Orofacial Clefts

Abstract

During embryogenesis, orofacial development takes place whereby cells migrate to their intended destinations to form the face of the fetus. Sometimes, cells of the palatal shelves and the lip buds do not properly migrate, leaving splits in the lip and/or palate. This can happen due to a multitude of reasons under the categories of genetic and environmental factors. We aimed to investigate the genetic and molecular basis underlying a case of non-syndromic cleft lip/palate. To do so, we extracted primary cells from a cleft patient and primary cells from a healthy individual. We were interested in looking at primary fibroblastic cells from the upper right gingiva of those individuals respectively. Through whole genome sequencing, we identified three variant genes, ARHGEF18, CUL7, and EPDR1, that we could link to the cleft phenotype. Through functional assays to test the discrepancy in the behavior of both sets of cells, we assessed proliferation, motility, adhesion, and Rho GTPase activity. We found no significant differences in the proliferative abilities of the patient-derived cells and the healthy cells. The striking difference in behavior was related to cell migration, whereby the patient-derived cells showed decreased motility, increased adhesion, and a general loss of polarity compared to the healthy cells. We found markers of focal adhesions to be heavily localized at the tail regions of patient-derived cells that could explain this behavior. We also found an increased RhoA activation in these cells which could be causative of the observed increase in focal adhesions as well as a decreased Cdc42 activation which could further explain the loss in cell polarity. Our results provide evidence that the ARHGEF18, CUL7, and EPDR1 variants could be at the root of the non-syndromic cleft lip/palate phenotype and are consistent with a dysregulation of Rho GTPase activity.