Patient health status assessment with FIQ, MFI, and SF-36 (Burckhardt et al., 1991; Mchorney et al., 1993; Smets et al., 1995) questionnaires.

Abbreviations: FIQ, Fibromyalgia Impact Questionnaire; MFI, Multi Fatigue Inventory; SF-36, Short-Form-36 Health Survey; SD, standard deviation; SE, standard error. Range refers to the possible values in the studied group.

DE of HERV elements discriminates ME/CFS, FM, and co-diagnosis.

(A) Volcano plots showing Log2(fold changes) and the adjusted p-values for all HERV assessed with HERV-V3 microarray for each set of groups, as indicated. Red dots indicate DE HERV (FDR<0.1 and |log2FC|>1). Grey boxes show numbers of overexpressed or underexpressed HERV elements. (B) HERV expression heatmap and cluster analysis of ME/CFS (n=8, green), FM (n=10, orange), co-diagnosed (n=16, yellow), and healthy control (n=9, blue) samples. The heatmap includes all HERV probes displaying significant DE between at least two of the compared groups (FDR<0.1 and |log2FC|>1). Clusters 1-4 correspond to groups of HERV probes displaying significant over/under-expression in at least one group. Box and violin plots summarize the distribution and expression levels of the DE probes in each cluster per study group. The scaled mean expression value (z score) for each HERV probe is plotted. Box plots show the median z-score value and the first and third quartiles. (C) Box and violin plots for Clusters 1 and 2 differentiating ME/CFS subgroups 1 and 2. (D) Principal component analysis of DE HERV. (E) Bar plot of top ten DE HERV with greater influence/contribution to principal component 1 (PC1) or principal component 2 (PC2). Statistical tests: unpaired two-sample Wilcoxon Test with Benjamin-Hochberg p-value correction (***p<0.001, ****p < 0.0001).

Diverse solitary LTR families are deregulated in ME/CFS, FM, and co-diagnosed conditions.

(A) Relative contribution of HERV families to each cluster, calculated as the proportion of HERV loci assigned to each family relative to total HERV loci in each cluster. Families with a representation of at least 2.5% are shown. Main HERV families (B) downregulated in ME/CFS, FM and co-diagnosed groups (Cluster 4), (C) upregulated in ME/CFS (Cluster 1), (D) downregulated in ME/CFS (Cluster 2), and (E) upregulated in FM (Cluster 3). Box and violin plots summarize distributions and expression levels of the different HERV probes belonging to the same family in the different study groups. The scaled mean expression value (z score) for each HERV probe is plotted. Box plots show the median z-score value and the first and third quartiles. (F) Proportion of HERV subdomains expressed by cluster. (G) Genomic context of the DE HERV loci by cluster. Statistical tests: Fisher’s exact test, T-test or Wilcoxon test with Benjamin-Hochberg p-value correction (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

HERV expression correlates with immune response genes.

Hierarchical clustering of DE HERV and genes (FDR<0.1 and |log2FC|>1) providing eight modules with highly correlated expression levels. Box and violin plots summarize the distribution and expression level of the different HERV (blue) and gene (green) probes in modules (A) 1 and 6, (B) 3 and 4, and (C) 2, 5, 7, and 8, per study group. The scaled mean expression value (z score) for each HERV and gene probe sets are plotted. Box plots show the median z score values with first and third quartiles. Statistical tests: Wilcoxon test (*p < 0.05; **p<0.01, ***p<0.001, ****p < 0.0001).

DE HERV sequences and DE immune-response genes are majorly independently transcribed.

(A) Genomic distribution of HERV, and genes encompassed in modules one through eight. (B) Genomic distance between DE HERV and their nearest DE genes per Module, plotted as density curves illustrating the distribution of distances from 0 to 100 million base pair (Mbp). (C) Violin dot plots showing the distribution and expression levels of some examples of co-localized HERV loci and genes in ME/CFS subgroup 1 (n=3), ME/CFS subgroup 2 (n=5), FM (n=10), co-diagnosed (n=16) and healthy control (n=9) samples. Blue and orange highlighted headings indicates the pair HERV/gene belonged to modules 1 or 6, or modules 2, 5, 7, or 8, respectively. (D) Heatmap of enriched transcription factor binding sites (TFBS) in HERV loci of modules one through seven. Module eight did not show significant enrichment of TFBS.

HERV fingerprints correlate with immune cell profiles.

(A) Measurements of immune cell proportions by CIBERSORTx per study group (n=8 ME/CFS, n=10 FM, n=16 co-diagnosed, n=9 controls). (B) Association between modules and immune cell proportions was evaluated by correlating eigengenes from each module with cell proportion values obtained from CIBERSORTx analysis. Boxes show Pearson correlation values and associated p-values (*, p<0.05; **, p<0.01; ***, p<0.001) between gene expression levels of each module and quantity of cells from each specific type. A value of 1 (green) and -1 (blue) quantify strongest positive and negative correlations, respectively, while 0 (white) shows no correlation. (C) Scatter plots between top HERV loci expression levels and immune cell proportion as measured by CIBERSORTx. Pearson correlation coefficients and associated p-values are shown in plots. Correlation 95% confidence intervals are: 0.33 to 0.74 for LTR65_20p11.21 vs. γδ T cells, -0.81 to -0.46 for MER49_11q13.1 vs. resting CD4 memory T cells, and -0.70 to -0.25 for MER72_4q34.3 vs. plasma cells.

HERV and gene deregulation associates with disease symptomatology.

(A) Association between modules and disease determined by correlations of eigengenes from each module with binarized disease traits. Boxes show Pearson correlation values and associated p-values (*, p<0.05; **, p<0.01; ***, p<0.001) by module and each disease trait. A value of 1 (orange) and -1 (blue) quantify strongest positive and negative correlations, respectively, while 0 (white) shows no correlation. (B) Barplots showing top ten HERV loci (upper) and top ten genes (lower) with highest correlations with ME/CFS disease. Bar height and color display the strength of each correlation, being high orange bars more correlated than lower blue bars. (C) Association between top ten HERV loci (left) and top ten genes (right) with disease traits as determined by their correlation with ME/CFS patient symptoms as assessed by questionnaire scores. Boxes show Pearson correlation values and associated p-values (*, p<0.05; **, p<0.01) by DE HERV or DE gene as indicated for FIQ subdomains (Function, Overall and Symptoms) or total FIQ; for MFI subdomains (GF: general fatigue, Pfa: physical fatigue, RA: reduced activity, RM: reduced motivation, and MF: mental fatigue), and for SF-36 subdomains (PF: physical functioning, RP: role physical, BP: bodily pain, GH: general health, VT: vitality, SF: social functioning, RE: role emotional, and MH: mental health).