Development of gene expression assay to stratify autoimmune connective tissue diseases

SLE is a complex multisystem autoimmune disease, causing difficulties when diagnosing and treating. IFN dysregulation is a hallmark of SLE pathogenesis which leads to the activation of self-antigens in autoimmunity. Subtypes of IFN signal through specific receptors, however, downstream activation produces pleiotropic downstream effects, with distinct overlap, making the IFN pathway a challenge to study. Quantification of IFN stimulated gene expression acts as an indirect measurement of IFN protein levels and is known as IFN signature. Previously, the Leeds Lupus group have established a clinically relevant two-scoring system to measure IFN activity in PBMCs and were named IFN-ScoreA and IFN-Score-B, however, IFN signatures have not yet reached routine clinical practice. In 2019 a EULAR task force highlighted points to consider and terminology consensus when reporting IFN assays, including reasonable justification of assay choices. ISG measurement in a whole blood (WB) sample type is more feasible than PBMC so development of a WB assay was an aim of this report. Firstly, quantified IFN-Score-A and -B determined by TaqMan array were analysed in 45 matched PBMC and WB sample types which showed the most clinically useful PBMC derived IFN-Score-B cannot be interchanged between sample types. Secondly, factor analysis reinterrogation of 31 ISGs in WB derived two-IFN-Scores: IFN-Score-C (CCL8, CXCL10, IFI27, ISG15 and LAMP3) and IFN-Score-D (CASP1, CEACAM1, SOCS1 and TRIM38) and explained 97% of the variation. IFN-Score-A best defined SLE, RA and healthy patient groups compared to other IFN-Scores and IFN-Score-B and -D expressed strong correlations within WB suggesting the potential for future clinical utility of IFN-Score-D. Finally, RefFinder identified YWHAZ, PGK1 and GUSB to be the most stable reference genes from a group of 16 candidate genes in a WB SLE and healthy cohort. In conclusion, novel whole blood IFN scores warrant further evaluation in clinical validation studies.