismo/metabolismo , Transcriptoma , Receptores de Dopamina D1/metabolismo , Consumo de Bebidas Alcoólicas , Receptores de Dopamina D2/metabolismo , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Women are 1.5-3 times more likely to suffer from depression than men. This sex bias first emerges during puberty and then persists across the reproductive years. As the cause remains largely elusive, we performed a methylation-wide association study (MWAS) to generate novel hypotheses. METHODS: We assayed nearly all 28 million possible methylation sites in blood in 595 blood samples from 487 participants aged 9-17. MWASs were performed to identify methylation sites associated with increasing sex differences in depression symptoms as a function of pubertal stage. Epigenetic deconvolution was applied to perform analyses on a cell-type specific level. RESULTS: In monocytes, a substantial number of significant associations were detected after controlling the FDR at 0.05. These results could not be explained by plasma testosterone/estradiol or current/lifetime trauma. Our top results in monocytes were significantly enriched (ratio of 2.48) for genes in the top of a large genome-wide association study (GWAS) meta-analysis of depression and neurodevelopment-related Gene Ontology (GO) terms that remained significant after correcting for multiple testing. Focusing on our most robust findings (70 genes overlapping with the GWAS meta-analysis and the significant GO terms), we find genes coding for members of each of the major classes of axon guidance molecules (netrins, slits, semaphorins, ephrins, and cell adhesion molecules). Many of these genes were previously implicated in rodent studies of brain development and depression-like phenotypes, as well as human methylation, gene expression and GWAS studies. CONCLUSIONS: Our study suggests that the emergence of sex differences in depression may be related to the differential rewiring of brain circuits between boys and girls during puberty.
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Estudo de Associação Genômica Ampla , Caracteres Sexuais , Encéfalo , Metilação de DNA , Depressão/genética , Feminino , Humanos , Masculino , PuberdadeRESUMO
BACKGROUND: Longitudinal studies are needed to clarify whether early adversities are associated with advanced methylation age or if they actually accelerate methylation aging. This study test whether different dimensions of childhood adversity accelerate biological aging from childhood to adulthood, and, if so, via which mechanisms. METHODS: 381 participants provided one blood sample in childhood (average age 15.0; SD = 2.3) and another in young adulthood (average age 23.1; SD = 2.8). Participants and their parents provided a median of 6 childhood assessments (total = 1,950 childhood observations), reporting exposures to different types of adversity dimensions (i.e. threat, material deprivation, loss, unpredictability). The blood samples were assayed to estimate DNA methylation age in both childhood and adulthood and also change in methylation age across this period. RESULTS: Cross-sectional associations between the childhood adversity dimensions and childhood measures of methylation age were non-significant. In contrast, multiple adversity dimensions were associated with accelerated within-person change in methylation age from adolescence to young adulthood. These associations attenuated in model testing all dimensions at the same time. Accelerated aging increased with increasing number of childhood adversities: Individuals with highest number of adversities experienced 2+ additional years of methylation aging compared to those with no exposure to childhood adversities. The association between total childhood adversity exposure and accelerated aging was partially explained by childhood depressive symptoms, but not anxiety or behavioral symptoms. CONCLUSIONS: Early adversities accelerate epigenetic aging long after they occur, in proportion to the total number of such experiences, and in a manner consistent with a shared effect that crosses multiple early dimensions of risk.
Assuntos
Envelhecimento , Transtornos de Ansiedade , Adolescente , Humanos , Criança , Adulto Jovem , Adulto , Estudos Transversais , Fatores de Risco , Envelhecimento/genética , Epigênese GenéticaRESUMO
Using an integrative, multi-tissue design, we sought to characterize methylation and hydroxymethylation changes in blood and brain associated with alcohol use disorder (AUD). First, we used epigenomic deconvolution to perform cell-type-specific methylome-wide association studies within subpopulations of granulocytes/T-cells/B-cells/monocytes in 1132 blood samples. Blood findings were then examined for overlap with AUD-related associations with methylation and hydroxymethylation in 50 human post-mortem brain samples. Follow-up analyses investigated if overlapping findings mediated AUD-associated transcription changes in the same brain samples. Lastly, we replicated our blood findings in an independent sample of 412 individuals and aimed to replicate published alcohol methylation findings using our results. Cell-type-specific analyses in blood identified methylome-wide significant associations in monocytes and T-cells. The monocyte findings were significantly enriched for AUD-related methylation and hydroxymethylation in brain. Hydroxymethylation in specific sites mediated AUD-associated transcription in the same brain samples. As part of the most comprehensive methylation study of AUD to date, this work involved the first cell-type-specific methylation study of AUD conducted in blood, identifying and replicating a finding in DLGAP1 that may be a blood-based biomarker of AUD. In this first study to consider the role of hydroxymethylation in AUD, we found evidence for a novel mechanism for cognitive deficits associated with AUD. Our results suggest promising new avenues for AUD research.
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Alcoolismo , Consumo de Bebidas Alcoólicas , Alcoolismo/genética , Encéfalo , Metilação de DNA , Epigenoma , HumanosRESUMO
BACKGROUND: To avoid false-positive findings and detect cell-type specific associations in methylation and transcription investigations with bulk samples, it is critical to know the proportions of the major cell-types. RESULTS: We present a novel approach that allows for precise estimation of cell-type proportions using only a few highly informative methylation markers. The most reliable estimates were obtained with 17 amplicons (34 CpGs) using the MuSiC estimator, for which the average correlations between the estimated and the true cell-type proportions were 0.889. Furthermore, the estimates were not significantly different from the true values (P = 0.95) indicating that the estimator is unbiased and the standard deviation of the estimates further indicate high precision. Moreover, the overall variability of the estimates as measured by the Root Mean Squared Error (RMSE), which is a function of both bias and precision, was low (mean RMSE = 0.038). Taken together, these results indicate that the approach produced reliable estimates that are both unbiased and highly precise. CONCLUSION: This cost-effective approach for estimating cell-type proportions in bulk samples allows for enhanced targeted analysis, which in turn will minimize the risk of reporting false-positive findings and allowing for detection of cell-type specific associations. The approach is applicable across platforms and can be extended to assess cell-type proportions for various tissues.
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We present the first large-scale methylome-wide association studies (MWAS) for major depressive disorder (MDD) to identify sites of potential importance for MDD etiology. Using a sequencing-based approach that provides near-complete coverage of all 28 million common CpGs in the human genome, we assay methylation in MDD cases and controls from both blood (N = 1132) and postmortem brain tissues (N = 61 samples from Brodmann Area 10, BA10). The MWAS for blood identified several loci with P ranging from 1.91 × 10-8 to 4.39 × 10-8 and a resampling approach showed that the cumulative association was significant (P = 4.03 × 10-10) with the signal coming from the top 25,000 MWAS markers. Furthermore, a permutation-based analysis showed significant overlap (P = 5.4 × 10-3) between the MWAS findings in blood and brain (BA10). This overlap was significantly enriched for a number of features including being in eQTLs in blood and the frontal cortex, CpG islands and shores, and exons. The overlapping sites were also enriched for active chromatin states in brain including genic enhancers and active transcription start sites. Furthermore, three loci located in GABBR2, RUFY3, and in an intergenic region on chromosome 2 replicated with the same direction of effect in the second brain tissue (BA25, N = 60) from the same individuals and in two independent brain collections (BA10, N = 81 and 64). GABBR2 inhibits neuronal activity through G protein-coupled second-messenger systems and RUFY3 is implicated in the establishment of neuronal polarity and axon elongation. In conclusion, we identified and replicated methylated loci associated with MDD that are involved in biological functions of likely importance to MDD etiology.
Assuntos
Encéfalo/metabolismo , Metilação de DNA , Transtorno Depressivo Maior/sangue , Epigenoma , Cromossomos Humanos Par 2/genética , Ilhas de CpG/genética , Proteínas do Citoesqueleto/genética , Metilação de DNA/genética , DNA Intergênico/genética , Transtorno Depressivo Maior/genética , Epigenoma/genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de GABA-B/genéticaRESUMO
Recurrent and chronic major depressive disorder (MDD) accounts for a substantial part of the disease burden because this course is most prevalent and typically requires long-term treatment. We associated blood DNA methylation profiles from 581 MDD patients at baseline with MDD status 6 years later. A resampling approach showed a highly significant association between methylation profiles in blood at baseline and future disease status (P = 2.0 × 10-16). Top MWAS results were enriched specific pathways, overlapped with genes found in GWAS of MDD disease status, autoimmune disease and inflammation, and co-localized with eQTLS and (genic enhancers of) of transcription sites in brain and blood. Many of these findings remained significant after correction for multiple testing. The major themes emerging were cellular responses to stress and signaling mechanisms linked to immune cell migration and inflammation. This suggests that an immune signature of treatment-resistant depression is already present at baseline. We also created a methylation risk score (MRS) to predict MDD status 6 years later. The AUC of our MRS was 0.724 and higher than risk scores created using a set of five putative MDD biomarkers, genome-wide SNP data, and 27 clinical, demographic and lifestyle variables. Although further studies are needed to examine the generalizability to different patient populations, these results suggest that methylation profiles in blood may present a promising avenue to support clinical decision making by providing empirical information about the likelihood MDD is chronic or will recur in the future.
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Metilação de DNA , Depressão , Transtorno Depressivo Maior , Suscetibilidade a Doenças , Encéfalo/metabolismo , Doença Crônica , Ilhas de CpG/genética , Metilação de DNA/genética , Depressão/sangue , Depressão/genética , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , HumanosRESUMO
The transcription factor 4 (TCF4) locus is a robust association finding with schizophrenia (SCZ), but little is known about the genes regulated by the encoded transcription factor. Therefore, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) of TCF4 in neural-derived (SH-SY5Y) cells to identify genome-wide TCF4 binding sites, followed by data integration with SCZ association findings. We identified 11 322 TCF4 binding sites overlapping in two ChIP-seq experiments. These sites are significantly enriched for the TCF4 Ebox binding motif (>85% having ≥1 Ebox) and implicate a gene set enriched for genes downregulated in TCF4 small-interfering RNA (siRNA) knockdown experiments, indicating the validity of our findings. The TCF4 gene set was also enriched among (1) gene ontology categories such as axon/neuronal development, (2) genes preferentially expressed in brain, in particular pyramidal neurons of the somatosensory cortex and (3) genes downregulated in postmortem brain tissue from SCZ patients (odds ratio, OR = 2.8, permutation P < 4x10-5). Considering genomic alignments, TCF4 binding sites significantly overlapped those for neural DNA-binding proteins such as FOXP2 and the SCZ-associated EP300. TCF4 binding sites were modestly enriched among SCZ risk loci from the Psychiatric Genomic Consortium (OR = 1.56, P = 0.03). In total, 130 TCF4 binding sites occurred in 39 of the 108 regions published in 2014. Thirteen genes within the 108 loci had both a TCF4 binding site ±10kb and were differentially expressed in siRNA knockdown experiments of TCF4, suggesting direct TCF4 regulation. These findings confirm TCF4 as an important regulator of neural genes and point toward functional interactions with potential relevance for SCZ.
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Redes Reguladoras de Genes/genética , Genoma Humano/genética , Esquizofrenia/genética , Fator de Transcrição 4/genética , Sítios de Ligação/genética , Encéfalo/metabolismo , Encéfalo/patologia , Imunoprecipitação da Cromatina , Ontologia Genética , Predisposição Genética para Doença , Humanos , Neurogênese/genética , Mudanças Depois da Morte , Células Piramidais/metabolismo , Células Piramidais/patologia , Esquizofrenia/fisiopatologia , Córtex Somatossensorial/metabolismo , Córtex Somatossensorial/patologiaRESUMO
Motivation: Enrichment-based technologies can provide measurements of DNA methylation at tens of millions of CpGs for thousands of samples. Existing tools for methylome-wide association studies cannot analyze datasets of this size and lack important features like principal component analysis, combined analysis with SNP data and outcome predictions that are based on all informative methylation sites. Results: We present a Bioconductor R package called RaMWAS with a full set of tools for large-scale methylome-wide association studies. It is free, cross-platform, open source, memory efficient and fast. Availability and implementation: Release version and vignettes with small case study at bioconductor.org/packages/ramwas Development version at github.com/andreyshabalin/ramwas. Supplementary information: Supplementary data are available at Bioinformatics online.
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Biologia Computacional/métodos , Metilação de DNA , Software , Animais , Estudos de Associação Genética/métodos , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
In 1918, Fisher suggested that his research team had consistently found inflated cousin correlations. He also commented that because a cousin sample with minimal selection bias was not available the cause of the inflation could not be addressed, leaving this inflation as a challenge still to be solved. In the National Longitudinal Survey of Youth (the NLSY79, the NLSY97, and the NLSY-Children/Young Adult datasets), there are thousands of available cousin pairs. Those in the NLSYC/YA are obtained approximately without selection. In this paper, we address Fisher's challenge using these data. Further, we also evaluate the possibility of fitting ACE models using only cousin pairs, including full cousins, half-cousins, and quarter-cousins. To have any chance at success in such a restricted kinship domain requires an available and highly-reliable phenotype; we use adult height in our analysis. Results provide a possible answer to Fisher's challenge, and demonstrate the potential for using cousin pairs in a stand-alone analysis (as well as in combination with other biometrical designs).
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Biometria , Estatura/genética , Família , Feminino , Humanos , Estudos Longitudinais , Masculino , Adulto JovemRESUMO
Methylome-wide association studies are typically performed using microarray technologies that only assay a very small fraction of the CG methylome and entirely miss two forms of methylation that are common in brain and likely of particular relevance for neuroscience and psychiatric disorders. The alternative is to use whole genome bisulfite (WGB) sequencing but this approach is not yet practically feasible with sample sizes required for adequate statistical power. We argue for revisiting methylation enrichment methods that, provided optimal protocols are used, enable comprehensive, adequately powered and cost-effective genome-wide investigations of the brain methylome. To support our claim we use data showing that enrichment methods approximate the sensitivity obtained with WGB methods and with slightly better specificity. However, this performance is achieved at <5% of the reagent costs. Furthermore, because many more samples can be sequenced simultaneously, projects can be completed about 15 times faster. Currently the only viable option available for comprehensive brain methylome studies, enrichment methods may be critical for moving the field forward.
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Encéfalo/metabolismo , Metilação de DNA , Análise de Sequência de DNA , Ilhas de CpG , Feminino , Loci Gênicos , Humanos , Pessoa de Meia-Idade , Especificidade de ÓrgãosRESUMO
BACKGROUND: Recent reviews have highlighted the potential use of blood-based methylation biomarkers as diagnostic and prognostic tools of current and future alcohol use and addiction. Due to the substantial overlap that often exists between methylation patterns across different tissues, including blood and brain, blood-based methylation may track methylation changes in brain; however, little work has explored the overlap in alcohol-related methylation in these tissues. METHODS: To study the effects of alcohol on the brain methylome and identify possible biomarkers of these changes in blood, we performed a methylome-wide association study in brain and blood from 40 male DBA/2J mice that received either an acute ethanol (EtOH) or saline intraperitoneal injection. To investigate all 22 million CpGs in the mouse genome, we enriched for the methylated genomic fraction using methyl-CpG binding domain (MBD) protein capture followed by next-generation sequencing (MBD-seq). We performed association tests in blood and brain separately followed by enrichment testing to determine whether there was overlapping alcohol-related methylation in the 2 tissues. RESULTS: The top result for brain was a CpG located in an intron of Ttc39b (p = 5.65 × 10-08 ), and for blood, the top result was located in Espnl (p = 5.11 × 10-08 ). Analyses implicated pathways involved in inflammation and neuronal differentiation, such as CXCR4, IL-7, and Wnt signaling. Enrichment tests indicated significant overlap among the top results in brain and blood. Pathway analyses of the overlapping genes converge on MAPKinase signaling (p = 5.6 × 10-05 ) which plays a central role in acute and chronic responses to alcohol and glutamate receptor pathways, which can regulate neuroplastic changes underlying addictive behavior. CONCLUSIONS: Overall, we have shown some methylation changes in brain and blood after acute EtOH administration and that the changes in blood partly mirror the changes in brain suggesting the potential for DNA methylation in blood to be biomarkers of alcohol use.
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Encéfalo/metabolismo , Depressores do Sistema Nervoso Central/sangue , Depressores do Sistema Nervoso Central/farmacologia , Metilação de DNA/genética , Etanol/sangue , Etanol/farmacologia , Metaboloma , Animais , Biomarcadores/sangue , Diferenciação Celular/genética , Ilhas de CpG/genética , Inflamação/genética , Íntrons/genética , Lipoproteínas HDL/genética , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos DBA , Via de Sinalização Wnt/genéticaRESUMO
BACKGROUND: Previous genomewide association studies (GWASs) have identified a number of putative risk loci for alcohol dependence (AD). However, only a few loci have replicated and these replicated variants only explain a small proportion of AD risk. Using an innovative approach, the goal of this study was to generate hypotheses about potentially causal variants for AD that can be explored further through functional studies. METHODS: We employed targeted capture of 71 candidate loci and flanking regions followed by next-generation deep sequencing (mean coverage 78X) in 806 European Americans. Regions included in our targeted capture library were genes identified through published GWAS of alcohol, all human alcohol and aldehyde dehydrogenases, reward system genes including dopaminergic and opioid receptors, prioritized candidate genes based on previous associations, and genes involved in the absorption, distribution, metabolism, and excretion of drugs. We performed single-locus tests to determine if any single variant was associated with AD symptom count. Sets of variants that overlapped with biologically meaningful annotations were tested for association in aggregate. RESULTS: No single, common variant was significantly associated with AD in our study. We did, however, find evidence for association with several variant sets. Two variant sets were significant at the q-value <0.10 level: a genic enhancer for ADHFE1 (p = 1.47 × 10-5 ; q = 0.019), an alcohol dehydrogenase, and ADORA1 (p = 5.29 × 10-5 ; q = 0.035), an adenosine receptor that belongs to a G-protein-coupled receptor gene family. CONCLUSIONS: To our knowledge, this is the first sequencing study of AD to examine variants in entire genes, including flanking and regulatory regions. We found that in addition to protein coding variant sets, regulatory variant sets may play a role in AD. From these findings, we have generated initial functional hypotheses about how these sets may influence AD.
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Alcoolismo/diagnóstico , Alcoolismo/genética , Estudos de Associação Genética/métodos , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Adulto , Alcoolismo/epidemiologia , Feminino , Humanos , Masculino , Adulto JovemRESUMO
The central importance of epigenetics to the aging process is increasingly being recognized. Here we perform a methylome-wide association study (MWAS) of aging in whole blood DNA from 718 individuals, aged 25-92 years (mean = 55). We sequenced the methyl-CpG-enriched genomic DNA fraction, averaging 67.3 million reads per subject, to obtain methylation measurements for the â¼27 million autosomal CpGs in the human genome. Following extensive quality control, we adaptively combined methylation measures for neighboring, highly-correlated CpGs into 4 344 016 CpG blocks with which we performed association testing. Eleven age-associated differentially methylated regions (DMRs) passed Bonferroni correction (P-value < 1.15 × 10(-8)). Top findings replicated in an independent sample set of 558 subjects using pyrosequencing of bisulfite-converted DNA (min P-value < 10(-30)). To examine biological themes, we selected 70 DMRs with false discovery rate of <0.1. Of these, 42 showed hypomethylation and 28 showed hypermethylation with age. Hypermethylated DMRs were more likely to overlap with CpG islands and shores. Hypomethylated DMRs were more likely to be in regions associated with polycomb/regulatory proteins (e.g. EZH2) or histone modifications H3K27ac, H3K4m1, H3K4m2, H3K4m3 and H3K9ac. Among genes implicated by the top DMRs were protocadherins, homeobox genes, MAPKs and ryanodine receptors. Several of our DMRs are at genes with potential relevance for age-related disease. This study successfully demonstrates the application of next-generation sequencing to MWAS, by interrogating a large proportion of the methylome and returning potentially novel age DMRs, in addition to replicating several loci implicated in previous studies using microarrays.
Assuntos
Envelhecimento/genética , Ilhas de CpG , Metilação de DNA , Epigenômica , Adulto , Idoso , Idoso de 80 Anos ou mais , Biologia Computacional , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Mapas de Interação de Proteínas , Fatores Sexuais , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The National Longitudinal Survey of Youth datasets (NLSY79; NLSY-Children/Young Adults; NLSY97) have extensive family pedigree information contained within them. These data sources are based on probability sampling, a longitudinal design, and a cross-generational and within-family data structure, with hundreds of phenotypes relevant to behavior genetic (BG) researchers, as well as to other developmental and family researchers. These datasets provide a unique and powerful source of information for BG researchers. But much of the information required for biometrical modeling has been hidden, and has required substantial programming effort to uncover-until recently. Our research team has spent over 20 years developing kinship links to genetically inform biometrical modeling. In the most recent release of kinship links from two of the NLSY datasets, the direct kinship indicators included in the 2006 surveys allowed successful and unambiguous linking of over 94 % of the potential pairs. In this paper, we provide details for research teams interested in using the NLSY data portfolio to conduct BG (and other family-oriented) research.