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Genetic Analysis of Crown Size in the First Molars Using SMXA Recombinant Inbred Mouse Strains

Department of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan;

^* corresponding author, takehiko{at}mascat.nihon-u.ac.jp

	ABSTRACT

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Tooth crown size may be determined by both genetic and environmental factors. The aim of this study was to identify quantitative trait loci (QTLs) affecting dental crown size and determine whether there is genetic independence between upper and lower teeth, using SMXA recombinant inbred strains of mice. Mesiodistal and buccolingual crown diameters (MD and BL, respectively) of the upper and lower first molars (M¹ and M₁, respectively) were measured. For each trait, mean values of substrains showed a continuous spectrum of distribution. Genome-wide scan detected QTLs exceeding suggestive threshold levels for MD of M¹ (chromosomes 7, 13, and 17), BL of M¹ (chromosomes 8 and 13), MD of M₁ (chromosomes 7 and 13), and BL of M₁ (chromosomes 3 and 15). These findings suggest that tooth crown size is controlled by multiple genes, and that there is some independence of genetic control between M¹ and M₁.

KEY WORDS: tooth size • SMXA recombinant inbred mice • QTL analysis • interval mapping

	INTRODUCTION

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Tooth crown size is one of the most important factors for the determination of dentition and occlusion. A discrepancy between tooth size and arch length causes orthodontic and subsequent oral functional problems. Thus, the molecular mechanisms for the determination of tooth size are of great interest to dentists as well as developmental biologists. Dental crowns are complex structures, and crown size may be determined by both genetic and environmental factors (Riesenfeld, 1970; Garn et al., 1980; Dempsey and Townsend, 2001). Studies using twin pairs (Dempsey et al., 1995; Hughes et al., 2000) or different ethnic groups (Hattab et al., 1996; Harris et al., 2001) have shown that variation in crown size includes a strong genetic component. Studies of individuals with sex chromosome aneuploidies or anomalies have found that sex-linked gene(s) on both X and Y chromosomes (Chrs) modulate tooth size (Alvesalo, 1997). Certain autosomal syndromes, including Down’s syndrome, are associated with reduction in size of permanent teeth (Peretz et al., 1996), indicating involvement of autosomal factors in dental crown growth. Although a great many genes have been identified as being expressed at sequential stages of tooth development in epithelial and/or mesenchymal cells (http://bite-it.helsinki.fi/), very little is known about the genetics of specific tooth size.

Animal models, in particular mouse models, have contributed to the understanding of tooth formation (Jernvall and Thesleff, 2000; Thesleff and Mikkola, 2002). Normal mouse dentition is composed of 1 continuously growing incisor and 3 molars of limited growth in each quadrant. A recent study where the investigators used interval mapping of the F2 progeny from a cross between the LG/J and SM/J strains of mice has revealed 3 quantitative trait loci (QTLs) for mandibular molar centroid size and 18 QTLs for mandibular molar shape (Workman et al., 2002). In mutant mouse stocks, crinkled (cr, Chr 13), tabby (Ta, Chr X) (Sofaer, 1979), and crooked (cd, Chr 6) (Sofaer, 1977) genes reduce molar size. However, in such mutants, variation of crown size (which affects morphological structure) is part of pleiotropic phenotypes that are analogous to human hypohidrotic ectodermal dysplasia, distinguishing these genes from genetic factors that influence crown growth in general populations of mice.

The SMXA recombinant inbred (RI) mouse strain set has been proven to be a powerful tool for the analysis of multifactorial genetic traits (Pataer et al., 1997; Suzuki et al., 2000; Kobayashi et al., 2003). SMXA RI strains were produced by systematic inbreeding from the F2 generation of a cross between A/J and SM/J inbred strains (Nishimura et al., 1995). The parental strains show phenotypic differences in a variety of traits, including body weight, blood insulin, and lipid levels (Anunciado et al., 2000). A detailed genetic profile of the SMXA RI strains has been reported (Mori et al., 1998).

In the present study, we performed linkage analysis to identify the QTLs involved in determination of the mesiodistal and buccolingual crown diameters of the upper and lower first molars, using SMXA RI mouse strains. We attempted to determine if there are multiple QTLs for tooth size, and whether these QTLs represent a degree of independence of genetic control between upper and lower molars.

	MATERIALS & METHODS

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Mice
In total, 226 mice (106 females and 120 males) were used, from parental strains A/J and SM/J (n = 10 each) and 21 of the 26 substrains of the SMXA RI set (n = 7–12 each). Five SMXA RI strains (SMXA-3, -6, -11, -21, and -23) were excluded from this study because of an insufficient number of samples. All mice were obtained from the Institute for Experimental Animals, Hamamatsu University School of Medicine (Hamamatsu, Japan) and were maintained under conventional conditions: 25 ± 2°C, 55 ± 5% humidity, and 12-hour light/dark cycle. The mice were fed a commercial diet (MR Breeder, Nihon Nohsan Co., Kanagawa, Japan) and tap water ad libitum. All procedures were approved by the Institutional Animal Care Committee, and were performed according to Nihon University School of Dentistry at Matsudo guidelines for the care and use of laboratory animals.

Measurement of Crown Diameters of the First Molars
The mice were killed under anesthesia at 40 days of age, and the skulls were then de-fleshed and macerated in 1% potassium hydroxide at 42°C for 48 hrs. The bilateral upper and lower first molars (M¹ and M₁, respectively) were extracted from the maxilla and mandible. In total, 392 M¹ (n = 12–22 for each strain) and 425 M₁ (n = 12–23 for each strain) were used, excluding 97 teeth that were lost or broken during preparation. Maximum mesiodistal diameter (MD) and buccolingual diameter (BL) parallel to the occlusal plane of the crowns of M¹ and M₁ were measured (32x magnification) under a stereoscopic microscope equipped with an ocular micrometer (Kristenová-ermáková et al., 2002). All measurements were performed under standard conditions. The subjective error of measurements was determined to be non-significant, based on a t test and three sets of 10 independent measurements.

QTL Analysis
For genome-wide scan, we used more than 1000 polymorphic markers (well-distributed on autosomes and X chromosome) that exhibited informative strain distribution patterns (SDPs) (Mori et al., 1998; http://www.med.nagoya-u.ac.jp/sisetu/index.htm). Interval mapping was performed by means of Map Manager QTXb15 (Manly et al., 2001), for the detection of QTLs for each crown diameter, and the significance of each QTL was represented as a likelihood ratio statistic (LRS). We obtained logarithm-of-odds (LOD) scores by dividing the LRS by 4.605 (Anunciado et al., 2000). The significance threshold for the interval mapping was computed by 1000x permutation tests (Doerge and Churchill, 1996). The following LOD scores calculated by permutation tests were used for detecting suggestive/significant associations for the dental crown diameters in male mice (MD of M¹, 1.95/3.21; BL of M¹, 2.02/3.45; MD of M₁, 2.13/3.67; BL of M₁, 2.15/3.76), female mice (MD of M¹, 2.02/3.37; BL of M¹, 1.98/3.52; MD of M₁, 2.10/3.88; BL of M₁, 2.21/4.06), and all mice (pooled) (MD of M¹, 1.98/3.30; BL of M¹, 2.00/3.54; MD of M₁, 2.13/3.71; BL of M₁, 2.21/4.04).

	RESULTS

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Measurement of Crown Diameters of the First Molars
The mean values of all crown diameters in the SMXA RI and parental strains did not show a significant difference between the sexes; therefore, males and females were pooled. For the sex-pooled population, the highest and lowest values of each diameter showed a significant difference (p < 0.01, t test), and the mean values of all traits showed a continuous distribution pattern (Fig.). The distribution patterns of MD for M¹ and M₁ were similar in terms of positions of the substrains and parental strains. SM/J was positioned at the higher end of the histogram, whereas A/J was positioned in the middle. For BL of M¹ and M₁, both parental strains were positioned at or near the middle of the histogram.

View larger version (48K): [in this window] [in a new window]   Figure. Distribution of mesiodistal and buccolingual crown diameters (MD and BL, respectively) of the upper and lower first molars (M1 and M1, respectively) in 21 of the SMXA RI strains and the 2 parental strains (SM/J and A/J). (a) MD of M1; (b) BL of M1; (c) MD of M1; and (d) BL of M1. Data are expressed as means ± SD. Numbers in parentheses represent the numbers of the sex-pooled population used.

	DISCUSSION

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Although the main purpose of the present study was to discover QTLs affecting tooth size in mice, we were also very interested in determining whether M¹ and M₁ are genetically independent in determination of crown size. In QTL analysis results, only 1 QTL (on Chr 13) had an effect on crown diameters of both M¹ and M₁; all other QTLs influenced the 4 traits independently. Although many genes that are known to influence the development of teeth may have effects that are basic to the formation of all kinds of teeth, our results suggest that the growth of different teeth in the upper and lower jaws is determined by partially independent genetic pathways. It has been reported that mice with targeted null mutations of both Dlx-1 and Dlx-2 genes did not develop maxillary molar teeth, but had normal incisors and mandibular molars (Thomas et al., 1997). Previous analysis of tooth development in activin betaA mutant embryos showed that incisor and mandibular molar teeth fail to develop, although development of maxillary molars was unaffected in the mutants (Ferguson et al., 1998). These findings support the hypothesis that there is a degree of genetic independence between M¹ and M₁.

In humans, males have larger tooth dimensions than females, on average, and there is statistically significant sexual dimorphism in both the primary and secondary dentitions (Harris et al., 2001), suggesting the influence of sex-linked gene(s) on crown growth. In addition, studies of individuals with various sex chromosome aneuploidies and their normal relatives demonstrated that gene(s) on the human sex chromosomes had differential direct effects on tooth crown growth (Alvesalo, 1997). In contrast, there is no significant sexual dimorphism in the crown lengths of molars in wild-type mice (Kristenova-Cermakova et al., 2002). In the present study, there was no significant difference in the 4 traits of SMXA RI strains between the sexes, and LOD scores exceeding suggestive threshold levels were not detected at any locus on Chr X. Chr Y was not examined without SDPs of the SMXA RI strains. We presumed that sex-linked gene(s) affecting crown size may exist in mice as well as humans, because some female- and male-specific QTLs were detected based on slight differences between the sexes in the histograms. Also, we presumed that autosomal genes may have stronger effects on crown growth of mice than sex-linked factor(s).

A recent study has shown that QTLs influencing mandible length (distance between menton and gonion) in SMXA RI strains are located on Chr 10 and 11, with the smallest mandible in SM/J mice and the largest mandible in A/J mice (Dohmoto et al., 2002). In the present study, QTLs were not detected on Chr 10 and 11 for crown diameters of M₁, and SM/J mice had the longest MD diameters for M₁, in contrast to findings for mandible length. It is conceivable that genetic determination of crown size of M₁ is independent from the growth of mandible length. In humans, a heritable pattern of small teeth in well-developed jaws has been reported (Peck et al., 1998), suggesting that crown growth does not genetically parallel jaw growth.

We searched for potential candidate genes that map close to the QTLs we found here, based on the Mouse Genome Database. Candidate genes involved in tooth development were located within 1-LOD support interval of the QTL for MD of M₁ on Chr7 (Tgfb1) (Chai et al., 1994) and the QTL for BL of M₁ on Chr3 (Fgf2) (Cam et al., 1992). Ccnd1, which has been shown to influence tooth alignment (Fantl et al., 1995), maps near the present Chr7 QTL for MD of M¹. Hexb maps 1 cM distal from D13mit191, where we found a QTL affecting both M¹ and M₁ size. Hexb-deficient mice exhibit post-natal growth abnormality and craniofacial defects (Sango et al., 1996). There were no candidate genes for other QTLs; therefore, at those QTLs, novel genes may be involved in the regulation of specific processes affecting crown development.

Confirmation of the candidate loci and further definition of chromosomal location requires a fine-mapping study with a F2(SM/JxA/J) intercross. The results of the present study and subsequent fine-mapping will help clarify the underlying mechanisms of dental crown growth in man, because mouse and human genes are highly syntenic.

	ACKNOWLEDGMENTS

 
We appreciate Dr. M. Nishimura (Nagoya University) for providing the SMXA RI strain mice. This investigation was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology, 13771283.

Received April 11, 2003; Last revision October 7, 2003; Accepted October 9, 2003

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