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DNA Methylation Analysis of Radiated Skin Reveals Chonic Inflammation with a Predominance of Polymorphonuclear Granulocytes
Hui Shen, Ph.D., Solmaz N. Leilabadi, M.D., Peter W. Laird, Ph.D., Alex K. Wong, M.D..
Keck School of Medicine of USC, Los Angeles, CA, USA.
PURPOSE: Although radiation therapy is a useful adjunct to surgical resection in the management of a variety of solid tumors, radiation-induced injury to normal skin is an unintended consequence that can lead to chronic non-healing wounds and increased risk of complications during reconstructive surgery. Insight into the damage pathways of radiated skin may reveal opportunities to better understand radiation delayed wound healing. Radiation-induced epigenetic changes in DNA methylation has not been well characterized for human skin.
METHODS: Pairs of matched radiated and normal skin were collected at the Keck Hospital of USC using an IRB-approved protocol from patients that had adjuvant chest wall irradiation > 10 weeks prior to collection. Samples were assayed on the Infinium HumanMethylation450 Beadarray. This platform measures the DNA methylation pattern at 482,421 CpG sites which cover 99% of RefSeq genes, with an average of 17 CpG sites per gene region distributed across the promoter, 5’UTR, first exon, gene body, and 3’UTR. It covers 96% of CpG islands, with additional coverage in island shores and the regions flanking them. The R-based methylumi package was used for data preprocessing, including background correction and dye-bias normalization. The level of DNA methylation at each CpG locus is summarized as beta (β) value calculated as (M/(M+U)), ranging from 0 to 1, which represents the ratio of the methylated probe intensity to the overall intensity at each CpG locus. A p value comparing the intensity for each probe to the background level was calculated at the same time, and data points with a detection P value >0.05 were deemed not significantly different from background measurements, and therefore were masked from the analysis.
RESULTS: Unsupervised clustering of the eight samples on 1,540 most variable probes (SD>0.15) revealed intra-sample heterogeneity in terms of DNA methylation. We compared the skin samples to the DNA methylation patterns of sorted blood components on the same loci, including polymorphonuclear granulocytes (PMN), CD3+ T cells, CD19+ B cells, and CD34+CD38- cells, and saw a clear and specific PMN signature in two of the four radiated skin samples, while none of the matched normal skins showed such signature. The PMN infiltration seemed to be extensive, with estimated PMN component of 80% in one sample.
CONCLUSION: DNA methylation array of radiation damaged skin revealed significant infiltration by polymorphonuclear (PMN) granulocytes. This data supports a pathophysiologic role of PMNs in chronic inflammation related to radiation damaged skin. We hypothesize that a deregulated inflammatory process might be associated with impaired healing in radiated skin.
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