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Quantitative Trait Loci on Chromosomes 10 and 11 Influencing Mandible Size of SMXA RI Mouse StrainsDepartment of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan; Correspondence: * corresponding author, maeda{at}mascat.nihon-u.ac.jp
Predicting the mandible size before the termination of growth of the maxillofacial bones is essential in pedodontics as well as for the predictions needed for genetic analysis. Here, Quantitative Trait Locus (QTL) analysis was used to detect the chromosomal regions responsible for the mandible length between the menton and gonion in an SMXA recombinant inbred strain of mice. Around the region 60 cM from the centromere in chromosome 10, the logarithm of the odds score showed a higher than suggestive level. Around the regions 13 cM and 16 cM in chromosome 11, two significant QTLs were detected. Analysis of genotypes from loci corresponding to those QTLs revealed a large mandible when the region between the markers Hba and D11Mit163 and D10Mit70 and D10Mit136 indicated the genotype from the A/J and SM/J alleles, respectively. These results suggest that the major gene(s) responsible for mandible length are located in these regions.
Key Words: mandible length • QTL analysis • SMXA recombinant inbred strain
The analysis of mandible shape has long been utilized for strain identification by discriminant analysis (Festing, 1972). In such studies, 11 to 13 measurement points set on the outline of the mandible are used for the characterization of mandible shape. Statistical analysis is carried out for the identification of the strain or sub-line (Goto et al., 1993). However, genetic analysis has not been added to the discriminant analysis. Previous research has suggested that the effects of genes on the mandible should be spatially patterned (Klingenberg et al., 2001). Signaling interactions coordinate the outgrowth of the facial primordia from buds of undifferentiated mesenchyme into the intricate series of bones and cartilage structures that, together with muscle and other tissues, form the adult face (Francis-West et al., 1998). The relationship between phenotypes and the genes responsible for the mandible shape is difficult to reveal, because polygenes are involved during facial development, including the growth of the mandible. Quantitative trait locus (QTL) analysis has been very successful in identifying chromosomal regions, with quantitative effects depending on the polygene such as body weight, alcoholism susceptibility, etc. (Nadeau and Frankel, 2000; Cheverud et al., 2001). Recombinant inbred (RI) strains of mice are valuable tools for the study of complex traits such as body weight (Liu et al., 2001). RI strains are derived from systematic inbreeding of randomly selected pairs of the F2 generation of a cross between two different inbred strains of mice. The SMXA RI strain is an existing RI strain derived from the mouse SM/J and the mouse A/J strains as progenitor strains. Both strains have been well-characterized and show differences in a variety of phenotypes, such as body weight (Nishimura et al., 1995). Presently, 26 SMXA RI strains have been generated (Mori et al., 1998). When RI strains are taken as a set, the segregation and gene mapping of a given trait can be analyzed based on the linkage of known marker genes (Anunciado et al., 2000). In this study, the focus is on the identification of the chromosomal regions involved in the regulation of the anteroposterior length of the mandible, as indicated by the distance between the sites corresponding to the menton and the gonion. We report on the genetic analysis of mandible size in the SMXA RI strain using QTL analysis.
Mice A total of 230 mice obtained from parental strains (5 males and 5 females of each of A/J and SM/J) and 21 out of the 26 SMXA RI strains (5 males and 5 females of each of the 21 RI strains) was used. Five SMXA RI strains (SMXA-3, -6, -11, -21, and -23) were excluded from this study due to an insufficient number of samples being available. 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 12L/12D light. The mice were fed a commercial diet (MR Breeder, Nihon Nohsan Co., Kanagawa, Japan) and tap water ad libitum. The animal-use protocol in this study was reviewed and approved by the Nihon University Institutional Review Board.
Preparation and Measurement of Mandibles
The left and right sides of the dried mandible were put on sectional paper with a 1 mm graduation, and the size of the mandible was scaled up to double its original size by means of a duplicator (Canon Co., Japan). The distance between the menton and gonion points was measured as shown in Fig. 1
QTL Analysis The Strain Distribution Pattern (SDP) of 789 polymorphic markers reported in a previous study (Mori et al., 1998) was used in the QTL analysis, but because of the clustering of the marker loci, the net number of loci showing a different SDP was 400. Interval mapping was performed with the use of Map Manager QT b28 (Manly, 1993). With the results of interval mapping, the Likelihood Ratio Statistic may be obtained, a value of additive effect and trait variance. At each marker locus, the significance of the trait association was tested by the logarithm of the odds (LOD) statistic. We obtained the LOD score by dividing the Likelihood Ratio Statistic by 4.605 (Anunciado et al., 2000). The significance threshold for the genome-wide scan was computed by means of a permutation test with 1000 permutated datasets (Doerge and Churchill, 1996). The permutation test is a method of establishing the significance of the Likelihood Ratio Statistic generated by interval mapping. As a result of these tests, the LOD scores used for detecting suggestive/significant associations for the mandible length were 2.2/3.8 in males and 2.3/4.0 in females.
Measurements of Mandible Size in Each Strain The mean values of the menton-gonion measurements in each strain are shown in Fig. 2  . The mandible was extremely large in males of the SMXA-25 strain (18.2 ± 0.1 mm) and small in females of the SMXA-1 strain (15.7 ± 0.1 mm). For the mean values for males and females, the mandible was large in A/J (17.9 ± 0.1 mm) and small in SM/J (15.9 ± 0.2 mm). The mandible sizes formed a continuous size distribution.
QTL Analysis Fig. 3 shows the results of the QTL analysis for mandible length. Three suggestive QTLs and two significant QTLs were detected in males and females. Around the region 60 cM from the centromere in chromosome 10, the LOD score showed a higher than suggestive level (LOD, 2.9; additive effect, 0.43; trait variance, 40% in females). The 95% confidence interval was located between the markers D10Mit2 and D10Mit14.
In the proximal region of chromosome 11, two significant QTLs were detected in females. The first peak LOD score was located at the region 13 cM from the centromere between the markers D11Mit152 and Hba, and the second peak LOD score was located at the region 16 cM between the markers D11Mit229 and D11Mit163. These regions correspond to the 95% confidence interval. The LOD scores were 5.1 at the two peaks in females (additive effect, -049; trait variance, 66%). The LOD scores from males, 2.6 and 2.3 at the two peaks, indicate suggestive levels in the same regions of chromosome 11 (additive effect, -032; trait variance, 36%). Significant or suggestive QTLs were not obtained in the other chromosomes.
We have reported that F1 mice obtained by the crossbreeding of mice with small and large mandibles showed the characteristics of the parent strain mice with larger mandibles. From measurement of 24 reference points in the mandible, the distance between the menton and gonion showed a significant dominant inheritance compared with other distances between reference points. This finding suggests that the distance between the menton and gonion was available as a phenotype for analyzing the genes that determined mandible length (Okamoto et al., 1997).
The SDP corresponding to this genotype was reported in a previous study (Mori et al., 1998). Fig. 4
In chromosome 10 of the SMXA-26, -7, and –30, which had large mandibles, it was indicated that the genotype was derived from the SM/J allele. In the SMXA-1, -4, and –12, which had small mandibles, it was indicated that the genotype was derived from the A/J allele between the markers D10Mit70 and D10Mit136. The effect of this region was opposite that of chromosome 11. The SM/J allele on chromosme 10 for the QTL is associated with a large mandible size.
The mandible size was determined not only by genes located in chromosomes 10 and 11, since there were also several effects that were weak in other chromosomes. Because the number of strains in an RI set is limited in the mouse (26 strains for the SMXA), with use of a more stringent
The Mouse Genome Database (http://www.informatics.jax.org/) was searched for candidate genes according to their position at around 60 cM of chromosome 10 and between 13 cM and 16 cM of chromosome 11. The Mouse Genome Database scan revealed more than 10 genes as candidates for mandible size in chromosomes 10 and 11 (for example, Syt, Myf5, Myf6, Kera, Lum, Kcnc2, and Kifc4b on chromosome 10; Mor2, Otx1, Cct4, Spnb2, Gek1 Hba, and Stk10 on chromosome 11). It is of interest that a candidate gene near the QTL for mandible size on chromosome 11 is Otx1 (orthodenticle), a gene highly related to Otx2. Mouse embryos homozygous for a knockout allele of Otx2 display a striking phenotype in which the entire brain rostral to rhombomere 3 is missing (Ang et al., 1996). This clearly demonstrates the importance of this gene in rostral head development. The knockout mice of Otx1 display a less severe phenotype, but nonetheless indicate a critical role for Otx1 in vertebrate head development (Acampora et al., 1997). Interestingly, Otx1 is also post-natally transcribed and translated in the pituitary gland. Cell culture experiments indicate that Otx1 may activate transcription of the growth, follicle-stimulating, and luteinizing hormones, and of The positions around 60 cM of the mouse chromosome 10 and between 13 cM and 16 cM of the mouse chromosome 11 correspond to regions 12q21 and 2p13 in human chromosomes, respectively. In this study, the experimental conditions were simplified by use of the SMXA RI strain whose chromosome was homozygous. If the result of this study is to be applied to clinical diagnoses, the effects of heterozygous chromosomes must be analyzed. However, focus can be placed on the two chromosomal regions 12q21 and 2p13. It might be possible to predict the mandible size of a patient before the termination of the growth of the maxillofacial bones by searching for the polymorphisms of these chromosomal regions, whether derived from large or small mandibles.
We thank Dr. M. Nishimura (Hamamatsu University School of Medicine) for providing the SMXA RI strain mice. We also thank the members of the Department of Pediatric Dentistry for helpful discussions. This work was supported by a grant from Research for Frontier Science (The Ministry of Education, Science, Sports and Culture). Received for publication November 12, 2001. Revision received May 6, 2002. Accepted for publication May 15, 2002.
Journal of Dental Research, Vol. 81, No. 7,
501-504 (2002) This article has been cited by other articles:
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level (which reduces the acceptable false-positive risk; 
