Gene Expression Profiles from Disease Discordant Twins Suggest Shared Antiviral Pathways and Viral Exposures among Multiple Systemic Autoimmune Diseases

<div><p>Viral agents are of interest as possible autoimmune triggers due to prior reported associations and widely studied molecular mechanisms of antiviral immune responses in autoimmunity. Here we examined new viral candidates for the initiation and/or promotion of systemic autoimmune diseases (SAID), as well as possible related signaling pathways shared in the pathogenesis of those disorders. RNA isolated from peripheral blood samples from 33 twins discordant for SAID and 33 matched, unrelated healthy controls was analyzed using a custom viral-human gene microarray. Paired comparisons were made among three study groups—probands with SAID, their unaffected twins, and matched, unrelated healthy controls—using statistical and molecular pathway analyses. Probands and unaffected twins differed significantly in the expression of 537 human genes, and 107 of those were associated with viral infections. These 537 differentially expressed human genes participate in overlapping networks of several canonical, biologic pathways relating to antiviral responses and inflammation. Moreover, certain viral genes were expressed at higher levels in probands compared to either unaffected twins or unrelated, healthy controls. Interestingly, viral gene expression levels in unaffected twins appeared intermediate between those of probands and the matched, unrelated healthy controls. Of the viruses with overexpressed viral genes, herpes simplex virus-2 (HSV-2) was the only human viral pathogen identified using four distinct oligonucleotide probes corresponding to three HSV-2 genes associated with different stages of viral infection. Although the effects from immunosuppressive therapy on viral gene expression remain unclear, this exploratory study suggests a new approach to evaluate shared viral agents and antiviral immune responses that may be involved in the development of SAID.</p></div

Comparisons of differential gene expression values determined by relative quantitative-polymerase chain reaction and microarray analyses^*.

<p>* Fold change (FC) values indicating relative expression levels of two HSV-2 genes (<i>UL36</i> and <i>RS1</i>) between two study groups: probands (P) and matched, unrelated healthy controls (C). qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative reverse-transcription PCR; Positive, the affected probands with probe values above the proposed normal range (mean plus three standard deviation of control group); Negative, the affected probands with probe values within the proposed normal range.</p><p>Comparisons of differential gene expression values determined by relative quantitative-polymerase chain reaction and microarray analyses^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#t004fn001" target="_blank">*</a>}.</p

Dot plots of three HSV-2 gene expression levels in SAID affected probands, unaffected twins, and matched, unrelated healthy controls.

<p>The individual normalized signal data for <i>UL19</i> (A, probe 1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#pone.0142486.t003" target="_blank">Table 3</a>), <i>UL36</i> (B) and <i>RS1</i> (C) were plotted according to the three study groups: probands, unaffected twins, and matched, unrelated healthy controls. Statistically significant <i>p</i> values between any two groups are shown (*) and were calculated using one-way ANOVA with corrections for multiple comparisons. The proposed “normal range” values were derived from the mean plus three standard deviations (SD, dash line) of healthy control expression values.</p

IFN-regulated genes that were significantly differentially expressed between SAID probands and their unaffected twins^*

<p>*Listed are the 41 IFN-regulated genes of 107 viral infection-related genes identified by the INTERFEROME program that were statistically significant (<i>q</i> < 0.05) and had a fold change >1.5 between disease discordant twins. Fold change values indicate increase (positive) or decreased (negative) levels of gene expression in probands related to unaffected twins.</p><p>IFN-regulated genes that were significantly differentially expressed between SAID probands and their unaffected twins^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#t002fn001" target="_blank">*</a>}</p

Four distinct viral oligo probes from three HSV-2 genes that were differentially expressed between probands and unaffected twins, as well as between probands and matched, unrelated healthy controls^*.

<p>*Paired comparisons were made between probands (P) and unaffected twins (U) or matched, unrelated healthy controls (C). Statistically significant viral probes (fold change, FC >1.5, <i>q</i> <0.05) were selected.</p><p>Four distinct viral oligo probes from three HSV-2 genes that were differentially expressed between probands and unaffected twins, as well as between probands and matched, unrelated healthy controls^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#t003fn001" target="_blank">*</a>}.</p

Baseline sample characteristics, their age-adjusted associations with ANA prevalence, and their unadjusted associations with all-cause mortality.

<p>Baseline sample characteristics, their age-adjusted associations with ANA prevalence, and their unadjusted associations with all-cause mortality.</p

Ingenuity pathway analysis of differentially expressed human genes between disease discordant twins.

<p>The analysis compared 789 oligo probes corresponding to 537 human genes significantly differentially expressed (fold change >1.5, <i>q</i> <0.05) between twins discordant for SAID. The upper horizontal axis (blue bars) describe the association of the data set with a given pathway (-log (<i>p</i> value)). The cutoff threshold value (defined as <i>p</i> = 0.05) is shown by the vertical red line. The ratio of the number of genes from the data set that map to a given pathway divided by the total number of molecules that comprise the pathway is shown on the horizontal axis (green triangles). Previously reported viral-related signaling pathways are in bold print.</p

Flow chart of the experimental design and data analyses.

<p>Flow chart of the experimental design and data analyses.</p

Covariate-adjusted associations between baseline ANA status and all-cause mortality within selected groups.

<p>Covariate-adjusted associations between baseline ANA status and all-cause mortality within selected groups.</p

Covariate-adjusted ANA associations with mortality by cause of death, group analyzed, and analysis performed.

<p>Covariate-adjusted ANA associations with mortality by cause of death, group analyzed, and analysis performed.</p