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Clinical and Molecular Investigation of Rare CLINICAL AND MOLECULAR INVESTIGATION OF RARE CONGENITAL DEFECTS OF THE PALATE RIMANTE SESELGYTE A thesis submitted for the degree of Doctor of Philosophy to University College London August 2019 page Title 1 DECLARATION I, Rimante Seselgyte, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed……………………….. Declaration 3 ABSTRACT Cleft palate (CP) affects around 1/1500 live births and, along with cleft lip, is one of the most common forms of birth defect. The studies presented here focus on unusual defects of the palate, especially to understand better the rarely reported but surprisingly common condition called submucous cleft palate (SMCP). The frequency and consequences of SMCP from a surgical perspective were first investigated based on the caseload of the North Thames Cleft Service at Great Ormond Street Hospital and St Andrew's Centre, Broomfield Hospital, Mid Essex Hospitals Trust. It was previously reported that up to 80% of individuals with unrepaired SMCP experience speech difficulties as a consequence of velopharyngeal insufficiency (VPI). Attempted repair of the palatal defect can sometimes give poor results, so controversies still exist about the correct choice of surgical technique to use. Over 23 years, 222 patients at The North Thames Cleft Service underwent operations to manage SMCP. Nearly half of them (42.8%) were diagnosed with 22q11.2 deletion syndrome (22q11.2 DS). The first operation was palate repair, with an exception of one case, followed by a second surgical intervention required in approximately half of the patients. A third procedure to manage VPI was carried out in 6% of patients. To better understand the histological anatomy of the palatal muscles in cleft patients, biopsies were taken from levator veli palatini (LVP) and/or palatopharyngeus (PP) muscles during surgical correction of CP. Muscles were compared from patients with SMCP to those with overt CP and also to controls. The controls consisted of descending PP muscle fibres from healthy children who underwent a tonsillectomy operation for obstructive sleep apnoea or recurrent chronic tonsillitis. Fifty-seven biopsy samples were available from children between 10 months to 9 years of age. Individual biopsy samples were also available from patients with achondroplasia, Apert, Cornelia de Lange and Kabuki syndromes. The study showed a prevalence of fast fibres in both muscles in all CP types. However, in both SMCP LVP and SMCP 22q11.2 DS LVP, this trend was reversed in favour of slow fibres. Single cases with syndromes did not reveal any obvious differences compared to more common cleft types. Abstract 5 Mutations in TBX22 are a frequent genetic cause of cleft palate and SMCP. The functional role of the encoded TBX22 transcription factor was investigated in a mouse model with SMCP. Cell lineage-specific fluorescence activated cell sorting of a conditional allele of Tbx22, was used to look at the RNA-Seq transcriptome in developing palatal shelves, with a view to identify downstream target genes. Eleven up regulated genes reached statistical significance after multiple testing correction in cranial mesoderm (CM) derived cells when comparing Tbx22null/Y and WT samples (Cspg4, Foxp2, Reln, Bmpr1b, Adgrb3, Sox6, Zim1, Scarna13, Fat1, Notch3, Peg3). Eleven genes were down regulated in the same comparison (Nr2f2, Lars2, Ahr, Aplnr, Emcn, Npnt, Apln, Ccr2, Tll1, Snord34, Snord99). Comparing Tbx22null/Y and WT in cranial neural crest (CNC) derived cells, only Cxcl14 was up regulated, while Tbx22 was down regulated. Osteoclast differentiation, calcium signalling, focal adhesion, Wnt signalling and cell adhesion molecule pathways were the most enriched pathways in functional annotation of significantly differentially expressed genes analysis. Finally, a family with an unusual velopharyngeal anatomy was investigated in order to determine the likely genetic cause. This involved the implementation of genetic technologies in an autosomal dominant multigeneration Egyptian family with 8 affected individuals who presented with absent uvula, short posterior border of the soft palate and abnormal pillars of the fauces. Using a combination of cytogenetic, linkage analysis and exome sequencing, followed by more detailed segregation and functional analysis, a dominantly acting missense mutation in the activation domain of FOXF2 was revealed. This variant was found to co-segregate with a copy number variant of unknown significance that could not at this stage be causally distinguished from the point mutation. Abstract 6 IMPACT STATEMENT Orofacial clefts are among the most common birth defects worldwide, affecting 1 in 700 births. Facial clefts, including cleft lip and/or cleft palate, have a very serious impact on the quality of life, requiring repeated surgical interventions in childhood and specialist medical attention throughout life. Recent research has emphasized the importance for a better understanding of cleft pathogenesis and genetic background using a combination of epidemiology, genome-wide association studies, linkage analysis, candidate gene sequencing and the study of animal models. Despite intensive research worldwide, much of the genetic basis of cleft lip and palate still remains unknown. It is an important goal to develop these research methods with a view to the gradual transition and application in the everyday clinical cleft patient management. The main focus of this work was the study of rare forms of cleft palate (CP), a group of congenital abnormalities that present a significant challenge since treatment outcomes are on the whole less successful. In this PhD, extensive palate muscle histology was analysed in patients with submucous cleft palate for the first time. The findings add to our understanding why surgical cleft repair is not always successful and provides evidence which can assist surgeons to manage expectations of patients and their families. At the same time, these results can form the basis of a larger prospective study into palatal muscle repair. A second aim was to examine the molecular basis of Tbx22 loss in a mouse model of CP. Mutations in TBX22 cause X-linked CP, which ranks as one of the most common genetic causes of CP in humans. Experiments were performed to identify consequential gene expression disbalances that occur in embryonic mouse palatal shelves, when the TBX22 transcription factor is silenced. This work has revealed several likely downstream target genes and highlighted biochemical pathways that are likely to be required for normal palatogenesis. This is of interest to a large international group of orofacial biologists and provides an important platform for future studies. t Finally, a family with an unusual, and possibly unique, inherited palate phenotype, characterised by an absent uvula, short posterior border of the soft palate and statemen Impact 7 abnormal pillars of the fauces was studied in order to identify the molecular cause. Using a combination of technologies including cytogenetic analysis, linkage, exome sequencing and cell-based functional studies identified FOXF2 as playing an important role in palatal, oropharyngeal and uvula development. Recent publication of this work benefits the broad society of researchers and clinicians, particularly raising awareness of this rare condition. The family has been fully informed for the benefit of future genetic counselling, especially since our findings also have implications for preventable late-onset glaucoma. The work in this PhD was funded by ‘CLEFT-bridging the gap’, which included a generous donation by the Childwick Trust and the results will be reported to the charity. Dissemination of the PhD project’s results via the Charity’s events, publications and website, helps to raise awareness about the importance of cleft lip and palate research. Much of the research reported in this thesis has been presented at national or international conferences. The genetic study of absent uvula and hypernasality has been published as a peer review article (Seselgyte et al., 2019), with a follow up clinical report currently under revision prior to final acceptance (The Cleft Palate-Craniofacial Journal). Other manuscripts are currently being prepared for publication. Impact statement Impact 8 ACKNOWLEDGEMENTS My recent years have been a profound journey accompanied by outstanding support that I am very grateful for. Without my supervisors and mentors Prof Philip Stanier, Dr Erwin Pauws and Mr Brian Sommerlad this work would have never reached the daylight. My PhD was funded by ‘CLEFT - bridging the gap’, and I highly appreciate everyone who has been involved in fund raising. Huge thanks to Prof Gudrun Moore for constant inspiration throughout my PhD. Our conversations enlightened me well beyond the research. Dale Bryant, Emma Peskett, Miho Ishida, Charalambos Demetriou, Melissa Riachi and Nita Solanky patiently shared their expertise in research and along with others, were my best friends when support, understanding and encouragement were needed. I am grateful to my mentors and colleagues at Great Ormond Street Hospital Ms Lesley Cochrane, Mr Norman Hay, Mr Loshan Kangesu, Mr Paul Morris, Ms Marie Pinkstone, Dr Debbie Sell, Mr Marc Swan and Mr Guy Thorburn. Also, very sincere thanks to the Clinical Nurse Specialists Julie, Helen, Clair and all others who were not only wonderful to work with but also are ‘GOSH family’. I would like to express
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