A potential osteogenic role for microRNA-181a-5p during palatogenesis.

BACKGROUND: In a previous study, we found that the highly conserved hsa-miR-181a-5p is downregulated in palatal fibroblasts of non-syndromic cleft palate-only infants. OBJECTIVES: To analyze the spatiotemporal expression pattern of mmu-miR-181a-5p during palatogenesis and identify possible mRNA targets and their involved molecular pathways. MATERIAL AND METHODS: The expression of mmu-miR-181a-5p was analyzed in the developing palates of mouse embryos from E11 to E18 using qPCR and ISH. Mouse embryonic palatal mesenchyme cells from E13 were used to analyze mmu-miR-181a-5p expression during osteogenic differentiation. Differential mRNA expression and target identification were analyzed using whole transcriptome RNA sequencing after transfection with a mmu-miR-181a-5p mimic. Differentially expressed genes were linked with underlying pathways using gene set enrichment analysis. RESULTS: The expression of mmm-miR-181a-5p in the palatal shelves increased from E15 and overlapped with palatal osteogenesis. During early osteogenic differentiation, mmu-miR-181a-5p was upregulated. Transient overexpression resulted in 49 upregulated mRNAs and 108 downregulated mRNAs (adjusted P-value < 0.05 and fold change > ± 1.2). Ossification (Stc1, Mmp13) and cell-cycle-related GO terms were significantly enriched for upregulated mRNAs. Analysis of possible mRNA targets indicated significant enrichment of Hippo signaling (Ywhag, Amot, Frmd6 and Serpine1) and GO terms related to cell migration and angiogenesis. LIMITATIONS: Transient overexpression of mmu-miR-181a-5p in mouse embryonic palatal mesenchyme cells limited its analysis to early osteogenesis. CONCLUSION: Mmu-miR-181-5p expression is increased in the developing palatal shelves in areas of bone formation and targets regulators of the Hippo signaling pathway.

Differential microRNA expression in cultured palatal fibroblasts from infants with cleft palate and controls.

Background: The role of microRNAs (miRNAs) in animal models of palatogenesis has been shown, but only limited research has been carried out in humans. To date, no miRNA expression study on tissues or cells from cleft palate patients has been published. We compared miRNA expression in palatal fibroblasts from cleft palate patients and age-matched controls. Material and Methods: Cultured palatal fibroblasts from 10 non-syndromic cleft lip and palate patients (nsCLP; mean age: 18 ± 2 months), 5 non-syndromic cleft palate only patients (nsCPO; mean age: 17 ± 2 months), and 10 controls (mean age: 24 ± 5 months) were analysed with next-generation small RNA sequencing. All subjects are from Western European descent. Sequence reads were bioinformatically processed and the differentially expressed miRNAs were technically validated using quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Results: Using RNA sequencing, three miRNAs (hsa-miR-93-5p, hsa-miR-18a-5p, and hsa-miR-92a-3p) were up-regulated and six (hsa-miR-29c-5p, hsa-miR-549a, hsa-miR-3182, hsa-miR-181a-5p, hsa-miR-451a, and hsa-miR-92b-5p) were down-regulated in nsCPO fibroblasts. One miRNA (hsa-miR-505-3p) was down-regulated in nsCLP fibroblasts. Of these, hsa-miR-505-3p, hsa-miR-92a, hsa-miR-181a, and hsa-miR-451a were also differentially expressed using RT-PCR with a higher fold change than in RNAseq. Limitations: The small sample size may limit the value of the data. In addition, interpretation of the data is complicated by the fact that biopsy samples are taken after birth, while the origin of the cleft lies in the embryonic period. This, together with possible effects of the culture medium, implies that only cell-autonomous genetic and epigenetic differences might be detected. Conclusions: For the first time, we have shown that several miRNAs appear to be dysregulated in palatal fibroblasts from patients with nsCLP and nsCPO. Furthermore, large-scale genomic and expression studies are needed to validate these findings.

Identification of a de novo variant in CHUK in a patient with an EEC/AEC syndrome-like phenotype and hypogammaglobulinemia.

The cardinal features of Ectrodactyly, Ectodermal dysplasia, Cleft lip/palate (EEC), and Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndromes are ectodermal dysplasia (ED), orofacial clefting, and limb anomalies. EEC and AEC are caused by heterozygous mutations in the transcription factor p63 encoded by TP63. Here, we report a patient with an EEC/AEC syndrome-like phenotype, including ankyloblepharon, ED, cleft palate, ectrodactyly, syndactyly, additional hypogammaglobulinemia, and growth delay. Neither pathogenic mutations in TP63 nor CNVs at the TP63 locus were identified. Exome sequencing revealed de novo heterozygous variants in CHUK (conserved helix-loop-helix ubiquitous kinase), PTGER4, and IFIT2. While the variant in PTGER4 might contribute to the immunodeficiency and growth delay, the variant in CHUK appeared to be most relevant for the EEC/AEC-like phenotype. CHUK is a direct target gene of p63 and encodes a component of the IKK complex that plays a key role in NF-κB pathway activation. The identified CHUK variant (g.101980394T>C; c.425A>G; p.His142Arg) is located in the kinase domain which is responsible for the phosphorylation activity of the protein. The variant may affect CHUK function and thus contribute to the disease phenotype in three ways: (1) the variant exhibits a dominant negative effect and results in an inactive IKK complex that affects the canonical NF-κB pathway; (2) it affects the feedback loop of the canonical and non-canonical NF-κB pathways that are CHUK kinase activity-dependent; and (3) it disrupts NF-κB independent epidermal development that is often p63-dependent. Therefore, we propose that the heterozygous CHUK variant is highly likely to be causative to the EEC/AEC-like and additional hypogammaglobulinemia phenotypes in the patient presented here.

Retinoic acid signalling in the development of the epidermis, the limbs and the secondary palate.

Retinoic acid (RA), the active derivative of vitamin A, is one of the major regulators of embryonic development, including the development of the epidermis, the limbs and the secondary palate. In the embryo, RA levels are tightly regulated by the activity of RA synthesizing and degrading enzymes. Aberrant RA levels due to genetic variations in RA metabolism pathways contribute to congenital malformations in these structures. In vitro and in vivo studies provide considerable evidence on the effects of RA and its possible role in the development of the epidermis, the limbs and the secondary palate. In conjunction with other regulatory factors, RA seems to stimulate the development of the epidermis by inducing proliferation and differentiation of ectodermal cells into epidermal cells. In the limbs, the exact timing of RA location and level is crucial to initiate limb bud formation and to allow chondrogenesis and subsequent osteogenesis. In the secondary palate, the correct RA concentration is a key factor for mesenchymal cell proliferation during palatal shelf outgrowth, elevation and adhesion, and finally to allow bone formation in the hard palate. These findings are highly relevant to understanding the mechanism of RA signalling in development and in the aetiology of specific congenital diseases.

Effects of Remote Ischemic Preconditioning on Heme Oxygenase-1 Expression and Cutaneous Wound Repair.

Skin wounds may lead to scar formation and impaired functionality. Remote ischemic preconditioning (RIPC) can induce the anti-inflammatory enzyme heme oxygenase-1 (HO-1) and protect against tissue injury. We aim to improve cutaneous wound repair by RIPC treatment via induction of HO-1. RIPC was applied to HO-1-luc transgenic mice and HO-1 promoter activity and mRNA expression in skin and several other organs were determined in real-time. In parallel, RIPC was applied directly or 24h prior to excisional wounding in mice to investigate the early and late protective effects of RIPC on cutaneous wound repair, respectively. HO-1 promoter activity was significantly induced on the dorsal side and locally in the kidneys following RIPC treatment. Next, we investigated the origin of this RIPC-induced HO-1 promoter activity and demonstrated increased mRNA in the ligated muscle, heart and kidneys, but not in the skin. RIPC did not change HO-1 mRNA and protein levels in the wound 7 days after cutaneous injury. Both early and late RIPC did not accelerate wound closure nor affect collagen deposition. RIPC induces HO-1 expression in several organs, but not the skin, and did not improve excisional wound repair, suggesting that the skin is insensitive to RIPC-mediated protection.

Systematic analysis of copy number variants of a large cohort of orofacial cleft patients identifies candidate genes for orofacial clefts.

Orofacial clefts (OFCs) represent a large fraction of human birth defects and are one of the most common phenotypes affected by large copy number variants (CNVs). Due to the limited number of CNV patients in individual centers, CNV analyses of a large number of OFC patients are challenging. The present study analyzed 249 genomic deletions and 226 duplications from a cohort of 312 OFC patients reported in two publicly accessible databases of chromosome imbalance and phenotype in humans, DECIPHER and ECARUCA. Genomic regions deleted or duplicated in multiple patients were identified, and genes in these overlapping CNVs were prioritized based on the number of genes encompassed by the region and gene expression in embryonic mouse palate. Our analyses of these overlapping CNVs identified two genes known to be causative for human OFCs, SATB2 and MEIS2, and 12 genes (DGCR6, FGF2, FRZB, LETM1, MAPK3, SPRY1, THBS1, TSHZ1, TTC28, TULP4, WHSC1, WHSC2) that are associated with OFC or orofacial development. Additionally, we report 34 deleted and 24 duplicated genes that have not previously been associated with OFCs but are associated with the BMP, MAPK and RAC1 pathways. Statistical analyses show that the high number of overlapping CNVs is not due to random occurrence. The identified genes are not located in highly variable genomic regions in healthy populations and are significantly enriched for genes that are involved in orofacial development. In summary, we report a CNV analysis pipeline of a large cohort of OFC patients and identify novel candidate OFC genes.

Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis.

PURPOSE: 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. METHODS: WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. After deleterious mutations were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls. RESULTS: We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) in LRP6, respectively, in the patient with TA and OFC and in the patient with severe TA only. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo. CONCLUSION: Mutations in LRP6 cause TA in humans.Genet Med 18 11, 1158-1162.

AGORA, a data- and biobank for birth defects and childhood cancer.

BACKGROUND: Research regarding the etiology of birth defects and childhood cancer is essential to develop preventive measures, but often requires large study populations. Therefore, we established the AGORA data- and biobank in the Netherlands. In this study, we describe its rationale, design, and ongoing data collection. METHODS: Children diagnosed with and/or treated for a structural birth defect or childhood cancer and their parents are invited to participate in the AGORA data- and biobank. Controls are recruited through random sampling from municipal registries. The parents receive questionnaires about demographics, family and pregnancy history, health status, prescribed medication, lifestyle, and occupational exposures before and during the index pregnancy. In addition, blood or saliva is collected from children and parents, while medical records are reviewed for diagnostic information. RESULTS: So far, we have collected data from over 6,860 families (3,747 birth defects, 905 childhood cancers, and 2,208 controls). The types of birth defects vary widely and comprise malformations of the digestive, respiratory, and urogenital tracts as well as facial, cardiovascular, kidney, skeletal, and central nervous system anomalies. The most frequently occurring childhood cancer types are acute lymphatic leukemia, Hodgkin and non-Hodgkin lymphoma, Wilms' tumor, and brain and spinal cord tumors. Our genetic and/or epidemiologic studies have been focused on hypospadias, anorectal malformations, congenital anomalies of the kidney and urinary tract (CAKUT), and orofacial clefts. CONCLUSION: The large AGORA data- and biobank offers great opportunities for investigating genetic and nongenetic risk factors for disorders in children and is open to collaborative initiatives. Birth Defects Research (Part A) 106:675-684, 2016. © 2016 Wiley Periodicals, Inc.

Identification of germline mutations in the cancer predisposing gene CDH1 in patients with orofacial clefts.

Orofacial clefts (OFC) are among the most common birth defects worldwide. The etiology of non-syndromic OFC is still largely unknown. During embryonic development, the cell adhesion molecule E-cadherin, encoded by CDH1, is highly expressed in the median edge epithelium of the palate. Furthermore, in multiple families with CDH1 mutations, OFC cases are observed. To determine whether CDH1 is a causative gene for non-syndromic OFC and to assess whether CDH1 mutation screening in non-syndromic OFC patients enables identification of families at risk of cancer, direct sequencing of the full coding sequence of CDH1 was performed in a cohort of 81 children with non-syndromic OFC. Eleven children had heterozygous CDH1 sequence variants, 5 cases with 4 distinct missense mutations and 8 cases with 4 intronic variants. Using a combination of in silico predictions and in vitro functional assays, three missense mutations in four non-syndromic OFC patients were predicted to be damaging to E-cadherin protein function. The intronic variants including one tested in an in vitro assay appeared to be benign, showing no influence on splicing. Functionally relevant heterozygous CDH1 missense mutations were found in 4 out of 81 (5%) patients with non-syndromic OFC. This finding opens a new pathway to reveal the molecular basis of non-syndromic OFC. Cancer risk among carriers of these mutations needs to be defined.

Delayed cutaneous wound closure in HO-2 deficient mice despite normal HO-1 expression.

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO-1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO-2 deficient mice is impaired with exorbitant inflammation and absence of HO-1 expression. This study addresses the role of HO-2 in cutaneous excisional wound healing using HO-2 knockout (KO) mice. Here, we show that HO-2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO-2 KO mice compared to WT controls. Surprisingly, wound closure in HO-2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO-1 induction in HO-2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C-X-C) ligand-11 (CXCL-11) in wounds of HO-2 KO mice. Abnormal regulation of CXCL-11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL-11 expression in HO-2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.

Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Genomic approaches for studying craniofacial disorders.

Fast developing technologies in genomics have driven genetic studies of human diseases from classical candidate approaches toward hypothesis-free and genome-wide screening methods. Compared to the low-resolution cytogenetic techniques that were the only available methods to visualize genomic changes at the chromosomal level until some 15 years ago, genome-wide studies including analyses of copy number variation (CNV), genome-wide association and linkage studies, and exome sequencing (ES) provide more accurate information for unraveling the genetic causes of diseases. Moreover, genome sequencing (GS) which interrogates the genome of a single individual at the nucleotide resolution has also been applied in genetic studies. Here we review genomic approaches in craniofacial disorders, with the emphasis on orofacial clefts, and discuss the applications, advantages, limitations, challenges, and future perspectives.

Targeting the redox balance in inflammatory skin conditions.

Reactive oxygen species (ROS) can be both beneficial and deleterious. Under normal physiological conditions, ROS production is tightly regulated, and ROS participate in both pathogen defense and cellular signaling. However, insufficient ROS detoxification or ROS overproduction generates oxidative stress, resulting in cellular damage. Oxidative stress has been linked to various inflammatory diseases. Inflammation is an essential response in the protection against injurious insults and thus important at the onset of wound healing. However, hampered resolution of inflammation can result in a chronic, exaggerated response with additional tissue damage. In the pathogenesis of several inflammatory skin conditions, e.g., sunburn and psoriasis, inflammatory-mediated tissue damage is central. The prolonged release of excess ROS in the skin can aggravate inflammatory injury and promote chronic inflammation. The cellular redox balance is therefore tightly regulated by several (enzymatic) antioxidants and pro-oxidants; however, in case of chronic inflammation, the antioxidant system may be depleted, and prolonged oxidative stress occurs. Due to the central role of ROS in inflammatory pathologies, restoring the redox balance forms an innovative therapeutic target in the development of new strategies for treating inflammatory skin conditions. Nevertheless, the clinical use of antioxidant-related therapies is still in its infancy.

Targeting fibroblast growth factor receptors causes severe craniofacial malformations in zebrafish larvae.

Background and Objective: A key pathway controlling skeletal development is fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling. Major regulatory functions of FGF signaling are chondrogenesis, endochondral and intramembranous bone development. In this study we focus on fgfr2, as mutations in this gene are found in patients with craniofacial malformations. The high degree of conservation between FGF signaling of human and zebrafish (Danio rerio) tempted us to investigate effects of the mutated fgfr2 sa10729 allele in zebrafish on cartilage and bone formation. Methods: We stained cartilage and bone in 5 days post fertilization (dpf) zebrafish larvae and compared mutants with wildtypes. We also determined the expression of genes related to these processes. We further investigated whether pharmacological blocking of all FGFRs with the inhibitor BGJ398, during 0-12 and 24-36 h post fertilization (hpf), affected craniofacial structure development at 5 dpf. Results: We found only subtle differences in craniofacial morphology between wildtypes and mutants, likely because of receptor redundancy. After exposure to BGJ398, we found dose-dependent cartilage and bone malformations, with more severe defects in fish exposed during 0-12 hpf. These results suggest impairment of cranial neural crest cell survival and/or differentiation by FGFR inhibition. Compensatory reactions by upregulation of fgfr1a, fgfr1b, fgfr4, sp7 and dlx2a were found in the 0-12 hpf group, while in the 24-36 hpf group only upregulation of fgf3 was found together with downregulation of fgfr1a and fgfr2. Conclusions: Pharmacological targeting of FGFR1-4 kinase signaling causes severe craniofacial malformations, whereas abrogation of FGFR2 kinase signaling alone does not induce craniofacial skeletal abnormalities. These findings enhance our understanding of the role of FGFRs in the etiology of craniofacial malformations.

Tooth agenesis patterns in bilateral cleft lip and palate.

Individuals with cleft lip and palate present significantly more dental anomalies, even outside the cleft area, than do individuals without clefts. Our aim was to evaluate the prevalence of tooth agenesis and patterns of hypodontia in a large sample of patients with complete bilateral cleft lip and palate (BCLP). Serial panoramic radiographs (the first radiograph was taken at 10.5-13.5 yr of age) of 240 patients with BCLP (172 male patients, 68 female patients) were examined. Third molars were not included in the evaluation. Agenesis of at least one tooth was present in 59.8% of patients. Upper laterals and upper and lower second premolars were missing most frequently. Using the tooth agenesis code (TAC), 52 different agenesis patterns were identified, of which simultaneous agenesis of 12, 22, 15, 25, 35, and 45 was the most frequent pattern. Nine of the 240 patients showed combined BCLP and oligodontia.

Retinoic acid disrupts osteogenesis in pre-osteoblasts by down-regulating WNT signaling.

The skull bones are formed by osteoblasts by intramembranous ossification. WNT signaling is a regulator of bone formation. Retinoic Acid (RA) act as a teratogen affecting craniofacial development. We evaluated the effects of RA on the differentiation and mineralization of MC-3T3 cells, and on the expression of WNT components. MC-3T3 were cultured with or without 0.5 µM RA in osteogenic medium and mineralization was assessed by alizarin red staining. The expression of osteogenic marker genes and WNT genes was evaluated at several time points up to 28 days. RA significantly inhibited MC-3T3 mineralization (p < 0.01), without affecting ALP activity or Alp gene expression. Both parameters gradually increased in time. During culture, RA stimulated Runx2 expression at 14 and 28 days compared to the respective controls (p < 0.05). Also, RA significantly reduced Sp7 expression at days 14 and 21 (p < 0.05). Simultaneously, RA significantly reduced the expression of the WNT genes cMyc, Lef1, Lrp5, Lrp6 and Wnt11 compared to the controls (p < 0.05). In contrast, RA increased the expression of the WNT inhibitors Dkk1 at day 21 and Dkk2 at days 14 and 21 (p < 0.01). Our data indicate that RA disrupts osteogenic differentiation and mineralization by inhibiting WNT signaling.

Deregulated Adhesion Program in Palatal Keratinocytes of Orofacial Cleft Patients.

Orofacial clefts (OFCs) are the most frequent craniofacial birth defects. An orofacial cleft (OFC) occurs as a result of deviations in palatogenesis. Cell proliferation, differentiation, adhesion, migration and apoptosis are crucial in palatogenesis. We hypothesized that deregulation of these processes in oral keratinocytes contributes to OFC. We performed microarray expression analysis on palatal keratinocytes from OFC and non-OFC individuals. Principal component analysis showed a clear difference in gene expression with 24% and 17% for the first and second component, respectively. In OFC cells, 228 genes were differentially expressed (p < 0.001). Gene ontology analysis showed enrichment of genes involved in ß1 integrin-mediated adhesion and migration, as well as in P-cadherin expression. A scratch assay demonstrated reduced migration of OFC keratinocytes (343.6 ± 29.62 µm) vs. non-OFC keratinocytes (503.4 ± 41.81 µm, p < 0.05). Our results indicate that adhesion and migration are deregulated in OFC keratinocytes, which might contribute to OFC pathogenesis.

Deletions and loss-of-function variants in TP63 associated with orofacial clefting.

We aimed to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy number variants were assessed in three OFC families using microarray analysis. Subsequently, we analyzed TP63 in a cohort of 1072 individuals affected with OFC and 706 population-based controls using molecular inversion probes (MIPs). We identified partial deletions of TP63 in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant c.569_576del (p.(Ala190Aspfs*5)) and a nonsense variant c.997C>T (p.(Gln333*)) that introduces a premature stop codon in the DNA-binding domain. In addition, we identified the first missense variants in the oligomerization domain c.1213G>A (p.(Val405Met)), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant-negative. All of these variants were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Our data indicate that loss-of-function alleles in TP63 can also give rise to OFC as the main phenotype. We have uncovered the dosage-dependent functions of p63, which were previously rejected.

MicroRNAs in Palatogenesis and Cleft Palate.

Palatogenesis requires a precise spatiotemporal regulation of gene expression, which is controlled by an intricate network of transcription factors and their corresponding DNA motifs. Even minor perturbations of this network may cause cleft palate, the most common congenital craniofacial defect in humans. MicroRNAs (miRNAs), a class of small regulatory non-coding RNAs, have elicited strong interest as key regulators of embryological development, and as etiological factors in disease. MiRNAs function as post-transcriptional repressors of gene expression and are therefore able to fine-tune gene regulatory networks. Several miRNAs are already identified to be involved in congenital diseases. Recent evidence from research in zebrafish and mice indicates that miRNAs are key factors in both normal palatogenesis and cleft palate formation. Here, we provide an overview of recently identified molecular mechanisms underlying palatogenesis involving specific miRNAs, and discuss how dysregulation of these miRNAs may result in cleft palate.

MSX1 mutations and associated disease phenotypes: genotype-phenotype relations.

The Msx1 transcription factor is involved in multiple epithelial-mesenchymal interactions during vertebrate embryogenesis. It has pleiotropic effects in several tissues. In humans, MSX1 variants have been related to tooth agenesis, orofacial clefting, and nail dysplasia. We correlate all MSX1 disease causing variants to phenotypic features to shed light on this hitherto unclear association. MSX1 truncations cause more severe phenotypes than in-frame variants. Mutations in the homeodomain always cause tooth agenesis with or without other phenotypes while mutations outside the homeodomain are mostly associated with non-syndromic orofacial clefts. Downstream effects can be further explored by the edgetic perturbation model. This information provides new insights for genetic diagnosis and for further functional analysis of MSX1 variants.