Nani, D.; Alvizi, L.; Bueno, H. M. S.; Lacerda, E. C. M.; Passos-Bueno, M. R.

Cleft lip with or without palate (CLP) is a multifactorial trait associated with environmental exposures such as smoking, alcohol consumption, and pro-inflammatory conditions during early pregnancy, as well as with both common and rare genetic variants. Our group identified CDH1 loss-of-function variants segregating in families with CLP, with incomplete penetrance linked to promoter hypermethylation. Previously, we demonstrated that gene-environment interactions driven by pro-inflammatory factors can induce this methylation, downregulate E-cadherin expression, and impair neural crest migration, thereby contributing to the etiology of CLP. However, the embryonic consequences of the two-hit CLP model (CDH1 haploinsufficiency combined with pro-inflammatory insult) have not yet been explored in mammals. Here, under single dose of floxed-Cdh1, we investigated the pro-inflammatory response at the maternal-fetal interface, its impact on cytokine and Cdh1 expression along the anteroposterior axis, the associated epigenetic landscape in the embryonic head, and the effects on cranial NC-derived structures. We found that pro-inflammatory activation differentially signals to the embryo along the anteroposterior axis, impairing Cdh1/E-cadherin expression in NC cells of the head, accompanied by Cdh1 promoter hypermethylation and other differentially methylated genes involved in cell-junction maintenance. Our findings support a model in which maternal pro-inflammatory responses act as environmental factors that repress NC CDH1 expression through epigenetic mechanisms, contributing to CLP development by downregulating E-cadherin and potentially compromising overall epithelial integrity. Animal Ethics Committee (CEUA IB/USP) number approval: 394/2022