Longitudinal Single‐Cell Transcriptomic Profiling Reveals Dynamic Immune Cell Alterations During Burosumab Therapy in X‐Linked Hypophosphatemia

ABSTRACT

X‐linked hypophosphatemia (XLH) is a rare hereditary disorder characterized by PHEX gene mutations, elevated FGF23 levels, and impaired bone mineralization. Burosumab, a monoclonal antibody targeting FGF23 , has demonstrated clinical efficacy; however, the immunological dynamics during treatment remain unexplored. This study employed longitudinal single‐cell RNA sequencing (scRNA‐seq) to characterize peripheral blood immune cell alterations across multiple treatment stages in pediatric XLH. We performed scRNA‐seq on peripheral blood mononuclear cells from pediatric patients with XLH at five time points spanning pretreatment and burosumab therapy phases, along with healthy pediatric controls. A total of 93,112 cells were analyzed using comprehensive bioinformatic pipelines, including unsupervised clustering, pseudotime trajectory analysis, temporal gene expression profiling, and cell–cell communication inference. Eleven major immune cell populations were identified, with notable dynamic alterations in T cells and natural killer (NK) cell subtypes across treatment stages. The cellular proportion of T helper 2 (Th2) cells and regulatory T (Treg) cells were elevated before treatment and normalized during therapy, whereas T helper 17 (Th17) cells exhibited reciprocal patterns. Genes upregulated in Treg cells during early treatment were enriched in osteoclast differentiation pathway. Natural killer subtype 2 cells showed enrichment in osteoclast differentiation and interleukin‐12 response pathways. Cell–cell communication analysis identified dynamic interactions among Th2 cells, Th17 cells, Treg cells, and NK cell subtypes mediated by KLRB1–CLEC2D and SELL–SELPLG ligand‐receptor pairs. This longitudinal transcriptomic study provides the first comprehensive characterization of peripheral immune dynamics during burosumab therapy in XLH, offering new insights into the immunological mechanisms underlying treatment response.