Mutation Screening And Functional Analysis On The Molecular Mechanisms Underlying Tooth Agenesis & The Study Of TARP In The Pathogenesis Of Salivary Adenoid Cystic Carcinoma

Part ?:Mutation screening and functional analysis on the molecular mechanisms underlying tooth agenesisTooth agenesis,an abnormality of tooth number,is a congenital anomaly frequently occurred in humans.According to the number of missing teeth,tooth agenesis is grouped into hypodontia(missing up to five permanent teeth excluding third molar),oligodontia(missing six or more permanent teeth excluding third molar),and anodontia(missing all teeth).Tooth agenesis can be categorized into non-syndromic and syndromic tooth agenesis based upon whether there are other symptoms.Tooth agenesis may occur as sporadic or familial;the familial kind could be triggered by autosomal dominant,autosomal recessive or X-linkage inheritance.To date,mutations in MSX1,PAX9,AXIN2,EDA and WNT10A have been discovered to cause non-syndromic tooth agenesis in humans.EDA,IRF6,PITX2,FGFR1 and SHH have been associated with syndromic tooth agenesis.Our team unceasingly collects families and sporadic cases with tooth agenesis.Written consent was acquired from all participants or their guardians,which was approved by the Institutional Review Board of Hospital and School of Stomatology,Wuhan University.Each participant underwent oral examinations and a thorough check-up.The diagnosis of patients was ascertained by panoramic radiographs.Objective:A detailed pedigree analysis and genetic analysis of family members were conducted to analyze the disease-causing genes of tooth agenesis and the underlying pathogenic mechanisms in Chinese families and sporadic cases with congenital tooth agenesis.Methods:Genomic DNA was extracted from peripheral blood lymphocytes of the patients,all available unaffected family members and 800 normal controls.Mutation analysis was performed by amplifying the entire exons,including exon-intron boundaries of MSX1,PAX9,AXIN2,EDA,WNT10A and directly sequencing.As to the analysis of mutated PAX9,restriction enzyme analysis was performed to confirm the mutation since the heterozygous c.2T>A mutation destroys a BsrD1 restriction site.As to the functional study of mutated AXIN2,site-directed mutagenesis was performed after detecting the mutation.Vectors expressing wild-type and mutant AXIN2[novel AXIN2 mutant discovered in our study:c.1978C>T(p.His660Tyr);two reported AXIN2 mutants:c.1966C>T(p.Arg656Stop)and c.1994delG(p.Leu688Stop)]were transiently transfected into 293T cells.Western blot and dual-luciferase reporter assay were performed to investigate the effects of AXIN2 mutation on protein function.Results:Our present study identified a novel initiation codon mutation in the PAX9 gene and a novel missense mutation in the AXIN2 gene.The heterozygous c.2T>A mutation in PAX9,changing the ATG initiation codon to AAG,was detected in all available affected individuals but not present in any of the unaffected relatives and the controls.Further in vitro experiments were performed to investigate the effect of the mutated AXIN2 on Wnt/?-catenin pathway.Western blot showed lower expression of p.His660Tyr mutant and truncated p.Arg656Stop and p.Leu688Stop mutants.Expression of ?-catenin was detected to study whether mutant AXIN2 influences the level of activation of canonical Wnt/?-catenin pathway.Western blot analysis of cytoplasmic and nuclear proteins indicated that p.His660Tyr caused decreased ?-catenin,while p.Arg656Stop and p.Leu688Stop caused increased P-catenin.Dual-luciferase assay revealed that,compared with wild-type AXIN2,p.His660Tyr reduced luciferase activity,while both p.Arg656Stop and p.Leu688Stop increased luciferase activity.Conclusions:A novel initiation codon mutation in the PAX9 gene and a novel missense mutation in AXIN2 gene have been identified,which led to tooth agenesis in the two unrelated families.Our study enlarges the mutation spectrum of PAX9 and AXIN2 among patients with tooth agenesis.Mutant p.His660Tyr led to non-syndromic tooth agenesis in this Chinese family by causing inhibition of Wnt/?-catenin pathway,and mutant p.Arg656Stop resulted in syndromic tooth agenesis in the Finnish family through overactivation of Wnt/?-catenin pathway.Our study suggests that different types of AXIN2 mutations may have different influence on Wnt/?-catenin pathway and disease phenotype.Our research offers supporting functional evidence for the view that both inhibition and overactivation of Wnt pathway may cause tooth agenesis.Part ?:The study of TARP in the pathogenesis of salivary adenoid cystic carcinomaSalivary adenoid cystic carcinoma(SACC)is one of the most common malignancies of the salivary gland.Its clinical features include neural invasiveness,frequent recurrence rates and distant metastasis.The current treatment options for SACC patients are mainly surgery and post-operative radiotherapy.The long-term prognosis is still unsatisfactory despite the advance in treatments.The exact biologic mechanism underlying SACC is not clear,further understanding of which can provide new insights into therapeutic strategies.Recognizing crucial regulatory factors associated in the pathogenesis and progression of SACC is conducive to early discovery and prognosis improvement.Current factors,including c-KIT,EGFR and HER2,are potential molecular targets of SACC therapy.Considering the uninspiring application of these targets in cancer treatment,we need to explore more effective targets in order to achieve great breakthroughs in treatment for cancer.T-cell receptor gamma chain alternate reading frame protein(TARP)is a 7 kDa mitochondrial outer membrane protein.In addition to benign prostate gland tumors and prostate cancers,high expression of TARP was also described in breast cancers,and endometrial carcinomas.As a tumour-associated antigen,TARP was expected to provide therapeutic targets for cytotoxic T lymphocytes and helper T lymphocytes in immunotherapy of tumors.So far,the downstream target genes of TARP stay unidentified and the pathogenic mechanisms of TARP in the tumorigenesis and progression need further investigation.To our knowledge,no studies have explored the influence of TARP on the migration and invasion of cells.No previous studies have investigated the clinical expression and functional characteristics of TARP in SACC and its metastases.Objective:To determine the expression and role of TARP in SACC and its distant metastases.Methods:50 primary SACCs,13 metastatic ACC of salivary gland origin were analyzed in our study.Immunohistochemistry was used to assess the expression of TARP.?2 test was performed to explore the relationship between clinicopathologic features and TARP expression.Cell Counting Kit-8 assay,wound healing assay,and transwell assays were carried to examine the impact of TARP on cytobiological characters of stably transfected cells that overexpress TARP.Results:TARP expression was obviously higher in primary SACCs than that in normal tissue around the cancer.Moreover,the expression of TARP was further upregulated in metastases than that in primary SACCs.It was also found that the expression level of TARP was related with tumor size,TNM stage,perineural invasion,histopathological subtypes and distant metastasis.Overexpressed TARP facilitated cell proliferation,migration and invasion.Conclusions:TARP plays an important role in and may be used as a marker to indicate the development and progression of SACC.