A novel variant of NOTCH2 causes skeletal fragility

A 2-month-old female child presented with a low-trauma femoral fracture, low bone mass, bowing of long bones, loss of height of a thoracic vertebra, and Wormian bones. Exome sequencing revealed the presence of a novel heterozygous 4006G > C pG1336R mutation in exon 25 of NOTCH2 in the child and her father. In silico analysis considered the variant as likely deleterious. CRISPR/Cas9 was used to introduce the Notch2 4006G>C mutation into Notch2 to create Notch2 em1Ecan mutant mice. Homozygous Notch2 em1Ecan mutant mice were active, appeared healthy, had normal femoral length, but lower weights than controls. μCT of the distal femur revealed a 25 % decrease in trabecular bone volume, and a decrease in total, bone and marrow area, in periosteal perimeter and polar moment of inertia, revealing the presence of small and potentially fragile bones. Three-point bend testing demonstrated decreased toughness in Notch2 em1Ecan femurs. Cancellous bone histomorphometry demonstrated decreased eroded surface, and Raman spectroscopy revealed normal mineral to matrix ratios, carbonate:phosphate and collagen peak ratios. A structure homology model of NOTCH2 EGF33–36 repeats suggests that the G1336R mutation may disrupt the local structure, reducing the flexibility of the extracellular domain and thereby affecting receptor activation and signaling. Indeed, there was a modest decrease in Notch canonical target genes in osteoblasts from Notch2 em1Ecan mice. Osteoblast and osteoclast differentiation were diminished in cells from Notch2 em1Ecan mice. In conclusion, a novel mutation affecting the NOTCH2 extracellular domain is associated with small and apparently fragile bones, possibly due to altered Notch signaling. • A novel NOTCH2 4006G>C pG1336R variant predicted to be deleterious is associated with fragility fractures. • Homozygous Notch2 em1Ecan mice harboring the 4006G > C mutation have small and potentially fragile bones. • Type I collagen is not affected in Notch2 em1Ecan mice. • The G133R substitution disrupts the NOTCH2 structure, reducing its flexibility, affecting Notch signaling.