Nowadays there is an emphasis on detecting
de novo gene changes. The aim of this project was to identify novel
de novo gene mutations in children with orofacial clefts. To achieve this, I targeted cleft children with unknown aetiology at the South Thames regional cleft centre, London, UK. Using a trio-based design, I recruited 90 child probands and 159 relatives and used parental tooth anomalies as a subclinical marker to categorise probands into those with potentially inherited or
de novo genetic risks. I identified children with ‘cleft-only’, ‘cleft-tooth anomaly’ and ‘cleft-medical condition +/- tooth anomaly’. Of those dentally examined, (55/127) relatives in 45 trios were found to have dental anomalies, especially hypodontia outside their child’s cleft side, suggesting Mendelian inheritance. In Chapter 3, I reported findings from the Clinical Study and described the cohort with non-syndromic clefts. I showed that 16 probands had a ‘cleft-only’ condition and only six of them had parents with no dental anomalies or family history of clefting. This chapter highlighted that the clinical diagnosis of ‘isolated’ clefts needs to be more precise. The Clinical Study further identified (28/90) probands who also had other congenital anomalies or medical co-morbidities associated with their orofacial clefts. Family trios from the ‘cleft-medical condition +/- tooth anomaly’ group were explored further and became the focus of this thesis. Whole exome sequencing was carried out on several trios from this group. In Chapter 4, a novel
de novo mutation in the catenin delta-1 (CTNND1) gene was identified. This gene encodes the p120-catenin protein known for its role in cell-cell adhesion and the regulation of epithelial-to-mesenchymal transition. I expanded on the developmental roles for p120-catenin demonstrated through the phenotypes I described in the human patients and in mouse and Xenopus models. I used the Deciphering Developmental Disorders (DDD) dataset to search and recruit further subjects with CTNND1 mutations and identified 12 more individuals whom I found to have characteristic craniofacial and dental features as well as heart, limb and neurodevelopmental anomalies. Using loss-offunction genetic approaches in mouse and Xenopus, I demonstrated novel roles for CTNND1 in the vocal cords and the velopharynx, craniofacial cartilages and the heart. I suggest that CTNND1 is a candidate neurocristopathy gene, highlighting both epithelial and mesenchymal roles for p120-catenin. My work expands upon the spectrum of abnormalities associated with CTNND1 variants beyond those previously described in non-syndromic cleft lip/palate (CLP) and blepharocheilodontic syndrome (BCD1) and that variations in this gene may be expanded to a broader velocardiofacial-like syndrome. In Chapter 5, I conducted exploratory research into phenotypically different monozygotic twins who had also been recruited through my Clinical Study. Whole exome sequencing identified a novel
de novo copy number variation in the AGAP6 gene. I confirmed that AGAP6 transcripts are strongly expressed during human embryonic development in craniofacial structures. In order to validate the pathogenicity of AGAP6, future work involves its implication in other unrelated individuals with rare craniofacial anomalies. Finally, in Chapter 6, I demonstrated the utility of a publicly available dataset (DECIPHER, www.decipher.org, DDD) in gene discovery. I developed a protocol to interrogate and analyse the dataset that included individuals with neurocristopathic anomalies using Human Phenotype Ontology (HPO) search terms honed from the medical conditions that I found in my Clinical Study. I assembled a list of novel putative genetic variants in DIP2C, ABCA2 and CELSR1. The protocol I developed could be used for future studies. The segregation of cleft subjects based on ‘associated anomalies’, be it dental or medical, and on whether family members were affected with subclinical anomalies, emphasizes the genetic status underlying their conditions. In conclusion, I found novel
de novo gene mutations in patients with orofacial clefts particularly in CTNND1 and suggest a list of other potential candidate genes for future study.