DeepFace: Deep-learning-based framework to contextualize orofacial-cleft-related variants during human embryonic craniofacial development.

Orofacial clefts (OFCs) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the "casual" variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson correlation coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50-0.83. Specifically, our model revealed that SNPs significantly associated with OFCs tended to exhibit higher SAD scores across various variant categories compared to less related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFCs. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFCs, and the model can be extended to study other complex diseases or traits.

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.

Fisura labio palatina: revisión de la literatura / Cleft lip and palate: literature review

La formación del paladar ocurre entre la quinta y undécima semana de vida intrauterina producto de la unión del paladar primario y secundario. Por otra parte, la formación del labio superior ocurre entre la quinta y sexta semana del desarrollo, y se configura en su parte media por la fusión de los procesos nasales mediales y lateralmente, a expensas de los procesos maxilares. La prevalencia de las fisuras labiales y/o fisura palatina varía según las distintas etnias, con cifras entre 0,7 hasta 1,1 casos por 1000 nacidos vivos. El objetivo de este trabajo fue realizar una revisión bibliográfica sobre aspectos epidemiológicos, mecanismos genéticos moleculares y ambientales que influyen en la ocurrencia de la fisura labial, fisura palatina y fisura labio palatina. La búsqueda bibliográfica se realizó en las bases de datos PUBMED, SCOPUS, SPRINGER, SCIENCEDIRECT utilizando los términos en inglés "cleft lip and palate", "cleft lip", "cleft palate" y "embriology". Entre los criterios de inclusión se consideraron estudios realizados en humanos y animales, publicados entre los años 2015 y 2021. La búsqueda arrojó un total de 407 trabajos, de los cuales tras un filtro por título y resumen quedaron un total de 38 artículos, en los cuales se realizó un análisis de texto completo para finalmente seleccionar 26 artículos que abarcan temas genéticos-moleculares, ambientales, epidemiológicos y sindrómicos. Además se incorporaron por búsqueda manual, 6 documentos asociados a libros de texto, y artículos científicos, sin considerar el criterio inclusión de tiempo. Dentro de esta revisión se describe la fuerte asociación entre las fisuras orales y las mutaciones de genes Msx1, sonic hedgehog, proteínas morfogenéticas óseas y factor de crecimiento fibroblástico durante la migración de las células de la cresta neural y la modelación y formación del paladar. La ausencia de ácido fólico durante el desarrollo del paladar y la presencia de hipoxia por exposición a humo, son los factores ambientales observados con mayor frecuencia en malformaciones orofaciales.

SUMMARY: Palate formation occurs between the fifth and eleventh week of intrauterine life as a result of the union of the primary and secondary palate. On the other hand, the formation of the upper lip occurs between the fifth and sixth week of development, and is configured in its middle part by the fusion of the medial and lateral nasal processes, at the expense of the maxillary processes. The prevalence of cleft lips and / or cleft palate varies according to the different ethnic groups, with figures ranging from 0.7 to 1.1 cases per 1000 live births. The aim of this work was to carry out a literature review on epidemiological aspects, molecular and environmental genetic mechanisms that influence the occurrence of cleft lip, cleft palate and its embriology. The literature search was carried out in the databases PUBMED, SCOPUS, SPRINGER, SCIENCEDIRECT using the English terms "cleft lip and palate", "cleft lip", "cleft palate" and "embryology". Inclusion criteria included studies carried out in humans and animals, published between 2015 and 2021. The search yielded a total of 407 works, of which after a filter by title and abstract, a total of 38 articles remained, in which a text analysis was carried out complete to finally select 26 articles that cover genetic-molecular, environmental, epidemiological and syndromic topics. In addition, 6 documents associated with textbooks and scientific articles were incorporated by manual search, without considering the inclusion criterion of time. This review describes the strong association between oral fissures and mutations of genes Msx1, sonic hedgehog, bone morphogenetic proteins and fibroblast growth factor during migration of neural crest cells and palate shaping and formation. Lack of folic acid during palae development dar and the presence of hypoxia due to exposure to smoke, are the environmental factors most frequently observed in orofacial malformations.

RERE deficiency contributes to the development of orofacial clefts in humans and mice.

Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7 and GRHL3. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH). Cleft lip has previously been described in one individual with NEDBEH. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest (CNC) cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox; Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of orofacial clefts in individuals with proximal 1p36 deletions and NEDBEH and that RERE expression in CNC cells and their derivatives is required for normal palatal development.

SPECC1L-deficient primary mouse embryonic palatal mesenchyme cells show speed and directionality defects.

Cleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.

Webpalestra: amamentação nos casos de fissura labiopalatina

Estamos comemorando o AGOSTO DOURADO! Veja mais vídeos sobre amamentação no link: https://www.youtube.com/playlist?list... Acesse os slides das nossas palestras na Biblioteca Virtual do Telessaúde ES! Confira a data da exibição e encontre o material desejado. Faça download e tenha o material preparado pelos nossos palestrantes. https://telessaude.ifes.edu.br/biblio...

An overview of cleft lip and palate.

Cleft lip and palate are types of craniofacial birth defects that affect thousands of children worldwide each year. These conditions are sensitive topics of conversations, often affected by the stigma of physical birth deformities and cultural myths. This article reviews the pathophysiology of cleft lip and palate, and describes the traditional management of patients with oral-facial clefts, including the extensive supportive care and an interprofessional team or cleft team approach that extends beyond the surgical correction.

Msx1 deficiency interacts with hypoxia and induces a morphogenetic regulation during mouse lip development.

Nonsyndromic clefts of the lip and palate are common birth defects resulting from gene-gene and gene-environment interactions. Mutations in human MSX1 have been linked to orofacial clefting and we show here that Msx1 deficiency causes a growth defect of the medial nasal process (Mnp) in mouse embryos. Although this defect alone does not disrupt lip formation, Msx1-deficient embryos develop a cleft lip when the mother is transiently exposed to reduced oxygen levels or to phenytoin, a drug known to cause embryonic hypoxia. In the absence of interacting environmental factors, the Mnp growth defect caused by Msx1 deficiency is modified by a Pax9-dependent 'morphogenetic regulation', which modulates Mnp shape, rescues lip formation and involves a localized abrogation of Bmp4-mediated repression of Pax9 Analyses of GWAS data revealed a genome-wide significant association of a Gene Ontology morphogenesis term (including assigned roles for MSX1, MSX2, PAX9, BMP4 and GREM1) specifically for nonsyndromic cleft lip with cleft palate. Our data indicate that MSX1 mutations could increase the risk for cleft lip formation by interacting with an impaired morphogenetic regulation that adjusts Mnp shape, or through interactions that inhibit Mnp growth.

Cleft lip and cleft palate in Esrp1 knockout mice is associated with alterations in epithelial-mesenchymal crosstalk.

Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1-/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients.

Prenatal Imaging for Cleft Lip and Palate.

Cleft lip (CL) and cleft palate (CP) are common orbitofacial birth defects that vary in severity and effect on anatomical function. They might occur as an isolated event, be associated with a syndrome, or result from genetic and environmental factors. Diagnosis and treatment of individuals begin prenatally and can extend through adulthood. Sonography and magnetic resonance imaging are used prenatally to evaluate and monitor treatment outcomes and complications associated with CL and CP. This article discusses the anatomical development of CL and CP and the imaging techniques used to diagnose and manage them.

The molecular anatomy of mammalian upper lip and primary palate fusion at single cell resolution.

The mammalian lip and primary palate form when coordinated growth and morphogenesis bring the nasal and maxillary processes into contact, and the epithelia co-mingle, remodel and clear from the fusion site to allow mesenchyme continuity. Although several genes required for fusion have been identified, an integrated molecular and cellular description of the overall process is lacking. Here, we employ single cell RNA sequencing of the developing mouse face to identify ectodermal, mesenchymal and endothelial populations associated with patterning and fusion of the facial prominences. This analysis indicates that key cell populations at the fusion site exist within the periderm, basal epithelial cells and adjacent mesenchyme. We describe the expression profiles that make each population unique, and the signals that potentially integrate their behaviour. Overall, these data provide a comprehensive high-resolution description of the various cell populations participating in fusion of the lip and primary palate, as well as formation of the nasolacrimal groove, and they furnish a powerful resource for those investigating the molecular genetics of facial development and facial clefting that can be mined for crucial mechanistic information concerning this prevalent human birth defect.

Prenatal diagnosis of cleft lip and cleft lip palate - a case series.

AIM: Orofacial clefts comprise cleft lip (CL) or cleft lip-palate (CLP) and are the most frequently encountered malformation of the facial region, accounting for approximately 1-2.2/1,000 live births. The aim of this study was to reveal the particularities regarding the prenatal diagnosis of orofacial clefts in a series of 11 cases diagnosed in a tertiary center.Material and methods: The study was performed in a tertiary diagnostic center for a period of 8 years (January 2010 - December 2017), on8125 patients that were assessed for screening or suspicion of malformations. RESULTS: During the assessed period a number of 11 fetuses (0.13%) were diagnosed by 2D and 3D ultrasound with CL (4 cases) or CLP (7 cases). The smallest gestational age at diagnosis was of 14 weeks, whereas the highest was 35 weeks. Of the 7 cases diagnosed with CLP, 4 presented also other associated anomalies that involved the central nervous system, the kidney, the skeleton and the stomach. All 4 cases of CL had identifiable associated anomalies. Termination of pregnancy was encountered in 3 cases with CLP. CONCLUSIONS: CLP can be diagnosed even at the end of the 1st trimester of pregnancy. CL is usually diagnosed during the 2nd trimester ultrasound exam and is commonly an isolated anomaly.

[Genetic analysis of 100 fetuses with cleft lip with or without palate].

OBJECTIVE: To explore the genetic basis for fetuses with cleft lip and palate. METHODS: For 100 fetuses diagnosed with cleft lip with or without palate, G-banding chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out on chorionic villi, amniotic fluid or cordocentesis samples. RESULTS: No genomic abnormality was found among 49 fetuses with isolated cleft lip and palate, while 12 genomic aberrations were found among 51 fetuses with syndromic cleft lip and palate, which included 4 cases with trisomy 13, 2 cases with trisomy 18, 1 with X chromosome aneuploidy, 2 with other chromosomal aneuploidies and 3 with pathogenic CNVs. CONCLUSION: The incidence of genomic abnormalities in fetuses with cleft lip and palate was high. In addition to chromosomal abnormalities, attention should also be paid to pathogenic CNVs.

A score-based method for quality control of fetal hard palate assessment during routine second-trimester ultrasound examination.

INTRODUCTION: When an orofacial cleft lip is discovered, precise characterization of this malformation is necessary, especially the extension of this cleft to the secondary palate. We aimed to develop and evaluate the feasibility/reproducibility of a score-based quality control for the visualization of the fetal hard palate during the second-trimester scan. MATERIAL AND METHODS: All ultrasound images of fetal hard palate assessed routinely during second-trimester scan were retrospectively retrieved for a 6-month period. One hundred of these images were randomly selected and analyzed by two blinded reviewers, according to a scoring system (0-6 points). Criteria retained in the score were complete palate bone horizontal plate, presence of two pterygoid processes, visible alveolar ridge, and horizontal axis of insonation. A score ≥4 defined images of good quality. Inter- and intra-reviewer reproducibility was assessed. RESULTS: Inter-reviewer reproducibility was excellent with significant correlation (Pearson coefficient 0.953; P < .0001), global adjusted κ coefficient (0.86, 95% CI 0.79-0.94) and individual criteria adjusted κ coefficient always > 0.8. Rates of images of good quality (score ≥ 4) were 75%-77%, also with excellent agreement (κ coefficient 0.89, 95% CI 0.79-0.99). Intra-reviewer reproducibility retrieved the same results (excellent agreement) except for the axis of insonation (satisfactory agreement). CONCLUSIONS: This simple image scoring system for the fetal palate is easy, has excellent inter- and intra-reviewer reproducibility and could also help sonographers to correctly identify the palate structure.

Assessing the association between hypoxia during craniofacial development and oral clefts.

Objectives To evaluate the association between hypoxia during embryo development and oral clefts in an animal model, and to evaluate the association between polymorphisms in the HIF-1A gene with oral clefts in human families. Material and Methods The study with the animal model used zebrafish embryos at 8 hours post-fertilization submitted to 30% and 50% hypoxia for 24 hours. At 5 days post-fertilization, the larvae were fixed. The cartilage structures were stained to evaluate craniofacial phenotypes. The family-based association study included 148 Brazilian nuclear families with oral clefts. The association between the genetic polymorphisms rs2301113 and rs2057482 in HIF-1A with oral clefts was tested. We used real time PCR genotyping approach. ANOVA with Tukey's post-test was used to compare means. The transmission/disequilibrium test was used to analyze the distortion of the inheritance of alleles from parents to their affected offspring. Results For the hypoxic animal model, the anterior portion of the ethmoid plate presented a gap in the anterior edge, forming a cleft. The hypoxia level was associated with the severity of the phenotype (p<0.0001). For the families, there was no under-transmitted allele among the affected progeny (p>0.05). Conclusion Hypoxia is involved in the oral cleft etiology, however, polymorphisms in HIF-1A are not associated with oral clefts in humans.

Subphenotyping and Classification of Cleft Lip and Alveolus in Adult Unoperated Patients: A New Embryological Approach.

OBJECTIVE: Previously, a new embryological classification was introduced subdividing oral clefts into fusion and/or differentiation defects. This subdivision was used to classify all subphenotypes of cleft lip with or without alveolus (CL±A). Subsequently, it was investigated whether further morphological grading of incomplete CLs is clinically relevant, and which alveolar part is deficient in fusion/differentiation defects. DESIGN: Observational cohort study. SETTING: Three hundred fifty adult unoperated Indonesian cleft patients presented themselves for operation. Cephalograms, dental casts, and intraoral and extraoral photographs-eligible for the present study-were used to determine morphological severity of CL±A. PATIENTS: Patients with unilateral or bilateral clefts of the primary palate only were included. MAIN OUTCOME MEASURES: Clefts were classified-according to developmental mechanisms and timing in embryogenesis-as fusion and/or differentiation defects. Grades of incomplete CLs were related to the severity of alveolar clefts (CAs) and hypoplasia, and permanent dentition was used to investigate which alveolar part is deficient in fusion/differentiation defects. RESULTS: One hundred eight adult patients were included. All subphenotypes-96 unilateral and 12 bilateral clefts-could be classified into differentiation (79%), fusion (17%), fusion-differentiation (2%), or fusion and differentiation (2%) defects. The various grades of incomplete CLs were related to associated CAs and hypoplasia, and all alveolar deformities were located in the premaxillae. CONCLUSIONS: This study showed that all CL±A including the Simonart bands can be classified, that further morphological grading of incomplete CLs is clinically relevant, and that the premaxilla forms the deficient part in alveolar deformities.

Face morphogenesis is promoted by Pbx-dependent EMT via regulation of Snail1 during frontonasal prominence fusion.

Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.