Selection of candidate genes using RNA sequencing
Further analysis was conducted on RNA sequencing data from 7 samples; 2 ACPA+ RA patients, 2 ACPA- RA patients and 3 healthy controls. Differentially expressed genes (DEGs) were analyzed in the two different sample types, and three groups of DEGs were identified (ACPA+ vs. control, ACPA+ vs. ACPA-, ACPA- vs. control). We identified 1007 DEGs (522 up-regulated and 485 down-regulated) between ACPA+ and control, 628 DEGs (344 up-regulated and 284 down-regulated) between ACPA+ and ACPA-, and 1097 DEGs (549 up-regulated and 548 down-regulated) between ACPA- and control (Fig. 1A and 1B). All these DEGs exhibited distinct expression patterns in the three sample types (Fig. 1C).
Pathway enrichment analysis/ingenuity pathway analysis of DEGs yielded 19 canonical pathways that were significantly enriched between ACPA+ and control, 24 between ACPA+ and ACPA-, and 35 between ACPA- and control (see supplementary information). Some of these pathways were enriched in more than one comparison (Fig. 1D). The agranulocyte adhesion and diapedesis pathway was particularly enriched in ACPA+ vs. control and ACPA+ vs. ACPA- (Fig. 1D). Different DEGs were involved in the agranulocyte adhesion and diapedesis pathway in two comparisons (Fig. 1E and 1F), and of these, CXCL2 and C-X-C motif chemokine ligand 7 (CXCL7) were exclusively up-regulated in ACPA+ samples (Fig. 1E).