AAV6-Mediated Gene Therapy Prevents Developmental Dentin Defects in a Dentinogenesis Imperfecta Type â ¢ Mouse Model.

Dentin is a major type of hard tissue of teeth and plays essential roles for normal tooth function. Odontoblasts are responsible for dentin formation. Mutations or deficiency in various genes affect the differentiation of odontoblasts, leading to irreversible dentin developmental defects in animals and humans. Whether such dentin defects can be reversed by gene therapy for odontoblasts remains unknown. In this study, we compare the infection efficiencies of six commonly used adeno-associated virus (AAV) serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVDJ) in cultured mouse odontoblast-like cells (OLCs). We show that AAV6 serotype infects OLCs with the highest efficiency among the six AAVs. Two cellular receptors, which are able to recognize AAV6, AAV receptor (AAVR), and epidermal growth factor receptor (EGFR), are strongly expressed in the odontoblast layer of mouse teeth. After local administration to mouse molars, AAV6 infects the odontoblast layer with high efficiency. Furthermore, AAV6-Mdm2 was successfully delivered to teeth and prevents the defects in odontoblast differentiation and dentin formation in Mdm2 conditional knockout mice (a mouse model of dentinogenesis imperfecta type Ⅲ). These results suggest that AAV6 can serve as a reliable and efficient vehicle for gene delivery to odontoblasts through local injection. In addition, human OLCs were also successfully infected by AAV6 with high efficiency, and both AAVR and EGFR are strongly expressed in the odontoblast layer of extracted human developing teeth. These findings suggest that AAV6-mediated gene therapy through local injection may be a promising treatment approach for hereditary dentin disorders in humans.

Mouse Dspp frameshift model of human dentinogenesis imperfecta.

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

Phenotypic Properties of Collagen in Dentinogenesis Imperfecta Associated with Osteogenesis Imperfecta.

INTRODUCTION: Dentinogenesis imperfecta type 1 (OIDI) is considered a relatively rare genetic disorder (1:5000 to 1:45,000) associated with osteogenesis imperfecta. OIDI impacts the formation of collagen fibrils in dentin, leading to morphological and structural changes that affect the strength and appearance of teeth. However, there is still a lack of understanding regarding the nanoscale characterization of the disease, in terms of collagen ultrastructure and mechanical properties. Therefore, this research presents a qualitative and quantitative report into the phenotype and characterization of OIDI in dentin, by using a combination of imaging, nanomechanical approaches. METHODS: For this study, 8 primary molars from OIDI patients and 8 primary control molars were collected, embedded in acrylic resin and cut into longitudinal sections. Sections were then demineralized in 37% phosphoric acid using a protocol developed in-house. Initial experiments demonstrated the effectiveness of the demineralization protocol, as the ATR-FTIR spectral fingerprints showed an increase in the amide bands together with a decrease in phosphate content. Structural and mechanical analyses were performed directly on both the mineralized and demineralized samples using a combination of scanning electron microscopy, atomic force microscopy, and Wallace indentation. RESULTS: Mesoscale imaging showed alterations in dentinal tubule morphology in OIDI patients, with a reduced number of tubules and a decreased tubule diameter compared to healthy controls. Nanoscale collagen ultrastructure presented a similar D-banding periodicity between OIDI and controls. Reduced collagen fibrils diameter was also recorded for the OIDI group. The hardness of the (mineralized) control dentin was found to be significantly higher (p<0.05) than that of the OIDI (mineralized) dentine. Both the exposed peri- and intratubular dentinal collagen presented bimodal elastic behaviors (Young's moduli). The control samples presented a stiffening of the intratubular collagen when compared to the peritubular collagen. In case of the OIDI, this stiffening in the collagen between peri- and intratubular dentinal collagen was not observed and the exposed collagen presented overall a lower elasticity than the control samples. CONCLUSION: This study presents a systematic approach to the characterization of collagen structure and properties in OIDI as diagnosed in dentin. Structural markers for OIDI at the mesoscale and nanoscale were found and correlated with an observed lack of increased elastic moduli of the collagen fibrils in the intratubular OIDI dentin. These findings offer an explanation of how structural changes in the dentin could be responsible for the failure of some adhesive restorative materials as observed in patients affected by OIDI.

Dentinogenesis imperfecta type II in Swedish children and adolescents.

BACKGROUND: Dentinogenesis imperfecta (DGI) is a heritable disorder of dentin. Genetic analyses have found two subgroups in this disorder: DGI type I, a syndromic form associated with osteogenesis imperfecta (OI), and DGI type II, a non-syndromic form. The differential diagnosis between types I and II is often challenging. Thus, the present cross-sectional study had two aims: to (i) investigate the prevalence and incidence of DGI type II among Swedish children and adolescents and (ii) search out undiagnosed cases of DGI type I by documenting the prevalence of clinical symptoms of OI in these individuals. We invited all public and private specialist pediatric dental clinics (n = 47) in 21 counties of Sweden to participate in the study. We then continuously followed up all reported cases during 2014-2017 in order to identify all children and adolescents presenting with DGI type II. Using a structured questionnaire and an examination protocol, pediatric dentists interviewed and examined patients regarding medical aspects such as bruising, prolonged bleeding, spraining, fractures, hearing impairment, and family history of osteoporosis and OI. Joint hypermobility and sclerae were assessed. The clinical oral examination, which included a radiographic examination when indicated, emphasized dental variables associated with OI. RESULTS: The prevalence of DGI type II was estimated to be 0.0022% (95% CI, 0.0016-0.0029%) or 1 in 45,455 individuals. Dental agenesis occurred in 9% of our group. Other findings included tooth retention (17%), pulpal obliteration (100%), and generalized joint hypermobility (30%). Clinical and radiographic findings raised a suspicion of undiagnosed OI in one individual, a 2-year-old boy; he was later diagnosed with OI type IV. CONCLUSIONS: These results show a significantly lower prevalence of DGI type II than previously reported and point to the importance of excluding OI in children with DGI.

Osteogenesis imperfecta with ectopic mineralizations in dentin and cementum and a COL1A2 mutation.

We report a Thai father (patient 1) and his daughter (patient 2) affected with osteogenesis imperfecta type IV and dentinogenesis imperfecta. Both were heterozygous for the c.1451G>A (p.Gly484Glu) mutation in COL1A2. The father, a Thai boxer, had very mild osteogenesis imperfecta with no history of low-trauma bone fractures. Scanning electron micrography of the primary teeth with DI of the patient 2, and the primary teeth with DI of another OI patient with OI showed newly recognized dental manifestations of teeth with DI. Normal dentin and cementum might have small areas of ectopic mineralizations. Teeth affected with DI have well-organized ectopic mineralizations in dentin and cementum. The "French-fries-appearance" of the crystals at the cemento-dentinal junction and abnormal cementum have never been reported to be associated with dentinogenesis imperfecta, either isolated or osteogenesis imperfecta-associated. Our study shows for the first time that abnormal collagen fibers can lead to ectopic mineralization in dentin and cementum and abnormal cementum can be a part of osteogenesis imperfecta.

Bone Morphogenetic Protein 2 Coordinates Early Tooth Mineralization.

Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Odontoblast and ameloblast differentiation involves reciprocal and sequential epithelial-mesenchymal interactions. Similar to early tooth development, various Bmps are expressed during this process, although their functions have not been explored in detail. Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. In developing molars, Bmp2LacZ reporter mice revealed restricted expression of Bmp2 in early polarized and functional odontoblasts while it was not expressed in mature odontoblasts. Loss of Bmp2 in neural crest cells, which includes all dental mesenchyme, caused a delay in dentin and enamel deposition. Immunohistochemistry for nestin and dentin sialoprotein (Dsp) revealed polarization defects in odontoblasts, indicative of a role for Bmp2 in odontoblast organization. Surprisingly, pSmad1/5/8, an indicator of Bmp signaling, was predominantly reduced in ameloblasts, with reduced expression of amelogenin ( Amlx), ameloblastin ( Ambn), and matrix metalloproteinase ( Mmp20). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry showed that loss of Bmp2 resulted in increased expression of the Wnt antagonists dickkopf 1 ( Dkk1) in the epithelium and sclerostin ( Sost) in mesenchyme and epithelium. Odontoblasts showed reduced Wnt signaling, which is important for odontoblast differentiation, and a strong reduction in dentin sialophosphoprotein ( Dspp) but not collagen 1 a1 ( Col1a1) expression. Mature Bmp2-deficient teeth, which were obtained by transplanting tooth germs from Bmp2-deficient embryos under a kidney capsule, showed a dentinogenesis imperfecta type II-like appearance. Micro-computed tomography and scanning electron microscopy revealed reduced dentin and enamel thickness, indistinguishable primary and secondary dentin, and deposition of ectopic osteodentin. This establishes that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

Immunocytochemical detection of dentin matrix proteins in primary teeth from patients with dentinogenesis imperfecta associated with osteogenesis imperfecta.

Dentinogenesis imperfecta determines structural alterations of the collagen structure still not completely elucidated. Immunohistochemical analysis was used to assay Type I and VI collagen, various non-collagenous proteins distribution in human primary teeth from healthy patients or from patients affected by type I dentinogenesis imperfecta (DGI-I) associated with osteogenesis imperfecta (OI). In sound primary teeth, an organized well-known ordered pattern of the type I collagen fibrils was found, whereas atypical and disorganized fibrillar structures were observed in dentin of DGI-I affected patients. Expression of type I collagen was observed in both normal and affected primary teeth, although normal dentin stained more uniformly than DGI-I affected dentin. Reactivity of type VI collagen was significantly lower in normal teeth than in dentin from DGI-I affected patients (P<0.05). Expressions of dentin matrix protein (DMP)-1 and osteopontin (OPN) were observed in both normal dentin and dentin from DGI-I affected patients, without significant differences, being DMP1 generally more abundantly expressed. Immunolabeling for chondroitin sulfate (CS) and biglycan (BGN) was weaker in dentin from DGI-I-affected patients compared to normal dentin, this decrease being significant only for CS. This study shows ultrastructural alterations in dentin obtained from patients affected by DGI-I, supported by immunocytochemical assays of different collagenous and non-collagenous proteins.

Diseases of the tooth: the genetic and molecular basis of inherited anomalies affecting the dentition.

In humans, inherited variation in the number, size, and shape of teeth within the dentitions are relatively common, while rarer defects of hard tissue formation, including amelogenesis and dentinogenesis imperfecta, and problems associated with tooth eruption are also seen. In many cases, these anomalies occur in isolation, but they can also present as a feature of numerous well-characterized developmental syndromes. Complex reiterative signaling between the epithelium and mesenchyme is a feature of normal tooth development in the embryo, occurring from early patterning through morphogenesis, hard tissue formation and during root development. Significant events also occur during postnatal development of the dentition, including hard tissue maturation and tooth eruption. In the last decade, advances in human and mouse genetics have meant that in many cases candidate genes have been identified for these anomalies. These genes have provided a useful platform for developmental biologists, allowing them to begin elucidating how these signals interact to generate a functional dentition and understand the mechanisms underlying many of the anomalies that are seen in human populations. In this article, we review current concepts relating to the developmental biology of tooth number, size, and shape, formation of the dental hard tissues and eruption of the tooth into the oral cavity. We will focus on the molecular mechanisms underlying these processes in both health and disease.

Rough endoplasmic reticulum trafficking errors by different classes of mutant dentin sialophosphoprotein (DSPP) cause dominant negative effects in both dentinogenesis imperfecta and dentin dysplasia by entrapping normal DSPP.

Families with nonsyndromic dentinogenesis imperfecta (DGI) and the milder, dentin dysplasia (DD), have mutations in one allele of the dentin sialophosphoprotein (DSPP) gene. Because loss of a single Dspp allele in mice (and likely, humans) causes no dental phenotype, the mechanism(s) underling the dominant negative effects were investigated. DSPP mutations occur in three classes. (The first class, the mid-leader missense mutation, Y6D, was not investigated in this report.) All other 5' mutations of DSPP result in changes/loss in the first three amino acids (isoleucine-proline-valine [IPV]) of mature DSPP or, for the A15V missense mutation, some retention of the hydrophobic leader sequence. All of this second class of mutations caused mutant DSPP to be retained in the rough endoplasmic reticulum (rER) of transfected HEK293 cells. Trafficking out of the rER by coexpressed normal DSPP was reduced in a dose-responsive manner, probably due to formation of Ca2+-dependent complexes with the retained mutant DSPP. IPV-like sequences begin many secreted Ca2+-binding proteins, and changing the third amino acid to the charged aspartate (D) in three other acidic proteins also caused increased rER accumulation. Both the leader-retaining A15V and the long string of hydrophobic amino acids resulting from all known frameshift mutations within the 3'-encoded Ca2+-binding repeat domain (third class of mutations) caused retention by association of the mutant proteins with rER membranes. More 5' frameshift mutations result in longer mutant hydrophobic domains, but the milder phenotype, DD, probably due to lower effectiveness of the remaining, shorter Ca2+-binding domain in capturing normal DSPP protein within the rER. This study presents evidence of a shared underlying mechanism of capturing of normal DSPP by two different classes of DSPP mutations and offers an explanation for the mild (DD-II) versus severe (DGI-II and III) nonsyndromic dentin phenotypes. Evidence is also presented that many acidic, Ca2+-binding proteins may use the same IPV-like receptor/pathway for exiting the rER.

Molecular basis of human dentin diseases.

In recent years, substantial progress has been made regarding the molecular etiology of human structural tooth diseases that alter dentin matrix formation. These diseases have been classified into two major groups with subtypes: dentin dysplasia (DD) types I and II and dentinogenesis imperfecta (DGI) types I-III. Genetic linkage studies have identified the critical loci for DD-II, DGI-II, and DGI-II to human chromosome 4q21. Located within the common disease loci for these diseases is cluster of dentin/bone genes that includes osteopontin (OPN), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP). To date, only mutations within dentin sialophosphoprotein have been associated with the pathogenesis of dentin diseases including DGI types-II and -III and DD-II. In this article, we overview the recent literature related to these dentin genetic diseases, their clinical features, and molecular pathogenesis.

The importance of intrafibrillar mineralization of collagen on the mechanical properties of dentin.

It is widely held that the hardness and modulus of dentin increase in proportion to the mineral concentration. To test this belief, we measured hardness and modulus of normal dentin and an altered form of dentin without gap-zone mineralization in wet and dry conditions by AFM nanoindentation to determine if the modulus and hardness scale linearly with mineral concentration. Mineral concentrations in the mid-coronal location of the normal and altered dentins were 44.4 vol% and 30.9 vol%, respectively. Surrounding the pulp of the altered dentin was a region of higher mineralization, 40.5 vol%. The indentation modulus of normal dentin was 23.9 (SD = 1.1) GPa dry and 20.0 (SD = 1.0) GPa wet. In mid-coronal regions of the altered dentin, the indentation modulus was 13.8 (SD = 2.0) GPa dry and 5.7 (SD = 1.4) GPa wet. In the more mineralized regions of the altered dentin, the modulus was 20.4 (SD = 1.8) GPa dry and 5.3 (SD = 0.8) GPa wet; the properties of the altered wet dentin did not correlate with mineral concentration. The results of this study raise doubt as to whether mineral concentration alone is a sufficient endpoint for assessing the success or failure of remineralization approaches in restorative dentistry.

Dental structural diseases mapping to human chromosome 4q21.

Genetic diseases affecting tooth structure have been classified by the tissue affected enamel versus dentin, and their pattern of inheritance autosomal dominant, autosomal recessive, or X-linked. Advances in molecular genetics and the Human Genome Project have provided substantial progress regarding the identification of genes involved in the pathogenesis of human diseases. These include dental diseases affecting enamel and dentin formation: amelogenesis imperfecta (AI), dentinogenesis imperfecta (DGI) types II and III, and dentin dysplasia (DD) type II. Linkage studies using large informative families have provided insight identifying two proximal gene clusters on human chromosome 4q21 that contain the critical loci for five dental structural diseases. Studies related to the autosomal dominant forms of AI, representing approximately 85% of all cases, have established linkage to 4q21 for two forms: local hypoplastic and smooth hypoplastic AI. Two enamel matrix proteins, ameloblastin and enamelin, have been mapped within the critical regions for these diseases. Located more toward the telomere is another cluster containing loci for three dentin diseases: DGI type II, type III, and DD type II. Located within an overlapping segment of these diseases is a dentin/bone gene cluster that contains osteopontin, bone sialoprotein, matrix extracellular phosphoglycoprotein also known as osteoblast/osteocyte factor 45 or osteoregulin, dentin matrix protein 1, and dentin sialophosphoprotein. Continuing molecular genetic studies will facilitate the identification of novel tooth matrix proteins within these two tooth matrix gene clusters as well as the identification of additional autosomal dominant AI loci.

The non-collagenous dentin matrix proteins are involved in dentinogenesis imperfecta type II (DGI-II).

Dentinogenesis Imperfecta type II (DGI-II) is a localized form of mesodermal dysplasia of the dentin affecting both the primary and permanent dentitions. This is an autosomal-dominant disease in which there is a disorder in dentin mineralization. Several studies have localized DGI-II to human chromosome 4 in the region 4q 12-21. Many ECM genes-such as OPN, DMP1, DMP2, DMP3 (DSPP), and BSP-have been mapped to the same locus. Biochemical studies indicated that dentin phosphophoryn (DMP2) might be a candidate gene in DGI-II. In this study, we have used histological and RFLP analyses of tissues from a DGI-II-affected patient, as compared with two normal controls, to determine if DMP1, 2, or 3 was linked to DGI-II. The histology of the affected tooth was very different in the DGI-II patient as compared with the normals. In particular, the dentinal tubules in the DGI-II patient were very irregular, which could be the result of perturbations in the process of dentin formation. Patient and control DNA samples were digested with EcoRI or PstI and Southern-hybridized with the DMP1, DMP2, and DMP3 cDNAs. Few differences in the restriction pattern were observed between affected and normal samples for DMP1 and DMP3-3' region (phosphophoryn-like sequences) probes. On the other hand, DMP2 showed a dramatic shift in the restriction pattern in DGI-II. This study suggests that the different restriction enzyme digestion profiles of the DNA from the DGI-II patient, as probed by DMP2, might be related to the defective mineralization of dentin in DGI-II.

Dental manifestations of osteogenesis imperfecta and abnormalities of collagen I metabolism.

The in vitro protein-chemical features and the molecular background of osteogenesis imperfecta (OI), a heritable disorder of collagen I metabolism, have been elucidated in recent years. The aim of our study was to find the prevalence of dentinogenesis imperfecta (DI) and other dental anomalies in 88 patients with OI, to compare clinical with radiologic abnormalities, and to correlate these clinical/radiologic findings with the results of gel electrophoresis and molecular studies of collagen I. Twenty-eight percent of OI patients had DI. Most patients with DI had radiologic abnormalities, but some patients had radiologic signs compatible with DI, but no clinical signs of DI. OI type I patients with DI were more severely affected by OI than those without DI. In OI type III and IV, in contrast, there was no difference in overall severity between patients with and without DI. DI was not associated with any particular molecular aberration in any OI type. If defining DI from the presence of both clinical and radiologic signs, collagen I produced by cultured fibroblasts was qualitatively abnormal from all OI patients with DI. Some OI patients had dental abnormalities not resembling DI. A qualitative collagen abnormality could not be found in any of these patients. Denticles, i.e., calcifications within the pulpal cavity, were found more frequently in OI patients than in control subjects.

Unusual forms of collagen in human dentin.

Unusual fibrillar and segmental forms of collagen have been documented in several tissues, including rodent dentin - but not human dentin, which was analyzed here for the presence of such structures. Of six normal human permanent and deciduous teeth examined, cusps of two deciduous molars displayed atypical formations, designated here symmetrical collagen segments (SCSs). They were detected inside of dentinal tubules. It was evident that the 264-nm long, cross-banded SCSs were occasionally arranged vertically and laterally in a staggered manner into thick, irregularly shaped aggregates. Two deciduous incisors from patients with dentinogenesis imperfecta associated with osteogenesis imperfecta were also studied. SCSs were not observed, but fibrous long-spacing-like collagen was rarely found intratubularly in one tooth.

Altered collagen expression in human dentin: increased reactivity of type III and presence of type VI in dentinogenesis imperfecta, as revealed by immunoelectron microscopy.

We used transmission immunoelectron microscopy and polyclonal antibodies to study the reactivities of Types III and VI collagen in dentin of normal human permanent and primary teeth and in primary teeth from five patients with dentinogenesis imperfecta (DI) associated with osteogenesis imperfecta and occurring as a single trait. In the normal permanent tooth, reactivity of Type III collagen was occasional and, where present, peritubular. Staining of normal primary teeth was less occasional but still rare, whereas the abnormal dentin stained more uniformly. Atypical, non-striated fibrillar structures that also showed Type III collagen reactivity were observed in dentin of two of the three patients with DI as a single trait. Later, these two patients proved to be first cousins. Unlike antibodies to the N-terminal pro-peptide of Type I pro-collagen, antibodies to the C-terminal telopeptide of Type I collagen, used for comparison stained the affected dentin homogeneously. Reactivity of Type VI collagen, not detected in normal teeth, was seen in the dentin of all abnormal teeth, in association with non-fibrillar delicate material. This study also shows that although readily detectable in dentin affected by DI, Type III collagen is a minor constituent of normal human dentin matrix.

ED-A region-containing isoform of cellular fibronectin is present in dentin matrix in dentinogenesis imperfecta associated with osteogenesis imperfecta.

To elucidate the defective dentin formation in osteogenesis imperfecta (OI), we analyzed the expression of selected fibronectin (FN) isoforms in the dentin matrix of a patient with dentinogenesis imperfecta (DI) associated with OI, and in normal teeth. Frozen tooth sections were immunostained with three monoclonal antibodies (MAbs). The MAb recognizing the major cell-binding region (f-33), shared by plasma FN (pFN) and cellular FN (cFN), stained the pulp of normal adult permanent teeth intensely, while no reactivity was present in predentin, (demineralized) dentin, or dental cementum. The periodontal ligament stained unevenly. The dentin matrix of the patient with OI displayed reactive zones, alternating layerwise or concentrically with non-reactive ones. Staining throughout the connective tissue of adult oral mucosa, analyzed for the form of FN present, was intense, and in dermis, which was also studied, it was moderate. Reactivities in dental tissues with the MAb specific for the ED-A region (IST-9), included in cFN but not pFN, were similar to those with MAb f-33. The mucosal connective tissue stained weakly and dermis was negative, except that nerves and endothelia of some large blood vessels stained clearly. The MAb specific for the ED-B segment (BC-1), also included in cFN only, did not stain any of the tissues analyzed. The results suggest that, unlike mucosal and dermal FNs, FNs in the dental tissues are largely cellular, and also that dentin formation in OI may be completed by successive generations of pulpal fibroblasts differentiated into hard-tissue-forming cells.

Presence of dentin phosphoprotein in molars of a patient with dentinogenesis imperfecta type II.

Dentin phosphoprotein (DPP) is the major noncollagenous protein component of the dentin extracellular matrix. This highly acidic phosphorylated protein is solely expressed by the ectomesenchymal-derived odontoblast cells of the tooth organ. Several biochemical studies have suggested diminished levels of, or even the absence of, this protein, which is associated with the human genetic disease dentinogenesis imperfecta (DGI) type II. However, more recent molecular studies have established that the DPP gene locus is not localized to the region of human chromosome 4 (4q13-q21), where several previous linkage analysis studies have mapped DGI types II and III. The purpose of this study was to determine the presence or absence of DPP in the dentition of a patient affected with DGI type II using a sensitive and specific immunodetection method with a polyclonal antibody against mouse DPP. Our results indicate that a 95-kDa protein, immunologically crossreactive with the DPP antibody, was detected within the dentin extracellular matrix of molars isolated from both a proband affected with DGI-II and from an age-matched normal individual. In addition, both DGI-II and normal individuals showed comparable DPP in situ degradation associated with dentin extracellular matrix maturation. These results strongly support the hypothesis that the DPP structural gene does not produce the gene product primarily responsible for the human genetic disease DGI type II.

Type II collagen defect in two sibs with the Goldblatt syndrome, a chondrodysplasia with dentinogenesis imperfecta, and joint laxity.

We report on a syndrome of spondylo-epimetaphyseal dysplasia, dentinogenesis imperfecta, and ligamentous hyperextensibility in two sibs born to nonconsanguineous parents. This chondrodysplasia was characterized by severe shortness of stature and an osteoporosis without fractures. Electron microscopic examination of the cartilage documented large vacuoles of dilated rough endoplasmic reticulum within the cytoplasm of chondrocytes. Gel electrophoresis of pepsin-soluble collagen extracted from cartilage demonstrated the presence of type II collagen chains with an abnormal mobility. Prolyl and lysyl hydroxylations were slightly increased. The abnormal molecules melted at a higher temperature than the normal ones. CNBr peptide mapping of type II collagen showed an altered electrophoretic migration of peptides CB 11, CB 8, and CB 10,5 whereas CB 9,7 looked normal. In addition, two small non-collagenous proteins isolated from cartilage were not found in an age-matched control individual but were detected in a normal newborn infant. The quantitation of proline-labelled collagen synthesized by dermal fibroblasts demonstrated a 50% reduction of total collagen. This decrease essentially affected the amount of extracellular type I collagen, which was secreted less efficiently than in control cells. Nevertheless, type I collagen chains behaved normally on 5% polyacrylamide gels. The reduced mRNA levels of alpha 1I and alpha 2I chains might reflect either a transcriptional defect or a decreased stability of mRNA transcripts. We suggest that the association of both pathological chondrocytes producing altered collagen type II and decreased synthesis of type I could be responsible for this peculiar phenotype. The overmodification of alpha 1II CNBr peptides is consistent with the presence of a single-base substitution in the COL2A1 gene. Whether there is a direct causal relationship between the type II collagen defect and the underexpression of type I collagen will require clarification.

Dentin phosphoprotein in dentin development: implications in dentinogenesis imperfecta.

Dentin phosphoprotein (DPP, phosphophoryn) is the major non-collagenous protein component of the dentin extracellular matrix. This highly acidic phosphorylated protein is solely expressed by ectomesenchymal-derived odontoblast cells of the tooth organ. Previous biochemical studies have suggested the absence of this protein associated with the human genetic disease dentinogenesis imperfecta (DGI) Types I and II. However, due to the normal degradation of human DPP during dentin maturation, it has not been possible to establish if these reported differences were due to changes in DPP expression or secondary degradation rates in DGI affected versus normal teeth. Recently, we have taken both a molecular and biochemical approach to address this problem. Molecular studies have utilized genetic linkage studies performed on several multi-generation informative DGI kindreds. These studies have determined linkage between DGI Types II and III and two markers localized to the long arm of human chromosome 4 in the region 4q11-4q21. The strategy used in our study was to map the DPP gene locus to the long arm of human chromosome 4, in the same region as DGI, using a DPP oligonucleotide probe and somatic hybrid cell lines. The results indicate DPP is not localized to any region of human chromosome 4. Our data indicates that a mutation within the DPP gene locus is not associated with DGI Types II or III. This data is supported by the identification of human DPP (95 kDa) within the dentin extracellular matrix of molars isolated from an affected DGI type II patient using a mouse anti-DPP antibody. However, this does not exclude the possibility that enzymes associated with DPP post-translational modifications (ie. phosphorylation or degradation) might be responsible for this genetic disease.