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.

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.

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.

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.

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.