Long-read single-cell RNA transcriptomics reveals a signature of PIK3CA mutation in a human capillary malformation

PIK3CA-related overgrowth syndromes (PROS) are caused by somatic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, however identification of an appropriate agent can be hampered by the mosaic nature and low variant allele frequency of the causal variant. Moreover, our understanding of the molecular repercussions of these variants at the single cell level remain limited. Here we report in vitro expansion of affected tissue followed by exome sequencing and combined 3' whole transcriptome and targeted long-read single cell RNA-sequencing in a patient with clinical symptoms consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). This approach identified a targetable PIK3CA variant restricted to a PAX3+ fibroblast population. These studies highlight the utility of novel next-generation sequencing strategies in the management of suspected syndromes of somatic mosaicism and provide insight into the underlying pathophysiology of a debilitating genetic syndrome. Overall design: In this study, we use exome sequencing, 3'-based single cell RNA-sequencing, and long-read single cell RNA-sequencing of in vitro expansion of affected tissue from a patient with clinical symptoms consistent with MCAP to diagnose and reveal disease specific transcriptional changes.