To Örebro University

Background: Connective tissue diseases (CTDs) including systemic lupus erythematosus (SLE), primary Sjögren’s syndrome (pSS) and systemic sclerosis (SSc) frequently share clinical and serological features, rendering precise differentiation challenging. Currently used autoantibodies (Abs) either demonstrate high sensitivity across all CTDs (e.g., ANA) or high specificity yet low sensitivity (e.g., anti-dsDNA, anti-Smith, anti-Scl70, anti-centromere). Better biomarkers for early and accurate diagnosis of CTDs are needed.

Objectives: To perform a broad explorative screen of IgG and IgA antibodies to autoantigen specificities in SLE vs pSS and SSc to identify novel Abs that potentially could aid in distinguishing across CTDs.

Methods: We analysed plasma samples from patients with SLE (n=289), pSS (n=208), and SSc (n=187) from the European PRECISESADS project (NTC02890121). Samples were screened for IgG and IgA seroreactivity against a panel of >1,600 protein autoantigens using KREX-based i-Ome arrays (Sengenics). Differential expression analysis was performed with the limma R package adjusting for e.g., polyspecific antibody reactivity. The level of significance for differentially expressed Abs (DEAbs) was set at p<0.05 and |fold change| >1.5. ROC analysis was carried out for each DEAb, yielding discriminative performance metrics e.g., sensitivity, specificity, area under the curve (AUC), and accuracy.

Results: DEAb analysis revealed 10 IgG (8 upregulated; 2 downregulated) and 1 downregulated IgA DEAb in SLE vs pSS. Of upregulated IgG DEAbs in SLE, anti-LIN28A (sen=0.71, spe=0.75, AUC=0.78), anti-PCBP2 (sen=0.56, spe=0.80, AUC=0.74), anti-HNRNPA2B1 (sen=0.74, spe=0.64, AUC=0.72), anti-HMG20B (sen=0.57, spe=0.81, AUC=0.74), and anti-NRF1 (sen=0.78, spe=0.64, AUC=0.75) demonstrated the best ability to discriminate between SLE and pSS. As expected, anti-TROVE2 IgG (anti-SSA), anti-SSB IgG, and anti-SSB IgA were significantly higher in pSS vs SLE, yet with modest accuracy in differentiating between the two groups (sen=0.64, spe=0.78, AUC=0.70 for anti-TROVE2 IgG; sen=0.57, spe=0.79, AUC=0.65 for anti-SSB IgG; sen=0.38, spe=0.84, AUC=0.59 for anti-SSB IgA). Analysis of SLE vs SSc revealed 15 IgG (9 upregulated; 6 downregulated) and 4 upregulated IgA DEAbs. Of upregulated IgG DEAbs in SLE, anti-LIN28A (sen=0.67, spe=0.84, AUC=0.81), anti-HBGB2 (sen=0.61, spe=0.85, AUC=0.77), anti-HNRNPA2B1 (sen=0.74, spe=0.64, AUC=0.72), anti-HMG20B (sen=0.66, spe=0.81, AUC=0.79), and anti-NRF1 (sen=0.64, spe=0.82, AUC=0.77) demonstrated the best discriminative ability. Moreover, upregulated IgA DEAbs in SLE vs SSc including anti-LIN28A (sen=0.61, spe=0.91, AUC=0.83), anti-HMG20B (sen=0.78, spe=0.69, AUC=0.80), anti-NOL4 (sen=0.68, spe=0.75, AUC=0.76), and anti-SSB (sen=0.67, spe=0.83, AUC=0.81) showed good accuracy in differentiating between the two groups. This analysis also revealed 6 upregulated DEAbs in SSc vs SLE, i.e., anti-CXB5, anti-GGPS1, anti-CRISP2, anti-HSPE1, anti-TEX101, and anti-KRT19. While these Abs showed low discriminative capacity, anti-HSPE1 showed the greatest sensitivity (84%) and anti-CBX5 the greatest specificity (89%), with AUCs ranging from 60% to 65%.

Conclusion: This study corroborated both classical IgG Ab specificities shared across CTDs (e.g., anti-SSB, anti-TROVE2) and specificities previously described (anti-LIN28A, anti-HBGB2, anti-HMG20B, anti-HNRNPA2B1)1 and identified novel IgG (anti-NRF1, anti-CCNB1) and novel IgA (anti-LIN28A, anti-NOL4, anti-HMG20B) Abs described for the first time in SLE, with robust accuracy in distinguishing SLE from pSS or SSc. Expression levels of anti-LIN28A, anti-HBGB2, anti-HMG20B, and anti-HNRNPA2B1 were significantly higher in SLE compared with both pSS and SSc, implying specificity for SLE. Importantly, many of these novel Abs outperformed the diagnostic and discriminative accuracy of currently used Abs, e.g., anti-dsDNA, anti-SSA, and anti-SSB.