Targeted Sequencing-Based Re-Evaluation of Candidate Immunogenetic Variants for Biologic Treatment Response and Difficult-to-Treat Rheumatoid Arthritis

Genetic determinants of response to biologic disease-modifying antirheumatic drugs (bDMARDs) in rheumatoid arthritis (RA) remain insufficiently characterized. We developed a targeted sequencing panel of candidate immunogenetic variants identified through a literature review and evaluated associations with response to TNF-α inhibitors, response to IL-6 inhibitors, and difficult-to-treat RA (D2TRA). Whole-blood DNA was sequenced using custom hybridization capture on a DNBSEQ-G400 platform (2 × 150 bp). Reads were aligned to hg38 using BWA-MEM, and variants were called with GATK HaplotypeCaller. After quality control, the D2TRA analysis included 154 patients (59 D2TRA and 95 non-D2TRA); drug-response analyses included 66 anti-TNF patients (26 responders, 40 non-responders) and 69 anti-IL-6 patients (29 responders, 40 non-responders). Treatment-response associations were assessed using unadjusted allelic chi-square tests with multiple-testing correction; D2TRA was analyzed using logistic regression adjusted for age, sex, and body mass index. For IL-6 inhibitor response, rs11656130/MAP2K6 (p = 0.0163; FDR = 0.3704) and rs4910008/GALNT18 (p = 0.0212; FDR = 0.3704) were identified, while rs7767069/LINC02549 was associated with TNF-α inhibitor response (p = 0.0171; FDR = 0.5968). For D2TRA, rs1813443 in CNTN5 (OR = 1.85, p = 0.017) and rs12081765, an intergenic variant near LMX1A/RXRG (OR = 0.59, p = 0.028), were nominally associated; neither association remained significant after multiple-testing correction. With the current sample sizes, the study could detect only large genetic effects, and was underpowered for these after Bonferroni correction (α = 0.00143). The findings highlight the limited reproducibility of candidate pharmacogenetic markers in RA and provide a targeted panel and analytical framework for larger replication studies.