Study On Genes For Dentinogenesis Imperfecta, Amelogenesis Imperfecta And Odontogenic Keratocysts

Genetic disorders on dental hard tissue are mainly focused on identifying causative genes and prevention of the diseases. Odontogenic keratocysts (OKC) arise from the dental laminal epithelium during the dental development. There is a potential relation between OKC and amelogenesis as well as dentinogenesis. The present study involved in exploring and analyzing the causive genes for amelogenesis imperfecta, dentinogenesis imperfecta and odontogenic keratocysts by genetic methods. The study includes the following four parts:Part â…  Phenotypes, genotypes and dentin ultrastructure in families with dentinogenesis imperfectaDentin defects associated with genetic agenesis have been classified as dentinogenesis imperfecta (DGI) type â… , type â…¡, type â…¢ and dentin dysplasia (DD) type â… , type â…¡. DGI type â…  is one of phenotypes of osteogenesis imperfecta (OI), an autosomal dominant disorder that affects bone. Mutations in type I procollogen gene were identified to cause more than 90% of OI. While DGI-â…¡, â…¢ and DD-â…¡ with various dental phenotypes appear to occur as an isolated trait, which usually inherited by an autosomal dominant hereditary transmission. DGI-â…¡, â…¢ and DD-â…¡ have been mapped by linkage analysis to chromosome 4q21.3. Mutations in dentinsialophophoproprotein gene (DSPP) have been demonstrated to be causative for them. DSP and DPP , the main non-collagenous proteins in dentin extracellular matrix, are cleavage products from the single transcript of DSPP gene. To date, 8 mutations in DSP coding region associated with DGI-â…¡ or DD-â…¡ and one compound mutation in DPP domain causative for DGI-â…¢ were reported. DSPP-knockout mice also presented dentin defects similar to those seen inhuman DGI-I1I. Although specific DSPP gene mutations have been detailed investigated, there has been limited characterization of the resulting phenotype with regarding to dentin structure and composition and phenotype discrepancy between different disease-causing mutations. The purpose of the present study was to characterize specific dentin compositional and structural features resulting from specific DSPP gene mutation as well as to define various phenotypes associated with the same or different mutations in families with dentinogenesis imperfecta type II (DGI-II).5 Chinese families with DGI-II or DD-II were investigated for clinical manifestations. Genomic DNA was extracted from peripheral blood samples of members of the five families. Mutation analysis was performed by amplifying the coding regions and splicing junctions of DSP in DSPP gene, sequencing the products, and digesting the products with specific restrictive enzyme. Dentin composition and structure associated with the specific mutation was examined with scanning electronic microscope (SEM), transmission electronic microscopy (TEM) and energy dispersive X-ray spectrometer (EDS). Discoloration, attrition, obliterated pulp chambers were showed in teeth of affected members in 5 families. In addition, "shell" teeth phenotypes were also presented in deciduous teeth of family I. A nonsense mutation (c.133C-*T) in family I and a missense mutation (c.52G-*T) in family III were identified in DSPP gene. The same mutations were reported previously by other authors in different populations with DGI-II. In other three families, no mutation was found in coding regions and splice junctions of DSP. SEM and TEM examination of affected teeth in family I showed that dentin tubules were greatly reduced in number and scattered irregularly; DEJ exhibited an obvious gap and lack of scallop structure; abnormal enamel structure were also presented; amounts of fibril bundles around dentin tubules or fiber-rich areas without surrounding tubules were manifested in the specimen. EDS examination revealed that the quantity of both Ca, P and Mg (wt%) in dentin for DGI-II specimen was lower than that for normal controls. In conclusion, we reported characteristic teeth ultrastructure resulting from a nonsense mutation in DSPP gene and supported that the c.133 C->T and c.52G-?T in DSPP couldbe the two mutation hotspots. The same DSPP mutation was causative for multipleunrelated DGI families with different clinical phenotypes.Part II Genes and infrastructure in amelogenesis imperfectaThe amelogenesis imperfectas (AI) are a clinically and genetically heterogeneous group of disorders that adversely affect enamel development causing abnormalities in the amount, structure, and composition of enamel. AI has been classified as hypoplastics, hypomineralization and hypomaturation with at least 14 different sub-types being recognized on the basis of clinical appearances. Taking all forms of AI into account, the prevalence has been reported to be as high as 1:700 in northern Swede. Extracellular matrix proteins in developing enamel include amelogenin, ameloblastin, enamelin, tuftelin and some proteinases such as kallikrein 4 and enamelysin. Genes coding some enamel matrix proteins have been associated with diverse forms of AI. Numerous mutations on amelogenin gene (Xp22) were identified in X-linked AI with various phenotypes. Genetic mutations on enamelin (4q21) gene were also revealed in some autosomal dominant and recessive forms of AI. Recent researches found mutations on kallikrein 4 (19ql3.4) and enamelysin (Ilq22.3-q23) responsible for autosomal recessive (AR) hypomaturation AI.To date, the defective genes for AI have not been identified in Chinese population. In the present study, we investigated AI families with different clinical phenotypes and hereditary modes, performed mutation analysis on candidate genes as well as ultrastructure examination on the affected deciduous tooth, and try to analyze the correlation between phenotypes and genotypes in different AI families. Four Chinese families with amelogenesis imperfecta were enrolled into the study: family I and II, AD AI; family III, AR AI; family IV, X-linked AI. Genomic DNA was extracted from peripheral blood samples of members of the five families. Based on respective inheritance form, mutation analysis was performed by amplifying all coding regions and splicing junctions of the selected relative candidate gene, examining enamelin gene in family I and II, detecting enamelin and kallikrein 4 gene in family III, screening amelogenin gene in family IV, sequencing the products, and digesting products with specific restrictiveenzyme. Infrastructure of the affected deciduous tooth was examined by SEM. The results showed that no disease-causing mutations were identified in 4 Al families. On SEM observation, enamel of the affected deciduous tooth is markedly reduced in thickness, lacks a prismatic structure in some areas and has an increased interval width between enamel rods. The results indicated that enamelin and kallikrein 4 genes were not causative for autosomal dominant and recessive AI in investigated Chinese families. Other genes may be responsible for AI in these families. AI affected deciduous teeth generated abnormalities in the amount as well as the structure of enamel. Part III Mutation analysis on PTCH gene in families with odontogenic keratocystsOdontogenic keratocysts (OKC) are common cystic lesions that have the potential to behave aggressively and destroy portions of the jaws. OKC can occur as an isolated condition in single or multiple forms or in association with nevoid basal cell carcinoma syndrome (NBCCS). NBCCS is a rare autosomal dominant disorder with a prevalence rate of 1 per 56,000. It is characterized by multiple basal cell carcinomas (BCCs), OKC, palmer or plantar pits, calcification of falx cerebra, skeletal anomalies, and occurrence of various types of tumors. OKC is one of the most prominent features in NBCCS. Familial nonsyndromic OKC has not been reported yet. In recent years, germline or somatic mutations in PTCH gene, a human homologue of the Drosophila segment polarity gene patched, have been identified in patients with NBCCS. PTCH is located on chromosome 9q22.3, consists of 23 exons, and is considered to be a tumor suppressor gene encoding a 1,447 amino acid protein, which functions as a receptor for the Hedgehog (Hh) ligand in the Hh signal transduction pathway. The PTCH protein contains 12 hydrophobic transmembrane domains, two large hydrophilic extracellular loops, and intracellular amino- and carboxyl-terminal regions. Mutations in the PTCH gene scattered irregularly in various domains and no mutation hot spots have been identified. Most alterations are nonsense or frameshift mutations which are predicted to cause truncation of the protein with premature stop codons. Missense or spicing site mutations were also detected. Loss of heterozygosity at 9q22.3-q31 and a somatic mutation of PTCH were also found insporadic OKC. However, the germline mutations in PTCH have not been described previously in families with non-syndrome-related OKC.In this study, we investigated germline mutations of PTCH in Chinese families with OKC in association with or without NBCCS. Three Chinese families with OKC were enrolled into the study. All 23 exons and the exon-intron boundaries of the PTCH gene were amplified by polymerase chain reaction. The direct DNA sequence analysis was used for mutation detection. Three novel germline mutations in PTCH were identified, including a missense mutation (p.S1089>P) in family 1 with isolated OKC, a nonsense mutation (p.Q160X) in family 2 and a de novo mutation of 10-bp deletion (c.768₇77delGACAAACTTC) in family 3.In conclusion, the results suggest that isolated OKC can be inherited in an autosomal dominant mode, and that the first germline mutation in the PTCH gene was identified in a Chinese family with isolated OKC. The first de novo 10-bp deletion associated with NBCCS have also been identified in a Chinese population. The study suggests that that the PTCH gene responsible for NBCCS also plays an important role in the formation of the cysts in jaws even when they are not syndrome-related.