Authors: Tetsutaro Yamaguchi, Kazuyoshi Hosomichi, Tatsuo Shirota, Yoichi Miyamoto, Wanida Ono, Noriaki Ono
Categories: Article, Animal model, Disease-specific induced pluripotent stem cells, Incomplete tooth eruption, Treatment
Source: The Japanese Dental Science Review
Primary failure of eruption (PFE) is a rare disorder defined as incomplete tooth eruption despite the presence of a clear eruption pathway. PFE is known to be caused by rare variants in the parathyroid hormone 1 receptor gene (PTH1R). Although several PTH1R variants have been reported, the etiology of PFE remains unclear. However, important studies that help elucidate the pathology of PFE have recently been published. The purpose of this review is to summarize current treatment options, clinical symptoms or phenotypes for diagnosis, genetic information including solid evidence in mouse disease models and disease-specific induced pluripotent stem cells, thus approaching the etiology of PFE from the perspective of the latest research.
Keywords: Incomplete tooth eruption, Animal model, Disease-specific induced pluripotent stem cells, Treatment
Primary failure of eruption (PFE) is defined as impaired tooth eruption despite the presence of an unobstructed eruption pathway (Fig. 1). PFE involves partial eruption or non-eruption of an initially non-ankylosed tooth due to a disturbed eruption mechanism, resulting in a posterior open bite [1]. The key manifestations were first described in 1981 by Proffit and Vig [2]. Selection of an appropriate treatment strategy depends on the correct diagnosis; however, orthodontic extrusion must be avoided as it can lead to ankylosis [1]. Unfortunately, diagnosis of PFE relies principally on a process of elimination in which all possible causative factors must be considered [3], [4], and many patients have received unsuccessful orthodontic treatment because PFE was not correctly diagnosed. For this reason, a good understanding of the clinical symptoms of PFE is necessary for correct diagnosis and effective treatment. Additionally, genetic information may be useful for diagnostic purposes. The discovery of causative variants of the parathyroid hormone 1 receptor gene (PTH1R) has led to research involving a mouse disease model and disease-specific induced pluripotent stem cells (iPSCs) that has deepened understanding of PFE etiology. Moreover, current studies are expanding the knowledge of PFE pathological conditions. Accordingly, this review summarizes current treatment options, clinical symptoms or phenotype and genetic information, and insights gained from research investigating PFE pathology.
Fig. 1 a. Oral photographs. The patient was a 29-year-old female with primary failure of tooth eruption. The mandibular first molar was impacted vertically in both sides, and mandibular second pre-molar on the left side was inclined distally, and the mandibular second molar on the right side was inclined mesially. Fig. 1b. Cephalograms. Lateral cephalograms revealed skeletal Class III. The posterior-anterior cephalogram revealed asymmetry with deviation to right side. Fig. 1c. Panoramic radiograph. Panoramic radiograph revealed impaction of the mandibular first molar in both sides. (The permission to reprint of these materials from the Showa Univ J Med Sci 2010;22:151-156.)
In PFE, tooth extrusion cannot be performed because orthodontic force applied to the erupted tooth can lead to ankylosis. Thus, orthodontic treatment is considered insufficient to improve malocclusion caused by PFE [2]. Treatment strategies for PFE vary depending on the clinical conditions. If only a few teeth have moderately abnormal eruption, prosthodontic restoration is recommended to establish satisfactory occlusion. In moderately severe cases, small-segment osteotomy and simultaneous elastic traction of the affected teeth will improve the level of the occlusal plane. Prosthetic treatment for these teeth has been shown to further improve malocclusion [5]. If the use of prosthetic teeth is not sufficient, occlusion may be effectively restored by extracting the affected teeth and replacing them with implants with alveolar ridge augmentation. In more severe cases with many incomplete erupted teeth and a significantly deformed alveolar ridge, prosthetic rehabilitation with a removable prosthetic appliance is effective in improving occlusion at an early stage [6]. In terms of surgical approaches to occlusion, bone grafting can be performed to improve occlusion. Specifically, segmental osteotomy of the alveolar bone affected by PFE can be performed to reposition the segment and subsequently improve the occlusal plane, and a bone graft can be placed between the bone segment and base of the alveolar bone [2], [7]. However, this technique may not be suitable for the maxilla because the thick and poorly elastic palate mucosa resists movement of the segment. This makes it difficult to reposition the segment at an appropriate location. Notably, there is high risk of causing inferior alveolar nerve damage when performing this procedure in the mandible. As such, studies have reported the use of distraction osteogenesis (DOG) of the alveolar bone in patients with severe lateral open bite [8], [9]. However, DOG of the alveolar bone is challenging in terms of controlling the direction of growth because the movement of the distraction divide can only be linear, and the segment tends to be displaced to the palatal side as the bone grows. In these cases, the floating bone concept may be more effective as it allows for 3-dimentional control of the position of the segment by continuously applying pressure to the segment before the bone heals completely [10]. Researchers previously performed DOG of the maxillary alveolar bone in a PFE case with severe lateral open bite, controlling the position of the segment with elastic traction and removing the device before the bone healed completely. This procedure successfully improved the morphology of the dental arch and achieved stable occlusion [11].
Evaluating the severity of clinical symptoms on an individual basis is needed to select the most appropriate treatment option for patients diagnosed with PFE. In other words, familiarity with the clinical symptoms of PFE is extremely important.
PFE is reported in approximately 0.06% of the population [12]. A prevalence among different populations has not been reported. Hanisch et al. compiled a systematic review of studies on PFE published until 2016 [1]. The review and subsequent articles are summarized in Table 1.
First and foremost, the easiest way to diagnose PFE is to check for occlusion in parents of patients suspected of having PFE. Almost 85% of patients with PFE had a family member affected by PFE (Table 1) [1]. However, parents may not recognize healthy occlusion since they experienced PFE at a young age; thus, it is strongly recommended to evaluate occlusion in the parents. Indeed, the father or mother may actually have severe malocclusion, even though they declared the absence of any dental problems. The next step in the diagnosis of PFE primarily relies on exclusion, in which all possible causative factors must be considered and eliminated. Table 2 lists the causes of eruption failure, divided into local and systemic causes. In many cases, local factors causing mechanical impairment are responsible for eruption failure. Infraocclusion, immobility, metallic sound on percussion, and radiographic obliteration of the periodontal ligament space are a clue to the diagnosis for mechanical failure of eruption (ankylosis). These characteristics might be differentiated from PFE. However, actuality, mechanical failure of eruption and PFE are often difficult to distinguish [1], [13], [14]. A genetic testing for variants in the PTH1R may be expected to avoid unnecessary treatment intervention [15], [16].
Eruption failure has been found to be a feature in many genetic disorders and syndromes as listed in Table 3. The gene(s) responsible for various disorders and syndromes associated with eruption failures have been identified. Moreover, Decker et al. [15] first reported that a variant in PTH1R was associated with PFE.
PTH1R variants have also been associated with diseases of bone and cartilage. Table 4 summarizes the PTH1R variants reported so far. A loss-of-function variant in PTH1R can result in Blomstrand osteochondrodysplasia (BOC; Orphanet # 50945; OMIM # 215045), a rare autosomal recessive skeletal dysplasia characterized by advanced endochondral bone maturation and premature ossification of skeletal elements. BOC causes short-limbed dwarfism and perinatal lethality [17]. Similarly, a homozygous PTHIR variant results in Eiken syndrome (Orphanet # 79106; OMIM # 600002), a rare autosomal recessive skeletal disorder characterized by extremely retarded ossification and multiple epiphyseal dysplasia [18]. Patients with Eiken syndrome exhibit abnormal bone modeling in their hands and feet, abnormal pelvic cartilage persistence, and mild growth retardation [18]. In contrast, gain-of-function PTH1R variants have been reported in patients with Jansen’s metaphyseal chondrodysplasia (Orphanet # 33067; OMIM # 156400), an autosomal dominant disorder characterized by pronounced short-limb dwarfism resulting from decelerated chondrocyte differentiation [19].
In PTH1R, 48 are registered as pathogenic or likely pathogenic in Human Gene Mutation Database (HGMD) [https://www.hgmd.cf.ac.uk]. In addition, the functional classification of these variants includes 24 missense variants, 9 nonsense variants, 6 frameshift deletions, 3 frameshift insertions, 1 non-frameshift deletion, and 5 intronic variants. Lollipops software [20] was used to confirm the accumulation of genetic regions and functional domains of these variants, and the locations of PTH1R variants associated with PFE and other diseases are shown in Fig. 2. The location of the pathogenic PTH1R variant was observed scattered throughout the gene, with no domain accumulation, and no phenotype-dependent features were observed.
Fig. 2 Location of variants in the PTH1R, schematized using Lollipops software [20].
One of the reasons for the large variety of phenotypes associated with PTH1R variants is allelic dose. Strong evidence supports that PFE is an autosomal dominant condition associated with heterozygous variants in PTH1R in most cases, whereas homozygous variants are present in extremely rare cases [21], [22], [23], [24]. Although the heterozygous missense PTH1R variant c 0.395 C>T (see Table 4) was reported in Japanese sporadic cases of PFE [25], the homozygous genotype has also been associated with the BOC phenotype [26]. Similarly, the heterozygous PTH1R variants c 0.310 C>T [27], [28], c 0.395 C>T [25], [26], [31], [52], c.1093delG [27], [29], [30], and c 0.1148 G>A [17], [25], [27], [31], have been reported in patients with PFE, while homozygous genotypes have also been associated with the BOC phenotype. Moreover, Jelani et al. [21] reported that the homozygous or biallelic PTH1R variant c 0.611 T > A (see Table 4), which caused a PFE phenotype that appeared to be unique to the family and also caused clinodactyly and nasal bridge deformity, was also identified as a heterozygous genotype associated with the Jansen’s metaphyseal chondrodysplasia phenotype. Moirangthem et al. [22] reported that a patient with autosomal recessive Eiken syndrome (c 0.103 G>A) (see Table 4) also had malposition and impaction in most teeth. In practice, similar to these examples, the disease overlap with PFE may not be a small number of patients. The reason may be due to unconversant oral examinations [15], [31].
As shown in Table 1, almost 15% of patients with PFE had no family members affected by PFE [1]. The patient of PFE without family history, may have spontaneous variant. Therefore, even in sporadic case, a genetic testing brings a valuable advantage for expecting the potential contribution of orthodontic intervention [32].
Although variable phenotypic expression can be observed, almost all PTH1R variants causing PFE are thought to exhibit complete penetrance [31]. However, one study documented a patient with severe PFE that carried the PTH1R variant c 0.505 G>T inherited from an unaffected mother, providing a typical example of incomplete penetrance [32]. This should be considered when interpreting the results of a genetic testing.
It remains unclear whether variants in only PTH1R cause PFE because PTH1R variants are not found in all patients with PFE [1], [27], [30]. The histone methyltransferase 2 C gene (KMT2C) is reportedly strongly associated with PFE [33]. However, affected families in which this gene was identified included family members without all permanent teeth, and the phenotype was somewhat more severe than that in simple PFE. In any case, it is clear that performing a genetic testing for PTH1R, whenever possible, provides useful information to confirm a diagnosis, even in sporadic case [24].
PTH1R encodes parathyroid hormone (PTH) receptor type 1 (PTH1R), a Class B G protein-coupled receptor (GPCR) with 7 transmembrane spanning helixes. PTH1R is activated by two similar ligands with distinct functions, PTH and PTH-related protein (PTHrP), which control the hormonal and local functions of the receptor, respectively. PTH and PTHrP bind to PTH1R in equivalent affinity [34], [35]. Upon ligand binding, PTH1R activates two major second messenger signaling systems, including the adenylyl cyclase/protein kinase A pathway and the phospholipase C/protein kinase C pathway [36]. As a locally acting autocrine and paracrine ligand, PTHrP exerts pleiotropic effects on cell proliferation and differentiation. During development, the PTHrP-PTH1R pathway mediates epithelial-mesenchymal interactions in various organs, such as the skin, hair follicles, mammary glands, pancreas, and developing teeth [37], [38], [39], [40], [41]. This pathway also regulates bone formation in the endochondral pathway [42].
As a result, the PTHrP-PTH1R pathway regulates tooth eruption in multiple aspects. Tooth eruption occurs in three distinct pre-eruptive tooth movement (Phase 1), intra-osseous and supra-osseous eruptive tooth movement (Phase 2), and post-eruptive tooth movement (Phase 3) [43], [44]. Phase 1 (pre-eruptive tooth movement) occurs during the early stages of tooth development, lasting until the commencement of tooth root formation. Enamel and dentin formation at this stage prepares the tooth crown for emergence into the oral cavity. Phase 2 (eruptive tooth movement) occurs during tooth root formation, lasting until the tooth crown reaches the occlusal plane. This phase is divided into two intra-osseous and supra-osseous eruptive tooth movement, and involves the formation of the tooth root, alveolar bone, and periodontal ligament. It also involves the osteoclast-mediated bone resorption of the cortical shell overlaying the tooth crown, which jointly facilitate the emergence of the tooth crown into the oral cavity. Phase 3 (post-eruptive tooth movement) maintains the tooth crown in occlusion as the alveolar bone continues to grow and remodel, which requires continuous maturation of the periodontal attachment apparatus. In this phase, the PTHrP-PTH1R pathway directly regulates the proliferation of dental mesenchymal progenitor cells and their differentiation into cementoblasts, periodontal ligament cells, and alveolar bone osteoblasts. It also indirectly regulates bone-resorbing osteoclasts through the RANKL-RANK axis.
Importantly, tooth eruption requires the formation of the eruption path and the motive force. The sources of the motive force include the dental follicle surrounding the tooth bud, osteogenic activities in the alveolar bone surrounding the tooth root, the formation of the periodontal ligament (PDL) and traction forces generated by the fibers therein, and the development of the tooth root and the cementum in the apical region. Traditionally, tooth eruption has been regarded as a separate and distinct process from tooth root formation, as teeth can emerge into the oral cavity without roots or PDLs [45], [46]. However, recent studies support the emerging theory that these two processes are intertwined [36], [39]. Because the eruption path is cleared in PFE, defective tooth eruption in this condition is likely to be induced by deficiency motive forces, many of which are regulated by the PTHrP-PTH1R pathway.
Various putative loss-of-function mutations in PTH1R are present in patients with PFE [15], [25], [27], [31], [47]. The majority of these PTH1R mutations are heterozygous, while in extremely rare cases, these mutations are also present in homozygous forms [21], [22], [23], [24]. Studies with genetically engineered mice present solid evidence that loss-of-function mutations in Pth1r cause similar skeletal abnormalities as those observed in humans. For example, mice harboring two copies of the Pth1r^tm1Hmk^ allele (Pth1r^-/-^), which lacks most of the coding region of the Pth1r gene, exhibit perinatal lethality that recapitulates Blomstrand osteochondrodysplasia (BOC) [48]. Interestingly, heterozygous Pth1r mutant mice (Pth1r^+/-^) do not show overt defects in tooth eruption [49], indicating that Pth1r haploinsufficiency is not sufficient to induce PFE in mice. It is possible that mouse molars are less sensitive to Pth1r gene dosage changes than human molars. Therefore, a better mouse disease model for PFE is needed to understand mechanisms that exclusively affect tooth eruption in molars.
A conditional gene deletion approach based on the Cre-loxP recombination system provides a practical modality to circumvent perinatal lethality of Pth1r^-/-^ mice and test the function of PTH1R in tooth eruption that exclusively occur during postnatal stages. In the *Pth1r-*floxed allele (Pth1r^tm2Hmk^), exon 1 of the Pth1r gene, which is essential for ligand binding, is flanked by loxP sites [50]. This allele can be rendered nonfunctional upon Cre-loxP recombination and inactivate PTH1R in a cell type-specific manner. For example, Pth1r can be specifically deleted in dental mesenchymal cells that express osterix (Osx, also known as Sp7) using Osx-cre and *Pth1r-*floxed alleles. This causes severe failure of tooth eruption in molars associated with truncated roots lacking periodontal ligaments [49], representing an extreme phenotype induced by PTH1R loss-of-function variants. However, these mutant mice do not survive beyond weaning owing to the inability to consume food. A delayed molar eruption phenotype is also observed by Pth1r deletion in Prrx1-expressing progenitor cells using Prrx1-cre [51]. Notably, cells marked by Osx-cre are distributed throughout the alveolar bone and the periodontal tissue surrounding the incisor and molars, while cells marked by Prrx1-cre are predominantly observed in the alveolar bone surrounding incisors and at the base of molars. Delayed eruption, instead of complete failure of eruption, observed in Prrx1-cre-mediated Pth1r mutant mice, is likely due to the restricted distribution of cells marked by Prrx1-cre. Reduced formation of the alveolar bone and PDL at the base of molars, instead of the unresorbed overlaying cortical shell, is likely to explain delayed eruption of Prrx1-cre-mediated Pth1r mutant molars. These findings emphasize the role of the alveolar bone formation in providing the motive force for tooth eruption. The major limitation of these models is that PTH1R is inactivated in the given cell types from embryogenesis. As a result, other bone compartments, including the mandible, skull and long bones, are also severely affected, making it difficult to discern the contribution of each component to the failure to tooth eruption. Therefore, these models lack the phenotypic specificity for tooth eruption in molars and present as conditions that are far more severe than those typically manifested in human PFE.
To inactivate PTH1R postnatally in a tooth root-specific manner, a dental follicle-specific, tamoxifen-inducible PTHrP-creER line can be used to delete PTH1R using PTHrP-creER and Pth1r-floxed allele [52]. By administering tamoxifen at a specific postnatal time point (in this case, postnatal day 3), this inducible cell type-specific approach enables biallelic inactivation of PTH1R in a small number of specific cell types (in this case, PTHrP^+^ dental follicle cells) at the onset of tooth root formation. This approach can create a cell type-specific “low mosaicism” of PTH1R-deficient cells during tooth root formation, providing substantial mechanical insights into the altered cellular dynamics underlying PFE. Postnatal PTH1R deletion in dental follicle cells exclusively induces failure of tooth eruption in molars, which essentially recapitulate human PFE. The mutant molars exhibit a significant delay in tooth eruption especially first molars, without involving other bone components. Furthermore, the affected molars exhibit dilacerated tooth roots and cementum anomalies, which are often observed in human PFE molars [30]. Interestingly, the mutant mouse PFE phenotype, particularly PFE in first molars, demonstrates aggravated open bite in adult stages [53], which is similar to human cases of PFE reported during a long-term follow-up study [54]. A mouse PFE phenotype is also observed when Pth1r is postnatally inactivated in a much broader type of Osx^+^ cells using Osx-creER [52], indicating that PTHrP^+^ dental follicle cells represent an important functional subset of dental mesenchymal cells that orchestrate tooth eruption.
Although these mutant mice do not completely represent the same genetic condition as human patients with PFE, these studies provide important insights into the pathogenesis of PFE (Table 5). PTH1R inactivation drives dental follicle cells to shift from physiological periodontal ligament fibroblasts and cryptal bone alveolar bone osteoblasts to non-physiological precocious cellular cementoblasts. It remains to be determined whether similar mechanisms of cell fate shift also occur in human PFE.
Yamanaka and his colleagues created mouse and human iPSCs by inducing the reprogramming of somatic cells through the ectopic expression of the four factors (Yamanaka factors), namely, OCT3/4, KLF4, SOX2, and c-Myc [55], [56]. Since iPSCs are free from the ethical problem of using a fertilized egg and the immunological rejection accompanied by embryonic stem cells, human iPSCs are regarded as promising tools for regenerative medicine. Also, disease-specific iPSCs are developing new research tactics in medical sciences. It made it possible to investigate the diseases' pathogenic mechanisms and find therapeutic methods using the disease-specific iPSCs [57].
Several heterozygous variants in the PTH1R have been identified in PFE patients, indicating a close relationship between these genetic variations and the pathogenesis of PFE [25], [31], [47], [58]. Examination of the effects of the introduction of variations in the PTH1R on the cellular functions and observation of the phenotypes of the animals having the modified Pth1r are considered adequate to demonstrate the pathogenic role of PTH1R variation in PFE patients. In addition to these measures, the study using PFE-specific iPSCs is regarded as another potent approach to clarify the pathogenesis of PFE. We established iPSC clones from hematopoietic progenitor cells obtained from a patient with the heterozygous 395 C>T substitution in the PTH1R [59] by introducing the Yamanaka factors using a Sendai virus vector [60]. Using the PFE-specific iPSCs, we tried to explain the role of the genetic variation in PTH1R in the pathogenesis of PFE in in-vitro studies [59].
Alveolar bone resorption is one of the critical events of tooth eruption [61]. Furthermore, it is known that the PTH1R-expressing osteoblasts are responsible for the differentiation and activation of osteoclasts, the bone-resorbing multinucleated giant cells [61]. Hence, we examined the differentiation of the PFE-specific iPSCs and the control iPSCs derived from a healthy volunteer into osteoblast-like cells after cultivation in the medium containing the osteoblast differentiation-inducing factors (β-glycerophosphate, ascorbic acid, and dexamethasone). Differentiation of PFE-specific iPSCs to osteoblast-like cells evaluated by mineralization of the extracellular matrix and the expression of the osteoblast differentiation-related genes (RUNX2, SP7, and BGLAP) was comparable to that of the control iPSCs. Notably, both the osteoblast-like cells derived from PFE-specific iPSCs and those from the control iPSCs expressed equivalent levels of the mRNA and protein of PTH1R [59].
On the other hand, the expression of the mRNA and protein of receptor activator of nuclear factor-κB ligand (RANKL), the essential molecule for osteoclast differentiation and activation [62], in response to PTH was significantly suppressed in the osteoblast-like cells from PFE-specific iPSCs compared to those from the control iPSCs. Active vitamin D3 induced the expression of the mRNA and protein of RANKL comparably in osteoblast-like cells derived from PFE-specific and those from the control iPSCs, indicating that the PTH-PTH1R signaling system leading to the RANKL expression is affected in the osteoblast-like cells derived from PFE-specific iPSCs [59].
The heterozygous 395 C>T variant in the PTH1R is supposed to result in the expression of both standard and P132L amino acid substituted PTH1R in osteoblasts. Our study above indicated that the PTH1R with P132L amino acid substitution in osteoblasts possibly contributed at least in part to the retarded tooth eruption through the incomplete induction of differentiation and activation of osteoclasts in the PFE patient. Detection of gene variants in patients with rare diseases, including PFE, through the techniques such as whole-exome sequencing coupled with studies employing the disease-specific iPSCs, may be a promising approach to clarify the etiology of the diseases.
The patient’s clinical symptoms need to be carefully scrutinized. Moreover, genetic testing for PTH1R variants is recommended to avoid unnecessary and ultimately unsuccessful orthodontic intervention. Screening for such variants in a patient with PFE will help in the selection of an appropriate management strategy and allow more realistic treatment options to be offered to the patient. Treatment options must be evaluated on an individual basis, depending on the patient’s age, background, and preferences. Many aspects of PFE pathophysiology remain unclear; however, disease-specific iPSCs present an unprecedented opportunity to replicate both normal and pathological human tissue formation in vitro, thereby enabling the disease to be investigated and an innovative treatment to be developed to be achieved. Use of a mouse disease model is also expected to offer a way to investigate PFE and act as a guide to the discovery of an effective drug.