Introduction: Cleft lip ± cleft palate (CL/P) is one of the most common birth defects. Although research has identified multiple genetic risk loci for different types of CL/P (i.e., syndromic or non-syndromic forms), determining the respective causal genes and understanding the relevant functional networks remain challenging. The recent introduction of single-cell RNA sequencing (scRNA-seq) has provided novel opportunities to study gene expression patterns at cellular resolution. The aims of our study were to: (i) aggregate available scRNA-seq data from embryonic mice and provide this as a resource for the craniofacial community; and (ii) demonstrate the value of these data in terms of the investigation of the gene expression patterns of CL/P candidate genes. Methods and Results: First, two published scRNA-seq data sets from embryonic mice were re-processed, i.e., data representing the murine time period of craniofacial development: (i) facial data from embryonic day (E) E11.5; and (ii) whole embryo data from E9.5-E13.5 from the Mouse Organogenesis Cell Atlas (MOCA). Marker gene expression analyses demonstrated that at E11.5, the facial data were a high-resolution representation of the MOCA data. Using CL/P candidate gene lists, distinct groups of genes with specific expression patterns were identified. Among others we identified that a co-expression network including Irf6, Grhl3 and Tfap2a in the periderm, while it was limited to Irf6 and Tfap2a in palatal epithelia, cells of the ectodermal surface, and basal cells at the fusion zone. The analyses also demonstrated that additional CL/P candidate genes (e.g., Tpm1, Arid3b, Ctnnd1, and Wnt3) were exclusively expressed in Irf6+ facial epithelial cells (i.e., as opposed to Irf6- epithelial cells). The MOCA data set was finally used to investigate differences in expression profiles for candidate genes underlying different types of CL/P. These analyses showed that syndromic CL/P genes (syCL/P) were expressed in significantly more cell types than non-syndromic CL/P candidate genes (nsCL/P). Discussion: The present study illustrates how scRNA-seq data can empower research on craniofacial development and disease.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.

To determine if nonsyndromic cleft lip with or without cleft palate (nsCL/P) genetic risk variants influence liability to nsCL/P through gene regulation pathways, such as those involving DNA methylation.

It has been hypothesised that nonsyndromic cleft lip/palate (nsCL/P) and cancer may share aetiological risk factors. Population studies have found inconsistent evidence for increased incidence of cancer in nsCL/P cases, but several genes (e.g., CDH1, AXIN2) have been implicated in the aetiologies of both phenotypes. We aimed to evaluate shared genetic aetiology between nsCL/P and oral cavity/oropharyngeal cancers (OC/OPC), which affect similar anatomical regions. Using a primary sample of 5,048 OC/OPC cases and 5,450 controls of European ancestry and a replication sample of 750 cases and 336,319 controls from UK Biobank, we estimate genetic overlap using nsCL/P polygenic risk scores (PRS) with Mendelian randomization analyses performed to evaluate potential causal mechanisms. In the primary sample, we found strong evidence for an association between a nsCL/P PRS and increased odds of OC/OPC (per standard deviation increase in score, odds ratio [OR]: 1.09; 95% confidence interval [CI]: 1.04, 1.13; p = .000053). Although confidence intervals overlapped with the primary estimate, we did not find confirmatory evidence of an association between the PRS and OC/OPC in UK Biobank (OR 1.02; 95% CI: 0.95, 1.10; p = .55). Mendelian randomization analyses provided evidence that major nsCL/P risk variants are unlikely to influence OC/OPC. Our findings suggest possible shared genetic influences on nsCL/P and OC/OPC.

There is increasing evidence that genetic risk variants for non-syndromic cleft lip/palate (nsCL/P) are also associated with normal-range variation in facial morphology. However, previous analyses are mostly limited to candidate SNPs and findings have not been consistently replicated. Here, we used polygenic risk scores (PRS) to test for genetic overlap between nsCL/P and seven biologically relevant facial phenotypes. Where evidence was found of genetic overlap, we used bidirectional Mendelian randomization (MR) to test the hypothesis that genetic liability to nsCL/P is causally related to implicated facial phenotypes. Across 5,804 individuals of European ancestry from two studies, we found strong evidence, using PRS, of genetic overlap between nsCL/P and philtrum width; a 1 S.D. increase in nsCL/P PRS was associated with a 0.10 mm decrease in philtrum width (95% C.I. 0.054, 0.146; P = 2x10-5). Follow-up MR analyses supported a causal relationship; genetic variants for nsCL/P homogeneously cause decreased philtrum width. In addition to the primary analysis, we also identified two novel risk loci for philtrum width at 5q22.2 and 7p15.2 in our Genome-wide Association Study (GWAS) of 6,136 individuals. Our results support a liability threshold model of inheritance for nsCL/P, related to abnormalities in development of the philtrum.

Previous studies have found that children born with a non-syndromic orofacial cleft have lower-than-average educational attainment. Differences could be due to a genetic predisposition to low intelligence and academic performance, factors arising due to the cleft phenotype (such as social stigmatization, impaired speech/language development) or confounding by the prenatal environment. A clearer understanding of this mechanism will inform interventions to improve educational attainment in individuals born with a cleft, which could substantially improve their quality of life. We assessed evidence for the hypothesis that common variant genetic liability to non-syndromic cleft lip with or without cleft palate (nsCL/P) influences educational attainment.

Previous research suggests a genetic overlap between nonsyndromic cleft lip with or without cleft palate (NSCL/P) and cancer. The aim of the present study was to identify common genetic risk loci for NSCL/P and cancer entities that have been reported to co-occur with orofacial clefting. This was achieved through the investigation of large genome-wide association study datasets. Investigations of 12 NSCL/P single nucleotide polymorphisms (SNPs) in 32 cancer datasets, and 204 cancer SNPs in two NSCL/P datasets, were performed. The SNPs rs13041247 (20q12) and rs6457327 (6p21.33) showed suggestive evidence for an association with both NSCL/P and a specific cancer entity. These loci harbor genes of biological relevance to oncogenesis (MAFB and OCT4, respectively). This study is the first to characterize possible pleiotropic risk loci for NSCL/P and cancer in a systematic manner. The data represent a starting point for future research by identifying a genetic link between NSCL/P and cancer.

Non-syndromic cleft lip with palate (NSCLP) is the most serious sub-phenotype of non-syndromic orofacial clefts (NSOFC), which are the most common craniofacial birth defects in humans. Here we conduct a GWAS of NSCLP with multiple independent replications, totalling 7,404 NSOFC cases and 16,059 controls from several ethnicities, to identify new NSCLP risk loci, and explore the genetic heterogeneity between sub-phenotypes of NSOFC. We identify 41 SNPs within 26 loci that achieve genome-wide significance, 14 of which are novel (RAD54B, TMEM19, KRT18, WNT9B, GSC/DICER1, PTCH1, RPS26, OFCC1/TFAP2A, TAF1B, FGF10, MSX1, LINC00640, FGFR1 and SPRY1). These 26 loci collectively account for 10.94% of the heritability for NSCLP in Chinese population. We find evidence of genetic heterogeneity between the sub-phenotypes of NSOFC and among different populations. This study substantially increases the number of genetic susceptibility loci for NSCLP and provides important insights into the genetic aetiology of this common craniofacial malformation.

Non-syndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations, and has a multifactorial background in which both exogenous and genetic risk factors act in concert. The present report describes a genome-wide association study (GWAS) involving a total of 285 nsCL/P patients and 1212 controls from the Netherlands and Belgium. Twenty of the 40 previously reported nsC/LP susceptibility loci were replicated, which underlined the validity of this sample. SNV-based analysis of the data identified an as yet unreported suggestive locus at chromosome 16p12.1 (p-value of the lead SNV: 4.17 × 10-7). This association was replicated in two of three patient/control replication series (Central European and Yemeni). Gene analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS was the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology.

Cranial neural crest (CNC) is a multipotent migratory cell population that gives rise to most of the craniofacial bones. An intricate network mediates CNC formation, epithelial-mesenchymal transition, migration along distinct paths, and differentiation. Errors in these processes lead to craniofacial abnormalities, including cleft lip and palate. Clefts are the most common congenital craniofacial defects. Patients have complications with feeding, speech, hearing, and dental and psychological development. Affected by both genetic predisposition and environmental factors, the complex etiology of clefts remains largely unknown. Here we show that Fas-associated factor-1 (FAF1) is disrupted and that its expression is decreased in a Pierre Robin family with an inherited translocation. Furthermore, the locus is strongly associated with cleft palate and shows an increased relative risk. Expression studies show that faf1 is highly expressed in zebrafish cartilages during embryogenesis. Knockdown of zebrafish faf1 leads to pharyngeal cartilage defects and jaw abnormality as a result of a failure of CNC to differentiate into and express cartilage-specific markers, such as sox9a and col2a1. Administration of faf1 mRNA rescues this phenotype. Our findings therefore identify FAF1 as a regulator of CNC differentiation and show that it predisposes humans to cleft palate and is necessary for lower jaw development in zebrafish.

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.

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13 × 10(-14) for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32-1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94-1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47-9.61, Pdiff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.

Nonsyndromic oral clefts are craniofacial malformations, which include cleft lip with or without cleft palate. The etiology for oral clefts is complex with both genetic and environmental factors contributing to risk. Previous genome-wide association (GWAS) studies have identified multiple loci with small effects; however, many causal variants remain elusive.

We aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients.

The transcription factor p63 is a key mediator of epidermal development. Point mutations in p63 in patients lead to developmental defects, including orofacial clefting. To date, knowledge on how pivotal the role of p63 is in human craniofacial development is limited. Using an inducible transdifferentiation model, combined with epigenomic sequencing and multicohort meta-analysis of genome-wide association studies data, we show that p63 establishes enhancers at craniofacial development genes to modulate their transcription. Disease-specific substitution mutation in the DNA binding domain or sterile alpha motif protein interaction domain of p63, respectively, eliminates or reduces establishment of these enhancers. We show that enhancers established by p63 are highly enriched for single-nucleotide polymorphisms associated with nonsyndromic cleft lip ± cleft palate (CL/P). These orthogonal approaches indicate a strong molecular link between p63 enhancer function and CL/P, illuminating molecular mechanisms underlying this developmental defect and revealing vital regulatory elements and new candidate causative genes.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a frequent congenital malformation with multifactorial etiology. While recent genome-wide association studies (GWAS) have identified several nsCL/P risk loci, the functional effects of the associated non-coding variants are largely unknown. Furthermore, additional risk loci remain undetected due to lack of power. As genetic variants might alter binding of transcription factors (TF), we here hypothesized that the integration of data from TF binding sites, expression analyses and nsCL/P GWAS might help to (i) identify functionally relevant variants at GWAS loci, and (ii) highlight novel risk variants that have been previously undetected. Analysing the craniofacial TF TFAP2A in human embryonic palatal mesenchyme (HEPM) cells, we identified 2845 TFAP2A ChIP-seq peaks, several of which were located near nsCL/P candidate genes (e.g. MSX1 and SPRY2). Comparison with independent data suggest that 802 of them might be specific to craniofacial development, and genes near these peaks are enriched in processes relevant to nsCL/P. Integration with nsCL/P GWAS data, however, did not show robust evidence for co-localization of common nsCL/P risk variants with TFAP2A ChIP-seq peaks. This data set represents a new resource for the analyses of craniofacial processes, and similar approaches with additional cell lines and TFs could be applied to generate further insights into nsCL/P etiology.

Mutations in components of the major spliceosome have been described in disorders with craniofacial anomalies, e.g., Nager syndrome and mandibulofacial dysostosis type Guion-Almeida. The U5 spliceosomal complex of eight highly conserved proteins is critical for pre-mRNA splicing. We identified biallelic mutations in TXNL4A, a member of this complex, in individuals with Burn-McKeown syndrome (BMKS). This rare condition is characterized by bilateral choanal atresia, hearing loss, cleft lip and/or palate, and other craniofacial dysmorphisms. Mutations were found in 9 of 11 affected families. In 8 families, affected individuals carried a rare loss-of-function mutation (nonsense, frameshift, or microdeletion) on one allele and a low-frequency 34 bp deletion (allele frequency 0.76%) in the core promoter region on the other allele. In a single highly consanguineous family, formerly diagnosed as oculo-oto-facial dysplasia, the four affected individuals were homozygous for a 34 bp promoter deletion, which differed from the promoter deletion in the other families. Reporter gene and in vivo assays showed that the promoter deletions led to reduced expression of TXNL4A. Depletion of TXNL4A (Dib1) in yeast demonstrated reduced assembly of the tri-snRNP complex. Our results indicate that BMKS is an autosomal-recessive condition, which is frequently caused by compound heterozygosity of low-frequency promoter deletions in combination with very rare loss-of-function mutations.