Evidence that disruption of Discoidin domain receptor 2 contributes to palate malformations through effects on the extracellular matrix.

Orofacial clefting (OFC) is among the most common birth defects and can occur either as part of a syndrome or in isolation (nonsyndromic, ns). Cleft palate only (CPO) is an OFC subtype. Here, we searched for novel nsCPO risk genes carrying homozygous and compound-heterozygous variants, by analyzing exome data from six sibling pairs with nsCPO born to unaffected parents. After stringent quality control and filtering, we identified 6 homozygous variants and 32 compound-heterozygous variants in 5 and 16 candidate genes, respectively. We prioritized DDR2, a collagen-activated receptor-tyrosine-kinase influencing extracellular matrix composition, as our top candidate for functional follow-up, since variants in this gene can cause Warburg-Cinotti syndrome, the phenotypic spectrum of which includes palatal abnormalities. Knock-down and knock-out of DDR2-orthologs in zebrafish caused craniofacial abnormalities resembling CPO in humans. Zebrafish immunostaining indicated that DDR2-orthologs were expressed in mature head muscle cells, while murine single-cell RNA-Sequencing data detected Ddr2 expression only in head muscle progenitor cells; the latter finding was confirmed in human embryo sections stained for DDR2. DDR2-expressing head muscle progenitor cells may influence extracellular matrix composition through DDR2-mediated signaling, thereby affecting outgrowth, elevation, and fusion of the palatal shelves, a previously postulated mechanism involved in palatogenesis. Most established OFC genes (e.g. CDH1, CTNND1, IRF6, and GRHL3) act via mechanisms related to epithelial integrity and periderm differentiation, whereas our data provide evidence supporting DDR2 as a risk gene for nsOFC that functions by influencing extracellular matrix composition.