Background/Purpose: While skin fibrosis is a hallmark of systemic sclerosis (SSc), internal organ involvement is the primary cause of morbidity and mortality, often related to pulmonary disease and gastrointestinal (GI) dysfunction. Here we tested the hypothesis, generated from a meta-analysis of ten different SSc datasets, that any single patient with SSc would have the same deregulated molecular signatures across multiple organ systems, consistent with the systemic nature of the disease.

Methods: RNA sequencing (RNA-seq) was performed on at least four organ biospecimens (skin, esophagus, fundus, duodenum, and/or blood) from 14 patients who met 2013 ACR/EULAR criteria for SSc. RNA was sequenced by 75bp paired-end RNA-seq at >80 million reads per sample and aligned to the reference genome (hg19). Each sample was assigned to an intrinsic subset (inflammatory, proliferative, or normal-like) using Support Vector Machine (SVM) classification and unsupervised hierarchical clustering with profiles of normalized Reads Per Kilobase of transcript per Million mapped reads (RPKM) values.

Results: Hierarchical clustering of the RPKM data showed that samples from a single tissue type cluster tightly together. Analysis of skin and esophagus individually also recapitulated the intrinsic subsets previously observed in these organs. Furthermore, for the first time, the inflammatory, proliferative, and normal-like intrinsic subsets were identified in the fundus and duodenum. The blood of patients with SSc also displayed gene expression signatures of the inflammatory, proliferative, and normal-like intrinsic subsets. Subsequent normalizing for tissue type in an agglomerative analysis of all tissues results in the identification of intrinsic gene expression subsets across organ systems. Importantly, all five organs assessed were represented in each intrinsic subset. The majority of patients (9/14) showed >60% concordance of intrinsic subset assignment across the multiple organs analyzed. Moreover, tissues from the gastrointestinal tract tended to belong to the same subset (5/14 with 100% concordance).

Conclusion: We have performed the first molecular analysis of biospecimens from multiple organs within an individual patient with SSc. Our data suggest that SSc molecular subsets are reproducible in different tissues and that deregulated molecular pathways are conserved across organ systems within the same patient. These data indicate that intrinsic subsets are common feature of end-organ pathology in SSc. We cannot rule out the possibility that pathogenic mechanisms of disease activity and progression are discordant in time in each organ, consistent with clinical observation. Further work is focused on using functional genomic networks to assess consistent and organ-specific mechanisms of disease. A subset of the samples have been queued for DNA methylation array and ATAC-seq.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/multi-organ-rna-sequencing-of-patients-with-systemic-sclerosis-ssc-finds-that-intrinsic-subsets-are-conserved-across-organ-systems/