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Tooth Agenesis: from Molecular Genetics to Molecular Dentistry
1 Laboratory of Animal Embryology, Institute of Animal Physiology and Genetics, Academy of Sciences, Brno, Czech Republic; Correspondence: * corresponding author, matalova{at}iach.cz
Tooth agenesis may originate from either genetic or environmental factors. Genetically determined hypodontic disorders appear as isolated features or as part of a syndrome. Msx1, Pax9, and Axin2 are involved in non-syndromic hypodontia, while genes such as Shh, Pitx2, Irf6, and p63 are considered to participate in syndromic genetic disorders, which include tooth agenesis. In dentistry, artificial tooth implants represent a common solution to tooth loss problems; however, molecular dentistry offers promising solutions for the future. In this paper, the genetic and molecular bases of non-syndromic and syndromic hypodontia are reviewed, and the advantages and disadvantages of tissue engineering in the clinical treatment of tooth agenesis are discussed.
Key Words: Tooth development • syndromic • non-syndromic • hypodontia • tooth engineering
Tooth agenesis is the most prevalent craniofacial congenital malformation in humans. Up to 25% of the population may lose at least one third molar. Agenesis of other permanent teeth, excluding third molars, ranges from 1.6 to 9.6%, depending on the population studied. Primary dentition may also be affected, but with lower prevalence (from 0.5 to 0.9%) (Vastardis, 2000). As a rule, when a primary tooth does not develop, the secondary tooth is also missing. The majority of persons with hypodontia (80%) lack only one or two teeth (Lidral and Reising, 2002), permanent second premolars and upper lateral incisors being predominantly affected (Symons et al., 1993). However, about 1% (0.08–1.1%) of the population suffers from oligodontia—the agenesis of more than 6 teeth (Schalk-Van der Weide et al., 1992; Stockton et al., 2000; Gabris et al., 2001). Loss of all teeth is known as anodontia. How tooth loss comes about is thus an important question. Tooth development is a complex process that involves signaling interplay between the embryonic stomodeal epithelium facing the oral cavity and the underlying neural-crest-derived mesenchyme. First, signaling molecules expressed in the dental epithelium signal to the underlying mesenchyme, establishing the dental mesenchyme (dental placode stage). Subsequently, the odontogenic potential shifts to the dental mesenchyme, and mesenchymal factors direct tooth bud morphogenesis, including formation of the primary enamel knot signaling center that directs future tooth crown shaping. Odontogenic epithelio-mesenchymal interactions repeatedly recruit basic organogenic cascades involving Fgf, Bmp, Shh, and Wnt signaling. Any disturbances in the tightly balanced signaling cascades may result in dental defects, including changes in tooth number, size, morphology, and cytodifferentiation. Lessons from animal models, especially the mouse, are crucial for the understanding of the basic genetic principles of hypodontia (Fleischmannová et al., 2008). On the surface, it would appear difficult to mimic a complex hypodontia phenotype in an oligodont animal with only one tooth generation. However, despite these substantial differences between the mouse and human dentitions, early stages of tooth development are largely similar in both species, and the basic principles of tooth development originally discovered in the mouse have been confirmed in humans. Exploiting the well-established techniques of mouse molecular genetics, investigators have identified several knockouts that display a tooth agenesis phenotype. Tooth development is arrested at the bud stage in Pax9, Msx1, Pitx2, Gli2/3, p63, and Lef1 knockout mice, while in the case of the Dlx1/2 knockout, maxillary molars alone are lost. In contrast, Activinβa deficiency results in an inverse phenotype, with loss of the mandibular molars and incisors and preservation of the maxillary molars. Disruption of several of these same molecules has been confirmed to result in tooth agenesis in humans as well.
Non-syndromic hypodontia is a common term, including different phenotypes ranging from hypodontia of one tooth (excluding third molars) to oligodontia and anodontia (Fig. 1  ). The usual mode of inheritance is autosomal-dominant, but autosomal-recessive inheritance and X-linked and polygenic or multifactorial models of inheritance have been reported (Suarez and Spence, 1974; Brook, 1984; Peck et al., 1993). Hypodontia itself is a complex phenotype, with variable penetrance and expressivity affecting various numbers of teeth in different regions. Differences in tooth size and the whole distribution of the dentition may also be observed, especially in more severe cases of oligodontia. The molecular basis of the defect is not completely understood, despite identification of several mutations in MSX1 and PAX9 genes that seem to be crucial for tooth agenesis (recently reviewed in Kapadia et al., 2007), and mutations in the AXIN2 gene that cause oligodontia together with a predisposition to colorectal cancer.
Msx1 and Pax9 are transcription factors necessary for normal development. Msx1 is a member of the muscle segment homeobox family, members of which act repetitively during organogenesis. Pax9 belongs to the paired box domain gene family that is named according to the presence of a DNA-binding "paired" domain. Pax9 plays an important role as a regulator of cellular pluripotency and differentiation during embryonic patterning and organogenesis and in post-natal life. Both Msx1 and Pax9 interact during the tooth-bud-to-cap transition. Their expression profiles during early tooth development are largely overlapping, and Pax9 is known to activate transcription of Msx1 at the bud stage. Recently, it has been shown that both molecules may dimerize and synergistically activate Bmp4 transcription (Ogawa et al., 2005). In the mouse, in the absence of either Msx1 or Pax9, tooth development is arrested at the bud stage. The human MSX1 gene consists of two exons. The second exon includes a homeodomain that binds DNA and facilitates protein-protein interaction of MSX1 with PAX9 and other odontogenetic molecules, like DLX family members (Zhang et al., 1997; Ogawa et al., 2005). The first indication that the MSX1 gene is connected with isolated hypodontia came from genetic linkage analyses in a family with agenesis of second premolars and third molars that pointed out a 4p16.1 locus where the MSX1 gene is located. Subsequent sequence analysis confirmed an arg31-to-pro missense mutation in the homeodomain of the MSX1 gene (Vastardis et al., 1996). Functional biochemical tests revealed that the ability of the mutated MSX1 to bind DNA and interacting proteins was virtually lost, and MSX1 was not able to exhibit its functions in vivo (Hu et al., 1998). To date, three mutations in exon 1 and four mutations in exon 2 have been associated with hypodontia that predominantly affects second premolars and third molars (Vastardis et al., 1996; DeMuynck et al., 2004) or hypodontia associated with clefting (van den Boogaard et al., 2000). Moreover, MSX1 mutations cause isolated clefts and Witkop syndrome (Stimson et al., 1997; Jumlongras et al., 2001; Jezewski et al., 2003; Suzuki et al., 2004). Individuals with Witkop syndrome are characterized by hypoplastic nails and hypodontia. The condition is usually not detected until the permanent teeth fail to erupt. Mandibular incisors, second molars, and maxillary canines are most often absent. The mode of inheritance of MSX1-caused hypodontia is predominantly autosomal-dominant, due to haploinsufficiency (Kim et al., 2006). However, an autosomal-recessive mutation in MSX1 has also been reported recently (Chishti et al., 2006).
PAX9 has been found to be the gene causing isolated molar oligodontia, an autosomal-dominant disorder with agenesis of most permanent molars, sometimes in combination with other types of teeth (Fig. 2
Recently, a nonsense mutation in AXIN2, an essential component of the WNT/β-catenin pathway, has been reported to cause familial oligodontia with a phenotype more severe than that described for MSX1 and PAX9 mutations. Mutations in AXIN2 lead to a lack of most permanent molars, premolars, lower incisors, and upper lateral incisors. In contrast, the upper central incisors are always present. In addition to oligodontia, mutation in AXIN2 also predisposes the individual to colorectal cancer (Lammi et al., 2004). Oligodontia together with a predisposition to colorectal cancer has been found as a consequence of 1966C>T transition in the AXIN2 gene in members of a Finnish family (Lammi et al., 2004). Additionally, a de novo germ line mutation (1994 ins G) has been identified in one proband. Recently, two novel AXIN2 gene variants (956+16 A>G and 2062 C>T polymorphisms) have been associated with higher risk of hypodontia—especially C to T transversion, which is likely to interfere with the splicing process and may therefore negatively affect the availability of AXIN2 in the cell (Mostowska et al., 2006). There are still many persons, however, with tooth agenesis but having no identified mutation in either the Pax9, Msx1, or Axin2 gene. Promising candidates are molecules causing syndromes including tooth agenesis or other craniofacial defects. IRF6, FGFR1, and TGFβ3 have been shown to be associated with non-syndromic hypodontia. Specific allelic variants of these genes (SNPs) were reported to be more prevalent in persons with hypodontia compared with healthy control individuals.
Teeth develop in the context of the whole craniofacial region and recruit conserved developmental cascades common to the morphogenesis of other orofacial structures and ectodermal derivatives. Hence, several syndromes involving hypodontia as a primary feature display various dysplasias and syndromic clefts. To date, studies have confirmed involvement of the same genes in both syndromic and non-syndromic hypodontia (Table ). The MSX1 mutations cause a wide spectrum of phenotypes, ranging from Witkop syndrome, hypodontia associated with clefts, to non-syndromic hypodontia and clefts only (discussed above). Mutations in Ectodysplasin, well-known to cause hypohydrotic ectodermal dysplasia (HED), have been reported also to cause hypodontia without any other syndromic features. These overlaps may indicate that genes involved in hypodontia associated with other syndromic features are also promising candidates for non-syndromic hypodontia.
Mutations in genes necessary for either initial stages of tooth development or bud-stage morphogenesis, especially Shh, Pitx2, Msx1, Irf6, p63, and Eda pathway genes, are of particular interest.
The Eda pathway acts to determine the size of the tooth field during tooth initiation, and later contributes to tooth morphogenesis and cusp formation. Mutations in the EDA pathway (EDA, EDAR, EDARADD, NEMO), together with defects in its main downstream target, the NF- If one excludes the Eda-pathway-connected ectodermal dysplasias, hypodontia is often connected with ectodermal dysplasia phenotypes caused by mutations in NECTIN-2 and p63. NECTIN-2 (PVRL1) codes for a cell adhesion molecule that is expressed in mouse tooth buds. Persons with Zlotogora-Ogur syndrome (Zlotogora, 1994) or the Margarita Island form of ectodermal dysplasia (Bustos et al., 1991) caused by a PVRL1 mutation have syndactyly, cleft lip/palate, nail and hair dysplasia, and hypodontia. Hypodontia affects mainly the upper lateral incisors, and additionally the sizes and shapes of tooth crowns differ (Bustos et al., 1991). p63 is necessary for the development of several ectodermal epithelial structures. The complexity of its functions is apparent from the wide range of phenotypes that are known to result from mutations in p63. Mammary gland, limb, and orofacial structures are often affected. SHH is a crucial signaling molecule acting during organogenesis, in dorso-ventral neural tube patterning, antero-posterior patterning of the limb, gut development, tooth initiation, and tooth morphogenesis. Its role in cancer is also under intensive investigation (Chari and McDonnell, 2007). Mutations in the SHH gene cause developmental disorders ranging from only mild microcephaly or dental defects to a very severe autosomal-dominant syndromic phenotype, holoprosencephaly. Moreover, craniofacial abnormalities are involved in syndromes caused by the Shh downstream transcription factor GLI3 (Pallister-Hall syndrome) (Radhakrishna et al., 1999). The tooth phenotype seems to result from a mid-facial fusion defect. Incisor tooth buds initially form normally; then the lateral growth of the maxillary processes is reduced and results in a premature fusion of the left and right parts of the dental lamina, leading to fusion of the incisor tooth buds and a single central incisor (Hardcastle et al., 1998). The homeobox gene Pitx2 is expressed in the oral epithelium at the sites of future tooth formation (Mucchielli et al., 1997) and is necessary for the maintenance of the balance of Bmp4/Fgf8 expressed in the oral epithelium (Lin et al., 1999; Lu et al., 1999). Mutations in this gene are responsible for some cases of Rieger syndrome, together with PAX6 (Riise et al., 2001). Rieger syndrome is an autosomal-dominant inherited disorder characterized by hypodontia, malformations of the anterior chamber of the eyes, and umbilical abnormalities. Affected individuals suffer from midfacial hypoplasia and underdevelopment of the premaxillary area. Both the primary and secondary dentition may be affected, with upper incisors and second upper premolars most commonly missing. Additionally, lower anterior teeth are often conical, and cleft palate may be present.
Hypodontia is associated with both syndromic and isolated clefts. The frequency of hypodontia (both in and outside the cleft region) is significantly increased in persons with clefts compared with the control population (Shapira et al., 2000; Slayton et al., 2003). Moreover, it has been shown that the number of affected teeth increases with the severity of the cleft phenotype (Dewinter et al., 2003; Slayton et al., 2003). ‘Oral clefts’ is a common term for two anatomically distinct features: cleft lip with or without cleft palate, and cleft palate only with a proposed oligogenic mode of inheritance. Msx1, p63, IRF6, FGFR1, and TGF family growth factors may contribute to oral clefts at the molecular level. Van der Woude syndrome, the most common form of syndromic cleft palate, is, together with popliteal pterygium, caused by a mutation in the IRF6 gene. Additional to clefts, persons often suffer from agenesis of incisors and premolars. Recently, non-syndromic clefts have been associated with specific allelic variants (SNPs) of the IRF6 gene. A similar association has also been reported with non-syndromic hypodontia, most preferentially in the premolar region. FGFR1 causes several developmental disorders, including Kallmann syndrome, that may involve clefting and, with lower frequency, hypodontia (Vieira et al., 2007). Finally, TGF
Beyond these genetic disorders, tooth loss is triggered by a variety of oral diseases, such as periodontitis and dental caries, and by traumatic and age-related alterations. Lack of permanent teeth, for example, may be a consequence of trauma, chemotherapy, or radiotherapy if this occurs at an age when teeth develop (Kaste et al., 1997; Marec-Berard et al., 2005). Tooth loss is also related to poor skeletal status, such as observed in post-menopausal women (Drozdzowska et al., 2006). Hypodontia does not represent a life-threatening condition; however, it is connected with masticatory, speech, and esthetic problems. Oligodontia, especially, has a critical and often life-long impact on oral health and emotional and social well-being of the affected individual. Therefore, the possible replacement of teeth presents a challenge to researchers. Multiple treatment options are currently available for persons with tooth agenesis. The replacement of lost or deficient tissues in general is based on drug therapies, use of different prosthetic materials and implants, and tissue/organ transplantations (Baum and Mooney, 2000). Synthetic dental implants are a widespread standard procedure to replace missing teeth, and its success has increased in recent decades, as documented by the longevity of those replacements (Adell et al., 1990; Dodson, 2006). Different fixtures—such as implant, crown, bridge, fixed or removable, complete and partial dentures—have been shown to be sufficient over the long term (Bartlett, 2007). The negative outcomes include possible infection causing failure of the implant to integrate with the bone, resulting in implant loss and possible bone loss (Callan, 2007). Implants placed into developing alveolar ridges have been shown to inhibit ridge formation (van Steenberghe et al., 1999). Infectious complications of dental implants may affect not only the function and longevity of the implant, but also the individual’s systemic health (Callan, 2007). Pre-operative antibiotic prophylaxis has been used to prevent bacterial infiltration (Schwartz and Larson, 2007). The consequences of an immune reaction against artificial materials, followed by inflammation and possible rejection, are also faced in dental implant treatment (Nowzari et al., 2008). The titanium surface of an implant is biocompatible; however, this can still be enhanced by the coating of synthetic implant surfaces with molecules that can influence basic host responses, enhance subsequent tissue integration, and increase the chances of implantation success (Scheideler et al., 2007). For example, the protein laminin is being tested for its ability to improve gingival adhesion to dental implants (Morra, 2007). When molecular signals are used in general, certain difficulties must be resolved, such as the short half-times of these molecules and the need for their presence over an extended period for effective treatment. The disadvantages of dental implants may also relate to adolescents, in whom craniofacial growth is incomplete, and may result in a change in position and angulation of the osseointegrated implant (Thilander et al., 2001). Moreover, implants do not have a periodontal ligament; thus, the shock-absorbing properties are absent. Implant-supported restorations are still limited; therefore, supportive therapies are currently under study to enhance the success of the implant strategy (Bartlett, 2007). In all cases, the inability of synthetic prostheses to replace any physiological functions of the tissue remains a major disadvantage to such implant procedures. Teeth grow in the context of surrounding tissues, particularly the jawbone, and the implants fail to follow the continuous remodeling of such tissues. The quality of the bone is an essential factor for the success of the implantation and does not necessarily correlate with the person’s age (Young et al., 2007). Therefore, corrective therapies for tooth-bone loss and recovery of the surrounding soft tissue have been investigated (Young et al., 2005; Grusovin et al., 2008). These troubles can be partially overcome by autotransplantation of teeth, whereby a person’s own tooth is transferred from one position in the jaw to another. The autotransplanted tooth prevents closure of the interdental space between remaining teeth (Steigmann et al., 2007), supplying space for the maintenance of orthodontic appliances and skeletal anchorages (Choonara, 2005; Gracco et al., 2007). The autotransplanted tooth also keeps the alveolus more or less functional, and supports the volume and physiological remodeling of bone tissue. The survival rate of transplanted teeth is high, with premolar-to-premolar autotransplants showing over 90% success over the long term (Jonsson and Sigurdsson, 2004).
The methods of molecular stomatology are based on knowledge obtained from embryonic in vivo research and are based around the use of stem cells (Fig. 3 ). The main sources of such "smart cells" are the stem cell niches. ‘Niche’ refers to a specific environmental compartment, consisting of supporting cells, stem cells, and molecular factors able to maintain the basic potential of the stem cells, i.e., to create daughter cells with different fates—one for self-renewal of the stem cell pool and the other one undergoing differentiation into various kinds of tissue-specific cells. Stem cell niches in bone marrow, but also hair follicles and incisor teeth, seem the most promising stem cell sources for tooth engineering. Other sources of craniofacially localized stem cells have been reported as periodontal ligament stem cells, stem cells from human exfoliated deciduous teeth, and dental pulp stem cells. These have been recently reviewed with respect to their potential for craniofacial tissue engineering (Mao et al., 2006). Although the bone-marrow-derived stem cells differ from tooth and hair follicle stem cells, they share some similarities in the molecular program involved in maintaining stem cell niches in different tissues. There are also some differences, however, since FGF is reported to regulate stem cells in the rodent incisor stem cell niche, but such a role is not demonstrated in bone marrow stem cell populations. Wnt signaling seems to play an important role in both mesenchymal and epidermal stem cell niches (Harada et al., 2002).
The re-organization of tissues in scaffolds to form new tissues/organs is one recent approach. This technique was successfully applied in periodontal regeneration (Duailibi et al., 2004; Taba et al., 2005) and has been tested in tooth engineering (e.g., Yelick’s group in the USA). So far, embryonic cells from fetuses have been used to form a new tooth de novo. The experiments support mammalian cell plasticity and suggest the existence of a cell-specific developmental program, even after tissue dissociation and growth in culture conditions (reviewed, e.g., in Nakahara and Ide, 2007). However, much more attractive is the challenge to form a mineralized tooth without any scaffold. Tooth germs can develop successfully following dissociation and re-association without a scaffold support (Hu et al., 2006). This then provides the opportunity to replace embryonic odontogenic cells with stem cells, with adult stem cells providing the best alternative. This technique would enable one to collect an individual’s cells from any stem cell niche, grow these in the culture, induce them to progress along a tooth-developmental pathway, and create a de novo tooth. Great steps have already been made in this direction, a tooth having been created where the mesenchymal parts were stem-cell-derived (Ohazama et al., 2004). Therefore, the only non-stem-cell-derived parts of the resulting tooth crown were the ameloblasts (reviewed in Yen and Sharpe, 2006).
These are challenging but exciting times in dental research. The translation of basic research discoveries into new clinical therapies is an often-repeated theme in medicine. The non-life-threatening nature of most dental disorders, together with the accessibility of the oral cavity, should not be underestimated. Translation in dentistry is, in general, easier than in many other areas of medicine. Dentistry can thus, for once, provide the lead in bringing stem-cell-based treatments into the clinic.
Research into molecular odontogenesis is supported by the Grant Agency of the Academy of Sciences, Czech Republic, B500450802. Investigation of tooth-bone interaction is supported by the Grant Agency of the Czech Republic (524/08/J032). Research in the Brno lab runs under IRP IAPG No. AVOZ 50450515. Received for publication January 23, 2008. Revision received March 19, 2008. Accepted for publication April 1, 2008.
Journal of Dental Research, Vol. 87, No. 7,
617-623 (2008)
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ABSTRACT
MOLECULAR BASIS OF TOOTH...
B transcription factor pathway, result in an HED phenotype. HED is a common name for a group of syndromes with different causal mutations and modes of inheritance, but with a typical phenotype. Mutations in ectodysplasin A1 cause X-linked HED; mutations in Nemo cause X-linked HED with immunodeficiency; while mutations in other components of the Eda signaling complex cause either autosomal-recessive (EDAR, EDARADD) or autosomal-dominant (EDAR) HED. Affected individuals have dysplastic nails, hair, and glands. Virtually all persons with hypohydrotic ectodermal dysplasia exhibit hypodontia (
seems to contribute significantly to oral clefts (