P22 Micro-RNA enriched pathway impact analysis applied to synovial RNA-seq in early rheumatoid arthritis identifies response prediction pathways
Authors: Sciacca Elisabetta , Alaimo Salvatore , Pulvirenti Alfredo , Latora Vito , Humby Frances , Ferro Alfredo , Lewis Myles J , Pitzalis Costantino 
Published: April 2020

Abstract

Background
We use a new pathway analysis tool MITHrIL (Mirna enriched paTHway Impact anaLysis) to analyse RNA-seq patterns in synovial biopsies from patients with rheumatoid arthritis from the Pathobiology of Early Arthritis Cohort (PEAC). MITHrIL augments pathways with missing regulatory elements, such as microRNAs, and their interactions with genes to enhance quantification of immunological pathway activation in bulk RNA-seq. MITHrIL pathways were compared with three major pathotypes (lympho-myeloid, diffuse myeloid and pauci-immune fibroid) in early treatment-naïve RA patients and responders vs non-responders to methotrexate-based DMARD regimens.

Methods
A differential gene expression analysis is performed on the PEAC observational cohort. The Log-Fold Changes retrieved from the pairwise comparisons between responders/non responders and across different pathotypes are used as input to the MITHrIL software. Using the KEGG biological pathways, MITHrIL proportionally spreads the known differential perturbation of a gene to the downstream nodes in the pathway. In so doing, a perturbation factor is assigned to each gene and, as a result, a list of differentially altered pathways is returned together with the corresponding statistical significance (p-values). Particular attention is given to immunological pathways. We show a list of differentially altered pathways for each comparison and provide specific insight to those more characterising pathways.

Results
The results show coherence with the previous findings providing greater granularity on how the gene level alterations can propagate through biological pathways. In particular, we found different gene expression levels in the pairwise comparisons across pathotypes highlighting different perturbations in the following pathways: chemokine signalling (adjusted P = 3.3E-13), Jak-STAT signalling (adjusted P = 6.5E-04), Leukocyte transendothelial migration (adjusted P = 3.7E-02), PI3K-Akt signalling (adjusted P = 4.6E-04), T cell receptor signalling (adjusted P = 3.7E-02), Antigen processing and presentation (adjusted P = 1.1E-08) and NF-kappa B signalling (adjusted P = 4.1E-02).
We also found confirmation of changes in lymphoid and myeloid subtypes over time, while fibroid RA patients present no significant alteration in their expression levels after the methotrexate-based DMARD therapy. Finally, we found different levels of activation in MAPK and AMPK signalling pathways for patients that do not respond to the DMARD therapy in contrast to those who do.

Conclusion
A novel pathway analysis approach is used to show the most differentially active biological pathways between different RA pathotypes and responder-resistant patients to methotrexate-based DMARD therapy. The results identify responder-resistant gene expression pathway patterns in early RA which may help to stratify patients to biologic therapy at an earlier stage.

Disclosures
E. Sciacca None. S. Alaimo None. A. Pulvirenti None. V. Latora None. F. Humby None. A. Ferro None. M.J. Lewis None. C. Pitzalis None.