Anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibodies are critical biomarkers in myositis, associated with distinct clinical features and prognosis. This study aimed to evaluate the proportion of anti-MDA5 positivity and compare the diagnostic performance of local immunoblotting (IB) with gold-standard immunoprecipitation (IP). We performed a retrospective analysis of 3272 physician-requested anti-MDA5 IB determinations over a five-year period (2019–2023). A subsequent exploratory pilot study of ten Hungarian patients with myositis was conducted to compare IB results with radiolabeled protein IP. Confirmatory in-house enzyme-linked immunosorbent assay (ELISA) was used to distinguish between 140 kDa bands (anti-MDA5 vs. anti-NXP2). Indirect immunofluorescence (IIF) on HEp-2 cells was also evaluated. In the retrospective cohort, 3.7% (n = 121) of samples were non-negative. Among 64 borderline patients, only one (1.6%) had a definitive diagnosis of dermatomyositis (DM). Conversely, the proportion of confirmed myositis cases was notably higher among patients with strong positive IB results. In our exploratory cross-sectional pilot study, complete concordance between the two assays was observed for negative and strong positive results. Discrepancies were noted in borderline and weak positivity ranges, where anti-MDA5 was not detected by IP; instead, alternative autoantibodies were identified. The three IP-confirmed MDA5 positive samples were all validated by ELISA. The characteristic IIF cytoplasmic staining was identifiable in 2 out of 10 cases (20%). In our cohort, borderline IB cases were frequently potential false positives, highlighting the need for careful clinical evaluation. Borderline and weak results require clinical correlation or confirmatory testing to avoid misdiagnosis.

Systemic sclerosis (SSc) is a severe autoimmune rheumatic disease with high mortality. Epigenetic factors, particularly micro-RNAs (miRNAs), may contribute to its pathogenesis by regulating gene expression. In this cross-sectional study, we assessed altered miRNA–mRNA regulatory networks in SSc and associated them with disease-related biological processes. We analyzed the miRNA profiles and differentially expressed genes (DEGs) of peripheral blood mononuclear cells (PBMCs) from 52 SSc patients (42 women and 10 men; mean age: 59.1 years) and 24 age- and gender-matched healthy controls. Total RNA was isolated and subjected to high-throughput next-generation sequencing for both miRNA and mRNA profiling. We identified 58 differentially expressed miRNAs (DEMs), 33 upregulated and 25 downregulated in SSc. In parallel, 6610 DEGs were detected (Mann–Whitney U-test, p < 0.05); 31 remained upregulated and nine downregulated after false discovery rate (FDR) correction. Integration of miRNA and mRNA data revealed 180 validated inverse miRNA–mRNA interactions. Notably, 22 of 31 upregulated DEGs corresponded to targets of downregulated miRNAs, indicating coordinated derepression. Functional enrichment analyses highlighted pathways related to extracellular matrix (ECM) remodeling, immune responses, fibrosis, and transcriptional regulation. Our findings suggest that altered miRNA expression contributes to widespread transcriptional dysregulation in SSc, promoting pro-fibrotic and immune-activated molecular pathways through coordinated miRNA–mRNA interactions.