methylation
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2. Results
2. Results
2.1. Whole-Genome DNA Methylation Patterns:
2.1. Whole-Genome DNA Methylation Patterns
Genome-scale DNA methylation changes were derived from the samples from each of five Healthy Controls (HC), five ME/CFS patients, and five LC patients. There were 342,055 fragments that covered 2,151,222 CpG sites in the 15 samples, from a total of 3.1×108 sequence reads (9.2×107 for HC, 10.8×107 for ME/CFS, and 10.9×107 for LC) allowing for comprehensive genome-wide analysis (Table S1). The global methylation genomic patterns were assessed and the median value obtained for global methylation with all three cohorts was similar (median methylation = 89.3% for HC, 89.9% for ME/CFS, and 88.7% for LC) showing hypermethylation was predominant. The promoter regions were, by contrast, predominantly hypomethylated in all three cohorts (Table S2). The intronic and exonic regions were predominantly hypermethylated with a similar median in all cohorts and with a slight increase in the first quartile in the ME/CFS cohort. A flow diagram of the decisions for analysis of the DMFs between ME/CFS and LC is shown in Figure 1.
Genome-scale DNA methylation changes were derived from the samples from each of five Healthy Controls (HC), five ME/CFS patients, and five LC patients. There were 342,055 fragments that covered 2,151,222 CpG sites in the 15 samples, from a total of 3.1 × 108 sequence reads (9.2 × 107 for HC, 10.8 × 107 for ME/CFS, and 10.9 × 107 for LC), allowing for comprehensive genome-wide analysis (Table S1). The global methylation genomic patterns were assessed, and the median value obtained for global methylation with all three cohorts was similar (median methylation = 89.3% for HC, 89.9% for ME/CFS, and 88.7% for LC), showing hypermethylation was predominant. The promoter regions were, by contrast, predominantly hypomethylated in all three cohorts (Table S2). The intronic and exonic regions were predominantly hypermethylated with a similar median in all cohorts and with a slight increase in the first quartile in the ME/CFS cohort. A flow diagram of the decisions for analysis of the DMFs between ME/CFS and LC is shown in Figure 1.
Figure 1. Flow diagram of the analysis of the DMFs in the ME/CFS, LC cohorts compared with age/sex matched HCs. Bam files were created from the Reduced Representation Bisulphite Sequencing (RRBS) data, the 15 samples analysed by DMAP2 [31] to give 342055 methylated fragments, those fragments found in all five patients of each of the three cohorts made were sub-selected (73239 fragments ), and those significantly different (p<0.05) comparing the ME/CFS and LC cohorts with HCs identified (3663 DMFs). Applying a limit of 10% change in differential methylation from the HCs gave 429 LC DMFs and 214 ME/CFS DMFs of which 118 were in common between the two disease cohorts. Twenty-six of these 118 had a >10 % methylation difference between the two patient cohorts.
Figure 1. Flow diagram of the analysis of the DMFs in the ME/CFS, LC cohorts compared with age/sex matched HCs. Bam files were created from the Reduced Representation Bisulphite Sequencing (RRBS) data, the 15 samples analysed by DMAP2 [31] to give 342,055 methylated fragments, those fragments found in all five patients of each of the three cohorts made were sub-selected (73,239 fragments), and those significantly different (p < 0.05) comparing the ME/CFS and LC cohorts with HCs identified (3363 DMFs). Applying a limit of 10% change in differential methylation from the HCs gave 429 LC DMFs and 214 ME/CFS DMFs, of which 118 were in common between the two disease cohorts. Twenty-six of these 118 had a >10% methylation difference between the two patient cohorts.
To begin the evaluation of the methylation landscape, the fragments were first sub-selected with a strict criterion as being present in all five patients of each of the three cohorts, and 73,239 fragments were then available for further analysis (Figure 1). A Principal Component Analysis (PCA) separated the cohorts into three distinct clusters (Figure 2). This indicated that both the ME/CFS and LC cohorts showed differential methylation within these common fragments compared with the healthy controls. The separation of the ME/CFS and LC cohorts into individual clusters (Figure 2) suggested that there might be differences between the extent of the methylation change at specific sites between the two cohorts, or changes at specific sites in only one of the disease cohorts. For this analysis a significance of P<0.05 was imposed for the methylation change, but no limits on the degree of methylation change. For this analysis, 3363 fragments met that criterion.
To begin the evaluation of the methylation landscape, the fragments were first sub-selected with a strict criterion as being present in all five patients of each of the three cohorts, and 73,239 fragments were then available for further analysis (Figure 1). A Principal Component Analysis (PCA) separated the cohorts into three distinct clusters (Figure 2). This indicated that both the ME/CFS and LC cohorts showed differential methylation within these common fragments compared with the healthy controls. The tight clustering of all patients of each cohort in the plot suggested most of the DMFs and their characteristics were common to all patients within the cohort. The separation of the ME/CFS and LC cohorts into individual clusters (Figure 2) suggested that there might be differences between the extent of the methylation change at specific sites between the two cohorts, or changes at specific sites in only one of the disease cohorts. For this analysis, a significance of p < 0.05 was imposed for the methylation change, but no limits on the degree of methylation change and 3363 fragments met that criterion.
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Figure 2. PCA of differentially methylated fragments in all members of the HC, LC, ME/CFS cohorts. PCA plot illustrating three distinct clusters representing HC, ME/CFS, and LC based on 3,363 DMFs common to all cohorts filtered by P < 0.05, without considering methylation difference.
Figure 2. PCA of differentially methylated fragments in all members of the HC, LC, and ME/CFS cohorts. PCA plot illustrating three distinct clusters representing HC, ME/CFS, and LC based on 3363 DMFs common to all cohorts filtered by p < 0.05, without considering methylation difference.
2.2. Characteristics of the Differential Methylation Changes in LC and ME/CFS
2.2. Characteristics of the Differential Methylation Changes in LC and ME/CFS
The analysis identified 429 DMFs between LC vs HC (Table S3, Figure 1) and 214 DMFs between ME/CFS vs HC (Table S4, Figure 1) (p-value <0.05, minimum 10% methylation difference). Of the 429 DMFs between LC and HC, 148 were hypomethylated and 281 were hypermethylated, and among the 214 DMFs between ME/CFS and HC, 69 were hypomethylated and 145 were hypermethylated. Thus, both cohorts showed methylation patterns shifting towards hypermethylation when compared to HCs and in this study, LC patients show more abundant methylation changes than the age/sex matched ME/CFS patients. Of the 429 DMFs in LC and 214 DMFs in ME/CFS when compared to HC, 118 DMFs were common to both disease datasets, as shown in the Venn diagram (Figure 3A). The Pearson R score was 0.88 when the methylation data within the common fragments of the two cohorts were compared, indicating a high correlation between the data at the differentially methylated sites from the LC and ME/CFS cohorts and emphasizing the similarity between the two conditions. Heat maps of LC vs HC and ME/CFS vs HC are shown in Figure 3B,C. Unsupervised hierarchical clustering of DMFs between HCs and LCs (Figure 3B) and between HCs and ME/CFS (Figure 3C) has separated the differential methylation patterns so that all members of each of the three cohorts (HC-green, LC-red, or ME/CFS -purple) group together. The regions of the genome from which the DMFs are derived (intergenic, promoter, exon, intron or the boundary between intron and exon) are shown on the axis at left, colour coded in the key. The heatmaps showed distinct patterns of the DMFs: (i) most hypermethylated fragments in the HC cohort became more strongly hypermethylated in both LC and ME/CFS cohorts, and a minority became more hypomethylated; (ii) most hypomethylated fragments in the HC generally also became more hypermethylated but with a minority becoming more hypomethylated. This was a common pattern for both disease cohorts. The minority group of DMFs was larger in the LC cohort than in the ME/CFS cohort (iii) Some fragments with mid-range methylation were more variable in their methylation among the individual members of the HC cohort but became predominantly more hypermethylated in the patient cohorts, again with a minority showing greater hypomethylation. A heatmap illustrating the methylation values and genomic location of the 118 common fragments (Table S6) found in each cohort compared with the HC cohort was generated and showed the majority of these DMFs are similarly differentially methylated (Figure 3D). In contrast to the heat maps in Figure 3B,C, unsupervised hierarchical clustering of DMFs has separated one ME/CFS patient (ME028) in the ME/CFS group from the other ME/CFS members into a ‘group of one’ whereas all five members of each of the other two cohorts (HC-green, LC-red) group together. It is interesting to note that in the PCA analysis shown in Figure 2 this patient ME028 was more widely separated from the other members of the ME/CFS cluster. The heat map showed a wide range of methylation levels in the HC of the 118 fragments and both changes towards hypermethylation and hypomethylation in the LC and ME/CFS cohorts.
The analysis identified 429 DMFs between LC vs. HC (Table S3, Figure 1) and 214 DMFs between ME/CFS vs. HC (Table S4, Figure 1) (p-value < 0.05, minimum 10% methylation difference). Of the 429 DMFs between LC and HC, 148 were hypomethylated and 281 were hypermethylated, and among the 214 DMFs between ME/CFS and HC, 69 were hypomethylated and 145 were hypermethylated. Thus, both cohorts showed methylation patterns shifting towards hypermethylation when compared to HCs, and in this study, LC patients show more abundant methylation changes than the age/sex matched ME/CFS patients. Of the 429 DMFs in LC and 214 DMFs in ME/CFS when compared to HC, 118 DMFs were common to both disease datasets, as shown in the Venn diagram (Figure 3A). The Pearson R score was 0.88 when the methylation data within the common fragments of the two cohorts were compared, indicating a high correlation between the data at the differentially methylated sites from the LC and ME/CFS cohorts and emphasizing the similarity between the two conditions. Heat maps of LC vs. HC and ME/CFS vs. HC are shown in Figure 3B,C. Unsupervised hierarchical clustering of DMFs between HCs and LCs (Figure 3B) and between HCs and ME/CFS (Figure 3C) has separated the differential methylation patterns so that all members of each of the three cohorts (HC-green, LC-red, or ME/CFS-purple) group together. The regions of the genome from which the DMFs are derived (intergenic, promoter, exon, intron, or the boundary between intron and exon) are shown on the axis at left, colour coded in the key. The heatmaps showed distinct patterns of the DMFs: (i) most hypermethylated fragments in the HC cohort became more strongly hypermethylated in both LC and ME/CFS cohorts, and a minority became more hypomethylated; (ii) most hypomethylated fragments in the HC generally also became more hypermethylated but with a minority becoming more hypomethylated. This was a common pattern for both disease cohorts. The minority group of DMFs was larger in the LC cohort than in the ME/CFS cohort (iii). Some fragments with mid-range methylation were more variable in their methylation among the individual members of the HC cohort but became predominantly more hypermethylated in the patient cohorts, again with a minority showing greater hypomethylation. A heatmap illustrating the methylation values and genomic location of the 118 common fragments (Table S6) found in each cohort compared with the HC cohort was generated and showed that the majority of these DMFs are similarly differentially methylated (Figure 3D). In contrast to the heat maps in Figure 3B,C, unsupervised hierarchical clustering of DMFs has separated one ME/CFS patient (ME028) in the ME/CFS group from the other ME/CFS members into a ‘group of one’ whereas all five members of each of the other two cohorts (HC-green, LC-red) group together. It is interesting to note that in the PCA analysis shown in Figure 2, this patient, ME028, was more widely separated from the other members of the ME/CFS cluster. The heat map showed a wide range of methylation levels in the HC of the 118 fragments and both changes towards hypermethylation and hypomethylation in the LC and ME/CFS cohorts.
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Figure 3. Heatmaps of differential methylation on fragments in LC and ME/CFS patient cohorts compared with HCs. (A)Venn diagram showing overlapping DMFs in LC and ME (P<0.05, >10% methylation difference). Heatmaps show DMFs between (B) LC and HC, (C) ME/CFS and HC (P<0.05, >10% methylation difference). The annotation column bar at the top of each heat map represents the cohort group (LC-red, HC-green, ME/CFS -purple), and the annotation on the Y axis shows the genome region of the DMF (color coded in the key). The colour gradient shown in the key indicates the methylation level of the fragments with the darker colours representing the higher degree of methylation. The locations of the DMFs are annotated beside the heatmaps, gene promoters (-1kb to +5kb from the TSS), exons, introns, and intergenic elements (>5kb upstream from the nearest TSS), and intron-exon boundary elements. (D) Heatmap showing the methylation values of the 118 common DMFs in the ME/CFS and LC cohorts and illustrating the methylation differences in LC and ME/CFS patient groups compared with the HC group. The annotation column bar at the top of the heat map represents the cohort groups (LC-red, HC-green, ME/CFS-purple), and the annotation row bar on the left side shows the genome region of the DMFs (colour coded in the key). The colour gradient yellow to indigo shown in the key ranges from hypermethylation (yellow) to hypomethylation (indigo). It indicates the colours matching specific methylation values.
Figure 3. Heatmaps of differential methylation on fragments in the LC and ME/CFS patient cohorts compared with HCs. (A) Venn diagram showing overlapping DMFs in LC and ME (p < 0.05, >10% methylation difference). Heatmaps show DMFs between (B) LC and HC, (C) ME/CFS and HC (p < 0.05, >10% methylation difference). The annotation column bar at the top of each heat map represents the cohort group (LC-red, HC-green, ME/CFS-purple), and the annotation on the Y axis shows the genome region of the DMF (colour coded in the key). The colour gradient shown in the key indicates the methylation level of the fragments, with the darker colours representing the higher degree of methylation. The locations of the DMFs are annotated beside the heatmaps, gene promoters (−1 kb to +5 kb from the TSS), exons, introns, and intergenic elements (>5 kb upstream from the nearest TSS), and intron-exon boundary elements. (D) Heatmap showing the methylation values of the 118 common DMFs in the ME/CFS and LC cohorts and illustrating the methylation differences in the LC and ME/CFS patient groups compared with the HC group. The annotation column bar at the top of the heat map represents the cohort groups (LC-red, HC-green, ME/CFS-purple), and the annotation row bar on the left side shows the genome region of the DMFs (colour coded in the key). The colour gradient from yellow to indigo shown in the key ranges from hypermethylation (yellow) to hypomethylation (indigo). It indicates the colours matching specific methylation values.
2.3. DMFs Associated with Gene Promoters and Gene Exons.
2.3. DMFs Associated with Gene Promoters and Gene Exons
Among the 118 common DMFs, twelve were associated with gene promoters. Nine (associated genes-LGALS3, SLC38A8, SLFN13, CCDC130, HSPB6, CTSZ, MYL9, DOM3Z, CACNA2D4) showed hypermethylation in both LC and ME/CFS, and two (associated genes -IRF2BPL, NMRAL1) were hypomethylated in both conditions. In contrast, one, associated with FGD2 was hypermethylated in the LC patients and hypermethylated in the ME/CFS patients when compared with healthy controls. Six fragments were associated with gene exons (ITPKB, KIF26B, CHD7, STAT5A, ABCA7, HSPA12B) among the 118 common DMFs. Three (ITPKB, KIF26B, CHD7) were hypomethylated in both the LC and ME patients, and two (STAT5A, ABCA7) were hypermethylated in both conditions. By contrast, one HSPA12B showed hypermethylation in LC and hypomethylation in ME/CFS compared to HC (Table S5). The twelve identified DMFs in promoter regions associated with specific genes are shown as box plots in Figure 4A, indicating the individual values of the five patients from each disease cohort and the controls. In most cases the changes were found in all patients of the cohort and variation among the individual patients was relatively small, apart from two sites for the LC cohort. The HC median values of the two promoter sites that were hypomethylated in both cohorts (IRF2BPL and NMRAL1) were significantly different, 25% and 75% methylation respectively. For the eight sites that were hypermethylated in both LC and ME/CFS compared with HCs, the median values of the methylation in the HCs varied from 9-75%. The promoter site associated with FGD2 that was hypermethylated only in the LC patients compared with the HC increased from 52% to 81% whereas it was unchanged at 50% in the ME/CFS cohort.
Among the 118 common DMFs, twelve were associated with gene promoters. Nine (associated genes-LGALS3, SLC38A8, SLFN13, CCDC130, HSPB6, CTSZ, MYL9, DOM3Z, CACNA2D4) showed hypermethylation in both LC and ME/CFS, and two (associated genes -IRF2BPL, NMRAL1) were hypomethylated in both conditions. In contrast, one, associated with FGD2, was hypermethylated in the LC patients and relatively unchanged in the ME/CFS patients when compared with healthy controls. Six fragments were associated with gene exons (ITPKB, KIF26B, CHD7, STAT5A, ABCA7, HSPA12B) among the 118 common DMFs. Three (ITPKB, KIF26B, CHD7) were hypomethylated in both the LC and ME patients, and two (STAT5A, ABCA7) were hypermethylated in both conditions. By contrast, one HSPA12B showed hypermethylation in LC and hypomethylation in ME/CFS compared to HC (Table S5). The twelve identified DMFs in promoter regions associated with specific genes are shown as box plots in Figure 4A, indicating the individual values of the five patients from each disease cohort and the controls. In most cases, the changes were found in all patients of the cohort as inferred from the PCA plot (Figure 2), and variation among the individual patients was relatively small, apart from two sites for the LC cohort. The HC median values of the two promoter sites that were hypomethylated in both cohorts (IRF2BPL and NMRAL1) were significantly different, 25% and 75% methylation, respectively. For the eight sites that were hypermethylated in both LC and ME/CFS compared with HCs, the median values of the methylation in the HCs varied from 9–75%. The promoter site associated with FGD2 that was hypermethylated only in the LC patients compared with the HC increased from 52% to 81% whereas it was unchanged at 50% in the ME/CFS cohort.
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Figure 4. Box plots showing individual patient differential methylation characteristics at gene-associated sites Box plots of (A) the twelve genes associated with differentially methylated promoters and (B) six genes associated with differentially methylated exons (B) identified within the 118 fragments in common between LC and ME/CFS patients. The plots show the individual patient values of the LC, ME/CFS and the HC cohorts. In both A & B, the HC are coloured green, the LC red and ME/CFS purple Individual patients are indicated by the points on the plots. The medians are indicated by the bold line and the box indicates the first and third quartiles. Associated gene IDs are displayed at the top of each plot.
Figure 4. Box plots showing individual patient differential methylation characteristics at gene-associated sites. Box plots of (A) DMFs associated with gene promoters; (B) DMFs associated with gene exons within the 118 fragments in common between LC and ME/CFS patients. The plots show the individual patient values of the LC, ME/CFS, and the HC cohorts. In both (A,B), the HC is coloured green, the LC red, and ME/CFS purple. Individual patients are indicated by the points on the plots. The medians are indicated by the bold line, and the box indicates the first and third quartiles. Associated gene IDs are displayed at the top of each sub plot.
The box plots of the six DMFs associated with exons of specific genes, ITPKB, KIF26B, CHD7, STAT5A, ABCA7, and HSPA12B are shown in Figure 4B. Most had HC methylation median values of ~40%. The variations in the methylation values among individual patients, while greater than in the controls, were still relatively small.
The box plots of the six DMFs associated with exons of specific genes, ITPKB, KIF26B, CHD7, STAT5A, ABCA7, and HSPA12B, are shown in Figure 4B. Most had HC methylation median values of ~40%. The variations in the methylation values among individual patients, while greater than in the controls, were still relatively small.
2.4. Methylation Differences Between Long COVID and ME/CFS
2.4. Methylation Differences Between Long COVID and ME/CFS
There were also differences in the degree of methylation change between the two disease cohorts. There were 26 DMFs among the 118 DMFs with >10% methylation difference between LC and ME/CFS (Table S6). Fifteen of the 26 DMFs lie within the intergenic regions and 11 are in more defined gene regions and therefore easier to interpret. Of these 11 DMFs, three of the genes corresponding to sites within exons (CHF7, ABCA7 and HSPA12B) and three within promoter regions (associated with FGD2, NMRAL1, LGALS3).
There were also differences in the degree of methylation change between the two disease cohorts. There were 26 DMFs among the 118 DMFs with >10% methylation difference between LC and ME/CFS (Table S6). Fifteen of the 26 DMFs lie within the intergenic regions, and 11 are in more defined gene regions and therefore easier to interpret. Of these 11 DMFs, three of the genes correspond to sites within exons (CHF7, ABCA7, and HSPA12B) and three within promoter regions (associated with FGD2, NMRAL1, and LGALS3).
In Table 1 the chromosome and the genomic start and end sites of the 26 DMFs where there was a >10% difference in the methylation changes between LC and ME/CFS cohorts are shown. The genomic locations of the DMFs (promoter, exon, intron or intergenic) are documented and the percentage differences in methylation between LC vs HC, ME/CFS vs HC and then LC vs ME/CFS indicated. GeneIDs linked to differentially methylated promoters, exons, and introns are given but not the possible gene linkages for intergenic regions. The DMF changes between the two cohorts are sorted according to the degree of difference between the LC and ME/CFS ( -29% to + 37% - shown in last column). It is to be noted that the exons and promoters in Table 1 are already represented in the box plots in Figure 4A,B.
In Table 1, the chromosome and the genomic start and end sites of the 26 DMFs where there was a >10% difference in the methylation changes between the LC and ME/CFS cohorts are shown. The genomic locations of the DMFs (promoter, exon, intron, or intergenic) are documented, and the percentage differences in methylation between LC vs. HC, ME/CFS vs. HC, and then LC vs. ME/CFS are indicated. GeneIDs linked to differentially methylated promoters, exons, and introns are given, but not the possible gene linkages for intergenic regions. The DMF changes between the two cohorts are sorted according to the degree of difference between the LC and ME/CFS (−29% to +37%-shown in last column). It is to be noted that the exons and promoters in Table 1 are already represented in the box plots in Figure 4A,B.
Table 1. Twenty-six DMFs segregating HC, LC and ME/CFS cohorts from each other.
Table 1. Twenty-six DMFs segregating HC, LC, and ME/CFS cohorts from each other.
In the eight of the 26 DMFs where there was hypomethylation in both cohorts, it was greater in seven in the LC patient group, and in only one fragment in the ME/CFS patient group. In the 12 of the 26 DMFs that were hypermethylated in both cohorts, nine showed greater differential methylation in the LC cohort and three in the ME/CFS cohort. Hence, the LC group of patients generally have a greater change in their methylation status in 16 of the 20 DMFs compared with the ME./CFS patients when the change is in the same direction. By contrast, six of the 26 DMFs showed opposite changes in their differential methylation in the two cohorts (five hypermethylated in LC but hypomethylated in ME/CFS; one hypermethylated in ME/CFS and hypomethylated in LC).
In 8 of the 26 DMFs where there was hypomethylation in both cohorts, it was greater in seven in the LC patient group, and in only one fragment in the ME/CFS patient group. Of the 12 of the 26 DMFs that were hypermethylated in both cohorts, nine showed greater differential methylation in the LC cohort and three in the ME/CFS cohort. Hence, the LC group of patients generally has a greater change in their methylation status in 16 of the 20 DMFs compared with the ME./CFS patients when the change is in the same direction. By contrast, six of the 26 DMFs showed opposite changes in their differential methylation in the two cohorts (five hypermethylated in LC but hypomethylated in ME/CFS; one hypermethylated in ME/CFS and hypomethylated in LC).
2.4. Functional Pathway Analysis of the DMFs of Long COVID and ME/CFS
2.5. Functional Pathway Analysis of the DMFs of Long COVID and ME/CFS
Among the 429 DMFs from HC vs LC and 214 DMFs between HC vs ME, the unique associated GeneIDs were isolated and tabulated, except from sites within the intergenic regions where gene linkages are much less certain. There were 215 unique genes linked to LC (Table S8) and 111 genes linked to ME/CFS (Table S9). To identify the functional pathways associated with the DMFs in LC and ME/CFS, pathway enrichment analysis was performed using Metascape [32]. This analysis revealed several shared pathways between the two conditions (Figure S1A & S1B), suggesting common biological mechanisms. Notably, Response to Wounding (GO:0009611) emerged as a key pathway involved in tissue repair and inflammatory processes, aligning with the immune dysregulation and chronic inflammatory responses observed in both conditions. Additionally, Regulation of System Process (GO:0044057) was significantly enriched, highlighting disruptions in physiological processes such as circulation and metabolism that may contribute to cardiovascular and neurological dysfunctions. Cellular Response to Cytokine Stimulus/Acid Chemical (GO:0071345/GO:0071229) was identified, which likely reflects the important role in immune signaling and inflammation, both of which are known to be altered in ME/CFS and LC. Furthermore, Regulation of Small GTPase-Mediated Signal Transduction (GO:0051057/GO:0051056) was enriched, implicating intracellular signaling pathways that modulate immune responses, cell migration, and tissue repair. Growth Regulation (GO:0040007/GO:0040008) was a prominent pathway, suggesting that aberrant growth signaling could contribute to impaired tissue regeneration in both conditions. Other than these similarities, the functional pathway analysis showed blood vessel morphogenesis, muscle organ development, AGE RAGE pathway, Neutrophil degranulation as other notable pathways in LC, whereas thyroid hormone production, leukocyte differentiation, negative regulation of T cell receptor pathway, heart development and blood circulation were highlighted in ME/CFS.
Among the 429 DMFs from HC vs. LC and 214 DMFs between HC vs. ME, the unique associated GeneIDs were isolated and tabulated, except for sites within the intergenic regions where gene linkages are much less certain. There were 215 unique genes linked to LC (Table S8) and 111 genes linked to ME/CFS (Table S9). To identify the functional pathways associated with the DMFs in LC and ME/CFS, pathway enrichment analysis was performed using Metascape [32]. This analysis revealed several shared pathways between the two conditions (Figure S1A,B), suggesting common biological mechanisms. Notably, Response to Wounding (GO:0009611) emerged as a key pathway involved in tissue repair and inflammatory processes, aligning with the immune dysregulation and chronic inflammatory responses observed in both conditions. Additionally, Regulation of System Process (GO:0044057) was significantly enriched, highlighting disruptions in physiological processes such as circulation and metabolism that may contribute to cardiovascular and neurological dysfunctions. Cellular Response to Cytokine Stimulus/Acid Chemical (GO:0071345/GO:0071229) was identified, which likely reflects the important role in immune signalling and inflammation, both of which are known to be altered in ME/CFS and LC. Furthermore, Regulation of Small GTPase-Mediated Signal Transduction (GO:0051057/GO:0051056) was enriched, implicating intracellular signalling pathways that modulate immune responses, cell migration, and tissue repair. Growth Regulation (GO:0040007/GO:0040008) was a prominent pathway, suggesting that aberrant growth signalling could contribute to impaired tissue regeneration in both conditions. Other than these similarities, the functional pathway analysis showed blood vessel morphogenesis, muscle organ development, AGE RAGE pathway, Neutrophil degranulation as other notable pathways in LC, whereas thyroid hormone production, leukocyte differentiation, negative regulation of T cell receptor pathway, heart development, and blood circulation were highlighted in ME/CFS.