A Study On Molecular Mechanism Of SSUH2 In Tooth Development Mutation Screening Of CTSC Gene And Prenatal Diagnosis For A Chinese Pedigree With Papillon-Lefevre Syndrome

BackgroundTeeth are almost present in all vertebrates. From a predetermined site in early embryo to a complete tooth of organism, it is a continuous, long-term and extremely complex biological processe, which including interaction between epithelium and mesenchyme, cell differentiation, tissue mineralization, maturity and tooth eruption. Teeth are the hardest body organ, which consists of enamel, dentin and pulp. Series of research are more likely to perform because tooth is a relatively independent whole. Therefore, the development of the teeth is a great model of organ development and evolution of the epithelial tissue, which has become one of research highlights in recent years. Although many scientists have been working on the regulation mechanism of tooth development, there are still a lot of tooth development mechanism has not been revealed up to now, such as hereditary dentin dysplasia causative genes has not been found, the specific expression mechanism of DSPP gene mineralization is still not clear. Figuring out the regulatory mechanism of tooth development is also a hot but difficult topic, which can provide molecular basis for tooth regeneration research and a vital theoretical and clinical meaning.Tooth development is the result of the interaction between the many signaling molecules, including BMP (Bone morphogenetic protein), FGF, Hh and Wnt. Except for BMP-1, others BMP belong to the transforming growth factor-β (TGF-β) superfamily. BMP members are involved in the early tooth development, regulation of tooth morphogenesis, cell proliferation, differentiation processes and germination. What’s more, BMP play a crucial role in the maintenance of normal tooth development. BMP2 and BMP4 not only play the most important role in the early steps of tooth Development, but also play a key role for the formation of molars. FGF (Fibroblast growth factor) is a peptide-like molecule, which binding to specific receptors on the cell membrane, in order to regulate cell growth. FGF signaling proteins has been expressed in early steps of tooth development, which can determine the site of the formation of teeth by inducing the expression of Pax9, Paxl and Pix2. Shh is a member of the Hedgehog (Hh) protein, which began to express in the dental lamina of the epithelium and participate in the early development of teeth. Shh signaling pathway consist of shh glycoprotein ligand, a transmembrane receptor Patched (Ptc), Smoothened (Smo), nuclear transcription factor Gli (Gli1, Gli2 and Gli3) and so on. Shh signaling pathway plays an important role in early tooth development of signal transduction from epithelium to mesenchyme, especially in the regulation of proliferation of epithelial and mesenchymal cells and inducing early tooth morphogenesis. Wnt is a class of secreted glycoprotein, its signaling pathways including the classic Wnt-P-catenin and non-canonical Wnt signaling pathway (Wnt/ JNK pathway and Wnt/ca2+ pathway). Studies have shown that almost all organs development are under the regulation of the Wnt signaling pathway. Wnt signaling plays an important role in dental induction between epithelium and mesenchyme.In our previous study, we found a new DDI causative genes SSUH2 (C3orf32, fls184), which located 3P26.1. The literature reports that SSUH2 as chaperones whose expression associated with celiac disease, except that research, the study of SSUH2 gene is still a blank. We find that a large Chinese family with 14 DDI patients all with a missense mutation in SSUH2 c.353 C>A (p.P118Q). Through the molecular research and animal model, we found that SSUH2 mutations can cause hereditary dentin dysplasia. However, SSUH2 how to participate in tooth development and involved in what signaling pathway of tooth development has not been disclosed. Therefore, in this study we will focus on the study on tooth development molecular mechanism of SSUH2.PurposeOur purpose is to reveal the disease dentin dysplasia molecular mechanisms of SSUH2, screening of protein interactions with SSUH2 and clarifying what tooth development signal pathway SSUH2 involved in, perfecting the study of SSUH2, complementing theoretical knowledge about dental such as root and dentin development mechanism and their signaling pathways.Materials and Methods1. Materials:HEK293T cells, Human gingival fibroblasts (HGF), Ssuh2 knock out mice2. SSUH2 recombinant plasmid construction. The eukaryotic expression vector pcDNA-3.1(+)-flag and the prokaryote expreesion vector pET-41a(+)(GST tag) were used to construct ssuh2 overexpression plasmids. BamH I and HindⅢ restriction sites were used to add SSUH2 CDS to the recombinant vector.3. SSUH2 gene overexpression HGF stably transfected cell lines Construction. Wild-type and mutant SSUH2 were constructed on the C-terminal fusion myc tag lentiviral vectors. Packaging lentivirus and screening SSUH2 wild-type and mutant cell lines stably transfected HGF and HFG load control cell lines. And then, using quantitative PCR to verify SSUH2 mRNA expression.4. Using co-immunoprecipitation to screen interactions protein with SSUH2. Then recombinant WT or mutant pcDNA-3.1(+)-flag-SSUH2 vector was transfected into HEK-293T cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Co-immunoprecipitation was performed by Flag antibody. SDS-PAGE, Silver staining and Mass spectrometry were used to identify differentially expressed protein. And employ immunoprecipitation and immunoblotting for review the result.5. RNA-seq was performed to screening differentially expressed genes which were affected by SSUH2.RNA-seq material were E15 ssuh2 knock out mice and wild type mice.6. Using quantitative PCR to validate the RNA-seq data.We selected E15 ssuh2 knockout mice and wild-type mice in the same litterand separated tooth germ organization for total RNA extraction.Using RT-PCR and QT-PCR to check the result of the RNA-seq.7. Take different genotypes of mandible from thrity days old mice, after fixation, dehydration, embedding, paraffin and slice thickness is 0.2 μm. After the section, dewaxing, water and a series of steps to HE staining and dyeing dry sealing piece, take pictures by inverted fluorescence microscope.8. Fixed jaw of four months old different genotype mice. After gradient dehydration and soaking in different gradient of resin, embed jaws in resin grinding. Finaly, Golding the grind samples and took photos by scanning electron microscope.9. Fixed wild type and mutant mice in different periods, using micro-CT to scan bone development, for composing three-dimensional images and calculating their bone mineral density. Compare different genotypes mice bone mineralization and development.10. Randomly selected several different genotype transgenic mice, carrying out tail suspension test, then observed its hind legs stretched condition 20S. Each mouse was scored for examining its leg strength.Results:1. We successfully constructed stable HGF cell line of widetype and mutant SSUH2. And qPCR result showed the mRNA level in over expression widetype and mutant sshu2 cell lines is 20 folds higher than control cells.2. The eukaryotic expression vector pcDNA-3.1(+)-flag and the prokaryote expreesion vector pET-41a(+)(GST tag) were used to construct ssuh2 overexpression plasmids. All the vectors were confirmed by sanger sequencing and double enzyme digestion.3. HEK293T cells were transfected with pcDNA3.1(+)-flag vector and pcDNA3.1(+)-flag-SSUH2 plamids, respectively.Immunoprecipitation was carried out by using anti-flag antibodies follow by silver nitrate staining. We discover a 70kDa protein band which differentially expressed in SSUH2 SDS-PAGE lane. Then, we identified GRP78 gene by mass spectrometry, which has been reported to mediate DMP1 translocates into the nucleus.4. Twenty-nine differentially expressed genes have been found in the transcriptome sequencing of wild type and homozygous knock-out mice. Among which, Col2a1, Col10a1 and Matnl were related to teeth and skeletal development. These genes were down-regulated in ssuh2 knock out mice, and we validated the consequence of RNA-seq by qPCR. Moreover, among the 29 genes, the expression of transcription factor Fos is increased. Previously studies have suggested FOS modulates the expression of downstream target genes such as DSPP, DMP1 as well as calcitonin.5. Jaw HE staining of different genotypes mice shows:Two groups of mice with no obvious differences in tooth phenotypes, such as the former dentin has not changed.6. By scanning electron microscope observation, there was no significant difference between wild type and Ssuh2 deficient mice and the arrangement of dentinal tubules of two group mice were relatively close.7. After microscopic CT scanning, the three dimensional synthesis results showed that bone development of mutant mice were not abnormal. Such as their bone growth and mineralization were similar to wild type mice.8. Tail suspension test shows that no significant difference in the score between different genotype mice, indicating that leg strength in Ssuh2 deficient mice were not affected.Conclusion1. Results of RNA-seq analysis indicate that the expression of Ssuh2 influence the expression of Col2a1, Col10a1, Matn1, and Fos. We supposed the decreasing expression level of Clo2a1 and Col10a1 in Ssuh2 knock-out mice may lead to dentin’s abnormal mineralization through collagen decreasing, and finally make Dentin dysplasia. Futhermore, we deduced that FOS may be the transcription factors of SSUH2. The expression level of Fos increases with knockout of Ssuh2 due to negative feedback regulation mechanisms.2. CO-IP experiments find GRP78, which is one of Hsp70 heat-shock protein family, may interact with SSHU2. Through functional prediction and amino acid sequence analysis of SSHU2, we find a DnaJ molecular chaperone in SSUH2 can bind to Dank(Hsp70 protein) specifically, so that SSHU2 can participate in protein transportation, protein folding and degradation of misfolded protein. What’s more, GRP78 not only participate in the endocytosis of dentine matrix protein DMP1, but also promote accumulation of calcium and phosphorus through combining with Type I collagen and DMP1. From the above, we speculate combining SSHU2 and GRP78 together via the region DnaJ may influence teeth development through theabnormal transpotation of type I collagen and DMP1.3. The successful establishment of wild-type and mutant SSUH2 overexpression stable transfection HGF cell model provides samples for validation experiments afterward.4. After a series of experiments in mice phenotypes, including jaw HE staining, scanning electron microscopy analysis of the molars, bone scanning microscopy CT and tail suspension test, we did not find any difference in tooth and bone between wild type and Ssuh2 deficient mice. In view of the Ssuh2 deficient mice phenotype inconsistent with our previous studies, we discussed that is due to racial differences, there is a big difference between the human species and mice, their developmental regulatory mechanisms vary greatly. Different stages of development in mice, genes expression exist certain changes, it is difficult to grasp particular stage of genes have a profound impact on the growth and development. What’s more, the gene expression systems in creatures are extremely complex. Down-regulate of any gene expression level in an individual may be adjusted and remedied by other regulatory mechanisms, which are unknown. We supposed, that is the reason why the pathogenic phenotype had not been found in our study.5. We find GPR78, COL2A1, COL10A1, MATN1, FOS which associated with tooth development by a series of molecular experiments such as Co-Immunoprecipitation and RNA-seq experiment, revealing the association between SSUH2 and tooth development. Our study provides further strategies for the study on tooth development molecular mechanism of SSUH2.BackgroundPalmoplantar keratoderma with periodontitis which is also known as Papillon-Lefevre syndrome(PLS), was firstly reported by Papillon and Lefevre in 1924. PLS is characterized by Hyperkeratosis at palms and soles of feet and severe destruction at periodontium of primary teeth and permanent teeth. Some cases are associated with calcification of the dura mater. In general, patients are healthy and with normal intelligence. PLS is a rare autosomalrecessivegenetic disorder present in 1-4 in 1000,000. The clinical feature of PLS is severe destruction of periodontium, loss of tooth at early age, and hyperkeratosis at palms and soles of feet. PLS is resulted from the mutation of CTSC, which lead to immunoregulation disorder and severe inflammatory response by a deficiency in cathepsin C.CTSC is located at I1q14.1-q14.3, encoding cathepsin C. CTSC also known as dipeptidyl peptidase, is a lysosomal exo-cysteine protease. Previous study showed that cathepsin C can active some proenzyme and participate in various immune response and inflammatory reaction, such as active phagocyte and lymphocyte to remove pathogenic microorganism.PLS is a rare monogenic disease, whoes reports are more about cases rather than researches. There is also few advances in molecular genetics of PLS, such as mutation screening of CTSC. Consequently, as the mutational profiling of CTSC needing consummates, the incomprehension of PLS in public will delay early protection and treatment for tooth, leading to severe tooth defects. Research shows that intermarriage increased the incidence of PLS. It is sure that prenatal diagnosis must be done to reduce the birth of afflicted child.PurposeIn our investigation, we worked on the potential pathogenic mechanism of PLS by analyzing the clinical feature and identify mutation at CTSC c.901G>A of a pedigree from Huizhou, Guangdong. Meanwhile, as the two-child policy open, a baby boom will come soon. Prenatal diagnosis and geneticcounselling is urgent for the patients to reduce the birth of affected infants which can improve the population quality.Materials and MethodsResearch object:a pedigree affected PLS from Huizhou. The proband is a four-year-old girl whose skin became rough and desquamative at palms and soles of feet when she was two. Now she is curing her tooth at Nanfang Hospital.Research methods:Clinical phenotypes identification and oral examination for the proband has been verified the clinical diagnosis of PLS. PCR and Sanger sequencing were performed to identify the mutation with CTSC gene. Prediction of functional effects of the missense mutation was performed with SIFT and PolyPhen-2. Swiss-Port is used to predict the tertiary structure of wild type and mutant protein. The mRNA and protein levels were analyzed by real-time PCR and Western blot.Results1. Result of phenotype:the patient was diagnosed to be suffered PLS, which is an autosomal recessive genetic disorder.2. Mutation at CTSC identification:7 pairs primers cover the CTSC exons were designed using PRIMER 3.0 and Oligo 7. PCR products were to be used for Sanger sequencing. Results showed that proband has a homozygous mutation c.901G>A (p.G301S) located at CTSC and 1-1,1-3, II-1, II-2 have a heterozygous mutation.3. mRNA analysis:we performed fluorescent quantitative analysis to quantify mRNA expression of proband and carriers to study the influence of homozygous or heterozygous mutation at CTSC. Resulted showed that there was no significant statistic differences in mRNA expression between proband and carriers.4. Protein analysis:to study protein expression of mutant, we transfected with wild-type or mutant PLS plasmids to HEK293. At 24 hours after transfection, cells were collected for western blot. Resulted showed that there was no significant statistic differences in protein expression between wild-type and mutant.5. Bioinformatics:mutant protein caused by c.901G>A at CTSC was predicted to be damaging with Polyphen-2. The score was 1 which was the most possible to be damaging. Sequence alignment showed that this mutation is located in evolutionarily conserved regions. The score predicted by SIFT was-5.599, which was thought to affect the phenotype as under -2.5. Besides, there has two case reports about c.901G>A at CTSC in foreign country. In addition,3D structure predicted by Swiss-Port showed that the mutated amino acid was located near the a-helix which suggested that mutant was different from widetype.6. Prenatal diagnosis:since there has a patient in this family, we advice the mother (II-1) collects the amniotic fluid to take a prenatal test when she’s pregnant. We exacted DNA using standard phenol/chloroform exaction from amniotic fluid which acquired by amniocentesis when she had been pregnant for 18 weeks. Sanger sequencing was performed to identify this mutation site. Result showed that this fetus had the homozygous mutation just like the proband.ConclusionIn our study, there are heritable homozygous mutation c.901G>A in CTSC of proband (III 1), and heterozygous mutation in CTSC ofrelatives (I1,13, II1,112). Fluorescence quantitative PCR shows that there is no statistically significant expression level difference in mRNA which are transcribed form CTSC gene in peripheral blood of patients (III 1) and their parents (II 1 and II 2). Furthermore, no significant expression level difference between wild-type and mutant-type CTSC protein has been confirmed by western blot, which means that the mutation does not affect the transcription and translation of CTSC gene. Prediction of functional effects of the missense mutation was performed with SIFT and PolyPhen-2 whose results are very consistent, which shows that mutations in CTSC is absolutely harmful. The mutation is located in the conserved region of the enzyme and predicted to be damaging and change the structure of the protein bySwiss-Prot. Our conclusion is that CTSC gene c.901G>A mutation changes the conformation of CTSC, which lead to the sharp decline in activity and the dysfunction of cathepsin C.Prenatal diagnosis is the key to rare disease prevention. After the amniocentesis of family member Ⅱ 1 being performed at 18 weeks of gestation in hospital, PCR and Sanger sequencing were performed to identify the mutation with CTSC gene. Unfortunately we found that the fetus also had homozygous c.901G>A missense mutation. The strategy of our study for diagnosis of monogenic disease is to use clinical diagnosis, literature reviewing, determining candidate gene, designing primers, PCR amplication and Sanger sequencing for determining the pathogenic mutations. Also we will perform prenatal diagnosis according to the result of monogenic diagnosis, which is conducive to the prevention of birth of children with monogenic diseases, improve the quality of births.The mutation with c.901G> A of CTSC gene is first reported in China, which extends the mutation spectrum of CTSC gene in Chinese populations. In addition, we provide specialprenatal diagnosis for the family with PLS.