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MSX1 and Orofacial Clefting with and without Tooth Agenesis
1 Dows Institute for Dental Research, College of Dentistry, University of Iowa; Correspondence: * corresponding author, andrew-lidral{at}uiowa.edu
MSX1 has been considered a strong candidate for orofacial clefting, based on mouse expression studies and knockout models, as well as association and linkage studies in humans. MSX1 mutations are also causal for hereditary tooth agenesis. We tested the hypothesis that individuals with orofacial clefting with or without tooth agenesis have MSX1 coding mutations by screening 33 individuals with cleft lip with or without cleft palate (CL/P) and 19 individuals with both orofacial clefting and tooth agenesis. Although no MSX1 coding mutations were identified, the known 101C>G variant occurred more often in subjects with both CL/P and tooth agenesis (p = 0.0008), while the *6C-T variant was found more often in CL/P subjects (p = 0.001). Coding mutations in MSX1 are not the cause of orofacial clefting with or without tooth agenesis in this study population. However, the significant association of MSX1 with both phenotypes implies that MSX1 regulatory elements may be mutated.
Key Words: MSX1 • cleft lip • cleft palate • tooth agenesis
Isolated cleft lip with or without cleft palate (CL/P) is a common birth defect that affects about 1/700 births, depending on the population. Asian or Amerindian populations have the highest frequencies (1/500 or higher), Caucasian populations have intermediate frequencies (1/1000), and African populations have the lowest reported frequencies (1/2500) (Vanderas, 1987; Wyszynski et al., 1996; Mossey and Little, 2002). CL/P presents with characteristics of a genetically complex trait. It has been suggested that from 3 to 14 genes, interacting multiplicatively, may be involved in the etiology of CL/P (Schliekelman and Slatkin, 2002). MSX1, a non-clustered homeobox gene, has been considered a strong candidate for clefting in humans, based on the biological evidence composed of expression studies (Robert et al., 1989) and a knockout mouse model (Satokata and Maas, 1994) as well as association studies (Lidral et al., 1998; Beaty et al., 2001; Fallin et al., 2003; Jugessur et al., 2003; Vieira et al., 2003), complete sequencing (Jezewski et al., 2003; Suzuki et al., 2004), and linkage studies (Moreno et al., 2004; Schultz et al., 2004) in humans. We have identified a susceptibility locus for isolated CL/P in the 4p16 region in families from Ohio (Moreno et al., 2004). The positive marker D4S2366, located 4.63 cM proximal from the MSX1 gene, presented a LOD score of 1.53 under the parametric recessive linkage analysis. Because of the important role that MSX1 plays in the etiology of orofacial clefting, we performed a MSX1 mutation screen in individuals with isolated CL/P from the same Ohio population. Furthermore, data from genetic studies are consistent with a contribution of MSX1 to CL/P and tooth agenesis (Slayton et al., 2003) and isolated tooth agenesis (Vieira et al., 2004). MSX1 mutations and rare variants have been previously described in individuals with CL/P and/or hereditary tooth agenesis (Vastardis et al., 1996; van den Boogaard et al., 2000; Jumlongras et al., 2001; Lidral and Reising, 2002; Jezewski et al., 2003; Suzuki et al., 2004; De Muynck et al., 2004). Also, Msx1-deficient mice have both cleft of the secondary palate and failure of tooth development (Satokata and Maas, 1994). Tooth agenesis affects 1.6% to 9.6% of the Caucasian general population, excluding third molars (Graber, 1978). This malformation is found more frequently in children affected with CL/P than in the general population (Ranta, 1986; Shapira et al., 1999). The prevalence of hypodontia, both in the vicinity of the cleft and outside the cleft area, in the permanent dentition is significantly higher in children with cleft lip, cleft palate, or both, and the prevalence of hypodontia increases markedly with the severity of cleft. We believe that the occurrence of both CL/P and tooth agenesis in some individuals is caused by the same genetic mutation, and that MSX1 is a very plausible candidate. Therefore, we hypothesized that mutations in MSX1 are causal for orofacial clefting with or without tooth agenesis.
Subjects The study group consisted of 52 unrelated individuals with orofacial clefting recruited from the Children’s Hospital in Columbus, OH, USA. The age range of these individuals was from 7 to 16 yrs old. The inclusion criterion was the diagnosis of orofacial clefting with or without congenital agenesis of at least one permanent tooth, not including third molars, as verified by radiographs and dental history. Instances of tooth agenesis adjacent to a cleft site were not included, because the absence of such teeth is likely the consequence of local developmental anomalies at the cleft site. To identify any syndromes or phenocopies, we examined individuals clinically and interviewed them using a clinical survey to gather information regarding medical history, family history, and gestational environmental exposures.
Thirty-three of the individuals had only isolated orofacial CL/P. They were included in this mutation search, however, because we have found suggestive linkage to the MSX1 region in this population. Nineteen of the individuals had both orofacial clefting and tooth agenesis, and they were included in this study to test the hypothesis that MSX1 is mutated in patients with both phenotypes. Six of these 19 had additional major anomalies or facial dysmorphology (Table 1
The study was approved by the institutional review boards at the Ohio State University and the University of Iowa, and written, informed consent was obtained from each person included in the study.
Direct Sequencing Clean-up of amplicons was performed through the AMPureTM PCR Purification system (Agencourt, Beverly, MA, USA), with the use of Agencourt’s solid-phase paramagnetic bead technology. Beads were washed with 85% ethanol to remove excess oligonucleotides, nucleotides, salts, and enzymes, and purified products were eluted from the magnetic beads with sterilized water and injected onto an Applied Biosystems 3700 sequencer.
Sequence Analysis
Case-Control Comparisons In this study, we used controls of the same ethnic group (Caucasian) from a population-based case-control study within the University of Iowa Craniofacial Anomalies Research Center (CARC), previously genotyped by Lidral et al.(1998). Using a pseudo-random number generator (Romitti et al., 1998), we selected controls from all Iowa live births (between 1 January 1987 and 31 December 1991) not reported to the Iowa Birth Defects Registry.
We also analyzed the group with both orofacial clefting and tooth agenesis, excluding six individuals with clefting, tooth agenesis, and additional major anomalies or facial dysmorphology (Table 1
Data were analyzed by means of 2 x n contingency tables, which were evaluated by either the Pearson
Phenotypes of the individuals who presented with both orofacial clefting and tooth agenesis are described in Tables 1  and 2 . Most individuals were missing only 1 tooth outside of the cleft area, and the highest number of missing teeth in an individual was 8 (Fig.). Of the 33 individuals with isolated CL/P, 20 had a positive family history for CL/P. Among the 11 individuals with both orofacial clefting and tooth agenesis, and for whom the family history was known, only four presented a positive family history for CL/P. Among the eight individuals with both orofacial clefting and tooth agenesis, and for whom the family history was known, one presented a positive family history for tooth agenesis.
Neither new mutations nor new variants were found in the MSX1 coding regions of the 52 individuals. However, 4 known polymorphic variants were observed in the study population (Table 3 ). The *6C>T variant, 6 nucleotides 3' of the stop codon, was observed more frequently in subjects with CL/P than in controls (p = 0.001). The exon 1 101C>G variant, causing an Ala34Gly substitution, was observed more frequently in subjects with both orofacial clefting and tooth agenesis than in controls (p = 0.0008) (Table 3). This difference was still significant after exclusion of the six individuals presenting additional major anomalies or facial dysmorphology (Table 3).
No specific variant of MSX1 has been directly implicated as a major causal allele for clefting thus far. In our study, we found association of MSX1 variants not only with clefting, but also with clefting and tooth agenesis (Table 3 ). The 101C>G variant is more frequent in individuals with both orofacial clefting and tooth agenesis than in controls. In contrast, the *6C>T variant is more common in individuals with isolated CL/P than in controls. Also, the frequency of the *6C>T variant is much lower in clefting with tooth agenesis cases. Similar results were found in the clefting and tooth agenesis study group, when individuals presenting additional major anomalies or facial dysmorphology were excluded from the analysis. Moreover, when all 52 individuals were compared with the controls, the p-value was not significant (p = 0.052) (data not shown). This provides evidence that future investigations should phenotypically distinguish clefting associated with tooth agenesis from clefting alone. It appears that the variant 101C>G is marking a specific genetic factor that contributes to the clefting and tooth agenesis phenotype, and the variant *6C>T is specific for isolated clefting. Interestingly, the MSX1-CA 169 base-pair allele (allele 4)—the most common allele in all populations, and one that has been associated with isolated clefting in populations of European descent (Lidral et al., 1998; Beaty et al., 2001; Vieira et al., 2003)—is in linkage disequilibrium with the *6C>T variant (Jezewski et al., 2003). It is possible, therefore, that the *6C>T variant is the actual functional variant contributing to clefting in the study population. The cytosine of the *6C>T variant is conserved with bovines, but not with mice or rats. The variant 101C>G could also be functionally relevant to the development of clefting with tooth agenesis. This variant produces an amino acid change, Ala34Gly, which is conserved among mice, rats, bovines, and chickens (Jezewski et al., 2003). Functional analysis of the variants observed in this study would be of interest in further investigations of their consequences and role in clefting, with or without tooth agenesis. In the present study, no mutations were found in MSX1 coding regions in individuals with isolated CL/P or both orofacial clefting and tooth agenesis. Similarly, De Muynck et al.(2004) found no mutations in 43 families with CL/P with or without tooth agenesis. To date, only one MSX1 mutation was reported in a family with both CL/P and hereditary tooth agenesis (van den Boogaard et al., 2000). One possible explanation could be that MSX1 mutations for this phenotype are in regulatory regions, which have not been well-characterized. Another explanation is that MSX1 microdeletions may be causal in our study participants, and such deletions would have been missed by our mutation screen. Previous reports have showed that oligodontia in individuals with Wolf-Hirschhorn syndrome is associated with deletion or inactivation of one copy of MSX1 (Hu et al., 1998; Nieminen et al., 2003), supporting the conclusion that hereditary tooth agenesis associated with mutations in MSX1 is caused by haploinsufficiency. Gene-gene interactions could be the mechanism for developing CL/P with tooth agenesis. There is genetic evidence that MSX1 interacts with PAX9 in isolated tooth agenesis (Vieira et al., 2004), and apparently Pax9 regulates Msx1 expression in the mouse (Peters et al., 1998). PAX9 is also a transcription factor expressed in the face and tooth buds, and mice lacking Pax9 also present with both cleft palate and oligodontia (Peters et al., 1998). Future studies focusing on the MSX1 variants observed in our study and PAX9 variants are recommended to test the hypothesis that these two genes play a joint role in CL/P with tooth agenesis.
Finally, the phenotypes of the study participants may not be caused by MSX1 coding mutations. There is a typical pattern of tooth agenesis and a large number of missing teeth among the families reported to have MSX1 mutations. Specifically, the average number of missing teeth has been reported to be 11/person (Vastardis et al., 1996), 8/person (van den Boogaard et al., 2000), 16/person (Jumlongras et al., 2001), 12/person (Lidral and Reising, 2002), and 17/person (De Muynck et al, 2004). In our study, only two cases (10.5%) presented oligodontia (6 or more missing teeth) (Fig.
We thank all the individuals who participated in this study. We also thank Jeff Murray for the use of his laboratory facilities and equipment under the auspices of the Craniofacial Anomalies Research Center. This work has benefited greatly from discussions with Alex Vieira. We appreciate the insightful technical suggestions from Steven Bullard. This work was supported by NIH grant R01DE14677, March of Dimes grant #6-FY01-616, and start-up funds from the Ohio State University.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication April 12, 2005. Revision received February 2, 2006. Accepted for publication February 9, 2006.
Journal of Dental Research, Vol. 85, No. 6,
542-546 (2006) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
2 test or Fisher’s exact test, when any of the cells had an expected frequency of 
5. P values < 0.05 were considered statistically significant. 
