3073Leveraging transcriptome sequencing for detecting novel disease-related pathways using human cardiac sarcoidosis myocardium biopsies

Abstract

Abstract Background/Introduction Cardiac sarcoidosis (CS) is one of the main causes of poor outcomes in patients with sarcoidosis, a systemic granulomatous disorder of complex etiology including a genetically susceptible host and specific exposure to disease-triggering antigens. Recently, transcriptome analysis using sarcoidosis peripheral monocytes was reported to be useful for exploring genetic susceptibility and novel disease-causing pathways. However, transcriptome sequencing has not been used to explore disease-related genes and pathways directly using human CS myocardial biopsies. Purpose This study aimed to identify transcriptome profiles and novel disease-related pathways of CS by comparing human CS myocardial biopsies with control samples using ribonucleic acid (RNA) sequencing (RNA-Seq). Methods We assessed 30 patients with suspected myocardial disorders who underwent transcatheter endomyocardial biopsies at our University Hospital, Japan. Of those, 7 were clinically diagnosed with CS, 9 with hypertrophic cardiomyopathy (HCM), and 14 with dilated cardiomyopathy (DCM). Messenger RNAs were extracted from cardiac muscle biopsies using the Ovation SoLo RNA-Seq System (NuGEN Technologies), according to the manufacturer's instructions. Sequencing was performed with coverage of approximately 20 million reads per sample using Illumina HiSeq 2000. Sequencing reads were mapped using the STAR 2-pass method with GRCh37 as the reference. The DESeq2 R package (version 3.8) was used for further analyses. Principal component analysis (PCA) on gene expression was conducted for detecting outliers such as non-muscular samples. Differential gene expression analysis was performed between the 7 patients with CS and 23 patients with cardiomyopathy (HCM and DCM, non-CS). Gene Ontology (GO) enrichment analysis was conducted to estimate possible disease-related pathways. Results We successfully sequenced 60 myocardial biopsy samples (original and biological duplicates) from 30 CS patients. Of these, 2 outlier samples shown by the PCA plot were removed, and 58 were used for further analyses. We found 243 genes that were differentially expressed between CS patients and non-CS patients. Top-rated genes were RP11–366M4.8, RELN, S100A6, WASF3and UCHL1. Pathway analysis using GO demonstrated enrichment oflymphocyte activation (P=4.8x10–16), organelle fission (P=6.1x10–14), the M phase of the mitotic cell cycle (P=2.2x10–13), nuclear division (P=2.4x10–13), mitosis (P=2.4x10–13) and T-cell activation pathways (P=1.2x10–12). Conclusions Our differential gene expression and pathway analysis results using human CS myocardial biopsies suggested that lymphocyte activation, specifically the T-cell activation pathway, is linked to CS pathogenesis. Further studies are needed to decipher the role of specific genes related to genetic susceptibility and/or pathways associated with CS occurrence.