Identification And Functional Study Of Novel Pathogenic Genes Of Non-syndromic Hypodontia

Research background and objectivesHypodontia is one of the most common abnormalities in tooth development,of which the non-syndromic hypodontia refers to the congenital loss of teeth without other developmental malformations.Hypodontia not only affects the mastication of patients,but also affects their appearance,pronunciation and even mental health in severe cases.Therefore,the search for its etiology has become a hot topic for many researchers nowadays.Thus far,there have been insufficient systematic researches on hypodontia from molecular level to cellular level and in animals.Therefore,the studies on its pathogenesis can lay the theoretical foundation in genetics for future gene therapy.A non-syndromic hypodontia family with five patients was recruited in this research.By whole exon sequencing(WES),short tandem repeat(STR)linkage analysis and family co-segregation,a new pathogenic gene,desmoplakin(DSP)was found,which has never been reported to be associated with non-syndromic hypodontia.The desmoplakin(DP)encoded by DSP is the main protein composing desmosome structure and it contains two subtypes DP I and DP II,which are mainly expressed in cardiomyocyte and skin cells.But the correlation between DSP and tooth development is still not clear,nor are there any relevant functional studies found.The objective of this study was to find out the molecular mechanism of DSP in non-syndromic hypodontia by analysing the mutated protein function,establishing cell models and zebrafish models in vivo and in vitro,and to provide new clues for the physiological development of teeth.Materials and Methods1.An investigation into the falimy with congenital hypodontia was perfomed,and collections for medical history and blood samples were carried out.2.Samples were examined by STR linkage analysis,and the new pathogenic genes and mutation sites were identified by WES technique and family isolation.3.Comparision was conduncted between the secondary structure and three-dimension structure of wild-type(WT)and mutant(MUT)proteins with prediction software SOPMA and I-TASSER.Polyphen-2 was used to predict the effects of the changes of amino acid on protein function.4.The cDNA of both WT and MUT transcripts of DSP were cloned into the plasmid pIRES-EGFP and transfected into HEK-293T cells.Expressions of the DSP mRNA and DP protein were detected by qPCR and Western Blot.The subcellular localization of DP was observed by confocal microscopy.5.After the transfection of pIRES2-DSP-WT and pIRES2-DSP-MUT with HEK-293T cells for 16 h,Cycloheximide(CHX)was used to deal with these two group cells at different time points(0h,1h,3h,6h,12h and 24h)and DP expression was determined by Western Blot.6.HEK-293T cells were co-transfected with three plasmids(pTOP/pFOP,pIRES2-DSP-WT/pIRES2-DSP-MUT and pRL-TK).After 48 hours of transfection,dual luciferase reporter assay was performed to detect the effect of DSP mutation on Wnt/β-catenin signaling pathway.7.Cell model(ShRNA-DSP)was constructed to inhibit the expression of DSP in human dental pulp stem cells(hDPSCs).There were three groups(MOCK,ShRNA-Ctrl,and ShRNA-DSP)in the research.The proliferation,migration and apoptoses of hDPSCs in these three groups were detected by CCK8,transwell,and flow cytometry.hDPSCs was induced to odontoblastic differentiation.Alkaline phosphatase staining and alizarin red staining were respectively used to detect the alkaline phosphatase activities and minerized nodules of hDPSCs in the three groups.The expressions of genes and proteins related with odontoblastic differentiation of hDPSCs were detected with qPCR and WB.8.Two morpholino(MO-1,MO-2)targeting the zebrafish homologous gene dspa were synthesized and injected into the fertilized eggs of zebrafish.Changes in mRNA levels of homologous gene dspa by morpholino were detected.The number of zebrafish with tooth agenesis was observed and recorded on the sixth day post-fertilization.9.Antisense RNA probes related with early tooth development of zebrafish were designed and synthesized.Whole-mount in situ hybridization was used to analyze the expressions of bmp2a,dlx2b,and pitx2,which were releted with early development of zebrafish,after knocking down the zebrafish homologous gene dspa.Results1.Clinical characteristics of the non-syndromic hypodontia familyThe proband’s(16 years old,female)deciduous teeth of 55,65,75 retained in her permanent teeth dentition with significant front interdental spaces in the mandible and the tooth 15 partially erupted titling towards the buccal.Panoramic radiograph showed congenital loss of permanent teeth 25,28,35,42 and 45.Her mother(II-9)had congenital absence of permanent teeth 32.Her family member III-3 lost the permanent teeth 42 and family members Ⅱ-3 lost the permanent teeth 15,25,38,45 and 48.2.Identification of mutation sitesWe conducted STR genetic linkage analysis for 12 members in this family and it showed the region 6p25.2-6p24.1 was positive area for pathogenic genes.We performed WES analysis on patients(Ⅱ-9,Ⅲ-9 and Ⅲ-3)and healthy controls(Ⅲ-4 and Ⅲ-10).Sequencing results showed that there were seven gene mutations existing in all the three patients.A missense mutation(rs6929069,c.5213 G>A)located in exon 23 of DSP gene was identified by family isolation which was detected in all patients but not in healthy family members.This mutation resulted in a mutation of the amino acid arginine to glutamine at position 1738 of the DPI protein(p.R1738Q).The mutation did not change the secondary structure and three-dimensional of DP protein,but the mutant amino acid was highly conserved among different species.3.Analyses of protein expression and subcellular localization of p.R1738QWestern blot indicated that the expression of MUT DP was significantly higher than that of WT DP(p<0.001)and cycloheximide chase analysis showed Mut DPI was more stable than WT DPI in cells.Subcellular localization suggested that both WT DP and MUT DP were located in cytoplasm,and the mutation did not affect the subcellular localization.4.DSP mutation interfers with the Wnt/β-catenin signaling pathwayThe expression of nuclear β-catenin protein was down-regulated by MUT DP(P<0.05).Consistent with this notion,upregulation of the TCF/LEF transcriptional activity was reduced by MUT DP expression in reporter assays(p<0.01)..5.Effects of inhibiting DSP expression on proliferation,migration and apoptoses of hDPSCs and odontoblastic differentiation.The prolieration activity of hDPSCs was significantly decreased on the fifth day(p<0.05)after knocking down the expression of DSP and the portions of cell apoptosis was distinctly higher than that in the control gruops(p<0.05).Transwell results demonstrated that the number of cells migrating through the membrane pores was greatly fewer than that in the in the control gruops(p<0.05)after knocking down the expression of DSP.After odontoblastic differentiation for 21 days,the number of mineralized nodules in ShRNA-DSP group was greatly increased.Results of qPCR and WB showed significant up-regulation in odontoblast related genes(BSP and DSPP)and proteins(DSPP and ON)(p<0.05)in ShRNA-DSP group.6.Effects after inhibition of zebrafish homologous gene dspa on tooth and tooth development-related genes in zebrafishIn MO-1 and MO-2 group,the average rate of tooth agenesis was 58.21%±1.82%(MO1)and 54.67%±4.12%(MO2),respectively,which is significantly lower than that of the control group,at 8.85%±0.73%(p<0.001)after the inhibition of the homologous gene dspa.The results of in situ hybridization showed that after the expression of homologous gene dspa was downregulated,the expression of tooth development related genes dlx2b and pitx2 were absent,and the expression of bmp2a was down-regulated.Conclusion1.We identified a missense mutation in the DSP gene(c.5213 G>A)in a family of non-syndromic hypodontia from Jiangxi Province.It is also the first report about the DSP mutation associated with non-syndromic hypodontia.2.DSP mutation did not affect the three-dimensional structure and subcellular localization of the protein,but the expression of the mutant protein in the cells increased significantly,and the half-life of mutant protein was increased.Its long-term stable existence inhibited the Wnt/β-Catenin signaling pathway;3.DP protein was highly expressed in mesenchymal derived hDPSCs,and its loss of function inhibited the normal development of biological characteristics of hDPSCs;4.The phenotype of tooth agenesis was duplicated and the expression of odontogenesis-related genes were downregulated by suppressing the expression of dspa gene in zebrafish,which meant DSP was very likely to be a novel pathogenic gene resulting in non-syndromic hypodontia and play an essential role in odontogenesis.