Phenotype Variations From Initial To Five Years Later And Ultrastructure Of Deciduous Teeth And Virulence Gene Associated With Dentin Dysplasia Type â… 

Hereditary dentin defects of the disease is a class of dentin abnormity autosomal dominant genetic disease, it can be divided into two types: imperfecta(dentinogenesis imperfecta, DGⅠ) and adverse developmental dentin(dentin dysplasia, DD), which is divided into DGⅠ DGⅠ-Ⅰ, DGⅠ-Ⅱ, DGⅠ-Ⅲ three types, DD is divided into DD-Ⅰ, DD-Ⅱ. The topic of our is DD-Ⅰ.Dentin dysplasia type Ⅰ(DD-Ⅰ) is a rare autosomal dominant hereditary disorder which seriously affects the root development of teeth among teenagers causing spontaneous tooth loss. And its incidence is low, only 1/100000, deciduous and permanent teeth are involved. Ⅰt was first reported by the Ballsehmiede in 1920. The salient features of DD-Ⅰ was abnormal roots which are conical or blunt, significantly shorter. The clinical manifestations of DD-Ⅰ was early loss of primary and permanent teeth. Shields describing the elements of DD-Ⅰ clinical diagnosis was that 1) normal crown, deciduous and permanent teeth involved; 2) normal crown dentin, root dentin defected; 3) X-ray showed crown canal occlusion or "thin-shaped", complete occlusion of the root canal, short or void roots. Some patients have unexplained low density apical teeth transmission shadow. The diagnostic elements become clinical diagnostic criteria in later researchers. At present, the study of DD-Ⅰ focuses on familial and phenotypic analyses and reports regarding the ultrastructural study of DD-Ⅰ are few. So far, the cause of DD-Ⅰ remains a mystery. Objective1. To compare phenotype variations of proband and his brother from initial to five years later.2. To observe and analyze the ultrastructure of deciduous teeth with DD-Ⅰ.3. To determine the virulence gene of DD-Ⅰ. Methods1. We recruited 31 members of a four-generation Chinese family, including eleven with dentin defects. We compare with clinical manifestations of the proband and his young brother affected by DD-Ⅰ from initial to five years later. Two deciduous teeth were obtained from individuals affected by DD-Ⅰ. At the same time, two caries-free like-numbered deciduous teeth served as controls. Analyses of these teeth were carried out using stereomicroscopy, light microscopy, and scanning and transmission electron microscopy, respectively.2. We extracted peripheral blood DNA with phenol / chloroform and collected 23 DNA samples of the family which included 10 persons with DD-Ⅰ and 13 normal individuals. And then these 23 DNA samples were analyzed by whole exome sequencing with Affymetrix Genome-wide Human SNP Arrays 6.0, and linkage analysis with Merlin v.1.0.3. According to the result of the above, candidate regions were selected multiple STR loci and explicit rates were set at 80%, 90%, 99% and 100% to determine which chromosome the virulence gene was in. Further analysis, we combined with the target region capture sequencing and SNP screening pedigrees to determine the virulence gene. Results1. By comparing the clinical manifestations and X-ray of the proband and his young brother affected by DD-Ⅰ from initial to five years later, we found that the shape, color and crown size of full mouth teeth with DD-Ⅰ were normal, anterior opening and occlusion disorder no matter in primary dentition or permanent dentition and mixed dentition. As time went on,the teeth were gradual loosening and anterior teeth were gradual loss and periapical radiolucencies were more and larger. Even the permanent tooth germ was also obliteration of all pulp chambers with crescent-shaped pulp remnants parallel to the cementoenamel junction.2. Ⅰn terms of the ultrastructure of deciduous teeth affected by DD-Ⅰ,we found that:(1) thinner dentin;(2) larger scalloped dentinoenamel junctions;(3) micropore in dentinoenamel junctions;(4) a characteristic “stream flowing around boulders” root dentin structure;(5) irregular collagen fibers.3. These 23 DNA samples were analyzed by whole exome sequencing with Affymetrix Genome-wide Human SNP Arrays 6.0, and linkage analysis with Merlin v.1.0. After calculation and graphing, we found the LOD value of chromosome 3 and chromosome 18 is greater than 3. And then two candidate regions were selected multiple STR loci. Combined with pedigree information, we conducted a linkage analysis detected candidate area, the families were two candidate area LOD value calculation. According to the current calculated for each candidate region LOD result, part of the section of chromosome 18 candidate areas LOD greater than 3, there is a very high probability of disease-chain; and the candidate region of chromosome 3 are all LOD is less than 0, the disease can be ruled out with chain. After our groups on chromosome 18 for further analysis, combined with the target region capture sequencing and SNP screening pedigrees determine virulence gene was VPS4 B. And then we discovered the forty-sixth nucleotide intron seventh of the VPS4 B gene heterozygous mutation was C>G. ConclusionsThe results exhibited defined features of DD-Ⅰ in the family and further confirmed that abnormal dentin structure affected both the deciduous and permanent dentitions. Ⅰn addition, these findings may contribute to a better understanding of the pathogenesis of DD-Ⅰ. Through the experimental analysis, we discovered the forty-sixth nucleotide intron seventh of the VPS4 B gene heterozygous mutation was C>G on chromosome 18.And then, we can study the pathogenesis of DD-Ⅰ from the molecular level on this basis. This is another breakthrough in rare disease DD-Ⅰ, while continuing to collect DD-Ⅰ families to verify the pathogenic gene is also essential for the work.