Detection And Functional Analysis Of Disease-causing Gene In A Non-syndromic Hereditary Gingival Fibromatosis Family

Background and ObjectiveHereditary gingival fibromatosis(HGF)is a rare oral hereditary disease with a large of diffuse and progressive proliferation of the gingival connective tissue.The main manifestation of gum tissue is usually extensive,progressive hyperplasia,which can affect the marginal gingiva,attached gingiva and gingival papilla of the entire mouth.The enlarged gingiva may cover part or the entire clinical crown,and teeth are often displaced due to hyperplasia of gingival.Severe cases can cause abnormal permanent teeth eruption,chewing restrictions,aesthetic problems and psychological burden.The hyperplastic gingiva is usually normal in color,with firm consistency and obvious stippling.The genetic pattern of Hereditary gingival fibromatosis is mainly autosomal dominant inheritance,but there is also recessive inheritance.It has been reported that the incidence of the HGF is 1 in 175,000 internationally with no gender difference.However,there is no exact statistics in China.Some cases of HGF can co-exist with a variety of systemic symptoms,which belongs to Syndromic Hereditary Gingival Fibromatosis.SOS1 and REST genes have been currently identified as pathogenic genes abroad.But the pathogenic genes have not been verified in domestic families.What’s more,the Chinese scholars discovered a novel pathogenic gene ZNF862 for HGF for the first time this year.These studies fully indicate that HGF has a high degree of genetic heterogeneity.In our previous work,we collected a large pedigree of 48 patients,including 19 patients,who were definitely diagnosed as non-syndromic HGF,and excluded the reported SOS1,REST and ZNF862 genes by Whole Exome Sequencing(WES).In this study,our group mapped the pathogenic gene loci of the non-syndromic HGF family to chromosome 2 through SNP chip genome-wide linkage analysis,and combined with the Whole Exome Sequencing and co-segregation within the pedigree to identify a new disease-causing gene,temporarily named as HGF related gene(Hereditary gingival fibromatosis related gene,HGFR).What’s more,we intend to further study the new mutation site of the non-syndromic HGF through bioinformatics analysis and the cell model establishment.In addition,we will also try to figure out the pathogenic gene of non-syndrome HGF and explore the role of this new gene in the pathogenesis of non-syndromic hereditary gingival fibromatosis.Our study can provide new genetic clues and novel theoretical basis for the molecular mechanism of non-syndromic HGF.Materials and MethodsⅠ.Carry out family investigation,medical history collection and specimen collection for the non-syndromic HGF family;Ⅱ.Genome-wide SNP chip scanning was used for linkage analysis of the family to identify the region of the disease-causing gene loci.Ⅲ.Identify new mutation site by whole exome sequencing(WES)and co-segregation within the family.Ⅳ.Bioinformatics analysis:PROVEAN software was used to predict the harmfulness of mutant protein,and Polyphen-2 was used to predict the conservation analysis of target amino acid among multiple species.The physicochemical properties of proteins before and after mutation were predicted by ExPAsy database.Comparison was analyzed between the secondary and tertiary structures of wild-type and mutant-type proteins by SOPMA and I-TASSER prediction software,respectively.Ⅴ.RNA and protein expression analysis in vivo:The gingival tissues of patients and normal subjects were collected for RNA extraction and immunohistochemical staining,and the mRNA transcription level and protein translation level of HGFR gene were analyzed in vivo.Ⅵ.Subcellular localization:human HGFR wild-type(WT)and mutant-type(MUT)cDNA were cloned into vector pEGFP-N1,and transfected into cultured HEK-293T cells with Lipofectamine 3000 transfection reagent.The changes of subcellular localization of wild-type and mutant-type proteins were compared after transfection for 48h.Ⅶ.HGFR wild-type(WT)and mutant-type(MUT)pcDNA3.1-3xFlag overexpression plasmid were constructed and transfected into human gingival fibroblasts(HGF-1)with Lipofectamine 3000 transfection reagent.After 48h transfection,RNA and protein were extracted and the mRNA expression level and protein expression level of the gene were analyzed in vitro.ResultsⅠ.Clinical features:All the patients in this pedigree showed extensive and diffuse gingival hyperplasia,which could involve the whole mouth gingival tissue,reaching the mucogingival junction.Gingival hyperplasia ran through the deciduous dentition,mixed dentition and permanent dentition of the family.What’s more,the hyperplasia of the posterior teeth area was significant.The hyperplasia of gingival was mulberry-shaped or nodular,which can cover more than 2/3 of the dental crown.In some cases,the teeth were displaced,which could result in an irregular dentition.The color of the hyperplasia gingival was pink,the tissue was firm,and the stippling was obvious.Ⅱ.Determination of mutation sites:Genome-wide linkage analysis with SNP chip was performed on 25 members of this pedigree,and the positive regions of candidate pathogenic genes were located between 2p23.3 and 2p24.1.Combined with the Whole Exome Sequencing analysis results of affected(Ⅱ-6,Ⅳ-10)and healthy controls(Ⅲ-6)and co-segregation within the family,it was finally confirmed that HGFR had a missense mutation:c.4154G>T(p.W1385L).Ⅲ.Bioinformatics analysis:The PROVEAN predicted that the missense mutation was a deleterious mutation,and the Polyphen-2 indicated that the mutant amino acid was highly conserved among multiple species.The ExPAsy database predicted that physical and chemical properties of the mutant protein would be affected to a certain extent,and the I-TASSER prediction software showed that the missense mutation would lead to major changes in three-dimension structure of the protein.Ⅳ.The vivo experiments analysis indicated that transcriptional expression level of gingival tissue in affected individuals in the pedigree was increased compared with that unaffected(P<0.01).What’s more,the protein expression level of the patients was also increased correspondingly by immunohistochemistry analysis(P<0.01).Ⅴ.The results of in vitro experiments indicated that the subcellular localization of the mutant protein did not change,which the proteins were both localized in the cytoplasm and nucleus.However,the mRNA expression level was significantly increased compared with the control group(P<0.01).ConclusionsWe located the pathogenic gene loci at 2p23.3-2p24.1 by SNP linkage analysis within a large non-syndromic HGF family collected in eastern Henan area.This linkage region is close to but does not overlap with the reported GINGF3 isoform locus,which is a newly identified pathogenic locus.Then we combined with the Whole Exon Sequencing and co-segregation within the family to identify a missense mutation of HGFR gene:C.4154G>T(p.W1385L).We further found the missense mutation was a deleterious mutation,and the mutation site was highly conserved among multiple species.What’s more,the tertiary structure of the protein had also changed greatly after the mutation,which all indicated the pathogenicity of the missense mutation of the gene.In vivo experiments,we found that the mRNA and protein expression levels of HGF patients were higher than those of unaffected people;Although the result of vitro experiments showed that the subcellular localization of the mutant protein did not change,the mRNA expression level was higher than that of the control group.In a word,the missense mutation of HGFR gene c.4154G>T(p.W1385L)expands the mutation spectrum of non-syndromic HGF and provides a new research idea for molecular mechanism of non-syndromic HGF.