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Nahyoung G. Lee, Stephen R. Planck, Blake Acohido, Dongseok Choi, Christine A. Harr, David J. Wilson, James T. Rosenbaum; Gene Expression Profiling in Nonspecific Orbital Inflammation Using Microarray Analysis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3298.
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Non-specific orbital inflammation (NSOI) is one of the most common causes of an orbital mass lesion and causes significant pain, disabling double vision, and permanent visual loss. However, NSOI pathogenesis is poorly understood and there are no established treatment paradigms. In general, NSOI patients are placed on a regimen that consists of high dose corticosteroids or corticosteroid-sparing agents. To elucidate the pathogenesis and classify subtypes of this disease, we used microarray-based profiling to identify the genes that are differentially expressed in NSOI compared to tissue-matched controls.
Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) specimens from 6 orbital fat biopsies that received a diagnosis of NSOI and from 5 non-inflamed orbital fat biopsies. The NuGEN WT Ovation FFPE labeling protocol was used and the products were hybridized to the Affymetrix Human Gene 1.0 ST Arrays that interrogate 28,869 genes. After normalization of data among arrays, probe sets for differentially expressed genes between the two experimental groups were identified by Significance Analysis of Microarray software with a false positive rate threshold set at 5% and a minimum fold-change of 1.5. Principle coordinate analysis was used to look for clustering or global differences of the expression profiles of individual samples.
Increased gene expression in the NSOI group was detected in 94 probe sets that represent about 78 genes. Decreased expression was detected by 428 probe sets representing about 415 genes. Of the genes with increased expression, many encode immunoglobin or HLA components and many encode signaling molecules, e.g., CCL18, CCL19, Syk, LTβ, CSK, and CARD17. Principal coordinate analysis (PCA) shows clustering of the normal control samples and greater diversity among the NSOI samples.
This study demonstrates the feasibility of using RNA from FFPE ocular tissues for gene expression profiling. The increased expression of immunoglobulins and HLA molecules is compatible with the successful treatment of NSOI patients with agents like rituximab. The upregulation of chemokines and other signaling modulators present additional potential treatment modalities for NSOI that may be recalcitrant to current therapies. The greater variation in PCA plots for the NSOI vs. control tissues suggests that this technique would be applicable for identification of disease subsets after profiling a sufficient number of subjects.
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