B-cell repertoire sequencing reveals frequent rearrangements of IGHD5-5 in patients with systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disorder marked by fibrosis of the skin and internal organs, with B cells increasingly recognized as key players in its pathogenesis. However, the characteristics of the B cell receptor (BCR) repertoire in SSc remain insufficiently defined. In this study, we performed high-throughput sequencing of immunoglobulin heavy chain genes in 15 female anti-centromere antibody (ACA)-positive SSc patients and five age-matched healthy female controls to explore disease-specific repertoire biases. A total of 2,597,460 in-frame sequence reads and 384,111 unique reads were obtained. While diversity metrics, including the Shannon, Simpson, and Pielou indices, tended to be higher in the SSc group, the differences were not statistically significant. Notably, the average complementarity-determining region 3 (CDR3) length was significantly shorter in SSc patients compared to controls (16.91 ± 3.727 vs. 17.44 ± 3.836, p < 0.0001). Gene usage analysis revealed no significant differences in IGHJ or IGHC segments; however, several IGHV and IGHD segments displayed statistically significant differences. IGHD5-5 (1.636% vs. 0.547%, p = 0.0001) and IGHD5-18 (1.607% vs. 0.547%, p = 0.0001) were significantly overrepresented in the SSc group, whereas IGHV1/OR15-2 was significantly underrepresented (0.062% vs. 0.130%, p = 0.0080). Further analysis demonstrated that IGHD5-5 clones with a 15-nucleotide CDR3 length were more conserved and exhibited distinctive sequence patterns compared to other lengths or genes. Specific nucleotide lengths, including 15, 23, and 24 for IGHD5-5, and 18 for IGHV1/OR15-2, showed significant frequency differences between groups (p < 0.05). Sequence logo plots confirmed reduced variability in these conserved clones, suggesting antigen-driven clonal selection. These findings identify unique BCR repertoire features in ACA-positive SSc patients and suggest their potential utility as disease biomarkers or therapeutic targets.