Retrospective analysis of the clinical significance of Ro52/TRIM21 antibody and specific antinuclear antibody patterns by indirect immunofluorescence

Objective: To determine the clinical significance of Ro52 protein/tripartite motif-containing 21 antibody and specific antinuclear antibody patterns using indirect immunofluorescence technique. Method: The retrospective study was conducted at the clinical laboratory of the First Affiliated Hospital of Chongqing Medical University, China, and comprised data from January 2017 to December 2021 of patients who underwent antinuclear antibody and anti-extractable nuclear antigen antibody detection. Inpatients with Ro52 antibody-positive status were taken as the cases, while anti-Ro52 negative patients with clear clinical diagnosis were taken as the controls. Data was analysed using SPSS 19. Results: There were 1802 cases and 1211 controls. Positive Ro52 showed significantly greater frequency in patients with primary Sjogren’s syndrome, systemic lupus erythematosus, inflammatory myositis, dry eyes and interstitial lung disease (p<0.05). Ro52 antibody showed high positive predictive value for primary Sjogren’s syndrome 25(96.15%), systemic lupus erythematosus 259(91.20%), connective tissue disease-associated interstitial lung disease 45(86.67%) and inflammatory myositis 60(86.67%). Antinuclear antibody indirect immunofluorescence patterns most frequently detected were nuclear speckled 128(40.89%) and cytoplasmic speckled 126(40.26%) (p<0.05). Interstitial lung disease was associated with the presence of cytoplasmic speckled antinuclear antibody indirect immunofluorescence pattern 24(19.2%), while tumours 47(36.5%) and hepatitis B 26(20.3%) seemed to be more frequent with nuclear speckled pattern (p<0.05). The simultaneous reactivity extractable nuclear antigen antibodies most frequently detected were antinuclear antibody+Ro52+anti-Sjogren's syndrome A+ 558(33.96%). Conclusion: Ro52 antibody positivity was found to be associated with Sjogren’s syndrome, systemic lupus erythematosus, inflammatory myositis, dry eye and interstitial lung disease. The antinuclear antibody immunofluorescence pattern of Ro52 positive was single and primarily granular cytoplasm type. Antinuclear antibody negative and Ro52 positive in the serum of patients also had certain significance in auxiliary disease diagnosis.


Introduction
Ro52 protein/tripartite motif-containing 21 (TRIM21) belongs to the TRIM family of proteins and contains a RING (RING Finger Domain) and a B-box (B-Box zinc finger gene containing protein) motif, followed by a coiled-coil (CC) domain and a B30.2 (or PRYSPRY) region at the Cterminal end. 1 The anti-Ro52 antibody is one of the most widely distributed and common anti-nuclear antibodies (ANAs), which can be found in the sera of patients with various autoimmune diseases.Although its specificity is low, it is closely associated with certain specific clinical manifestations of autoimmune diseases, such as systemic lupus erythematosus (SLE), Primary Sjogren's syndrome (pSS), autoimmune hepatitis, primary biliary cholangitis, myositis, neonatal atrioventricular block and pulmonary interstitial fibrosis 2,3 .In addition, the Ro52 antibody is also detected in some non-autoimmune diseases, such as viral infections and neoplastic diseases 1,3,4 .Regarding the clinical significance of the Ro52 antibody, there have been more studies in European and American populations than in Asian populations 5 .As the clinical significance and diagnostic value of Ro52 antibody differ greatly from those reported in the literature, some immunological laboratories have stopped using Ro52 antibody detection 6 .
researchers.Many studies have found that Ro52 plays various roles in cells, including the regulation of cell proliferation and apoptosis, signal transduction, and interaction with nuclear deoxyribonucleic acid (DNA) 1,4,7 .
Ro52/TRIM21 is among the most common autoantibodies in systemic autoimmune rheumatic diseases, but the clinical association and specific ANA patterns by indirect immunofluorescence (IIF) remain poorly understood.The current study was planned to describe the clinical significance, serological associations and specific ANA-IIF patterns of Ro52/TRIM21 antibody in a large number of patients in a tertiary care setting.

Materials and Methods
The retrospective study was conducted at the clinical laboratory of the First Affiliated Hospital of Chongqing Medical University, China, and comprised data from January 2017 to December 2021 of patients who underwent ANA and anti-extractable nuclear antigen (ENA) antibody detection.All the participants were Han Chinese.Data of patients who had inconsistent repeated results, duplicate detections, data with missing elements and data with unclear information was excluded.Data included related to antibody-positive inpatients, outpatients and patients undergoing regular physical examination and screening at the institutional health management centre.
The diagnosis was confirmed by reviewing the patient's medical records and laboratory/histopathology results, according to the classification criteria present at the time of analysis [8][9][10][11][12][13][14][15][16][17][18] .Autoimmune disease was defined when a patient presented with one of the following: SLE, rheumatoid arthritis (RA), pSS, interstitial lung disease (ILD), mixed connective tissue disease (MCTD) and inflammatory myositis (IM).Other non-autoimmune pathologies included various non-autoimmune diseases, such as dry eyes, tumours, infections or tuberculosis (TB).The first-time result of Ro52 test was selected from the same patient with multiple admissions to the hospital.The fluorescence pattern and titre were analysed retrospectively.In addition, Ro52 antibody-negative patients were set as the control group for ANA and anti-ENA tests.
Approval was obtained from the institutional ethics review committee.
Euroimmun IF Sprinter (Euroimmun AG, Lübeck, Germany) was used for fully automated processing of IIF tests.EUROBlotMaster (Euroimmun AG, Lübeck, Germany) was used for fully automated processing of immunoblots.The incubated strips were digitized by EUROBlotCamera and used EUROLineScan to evaluate (EUROIMMUN, Lübeck, Germany).Data was analysed using SPSS 19.Qualitative data was expressed as frequencies and percentages, while quantitative data was expressed as mean and standard deviation.Analysis of variance (ANOVA) was used to compare the mean values of continuous variables, and chi-squared test was used for comparisons between qualitative variables.In addition, comparisons between independent groups were carried out by using chisquared or Fisher's exact tests for categorical variables, and the student's t-test or Mann-Whitney test for quantitative parameters.Data normality and assumption of homoscedasticity were evaluated using the Fisher-Snedecor test.Logistic regression analysis was applied to calculate the odds ratio (OR) and 95% confidence intervals (Cis).Two-tailed p<0.05 was considered statistically significant.Positive predictive value (PPV) and negative predictive value (NPV) for Ro52 antibody as screening tests were also worked out.
The frequency of associated clinical diagnosis and the difference of gray value (IB, Immunoblotting method) in isolated anti-Ro52 patients' group with cytoplasmic speckled ANA IIF pattern versus nuclear speckled pattern showed ILD to be more frequent in the isolated anti-Ro52 group with cytoplasmic speckled than the nuclear speckled pattern (19.2% versus 9.5%), and PM/DM (polymyositis and dermatomyositis) (12.8% versus 2.7%).Tumours, especially lung cancer, seemed to be more frequent in the isolated anti-Ro52 group with nuclear speckled patterns than with cytoplasmic speckled patterns (36.5% versus 16.7%) and hepatitis B (20.3% versus 9.0%).Ro52 may therefore regulate both cytoplasmic and nuclear substrates via ubiquitination.Anti-Ro52 demonstrated a number of associations with clinical features of diseases, which may be useful in identifying a subgroup of patients at risk of developing certain symptoms, biochemical profiles or prognoses.The Ro52/TRIM21 protein plays an important role in innate and adaptive immunity.Additionally, it plays an important role in regulating inflammation and is involved in the pathogenesis of autoimmune diseases.The Ro52 protein and its regulatory molecules and pathways are important targets for further research and targetted therapies.
The current study has certain limitations.Some diagnoses were based on the referring physician's decision and the classification and clinical stage of diseases remain to be further refined.Even though the results seem coherent with earlier results, these limitations should be kept in mind when interpreting the current findings.Prospective studies are needed to determine whether or not anti-Ro52 antibodies analysis contributes to the diagnosis of the patient's related diseases.

Conclusion
The findings confirmed some known association of Ro52/TRIM21 antibody with gender, age and AID (autoimmune disease)/non-AID status, and added some new details for diagnostic purposes that may lay the foundation for further studies on the clinical significance and molecular mechanisms of Ro52 in specific diseases and provide a basis for further classification of autoantibody fluorescent patterns.

Figure :
Figure: Typical ANA IIF pattern of Ro52 antibody.Panel A: ANA IIF pattern of Ro52 positive sample on human epithelial cell2 (HEp2) (Cytoplasmic speckled); Panel B: ANA IIF pattern of Ro52 positive sample on HEp2 cells (Nuclear speckled); Panel a, b: ANA IIF pattern of Ro52 positive sample on monkey liver tissue; and Panel C, c: Negative control (healthy individuals).ANA-IIF: Antinuclear antibody-indirect immunofluorescence.
Qualitative data were compared with chi-square test or, when not possible, the Fischer exact test.* Anti-Ro52 positive vs negative group, p<0.05.NS: Not significant, SLE: Systemic lupus erythematosus, CTD-ILD: Connective tissue disease-associated interstitial lung disease, IM: Inflammatory myositis, RA: Rheumatoid arthritis, MCTD: Mixed connective tissue disease: pSS: Primary Sjogren's syndrome.Other diseases were not included in the analysis owing to few and scattered cases.