Mutation analysis in patients with nonsyndromic tooth agenesis using exome sequencing.

BACKGROUND: Tooth agenesis (TA) is a congenital abnormality that may present as syndromic or nonsyndromic. Considering its complex genetic aetiology, the aim of this study was to uncover the pathogenic mutants in patients with nonsyndromic TA and analyse the characteristics of these mutants. METHODS: Exome sequencing was performed to detect pathogenic variants in 72 patients from 43 unrelated families with nonsyndromic TA. All candidate variants were validated using Sanger sequencing. Bioinformatics and conformational analyses were performed to determine the pathogenic mechanisms of the mutants. RESULTS: The following eight mutations (six novel and two known) in six genes were identified in eight families: WNT10A [c.742C > T (p.R248*)], LRP6 [c.1518G > A (p.W506*), c.2791 + 1G > T], AXIN2 [c.133_134insGCCAGG (p.44_45insGQ)], PAX9 [c.439C > T (p.Q147*), c.453_454insCCAGC (p.L154QfsTer60)], MSX1 [c.603_604del (p.A203GfsTer10)] and PITX2 [c.522C > G (p.Y174*)]. Bioinformatics and conformational analyses showed that the protein structures were severely altered in these mutants, and indicated that these structural abnormalities may cause functional disabilities. CONCLUSIONS: Our study extends the mutation spectrum in patients with nonsyndromic TA and provides valuable data for genetic counselling. The pathogenic mechanisms of TA in patients/families with unknown causative variants need to be explored further.

The introduction of team-based learning into the clinical pharmacology section of the endodontics clinical course.

Team-based learning (TBL) has been widely applied and evaluated to produce better student outcomes. TBL has been introduced into the clinical pharmacology section of the endodontics clinical course at the School of Stomatology, Wuhan University since 2021. Here, the teaching experience in this course was summarized. The TBL course consisted of a knowledge assignment, intrateam and interteam discussion, practicing, evaluation, cases discussion and examination. The topics of the TBL class included cavity preparation and filling for treatment of dental caries, disinfection, and shaping and filling of root canal for root canal therapy. A total of 64 students participated in the TBL course. The students completed course work and hands-on practice to the satisfaction of the instructor. Furthermore, most participants held positive attitudes toward the TBL course because TBL provided the opportunity for teamwork to enable them to acquire and understand the therapeutic drug and material more quickly and made them more confident in the following practice. Our experience suggested that the application of the TBL contributed to the authentic practice of the endodontics clinical course.

Mycn deficiency underlies the development of orofacial clefts in mice and humans.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common subphenotype of non-syndromic orofacial clefts arising from genetic and/or environmental perturbations during embryonic development. We previously identified 2p24.2 as a risk locus associated with NSCL/P in the Chinese Han population, and MYCN is a candidate risk gene in this region. To understand the potential function of MYCN in craniofacial development, we generated Wnt1-Cre;Mycnflox/flox mice that exhibited cleft palate, microglossia and micrognathia, resembling the Pierre Robin sequence (PRS) in humans. Further analyses indicated that the cleft palate was secondary to the delayed elevation of palatal shelves caused by micrognathia. The micrognathia resulted from impaired chondrogenic differentiation in Merkel's cartilage, which limited tongue development, leading to microglossia. In terms of mechanism, Mycn deficiency in cranial neural crest cells (CNCCs) downregulated Sox9 expression by inhibiting Wnt5a in a CNCC-derived chondrogenic lineage in Merkel's cartilage. To investigate whether MYCN deficiency contributed to NSCL/P, we performed direct sequencing targeting all exons and exon-intron boundaries of MYCN in 104 multiplex families with Mendelian NSCL/P and identified a novel pathogenic variant in MYCN. Taken together, our data indicate that ablation of Mycn in mouse CNCCs could resemble PRS by suppressing the Wnt5a-Sox9 signaling pathway in Merkel's cartilage and that mutations in MYCN may be novel potential causes of NSCL/P.

Novel IRF6 mutations in Chinese Han families with Van der Woude syndrome.

BACKGROUND: Interferon Regulatory Factor 6 (IRF6) gene encodes a member of the IRF family of transcription factors. Mutations in IRF6 cause Van der Woude Syndrome (VWS), which is the most common malformation of syndromic orofacial clefts in humans. METHODS: Here, we performed sequencing studies of six families with VWS in the Chinese Han population. The entire IRF6-coding region and the exon-intron boundaries including exons 3-8 and part of exon 9 were screened among all the collected family members by Sanger sequencing. RESULTS: We found a novel splice site variant c.175-6T>A, two novel missense variants (p.Lys66Arg and p.Pro107Thr), in addition with a previously reported missense variant (p.Leu87Phe), which were all located in and nearby exon 4 of IRF6. Meanwhile, a novel frameshift variant p.G257Vfs*46 in exon 7 of IRF6 was also detected. All the mutations presented to be co-segregated in each family. CONCLUSION: Our study has advanced the understanding of the genetic architecture of VWS and provides the basis for genetic counseling, antenatal diagnosis, and gene therapy of high risk groups.

Two novel mutations in MSX1 causing oligodontia.

Tooth agenesis is one of the most common developmental anomalies in humans and can affect dental occlusion and speech pronunciation. Research has identified an association between mutations in MSX1, PAX9, EDA, AXIN2, WNT10A, WNT10B and LRP6 and human tooth agenesis. Two unrelated individuals with non-syndromic tooth agenesis and their families were enrolled in this study. Using Sanger sequencing of the candidate genes, we identified two novel mutations: a missense mutation c.572 T>C and a frameshift mutation c.590_594 dup TGTCC, which were both detected in the homeodomain of MSX1. After identifying the mutations, structural modeling and bioinformatics analysis were used to predict the resulting conformational changes in the MSX1 homeodomain. Combined with 3D-structural analysis of other MSX1 mutations, we propose that there is a correlation between the observed phenotypes and alterations in hydrogen bond formation, thereby potentially affecting protein binding.

Elevated TARP promotes proliferation and metastasis of salivary adenoid cystic carcinoma.

OBJECTIVE: This study aims to analyze the expression of T-cell receptor γ chain alternate reading frame protein (TARP) in salivary adenoid cystic carcinoma (SACC) and its distant metastases and to investigate its influences on the development and progression of SACC. STUDY DESIGN: TARP expression was analyzed in 50 primary SACCs, 13 specimens of metastatic adenoid cystic carcinoma of salivary gland origin, and 20 noncancerous tissues around SACC via immunohistochemistry. Cell Counting Kit-8 tests, wound healing assay, and Transwell experiments were performed to evaluate the effects of lentivirus-mediated TARP overexpression on the proliferation, migration, and invasion of SACC cells. RESULTS: TARP expression was significantly increased in primary SACCs compared with adjacent noncancerous tissues, and this increase was further enhanced in metastases compared with primary SACCs. The expression level of TARP correlated significantly with tumor size, tumor-node-metastasis stage, perineural invasion, histologic type, and distant metastasis. Furthermore, TARP overexpression promoted the growth, migration, and invasion of SACC cells. CONCLUSIONS: TARP plays an important role in and may be used as a marker to indicate the development and progression of SACC.

Functional analysis of a novel missense mutation in AXIN2 associated with non-syndromic tooth agenesis.

Tooth agenesis is a congenital anomaly frequently seen in humans. Several genes have been associated with non-syndromic tooth agenesis, including msh homeobox 1 (MSX1), paired box 9 (PAX9), axis inhibition protein 2 (AXIN2), ectodysplasin A (EDA), and wingless-type MMTV integration site family member 10A (WNT10A). In this study, we investigated a Chinese family with non-syndromic tooth agenesis. A novel missense mutation (c.C1978T) in AXIN2 was identified in affected members. The mutation results in a His660Tyr substitution located between the Axin beta-catenin binding domain and the DIX domain of the axis inhibition protein 2 (AXIN2). We analysed this novel AXIN2 mutant, together with two reported AXIN2 mutants [c.1966C>T (p.Arg656Stop) and c.1994delG (p.Leu688Stop)] that cause colorectal cancer with and without oligodontia, to study the effect of the mutant p.His660Tyr on the Wnt/ß-catenin signaling pathway and to compare the molecular pathogenesis of different AXIN2 mutants in tooth agenesis and carcinogenesis. Further in vitro experiments indicated that the mutant p.His660Tyr caused inhibition of the Wnt/ß-catenin pathway, and the mutants p.Arg656Stop and p.Leu688Stop resulted in over-activation of the Wnt/ß-catenin pathway. In line with previous AXIN2 mutation studies, we suggest that AXIN2 mutations with different levels of severity may have distinct effects on the Wnt pathway and the phenotype of disease. Our study provides functional evidence supporting the notion that both inhibition and over-activation of the Wnt pathway may lead to tooth agenesis.

A novel initiation codon mutation of PAX9 in a family with oligodontia.

OBJECTIVE: Recent studies have attributed non-syndromic tooth agenesis to mutations in several genes, including MSX1, PAX9, AXIN2, WNT10A and EDA. In this study, mutation of PAX9gene was investigated in a four-generation Chinese family with oligodontia. DESIGN: Genomic DNA was isolated from the blood samples of all the available family members. Candidate genes MSX1 and PAX9 were amplified using polymerase chain reaction and then directly sequenced. RESULTS: A novel initiation codon mutation was identified; it consisted of a heterozygous c.2T>G mutation in the PAX9 gene which changed the ATG initiation codon to AGG. Restriction-enzyme analysis was performed to verify this mutation, which was segregated amongst the members with the oligodontia phenotype. CONCLUSIONS: Our results demonstrate a new initiation codon mutation in the PAX9 gene. This mutation probably caused the oligodontia in the investigated Chinese family through haplo-insufficiency.