Clinical and molecular investigation of rare congenital defects of the palate

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. 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

Principles of Head and Neck Reconstruction: An Algorithm to Guide Flap Selection

Advances in head and neck reconstruction have resulted in improved outcomes with single-stage repair of defects ranging from intraoral to pharyngoesophageal to skull base defects. Key to success of surgery is choosing an appropriate reconstructive option based on the patient's wishes and fitness for major surgery. Where possible, free tissue transfer provides the best functional and aesthetic outcomes for the vast majority of defects. In this article, we present an algorithm to guide choice of flap selection and review principles of reconstruction and secondary surgery for head and neck defects

Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, similar to 5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 x 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (p(combined) = 2.63 x 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 x 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO