Genomic Investigation of Lupus in the Skin Animesh A. Sinha, Rama Dey-Rao  Journal of Investigative Dermatology Symposium Proceedings  Volume 18, Issue 2, Pages S75-S80 (October 2017) DOI: 10.1016/j.jisp.2016.09.002 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Unsupervised clustering of CLE skin and blood microarray datasets. (a) Principal components analysis (PCA) displays the largest spatial separation based on gene expression variations in the skin and blood sample groups. In the three-dimensional plot, the three principle components, PC#1, #2, and #3 of all probe set IDs and their respective variations are expressed on the x-, y- and z-axes. The total percentage of PCA mapping variability is 66.4%. Each data point represents one sample. The ellipsoids highlight portioning of the different samples. Assignment of samples by color: lesional skin (pink) and nonlesional skin (green) from CCLE patients, blood samples from CLE patients (orange) and healthy control subjects (blue). (b) The DEGs in the skin and blood were established according to well-established protocols in the laboratory. Although we noted an 11% overlap in DEGs between the two environments (not shown), most genes in both lists are distinct to the respective environment (skin and blood) from which they are generated. CCLE, chronic cutaneous lupus erythematosus; CLE, cutaneous lupus erythematosus; DEG, differentially expressed gene; ID, identification; PC, principle component; PCA, principle components analysis. Journal of Investigative Dermatology Symposium Proceedings 2017 18, S75-S80DOI: (10.1016/j.jisp.2016.09.002) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Ontology enrichment analysis of CCLE blood and skin gene expression profiles. Differentially expressed genes were generated from the comparison between CLE lesional skin versus nonlesional skin and peripheral blood samples of CLE patients versus healthy control subjects. Ontology enrichment analysis of the DEGs from skin and blood transcriptional profiles shows several common disease-related pathways and processes. We broke down the number of DEGs (up- and down-regulated) included in some of the shared disease-related pathways and processes and express them as percentage of the up- and down-regulated genes observed in each. The prominent signatures for INF and apoptosis in both profiles are marked with a star. As an example, out of the total 28 DEGs in CLE blood profile that are associated with oxidation:reduction-related processes, 20 (71%) are up-regulated and 8 (29%) are down-regulated. B, blood; CCLE, chronic cutaneous lupus erythematosus; CLE, cutaneous lupus erythematosus; Cyto-chemo, cytokine-chemokine related; DC, dendritic cell related; DEG, differentially expressed gene; Lys/Pro, lysosome/proteasome related; NK cell, natural killer cell related; oxid:red, oxidation:reduction-related pathways and processes; S, skin; TLR, toll-like receptor signaling. Journal of Investigative Dermatology Symposium Proceedings 2017 18, S75-S80DOI: (10.1016/j.jisp.2016.09.002) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Coincidence with previously reported SLE-associated genes. A list of 152 CLE DEGs (skin and blood) were associated with previously reported SLE-linked expressed genes or potential susceptibility loci. Upon mapping the CLE skin and blood DEGs from this study on the SLE canonical pathway in Kyoto Encyclopedia of Genes and Genomes (KEGG) database (hsa05322), we noted an involvement of several dysregulated genes in all sections of the map (red starred). SLE is an autoimmune disease characterized by production of autoantibodies to dsDNA, nuclear proteins, and several cytoplasmic components. Immune complexes are deposited at the site of damage, which in CLE is the skin. These are composed of autoantibodies along with the self-antigen that can mediate a systemic inflammatory response by activating complement or via Fc (gamma) R-mediated neutrophil and macrophage activation. The prominent enrichment of apoptosis related biological processes in the CLE skin potentially indicates an initial trigger that causes a breakdown and death of cells, accompanied by an incomplete removal of breakdown products, leading to the initiation of the autoimmune reaction that causes target damage and further cell death in the skin. Subsequently, we removed the 152 SLE-associated genes from the CLE-skin and -blood DEGs and performed an ontology enrichment analysis. The pathways and process enriched among the CLE-specific DEGs are displayed in the blue rectangles. CLE, cutaneous lupus erythematosus; DEG, differentially expressed gene; dsDNA, double-stranded DNA; ECM, extracellular matrix; Jak, Janus kinase; SLE, systemic lupus erythematosus; STAT, signal transducer and activator of transcription. Journal of Investigative Dermatology Symposium Proceedings 2017 18, S75-S80DOI: (10.1016/j.jisp.2016.09.002) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Mapped transcriptional hot spots in CLE skin and blood. CCLE blood and skin gene expression data are leveraged to identify transcriptional hot spots in chromosomes where DEGs map with statistically increased frequency. Significant chromosomal stretches at 0.001 false discovery rate (Benjamini Hochberg), considered to be transcriptionally active hot spots, were shown. There were 13 CLE-skin (blue) and 16 CLE-blood (red) hot spots. Ten CLE-skin genes (blue boxes) and seven CLE-blood genes (red boxes) mapping within transcriptional hot spots have been previously reported to be putative disease loci for SLE. TNF and CSNK2B have also been reported as CLE-associated risk factors. CCLE, chronic cutaneous lupus erythematosus; Chr, chromosome; CLE, cutaneous lupus erythematosus; DEG, differentially expressed gene; SLE, systemic lupus erythematosus. Journal of Investigative Dermatology Symposium Proceedings 2017 18, S75-S80DOI: (10.1016/j.jisp.2016.09.002) Copyright © 2016 The Authors Terms and Conditions