Identification of epigenetically altered genes in sporadic amyotrophic lateral sclerosis - PubMed (original) (raw)

Identification of epigenetically altered genes in sporadic amyotrophic lateral sclerosis

Claudia Figueroa-Romero et al. PLoS One. 2012.

Abstract

Amyotrophic lateral sclerosis (ALS) is a terminal disease involving the progressive degeneration of motor neurons within the motor cortex, brainstem and spinal cord. Most cases are sporadic (sALS) with unknown causes suggesting that the etiology of sALS may not be limited to the genotype of patients, but may be influenced by exposure to environmental factors. Alterations in epigenetic modifications are likely to play a role in disease onset and progression in ALS, as aberrant epigenetic patterns may be acquired throughout life. The aim of this study was to identify epigenetic marks associated with sALS. We hypothesize that epigenetic modifications may alter the expression of pathogenesis-related genes leading to the onset and progression of sALS. Using ELISA assays, we observed alterations in global methylation (5 mC) and hydroxymethylation (5 HmC) in postmortem sALS spinal cord but not in whole blood. Loci-specific differentially methylated and expressed genes in sALS spinal cord were identified by genome-wide 5mC and expression profiling using high-throughput microarrays. Concordant direction, hyper- or hypo-5mC with parallel changes in gene expression (under- or over-expression), was observed in 112 genes highly associated with biological functions related to immune and inflammation response. Furthermore, literature-based analysis identified potential associations among the epigenes. Integration of methylomics and transcriptomics data successfully revealed methylation changes in sALS spinal cord. This study represents an initial identification of epigenetic regulatory mechanisms in sALS which may improve our understanding of sALS pathogenesis for the identification of biomarkers and new therapeutic targets.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow chart for genome-wide epigenetic and expression analysis.

DNA and RNA from postmortem human spinal cord samples were subjected to high-throughput epigenetic and gene expression screening, respectively. DMGs and DEGs representing concordant direction were further analyzed by bioinformatics analyses. Finally, identified candidate genes were experimentally confirmed in the spinal cord.

Figure 2. Global 5mC is increased in spinal cord of sALS.

Genomic DNA extracted from control (open bar) or sALS (filled bar) postmortem human spinal cord was analyzed with an ELISA colorimetric assay for methylation, (5mC; Ctrl = 11, sALS = 11). Outliers, outside of median ±1.5 x Inter-Quartile-Range, were excluded. The data is presented as mean ± SEM of percent (%) 5mC using box and whiskers vertical bars plotting minimum to maximum values. A two-sample equal variance t-test used; ***p<0.001 compared to control group (Ctrl) with an achieved power of 99%.

Figure 3. Biological functions of DMGs, DEGs, and concordant epigenes.

Overrepresented biological functions were identified using DAVID. The top 20 biological functions ordered by p-value were collected from each gene set, and redundant terms were combined. The values in the table correspond to -log10 (DAVID p-value), ranging from 0 (white) to 30 (bright red). The values are not normalized across different gene sets with variable numbers of genes. The order of biological terms was based on the log-transformed p-values of DMGs and DEGs.

Figure 4. Overlapping of DMGs and DEGs.

Identified DMGs and DEGs were compared for overlapping. Among 3,574 DMGs and 1,182 DEGs, 251 genes were common. Among these shared genes, 112 genes had concordant direction of change in methylation and expression (hyper-methylation with down-regulation or hypo-methylation with up-regulation) (shaded circle).

Figure 5. Literature-based association network of concordant epigenes.

Literature-derived information for potential associations among the concordant epigenes was obtained using Genomatix Pathway System (GePS). Among the 112 concordant epigenes, 53 genes were co-cited at least once in PubMed abstracts at sentence-level (solid lines) or abstract-level (dashed lines). These epigenes were grouped by their representative biological role: immune response (dark pink), antigen presentation (light pink), tumor/suppressor related (green), extracellular matrix repair (yellow), and others (gray). Neuronal development-related genes are enclosed by a square. The epigenes with more than 5 connections to other genes are enlarged. A diamond-shape represents a transcription factor.

Figure 6. RT-PCR confirmation of concordant epigenes in spinal cord.

RNA was extracted from the postmortem human spinal cord tissue that was used for the methylation analysis from sALS (n = 8–11) subjects and controls (n = 8–11) and subjected to RT-PCR. Results were normalized to glyceraldehyde-3- phosphate dehydrogenase (GAPDH) except for STAT5A which was normalized to TATA box binding protein (Tbp) and presented as fold changes calculated by the 2−ΔΔC T method. Similar results were obtained when using different housekeeping genes (Fig. S2); *p<0.05, **p<0.01, ***p<0.001 compared to the control group (Ctrl). Data mean ± SEM is plotted using box and whiskers vertical bars plotting minimum to maximum values.

Figure 7. Global 5 HmC is increased in spinal cord of sALS.

Genomic DNA extracted from control or sALS postmortem human spinal cord was analyzed with an ELISA colorimetric assay for hydroxymethylation (5 HmC; Ctrl = 9, sALS = 8). The data is presented as mean ± SEM of percent (%)5 HmC using a two-sample equal variance t-test and plotted using box and whiskers vertical bars with minimum to maximum values; ****p<0.0001 compared to the control group (Ctrl).

Figure 8. Changes in global 5 HmC and 5mC are not detected in ALS whole blood.

Genomic DNA extracted from control or sALS human whole blood was analyzed for 5mC (Ctrl n = 11, ALS n = 11_; p = 0.94_) and 5 HmC (Ctrl n = 11, sALS n = 11; p = 0.40). Percent (%) 5mC and 5 HmC is presented as mean ± SEM using a two-sample equal variance t-test and graphed using box and whiskers vertical bars plotting minimum to maximum values.

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Identification of epigenetically altered genes in sporadic amyotrophic lateral sclerosis - PubMed (original) (raw)