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A Genome Segment on Mouse Chromosome 12 Determines Maxillary Growth
1 Section of Orthodontics, UCLA School of Dentistry, 43-091 Center for the Health Sciences, Box 951668, Los Angeles, CA 90095-1668, USA; Correspondence: * corresponding author, epae{at}dentistry.ucla.edu
The primary and modifier genes that regulate normal maxillofacial development are unknown. Previous quantitative trait locus (QTL) analyses using the F2 progeny of 2 mouse strains, DBA/2J (short snout/wide face) and C57BL/6J (long snout/narrow face), revealed a significant logarithm-of-odds (LOD) score for snout length on mouse chromosome 12 at 44 centimorgan (cM). We further sought to validate this locus contributing to anterior-posterior dimensions of the upper mid-face at the D12Mit7 marker in a 44-centimorgan portion of chromosome 12. Congenic mice carrying introgressed DNA from DBA/2J on a C57BL/6J background were selected for submental vertex cephalometric imaging. Results confirmed QTLs, determining that short snout length (P < 0.05) and face width relative to snout length (P < 0.01) were present in the 44-cM region of chromosome 12. We conclude that one or more genes contributing to the shape of the maxillary complex are located near 44 cM of mouse chromosome 12.
Key Words: facial types • congenic mice • maxilla • quantitative trait loci (QTL)
Malocclusions associated with craniofacial imbalance due to retrognathic or prognathic maxillae, presumed to be inherited characteristics (Lauweryns , 1993; Townsend , 1998; Eguchi, 2004), are reported to affect 40% or more of the US population (Proffit et al., 1998). The association of facial dysmorphogenesis with various genetic disorders has increased information in the field of craniofacial genetics. However, the genetic component of subtle dysmorphisms commonly seen in orthodontic patients, such as prognathic or retrograthic maxillae, remains unclear. Structural variations of the face appear polygenic in origin (Shum et al., 2000). Linkage of quantitative measurements on genetic traits—i.e., phenotypic difference with genomic differences—is a basic strategy for mapping quantitative trait loci (QTL). Unlike other approaches involving mutants and segregation analyses (McBratney et al., 2003), or haplotype-tag SNP and linkage disequilibrium analysis (Coussens and van Daal, 2005), which are based on testing a specific candidate gene, QTL analysis surveys the entire genome to identify loci underlying phenotypic variation (Lander and Schork, 1994; Drake et al., 2001; Mackay, 2001). Previous QTL analysis of an F2 intercross between DBA/2J (short snout/wide inter-zygoma) and C57BL/6J (long snout/narrow inter-zygoma) strains identified a locus at marker D12Mit7 on chromosome 12 (Nishimura et al., 2003). Recombinant inbred strains carry approximately equal proportions of genes from both parental strains, which are scattered randomly over all chromosomes, and are considered excellent for gene mapping (Chesler et al., 2005). The detecting power of recombinant inbred strains for phenotype difference is significantly superior to that of intercrossed F2 analysis (Belknap et al., 1996). However, it is almost impossible, with a single recombinant inbred strain, to study phenotype effects of an isolated locus. Recently, a genome-wide library of congenic mice carrying DBA/2J alleles introgressed on a C57BL/6J background became available (Iakoubova et al., 2001; Estrada-Smith et al., 2004; Davis et al., 2005). These mice can be utilized to increase the accuracy of gene mapping. This is a technique used particularly when an improved genetic resolution of quantitative traits is required (Fridman et al., 2004). In this study, we used congenic strains to confirm the hypothesis that congenic mice carrying DBA/2J alleles at the target marker (D12Mit7 of DBA/2J) will exhibit the short snout/wide zygomatic arch of the donor strain.
Overlapping congenic strains covering the proximal (B6.D2.12P or 12P), middle (B6.D2.12M or 12M), and entire length of chromosome 12 (B6.D2.12C or 12C) were selected for study (Fig. 1a  ). The 12M and 12C strains carry the Mit7 locus, while the 12P strain was selected as a negative control. Mapping data for the congenic regions are shown in Table 1, modified from a previous publication (Davis et al., 2005). We waited until craniofacial growth of the mice ended, at 10 wks of age, then measured 4 male and 4 female mice from each congenic strain and the 2 parental strains (C57BL/6J and DBA/2J) for craniofacial dimension by submental-vertex radiography, utilizing dental intra-oral films (Kodak Ultra-Speed, DF-57, Kodak, Rochester, NY, USA), a custom-made cephalostat apparatus, and a dental x-ray unit (70 Kvp, 15 mA) (Gendex®, General Electric Co., Milwaukee, WI, USA). The cephalostat helped us orient the craniofacial sagittal plane perpendicular to the film, and allowed for consistent and reproducible distances among film, subject, and x-ray source. We used 10-week-old mice whose craniofacial growth was complete. All procedures in the study were carried out under protocols approved by the University of California, Los Angeles, Animal Research Committee.
The following landmarks were identified on the submental-vertex films, as previously reported (Nishimura et al., 2003): the anterior-most point of the mid-palatal suture (I), the right and left anterior junctions of the zygomatic arch and the maxillary process (Za), the right and left posterior junctions of the zygomatic arch and the maxillary process (Zb), and the right and left lateral junctions between zygomatic processes of maxillary and temporal bones (Zc) (Fig. 1b  ). Measurements were obtained by means of an electronic digital caliper with an accuracy of ± 0.02 mm. For each specimen, 2 measurements were taken (right and left) for snout length (I-Za) and zygomatic arch width (Zb-Zc). Zb-Zc, not Zc-Zc, was used, so that nasal width included in the measurement Zc-Zc would not act as a confounding factor. Each sample was measured 3 times and on 3 separate occasions. The first 2 measurement sets were taken by one operator. The third measurement set was performed by a blinded third-party operator. No significant difference in measurement values was found between operators or between occasions. We randomly selected a set of data from among the 3 sets for appropriate statistical analyses, and discarded the other 2 sets. We subsequently analyzed all values obtained from each strain, to determine the mean and standard error of each group. We used ANOVA to assess statistical significance of differences among the groups, and a post hoc Bonferroni test for evaluation of multiple pairs (SPSS v. 12.0). As for males vs. females, no inference test was attempted, due to small sample size, yet no significant trend was noted in either direction.
Congenic strains 12C and 12M, which carried the target Mit7 allele, showed a significantly shorter snout (P < 0.05) and a wider face relative to snout length (P < 0.05), compared with 12P and the background strain C57BL/6J (Table 2 , Fig. 2). The ratio between snout length and facial width between the groups showed that both 12M and 12C exhibited a short snout and a small snout length to facial width ratio of DBA/2J, while the snout length/facial width ratio more closely matched that of the background strain (Table 2, Fig. 2).
Size and shape variations of craniofacial bones compared with the size of the teeth are the significant underlying etiology of malocclusions. Many orthodontic patients have mild to moderate maxillary skeletal discrepancies, which are associated with unfavorable facial esthetics, occlusion, and psychosocial complications (Birkeland et al., 2000). Although human maxillofacial and dental morphology appears to be influenced by both genetic and environmental factors, an estimated 40% or more of dental and skeletal variations that lead to malocclusion may be ascribed to hereditary factors (Lauweryns et al., 1993; Townsend et al., 1998; Eguchi et al., 2004). Early studies with longitudinal cephalometric radiographs and dental casts of siblings showed that facial skeletal features had stronger heritability when compared with that of pure dental features. Therefore, it was concluded that the skeletal contribution of a malocclusion is apt to have a significant hereditary component, as opposed to the more environmentally determined dental contribution (Harris and Johnson, 1991). Our results clearly showed that the congenic strain 12M shows facial characteristics of the donor strain. However, while not statistically significant, the consomic mice (12C) carrying additional DBA/2J regions on chromosome 12 of D2 have trended toward a shorter snout than D2 (2.91 mm for 12C vs. 3.57 mm for D2). This suggests the potential existence of other cis-acting sequence variations located proximal to the 50,305,248-base-pair region at the proximal end of chromosome 12 in 12M mice. Because QTLs localize traits only to a large chromosomal span, additional strategies are required to identify the underlying genes. The use of congenic strains eliminates much of the genetic "background noise" that potentially conceals the effects of QTLs on a phenotype of interest (Iakoubova et al., 2001; Estrada-Smith et al., 2004), since congenic mice are strains that differ in genotype at, ideally, one contiguous locus only. After initial QTL mapping is performed, congenic strains can confirm and further fine-map a trait (Davis et al., 2005). These strains contain a distinct chromosomal segment harboring the QTL of interest from a donor parental strain, with the remainder of the genome deriving from the background recipient strain. Congenic strains can reveal the magnitude of effect of an isolated gene, or genes, on phenotype. Comparing phenotypes between congenic mice and controls from the recipient or background strain using more advanced imaging techniques, such as micro-CT, may corroborate our suspected QTL effects. Similar characterization of subcongenic strains carrying smaller regions of introgressed donor alleles can narrow the search for effector genes in the future. The size and position of the mandible can also influence the configuration of the maxilla, and thus, needs to be taken into consideration as a confounding factor. Several QTL studies have already been undertaken with the goal of identifying the loci that determine facial size and shape, and these experiments have focused primarily on the mandible. A major locus controlling shape and size of the mandible is suggested to be on chromosomes 10 and 11, which regulate the linear menton-gonion length of the mouse mandible body (Dohmoto et al., 2002). QTL analyses on an F2 cross between large mandible and small mandible suggested approximately 23 QTLs affecting distances between mandibular landmarks (Cheverud et al., 2004). QTLs affecting mandibular size and shape have been identified in separate analyses based on the Procrustes superimposition (Klingenberg et al., 2001, 2004); however, none of the QTLs on chromosome 12 was found to be associated with mandible size variations. Due to small sample size, the background environment—such as litter size, age of the mothers, and diet—could influence the results. To reduce type II errors, our mice were raised under very stringent conditions in a tightly controlled facility. The use of subcongenic strains, combined with rapidly improving technology, including Web-based databases in bioinformatics, in conjunction with high-throughput molecular techniques, is likely to enable us to resolve the present locus with much higher precision (Schadt et al., 2003; Pletcher et al., 2004). Genome-Tagged Mice, the pre-constructed overlapping sets of congenic mouse strains used in this study, are a valuable resource and provide an excellent starting point for analyzing sub-regions of this important locus (Davis et al., 2005). The current study validated QTLs, determining the shape of the maxilla on chromosome 12, and confirmed that sequence variation in the vicinity of D12Mit7 in DBA affects facial morphology. Further work will be directed toward identification of the affected genes and metabolic pathways. Utilizing a mouse animal model is a practical means for studying genetics of human craniofacial malformations, since functional information in one species (e.g., mice) can provide insights into functions for homologous genes in other species (e.g., humans). In fact, extensive linkage exists between the mouse and human genomes (Nagy et al., 2003). Previous studies have used mouse models to investigate myriad human craniofacial anomalies, such as facial clefting, hemifacial microsomia, and retinoic acid syndrome (Johnston and Bronsky, 1991). Gene function studies in mice led directly to further understanding of the HOX genes’ role in human craniofacial morphogenesis (Whiting, 1997). In the current study, we hypothesized that cis-acting gene variants located on introgressed segments derived from the donor DBA/2J include the primary candidate gene(s) for the phenotype. Thus, using gene-expression profiling in bone or other relevant tissue to find differentially expressed genes between the congenic and background strains will be an informative screen for candidate genes in the region. Ultimately, this work may lead to identification of the affected genes and metabolic pathway, and investigation of the human homologues may improve our understanding of dysmorphic faces.
This study was supported by the University of California School of Dentistry Dean’s Seed grant to E. Pae. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR014529 from the National Center for Research Resources, National Institutes of Health. Received for publication January 31, 2007. Revision received September 5, 2007. Accepted for publication September 12, 2007.
Journal of Dental Research, Vol. 86, No. 12,
1203-1206 (2007) This article has been cited by other articles:
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