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Novel Mutation of the Initiation Codon of PAX9 Causes Oligodontia
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; Correspondence: * corresponding author, mette_klein{at}mail.dk
Tooth development is under strict genetic control. Oligodontia is defined as the congenital absence of 6 or more permanent teeth, excluding the third molar. The occurrence of non-syndromic oligodontia is poorly understood, but in recent years several cases have been described where a single gene mutation is associated with oligodontia. Several studies have shown that MSX1 and PAX9 play a role in early tooth development. We screened one family with non-syndromic oligodontia for mutations in MSX1 and PAX9. The pedigree showed an autosomal-dominant pattern of inheritance. Direct sequencing and restriction enzyme analysis revealed a novel heterozygous A to G transition mutation in the AUG initiation codon of PAX9 in exon 1 in the affected members of the family. This is the first mutation found in the initiation codon of PAX9, and we suggest that it causes haploinsufficiency.
Key Words: PAX9 • hypodontia • oligodontia • teeth
The tooth is one of the vertebrate organs in which molecular regulation has been actively studied in recent years, mostly with mouse molar teeth as a model system. The development of teeth is regulated by inductive interactions between epithelial and mesenchymal cells and is under strict genetic control. More than 200 genes are known to be expressed during tooth development (Thesleff and Nieminen, 1996; Gene expression in tooth, http://bite-it.helsinki.fi/). 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.
This study included subjects from a family of Chinese descent. The family was referred to the Department of Pediatric Dentistry and Clinical Genetics at the University of Copenhagen for diagnostic evaluation and treatment of oligodontia. Pedigree construction was made by clinical examination of available family members and by interviews. We took orthopantomographic radiographs to verify the tooth agenesis. Blood samples were collected from available members of the family. The study was conducted with informed consent of the family members and under permission of the Ethical Committee of Copenhagen, Denmark.
DNA Extraction from Whole Blood Samples
Polymerase Chain-reaction and Sequencing of Products with Radioactive Terminators
Polymerase Chain-reaction and Sequencing of Products with Dye Terminator Chemistry
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®
Analysis of Sequencing Results
Clinical Examination The proband had oligodontia and was severely myopic. According to his father, the family had previously been genetically examined in China with respect to their dental problems and their myopia, and the father had the resultant pedigree in his possession. Pedigree analysis showed autosomal-dominant inheritance (Fig. 1  ). 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).
Mutation Analysis Exons 1–2 of MSX1 and exons 1–4 of PAX9 were examined. We found one mutation in PAX9, but no mutations in the MSX1 gene. PAX9 of the proband, the affected father, the affected grandfather, and the unaffected mother was sequenced. In the affected members, we observed a heterozygous A to G transition mutation in the AUG initiation codon of PAX9 in exon 1. The A to G transition mutation (A1G) was observed in the sequencing results with both the autoradiographic detection method and with dye terminator chemistry (ABI sequencer) (Figs. 3a , 3b). The unaffected mother did not show the mutation.
Restriction-enzyme Analysis After the PAX9 mutation in the family had been detected, we created a restriction-site map (www.firstmarket.com/cutter/cut2.html). The mutation destroys a BsrDI restriction site, and we performed a restriction-enzyme analysis. The DNA of the proband, the affected father, the affected grandfather, and the unaffected mother was amplified with specific primers PAX9ex1aF and PAX9ex1bR. Restriction digestion with BsrDI (5'-GCAATGNN-3') was then performed according to the manufacturer’s instructions (New England BioLabs Inc., Beverly, MA, USA). Digestion products were analyzed with the use of 3% agarose gel electrophoresis. The presence of the mutation in the affected family members was confirmed (Fig. 3c  ).
In this report, we described a novel mutation in the initiation codon of PAX9 as being responsible for non-syndromic oligodontia in one family. The patterns of missing teeth and autosomal-dominant inheritance in this family were similar to those of previously described cases in which oligodontia was connected with PAX9 mutations (Stockton et al., 2000; Nieminen et al., 2001; Das et al., 2002; Frazier-Bowers et al., 2002). The phenotype in the present family, however, was more severe in terms of the number of missing teeth, i.e., with the proband lacking all permanent molars, all second premolars, and upper first premolars, in combination with hypodontia in the primary dentition, which is very rare. The phenotype found in the present study most resembles that previously described in a family with a PAX9 deletion (Das et al., 2002). Several reports about other genes have demonstrated a similar mutation (AUG to GUG) in the initiation codon segregating with a disease (Isashiki et al., 1995). This supports our finding that an A to G mutation in the initiation codon of PAX9 causes oligodontia. 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).
We thank Sinikka Pirinen and Sirpa Arte for the rewarding stay in Helsinki, Kirsten Winther, Copenhagen University, for her laboratory assistance, and Dolrudee Jumlongras, Harvard Medical School, for her help with MSX1. This project was supported by grants from The Danish Medical Research Council (SSVF 22-02-0060). Received for publication March 1, 2004. Revision received August 23, 2004. Accepted for publication September 15, 2004.
Journal of Dental Research, Vol. 84, No. 1,
43-47 (2005) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
-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). 
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. 
