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.

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.

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.

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.

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.

Mechanical Stress Changes the Complex Interplay Between HO-1, Inflammation and Fibrosis, During Excisional Wound Repair.

Mechanical stress following surgery or injury can promote pathological wound healing and fibrosis, and lead to functional loss and esthetic problems. Splinted excisional wounds can be used as a model for inducing mechanical stress. The cytoprotective enzyme heme oxygenase-1 (HO-1) is thought to orchestrate the defense against inflammatory and oxidative insults that drive fibrosis. Here, we investigated the activation of the HO-1 system in a splinted and non-splinted full-thickness excisional wound model using HO-1-luc transgenic mice. Effects of splinting on wound closure, HO-1 promoter activity, and markers of inflammation and fibrosis were assessed. After seven days, splinted wounds were more than three times larger than non-splinted wounds, demonstrating a delay in wound closure. HO-1 promoter activity rapidly decreased following removal of the (epi)dermis, but was induced in both splinted and non-splinted wounds during skin repair. Splinting induced more HO-1 gene expression in 7-day wounds; however, HO-1 protein expression remained lower in the epidermis, likely due to lower numbers of keratinocytes in the re-epithelialization tissue. Higher numbers of F4/80-positive macrophages, αSMA-positive myofibroblasts, and increased levels of the inflammatory genes IL-1ß, TNF-α, and COX-2 were present in 7-day splinted wounds. Surprisingly, mRNA expression of newly formed collagen (type III) was lower in 7-day wounds after splinting, whereas, VEGF and MMP-9 were increased. In summary, these data demonstrate that splinting delays cutaneous wound closure and HO-1 protein induction. The pro-inflammatory environment following splinting may facilitate higher myofibroblast numbers and increase the risk of fibrosis and scar formation. Therefore, inducing HO-1 activity against mechanical stress-induced inflammation and fibrosis may be an interesting strategy to prevent negative effects of surgery on growth and function in patients with orofacial clefts or in patients with burns.

Further delineation of the KBG syndrome phenotype caused by ANKRD11 aberrations.

Loss-of-function variants in ANKRD11 were identified as the cause of KBG syndrome, an autosomal dominant syndrome with specific dental, neurobehavioural, craniofacial and skeletal anomalies. We present the largest cohort of KBG syndrome cases confirmed by ANKRD11 variants reported so far, consisting of 20 patients from 13 families. Sixteen patients were molecularly diagnosed by Sanger sequencing of ANKRD11, one familial case and three sporadic patients were diagnosed through whole-exome sequencing and one patient was identified through genomewide array analysis. All patients were evaluated by a clinical geneticist. Detailed orofacial phenotyping, including orthodontic evaluation, intra-oral photographs and orthopantomograms, was performed in 10 patients and revealed besides the hallmark feature of macrodontia of central upper incisors, several additional dental anomalies as oligodontia, talon cusps and macrodontia of other teeth. Three-dimensional (3D) stereophotogrammetry was performed in 14 patients and 3D analysis of patients compared with controls showed consistent facial dysmorphisms comprising a bulbous nasal tip, upturned nose with a broad base and a round or triangular face. Many patients exhibited neurobehavioural problems, such as autism spectrum disorder or hyperactivity. One-third of patients presented with (conductive) hearing loss. Congenital heart defects, velopharyngeal insufficiency and hip anomalies were less frequent. On the basis of our observations, we recommend cardiac assessment in children and regular hearing tests in all individuals with a molecular diagnosis of KBG syndrome. As ANKRD11 is a relatively common gene in which sequence variants have been identified in individuals with neurodevelopmental disorders, it seems an important contributor to the aetiology of both sporadic and familial cases.

Variability in dentofacial phenotypes in four families with WNT10A mutations.

This article describes the inter- and intra-familial phenotypic variability in four families with WNT10A mutations. Clinical characteristics of the patients range from mild to severe isolated tooth agenesis, over mild symptoms of ectodermal dysplasia, to more severe syndromic forms like odonto-onycho-dermal dysplasia (OODD) and Schöpf-Schulz-Passarge syndrome (SSPS). Recurrent WNT10A mutations were identified in all affected family members and the associated symptoms are presented with emphasis on the dentofacial phenotypes obtained with inter alia three-dimensional facial stereophotogrammetry. A comprehensive overview of the literature regarding WNT10A mutations, associated conditions and developmental defects is presented. We conclude that OODD and SSPS should be considered as variable expressions of the same WNT10A genotype. In all affected individuals, a dished-in facial appearance was observed which might be helpful in the clinical setting as a clue to the underlying genetic etiology.

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.