Research On The Disease-causing Gene In A Hereditary Dentin Dysplasia Type â…  Family

Background and PurposeHereditary dentin dysplasia is a common hereditary disease of human dentin, mainly confined to the teeth and dental papilla mesodermal dysplasia of a class of diseases, the incidence of 1/10000-1/6000 range. As early as in 1973,Shields divided the hypoplasia of dentin into two types1:dentin dysplasia with 2 subtypes, dentinogenesis with 3 subtypes. This study mainly focused on dentin dysplasia type I (DD-I).DD-I (OMIM:125400) is a rare autosomal dominant disease, whose penetrance less than 100%, the incidence of 1/1000002. Clinical examination revealed that the crown shape, color performance of normal, but the formation of dentin defects and marrow cavity block, often accompanied by periapicalradiolucencies. The disorder was described in 1920 by Ballschmiede, who called the condition "rootless teeth" because of the short blunted appearance of the roots and spontaneous exfoliation of teeth in 7 children from the same family3.Similar to human and other mammals, zebrafish have dental pulp cavity, dentin, enamel, and enamel organ between the teeth. The tooth development of zebrafish, namely the bud stage, cap stage, bell stage, is similar to human, although the period is relatively short. The zebrafish lacks oral teeth, but has teeth implanted on the fifth ceratobranchials, the pharyngeal jaws. Adult zebrafish pharyngeal arch skeleton and teeth are like bone tissue. The sclerous tissues structure of the zebrafish tooth is very similar to the enamel and dentin of human tooth. The zebrafish tooth also have pulp cavity, and there is also pulp tissue inside it. Under SEM, there are numerous dentinal tubules into the dentin wall of zebrafish teeth. Tissue structure and ultramicro structure of zebrafish tooth is similarity to the human tooth. Therefore, zebrafish can be used as a model organism of tooth development research.The present study related genes for the type DD-I is still a mystery. To solve this problem, we carried out the related basic research on one-person households in Zhengzhou, Henan China. We determined the VPS4B gene mutation leads to type DD-I disease using linkage analysis, exome sequencing and direct DNA sequencing etc. method, provided molecular basis for the DD-I-type disease diagnosis. Through the analysis of the detection of VPS4B gene, we expound the pathogenic mechanism of VPS4B gene mutation. At the cellular level, we analyzed the regulation of VPS4B on tooth development related to the classical pathway-WNT5A classical pathway with the VPS4B over-express and si-RAN knock-down experiments, provided important information to clarify the mechanism of tooth development. In this report, we focus on vps4b function in zebrafish embryos development using over-expressing and knock-down of vps4b and rescue studies. The aim of current study is to identify this gene in zebrafish and to evaluate the role of this gene in the development of zebrafish especially the development of tooth.Materials and Methods1.We use linkage analysis, whole exome sequencing, sanger sequencing and restriction endonuclease verification to analyze a Henan Zhengzhou Chinese family affected with DD-I and determine the pathogenic gene with mutation site. Total DNA from family DNA samples were extracted by the metheds of phenol-chloroform extraction method. Total RNA from gingival tissue of family was extracted with Kit (RNeasy Mini Kit. Qiagen). Reverse transcription was performed with a ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO).2. HRM technique was used to determine the mutation frequency of VPS4B gene IVS7+46C>G in 300 copies of the local population random samples.3. The spatiotemporal expression profiling of VPS4B gene:RT-PCR was used to determine the gene expression profile in human VPS4B different tissues, cell lines and different stages of brain tissue in mice. Total RNA tissue and cell lines were extracted with Trizol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed with a ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Amplification products were sequenced directly.4. Splicing mutation verification:The transcription of specific amplification primers were designed for us to identified splicing mutation. Identified splicing mutation using the family patients and normal people have been reversed transcriptase well cDNA through RT-PCR glue judgment and Sanger sequencing confirmed.5. The corresponding primers were designed by Olig7. Detected the differences between patients and normal total transcriptional level of VPS4B gene in gingival tissue and the the differences between the normal transcript and the abnormal transcript transcriptional level in gingival tissue of patients using the fluorescent quantitative PCR technology.6. Prediction of protein spatial structure:Three-dimensional structures of WT and mutant VPS4B were predicted with Swiss-Model by importing WT and mutant VPS4B amino acid sequences.7. The abnormal transcription (Jiangsu Suzhou Jin Weizhi Biological Technology Co, Ltd. China. Synthesis.) or normal transcription cDNA of human VPS4B gene was used to clone into the vector pcDNA3.1+-FLAG, then recombinant WT or mutant VPS4B was transfected into HEK-293T cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were collected and extracted total protein at 48 hours after transfection, the protein expression was determined by western blots.8. Cell sublocalization:The normal and abnormal transcription cDNA of human VPS4B gene were used to clone into the vector pEGFP-C1, then recombinant WT or mutant VPS4B was transfected into COS7 cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 48 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).9. To establish cell lines of the VPS4B overexpression and knock down effect on the expression of related gene CHMP4B and WNT5A, expression vector pcDNA3.1+-VPS4B or siRNA was transfected into Human Gingival fibroblasts(HGF) cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, the cells at 48 h post-transfection were collected for real-time RT-PCR. Transcription VPS4B, CHMP4B and WNT5A were analyzed using a SYBR green system. Gene expression levels were calculated using the 2 (-ΔΔCt)method.Target-gene expression was normalized to β-actin expression.10. We use Alizarin Red staining of zebrafish tooth development, for experimental research.11. Identification of homologous gene of human VPS4B in zebrafish Using the online sequence-comparing tools, BLAST from NCBI, the homologous gene of human VPS4B in zebrafish was identified and the similarity between this gene and human VPS4B was analyzed. We used GeneStar to compare12. RT-PCR was used to determine the expression levels of the homologous gene of human VPS4B transcripts in zebrafish. We generated the digoxigenin-labeled antisense cRNA probes according to the protocol of the manufacturer by using the cDNA templates of zebrafish vps4b transcripts.13. Design and synthesis of morpholino oligonucleotides:Promoter-inhibition, splice-blocking, and standard control morpholino oligonucleotides targeting the homologous gene of human VPS4B in zebrafish were designed and synthesized by Gene Tools (USA). One was proposed to suppress gene translation and the another aimed to interfere with the normal splicing of the mRNA of this gene to result in abnormal mRNA, followed by the generation of malfunction protein.14. Microinjection into the embryos of zebrafish:Morpholino oligos were injected into the egg yolk of 1-2-cell-stage Oregon AB embryos according to the standard protocol. To verify the evaluation of the effect of vps4b-MO2, we detected the splicing alterations for vps4b, the homologous gene of human VPS4B in zebrafish, at different time points after the injection of the morpholino. For rescue studies, MOs were coinjected into 1-2-cell-stage zygotes with either wildtype (WT) or mutant VPS4B mRNA; these zygotes were expressed in pCS2+ and were purified by mMSSAGE mMACHINE SP6 Kit (Ambion, USA).15. Observation of the effect of morpholinos on the development of zebrafish:The development of zebrafish embryos after injection of the above morpholinos was observed. Besides, choosing 6dpf as the time point, we observed the dentition by alizarin-staining, and then counted the visually significantly defective embryos and calculated the defective rate and survival rate.16. In situ hybridization to visualize expression of the homologous zebrafish gene vps4b used digoxigenin-labeled antisense cDNA probes generated according to the manufacturer’s protocol (Roche Applied Science, Mannheim, Germany) using zebrafish vps4b cDNA templates. dlx2b, bmp2a, pitx2, wnt5a and hoxb5a38 full-length cDNA was cloned into the pBSK plasmid. Expression of dlx2b, bmp2a and pitx2, wnt5a and hoxb5a were viewed in embryos 56hpf, respectively. Whole-mount in situ hybridization of albino embryos was as previously described 19,21. Probe hybridization was detected by color development with Pierce NBT/BCIP 1-Step Solution.ResultsThrough the analysis of DD-I pedigree genetic map, we found the genetic mode of the family genetic dentin dysplasia is dominance heredity. We use linkage analysis, whole exome sequencing, direct DNA sequencing and restriction endonuclease verification methods to analyze the members of the family. The results show that:In the patient’s Chr18q21.2-Chr18q21.33, the forty-sixth nucleotide intron seventh of the VPS4B gene Gene heterozygous mutation was C>G. flag:CACT C TGTCGâ†'CACT G TGTCGMutation screening frequency:300 copies of the local population random DNA samples were extracted using HRM technology analysis and sequencing. The results show that:the crowd was not found in the locus mutation.VPS4B transcribed in Different human tissues and Cell lines, blood, heart, small intestine, brain, spleen, mandibular, liver, ovary, vortex ear, gums, colon, kidney, gallbladder and DPSC,lovo,and different periods of brain tissue of mice:E7, E11, E15, E17, P1, P3, P7, P10, P15, P16, Adult.The family patients and normal people have been reversed transcriptase well cDNA were used to RT-PCR, glue judgment and Sanger sequencing identified the splicing mutation. Results show:The splicing mutation forming a new transcript. Total RNA from gingival tissue from 2 affected individuals and 5 unaffected individuals in the DDI family was purified with TRIzol method (Invitrogen) and reverse transcribed with a reverse transcription kit (TOYOBO, Osaka, Japan). For Total RNA leve of affected and unaffected individuals in the DDI family using specially primers in the public of two transcriptions. The RNA leve rang of the normally spliced VPS4B transcript between abnormally spliced VPS4B transcripts in the affected individuals of the DDI family with two pairs specially primers. Normally spliced VPS4B transcript real-time RT-PCR with primers spanning the intron7 variant (exon 7 to exon 8), Abnormal spliced VPS4B transcript real-time RT-PCR with primers was located in exon 7 and intron 7 variant. The results show that:Total RNA leve of unaffected (II-2, IV-6) higher than affected (â…¡-1,â…¢-8). In the affected individuals (P<0.001), the transcriptional level of normally spliced VPS4B transcript higher than abnormally spliced VPS4B transcript (P<0.001), aberrant transcriptional level transcripts showed an increasing trend with age (â…¡-2, â…£-4,â…¢-7, â…£-5).The prediction of three-dimensional VPS4B wild-type and mutant protein structure:Mutant VPS4B protein in random angle region into a 15aa (267-281 bit), the 132-134 turned into random angle beta sheet,237-239 turned into random angle alpha helix (Wiss-PdbViewer 4.1.0, http://swissmodel.expasy.org/).The secondary structure prediction of wild-type and mutant proteins of VPS4B:In addition to changes in both the amino acid sequence length, but also the secondary structure is changed after 250AA. The mutant type compared with wild type, the proportion of the secondary structure of the alpha helix and beta folding area reduced, but, the random coil region an increase of about 3%. (SOPMA, http: //nhjy.hzau.edu.cn/kech/swxxx/jakj/dianzi/Bioinf7/Expasy/Expasy8.htm.).In vitro protein level:The constructed vector pcDNA3.1+-FLAG-VPS4B-WT and pcDNA3.1+-FLAG-VPS4B-MUT was transfected into HEK-293T cells, the cells at 48 h post-transfection were collected for western blots. The result shows that: VPS4B-WT expression levels higher than VPS4B-MUT (p<0.005), VPS4B-WT+ VPS4B-MUT expression levels higher than VPS4B-MUT. Subcellular localization (p<0.005):The constructed vector pEGFP-VPS4B-WT and pEGFP-VPS4B-MUT was transfected into COS7cells, the cells at 48 h post-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). The results show that: VPS4B-WT expressed in the cytoplasm, nucleus, but VPS4B-MUT only expressed in cytoplasm.To establish cell lines of the VPS4B overexpression and knock down effect on the expression of related gene CHMP4B and WNT5A,expression vector pcDNA3.1+-VPS4B or siRNA was transfected into HGF cells, the cells at 24 h post-transfection were collected for real-time RT-PCR.The results show that:VPS4B upregulation of CHMP4B, WNT5A and β-catenin expression.The genes of human VPS4B in zebrafish genome, the code names of which in Gene Bank are Danio rerio vps4b. The gene is highly homologous with human VPS4B.vps4b was transcribed from 1 dpf to 5dpf in zebrafish development. The probe hybridization shows the transcript for vps4b was expressed in developing ceratobranchials at 48hpf and later stages.Designed and synthesized vps4b-MO, after microinjection results showed that: Injection of vps4b-MO can cause abnormal development of zebrafish tooth, vps4b-MO and human VPS4B mRNA co-injection showed abnormalities can be rescued by wildtype human VPS4B mRNA.Single injection of human VPS4B mRNA, the zebrafish tooth development has no obvious influence.After the injection of vps4b-MO made the early zebrafish tooth development related gene dlx2b, bmp2a, wnt5a, hoxb5a expression down regulation, pitx2 expression changes, gathered from two sides to the middle.ConclusionsThe linkage analysis, whole exome sequencing, direct DNA sequencing and restriction endonuclease verification methods were used to analyze the family affected with DD-I. The mutations of the first report in the world to determine pathogenic VPS4B gene, and the mutation (IVS7+46C>G) was successfully elucidate the splice mutation to a depth of intron mutation.VPS4B spatial and temporal expression profiling:VPS4B gene expression almost everywhere, the expressed in the gums and jaw tissues suggesting that VPS4B gene closely related to human tooth development.The total RNA leve of unaffected (II-2, IV-6) were higher than affected (II-1, III-8). In the affected individuals (P<0.001), the transcriptional level of normally spliced VPS4B transcript higher than abnormally spliced VPS4B transcript (p<0.001); the aberrant transcriptional level transcripts showed an increasing trend with age (II-2, IV-4, III-7, IV-5), the disease may exist certain relationship with age. Through the predicted results of the wild-type protein and the mutant protein three-dimensional structure and secondary structure, we found that the bigger change of mutant protein three-dimensional structure and secondary structure compared to the wild type protein. In vitro protein level:VPS4B-WT expressed in the cytoplasm, nucleus, VPS4B-MUT only expressed in cytoplasm. VPS4B-WT expression levels higher than VPS4B-MUT (P<0.005), VPS4B-WT+VPS4B-MUT expression levels higher than VPS4B-MUT, The molecular weight of VPS4B-MUT is bigger than VPS4B-WT 1.7KDa. It shows that the VPS4B-MUT proteins can exist stably in the body of a patient. Changes of expression level of the entire transcription mechanisms leading to tooth defects may prompt forhaplotype insufficient dosage.VPS4B gene binding with CHMP4B, promoting the expression of WNT5A, Inhibition of GSK3β promoting fl-catenin phosphorylation, tooth development related gene expression regulated by β-catenin regulate the development of tooth. It is suggested that VPS4B gene through up regulating tooth development related pathways of WNT5A signaling pathway affect tooth development.Both VPS4B gene expression in different stages and branchial archpart of zebrafish, indicates that vps4b may play a role during the development of zebrafish teeth.The antisense morpholino targeting Danio rerio vps4b might inhibit the transcription. The embryos treated with either vps4b -MO1 or vps4b -MO2 showed developmental delay and tooth defects compared to those treated with the control MOs. The phenotype of altered tooth development induced by either of the vps4b-specific morpholinos can be corrected when the embryos are coinjected with WT vps4b, but not with the mutant vps4b mRNA. It’s indicated that vps4b, the homologous gene of human vps4b in zebrafish genome, may play a critical role in tooth development of zebrafish.After the injection of vps4b-MO made the early zebrafish tooth development related gene dlx2b, bmp2a, wnt5a, hoxb5a expression down regulation, it is suggested that vps4b gene through up regulating tooth development related pathways affect zebrafish tooth development.