Alan Brook

Multilevel complex interactions between genetic, epigenetic and environmental factors in the aetiology of anomalies of dental development.

Dental anomalies are caused by complex interactions between genetic, epigenetic and environmental factors during the long process of dental development. This process is multifactorial, multilevel, multidimensional and progressive over time. In this paper the evidence from animal models and from human studies is integrated to outline the current position and to construct and evaluate models, as a basis for future work. Dental development is multilevel entailing molecular and cellular interactions which have macroscopic outcomes. It is multidimensional, requiring developments in the three spatial dimensions and the fourth dimension of time. It is progressive, occurring over a long period, yet with critical stages. The series of interactions involving multiple genetic signalling pathways are also influenced by extracellular factors. Interactions, gradients and spatial field effects of multiple genes, epigenetic and environmental factors all influence the development of individual teeth, groups of teeth and the dentition as a whole. The macroscopic, clinically visible result in humans is a complex unit of four different tooth types formed in morphogenetic fields, in which teeth within each field form directionally and erupt at different times, reflecting the spatio-temporal control of development. Even when a specific mutation of a single gene or one major environmental insult has been identified in a patient with a dental anomaly, detailed investigation of the phenotype often reveals variation between affected individuals in the same family, between dentitions in the same individual and even between different teeth in the same dentition. The same, or closely similar phenotypes, whether anomalies of tooth number or structure, may arise from different aetiologies: not only mutations in different genes but also environmental factors may result in similar phenotypes. Related to the action of a number of the developmental regulatory genes active in odontogenesis, in different tissues, mutations can result in syndromes of which dental anomalies are part. Disruption of the antagonistic balance between developmental regulatory genes, acting as activators or inhibitors can result in dental anomalies. There are critical stages in the development of the individual tooth germs and, if progression fails, the germ will not develop further or undergoes apoptosis. The reiterative signalling patterns over time during the sequential process of initiation and morphogenesis are reflected in the clinical association of anomalies of number, size and form and the proposed models. An initial step in future studies is to combine the genetic investigations with accurate recording and measurement of the phenotype. They also need to collate findings at each level and exploit the accurate definition of both human and murine phenotypes now possible.

Novel missense mutations and a 288-bp exonic insertion in PAX9 in families with autosomal dominant hypodontia

We describe the molecular analysis of three families with hypodontia involving primarily molar teeth and report two novel mutational mechanisms. Linkage analysis of two large families revealed that the hypodontia was linked to the PAX9 locus. These two families revealed missense mutations consisting of a glutamic acid substitution for lysine and a proline substitution for leucine within the paired domain of PAX9. A pair of identical twins affected with hypodontia in a third family demonstrated a 288‐bp insertion within exon 2 that resulted in a putative frameshift mutation and a premature stop codon. The insertion was associated with the loss of 7‐bp from exon 2. A block of 256‐bp of sequence within the insertion was completely identical to downstream sequence from the second intron of the PAX9 gene. These studies extend the spectrum of mutations in PAX9 associated with hypodontia to include heretofore undescribed categories, including missense mutations.

Genetic and environmental influences on human dental variation: A critical evaluation of studies involving twins

Utilising data derived from twins and their families, different approaches can be applied to study genetic and environmental influences on human dental variation. The different methods have advantages and limitations and special features of the twinning process are important to consider. Model-fitting approaches have shown that different combinations of additive genetic variance (A), non-additive genetic variance (D), common environmental variance (C), and unique environmental variance (E) contribute to phenotypic variation within the dentition, reflecting different ontogenetic and phylogenetic influences. Epigenetic factors are also proposed as important in explaining differences in the dentitions of monozygotic co-twins. Heritability estimates are high for most tooth size variables, for Carabelli trait and for dental arch dimensions, moderate for intercuspal distances, and low for some occlusal traits. In addition to estimating the contributions of unmeasured genetic and environmental influences to phenotypic variation, structural equation models can also be used to test the effects of measured genetic and environmental factors. Whole-genome linkage analysis, association analysis of putative candidate genes, and whole genome association approaches, now offer exciting opportunities to locate key genes involved in human dental development.

Epithelial histogenesis during tooth development

This paper reviews the current understanding of the progressive changes mediating dental epithelial histogenesis as a basis for future collaborative studies. Tooth development involves morphogenesis, epithelial histogenesis and cell differentiation. The consecutive morphological stages of lamina, bud, cap and bell are also characterized by changes in epithelial histogenesis. Differential cell proliferation rates, apoptosis, and alterations in adhesion and shape lead to the positioning of groups of cells with different functions. During tooth histo-morphogenesis changes occur in basement membrane composition, expression of signalling molecules and the localization of cell surface components. Cell positional identity may be related to cell history. Another important parameter is cell plasticity. Independently of signalling molecules, which play a major role in inducing or modulating specific steps, cell-cell and cell-matrix interactions regulate the plasticity/rigidity of particular domains of the enamel organ. This involves specifying in space the differential growth and influences the progressive tooth morphogenesis by shaping the epithelial-mesenchymal junction. Deposition of a mineralized matrix determines the final shape of the crown. All data reviewed in this paper were investigated in the mouse.

Morphogenetic fields within the human dentition: A new, clinically relevant synthesis of an old concept☆

This paper reviews the concept of morphogenetic fields within the dentition that was first proposed by Butler (Butler PM. Studies of the mammalian dentition. Differentiation of the post-canine dentition. Proc Zool Soc Lond B 1939;109:1–36), then adapted for the human dentition by Dahlberg (Dahlberg AA. The changing dentition of man. J Am Dent Assoc 1945;32:676–90; Dahlberg AA. The dentition of the American Indian. In: Laughlin WS, editor. The Physical Anthropology of the American Indian. New York: Viking Fund Inc.; 1951. p. 138–76). The clone theory of dental development, proposed by Osborn (Osborn JW. Morphogenetic gradients: fields versus clones. In: Butler PM, Joysey KA, editors Development, function and evolution of teeth. London: Academic Press, 1978. p. 171–201), is then considered before these two important concepts are interpreted in the light of recent findings from molecular, cellular, genetic and theoretical and anthropological investigation. Sharpe (Sharpe PT. Homeobox genes and orofacial development. Connect Tissue Res 1995;32:17–25) put forward the concept of an odontogenic homeobox code to explain how different tooth classes are initiated in different parts of the oral cavity in response to molecular cues and the expression of specific groups of homeobox genes. Recently, Mitsiadis and Smith (Mitsiadis TA, Smith MM. How do genes make teeth to order through development? J Exp Zool (Mol Dev Evol) 2006; 306B:177–82.) proposed that the field, clone and homeobox code models could all be incorporated into a single model to explain dental patterning. We agree that these three models should be viewed as complementary rather than contradictory and propose that this unifying view can be extended into the clinical setting using findings on dental patterning in individuals with missing and extra teeth. The proposals are compatible with the unifying aetiological model developed by Brook (Brook AH. A unifying aetiological explanation for anomalies of tooth number and size. Archs Oral Biol 1984;29:373–78) based on human epidemiological and clinical findings. Indeed, this new synthesis can provide a sound foundation for clinical diagnosis, counselling and management of patients with various anomalies of dental development as well as suggesting hypotheses for future studies.

Genetic, environmental and epigenetic influences on variation in human tooth number, size and shape

The aim of this review is to highlight some key recent developments in studies of tooth number, size and shape that are providing better insights into the roles of genetic, environmental and epigenetic factors in the process of dental development. Advances in molecular genetics are helping to clarify how epigenetic factors influence the spatial and temporal regulation of the complex processes involved in odontogenesis. At the phenotypic level, the development of sophisticated systems for image analysis is enabling new dental phenotypes to be defined. The 2D and 3D data that are generated by these imaging systems can then be analysed with mathematical approaches, such as geometric morphometric analysis. By gathering phenotypic data and DNA from twins, it is now possible to use ‘genome-wide’ association studies and the monozygotic co-twin design to identify important genes in odontogenesis and also to clarify how epigenetic and environmental factors can affect this process. Given that many of the common dental anomalies affecting the human dentition are interrelated, apparently reflecting pleiotropic genetic effects, the discoveries and new directions described in this paper should have important implications for clinical dental practice in the future.

Detection of a Novel Mutation in X-linked Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders of defective enamel formation. The major protein involved in enamel formation, amelogenin, is encoded by a gene located at Xp22.1-Xp22.3. This study investigated the molecular defect producing a combined phenotype of hypoplasia and hypomineralization in a family with the clinical features and inheritance pattern of X-linked amelogenesis imperfecta (XAI). Genomic DNA was prepared from buccal cells sampled from family members. The DNA was subjected to the polymerase chain-reaction (PCR) in the presence of a series of oligonucleotide primers designed to amplify all 7 exons of the amelogenin gene. Cloning and sequencing of the purified amplification products identified a cytosine deletion in exon VI at codon 119. The deletion resulted in a frameshift mutation, introducing a premature stop signal at codon 126, producing a truncated protein lacking the terminal 18 amino acids. Identifying mutations assists our understanding of the important functional domains within the gene, and finding another novel mutation emphasizes the need for family-specific diagnosis of amelogenesis imperfecta.

Tooth size patterns in patients with hypodontia and supernumerary teeth

AIMS Anomalies of tooth number may not be isolated conditions but may have wider associations in the development of the dentition including tooth size. This study aimed to examine links between hypodontia, supernumerary teeth and crown size, considering the effect on the development of the whole dentition and so increase understanding of the aetiology of these conditions. METHODS AND RESULTS The patients, who were all of European ancestry, were 60 young adults (30 males and 30 females) with hypodontia and 60 age and sex matched controls together with 60 young adults (39 males and 21 females) with supernumerary teeth and 60 age and sex matched controls. Hand measurements of mesiodistal and buccolingual dimensions were made of the teeth on dental study models using Mitutoyo electric callipers. The mean value of two measurements was used and intra-operator and inter-operator reliability determined. Patients with hypodontia had smaller teeth than the control group and this difference was statistically significant (p<0.05) for all teeth except the MD dimensions of 13, 23, 24 and 44. The difference in size was greatest for the BL dimensions in hypodontia patients. Further, the greater the number of missing teeth the smaller the tooth size. The hypodontia patients also showed higher variability in tooth dimensions than the control group. Patients with supernumerary teeth had larger teeth than the controls, with the greatest differences in the MD dimensions. In both hypodontia and supernumerary patients the differences in tooth size were generalised throughout the dentition. CONCLUSIONS In anomalies of tooth number the size of teeth is also involved. In patients with hypodontia and supernumerary teeth the crown size of the whole dentition is affected. These findings are compatible with a multifactorial aetiology of these conditions.

Variability and patterning in permanent tooth size of four human ethnic groups

AIMS Dental dimensions vary between different ethnic groups, providing insights into the factors controlling human dental development. This paper compares permanent mesiodistal crown diameters between four ethnic groups highlighting patterns of tooth size between these groups and considers the findings in relation to genetic and environmental influences. METHODS AND RESULTS Mesiodistal crown dimensions were recorded using standardised manual measurements on dental casts derived from four different human populations: Southern Chinese, North Americans of European ancestry, Modern British of European ancestry and Romano-British. Analyses based on double determinations showed that measurements in all study samples were reliable to an accuracy of 0.1mm. The Southern Chinese sample was found to have the largest teeth overall, whereas the Romano-British sample generally displayed the smallest mesiodistal crown dimensions (p<0.001). However, the Modern British sample had the largest maxillary central incisors, mandibular central and lateral incisors, and mandibular canines, while the North American sample had the largest maxillary first and second molars. Comparisons of coefficients of variation for teeth within each class showed that the later-forming teeth displayed greater variation in mesiodistal size than the earlier-forming teeth. CONCLUSION The different patterns of tooth size observed between the study samples are thought to reflect differences in the relative contributions of genetic, and environmental influences to dental development between the four populations. For example, it is proposed that major environmental insults during the early life of Romano-Britons, including recurrent illnesses, poor nutrition and excessive lead ingestion, contributed to the reduction in size and greater variability of their later-forming teeth. Using a standardised methodology, significant differences in mesiodistal crown diameters have been demonstrated between four human ethnic groups. There were also distinct differences in the patterns of crown size between the groups, with the later-forming teeth in each type generally showing greater size variation.

Tooth dimensions in hypodontia with a known PAX9 mutation

AIM Congenital absence of teeth is a complex condition affecting several parameters of oral development. This is the first study to measure tooth crown dimensions using image analysis in a family with hypodontia in whom the mutation has been identified, and compare them with a control group. METHODS AND RESULTS Study models were obtained from 10 family members from three generations affected by severe hypodontia with a missense mutation in PAX9 and 10 unaffected, unrelated controls. Using established image analysis techniques all teeth up to and including the first permanent molars were digitally imaged by two operators from the occlusal (O) and buccal (B) aspects three times and an average made for the mesio-distal (MDO and MDB) bucco-lingual (BL), area (A) and perimeter (P) measurements. Intra-class correlation coefficients (ICCC) were calculated to assess intra- and inter-operator reliability. Two-sample t-tests were then used to compare these dimensions with those of the controls. Reliability of the technique was high (mean r>0.95). The majority of tooth types throughout the dentition were significantly smaller in the family members with hypodontia than in the control group for all parameters measured. The levels of significance were very high for upper lateral incisors (p<0.0001) whilst the canines and first molars were less different. The greatest number of significant differences were found in BL and P, closely followed by MD and A measurements. CONCLUSIONS The significantly smaller tooth crown dimensions recorded in the affected family members show that the effect of the PAX9 mutation is seen not only in the congenitally missing teeth but also in smaller crown size throughout the dentition.