RESEARCH REPORTS Clinical S.A. Frazier-Bowers 1, D.C. Guo 2, A. Cavender 1, L. Xue 2, B. Evans 3, T. King 2, D. Milewicz 2, and R.N. D'Souza 1* 1 Department of Orthodontics, Dental Branch, Suite 371, and 2 Department of Internal Medicine, Medical School, University of Texas Health Science Center, 6516 M.D. Anderson Blvd., Houston, Texas 77030; 3 Specialists in Orthodontics, 817 9th Street, Rapid City, SD 57701; *corresponding author, rdsouza@mail.db.uth.tmc.edu A Novel Mutation in Human PAX9Causes Molar Oligodontia J Dent Res81(2):129-133, 2002 ABSTRACT Experimental and animal studies, as well as genetic mutations in man, have indicated that the development of dentition is under the control of several genes. So far, mutations in MSX1 and PAX9 have been associated with dominantly inherited forms of human tooth agenesis that mainly involve posterior teeth. We identified a large kindred with several individuals affected with molar oligodontia that was transmitted as an isolated autosomaldominant trait. Two-point linkage analysis using DNA from the family and polymorphic marker D14S288 in chromosome 14q12 produced a maximum lod score of 2.29 at = 0.1. Direct sequencing of exons 2 to 4 of PAX9 revealed a cytosine insertion mutation at nucleotide 793, leading to a premature termination of translation at aa 315. Our results support the conclusion that molar oligodontia is due to allelic heterogeneity in PAX9, and these data further corroborate the role of PAX9 as an important regulator of molar development. KEY WORDS: PAX9, frame shift mutation, tooth agenesis, oligodontia. Received September 5, 2001; Last revision December 14, 2001; Accepted December 18, 2001 A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Disturbances in tooth development lead to various dental anomalies, including tooth agenesis, or the congenital absence of primary and/or permanent teeth. Tooth agenesis occurs in up to 20% of the population, making it the most common craniofacial anomaly in man (for review, see Vastardis, 2000). The condition may occur as part of a genetic syndrome, but also as a familial non-syndromic disorder [described in the Online Mendelian Inheritance in Man database (http://www3.ncbi.nlm.nih.gov/omim/), 1999]. Tooth agenesis is typically inherited in an autosomal-dominant manner, but autosomal-recessive (Ahmad et al., 1998; Pirinen et al., 2001) and X-linked (Erpenstein and Pfeiffer, 1967) inheritance also occurs. A rare and non-syndromic form of molar oligodontia was reported to occur in an autosomal-dominant fashion in one large kindred (Goldenberg et al., 2000). The results of a genome-wide search led to the identification of an insertion mutation in the paired box domain (exon 2) of the PAX9 gene (Stockton et al., 2000). Recently, an inactivating (nonsense) mutation in the same region of PAX9 was shown to be associated with molar oligodontia in a single family (Nieminen et al., 2001). Previous studies have also associated inactivating mutations in the transcription factor MSX1 with familial non-syndromic second premolar agenesis (Vastardis et al., 1996). A substitution mutation in MSX1 was later shown to be associated with tooth agenesis in another kindred affected with premolar hypodontia, and orofacial clefts in some individuals (van den Boogard et al., 2000). Interestingly, in two affected individuals, permanent first and second molars were congenitally absent, suggesting that defects in MSX1 also contribute to molar agenesis. Moreover, in the Witkop s Tooth and Nail Syndrome (TNS), a mutation in MSX1 was found to cause tooth agenesis of predominantly anterior, and some posterior, teeth, but also nail dysplasia (Jumlongras et al., 2001). The importance of PAX9 in tooth development is further supported by the phenotype of an arrest at the bud stage that is seen in Pax9 (-/-) mice (Peters et al., 1998). Other molecular and genetic studies in mice have provided evidence for an odontogenic homeobox code of multiple genes that form the morphological gradients responsible for the patterning of dentition (Sharpe, 1995). Hence, it is likely that more than one gene may influence the development of molars in humans, and that conditions like molar oligodontia may involve locus heterogeneity. The primary objective of these studies was to assess whether a mutation in PAX9 is responsible for the molar oligodontia affecting several members in a large kindred. Our mutational analyses revealed a novel frameshift mutation in a less-defined region of PAX9, encoded by exon 4, that is associated with molar oligodontia. Our results thus confirm the importance of PAX9 in human molar 129
130 Frazier-Bowers et al. J Dent Res 81(2) 2002 Figure 1. Characterization of molar oligodontia. (A) Pedigree analysis by inspection reveals an autosomal-dominant form of molar oligodontia. Darkened symbols represent affected, clear symbols indicate normal unaffected, squares indicate male, circles indicate females, and (/) indicates deceased. (B) Panoramic radiograph of individual IV-5 depicts typical pattern of congenitally missing teeth in this family. development and support the hypothesis that similar patterns of tooth agenesis may arise from different mutations in the same gene. MATERIALS & METHODS Pedigree Construction and Clinical Diagnosis The family was first brought to our attention by the treating orthodontist. The index case was an individual with two affected siblings and an affected cousin. Through subsequent interviews, we extended the pedigree laterally and vertically for a total of 41 individuals, 18 affected and 23 unaffected (Fig. 1A). Several members were deceased; therefore, the family historian described the status of the dentition for these individuals based on personal recollection. The diagnosis of molar oligodontia for individuals in generations III, IV, and V was based on clinical examination of panoramic or periapical/bitewing dental radiographs. Cephalometric radiographs were also obtained for individuals who were receiving orthodontic care. For these individuals, a cephalometric analysis was performed to determine the presence of skeletal and/or dental malocclusion. Approval for this study was granted by the Committee for the Protection of Human Subjects (CPHS), University of Texas Health Science Center at Houston. Consent to participate in this study (including a release for dental records) was obtained from every adult participant or from a parental guardian in the case of minors. Figure 2. Analysis of human PAX9 gene. (A) Schematic of human PAX9 gene with boxes representing exons (1-4) as numbered. Boxes with hatched lines indicate 5 and 3 UTR regions, respectively, and solid lines represent introns. The region that encodes for the paired domain (PD) is shown in exon 2. Solid gray boxes represent the regions sequenced in this study. Arrow indicates location of mutation. (B) Representative chromatogram of normal family member showing absence of insertion mutation. (C) Representative chromatogram of affected family member showing an insertion mutation. Sequencing and Genotyping Peripheral blood samples or buccal swabs were obtained and DNA extracted from 11 affected and 13 unaffected individuals. DNA extractions were performed with the use of Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN, USA). DNA was amplified by polymerase chain reaction (PCR) with primer sets for exons 2 to 4 (Fig. 2A) of human PAX9 (primers listed in the Appendix, www.dentalresearch.org), which includes the entire coding region except for one methionine in exon 1. The PCR for PAX9 (NCBI accession number XM_048684) was carried out with the use of Fail Safe (Epicentre, Madison, WI, USA) buffer under the following conditions: 10 min 95 C activation/pre-melt step, followed by 35 cycles of 30 sec 94 C melt, 30 sec 60 C anneal, and 30 sec 72 C extension. Purification of PCR products was carried out by ExoSAP-IT (USB, Cleveland, OH, USA), followed by sequencing with ABI Big Dye terminator reagents (Applied Biosystems, Foster City, CA, USA) with the use of an ABI PRISM 377 DNA sequencer. To determine if the PAX9 locus was responsible for molar oligodontia in this family, we selected fluorescent-dye-labeled oligonucleotide primers flanking simple repeat polymorphisms for the region surrounding the PAX9 locus (14q12-q13). Genotyping was carried out with the
J Dent Res 81(2) 2002 PAX9 and Human Molar Agenesis 131 markers D14S288 and D14S1039 and an ABI 3100 PRISM Genetic Analyzer (Applied Biosystems). We carried out twopoint linkage analysis with the use of MLINK analysis software for members of this family to determine linkage at the PAX9 locus and the disease phenotype. In this analysis, we assumed a single, rare autosomal-dominant model that was completely penetrant. Marker allele frequencies were based on experimental data. Restriction-enzyme Analysis Genomic DNA was PCR-amplified with the primer set hpax9ex4f and hpax9ex4r and the conditions described earlier. Amplified DNA from 11 affected, 13 unaffected, and 50 unrelated ethnically matched controls was then subject to PvuII digestion, according to instructions from the manufacturer. Digestion products were analyzed with the use of 1.5-% agarose gel electrophoresis in 1% TAE buffer. RESULTS Clinical Diagnosis A review of the medical history did not reveal significant medical problems in affected individuals. There were no abnormalities of the toenails, fingernails, hair, or sweat glands. At least four affected and two unaffected individuals were determined to have a skeletal Class III malocclusion (Table) due to a prognathic mandible [data not shown]. A clinical examination by the treating orthodontist was initially performed to determine the status of the dentition for individuals (IV:5, IV:7, IV:8, and IV:9) (Fig. 1A). The Table illustrates the status of the dentition in nine affected individuals. We examined dental radiographs to confirm the diagnosis of oligodontia for affected individuals (Fig. 1B). The patterns of missing teeth among family members Figure 3. Restriction-enzyme analysis of PCR-amplified DNA fragment of exon 4 of PAX9. The PCR fragments 450 bp long were digested with PvuII. The mutant allele was not cleaved at this site, while the wild type was cleaved normally. showed considerable variability. In almost all cases, individuals were missing maxillary and mandibular second and third molars. Interestingly, while almost all individuals were missing maxillary first molars, less than half were missing mandibular first molars (Table). Two individuals (IV-7 and IV-8) lacked at least one maxillary lateral incisor and maxillary bicuspids. Moreover, two individuals were also missing mandibular central incisors, indicating that the pattern of missing teeth is not limited to posterior teeth. Some individuals reported the congenital absence of primary teeth, but dental records were not available for verification. Table. The Congenitally Missing Teeth Left Quadrant Right Quadrant M P C I I C P M Sk. cl III Upper 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 Lower 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 IV 7 IV 9 IV 8 IV 5 IV 11 III 12 III 7 III 9 III 11 All teeth are labeled in rows denoted upper and lower such that 1 indicates central incisors, 2 indicates laterals, etc. The star ( ) indicates a missing tooth, and () indicates a skeletal class III pattern.
132 Frazier-Bowers et al. J Dent Res 81(2) 2002 Linkage Analysis A previous study associated a mutation in PAX9 with molar oligodontia (Stockton et al., 2000). Therefore, we performed linkage analysis to determine if molar oligodontia was linked to polymorphic microsatellite markers surrounding the PAX9 locus, with the use of DNA from affected and unaffected family members. Twopoint linkage analysis is suggestive of linkage at marker D14S288 to the oligodontia phenotype (LOD score = 2.29, = 0.1). Mutation Analysis PCR of PAX9 with the use of intron-based, exon-specific primers, followed by direct sequencing, was performed on genomic DNA of five affected individuals (IV:7, IV:8, IV:9, III:9, and III:10). Sequence analysis revealed a cytosine insertion at nucleotide 793 in exon 4 for all individuals analyzed (Fig. 2B). The cytosine insertion creates a frame shift at amino acid 264 and premature truncation of the protein at amino acid 315, removing 25 amino acid residues and adding 51 nonsense amino acids. The insertion of a cytosine residue also destroys a PvuII restriction site; therefore, we performed a restrictionenzyme analysis to determine if the mutation co-segregates with the molar oligodontia phenotype. Our results confirmed the presence of a mutation in all affected family members but not in unaffected individuals (Fig. 3). A similar analysis of 50 unrelated controls showed the expected PvuII digest fragments of 120 and 330 base pairs, thus confirming the absence of a mutation in exon 4. Further, our results suggest that the mutation found in this family is completely penetrant, since all family members with the mutation had molar oligodontia. DISCUSSION Our findings indicate that a novel insertion mutation in PAX9, resulting in a truncated amino acid, is responsible for tooth agenesis in one family. Similar to the family reported previously (Goldenberg et al., 2000; Stockton et al., 2000), the molar oligodontia trait was inherited in an autosomal-dominant fashion. Results from our study further confirm that more than one mutation in PAX9 gives rise to the same phenotype. The novel insertion mutation we report here was most interesting, since it was identified outside of the critical paired domain located in exon 2. This highly conserved paired domain region, which is thought to be the most critical for the function of Pax proteins, is the same region where the first mutation leading to molar agenesis was identified. Identification of a mutation outside of the paired domain suggests that an evolutionarily conserved and Pax 9-specific region of PAX9 is also critical to its function in tooth development. Although there is no known function for the region of the protein encoded by exon 4 of PAX9, it is possible that the addition of 51 nonsense amino acids, including 5 new cysteine residues, may affect the proper folding of the protein, leading to loss of function. While these results were consistent with allelic heterogeneity, we cannot rule out that another gene may be responsible for other cases of molar oligodontia. For example, our recent studies of two families with a similar pattern of molar oligodontia did not reveal a causative mutation in PAX9 (Frazier-Bowers et al., 2002). Our clinical analysis revealed that the pattern of tooth agenesis seen in this family primarily affects permanent posterior teeth, while permanent anterior teeth were rarely affected. Interestingly, cephalometric analysis revealed that a Class III malocclusion, due primarily to mandibular prognathism, was present in some individuals who were affected or unaffected. The simplest explanation for this finding is that the trait for Class III malocclusion segregates independently from that of tooth agenesis or molar oligodontia. Future studies are warranted to help elucidate the genetic etiology of molar agenesis and other patterns of tooth agenesis. Understanding how PAX9 achieves its selective function during tooth development is critical to our understanding of the molecular pathogenesis of tooth agenesis. In summary, analysis of our data further underscores the importance of PAX9 in the development of the posterior dentition. ACKNOWLEDGMENTS We gratefully acknowledge the support of the family and dentists who participated in this study. The assistance of Amy Jackson, Dr. Puskas, and Dr. Needleman (DNA Sequencing Core Facility) is appreciated. 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