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Genetic Mapping of the Absence of Third Molars in EL Mice to Chromosome 3

¹ Department of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan; and
² Department of Pediatric Dentistry, Tsurumi University School of Dentistry, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan;

Correspondence: *corresponding author, nonchiku{at}mascat.nihon-u.ac.jp

	ABSTRACT

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We noted the absence of all 4 third molars (M3) in Epilepsy-Like disorder (EL) mice, an animal model for the study of epilepsy. This study was conducted to identify the major candidate chromosome and to detect the region that included the candidate gene causing the absence of M3 in EL mice. Linkage analysis was performed on genetic crosses of EL mice and MSM (Mus musculus molossinus) strain mice, which had a normal complement of teeth. Genome-wide screening by individual genotyping of F₂ intercross mice identified mouse chromosome 3 as one of the candidate chromosomes. Based on high linkage scores in detailed genotyping of F₂ intercross and N₂ backcross mice, the candidate locus for the absence of M3 in EL mice was mapped on the middle of chromosome 3.

Key Words: EL mice • absence of third molar • hypodontia • linkage analysis • interval mapping

	INTRODUCTION

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Hypodontia, which is defined as agenesis of one or more teeth, has been observed as one of the most common human dental developmental anomalies (Burzynski and Escobar, 1983). The misalignment, malocculusion, and oral functional problems caused by hypodontia emphasize the importance of our understanding of the underlying causes. The most commonly congenitally absent teeth are the third molars, followed by either upper lateral incisors or lower second premolars (Symons et al., 1993). So far, genetic mutations in MSX1 (Vastardis et al., 1996; van den Boogaard et al., 2000) and PAX9 (Stockton et al., 2000; Frazier-Bowers et al., 2002) in man have been associated with familial tooth agenesis.

Animal models, the mouse in particular, have contributed to our understanding of tooth formation (http://bite-it.helsinki.fi/). The mouse has a high homology with humans in both gene and chromosomal segments (http://www.ncbi.nlm.nih.gov/Omim/Homology/), so isolating a gene responsible for missing teeth in mice may provide us with a candidate gene in a homologous region for teeth agenesis in humans. The epilepsy-like disorder (EL) mouse was developed as an animal model for the study of epilepsy (Imaizumi and Nakano, 1964). Seizure in EL occurs spontaneously or with routine handling (Frankel et al., 1995). During studies on morphological anomalies of the dentition, we noted the absence of third molars (M3) in EL mice and reported that EL mice have a 97% incidence of absence of all 4 M3 (Asada et al., 2000). The absence of M3 in EL mice was recessively influenced by autosomal factor(s) in the genetic crosses, with wild-type mice showing 0% frequency for the F₁ progeny and 10% frequency for the F₂ progeny (Nakamura et al., 2000). Despite the complete absence of M3 in EL mice as parents, most of the F₂ progeny had partial absence of M3, suggesting a complex etiology.

The absence of M3 occurs at various frequencies in both inbred strains and mutant stocks. This phenotype occurs in about 18% of CBA/Gr mice (Grüneberg, 1951), 3% of CBA/J mice and 2% of A/J mice (Murai, 1975), and in about 92-100% in mutant stocks such as crinkled, Tabby (Miller, 1978), and Crooked-tail (Grewal, 1962) mice exhibiting abnormal crown morphology in the molars (Grüneberg, 1965). The lower rather than the upper M3 was more strongly affected in the mutant stocks, including EL mice. The crinkled and Tabby mutants have a hypohidrotic ectodermal dysplasia phenotype (Headon et al., 2001), and Crooked-tail mice have pleiotropic phenotypes such as anomalies of the axial skeleton, naked tail, and microphthalmia (Morgan, 1954). Since the EL mouse lacks M3 without any generalized polyanomaly, including abnormal molar crown forms, there is no obvious association with absences of M3 in previous mutant stocks (Asada et al., 2000).

We hypothesized that the absence of M3 in EL mice might be controlled by Msx1, because previous MSX1 mutation in humans (Vastardis et al., 1996; van den Boogaard et al., 2000) and knockout of Msx1 in mice (Satokata and Maas, 1994) resulted in tooth agenesis. However, our mutational analysis did not show any mutations in the coding region of Msx1 in EL (Asada et al., 2001), leaving the cause unknown. In the present study, the genetic factor(s) causing the absence of M3 in mice was studied by interval mapping (Taylor et al., 1994), with use of the EL mice and a wild-type strain having a normal dentition, and their genetic crosses. The purpose of this study was to identify a candidate chromosome and to detect the chromosomal region that included candidate gene(s) causing the absence of M3 in EL mice.

	MATERIALS & METHODS

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Mice
EL/Sea (EL) mice lacking all 4 M3 were obtained from Seac Yoshitomi Ltd. (Fukuoka, Japan). MSM/Msf (MSM) mice, which are a wild-type strain derived from Mus musculus molossinus progenitors, with 100% incidence of normal complement of teeth, were obtained from the animal facilities at the National Institute of Genetics (Shizuoka, Mishima, Japan). [F₁ (EL x MSM) x F₁ (EL x MSM)] F₂ intercross and [F₁ (EL x MSM) x EL] N₂ backcross mice were obtained by genetic crosses. 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. Fifteen F₂ and 7 N₂ mice without M3 and 15 control F₂ mice with normal dentition were used. The third molars in F₂ and N₂ were partially absent except for one F₂ with complete absence.

Polymerase Chain-reaction (PCR) Conditions
High-molecular-weight genomic DNA from the spleens of individual EL, MSM, F₂, and N₂ mice were used. The reaction mixtures for PCR were as follows: 0.05 µL (1 unit) of Ex Taq polymerase (TaKaRa, Tokyo, Japan), 1.0 µL 10 x reaction buffer, 0.8 µL dNTPs mixture (2.5 mM each), 2.0 µL (total 25 ng) template DNA, 3.0 µL MIT (Massachusetts Institute of Technology) primer mixture (1.5 µL each from forward and reverse primers, final concentration 0.52 µM), and 3.15 µL distilled water in a final volume of 10 µL. A TaKaRa 480 thermalcycler was used for PCR. Amplification conditions were as follows: DNA denaturation 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 2 min, extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min. The polyacrylamide gels after electrophoresis were stained with ethidium bromide for visualization of the PCR products under ultraviolet light.

Genome-wide Screening for Candidate Chromosome
To determine a candidate chromosome, we performed individual genotyping of 15 F₂ intercross mice with missing M3, using the 58 informative MIT markers that were polymorphic between EL and MSM mice in a previous study (Nomura et al., 2000). The polymorphic markers can distinguish clearly, on gel, between the homozygotes and heterozygotes for the two strains, based on microsatellite polymorphisms. These MIT markers were well-distributed for interval mapping throughout the autosomal genome (Table). Interval mapping distributes the DNA marker on the chromosome on the premise that interchromosomal recombinations occur, and map a responsible locus for a disease by linkage between the causative and marker loci. The loci of individual MIT markers were taken from the Mouse Genome Database (http://www.jax.org/). The sex chromosomes were excluded from genetic analysis because of autosomal inheritance (Nakamura et al., 2000). The linkage between the candidate locus for missing M3 and a marker locus was evaluated by the ² test with 2 degrees of freedom (Rise et al., 1991). The ² test was performed based on the expectation that if the DNA marker is not linked to the causative gene, the genotype ratio of F₂ mice would be EL homozygous:EL/MSM heterozygous:MSM homozygous = 1:2:1. This expected ratio was compared with the observed genotype ratio in F₂ progeny. High ² values associated with small probabilities (p < 0.05) indicated significant linkage.

Linkage Analysis on Candidate Chromosomes
A genome-wide scan suggested that mouse chromosomes 3, 14, 15, and 16 were interesting candidates for the absence of M3 (see RESULTS). To investigate these chromosomes in further detail, we placed additional polymorphic markers for genotyping of F₂ and N₂ progeny. First, genotyping of the 15 F₂ mice with absent M3 and the 15 control F₂ with normal complements of teeth was performed with the additional markers. The linkage was evaluated by ² test with 2 degrees of freedom. Genotyping of the 7 N₂ mice with missing M3 was then performed with the same markers. To evaluate the linkage, we compared the observed genotype ratio of EL homozygotes and EL/MSM heterozygotes among 7 N₂ mice with the expectation of 1:1.

	RESULTS

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Genome-wide Screening for Candidate Chromosome
If a marker locus is closely linked to the gene causing the absence of M3 in EL mice, the genotype will be EL-homozygous, because interchromosomal recombination occurring between two close loci would be rare. As shown in the Table, the highest score in the ² test with a high EL homozygote-genotype ratio was found at D3Mit141 on chromosome 3 (EL homozygous:EL/MSM heterozygous:MSM homozygous = 11:3:1, ² = 18.7, p < 0.0005). In addition, high scores in the ² test were found at D14Mit39 on chromosome 14 (genotype ratio = 8:4:3, ² = 6.6, p < 0.05), D15Mit10 on chromosome 15 (genotype ratio = 8:6:1, ² = 7.1, p < 0.05), and D16Mit1 on chromosome (genotype ratio = 8:7:0, ² = 8.6, p < 0.05).

Linkage Analysis on Candidate Chromosomes
Since the highest ² score on chromosome 3 was detected by genome screening, we focused on this chromosome for further analysis. As shown in Fig. AI, the significant ² scores for genotyping of the 15 F2 mice with missing M3 were found at a continuous chromosomal segment containing the loci D3Mit216, 290, 125, 194, 196, 38, and 17 (EL homozygous:EL/MSM heterozygous:MSM homozygous= 12:2:1, ² = 24.2, p < 0.0001). In addition, high ² scores were also found at neighboring chromosomal areas: D3Mit141, 233, 11, 103, 106, and 260 (genotype ratio = 11:3:1, ² = 18.7, p < 0.0005). In contrast, a low rate of EL homozygote in the control F₂ with normal dentition, compared with the F₂ mice with missing M3, would be expected to support the existence of a linkage. A low EL homozygote ratio was found at D3Mit125, 194, 38, 17, 260, and 147 (genotype ratio = 3:7:5, ² = 0.6), and 216 and 290 (genotype ratio = 4:6:5, ² = 0.7) (Fig. AII). The genotyping data of the 7 N₂ mice with missing M3 are shown in Fig. AIII. A high proportion of EL homozygotes compared with heterozygotes was associated with D3Mit216, 290, 125, 194, 196, 38, and 17 (genotype ratio = 5:2).

The genotyping results on chromosome 16 are shown in Fig. B. The suggestive linkage scores at D16Mit126 for genotyping of the F₂ mice with missing M3 (genotype ratio = 8:7:0, ² = 8.6, p < 0.05), the F2 with normal dentition (genotype ratio = 2:8:5, ² = 1.3), and the N₂ (genotype ratio = 4:3) were found. In contrast, we did not obtain evidence of a linkage for missing M3 on chromosomes 14 and 15, because genotyping results for the F₂ with normal dentition and N₂ conflicted with the linkage scores we expected, i.e., a high rate of EL homozygotes in F₂ mice with M3 and a low rate of EL homozygotes in N₂ mice were found at D14 Mit39, D15Mit10, and their neighboring loci (data not shown).

	DISCUSSION

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Seizures in EL mice are inherited as incompletely dominant or multigenic (Fueta et al., 1986). Previous genetic mapping studies have shown some quantitative trait loci (QTL)—El1 on chromosome (Chr) 9, El2 on Chr 2, El3 on Chr 10, El4 on Chr 9, El5 on Chr 14, and El6 on Chr 11—affecting seizures in EL mice (Rise et al., 1991; Frankel et al., 1995), but did not mention chromosome 3, which we identified as a candidate for the absence of M3. It may be presumed that the phenotypes of the seizure and the absence of M3 are controlled by different loci. Based on ² values in genotyping results of the F₂ with missing M3, there is the highest possibility for a candidate locus in the continuous chromosome areas from D3Mit216 to 17 (Fig. AI). Genotyping data of the F₂ with normal dentition supported the linkage to this area, because low rates of EL homozygotes were obtained from D3Mit125 to 147, where there was an overlap with the high score loci of F₂ with absence of M3 (Fig. AII). Moreover, genotyping of 7 N₂ backcross mice with missing M3 did not contradict the result of F₂ genotypes, exhibiting the highest proportion of EL homozygotes from D3Mit216 to 17 (Fig. AIII). Although we did not analyze the small sample of N₂ statistically, the coincidence of the peak for the genotype ratio between F₂ and N₂ mice supports the existence of a linkage. Taking together the results of F₂ and N₂ genotyping, we conclude that the strongest candidate locus affecting the molar phenotype was found on chromosome 3, and it maps to the middle of the chromosome.

There are some mismatches between phenotypes and genotypes in the candidate interval on chromosome 3 (i.e., MSM homozygous and EL/MSM heterozygous in F₂ with missing M3, EL homozygous in F₂ with normal dentition, and EL/MSM heterozygous in N₂ with missing M3). These findings suggest the involvement of other gene(s) in the occurrence of missing M3 and/or incomplete genetic penetrance. Therefore, we need to analyze in detail not only chromosome 3 but also chromosome 16, which might be another candidate for missing M3. In addition, our sample size is still quite small; thus, we could not completely eliminate chromosomes 14 and 15, and other chromosomes as well.

Although it is premature to consider candidate genes on chromosome 3 in detail, some of the potential candidates include Notch2, Ngfb, Pitx2, Lef1, and Egf (Fig. A). Notch2 is expressed in both the dental epithelium and mesenchyme during tooth development (Mitsiadis et al., 1995). Lef1 is normally expressed during early tooth development, and mice lacking Lef1 demonstrate arrest of tooth development at the bud stage (van Genderen et al., 1994). In explant experiments in vitro, EGF inhibits apoptosis in the dental mesenchyme (Vaahtokari et al., 1996), and antisense oligonucleotides to EGF mRNA inhibit tooth development (Kronmiller et al., 1991). Pitx2 was initially identified as one of the genes responsible for the human Rieger syndrome (Semina et al., 1996), an autosomal-dominant condition that causes developmental abnormalities including hypodontia. In Pitx2-gene-deleted mice, the tooth bud does not progress beyond the initiation stage (Lin et al., 1999). NGF mRNA appears in the mesenchymal field of the developing mouse tooth and supported odontogenesis in a first branchial arch explant culture of mice (Amano et al., 1999).

Mutational analysis of the potential candidate genes or their expression patterns during M3 development in EL mice will be examined in subsequent experiments. A histological examination to determine whether congenital absence of M3 in EL mice occurs because of failure of initiation of the tooth germ or subsequent arrest in development is presently under way. The results of this study will likely provide clues to our understanding of hypodontia and tooth development in mice as well as in humans.

View larger version (24K): [in this window] [in a new window]   Figure. Linkage analysis of chromosome 3 (A) and chromosome 16 (B). Genotype in 15 F2 mice with absence of M3 (I), in 15 F2 mice with normal dentition (II), and in 7 N2 mice with absence of M3 (III). Filled squares denote homozygosity for the EL allele; open squares denote homozygosity for the MSM allele; stippled squares denote heterozygosity for the EL/MSM allele; blank indicates that the marker was not tested. 2 values for genotyping ratio of F2 at each marker are shown to the right of the genotype panel; ap < 0.05; bp < 0.01; cp < 0.001; dp < 0.0005; ep < 0.0001. An illustration of the map position of the Mit markers in centimorgans (cM) from the centromere, relative to potential candidate genes, placed according to the Mouse Genome Database (MGD).

	ACKNOWLEDGMENTS

Received for publication May 17, 2001. Revision received June 18, 2003. Accepted for publication July 25, 2003.

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Journal of Dental Research, Vol. 82, No. 10, 786-790 (2003)
DOI: 10.1177/154405910308201005


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This Article Abstract Figures Only Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Nomura, R. Articles by Maeda, T. Search for Related Content PubMed PubMed Citation Articles by Nomura, R. Articles by Maeda, T. Social Bookmarking                         What's this?