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RESEARCH REPORT |
1 Dows Institute for Dental Research, College of Dentistry, University of Iowa;
2 Pediatric Dentistry Department, College of Dentistry, Federal University of Rio de Janeiro, Brazil;
3 Oral Science PhD Program, College of Dentistry, University of Iowa;
4 Pediatrics Department, College of Medicine, University of Iowa;
5 formerly of College of Dentistry, The Ohio State University and currently in private practice in Centerville, OH;
6 Orthodontics Department, College of Dentistry, 2174 Medical Laboratories, University of Iowa, Iowa City, IA 52242, USA; and
7 formerly of Section of Orthodontics, College of Dentistry, The Ohio State University
* corresponding author, andrew-lidral{at}uiowa.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: MSX1 • cleft lip • cleft palate • tooth agenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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
).
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Direct Sequencing
DNA was extracted from whole blood or cheek swabs with the use of a commercial kit (Puragene, Gentra, Minneapolis, MN, USA). The entire coding region of the MSX1 gene was direct-sequenced in both directions. Four primer pairs were used to amplify overlapping regions of the 2 exons of the MSX1 gene (Appendix). Genomic DNA was amplified by PCR under the following conditions: 0.24 µM each primer, 200 µM dNTPs, 50 mM KCl, 10 mM Tris Cl, 1 or 1.5 mM MgCl2, 0.01% gelatin, 0.045 U Taq polymerase, 10% (v/v) DMSO, and 20 ng/µL DNA in a 30-µL reaction volume. Templates included either PCR products purified by QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA) or QIAquick PCR Purification Kit (Qiagen). Cycle sequencing was performed in a 10-µL reaction with 1 µL of ABI Big Dye Terminator sequencing reagent (version 1.1, Applied Biosystems, Foster City, CA, USA), 0.35 µL of 20 µM/L sequencing primer, 0.5 µL DMSO, 1 µL of 5X buffer, and 2.5 ng/100 base pair of DNA template. Following a denaturation step at 96°C for 30 sec, reactions were cycle-sequenced at 96°C for 10 sec, at TM (melting temperature) of the primer – 5°C for 5 sec, and 60°C for 4 min for 35 cycles.
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
First-pass base-calling (Perkin Elmer) was performed with the ABI sequence software (version 2.1.2). Chromatograms were transferred to a Unix workstation (Sun Microsystems Inc., Mountain View, CA, USA), base-called with Phred (version 0.961028), assembled with Phrap (version 0.960731, scanned by PolyPhred (version 0.970312), and the results viewed with the Consed program (version 4.0) (Nickerson et al., 1997).
Case-Control Comparisons
The case-control comparisons in this study used individuals with orofacial clefting recruited from the Children’s Hospital in Columbus, OH, and included in our mutation search. Thirty-three unrelated individuals with only isolated CL/P, and 19 unrelated individuals with both orofacial clefting and tooth agenesis were used in the analysis.
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 
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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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). 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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). 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.). This suggests, again, that agenesis of only a few teeth is not associated with MSX1 mutations (Lidral and Reising, 2002).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received April 12, 2005; Last revision February 2, 2006; Accepted February 9, 2006
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REFERENCES |
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