Three-dimensional assessment of the pharyngeal airway in Japanese preschoolers with orofacial clefts.

OBJECTIVES/HYPOTHESIS: Individuals with orofacial clefts often experience respiratory problems because of nasopharyngeal abnormalities. Pharyngeal airway morphology is thought to differ among the various cleft types. We measured three-dimensional (3D) airway volume using cone-beam computed tomography (CBCT) analysis to evaluate and compare pharyngeal airways in Japanese preschoolers with and without orofacial clefts. STUDY DESIGN: Retrospective case-control study. METHODS: We enrolled 83 subjects (37 boys, 46 girls; mean age = 4.66 ± 0.56 years) with nonsyndromic orofacial clefts and 16 noncleft healthy subjects (seven boys, nine girls; mean age = 5.30 ± 0.52 years) as controls. The subjects were divided into five groups. Four groups were based on the cleft type: isolated cleft palate, unilateral cleft lip and alveolus), unilateral cleft lip and palate, and bilateral cleft lip and palate. The fifth group included the noncleft controls. All subjects were examined with CBCT, and the 3D airway volume was measured. We analyzed group differences statistically using analysis of covariance with the Bonferroni post hoc pairwise comparison tests for the corrected means. RESULTS: Compared with the noncleft group, each cleft group exhibited significantly decreased total and nasal airway volumes and increased superior and inferior pharyngeal airway volumes. The differences were all statistically significant. CONCLUSIONS: Our findings suggest that anatomical differences exist in pharyngeal airway volumes among various cleft groups and in those without a cleft. LEVEL OF EVIDENCE: 3b Laryngoscope, 130:533-540, 2020.

Cdc42 regulates cranial suture morphogenesis and ossification.

Cdc42 (cell division cycle 42) is ubiquitously expressed small GTPases belonging to the Rho family of proteins. Previously, we generated limb bud mesenchyme-specific Cdc42 inactivated mice (Cdc42 conditional knockout mice; Cdc42 fl/fl; Prx1-Cre), which showed short limbs and cranial bone deformities, though the mechanism related to the cranium phenotype was unclear. In the present study, we investigated the role of Cdc42 in cranial bone development. Our results showed that loss of Cdc42 caused a defect of intramembranous ossification in cranial bone tissues which is related to decreased expressions of cranial suture morphogenesis genes, including Indian hedgehog (Ihh) and bone morphogenetic proteins (BMPs). These findings demonstrate that Cdc42 plays a crucial role in cranial osteogenesis, and is controlled by Ihh- and BMP-mediated signaling during cranium development.

Exploration of genetic factors determining cleft side in a pair of monozygotic twins with mirror-image cleft lip and palate using whole-genome sequencing and comparison of craniofacial morphology.

OBJECTIVE: The aim of the present study is to explore genetic factors determining difference of cleft side using whole-genome sequencing and evaluation of craniofacial morphology using cephalometric analysis between Japanese monozygotic (MZ) twins with mirror-image cleft lip and palate (CLP). DESIGN: We selected a Japanese MZ twin pair (MZ-A and MZ-B) affected with unilateral CLP who are discordant for cleft side (left/right) and conducted whole-genome sequencing to identify genetic factors determining cleft side. Moreover, we compared their craniofacial morphologies using cephalograms. RESULTS: Whole-genome sequencing results suggested that no discordant DNA variants were found between MZ-A and MZ-B. The comparison of craniofacial morphology between the MZ twins revealed that MZ-B had maxillary deficiency and slightly more mandibular protrusion than MZ-A. CONCLUSIONS: It is indicated that environmental factors might be a critical factor that influences the determination of difference of cleft side in orofacial clefts. In addition, we found some differences in craniofacial morphology between MZ-A and MZ-B. Our findings suggest that various environmental factors, such as epigenetics, might be a critical factor that influences the determination of difference of cleft side in CLP rather than inherited genetic factors.

Whole-genome sequencing in a pair of monozygotic twins with discordant cleft lip and palate subtypes.

OBJECTIVE: Orofacial clefts (OFCs) are common and etiologically complex birth defects. This study explored potential genetic differences in a pair of Japanese monozygotic (MZ) twins with different forms of OFC using whole-genome sequencing. SUBJECTS AND METHODS: One co-twin (MZ-1) presented with nonsyndromic bilateral cleft lip and palate; the other co-twin (MZ-2) had nonsyndromic bilateral cleft lip and unilateral left-sided cleft alveolus. Neither parent had an OFC. Craniofacial morphologic features and potential genetic differences were compared using standard cephalometry and whole-genome sequencing, respectively. RESULTS: Morphologically, MZ-1 had a smaller vertical mandibular height, compared to MZ-2. However, no discordant genetic differences were detected. Moreover, both twins and their parents harbored rare candidate gene variants (GRHL3; TPM1) considered to be associated with OFCs. CONCLUSION: The observed differences between MZ-1 and MZ-2 in craniofacial morphology assessed by cephalograms might be directly attributable to the effects of the OFC on growth and/or differences in surgical history, given the lack of any differences in genetic background. However, comparisons of discordant MZ twins should continue to identify novel candidates that might control OFC or that might partly explain the missing heritability for this common birth defect, in addition to understanding craniofacial growth and development.

Orthodontic Treatment and Maxillary Anterior Segmental Distraction Osteogenesis of a Subject with Williams-Beuren Syndrome and Isolated Cleft Palate: A Long-Term Follow-Up from the Age of 5 to 24 Years.

Williams-Beuren syndrome (WBS) is a rare multisystem disorder caused by a hemizygous deletion of the elastin gene on chromosome 7q11.23. WBS patients have characteristic skeletal features and dental anomalies accompanied by mental retardation, a friendly outgoing personality, and mild to moderate intellectual disability or learning problems. In this case report, we present the combined orthodontic and surgical treatment of a WBS patient with an isolated cleft palate through a long-term follow-up from the age of 5 to 24 years. During the period of active treatment, comprehensive orthodontic treatment combined with maxillary anterior segmental distraction osteogenesis and prosthetic treatment using dental implants were effective in dramatically improving the patient's malocclusion. The patient's mental abilities and the cooperation shown by the patient and her family were crucial for the success of this complex and long-term treatment course.

Expression of nephronectin is enhanced by 1α,25-dihydroxyvitamin D3.

The extracellular matrix protein nephronectin (Npnt), also called POEM, is considered to play critical roles as an adhesion molecule in development and functions of various tissues, such as the kidneys, liver, and bone. In the present study, we examined the molecular mechanism of Npnt gene expression and found that vitamin D3 (1α,25-dihydroxyvitamin D3,VD 3) strongly enhanced Npnt mRNA expression in MC3T3-E1 cells from a mouse osteoblastic cell line. The VD 3-induced increase in Npnt expression is both time- and dose-dependent and is mediated by the vitamin D receptor (VDR).

Rho GTPase protein Cdc42 is critical for postnatal cartilage development.

Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 (fl/fl); Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 (fl/fl)) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages.

Association of aging with gene expression profiling in mouse submandibular glands.

Aging, also called senescence, is thought to be a physiological phenomenon that commonly occurs in various organs and tissues (Enoki et al., 2007 [1]). Many older adults experience dysfunction in their salivary glands, for example xerostomia, which is defined as dry mouth resulting from reduced or absent saliva flow (Nagler et al., 2004 [2]). In the present study, we investigated gene expression in submandibular glands of young (8 weeks old) and adult (50 weeks old) mice to analyze association of aging with gene expression profiling in mouse submandibular glands. Whole-genome gene expression profiles were analyzed using an Illumina Sentrix system with Mouse-WG-6 v.2 Expression BeadChips (Illumina). Of the genes screened, 284 showed detection values at a significance level of P < 0.01. Among those, the expression of 94 genes (33%) showed a greater decrease in adult mice as compared to young mice. On the other hand, that of 190 genes (77%) was increased in the adults more than in young mice. The data obtained in this study are publicly available in the Gene Expression Omnibus (GEO) database (accession number GSE66857).

Cdc42 is critical for cartilage development during endochondral ossification.

Cdc42 is a widely expressed protein that belongs to the family of Rho GTPases and controls a broad variety of signal transduction pathways in a variety of cell types. To investigate the physiological functions of Cdc42 during cartilage development, we generated chondrocyte-specific inactivated Cdc42 mutant mice (Cdc42(fl/fl); Col2-Cre). The gross morphology of mutant neonates showed shorter limbs and body as compared with the control mice (Cdc42(fl/fl)). Skeletal preparations stained with alcian blue and alizarin red also revealed that the body and the long bone length of the mutants were shorter than those of the control mice. Furthermore, severe defects were found in growth plate chondrocytes in the femur sections of mutant mice, characterized by a reduced proliferating zone height, wider hypertrophic zone, and loss of columnar organization in proliferating chondrocytes. The expression levels of chondrocyte marker genes, such as Col2, Col10, and Mmp13, in mutant mice were decreased as compared with the control mice. Mineralization of trabecular bones in the femur sections was also decreased in the mutants as compared with control mice, whereas osteoid volume was increased. Together these results suggested that chondrocyte proliferation and differentiation in growth plates in the present mutant mice were not normally organized, which contributed to abnormal bone formation. We concluded that Cdc42 is essential for cartilage development during endochondral bone formation.

Temperature-sensitive nonionic vesicles prepared from Span 80 (sorbitan monooleate).

Different types of nonionic vesicles were prepared from commercial Span 80 (also called sorbitan monooleate), as an inexpensive, biocompatible alternative to conventional phospholipid-based vesicles (liposomes). The vesicles were characterized by different techniques and comparison was made with vesicles formed from POPC (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine) or DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine). Dynamic light scattering measurements, electron microscopy analyses, and two types of fusion assays indicate that Span 80 vesicles are stable for at least 7 days at 4 or 25 degrees C, while storage at 42 degrees C causes irreversible vesicle fusion. This indicates that Span 80 vesicles are thermoresponsive with vesicle fusion occurring at elevated temperature. This property may be related to headgroup dehydration and is certainly not directly linked to the phase transition temperature (Tm) of the vesicles, since the Tm is below -30 degrees C, as determined by differential scanning calorimetry (DSC). The measured Tm value for Span 80 vesicles is lower than in the case of DOPC or POPC, correlating with a higher fluidity of Span 80 vesicles as compared to POPC or DOPC vesicles, as determined with DPH (1,6-diphenyl-1,3,5-hexatriene) as fluorescent membrane probe. High fluidity correlates with increased leakage of entrapped water-soluble dye molecules. Addition of cholesterol and soybean phosphatidylcholine lowers the extent of leakage, allowing a tuning of the bilayer permeability.