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, ∼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 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 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 × 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.

Non-syndromic cleft lip and/or palate (NSCLP) is a common congenital malformation with a multifactorial model of inheritance. Although several at-risk alleles have been identified, they do not completely explain the high heritability. We postulate that epigenetic factors as DNA methylation might contribute to this missing heritability. Using a Methylome-wide association study in a Brazilian cohort (67 NSCLP, 59 controls), we found 578 methylation variable positions (MVPs) that were significantly associated with NSCLP. MVPs were enriched in regulatory and active regions of the genome and in pathways already implicated in craniofacial development. In an independent UK cohort (171 NSCLP, 177 controls), we replicated 4 out of 11 tested MVPs. We demonstrated a significant positive correlation between blood and lip tissue DNA methylation, indicating blood as a suitable tissue for NSCLP methylation studies. Next, we quantified CDH1 promoter methylation levels in CDH1 mutation-positive families, including penetrants, non-penetrants or non-carriers for NSCLP. We found methylation levels to be significantly higher in the penetrant individuals. Taken together, our results demonstrated the association of methylation at specific genomic locations as contributing factors to both non-familial and familial NSCLP and altered DNA methylation may be a second hit contributing to penetrance.

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.

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo. Mutations in the human Sorting Nexin 14 (SNX14) gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20; OMIM 616354) 1. These mutations most often lead to complete loss or truncation of the SNX14 protein, resulting in early onset cerebellar atrophy, ataxia, developmental delay, intellectual disability and coarse facial features, with hearing loss, relative macrocephaly and seizures only reported in some patients 1-7. SNX14 is ubiquitously expressed among tissues, accounting for the clinically recognisable syndromic presentation characteristic of SCAR20 1,5,7. SNX14 belongs to the RGS-PX protein family, which includes SNX13, SNX19 and SNX25 8. No mutations in these other members have yet been identified as the cause of human diseases. Inside the cell, SNX14 mutations impact both autophagy and lipid metabolism 1,2,9. The most apparent subcellular phenotype is the accumulation of autolysosomes containing lipids 1,9. SNX14 is localised to the endoplasmic reticulum membrane via its N-terminal transmembrane domain where it is enriched in proximity to lipid

Risk, III.