Background/Purpose: MicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) that participate in post-transcriptional gene regulation. miRNAs have potential both as biomarkers for diagnosis and prognosis  and as therapies. Few studies have examined plasma miRNA expression in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV); those that have focused on microarray and PCR-based analyses of a limited number of candidate miRNAs. Our objective was to determine which plasma miRNAs are altered in AAV compared to patients with inflammatory autoimmune disease and also control subjects using an unbiased approach: high throughput small RNA sequencing.

Methods: This pilot study included 10 patients with AAV, 13 controls with inflammatory disease (4 with rheumatoid arthritis and 9 with systemic lupus erythematosus), and 5 controls without inflammatory disease. Stored plasma was used to extract RNA and construct RNA sequencing libraries. Sequencing was done on Illumina NextSeq500. High quality reads were mapped to the human GRCh38 genome using Bowtie. MiRBase21.0 was used to quantify miRNAs. The abundance of miRNA expression was compared between AAV and both groups of control subjects by DESeq2 with 5% false discovery rate and adjustment for multiple comparisons by the Benjamini and Hochberg method. Potential miRNA pathway targets were explored with TargetScanHuman v7.1 and Ingenuity Pathway Analysis.

Results: Mean age was 64 years ± 11 years among patients with AAV, 43 ± 12 years among inflammatory disease controls, and 58 ± 13 years among control subjects. All groups were predominately Caucasian. Active disease was present in 60% of patients with AAV and 69% of inflammatory disease control subjects. Among patients with AAV, 100% were ANCA positive, 60% were positive for anti-proteinase 3 antibody and 40% were positive for anti-myeloperoxidase antibody. Of 209 reliably mapped miRNAs, 15 were significantly differentially expressed (≥2-fold) in patients with AAV compared to both control groups (Table). Of these, miR-21-5p and miR-17-5p are both predicted to target TGFBR2, a gene associated with various vascular phenotypes; and miR-146a-5p is predicted to target TLR9, a gene containing variants associated with AAV.

Conclusion: Several plasma miRNAs are differentially expressed in patients with AAV compared to patients with inflammatory autoimmune disease and controls. Further validation is necessary to confirm these findings.

Table. Significant differential microRNA expression in patients with ANCA-associated vasculitis compared to controls with and without inflammatory disease.