|Oro- and craniofacial malformations comprise a heterogeneous group of congenital developmental disorders of the face and/or skull. Oro- and craniofacial malformations can present in different forms, and can occur as both, isolated malformations of the head or in combination with additional non-facial dysmorphic features as part of a syndromic disorder. Oro- and craniofacial malformations can be causes by different genetic alteration, but also non-hereditary forms caused for example by exposure of the unborn child to various noxious agents during pregnancy have been described. The aim of this work was the identification of novel genetic factors leading to orofacial and craniofacial malformations using different molecular genetic analyses, and to functional characterization of these alterations.
In the first part of my thesis, patients with syndromic and non-syndromic craniosynostosis were genetically characterized. For this, both classical molecular genetic methods and as well as next-generation sequencing-based analyses were performed. Using this strategy, I was able to identify causative genetic alterations in 18 out of 32 patients. Depended on the initial suspected diagnosis, detection rate varied between 100% (11/11 patients with suspected Apert syndrome) and 0% (0/7 patients with isolated craniosynostosis). In the course of this analysis, I identified a novel, pathogenic alteration in TWIST1 (c.80_92del; p.(Gln27Profs*94)) in a patient with the suspected diagnosis of Saethre-Chotzen syndrome, which has not been described, yet. Bioinformatic and molecular genetic analysis proved the causality of this alteration for the patient's disease.
Additionally, five families with non-syndromic cleft lip/palate were subjected to whole-exome sequencing to determine the underlying genetic cause of their disorder. The genetic data of affected and unaffected family members were compared, and detected variants were bioinformatically analyzed and classified with regard to their functional effects as well as their pathogenicity and their occurrence in the healthy general population. Selected variants in candidate genes were subsequently analyzed by molecular genetics methods regarding their co-segregation in the respective families. Using this strategy, variants in potentially pathogenic and causative variants in candidate genes were identified in two out of five families. Based on this strategy, I was able to identify the missense variants p.(Arg77Cys) (c.229C>T) in PAX7 in affected members of a family with isolated cleft lip/palate. Subsequent molecular genetic, bioinformatic and functional characterization using luciferase-based reporter gene assays supported pathogenicity of this variant. So far, pathogenic alterations in the PAX7 have only been described in a single patient with cleft lip/palate. The identification of an additional, second variant in PAX7 in additional patients in the course of this thesis support the role of PAX7 during oro-/craniofacial development and establishes PAX7 as a causative gene for non-syndromic cleft lip/palate. In a second family, I was able to identify the variant c.551C>T; p.Ser184Phe in NEDD4L. Pathogenic variants in NEDD4L have been associated with neurodevelopmental disorders associated with periventricular nodular heterotopia. The identification of a potentially causative variant in patients with isolated cleft lip/palate may provide initial evidence for genetic variability regarding alterations in this gene.
In summary, the data presented in this thesis expands our understanding of the genetic factors and the underlying cellular mechanisms leading to different forms of craniosynostosis and isolated, non-syndromic cleft lip/palate. The identification of a previously unknown pathogenic variant in TWIST1 and identification of two novel candidate genes, PAX7 and NEDD4L, as potential cause of isolated, non-syndromic cleft lip/palate will help to identify causative variants in affected patients and may provide novel perspectives on the underlying pathomechanisms leading to these disorders.