Research On Pathogenic Genes In Three Chinese Family With Developmental Disorders Of Dental Hard Tissues

ObjectiveDevelopmental disorders of dental hard tissues are important parts of non‐carious diseases in dental developmental diseases,including hereditary dentin disorders and amelogenesis imperfecta(AI).These disorders usually lead to defects of tooth structure and even tooth loss,which affect the masticatory function and facial aesthetic morphology.Developmental disorder of dental hard tissues is a kind of hereditary disease.Genetic factors play an important role in the pathogenesis of the disease.MethodsThree Chinese families including two families with dentinogenesis imperfecta and one family with dental enamel hypoplasia were studied.1.The whole-exome sequencing technology was used to identify genes in three families.The variants were annotated and filtered by db SNP138,Ex AC database,Gnom AD database,1000 Genomes Project and an in‐house WES sequencing variant database consisting of 1,092 healthy people to obtain candidate pathogenic sites.Sanger sequencing and TA-clone sequencing were used to verify the mutation sites.2.SIFT,Poly Phen2 and Provean software were used to predict the harmfulness of mutation sites;NCBI database was used to confirm the conservation of amino acids corresponding to variants;HOPE software was used to evaluate differences between the model of wild-type protein and mutant protein.3.The wild-type and mutant plasmids of DSP and DSPP were constructed for functional research in vitro.Immunocytochemistry(ICC)were performed to analysis localization of protein in 293 T cells.4.Human dental pulp stem cells(h DPSCs)were isolated from the pulp of primary teeth of the proband and three age-matched healthy children.The cells were stored in liquid nitrogen until use.The h DPSCs were identified by flow cytometry and adipogenic induction assay.Osteogenic differentiation assay was used to explore the difference of calcification ability between the two groups;q RT-PCR was used to detect the difference of DSPP,RUNX2 and ALP gene expression between the two groups.Results1.A heterozygous mutation c.53 T > G(p.V18G)in DSPP gene was identified in the proband of family 1 with DGI.The frequency of the variant was not found in Gnom AD and an in‐house WES sequencing variant database consisting of 1,092 healthy people,and the pathogenicity of the variant has not been reported.Sanger sequencing showed that the variant was heterozygous in the proband.The proband’father(patient)carried the heterozygous variant,while her mother(with normal phenotype)did not carry the variant.ICC results showed that the mutated DSP protein remained in the endoplasmic reticulum,while the wild-type DSP protein was located in Golgi apparatus.The osteogenic induction experiment of dental pulp stem cells showed that the patient group had lower calcification ability compared with control group.q RT-PCR showed that the expression of DSPP and RUNX2 in the patient group was lower,but the expression of ALP had no significant difference compared with control group.2.A heterozygous variant c.2470＿2479del p.(S824Vfs*487)in the DSPP gene was identified in the family 2 with DGI.The frequency of the variant was not found in Gnom AD and an in‐house WES sequencing variant database consisting of 1,092 healthy people,and the pathogenicity of the variant has not been reported.Sanger sequencing and TA-clonal sequencing were used to verify the variant in the family 2.The results showed that eight patients carried DSPP gene c.2470＿2479del but five healthy members did not carry the heterozygous variant,which was consistent with the co-segregation of genotype and phenotype in this pedigree.3.A heterozygous variant c.973 C > T(p.R325X)in the FAM83 H gene was identified in two families with AI.The frequency of the variant was not found in Gnom AD and an in‐house WES sequencing variant database consisting of 1,092 healthy people It has been reported that this variant is associated with AI.Sanger sequencing showed that eight patients carried the FAM83 H c.973 C > T(p.R325X)heterozygous variant,while eight healthy family members did not.The results showed that the variant was consistent with the co-segregation of genotype and phenotype in this pedigree.Conclusions1.The pathogenic variants of three families with developmental disorders of tooth hard tissue were successfully identified.The heterozygous variants of DSPP gene c.53 T >G(p.V18G)and c.2470＿2479del(p.S824 Vfs * 487)have not been reported.The pathogenic mutation spectrum of DSPP gene was expanded.2.The function of the c.53 T > G(p.V18G)in DSPP gene was studied.DSPP gene c.53 T > G can resulted in the mutant protein remaining in endoplasmic reticulum and ailing to secrete into Golgi apparatus.3.DSPP gene c.53 T > G variant affects the calcification ability of tooth tissue.The decreased mineralization ability of h DPSCs in patients with DGI may be related to the decreased expression of DSPP and RUNX2.