Hideto Imura

Replication of genome wide association identified candidate genes confirm the role of common and rare variants in PAX7 and VAX1 in the etiology of nonsyndromic CL(P).

Following recent genome wide association studies (GWAS), significant genetic associations have been identified for several genes with nonsyndromic cleft lip with or without cleft palate (CL(P)). To replicate two of these GWAS signals, we investigated the role of common and rare variants in the PAX7 and VAX1 genes. TaqMan genotyping was carried out for SNPs in VAX1 and PAX7 and transmission disequilibrium test (TDT) was performed to test for linkage and association in each population. Direct sequencing in and around the PAX7 and VAX1 genes in 1,326 individuals of European and Asian ancestry was done. The TDT analysis showed strong associations with markers in VAX1 (rs7078160, P = 2.7E−06 and rs475202, P = 0.0002) in a combined sample of Mongolian and Japanese CL(P) case–parent triads. Analyses using parent‐of‐origin effects showed significant excess transmission of the minor allele from both parents with the effect in the mothers (P = 6.5E−05, OR (transmission) = 1.91) more striking than in the fathers (P = 0.004, OR (transmission) = 1.67) for VAX1 marker rs7078160 in the combined Mongolian and Japanese samples when all cleft types were combined. The rs6659735 trinucleotide marker in PAX7 was significantly associated with all the US cleft groups combined (P = 0.007 in all clefts and P = 0.02 in CL(P)). Eight rare missense mutations found in PAX7 and two rare missense mutations in VAX1. Our study replicated previous GWAS findings for markers in VAX1 in the Asian population, and identified rare variants in PAX7 and VAX1 that may contribute to the etiology of CL(P). Determining the role of rare variants clearly warrants further investigation.

Cleft lip and palate in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: a morphological in vivo study

ABSTRACT    It is well‐known that TCDD (2,3,7,8, tetrachloridedibenzo‐p‐dioxin) induces cleft palates (CPs) in pregnant C57BL mice. However, it is unclear if TCDD is a possible teratogen for cleft lip. We examined maxillofacial malformations including cleft lip in three animal strains: A/J mice, C57BL/6J mice and ICR mice. The A/J mouse develops cleft lip and palate spontaneously at a 5–10% rate. TCDD was administered in olive oil on gestation day (GD) 12.5 with gastric tubes at 10 µg/kg, 20 µg/kg, or 40 µg/kg to examine the dose–response, and on a single day from GD 8.5–14.5 to examine the timing effects of TCDD administration on lip and palate formation. Furthermore, the palatal shelf movements during GD 8.5–14.5 were observed with a stereoscopic microscope. All embryos had cleft palates when the TCDD was administered just before palatogenesis (GD11.5‐GD12.5). With respect to the TCDD effects, there were large differences among the strains. In the A/J mice, the difference between a lethal dose and a dose that could induce a cleft palate was close. Cleft lips were not induced, even when the TCDD was given just before labiogenesis. Morphologically, both palatal shelves contacted perfectly along their lengths, but separated and formed cleft palates. In conclusion, TCDD is a strong inducer of cleft palates, and interferes with the fusion phase of the secondary palate, but has no effect on the lip.

Morphological and immunohistochemical studies on cleft palates induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice

ABSTRACT  Morphological and immunohistological examinations were performed to reveal the mechanisms of cleft palate induction by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). ICR strain mice 8–10 weeks of age were used in the study. TCDD was administered in olive oil on gestation day (GD) 12.5 with gastric tubes at 40 μg/kg. From GD 13.5 to 16.5, palates were examined by scanning electron microscopy (SEM), hematoxyline–eosin (HE) staining, and immunohistochemical staining of FGFR1/2, TGF‐β3, MSX1 and LHX8. In the control group, both of the palatal shelves began elevating on GD 14.0 and finished within 6 h. After the elevation, all of the shelves had completely fused with each other on GD 14.5. In the TCDD‐treated group, palatal shelves elevated 1 day later than in the control group. However, all palates had elevated by GD 15.0. After the elevation, the shelves contacted each other and fused; however, they were separated on GD16.0. HE staining showed that medial edge epithelium (MEE) was thinner in the TCDD group than in the control group. MEE observed under a high magnification (×2500) exhibited filopodia‐like filaments and the cells were bulged in the control group. In contrast, in the TCDD group, no filaments were observed and the cells were flat with unclear boundaries. Immunohistologically, there were no characteristic findings except for FGFR1. FGFR1 was not expressed in the TCDD group after the fusion phase (GD 14.5). TCDD induces many morphological and molecular changes to MEE cells and causes cleft palates.

Osteomyelitis of the mandible secondary to infantile osteopetrosis: A case report

A case of infantile malignant osteopetrosis with refractory mandibular osteomyelitis is reported. A female child was diagnosed with osteopetrosis at 2 years 8 months of age, and was scheduled to receive a bone marrow transplantation (BMT) in a pediatric department at 3 years 11 months of age. Her lower incisors were extracted at a dental clinic, after which she had recurring abscesses with progressive severity. She was referred to our department to control local infections before the BMT and was diagnosed with chronic mandibular osteomyelitis caused by osteopetrosis. Sequestrotomy and curettage were performed under general anesthesia. After surgery, daily saline irrigation was continued and osteomyelitis was well controlled. An uneventful BMT was performed, although it was refused and failed. A second BMT was planned, but during chemotherapy, cellulitis occurred after a recurrence of osteomyelitis caused by a newly erupted tooth. The local infection was controlled, but pneumonia recurred. She ultimately died of respiratory failure at 5 years of age.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin suggests abnormal palate development after palatal fusion

Mouse embryos exposed to 2,3,7,8‐tetrachloridedibenzo‐p‐dioxin (TCDD) develop cleft palates and hydronephrosis. Cleft palates occur after TCDD exposure due to contact and/or fusion failure. We investigated whether cleft palate can be induced by dissociation of the palatine process after fusion. Pregnant mice on gestational day (GD) 12 were randomly divided into two groups: one group was administered through gastric tubes one dose of olive oil (control group) and the other group was administered one dose of TCDD diluted with olive oil, both at a dose of 40 µg/kg body weight. Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13–18) and the palatal form was observed using a stereoscopic microscope. In TCDD‐exposed embryos, palatal fusion was observed on GD 14, 15 and 16 and the incidence of cleft palate was 100% on GD 18. Fusion rates were 17.5 ± 15.2% and 12.4 ± 11.8% on GD 15 and 16, respectively. Some palates from the TCDD‐exposed mouse embryos showed clearly developed cleft palate after fusion of the lateral palatine processes during palatal formation. A mass of cells, which were chiefly epithelial in the fused palates was observed in the TCDD‐exposed mouse embryos. A decrease in E‐cadherin expression was observed in this mass of cells, indicating its involvement in the development of cleft palate.

Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Development of Murine Palate in Organ Culture

Abstract Objective : 2,3,7,8-Tetrachlorodibenzo- p -dioxin (tetrachlorodibenzodioxin) is known to cause cleft palates in pregnant mice in vivo. This study aimed at investigating the effects of tetrachlorodibenzodioxin on murine palatogenesis in vitro using organ culture. Materials and Methods : Palatal shelves from ICR mice foetuses (gestational day, 12.5) were removed and dissected to isolate the palatal tissue. Tissues were cultured in a chemically defined, serumless medium using a suspension culture technique. Palatal fusion rates were assessed in suspension cultures containing tetrachlorodibenzodioxin at concentrations of 0.1, 1, 10 and 50 ng/mL and in non-treated controls. Palates from the control and 10 ng/mL groups were stained immunohistologically with antibodies against cleft palate-related factors (transforming growth factor-beta3, muscle segment homeobox 1, Lim-homeobox 8, fibroblast growth factor receptor 1, fibroblast growth factor receptor 2). Results : Over 90% of the palatal shelves completely fused in suspension culture after careful removal of the neural tissues. With tetrachlorodibenzodioxin addition, the palatal fusion rate decreased to 50% at a concentration of 1 ng/mL, however, the rate did not decrease any further at higher concentrations. Immunohistologically, transforming growth factor-beta3 disappeared earlier in the tetrachlorodibenzodioxin group. Conclusions : Tetrachlorodibenzodioxin contributes to the pathogenesis of cleftpalate in vitro; however, direct intracellular effects are insufficient to fully accountfor the observed effects of tetrachlorodibenzodioxin, and it is possible that indirect mechanisms are also in play in vivo.

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on cleft palate-related genes in mouse embryonic palatal mesenchymal cells

Abstract Objective: To investigate the possible involvement of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the pathogenesis of cleft palate in mouse palatal cells. Materials and Methods: Palatal shelves of gestation day 12.5 ICR mouse foetuses were removed and cultured with serum. One day after the second passage, the medium was changed to serum free and 2,3,7,8-tetrachlorodibenzo-p-dioxin was added at 1 ng/mL, 10 ng/mL, and 100 ng/mL. After culture for 1 and 3 hours, all cellular RNA was isolated from the cells. Expression of some cleft palate-related genes was analysed semiquantitatively using reverse transcriptase-polymerase chain reaction. Five genes (Tgfb3, Msx1, Lhx8, Fgfrl, and Fgfr2) were selected on the basis that functional disturbance of these genes causes cleft palate with high frequency. The data were compared with GAPDH. Results: In the first passage, many mouse embryonic palatal mesenchymal cells had spread out, and a number of monolayer spindle-shaped cells were identified. The cells were stable in the second passage and also in the serum free medium. One hour after addition of 2,3,7,8-tetrachlorodibenzo-p-dioxin, Tgfb3 mRNA expression was reduced at a concentration of 100 ng/mL. After 3 hours, Tgfb3, Msxl, and Lhx8 mRNA expression were also reduced. However, there was no effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the gene expression for Fgfr1 and Fgfr2 mRNA. Conclusion: 2,3,7,8-Tetrachlorodibenzo-p-dioxin may contribute to the induction of cleftpalate by suppressing Tgfb3, Msx1, and Lhx8 gene expression.

Association of MEOX2 Polymorphism with Nonsyndromic Cleft Palate Only in a Vietnamese population

To evaluate the association between the single nucleotide polymorphism (SNP) rs227493 in the MEOX2 gene and nonsyndromic cleft palate only, this research was conducted as a case–control study by comparing a nonsyndromic cleft palate only group with an independent, healthy, and unaffected control group who were both examined by specialists. Based on clinical examination and medical records, we analyzed a total of 570 DNA samples, including 277 cases and 293 controls, which were extracted from dry blood spot samples collected from both the Odonto and Maxillofacial Hospital in Ho Chi Minh City and Nguyen Dinh Chieu Hospital in Ben Tre province, respectively. The standard procedures of genotyping the specific SNP (rs2237493) for MEOX2 were performed on a StepOne Realtime PCR system with TaqMan SNP Genotyping Assays. Significant statistical differences were observed in allelic frequencies (allele T and allele G) between the non‐syndromic cleft palate only and control groups in female subjects, with an allelic odds ratio of 1.455 (95% confidence interval: 1.026–2.064) and P < 0.05. These study findings suggest that nonsyndromic isolated cleft palate might be influenced by variation of MEOX2, especially SNP rs2237493 in Vietnamese females.

Homeobox family Hoxc localization during murine palate formation

Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words)

Relationships between nasalance scores and nasopharyngeal shapes in cleft palate patients.

OBJECTIVES The aim of the present study is to clarify the relationship between nasalance scores and nasopharyngeal shapes obtained by lateral cephalograms. PATIENTS Eight patients who underwent a Wardill-Kilner push-back palatoplasty were included in this study. Perceptual judgment by a speech pathologist indicated that these patients had no hypernasality and no nasal emission at blowing. As normal controls, 33 non-cleft individuals, 4 boys and 10 girls aged 6 years old and 5 boys and 14 girls aged 7 years old, were investigated. METHODS Lateral cephalograms at rest were taken for both groups. For the cleft (palate) patients, lateral cephalograms at phonation /a/ and blowing were analyzed and nasometries were also performed using a kitsutsuki passage. RESULTS AND CONCLUSION There was no significant difference in the velar length, the pharyngeal depth, the ratio of the velar length to the pharyngeal depth and the velar angle between the cleft patients and the non-cleft individuals. Multiple regression analyses indicated that standardized regression coefficients of ratios for the velar length to the pharyngeal depth and the velar ascent at blowing had higher nasalance scores for sentences 1 and 3, which had high coefficients of determination, respectively.