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.

Concurrent de novo ZFHX4 variant and 16q24.1 deletion in a patient with orofacial clefting; a potential role of ZFHX4 and USP10.

A girl with a unilateral cleft lip, alveolus and palate, tooth agenesis, and mild dysmorphic features, without a specific underlying syndrome diagnosis, was genotypically characterized and phenotypically described. Cleft gene panel analysis, single-nucleotide polymorphism (SNP) array, whole genome sequencing (WGS), whole exome sequencing, and quantitative PCR (Q-PCR) analysis were used as diagnostic tests. SNP array revealed a maternal deletion at 16q24.1, encompassing the cleft candidate gene USP10. WES revealed an additional de novo Loss-of-Function variant (p.(Asn838fs)) in the Zinc-Finger-Homeobox-4 (ZFHX4) gene. Q-PCR was performed to explore the effect of the ZFHX4 variant and the deletion in 16q24.1. The mRNA expression of a selection of putative target genes involved in orofacial clefting showed a lowered expression of USP10 (52%), CRISPLD2 (31%), and CRISPLD1 (1%) compared to the control. IRF6 showed no difference in gene expression. This case supports ZFHX4 as a novel cleft gene and suggests USP10 may contribute to the etiology of orofacial clefts in humans.

Excess vitamin a might contribute to submucous clefting by inhibiting WNT-mediated bone formation.

OBJECTIVES: Cleft lip and/or palate (CLP) is a common craniofacial birth defect caused by genetic as well as environmental factors. The phenotypic spectrum of CLP also includes submucous clefts with a defect in the palatal bone. To elucidate the contribution of vitamin A, we evaluated the effects of the vitamin A metabolite all-trans retinoic acid (ATRA) on the osteogenic differentiation and mineralization of mouse embryonic palatal mesenchymal cells (MEPM). SETTING AND SAMPLE POPULATION: MEPM cells were isolated from the prefusion palates of E13 mouse embryos from three different litters. MATERIALS AND METHODS: MEPM cells were cultured with and without 0.5 µM ATRA in osteogenic medium. Differentiation was analysed by the expression of osteogenic marker genes and alkaline phosphatase (ALP) activity after 1, 2, and 7 days. The expression of Wnt marker genes was also analysed. Mineralization was assessed by alizarin red staining after 7, 14, 21, and 28 days. RESULTS: The bone marker genes Sp7, Runx2, Alpl, and Col1a1 were inhibited 10% ± 2%, 59% ± 7%, 79% ± 12% and 57% ± 20% (P < .05) at day 7. ALP activity was inhibited at days 1 and 7 by 35 ± 0% (P < .05) and 23 ± 6% (P < .001). ATRA also inhibited mineralization at 3 and 4 weeks. Finally, expression of the universal Wnt marker gene Axin2 was strongly reduced, by 31 ± 18% (P < .001), at day 7. CONCLUSION: Our data indicate that ATRA (vitamin A) inhibits bone formation by reducing Wnt signalling. This might contribute to the molecular aetiology of submucous clefting.

Retinoic acid effects on in vitro palatal fusion and WNT signaling.

Retinoic acid is the main active vitamin A derivate and a key regulator of embryonic development. Excess of retinoic acid can disturb palate development in mice leading to cleft palate. WNT signaling is one of the main pathways in palate development. We evaluated the effects of retinoic acid on palate fusion and WNT signaling in in vitro explant cultures. Unfused palates from E13.5 mouse embryos were cultured for 4 days with 0.5 µM, 2 µM or without retinoic acid. Apoptosis, proliferation, WNT signaling and bone formation were analyzed by histology and quantitative PCR. Retinoic acid treatment with 0.5 and 2.0 µM reduced palate fusion from 84% (SD 6.8%) in the controls to 56% (SD 26%) and 16% (SD 19%), respectively. Additionally, 2 µM retinoic acid treatment increased Axin2 expression. Retinoic acid also increased the proliferation marker Pcna as well as the number of Ki-67-positive cells in the palate epithelium. At the same time, the WNT inhibitors Dkk1, Dkk3, Wif1 and Sfrp1 were downregulated at least two-fold. Retinoic acid also down-regulated Alpl and Col1a2 gene expression. Alkaline phosphatase (ALP) activity was notably reduced in the osteogenic areas of the retinoic acid- treated palates. Our data suggest that retinoic acid impairs palate fusion and bone formation by upregulation of WNT signaling.

Highly variable rate of orthodontic tooth movement measured by a novel 3D method correlates with gingival inflammation.

OBJECTIVES: Individual orthodontic treatment duration is hard to predict. Individual biological factors are amongst factors influencing individual rate of orthodontically induced tooth movement (OTM). The study aim is to determine the rate of OTM by a novel 3D method and investigate parameters that may predict the rate of tooth movement. MATERIALS AND METHODS: In this prospective cohort study, rate of OTM was determined from 90 three-dimensional intra-oral scans in 15 patients (aged 12-15) undergoing orthodontic treatment. For each patient, intra-oral scans were taken every week for up to 6 weeks (T0-T5). The teeth were segmented from the scans and the scans were superimposed on the palatal rugae. The rate of OTM was calculated for each tooth. Other parameters were gingival inflammation, contact-point displacement and the biological markers, matrix metalloproteinases (MMP), MMP-9 and MMP-2 in gingival crevicular fluid (GCF). RESULTS: Our study showed a high variation in the rate of OTM, varying from 0.15 to 1.24 mm/week. Teeth in the anterior segment tended to move more compared with the posterior segment. The contact point displacement and gingival inflammation varied greatly amongst the patients. The MMPs measured did not correlate with tooth movement. However, the gingival inflammation index showed a significant correlation with OTM. Future studies should include other biological markers related to bone-remodeling. CONCLUSION: This novel and efficient 3D method is suitable for measuring OTM and showed large individual variation in rate of OTM. CLINICAL RELEVANCE: Patients show different rates of OTM. The rate of OTM in an individual patient can provide guidance in timing of follow-up appointments.

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.

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.

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.

Effects of retinoic acid on proliferation and gene expression of cleft and non-cleft palatal keratinocytes.

BACKGROUND: Retinoic acid (RA) is a key regulator of embryonic development and linked to several birth defects including cleft lip and palate (CLP). The aim was to investigate the effects of RA on proliferation and gene expression of human palatal keratinocytes (KCs) in vitro. METHODS: KCs from children with and without CLP were cultured with 2 and 5 µM RA. Proliferation was measured by quantification of DNA after 2, 4, 6, and 8 days. In addition, we analysed the effects of RA on messenger RNA expression of genes for proliferation, differentiation, apoptosis, and RA receptors. RESULTS: RA similarly inhibited proliferation of palatal KC from cleft and non-cleft subjects. The proliferation of KCs from cleft subjects was reduced to 59.8±13.4% (2 µM) and 41.5±14.0% (5 µM, Day 6), while that of cells from age-matched non-cleft subjects was reduced to 66.9±12.1% (2 µM) and 33.9±10.1% (5 µM). RA treatment reduced the expression of several of the investigated genes; the proliferating cell nuclear antigen (PCNA) was reduced in CLP KCs only. Keratins 10 and 16 were downregulated in keratinocytes from both cleft and non-cleft subjects. P63, a master regulator for epithelial differentiation, was only downregulated in KCs from cleft subjects, as was the RXRa receptor. Two P63 target genes (GJB6 and DLX5) were strongly downregulated by RA in all cell lines. None of the apoptosis genes was affected. CONCLUSION: Overall, RA similarly inhibits proliferation of palatal KCs from cleft and non-cleft subjects and reduces the expression of specific genes.

Collagenolytic fragments and active gelatinase complexes in periodontitis.

BACKGROUND: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). METHODS: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. RESULTS: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. CONCLUSIONS: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.

Mice lacking leukocyte common antigen-related (LAR) protein tyrosine phosphatase domains demonstrate spatial learning impairment in the two-trial water maze and hyperactivity in multiple behavioural tests.

Leukocyte common antigen-related (LAR) protein is a cell adhesion molecule-like receptor-type protein tyrosine phosphatase. We previously reported that in LAR tyrosine phosphatase-deficient (LAR-Delta P) mice the number and size of basal forebrain cholinergic neurons as well as their innervation of the hippocampal area was reduced. With the hippocampus being implicated in behavioural activity aspects, including learning and memory processes, we assessed possible phenotypic consequences of LAR phosphatase deficiency using a battery of rodent behaviour tests. Motor function and co-ordination tests as well as spatial learning ability assays did not reveal any performance differences between wildtype and LAR-Delta P mice. A spatial learning impairment was found in the difficult variant of the Morris water maze. Exploration, nestbuilding and activity tests indicated that LAR-Delta P mice were more active than wildtype littermates. The observed hyperactivity in LAR-Delta P mice could not be explained by altered anxiety or curiosity levels, and was found to be persistent throughout the nocturnal period. In conclusion, behavioural testing of the LAR-Delta P mice revealed a spatial learning impairment and a significant increase in activity.