Gene expression profiles from discordant monozygotic twins suggest that molecular pathways are shared among multiple systemic autoimmune diseases

Terrance P O'Hanlon, Lisa G Rider, Lu Gan, Rick Fannin, Richard S Paules, David M Umbach, Clarice R Weinberg, Ruchir R Shah, Deepak Mav, Mark F Gourley, Frederick W Miller, Terrance P O'Hanlon, Lisa G Rider, Lu Gan, Rick Fannin, Richard S Paules, David M Umbach, Clarice R Weinberg, Ruchir R Shah, Deepak Mav, Mark F Gourley, Frederick W Miller

Abstract

Introduction: The objective of this study is to determine if multiple systemic autoimmune diseases (SAID) share gene expression pathways that could provide insights into pathogenic mechanisms common to these disorders.

Methods: RNA microarray analyses (Agilent Human 1A(V2) 20K oligo arrays) were used to quantify gene expression in peripheral blood cells from 20 monozygotic (MZ) twin pairs discordant for SAID. Six affected probands with systemic lupus erythematosus (SLE), six with rheumatoid arthritis (RA), eight with idiopathic inflammatory myopathies (IIM), and their same-gendered unaffected twins, were enrolled. Comparisons were made between discordant twin pairs and these were also each compared to 40 unrelated control subjects (matched 2:1 to each twin by age, gender and ethnicity) using statistical and molecular pathway analyses. Relative quantitative PCR was used to verify independently measures of differential gene expression assessed by microarray analysis.

Results: Probands and unrelated, matched controls differed significantly in gene expression for 104 probes corresponding to 92 identifiable genes (multiple-comparison adjusted P values < 0.1). Differentially expressed genes involved several overlapping pathways including immune responses (16%), signaling pathways (24%), transcription/translation regulators (26%), and metabolic functions (15%). Interferon (IFN)-response genes (IFI27, OASF, PLSCR1, EIF2AK2, TNFAIP6, and TNFSF10) were up-regulated in probands compared to unrelated controls. Many of the abnormally expressed genes played regulatory roles in multiple cellular pathways. We did not detect any probes expressed differentially in comparisons among the three SAID phenotypes. Similarly, we found no significant differences in gene expression when comparing probands to unaffected twins or unaffected twins to unrelated controls. Gene expression levels for unaffected twins appeared intermediate between that of probands and unrelated controls for 6535 probes (32% of the total probes) as would be expected by chance. By contrast, in unaffected twins intermediate ordering was observed for 84 of the 104 probes (81%) whose expression differed significantly between probands and unrelated controls.

Conclusions: Alterations in expression of a limited number of genes may influence the dysregulation of numerous, integrated immune response, cell signaling and regulatory pathways that are common to a number of SAID. Gene expression profiles in peripheral blood suggest that for genes in these critical pathways, unaffected twins may be in a transitional or intermediate state of immune dysregulation between twins with SAID and unrelated controls, perhaps predisposing them to the development of SAID given the necessary and sufficient environmental exposures.

Figures

Figure 1
Figure 1
Heat map representing an unsupervised, hierarchical cluster analysis of the three study groups. The groups consisted of 20 probands with a systemic autoimmune disease, their 20 unaffected twins, and 40 unrelated, matched controls, respectively. The heat map uses 104 oligo probes that exhibited statistically significant differential gene expression (multiple comparison-adjusted P values (false discovery rate) of less than 0.1) between probands and unrelated, matched controls. Color codes to the immediate right of the dendrogram correspond to probands (yellow), unaffected twins (orange), and unrelated, matched controls (blue). The first major partition in the dendrogram (branches 1 and 2) is marked to the left of the heat map.
Figure 2
Figure 2
Principal component analysis of the three study groups. The groups consisted of 20 probands with a systemic autoimmune disease (green), their 20 unaffected twins (blue), or 40 unrelated, matched controls (red), respectively. The analysis uses 104 oligo probes that exhibited statistically significant differential gene expression (multiple comparison-adjusted P values (false discovery rate) of less than 0.1) between probands and unrelated, matched controls. C, unrelated, matched control; P, proband; PC, principal component; U, unaffected twin.
Figure 3
Figure 3
Ingenuity pathways analysis of 104 probes with false discovery rate-adjusted P values of less than 0.1. The analysis compares 20 twins affected with systemic autoimmune disease and 40 unrelated, matched controls. P values (blue bars) describing the confidence of the association of the data set with a given pathway are shown on the left vertical axis (-log(P value)). The cutoff threshold value (defined as P = 0.05) is also shown (horizontal yellow 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 (orange line connecting blue bars) is shown on the right vertical axis. IL-6, interleukin-6; NF-κB, nuclear factor-kappa-B.

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