A NOTCH2 pathogenic variant alters the osteoblast and vascular cell transcriptome of mouse femurs

Pathogenic variants of NOTCH2 that lead to a gain or loss of function are associated with serious clinical consequences. A recently reported novel NOTCH2 4006G > C variant was associated with decreased Notch signaling and skeletal fragility in humans and mouse ( Notch2 em1Ecan ) lines. In the present study, we analyze the transcriptome of femoral bones from mature control and Notch2 em1Ecan mice following the exclusion of hematological cells. Single cells were analyzed following microfluidic partitioning on a Chromium X instrument using a 3′ gene expression library. Uniform manifold approximation and projection (UMAP) for non-linear dimensional reduction defined 15 different cell clusters comprised of macrophages, red blood cells, endothelial, vascular and smooth and striated muscle cells and cells of the osteoblast lineage. Notch2 and Hes1 were the prevalent Notch receptor and target gene in most clusters. Independent clustering analysis of transcriptomes from control and Notch2 em1Ecan femurs revealed a 35–45% decrease in cells forming the osteogenic and a 50–55% decrease in cells comprising the vascular cluster in Notch2 em1Ecan femurs. Expression of Hes1 was decreased in Notch2 em1Ecan cells. Analysis of nascent transcripts (intron/exon sequences) using the scVelo pipeline revealed suppressed velocity in vascular and osteogenic clusters from Notch2 em1Ecan cells implying decreased transcription of genes constituting these clusters. In conclusion, a novel NOTCH2 deleterious variant associated with skeletal fragility alters the osteogenic and vascular transcriptome in femurs from adult mice. • A novel NOTCH2 pathogenic variant is associated with skeletal fragility • A NOTCH2 4006G > C variant impairs Notch signal activation • Single-cell RNA-Seq of adult femurs reveals 15 well defined cell clusters • A NOTCH2 4006G > C variant decreases osteoblast and vascular cell transcription