Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, Beirut, Lebanon.
School of Medicine, Lebanese American University, Beirut, Lebanon.
Cells Dev. 2021 Jun;166:203674. doi: 10.1016/j.cdev.2021.203674. Epub 2021 Mar 17.
Orofacial clefts are the most common congenital craniofacial birth defects. They occur from a failure in cell proliferation and fusion of neural crest cells of the lip buds and/or palatal shelves. In this study, we investigate the genetic basis and molecular mechanisms in primary cells derived from a cleft and lip palate patient presenting van der Woude syndrome (VWS). Since mutations in the integrin genes are widely correlated with VWS, Interferon Regulatory Factor 6 (IRF6) screening was conducted in a cohort of 200 participants presenting with orofacial anomalies. Primary fibroblastic cells derived from the upper right gingiva and palatal regions were isolated and two cellular populations from two participants were obtained: a control with no cleft phenotype and a patient with a cleft phenotype typical of van der Woude syndrome (VWS). IRF6 targeted sequencing revealed mutations in two distinct families. Our results showed no alteration in the viability of the CLP/VWS patient cells, suggesting the phenotype associate with the disease is not secondary to a defect in cell proliferation. We did however detect a significant decrease in the migratory ability of the CLP with Van der Woude syndrome (CLP/VWS) patient cells, which could account for the phenotype. When compared to normal cells, patient cells showed a lack of polarization, which would account for their lack of mobility. Patient cells showed protrusions all around the cells and a lack of defined leading edge. This was reflected with actin staining, WAVE2 and Arp2 around the cell, and correlated with an increase in Rac1 activation. Consistently with the increase in Rac1 activation, patient cells showed a loss in the maturation of focal adhesions needed for contractility, which also accounts for the lack in cell migration. Our findings give increased understanding of the molecular mechanisms of VWS and expands the knowledge of van der Woude syndrome (VWS) occurrence by providing a strong molecular evidence that CLP with Van der Woude syndrome (CLP/VWS) phenotype is caused by a defect in normal physiological processes of cells.
口腔颌面裂是最常见的颅面先天畸形。它们是由于唇芽和/或腭突的神经嵴细胞增殖和融合失败而发生的。在这项研究中，我们研究了来自唇腭裂和唇腭裂患者的原发性细胞的遗传基础和分子机制，该患者表现为范德沃特综合征（VWS）。由于整合素基因突变与 VWS 广泛相关，因此对 200 名有口腔颌面畸形的参与者进行了干扰素调节因子 6（IRF6）筛选。从右上牙龈和腭区分离出原代成纤维细胞，并从两名参与者中获得了两种细胞群：无裂孔表型的对照和具有典型范德沃特综合征（VWS）裂孔表型的患者。IRF6 靶向测序显示两个不同家族的突变。我们的结果表明，CLP/VWS 患者细胞的活力没有改变，这表明与疾病相关的表型不是细胞增殖缺陷的继发表现。然而，我们确实检测到 CLP 与范德沃特综合征（CLP/VWS）患者细胞的迁移能力显著下降，这可能是该表型的原因。与正常细胞相比，患者细胞显示出极化缺乏，这将解释其缺乏运动性。患者细胞在细胞周围显示出突起，并且缺乏明确的前缘。这反映在肌动蛋白染色、细胞周围的 WAVE2 和 Arp2 以及 Rac1 激活增加上。与 Rac1 激活增加一致，患者细胞显示出用于收缩性的成熟焦点黏附的丧失，这也解释了细胞迁移的缺乏。我们的发现增加了对 VWS 分子机制的理解，并通过提供强有力的分子证据，扩展了对范德沃特综合征（VWS）发生的认识，即具有范德沃特综合征（VWS）表型的唇腭裂（CLP/VWS）是由正常细胞生理过程的缺陷引起的。
