Investigation On The Disease-causing Gene And Molecular Mechanism For A Hereditary Dentin Dysplasia Type Ⅰ Family

Background and PurposeTooth development and morphogenesis is increasingly recognized as important for human health. Tooth development in vertebrate is a rather complex event. According to the current research progress, inherited dentin defects are divided into 5 types:3 types of dentinogenesis imperfecta (DGI), and 2 types of dentin dysplasia (DD). DGI type I is osteogenesis imperfecta(OI) with DGI. DD-Ⅱ, DGI-Ⅱ, and DGI-III, each with its own pattern of inherited defects limited to the dentition, have been found to be caused by various defects in DSPP (dentin sialophospho -protein), a gene encoding the major non-collagenous proteins of dentin. Hereditary dentin dysplasia is an autosomal dominant genetic disease, divided into Ⅰ,Ⅱ two subtypes, the incidence of type I dentin dysplasia at 1/1000000, belong to the rare diseases, and the causative gene has not been reported. DDI exhibits unique characteristics with phenotypes featuring obliteration of pulp chambers and diminutive root, thus providing a useful model for understanding the genetics of tooth formation. Using a large Chinese family with 14 DDI patients, we mapped the gene locus responsible for DDI to 3p26.2-3p24.3 and further identified a missense mutation, c.353 C>A (P118Q) in the ssuh2 gene on 3p26.1, which co-segregated with DDI. We researched that mRNA expression level of ssuh2 (P118Q) in vivo. The levels of mutant protein and mRNA compared to wild-type ssuh2 in vitro.This research found that genetic dentin dysplasia type I (DD-1) gene ssuh2 role in tooth development and function also need further exploration. Being similar to human and other mammals, zebrafish and mouse have dental pulp cavity, dentin, enamel, and enamel organ between the teeth. The tooth development of zebrafish and mouse, namely the bud stage, cap stage, bell stage, is similar to human, although the period is relatively short. And zebrafish and mice with a shortly breeding cycle and the advantage of large number of experiments, as a result, zebrafish and mice can be used as a model organism of tooth development research.Regulation of tooth development in zebrafish genes with BMPs, FGF, Alk8, Pitx2a, DLX and Pax9, they all have the corresponding homologous genes in human. In our early research, we found the ssuh2 homologous genes ZGC:153440 in zebrafish. Therefore, we through the morpholine modified antisense oligonucleotide (MO) reduce ssuh2 homologous gene expression in the zebrafish to study it affect other genes in tooth development, implementation complete route of tooth development related genes in the zebrafish, at the same time find ssuh2 correlation of tooth development in human.Accordingly inhibit the expression of genes, reduce or even eliminate certain genes are expressed in the organization, and then on the form, organization and biological molecules level to study its effect.we analyzed the ssuh2 homologous gene’s expression level in time and space and the gene expression level in organs in mice. Then, we through the knock-out technology constructed a genetic defect in mice, by PCR amplification technology and Sanger sequencing technology to identify the gene types in mice model. According to ssuh2 gene homologous gene expression spectrum in mice, we select the jaw tissue of different genotypes (wild type, the hybrid mutation, homozygous mutations) mice to analysize expressing quantity in 3 days old or three months old (d90).Therefore, this research on zebrafish and genetic defect mice as animal model, through patterns of adopting the method of "knock down expression" building gene defect animals, through the corresponding experiment means, preliminary study on the ssuh2 genes impact on the genes in the process of tooth development in the zebrafish and phenotypic characterization ssuh2 gene defects in mice.Samples and Methods1. Specimen collection and clinical data collection:Extract DNA and RNA from peripheral blood of every member pedigree; collect clinical medical records of patients.2. Linkage analysis:we defined the candidate region using genome-wide multipoint linkage analysis, we confirmed the results using microsatellite-based fine mapping of a potential locus.3. We applied sequencing of candidate segment with the genome DNA from the proband, identify the mutation Ssuh2 gene.4. Using High Resolution Melting technology screening 1000 random samples to identify ssuh2c.353C>A(P118Q) mutation frequency.5. Bioinformatics:Three-dimensional structures of WT and mutant C3orf32 were predicted with I-TASSER by importing WT and mutant C3orf32 amino acid sequences with sequence-breaking points identified by SOSUI. PolyPhen2 was used to estimate the functional effect of the p.Prol 18Gln mutation on the protein.6. Analysis the difference between affected and unaffected in mRNA expression level.7. Subcellular localization:The normal and abnormal transcription cDNA of human ssuh2 gene was cloned into the vector pEGFP-C1, then recombinant WT or mutant ssuh2 were transfected into COS7 cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 24 hours after transfection, cells were rinsed three times with PBS and cell nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO) and viewed under a fluorescence microscope (Nikon, Eclipse Ti-U, Tokyo, Japan).8. Analysis the expression level of mRNA and protein in vitro.9. Using BLAST in NCBI, searching for human Bmp2a Pitx2, Pax9 interact homologous genes in the zebrafish, to analyze their similarity. Through Primer3 software to design primers used to amplify genes mRNA CDS region.10. The CDS region of tooth development related gene cloning to the carrier pBSK, and then use T7 kit synthesis the homologous gene transcription of antisense RNA probe in vitro.11. By microinjection morpholino into zebrafish embryos, using the in situ hybridization technique to analyze the effects of the related homologous gene in Ssuh2 gene expression reduced.12. Using BLAST in NCBI to find homologous genes of ssuh2 in mice, using Primer3 software design primers, build mice ssuh2 homologous gene expression profiles at the RNA level13. Using TRIZOL extracting RNA from normal C57BL/6 mice different organs such as lheart, liver, spleen, lung, kidney, brain,jaw, small groups, then using reverse transcription kit transcription for cDNA as a template for PCR, observe ssuh2 gene expression in different organs in mice.14. Take the jaw of normal C57BL/6 mice in different periods, including the E1, E3, E5, E7, E11, P3, P7, P15 using TRIZOL extracting RNA, build ssuh2 space-time expression of genes in mice.15. Generating defected mice by knock-in technology, and after mating produce different genotypes (normal, hybrid, homozygous) mice.16. Eextracte DNA from cut the toes of mice and numbering, PCR products with Sanger sequencing to identify the genotypes in mice.17. Take different genotypes of mandible in three months old mice, after fixation, dehydration, embedding, paraffin and slice thickness is 0.2 μm. After the section, dewaxing, water and so on a series of steps to HE staining, dyeing dry sealing piece, after the completion of photograph and the inverted fluorescence microscope.18. Fixed jaw of three months old mice, the different genotype mice with micro-CT scan and three-dimensional composite images, comparing the difference between samples of different genotypes.19. Fixed jaw of three months old mice, the different genotype mice were then gradient dehydration, then soak in different gradient of resin, finally the embedding in resin grinding. Golding the grind samples, then scanning electron microscope photos, compare the difference between samples of different genotypes.20. Take jaw of the 15 days old mice, using liquid nitrogen grinding, TRIZOL extract tissue RNA, reverse transcription of cDNA.21.To compare different genotype Ssuh2 gene homologous genes in mice in RNA expression level, and compare with the tooth development related genes expressed in different genotype of the mice.ResultsWe defined candidate region on chromosome 3p26.1-3p24.3 (chr3:6,732,117-18,359,306) using genome-wide multipoint linkage analysis, and confirmed the results using microsatellite-based fine mapping of a potential locus in the DDI family. We applied sequencing of candidate segment with the genome DNA from the proband, identify the mutation c.353 C>A (P118Q) in ssuh2 gene. Then, the affected expression of ssuh.2 is less than unaffected in mRNA level. Subcellular localization analysis showed the c3orf32 protein is a nuclear protein and p.Pl 18Q did not affect nuclear. We detected significantly reduced expression of the p.P118Q variant at both the mRNA and protein levels compared to WT ssuh2 protein localization.Extract on of 56hpf zebrafish total RNA, and RT-PCR, do with well finished cDNA template for ordinary PCR, whether the tooth development related gene is expressed in this time period or not, the results showed:Bmp2a, Pitx2, Pax9 expressed in the early development of zebrafish embryos;In order to determine whether they are in the embryo is affected by Ssuh2 gene, by the development of embryo in situ hybridization experiment, the results showed:Bmp2a, Pitx2, Pax9 interact mainly concentrated in the gills arch expression, through morpholino reduce Ssuh2 gene expression, and tooth development related genes Bmp2a, Pitx2, Pax9 interact expression quantity shows the trend to decrease.Extract DNA from mice after cut toe to identify mice genetype.Extraction of RNA from the mandible after mice birth of 15 days and compare different genotype Ssuh2 gene expression differences in mice, the results showed that the heterozygous and homozygous mice Ssuh2 gene expression quantity are less than normal mice;Take jaw of one month old mice in different genotypes and HE staining, the result shows:the wild type mice have wide predentin zone, the heterozygous mice predentine zone narrowing, and homozygous predentine zone basic disappear; in mice in a months, After microscopic CT scanning, the three dimensional synthesis results showed that after heterozygous and homozygous compared with wild type mice of dental pulp cavity has a corresponding decrease;By scanning electron microscope observation, the results showed that wild type mice dentin tubule closely packed, hybrid type mouse dentin tubule decreased, homozygous dentin mice dentin tubule less than heterozygous mice.ConclusionsWe defined candidate region on chromosome 3p26.1-3p24.3 (chr3:6,732,117-18,359,306) using genome-wide multipoint linkage analysis and confirmed the results using microsatellite-based fine mapping of a potential locus in the DDI family. We applied sequencing of candidate segment with the genome DNA from the proband, identify the mutation c.353 C>A (PI 18Q) in ssuh2 gene. Then, the affected expression of ssuh2 is less than unaffected in mRNA level. Subcellular localization analysis showed the c3orf32 protein is a nuclear protein and p.P118Q did not affect nuclear. We detected significantly reduced expression of the p.P118Q variant at both the mRNA and protein levels compared to WT ssuh2 protein localization.In the zebrafish, ssuh2 homologous gene defect can lead to zebrafish tooth development defect.In this experiment, reduce zebrafish ssuh2 homologous gene expression by morpholino, using the in situ hybridization technique to determine the overall ssuh2 zebrafish lower expression of homologous gene will result in the expression of other genes related to tooth development, Bmp2a, Pitx2, Pax9 interact expression quantity in early zebrafish teeth development were reduced.Using PCR confirmed Ssuh2 homologous genes in embryonic development of mice and after the birth of the early stage of the development are expressed, and rising trend in embryogenesis evelopment time.In 15 days after the birth of expression quantity reaches its peak, and adult mice hardly express;Extraction of RNA from various organs of 15 days old mice to research Ssuh2 gene expression.Ssuh2 genes are expressed in most of the organs in mice, but the tongue, jaw, brain and intestine in higher quantity.Through knock-in techniqe to construct Ssuh2 gene defects mice, extract RNA from mandible in 15 days old mice.by RT-RCR to determine heterozygous mice Ssuh2 gene expression quantity is lower lower than the wild type mice, and homozygous mice Ssuh2 gene expression quantity is lower than the heterozygous gene expression, Ssuh2 gene defects dose effect cause the teeth defect.By Micro-computed tomography (CT) and scanning electron microscopy, found that ssuh2 genetic defects exist dose effect, a genetic defect teeth than normal mice genotypes had dental pulp cavity is small, less dentin tubule.Through the paraffin section, HE staining, observed the pathological morphology of teeth in mice, predentine zone decreases in genetic defect mice. Confimed ssuh2 homologous gene defect can lead to tooth development related gene expression changes in mice by RT-PCR technique, including DSPP, PAX9, Bmp2, Bmp4, Runx2, DMP1. Genetic defect mice teeth defect, developmental enamel, dental pulp cavity decreases, and dentin tubule decreased, predentine zone decreases, the defects mice phenotypes was similar to DD-1.