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

Area of Science:

• Genetics and Molecular Biology
• Skeletal Biology
• Transcriptomics

Background:

• Pathogenic NOTCH2 variants can cause significant health issues due to altered Notch signaling.
• A novel NOTCH2 variant (4006G>C) is linked to reduced Notch signaling and skeletal fragility.
• Understanding the molecular mechanisms underlying this variant's effects is crucial.

Purpose of the Study:

• To investigate the transcriptomic changes in femoral bone cells from mice with a novel NOTCH2 variant.
• To identify specific cell populations affected by the NOTCH2 variant.
• To elucidate the impact of the NOTCH2 variant on gene transcription in osteogenic and vascular cells.

Main Methods:

• Single-cell RNA sequencing (scRNA-seq) of murine femoral bone cells.
• Microfluidic partitioning using Chromium X technology for gene expression analysis.
• Uniform Manifold Approximation and Projection (UMAP) for cell clustering and identification.
• scVelo pipeline for analyzing nascent transcripts and transcriptional dynamics.

Main Results:

• Identification of 15 distinct cell clusters, including osteogenic and vascular cells.
• Significant reduction (35-55%) in osteogenic and vascular cell populations in Notch2^em1Ecan mice compared to controls.
• Decreased expression of Hes1, a downstream target of Notch signaling, in Notch2^em1Ecan cells.
• Suppressed transcriptional velocity in vascular and osteogenic clusters of Notch2^em1Ecan mice, indicating reduced gene transcription.

Conclusions:

• A novel deleterious NOTCH2 variant significantly impacts the transcriptome of adult mouse femurs.
• The variant leads to a marked decrease in osteogenic and vascular cell populations.
• Reduced Notch signaling and impaired gene transcription in key cell types contribute to skeletal fragility associated with this NOTCH2 variant.