Associations between Carabelli trait and cusp areas in human permanent maxillary first molars.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 129:196–203 (2006) Associations Between Carabelli Trait and Cusp Areas in Human Permanent Maxillary First Molars Shintaro Kondo1,2* and Grant C. Townsend2 1 Department of Oral Anatomy and Developmental Biology, Showa University School of Dentistry, Tokyo 142-8555, Japan 2 Dental School, University of Adelaide, Adelaide 5005, Australia KEY WORDS odontometry; sexual differences; tooth size; crown morphology ABSTRACT Few dental anthropological studies have investigated the associations between tooth crown size and crown traits in humans using quantitative methods. We tested several hypotheses about overall crown size, individual cusp areas, and expression of Carabelli cusps in human permanent ﬁrst molars by obtaining data from standardized occlusal photographs of 308 Australians of European descent (171 males and 137 females). Speciﬁcally, we aimed to calculate the areas of the four main molar cusps, and also Carabelli cusp, and to compare the relative variability of cusp areas in relation to timing of development. We also aimed to compare cusp areas between males and females and to describe how Carabelli cusp interacted with other molar cusps. Measurements included maximum crown diameters (mesiodistal and buccolingual crown diameters), the areas of the four main cusps, and the area of Carabelli cusp. The pattern of relative variability in absolute areas of molar cusps corresponded with their order of formation, the ﬁrstforming paracone displaying the least variation, and the last-forming Carabelli cusp showing the greatest. Overall crown size and areas of individual cusps all showed sexual dimorphism, with values in males exceeding those in females. Sexual dimorphism was smallest for paracone area and greatest for Carabelli cusp area. Overall crown size and cusp areas were larger in individuals displaying a Carabelli cusp, especially the hypocone area. Although the combined area of the protocone and a Carabelli cusp was greater in cuspal forms than noncuspal forms, protocone area alone was signiﬁcantly smaller in the former. Our ﬁndings lead us to propose that, in individuals with the genotype for Carabelli trait expression, larger molar crowns are more likely to display Carabelli cusps, whereas molars with smaller crowns are more likely to display reduced forms of expression of the trait. We suggest that the pattern of folding of the internal enamel epithelium in developing molar crowns, particularly in the protocone region, can be modiﬁed by a developing Carabelli cusp. Am J Phys Anthropol 129:196–203, 2006. V 2005 Wiley-Liss, Inc. Metric and nonmetric analyses of the human dentition have formed a central focus in the ﬁeld of dental anthropology for over a century (Keiser, 1990; Hillson, 1996; Scott and Turner, 1997). Many metric studies involved measurement of the maximum mesiodistal and buccolingual dimensions of dental crowns with calipers (e.g., Moorrees et al., 1957; Garn et al., 1967; Townsend and Brown, 1979) or more recently from standardized photographs (e.g., Reid et al., 1991, 1992; Townsend et al., 2003). Investigations of so-called nonmetric dental crown traits, including the Carabelli cusp, were usually based on scoring features with reference to standard plaques (e.g., Dahlberg, 1949; Turner et al., 1991), leading to calculations of their frequency of occurrence and degree of expression. Metric approaches conﬁrmed that sexual dimorphism exists in overall crown size, with values for males exceeding those for females on average. Sexual dimorphism was also reported for Carabelli trait occurrence and expression, but there seems to be no consistent pattern across different ethnic groups (Townsend and Brown, 1981; Hsu et al., 1997). Some studies included both metric and nonmetric data to make comparisons within and between populations, and some looked at the interactions or associations between these variables (Garn et al., 1966; Keene, 1968; Sasaki, 1968; Bang and Hasund, 1972; Lombardi, 1975; Noss et al., 1983; Reid et al., 1991, 1992; Hsu et al., 1997). More recently, researchers began analyzing intra- coronal components, e.g., cusp areas and intercuspal distances, which are thought to provide a better biological basis for describing tooth size variation (e.g., Biggerstaff, 1969; Corruccini, 1979; Townsend et al., 2003). These studies aimed to clarify the ontogenetic basis of crown size and shape, especially the role of enamel knots in inﬂuencing folding of the internal enamel epithelium, leading to the development of cusps (Jernvall and Jung, 2000). Each enamel knot represents the site of a future cusp tip, and it was suggested that evolutionary change in molar morphology is constrained to some extent by the relationship between when enamel knots start to form and when intercuspal growth ceases during odontogenesis (Polly, 1998). C V 2005 WILEY-LISS, INC. C Grant sponsor: National Health and Medical Research Council of Australia. *Correspondence to: Shintaro Kondo, Department of Oral Anatomy and Developmental Biology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo142-8555, Japan. E-mail: firstname.lastname@example.org Received 26 October 2004; accepted 6 January 2005 DOI 10.1002/ajpa.20271 Published online 1 December 2005 in Wiley InterScience (www.interscience.wiley.com). CARABELLI TRAIT AND MOLAR CUSP AREAS Although the relationship between lower molar crown size, cusp number, and groove pattern was ﬁrst addressed by Dahlberg (1961), very few studies since then have looked at associations between overall crown size, intracoronal components, and dental crown traits, with quantitative methods being used to describe observed variation (Reid et al., 1991, 1992). Our general aim in this study was therefore to quantify overall crown size and cusp areas of a sample of human maxillary ﬁrst molars, as well as expression of Carabelli cusps, by calculating areas from standardized occlusal photographs of dental casts. Speciﬁcally, we aimed to calculate the areas of the four main molar cusps, and also Carabelli cusp, and to compare relative variability of cusp areas in relation to timing of development. Further objectives included making comparisons between males and females and describing how Carabelli cusp interacted with other molar cusps. We then aimed to explain our ﬁndings in the light of current knowledge about crown morphogenesis, including the folding of the internal enamel epithelium in response to the formation of enamel knots. The speciﬁc hypotheses we sought to test were: That the pattern of relative variation of cusp areas reﬂects the ontogeny of maxillary ﬁrst molar crown development, with earlier-forming cusps showing less variation than later-forming cusps; That molar cusp areas, and the areas of Carabelli cusps, are larger in males on average than in females; That teeth with a Carabelli cusp have larger crowns on average than those without a Carabelli cusp; That the size of the protocone is reduced in teeth with a Carabelli cusp compared with those without a Carabelli cusp, reﬂecting an interaction between adjacent growth regions of the developing crown; and That the hypocone is larger in molars with a Carabelli cusp, reﬂecting a general increase in cusp expression on the lingual aspect of these teeth. MATERIALS AND METHODS In total, 308 dental casts of South Australian twins aged 8–29 years, with the majority being teenagers, were selected from a collection of over 600 pairs of dental casts housed in the Dental School at the University of Adelaide. The twins were all of European ancestry. Previous studies considered genetic aspects of tooth size and Carabelli trait expression (Pinkerton et al., 1999; Dempsey and Townsend, 2001), but in this study we concentrated on phenotypic associations, so only one member from each twin pair was included in the analysis. Only subjects with little or no evidence of wear on their maxillary ﬁrst molars, either occlusally or interproximally, were included in the study. Only molars with four main cusps were included. Our ongoing study of teeth and faces of Australian twins was approved by the Committee on the Ethics of Human Experimentation, University of Adelaide (approval no. H/07/84A), and all participants provided informed consent. Initially, Carabelli trait was scored according to the nine grades described by Reid et al. (1991, 1992), but these grades were then combined into four categories: grade 0, representing absence of the trait, formed cate- 197 gory 0; grades 1–4, including pits and furrows, became category 1; grade 5, deﬁned as a mesial and distal furrow, was grouped with teeth showing minor lingual prominences that could not be measured from photographs to form category 2; and grades 6–8, including examples with free apices and those forms of expression that could be measured on occlusal photographs, were combined to form category 3. Standardized photographs of the occlusal surfaces of maxillary ﬁrst molars were obtained from dental casts using a Nikon CoolPix 950 digital camera. The molar crowns were oriented so that the plane produced by the cusp tips of the three major cusps, excluding the hypocone, was perpendicular to the optical axis of the camera. The tooth was positioned in the center of the image, and a millimeter scale was placed next to the tooth in the same horizontal plane as the occlusal surface. Measurements on photographs were performed with manual image measurement software (Visual Measure 32, Version 1.2, Rise Co.) on a personal computer. The method described by Wood and Engleman (1988) was followed when measuring basal cusp areas, using the primary occlusal grooves to deﬁne the four main cusps, and the grooving associated with a Carabelli cusp to determine its area. Measurements were obtained normally of ﬁrst molars on the right side, but if a tooth on the right could not be measured because of absence, abnormality, heavy wear, or other reasons, the corresponding tooth on the left side of the arch was measured. Maximum mesiodistal and buccolingual crown diameters, and the areas of the four main cusps, i.e., the paracone (mesiobuccal), protocone (mesiolingual), metacone (distobuccal), and hypocone (distolingual), were measured to the nearest 1 mm2 (Fig. 1). Only those forms of Carabelli trait that appeared as distinct lingual prominences (category 3), and that could be distinguished clearly from the protocone, were measured separately. Total crown area was calculated by summing the areas of individual cusps. Previous studies to assess the replicability of the photographic procedure based on linear measurements of scanned images compared with those obtained directly from dental casts showed no systematic methodological errors (Townsend et al., 2003). Furthermore, Bailey et al. (2004) showed that the levels of interobserver error for cusp area measurements obtained from digitized images are similar to those for intraobserver comparisons. In the present study, measurement errors of the method were analyzed by a procedure in which double determination measurements were made on separate occasions for 40 subjects selected at random for whom the entire photographic and digitizing process was repeated. Differences between ﬁrst and second determinations were analyzed by computing the standard deviation of a single determination, or so-called technical error of measurement, using the following formula (Dahlberg, 1940): sﬃﬃﬃﬃﬃﬃﬃﬃﬃ Rd2 Error ¼ 2N In this formula, d ¼ difference between double determinations, and N ¼ number of double determinations. Paired t-tests were also used to detect systematic errors. To assess the observation error in classifying Carabelli 198 S. KONDO AND G.C. TOWNSEND Fig. 1. Measurement of overall crown dimensions and cusp areas of maxillary right molar. Individual cusps were distinguished by locating major occlusal grooves. Carabelli cusp area was only measured when it could be discerned clearly from protocone. MD, mesiodistal crown diameter; BL, buccolingual crown diameter; Pa, paracone area; Pr, protocone area; Me, metacone area; Hy, hypocone area; Ca, Carabelli cusp area. trait, the concordance rate in the double determination observations was calculated for 50 subjects selected at random. Sex differences were compared by calculating the percentage of sexual dimorphism (Garn et al., 1967), deﬁned as [(M F)/F] 3 100, where M and F are the mean values of males and females, respectively. Descriptive statistics, including distribution parameters, were calculated with JMP statistical software (SAS Institute, Version 4.02) on a personal computer. Differences in cusp area measurements between different categories of Carabelli trait were analyzed with one-way ANOVA followed by Dunnett’s test, with statistical signiﬁcance set at P < 0.05. RESULTS There was no indication of systematic methodological errors in the calculation of crown dimensions or areas between ﬁrst and second determinations, based on paired t-tests. Mean differences between ﬁrst and second determinations for mesiodistal and buccolingual diameters were 0.02 and 0.03 mm, and for each cusp area including Carabelli cusp area, values ranged from 0.07 to 0.07 mm2. Percentage errors for the areas of the four main cusps were less than 0.5%, while that for Carabelli cusp area was 1.5%. These percentage values for intraobserver errors were smaller than those of interobserver errors for molar cusp area in Pan reported by Bailey et al. (2004). The technical errors of measurement were 0.17 and 0.18 mm, respectively, for mesiodistal and buccolingual dimensions, and ranged from 0.22–0.29 mm2 for cusp areas. Error variances, calculated as the square of the Dahlberg statistic, were all less than 10% of total observed variation, conﬁrming that errors of the method were small and unlikely to bias the results. The concordance rate for scoring the Carabelli trait on two separate observations was 92% in the sample of 50 individuals, and all discordances between ﬁrst and second determinations were between categories 1 and 2. Thus, errors in scoring were small. Table 1 shows basic descriptive statistics for molar crown measurements. All mean values were signiﬁcantly larger in males than in females (P < 0.01). Dimorphism percentage values for absolute crown areas exceeded those for overall crown diameters, but no statistical tests were applied to compare these values, and care is needed in drawing any conclusions based on comparisons of this measure of sexual dimorphism. Carabelli trait area showed the largest dimorphism value, and the smallest value was for the paracone area. The hypocone was the most variable in absolute area of the four main molar cusps, whereas the least variable cusp in absolute area was the paracone. Carabelli cusp area showed the most variation in size of all molar cusps. To take account of overall tooth size, comparisons of relative crown size were also made between males and females. With the exception of the Carabelli cusp, sexual differences in the relative cusp areas were fairly small. Carabelli cusps still showed the largest sexual difference in the relative cusp area (P < 0.01). Table 2 shows crown measurements for each category of Carabelli trait. Around 70–80% of all subjects showed evidence of the trait in varying forms, with 19–25% exhibiting cusps (category 3). Approximately 13–19% of subjects showed category 2, and 35–38% showed category 1. These percentages are similar to those reported in our earlier studies of Australian twins (Townsend and Martin, 1992; Pinkerton et al., 1999). Although there was no statistically signiﬁcant sex difference in frequencies for each of the categories (v2 ¼ 6.126, P ¼ 0.106), the cuspal forms of Carabelli trait tended to be more common in males than in females (25.7% compared with 19.0%). Overall crown measurements were larger in cuspal forms than in noncuspal forms. For example, the mean mesiodistal diameter of ﬁrst molars without Carabelli trait was 10.1 mm in males and 9.9 mm in females, compared with 10.5 mm and 10.3 mm, respectively, in molars displaying category 3 expression of the Carabelli trait. Furthermore, the lingual cusps, i.e., protocone and hypocone, showed larger differences between cuspal forms and noncuspal forms than the buccal cusps, i.e., paracone and metacone. In males, lingual cusps showed signiﬁcant differences between cuspal forms and noncuspal forms (P < 0.05), but buccal cusps did not. For example, the mean area of the hypocone in molars without Carabelli trait was 17.4 mm2, compared with 19.5 mm2 in molars showing category 3 expression. Similar trends were also shown in females, but the results of statistical tests were not signiﬁcant. The combined area of the protocone and Carabelli trait was signiﬁcantly larger in cuspal forms (30.9 mm2 in males and 28.8 mm2 in females) than in noncuspal forms, where the areas averaged 28.1 mm2 and 27.1 mm2, respectively (P < 0.05). However, the protocone area alone was smaller in cuspal forms than in noncuspal forms (25.8 mm2 compared with 28.1 mm2 in males, and 25.4 mm2 compared with 27.1 mm2 in females), the difference in males being signiﬁcant at P < 0.05. Table 3 shows a correlation matrix of molar crown measurements, with values for males above the diagonal and values for females below. Correlation coefﬁcients calculated between crown diameters and total crown areas were all moderately high. However, values of correlation coefﬁcients between cusp areas were low to moderate in magnitude, especially between the hypocone area and other cusp areas. These ﬁndings are consistent with our previous results for intercuspal distances in molar teeth, 199 CARABELLI TRAIT AND MOLAR CUSP AREAS 1 TABLE 1. Basic statistics of maxillary ﬁrst molar crown diameters, and absolute and relative cusp areas Males N Mean Sexual difference Females SD Crown diameters (mm) MD 171 10.3 0.54 BL 171 11.3 0.52 2 Absolute cusp areas and total crown area (mm ) Pa 171 23.6 2.61 Pr þ Ca 171 29.6 3.62 Ca 44 5.1 2.79 Me 171 22.5 2.89 Hy 171 19.0 3.46 Total crown area 171 94.7 8.45 Relative cusp area to total crown area (%)2 Pa 171 25.0 2.20 Pr þ Ca 171 31.3 2.97 Ca 44 5.2 2.78 Me 171 23.7 2.04 Hy 171 20.0 2.78 CV3 N Mean SD CV3 t-test 5.3 4.6 137 137 10.1 11.0 0.51 0.59 5.0 5.4 ** ** 2.6 3.1 11.1 12.2 55.1 12.9 18.2 8.9 137 137 26 137 137 137 22.6 27.8 3.4 21.2 17.9 89.6 2.61 3.49 1.40 2.82 3.40 8.74 11.6 12.5 40.9 13.3 19.0 9.8 ** ** ** ** ** ** 4.4 6.6 48.3 5.8 6.1 5.8 8.8 9.5 53.5 8.6 13.9 137 137 26 137 137 25.3 31.1 3.7 23.7 20.0 2.19 2.64 1.49 2.00 2.89 8.7 8.5 40.0 8.4 14.5 NS NS ** NS NS 1.3 0.8 39.0 0.0 0.3 % 1 Abbreviations of crown dimensions are summarized in Figure 1. NS, not signiﬁcant. Relative cusp area ¼ (Cusp area)/(Total crown area) 3 100. 3 CV ¼ (SD/Mean) 3 100. **P < 0.01. 2 which showed that the values of correlation coefﬁcients between intercuspal dimensions were consistently lower than those between overall crown measurements, suggesting that much of the intracoronal covariation in cusp position and area is unexplained (Townsend et al., 2003). DISCUSSION The pattern of relative variation in absolute areas of the four main molar cusps mirrored the known ontogenetic sequence of cusp development, with earlier-forming cusps being more stable than later-forming cusps. This ﬁnding is consistent with the results of previous studies (Gingerich, 1974; Corruccini, 1979; Kondo et al., 2005). Relative cusp areas were more stable than absolute cusp areas, especially for the metacone (Table 1). Absolute cusp areas were also associated with greater sexual dimorphism percentage values than mesiodistal and buccolingual crown diameters, a result consistent with a previous ﬁnding based on molar cusp diameters (Kondo et al., 2005). However, because most researchers used categorical systems to classify the expression of Carabelli trait, little information has been available about its relative variation in size. Our method enabled accurate quantiﬁcation of the area of Carabelli cusps, and showed that it displays greater relative variation in its area than any of the main molar cusps, consistent with being the last cusp to appear during crown development (Kraus and Jordan, 1965). This quantitative approach also provided support for a previous ﬁnding in our sample of Australian twins: the cuspal form of Carabelli trait displays signiﬁcant sexual dimorphism in its expression. Some workers found signiﬁcant sexual differences in the expression of Carabelli trait (e.g., Townsend and Brown, 1981; Mizoguchi, 1985), but others reported no sex differences in this trait (Garn et al, 1966). Mizoguchi (1985) highlighted the inconsistencies one can encounter when assessing sex differences in crown trait expression. He presented frequency distributions for 12 different crown traits observed in two Japanese samples, and found that only Carabelli trait showed a signiﬁcant sex difference in both samples. Our earlier studies of Australian twins based on scoring the Carabelli trait showed differences in frequency of occurrence and degrees of expression between the sexes (Townsend and Martin, 1992; Pinkerton et al., 1999), whereas this quantitative study showed that the average area of a Carabelli cusp is signiﬁcantly greater in males than females. It seems that the extent of sexual dimorphism for Carabelli trait varies between different ethnic groups, so care is needed when making comparisons between groups using standardized scoring systems (e.g., Turner et al., 1991), especially if sex is unknown. We found that the mesiodistal and buccolingual crown diameters of ﬁrst molars were larger on average in those individuals who displayed Carabelli trait than in those who did not, a result consistent with many former studies (De Terra, 1905; Broekman, 1938; Keene, 1968; Sasaki, 1968; Lombardi, 1975; Noss et al., 1983; Hsu et al., 1997). Furthermore, cusp areas were larger in cuspal forms than in noncuspal forms, a trend that was particularly evident in the hypocone. Reid et al. (1991, 1992) also used a quantitative approach to show that molars with a Carabelli cusp were larger than those without, so it seems that this is a common positive association. Our ﬁnding that the hypocone area displayed the biggest difference between molars with and without a Carabelli cusp supports an earlier report by Mizoguchi (1985), and is also consistent with the trait being less common in three-cusped molars (Keene, 1968; Suzuki and Sakai, 1973; Scott, 1979). Dahlberg (1949) and Korenhof (1960) reported that protocone size tends to be reduced when Carabelli trait is strongly developed. Reid et al. (1991, 1992) contradicted these ﬁndings, but they included protocone area within their measurement of Carabelli trait, and so they were unable to separate the contributions of the two cusps. We measured Carabelli cusp and protocone areas independently, and found that combined protocone and Carabelli cusp area was signiﬁcantly larger in cuspal forms than in noncuspal forms. However, protocone area alone was signiﬁcantly smaller in cuspal forms than in noncuspal forms. Thus, it seems that protocone area is 200 S. KONDO AND G.C. TOWNSEND TABLE 2. Maxillary ﬁrst molar crown diameters (mm) and cusp areas (mm2) for various categories of Carabelli trait1 Category of Carabelli trait Males N (%) MD BL Pa Pr þ Ca Pr Ca Me Hy Total crown area Females N (%) MD BL Pa Pr þ Ca Pr Ca Me Hy Total crown area Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 0 1 2 3 33 (19.3%) 10.1 0.55 11.1 0.61 23.0 2.88 28.1 3.47 28.1 3.47 61 (35.7%) 10.4*** 0.57 11.3 0.50 23.5 2.89 29.7 3.29 33 (19.3%) 10.4 0.48 11.3 0.51 23.5 2.07 29.4 2.92 44 (25.7%) 10.5**** 0.50 11.5**** 0.45 24.3 2.29 30.9**** 4.23 25.8***** 4.08 5.1 2.79 22.7 2.50 19.5*** 3.55 97.3**** 7.97 22.2 2.90 17.4 3.20 90.8 9.27 40 (29.2%) 9.9 0.53 10.7 0.63 22.3 2.80 27.1 3.76 27.1 3.76 20.2 2.50 16.8 3.74 86.5 9.27 22.4 2.95 19.2*** 3.50 94.7 8.07 52 (38.0%) 10.1 0.43 11.0 0.48 22.6 2.31 27.6 3.21 21.5 2.16 18.2 3.38 90.0 7.49 22.6 3.30 19.7*** 3.15 95.1 7.76 19 (13.9%) 10.2 0.54 11.1 0.67 22.9 3.41 28.3 3.58 22.0 2.84 18.5 3.24 91.6 9.36 26 (19.0%) 10.3**** 0.52 11.2**** 0.55 22.8 2.30 28.8 3.44 25.4 3.33 3.4 1.40 21.6 3.98 18.7 2.69 92.0*** 8.85 ANOVA F-ratio P value F-ratio P value F-ratio P value F-ratio P value 3.503 0.017* 3.619 0.014* 1.664 0.177 3.902 0.010** F-ratio P value F-ratio P value F-ratio P value 0.184 0.907 3.286 0.022* 4.029 0.008** F-ratio P value F-ratio P value F-ratio P value F-ratio P value 3.496 0.017* 3.735 0.013* 0.290 0.832 1.505 0.216 F-ratio P value F-ratio P value F-ratio P value 2.567 0.057 2.241 0.086 2.857 0.040* 1 Abbreviations of crown dimensions are summarized in Figure 1. *P < 0.05. **P < 0.01 (ANOVA). ***P < 0.05. ****P < 0.01 (Dunnett’s test between category 0 and others for this and P < 0.05). *****Signiﬁcant difference between mean areas for category 0 and 3 at P < 0.05 level by t-test. reduced when the Carabelli trait is well-developed, and we believe that this can be explained by interactions between adjacent folding regions of the internal enamel epithelium during odontogenesis. Figure 2 gives an example of a molar with a well-developed Carabelli cusp and with the protocone displaced buccally and distally. The Carabelli trait originates from the lingual cingulum region of maxillary molar crowns, being evident particularly on the ﬁrst molar (Korenhof, 1960; Sakai and Hanamura, 1967). Kraus and Jordan (1965) noted that a Carabelli cusp is created by an extension of the calciﬁcation process from the protocone during tooth development, and that it appears after the other cusps have begun to calcify. It is evident, at least in its cuspal form, at the dentino-enamel junction of fully-formed teeth in modern humans (Sakai and Hanamura, 1971; Sasaki, 1997). Schwartz et al. (1998) also noted some correspondence between expression of Carabelli cusp on the tooth crown surface and the topography of the dentino- enamel junction in early hominid specimens, even though the nature of the relationship was variable. So even though the expression of the trait on the surface of the tooth and at the dentino-enamel junction may not coincide precisely, it appears that expression of Carabelli trait on molar crowns is related to folding of the internal enamel epithelium during odontogenesis, and is not produced merely by deposition of enamel. The Carabelli trait appears to be under strong genetic control, and there were suggestions of major gene involvement for its expression (Kolakowski et al., 1980; Nichol, 1989; Townsend and Martin, 1992). Some researchers reported low estimates of heritability (Biggerstaff, 1973; Alvesalo et al., 1975; Mizoguchi, 1977), but these results may reﬂect differences in methodologies between studies. In fact, Alvesalo et al. (1975) noted that the dichotomy of having a Carabelli cusp or not may have a genetic basis, but that there appeared to be large variation in the expression of the ‘‘cusp-genotype.’’ CARABELLI TRAIT AND MOLAR CUSP AREAS TABLE 3. Matrix of correlations between molar crown diameters and cusp areas1 MD BL Pa MD 0.81** 0.61** BL 0.84** 0.59** Pa 0.64** 0.68** Pr þ Ca 0.60** 0.72** 0.43** Me 0.71** 0.68** 0.44** Hy 0.63** 0.60** 0.18* TCA 0.91** 0.94** 0.68** Pr þ Ca Me Hy TCA 0.56** 0.58** 0.29** 0.63** 0.68** 0.42** 0.17* 0.64** 0.61** 0.12 0.15* 0.48** 0.91** 0.92** 0.63** 0.64** 0.74** 0.68** 0.39** 0.22** 0.74** 0.37** 0.76** 0.65** 1 Abbreviations of crown dimensions are summarized in Figure 1. TCA, total crown area. Upper right, males (N ¼ 171); lower left, females (N ¼ 137). *P < 0.05. **P < 0.01. Fig. 2. Example of permanent ﬁrst molar with well-developed Carabelli cusp. Protocone is displaced buccally and distally, and its size is reduced. Mesial marginal ridge is very well-developed. D, distal; L, lingual; Pa, paracone; Pr, protocone; Me, metacone; Hy, hypocone; Ca, Carabelli cusp. Hlusko and Mahaney (2003) found that cusp-like structures which derived from the lingual cingula of maxillary molars in baboons, similar to features referred to as Carabelli trait in humans, were signiﬁcantly heritable. However, several studies mentioned that Carabelli trait may be present on the deciduous second molar but not on the permanent ﬁrst molar of the same individual (Townsend and Brown, 1981; Saunders and Mayhall, 1982; Smith et al., 1987; Pinkerton et al., 1999). Interestingly, the reverse relationship is very rare. It was suggested that reduced expression in the permanent ﬁrst molar may result from environmental inﬂuences operating during its longer period of development. In other words, the Carabelli phenotype of deciduous second molars may represent a more faithful representation of the underlying genotype than its phenotypic expression in the permanent dentition. We plan to extend our study 201 of cusp areas and Carabelli trait expression to the deciduous dentition, but believe that analyses taking account of the timing and sequence of crown formation of the permanent ﬁrst molar crown can also provide valuable insights into the nature of variation in expression of the Carabelli trait. Developments in molecular biology are now providing a much clearer picture of the processes involved in odontogenesis, including the development of dental crown shape (Sperber, 2004). Reciprocal interactions between oral epithelium and neural crest-derived mesenchyme inﬂuence how the internal enamel epithelium, which represents the future dentino-enamel junction and provides a blueprint for completed crown structure, will fold. This folding is associated with the appearance of groups of nondividing cells, referred to as enamel knots, that act as signaling centers. The primary enamel knot is one such transient signaling center that seems to be an important regulator of overall tooth shape during the cap stage of odontogenesis (Jernvall and Jung, 2000). Secondary enamel knots form subsequently at the sites of future cusp tips, providing the ﬁrst signs of speciesspeciﬁc cusp patterns. Control of secondary enamel knot spacing must exist during morphogenesis, as this process determines correct cusp position and size, leading to a functional tooth shape (Jernvall and Thesleff, 2000). Although the spacing between enamel knots needs to be accurately controlled to produce the patterns of cusp position that distinguish different species, the results of our correlation analysis in this study, together with ﬁndings from previous analyses of intercuspal distances, suggest that the stochastic nature of the local epigenetic events involved in crown formation may contribute to variation in cuspal arrangements within human tooth classes (Townsend, 1985; Townsend et al., 2003). The recent ﬁndings of Hlusko et al. (2004) of genetic independence of mesial and distal loph variation in baboon molar crowns also support the view that minor variations in cusp position within species are determined intrinsically. No cusp-speciﬁc differences in homeobox gene expression have been reported within species (Zhao et al., 2000), and the development of individual cusps appears to use repeatedly the same set of developmental genes, forming a ‘‘developmental module.’’ In fact, the repeated activation of these developmental modules may explain the cumulative variation in later-developing cusps (Jernvall and Jung, 2000). It was proposed that the wave-like expression of signaling factors produced within developing teeth, reﬂecting dynamic interactions between molecules, may be responsible for crown patterns (Weiss, 1990; Weiss et al., 1998). Furthermore, a patterning cascade mode of cusp spacing may promote the evolution of new cusps (Polly, 1998; Jernvall, 2000). If a simple patterning cascade is applied to human molars, a larger Carabelli cusp would be expected to be present when the height of the protocone, relative to the paracone, is large. As the differences would be cumulative, only a very small increase in size of the protocone would be needed to have a large effect on the size of a Carabelli cusp. Putting together the various aspects of molar morphogenesis described above, and drawing on the morphogenetic triangle concept of Keene (1991), we propose the following explanation for observed relationships between overall molar crown size, cusp areas, and Carabelli trait (Fig. 3). In those individuals who have the genetic con- 202 S. KONDO AND G.C. TOWNSEND crown formation in males leading to larger teeth on average. Furthermore, the tendency for teeth with Carabelli cusps to have larger hypocones may also relate to an extended period of crown development, enabling stronger expression of this later-formed cusp. The reduction in area of the protocone in those molars with welldeveloped Carabelli cusps is thought to reﬂect the interaction occurring between closely developing cusp regions, as the inner enamel epithelium folds at the site of each of the ﬁve enamel knots. CONCLUSIONS We propose that molars with larger crowns will be more likely to display Carabelli cusps in genetically predisposed individuals, because the ﬁfth enamel knot that appears late during crown development will be more likely to be fully expressed. In contrast, smaller molars will tend to be associated with less developed forms of Carabelli trait. Furthermore, we suggest that the pattern of folding of the internal enamel epithelium in the protocone region can be modiﬁed by the presence of a ﬁfth enamel knot for Carabelli cusps, leading to displacement of the protocone on the fully calciﬁed crown. ACKNOWLEDGMENTS Fig. 3. Proposed relationship between maxillary molar crown size and shape, and Carabelli trait expression. G and E represent genetic and environmental factors. The assistance of the Australian Twin Registry is greatly appreciated, and we also thank the twins and their families for their enthusiastic participation. Sandy Pinkerton and Wendy Schwerdt assisted in data collection. LITERATURE CITED stitution for Carabelli trait expression, we suggest that molars with larger crowns are more likely to display Carabelli cusps. In these individuals, a ﬁfth enamel knot will tend to develop near to the protocone during the later stages of molar crown development. This genotypic potential will only be expressed to its full extent phenotypically if the period of crown growth is extended for a sufﬁcient time to enable an additional late folding of the inner enamel epithelium to occur in the protocone region. This may occur if the duration of mitotic activity of the developing tooth germ is extended and/or the coalescence of calciﬁcation between cusp tips is delayed. In either case, it is likely that the Carabelli trait will be expressed in its cuspal form, and also that the crown will tend to be larger overall. In contrast, smaller molars, in which the period of crown growth is shorter, will be less likely to express the more developed forms of Carabelli trait, because the extent of folding related to formation of the ﬁfth enamel knot will be reduced. Thus the positive association between crown size and presence of Carabelli cusp may not relate to the Carabelli cusp ‘‘causing’’ the crown to be bigger, but rather may occur because individuals whose ﬁrst molars tend to be bigger will consequently be more likely to display the Carabelli trait. 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