RSPO4 is a potential risk gene of stages III–IV, grade C periodontitis through effects on innate immune response and oral barrier integrity

Aim: R-spondin 4 (RSPO4) is a suggestive risk gene of stage III–IV, grade C periodontitis and upregulated in gingiva of mice resistant to bacteria-induced alveolar bone loss. We aimed to replicate the association, identify and characterize the putative causal variant(s) and molecular effects, and understand the downstream effects of RSPO4 upregulation. Materials and Methods: We performed a two-step association study for RSPO4 with imputed genotypes of a German–Dutch (896 stage III–IV, grade C periodontitis cases, 7104 controls) and Spanish sample (441 cases and 1141 controls). We analysed the allelic effects on transcription factor binding sites with reporter gene and antibody electrophoretic mobility shift assays. We used CRISPR/dCas9 activation and RNA sequencing to pinpoint RSPO4 as the target gene and to analyse downstream effects. Results: RSPO4 was associated with periodontitis (rs6056178, p_meta = 4.6 × 10⁻⁵). rs6056178 contains a GATA-binding motif. The rs6056178 T-allele abolished reporter activity (p =.004) and reduced GATA binding (−14.5%). CRISPRa of the associated region increased RSPO4 expression (25.8 ± 6.5-fold, p =.003). RSPO4 activation showed strongest induction of Gliomedin (439-fold) and Mucin 21 (178-fold) and of the gene set “response to interferon-alpha” (area under the curve [AUC] = 0.8, p < 5 × 10⁻⁶). The most repressed gene set was “extracellular matrix interactions” (AUC = 0.8, p_adj =.00016). Conclusion: RSPO4 is a potential periodontitis risk gene and modifies host defence and barrier integrity.