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RESEARCH REPORT |
1 Department of Pediatric Dentistry and Clinical Genetics, School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Nørre Allé 20, DK- 2200 Copenhagen, Denmark;
2 Institute of Dentistry and Institute of Biotechnology, University of Helsinki, Finland;
3 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Finland;
4 Department of Medical Biochemistry and Genetics, Faculty of Health Sciences, University of Copenhagen, Denmark; and
5 Center for Rare Oral Diseases, Copenhagen University Hospital, Denmark;
* corresponding author, mette_klein{at}mail.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: PAX9 • hypodontia • oligodontia • teeth
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS & METHODSRESULTSDISCUSSIONREFERENCES |
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Agenesis of one or more permanent teeth occurs in about 8% of the Danish population, excluding third molar agenesis, which is more prevalent (Rølling, 1980). Most often, 1 or 2 second premolars or lateral maxillary incisors are missing. The term ‘hypodontia’ is defined as the congenital absence of fewer than 6 teeth, whereas ‘oligodontia’ designates the congenital absence of 6 or more permanent teeth, excluding the third molars (Schalk van der Weide, 1992). Oligodontia is a rare condition that can occur in association with genetic syndromes, or as a non-syndromic isolated familial trait, or as a sporadic finding (Gorlin et al., 2001). The prevalence of non-syndromic oligodontia in Danish schoolchildren has been reported to be 0.16% (Rølling and Poulsen, 2001). Non-syndromic familial oligodontia has, in most cases, been shown to be inherited as an autosomal-dominant trait (Grahnén, 1956). Several cases where a single gene mutation is associated with oligodontia have been described in recent years. So far, mutations in MSX1 and PAX9 have been shown in families with non-syndromic familial oligodontia (Vastardis et al., 1996; Stockton et al., 2000). Furthermore, agenesis of a few teeth to the entire set of teeth in one large Chinese kindred has been mapped to a locus on chromosome 10q11 (Liu et al., 2001). MSX1 and PAX9 are both transcription factors expressed in the dental mesenchyme at the stage of initiation of tooth development, in both the dental papilla and the dental follicle (Jowett et al., 1993; Peters et al., 1998a).
MSX1 is situated on chromosome 4 and is a homeobox gene. Homeobox genes contain a homeodomain and constitute a large multigene family of developmentally regulated transcription factors (Jowett et al., 1993). Homozygous Msx1-deficient mice exhibit defects in craniofacial development, e.g., complete cleft of the secondary palate and failure of tooth morphogenesis, with an arrest in molar tooth development at the bud stage. Heterozygous Msx1 mutant mice do not exhibit this phenotype (Satokata and Maas, 1994). The cases reported where an MSX1 mutation is responsible for autosomal-dominant tooth agenesis are few, and in these cases, tooth agenesis may be combined with other phenotypic traits, e.g., cleft lip, cleft palate, or cleft lip and palate (van den Boogaard et al., 2000; Slayton et al., 2003). Reports on two families with MSX1 mutations, where tooth agenesis constitutes the only phenotypic trait, have been published (Vastardis et al., 1996; Lidral and Reising, 2002). A heterogenous nonsense mutation in the homeodomain of MSX1 (ser202stop) segregated with Witkop tooth and nail syndrome (Jumlongras et al., 2001). Another nonsense mutation, but in exon 1 outside the homeodomain, was detected in a large family where all affected members showed various combinations of cleft lip, cleft palate, and tooth agenesis (van den Boogard et al., 2000). In all families reported in the literature with tooth agenesis segregating with a MSX1 mutation, the missing teeth were predominantly second premolars and third molars.
PAX9 is situated on chromosome 14 and belongs to the PAX gene family, which encodes a group of transcription factors that play a role in early development. PAX proteins are defined by the presence of a DNA-binding domain, the ‘paired-domain’, which makes sequence-specific contact with DNA (Chi and Epstein, 2002). Pax9 expression has a highly specific pattern during mouse embryogenesis, in derivatives of the foregut endoderm, somites, limb mesenchyme, midbrain, and the cephalic neural crest (Peters et al., 1998b). Homozygous Pax9-deficient mice die shortly after birth, most likely as a consequence of cleft of the secondary palate; they lack pharyngeal pouch derivatives, and craniofacial/visceral skeletogenesis is disturbed. Heterozygous Pax9 mutant mice exhibit no obvious abnormalities (Peters et al., 1998b). PAX9 is expressed in the dental mesenchyme prior to the first morphological manifestation of odontogenesis (Neubüser et al., 1997). As in Msx1 knockout mice, the tooth development in homozygous Pax9-deficient mouse embryos is arrested at the bud stage, indicating that Pax9 is required for tooth development to proceed beyond this stage (Satokata and Maas, 1994; Peters et al., 1998b).
So far, the literature reports eight families in whom PAX9 mutations segregate with non-syndromic autosomal-dominant inherited oligodontia. The eight families have all shown different kinds of mutations in the PAX9 coding region. Stockton et al.(2000) identified a frameshift mutation in the paired domain in exon 2 (ins G219). A nonsense heterozygous mutation (A340T), also in the paired domain, was demonstrated in one Finnish family (Nieminen et al., 2001). Frazier-Bowers et al.(2002) described an insertion of cytosine in exon 4, leading to frameshift and premature stop codon (ins C793). A very severe case of non-syndromic oligodontia was associated with a large heterozygous deletion (44–100 bp) on chromosome 14, which included the whole PAX9 gene (Das et al., 2002). Three different missense mutations (Arg26Pro, glu91lys, and leu21pro) and one 288-bp insertion, leading to frameshift, were described recently (Das et al., 2003; Lammi et al., 2003). A heterozygous transition mutation (G151A) in the paired box of PAX9 was recently described in a sporadic case of oligodontia (Mostowska et al., 2003). In all of the eight families with PAX9 mutations and non-syndromic oligodontia, the missing teeth were predominantly permanent molars and premolars.
The purpose of the present study was to screen a family with non-syndromic oligodontia for mutations in MSX1 and PAX9. The phenotype correlated well with reports on families described earlier in the literature, and the oligodontia was inherited as an autosomal-dominant trait.
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SUBJECTS & METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS & METHODSRESULTSDISCUSSIONREFERENCES |
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DNA Extraction from Whole Blood Samples
DNA was extracted from peripheral blood samples with the use of the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN, USA).
Polymerase Chain-reaction and Sequencing of Products with Radioactive Terminators
DNA was amplified by polymerase chain-reaction (PCR). Exon 2 of MSX1 was amplified with two different sets of primers. Primers used were 570F/840R, as described elsewhere (Vastardis et al., 1996), and 61400F (5'-ACTTGGCGGCACTCAATATC-3')/2R (5'-CCCCAGAGCAAATGTTTTGT-3'). For PAX9, primer sets for exons 1–4 and the flanking sequences were as follows: exon 1F, 5'-GCCCACGTTGCTGCTTAGATTGAAA-3', and exon 1R, 5'-CTCCCTCCCTTCCCGGCTCT-3'; exon 2aF, 5'-AGGCAGCTGTCCCAAGCAGCG-3', and exon 2aR, 5'-TGTATCGCGCCAGGATCTTGCTG-3'; exon 2bF, 5'-ATCCGACCGTGTGACATCAGCC-3', and exon 2bR, 5'-GGAGGGCACATTGTACTTGTCGC-3'; exon 2cF, 5'-GCATCTTCGCCTGGGAGATCCG-3', and exon 2cR, 5'-GAGCCCCTACCTTGGTCGGTG-3'; exon 3F, 5'-TTTGGGTCCCGTCTCAAGAGTGG-3', and exon 3R, 5'-CCTAAATCCCCGCCGCCACG-3'; and exon 4F, 5'-GGAGAGTAGAGTCAGAGCATTGCTG-3', and exon 4R, 5'-GAGACCTGGGAATTGGGGGA-3'. The PCR was performed with use of the primers mentioned above with AmpliTaq Gold PCR Master Mix (AmpliTAq 2,5 mM MgCl2, dNTP 200 µM) (Applied Biosystems, Foster City, CA, USA). In addition, hox7R, previously described by Goldenberg et al.(2000), was used as an internal primer for the PCR products of primer set 61400F/2R. For MSX1, the following conditions were used: 5 min of denaturation at 95°C (enzyme activation and initial denaturation step) followed by 35 20-second cycles of 95°C denaturation, then 20 sec of 57°C and 20 sec of 72°C extension, followed by 7 min of 72°C final extension. For exons of PAX9, 55°C was used for annealing. PCR products were purified with Exo-SAP (USB, Cleveland, OH, USA) and then sequenced with the use of a Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit with dGTP Nucleotide Mastermix and [
-33P] ddNTPs, followed by gel electrophoresis in a gel containing 7% acrylamide/bis-acrylamide 19:1, 0.8 x GTG buffer, 7 M urea, and autoradiography by BioMax MR (Kodak, Eastman Kodak Company, Rochester, NY, USA).
Polymerase Chain-reaction and Sequencing of Products with Dye Terminator Chemistry
Primers used to amplify exon 1 of MSX1 were 361F and 920R, described by Vastardis et al.(1996). Primers used to amplify exon 2 of MSX1 were 500F and 1250R, as described elsewhere (Jumlongras et al., 2001). We used Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA) and PreMix G from the MasterAmp PCR amplification kit (Epicentre Technologies), which contains 1.5 mM Mg2+ and 4x Betaine, to amplify exon 1. For exon 2, we used Taq DNA polymerase and the buffer supplied by Sigma for the PCR reactions. The PCR was performed under the following conditions: 3 min at 95°C, 1 min at 94°C, 50 sec at 63°C (for exon 1) or 58°C (for exon 2), and 1 min at 72°C, with a final extension of 30 min after 30 cycles. PCR products were purified with the use of the Qiaquick PCR purification kit (Qiagen, Bothell, WA, USA). Internal primers 643F/1044R (Jumlongras et al., 2001) were used to sequence exon 2.
Exons 1–4 of PAX9 were amplified by PCR with DyNAzymeTM EXT DNA polymerase (Finnzymes, Espoo, Finland). PCR primers and conditions were as previously described (Lammi et al., 2003), except that, for exon 1, an annealing temperature of 61°C was used. PCR products were purified enzymatically with the ExoSap-IT reagent (USB, Cleveland, OH, USA) according to the instructions of the manufacturer. PCR products were sequenced with dye terminator chemistry (ABI Prism® 
BigDyeTM Terminator Cycle sequencing kit, version 2.0, Applied Biosystems, Foster City, CA, USA), and analyzed in 4% denaturing gels on the ABI 377 DNA sequencer.
Analysis of Sequencing Results
Sequencing results were compared by BLAST2 (http://ncbi.nlm.nih.com/gorf/bl2.html) with EMBL entries AJ238381, AJ238382, and AJ238383.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Oligodontia could be traced in four generations and myopia in three generations. Pedigree analysis showed that the myopia was inherited from the grandmother, whereas the oligodontia was inherited from the grandfather. The pedigree showed an autosomal-dominant transmission of both the gene for myopia and the gene for oligodontia. Therefore, we concluded that the proband showed non-syndromic oligodontia. Both the proband and his father reported missing teeth in the primary dentition; however, dental records were not available for verification. The proband lacked all permanent molars, all second premolars, and upper first premolars. Both upper lateral incisors were peg-shaped, and upper permanent canines and lower first premolars had an abnormal crown shape (Fig. 2a). The affected father lacked all permanent molars, second premolars, upper first premolars, upper permanent canines, one upper lateral incisor (possibly due to extraction), and both lower first permanent incisors (Fig. 2b).
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, 3b). The unaffected mother did not show the mutation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS & METHODSRESULTSDISCUSSIONREFERENCES |
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Mehdi et al.(1990) described the possibility of the existence of GUG as an additional non-AUG start codon in natural mRNAs of mammalian cells and their viruses, with the efficient utilization of GUG (20%) in Sendai virus P/C mRNA and inefficient (3–5%) utilization of GUG in chloramphenicol acetyltransferase mRNA. However, even if PAX9 could be translated to a similar extent, with GUG as the initiation codon, the amount of PAX9 transcription factor would be remarkably reduced and could be compared with the consequences of a deletion, i.e., haploinsufficiency. This is in accordance with the similarity of the severe dental phenotypes caused by the initiation codon mutation described here and in the family reported by Das et al.(2002), with a deletion of PAX9. Therefore, we suggest that a heterozygous A to G mutation in the initiation codon of PAX9 causes severe or complete inhibition of PAX9 translation at one allele, resulting in a reduced amount of PAX9 transcription factor. Furthermore, it could be hypothesized that the development of the dental lamina for premolars and permanent molars is sensitive to PAX9, as previously suggested by Das et al.(2002).
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ACKNOWLEDGMENTS |
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Received March 1, 2004; Last revision August 23, 2004; Accepted September 15, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS & METHODSRESULTSDISCUSSIONREFERENCES |
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Das P, Stockton DW, Bauer C, Shaffer LG, D’Souza RN, Wright T, et al. (2002). Haploinsufficiency of PAX9 is associated with autosomal dominant hypodontia. Hum Genet 110:371–376.[ISI][Medline]
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