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A Novel Mutation in the DSPP Gene Associated with Dentinogenesis Imperfecta Type II
1 Department of Cell and Developmental Biology, Dental Research Institute & BK21 program Correspondence: * corresponding author, pedoman{at}snu.ac.kr
Hereditary dentin defects are divided into dentinogenesis imperfecta and dentin dysplasia. We identified a family segregating severe dentinogenesis imperfecta. The kindred spanned four generations and showed an autosomal-dominant pattern of inheritance. The proband was a child presenting with a severely affected primary dentition, with wide-open pulp chambers and multiple pulp exposures, resembling a DGI type III (DGI-III) pattern. We hypothesized that a mutation in the DSPP gene is responsible for this severe phenotype. Mutational analyses revealed a novel mutation (c.53T>A, p.V18D) near the intron-exon boundary in the third exon of the DSPP gene. We analyzed the effect of the mutation by means of an in vitro splicing assay, which revealed that the mutation did not affect pre-mRNA splicing. Further studies are needed for a better understanding of the nature of the disease and the development of an appropriate treatment strategy.
Key Words: dentin sialophosphoprotein • DSPP • dentinogenesis imperfecta • dentin dysplasia • splicing assay
Dentin matrix is secreted by odontoblasts in developing dental tooth germs following a series of interactions between the oral ectoderm and underlying neural-crest-derived ectomesenchyme. The dentin matrix mineralizes following its secretion. Subsequently, odontoblasts migrate toward the pulp, leaving odontoblast processes within the matrix (Thesleff, 2003). The resulting dentin has unique structural and mechanical properties. The mineral content and microhardness of dentin are between those of enamel and bone, allowing it to support enamel and to endure masticatory forces. Odontoblast processes organize the structure of the mineralized matrix into peritubular and intertubular regions and project cellular capabilities, such as the reception of sensory information and responses against noxious stimuli through the entire thickness of dentin and to the dentino-enamel junction (Torneck, 1998). While hereditary conditions affecting dentin have long been observed, the term "hereditary opalescent dentin" was adopted to describe conditions of diseases consisting of only dental phenotypes (Hodge et al., 1936). Likewise, the term "dentinogenesis imperfecta" was introduced to describe the common dental phenotypes of osteogenesis imperfecta and hereditary opalescent dentin (Roberts and Schour, 1939). The classification system currently in use recognizes 3 types of dentinogenesis imperfecta (DGI-I, DGI-II, and DGI-III) and 2 types of dentin dysplasia (DD-I and DD-II). The class distinctions vary according to the clinical phenotype, radiographic findings, and severity and were proposed in the report of a dentin dysplasia type II family (Shields et al., 1973). When this classification system was first published, a detailed genetic basis for these diseases was unknown, and thus, some have suggested that it may be necessary to update the nomenclature for hereditary dentin defects after the underlying genetic defects have been identified (Bixler, 1976; Dean et al., 1997). Recent studies have shown that defects in the dentin sialophosphoprotein (DSPP) gene cause DGI-II (Xiao et al., 2001; Zhang et al., 2001; Kim et al., 2004; Malmgren et al., 2004; Holappa et al., 2006; McKnight et al., 2008; Song et al., 2008), DGI-III (Dong et al., 2005; Kim et al., 2005), and DD-II (Rajpar et al., 2002). To date, despite the existence of other candidate genes for hereditary dentin defects (Ye et al., 2004), no disease-causing mutations outside of the DSPP gene have been identified (Beattie et al., 2006). In this study, we identified a family having severe dentinogenesis imperfecta, with features such as wide-open pulp chambers and multiple pulp exposures reported in DGI-III. We performed mutational analyses to identify the underlying molecular genetic etiology.
Enrollment of Human Participants The protocols for the current study, as well as participant consents, were reviewed and approved by the Institutional Review Boards at Seoul National University Dental Hospital. Appropriate informed consent was obtained from all participants.
Primer Design, Polymerase chain-reaction (Pcr), and DNA sequencing
BamHI Digestion of Pcr Products
In vitro splicing Assay
Clinical Findings The proband (IV:20) first presented at 2.5 yrs of age at the Pediatric Dental Clinic at Seoul National University Dental Hospital. At the time of presentation, the deciduous dentition displayed severe attrition and marked discoloration. Periapical inflammation was observed in several teeth in which the pulp tissue was exposed by attrition. However, a treatment appointment was missed, and the individual did not appear for an additional 2 yrs. The oral condition of the proband by age 4.5 yrs had markedly declined. Several teeth were missing, and the remaining teeth were worn down to their roots and exhibited pulpal pathology. Only a few lower anterior teeth were without exposure of pulp and still had dentin covering their occlusal surfaces (Fig. 1  ). The lower deciduous incisors (#71, 72, 82) had some, but not complete, pulpal obliteration, and somewhat wide pulp chambers were still present in the lower deciduous canines (Fig. 2).
For the purposes of this study, 27 family members were recruited, consisting of 15 affected and 12 unaffected members (Fig. 3 ). There was no history of bone fragility or symptoms of progressive hearing loss in the family. Most affected members were edentulous and wore complete dentures, although one member (III:23) had overdentures over root rests (Fig. 1G ). The permanent teeth of affected members were also severely affected (IV:17, Fig. 1F).
Mutation Results Sequencing analysis revealed the existence of a T to A transversion in exon 3 (g.1198T>A, c.53T>A, and p.V18D, based on reference sequences NC_000004 and NM_104208) of the DSPP gene, and this sequence variation correlated perfectly with the presence of the disease (Fig. 3 ). Further, this sequence variation was not present in 100 unaffected control individuals from the Korean population (data not shown), suggesting that the mutation is not a common variant of the DSPP gene.
In vitro splicing assay of normal and mutated pSPL3 vectors showed a single band of normally spliced mRNA, which indicated that the mutation did not affect pre-mRNA processing (Fig. 4
The clinical phenotype in our proband was severe, with features reported in DGI-III. The DGI-III classification was developed to describe the dental phenotypes found in the Brandywine triracial isolate of Native American Indians, African Americans, and Caucasians of European decent in the USA. Clinically, the DGI-III-affected teeth varied in color and shape, like those described for DGI-I and DGI-II, but unlike these conditions, their deciduous teeth displayed multiple pulp exposures. Radiographically, the deciduous teeth showed considerable variation in appearance, ranging from total pulp obliteration, to normal, and even to shell-like teeth (Shields et al., 1973). Multiple pulp exposures and shell-like teeth were the two main features previously used to distinguish DGI-III from DGI-II, but are no longer considered unique to DGI-III (Kim and Simmer, 2007). ‘Shell-like teeth’ was a phenotypic variation originally found in children of the Brandywine isolate (Witkop et al., 1966); however, pulp chambers in the primary dentitions of persons with DGI-II and of multiple ethnicities are initially abnormally wide, but progressively show obliteration (Heimler et al., 1985; Tanaka and Murakami, 1998; Sapir and Shapira, 2001). Overlap of the phenotypic features in DGI-II and DGI-III presumably relates to their similar genetic causes: The same mutation of the DSPP gene (c.52G>T) causes DGI-II and DGI-III in different families (Kim et al., 2005; Holappa et al., 2006; Song et al., 2006). Furthermore, dental phenotypes in this family provide additional evidence that DGI-III is not a distinct entity, but a slightly varied phenotype with a reduced rate of dentin formation in the early deciduous dentition.
The mutation (g.1198T>A, c.53T>A, p.V18D) identified in this family was located adjacent to a proposed mutational ‘hotspot’ (g.1197G>T, c.52G>T) in the DSPP gene, occurring at the second nucleotide of exon 3. The Valine residue at this position is conserved across known species, such as human (NP_055023), mouse (NP_034210), rat (NP_036922), and pig (NP_998942). Given the proximity to the border of exon 3, the mutation was suspected to have some influence on normal pre-mRNA splicing. However, in silico analysis of the effect of this mutation on the pre-mRNA splicing was not enough to accept it as a mutation affecting splicing. There were only small changes in the splice acceptor site prediction value (0.97 To verify the effect of the mutation on the pre-mRNA splicing, we performed an in vitro splicing assay. The result revealed that the mutation did not affect the pre-mRNA splicing process, as indicated by in silico analysis. We conclude that the result of the mutation in this study is unlikely to be aberrant pre-mRNA splicing, but rather the consequence of the amino acid change (Valine to Aspartic acid at the codon position 18). Recently, it has been suggested that all the DSPP mutations reported in the DSP portion cause their phenotypes through disruption of signal peptide processing, resulting in interference with protein processing (McKnight et al., 2008). The novel mutation identified in this study replaced the otherwise-invariant Val18 with a hydrophilic Asp18 without an abnormal pre-mRNA splicing. It is possible that the signal peptide cleavage is impaired by this change. However, at this time, it is not known whether the signal peptide of the mutant protein is improperly processed and the mutant protein is accumulated in the odontoblast, or if the conserved Val18 is critical for proper DSPP folding or post-translational modification after signal peptide cleavage. It should be further investigated whether the mutant protein activates ER stress, which might affect other matrix protein processing (including DSPP itself) and/or cell function. Proper genotype-phenotype correlations as well as correct diagnosis are essential for an understanding of the nature of disease as well as the development of treatment strategy in gene therapy. In this study, we were able to analyze the effect of the mutation with the help of an in vitro splicing assay. Identification of the disease-causing mutation and characterization of the molecular pathogenesis may lead us to a better understanding of this disease and the development of a novel strategy of disease modulation.
We thank all the family members for their cooperation. We thank Dr. Alessandro Stella, University of Bari, Italy, for the pSPL3 splicing vector used in this study. This research was supported by grant 03-2005-003 from the Seoul National University Dental Hospital Research Fund. We thank Dr. James Simmer for his careful review of this manuscript. Received for publication February 15, 2007. Revision received September 24, 2008. Accepted for publication October 14, 2008.
Journal of Dental Research, Vol. 88, No. 1,
51-55 (2009)
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