STAT3 gain-of-function syndrome, characterized by early-onset autoimmunity and primary immune regulatory disorder, remains poorly understood in terms of its immunological mechanisms. We employed whole-genome sequencing of familial trios to elucidate the pivotal role of de novo mutations in genetic diseases. We identified 37 high-risk pathogenic loci affecting 23 genes, including a novel STAT3 c.508G>A mutation. We also observed significant down-regulation of pathogenic genes in affected individuals, potentially associated with inflammatory responses regulated by PTPN14 via miR378c. These findings enhance our understanding of the pathogenesis of STAT3 gain-of-function syndrome and suggest potential therapeutic strategies. Notably, combined JAK inhibitors and IL-6R antagonists may offer promising treatment avenues for mitigating the severity of STAT3 gain-of-function syndrome.