Mutation of PAX9 is associated with oligodontia

© 2000 Nature America Inc. • http://genetics.nature.com

brief communications

autosomes. A significant two-point lod score of 6.83 at θ=0 was obtained with marker D14S288, and the gene was localized to an 18.9-cM interval between D14S1060 and D14S276 (Fig. 2a). PAX9 was previously mapped to chromosome 14q12–q13 by fluorescence in situ hybridization and somatic cell hybrids7. Using radiation hybrid mapping, we placed PAX9 within our nonrecombinant region, 8.66 cR from marker WI-4859 with a lod score greater than 3.00. Mutational analysis on genomic DNA from affected individual III:11 by direct sequencing of the coding regions of exons 2–4 of PAX9 (primers available on request) revealed an insertion of a guanine at nt 219 (Fig. 2b). We used a singlestranded conformational polymorphism (SSCP) assay to assess the mutation in all family members, and saw an abnormal conformer in all affected individuals and one individual of unknown status. All of the remaining 22 unaffected individuals and 300 unrelated control chromosomes showed normal SSCP patterns (Fig. 2c). PAX9 encodes a member of a family of transcription factors that appear to function in development8. Pax9–/– mice lack

Mutation of PAX9 is associated with oligodontia


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second premolars; agenesis involving first and second molars is extremely rare3. The aetiology of tooth agenesis is largely unknown. The homeobox gene MSX1 has previously been associated with agenesis of the second premolars and third molars in a single family4. The involvement of MSX1, however, has been excluded in other forms of hypodontia involving both second premolars and lateral incisors5 or lateral incisors and canines6. We studied a family segregating a unique form of oligodontia in an autosomal dominant manner (Fig. 1a). The affected individuals had normal primary dentition but lacked most permanent molars. Some individuals also lacked maxillary and/or mandibular second premolars as well as mandibular central incisors (Fig. 1b,c). We performed a genome-wide search using microsatellite markers spaced at an average interval of 10 cM throughout the

e identified a frameshift mutation in the paired domain of PAX9 following genome-wide analysis of a family segregating autosomal dominant oligodontia. Affected members have normal primary dentition but lacked most permanent molars. Tooth agenesis is a common human anomaly that affects approximately 20% of the population. Oligodontia is the agenesis of six or more permanent teeth without associated systemic disorders, whereas absence of less than six teeth is referred to as hypodontia. Although tooth agenesis is associated with more than 49 syndromes (http://www3.ncbi.nlm.nih.gov/Omim/), several case reports describe non-syndromic forms that are either sporadic or familial in nature1,2. The incidence of familial tooth agenesis varies with each class of teeth. Most commonly affected are third molars (wisdom teeth), followed by either upper lateral incisors or lower

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Fig. 1 Clinical evaluations. a, Pedigree. All affected individuals and individual IV:4 (affection status unknown) carried the frameshift mutation in PAX9. Affected individual III:2 was not studied. b, A panoramic radiograph of mixed dentition in an affected individual (V:5) at 13 years. Arrowheads indicate the positions of missing teeth; stars indicate persistent primary teeth. c, Synopsis of the permanent dentition in affected family members. Filled stars represent absent teeth.

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nature genetics • volume 24 • january 2000


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a

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lod score (Z) at recombination fraction (θ)

Marker D14S262 D14S1071 D14S1060 D14S70 D14S988 D14S75 D14S1039 D14S288 D14S276 D14S63 D14S258

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pharyngeal pouch derivatives, have craniofacial and limb anomalies, and fail to form teeth beyond the bud stage9. The parathyroid glands, thymus and ultimobranchial bodies are not present and all mutants had a cleft secondary palate. The limb anomalies included preaxial digit duplications in all four limbs and a small supernumerary toe in the hindlimbs. Heterozygous mutants did not exhibit any obvious abnormalities. Affected individuals from the family studied here did not have any limb anomalies (X-rays of the hand were normal), or evidence for calcium metabolism abnormalities or compromised immune systems. Our results indicate that the frameshift mutation in PAX, resulting in an alteration in the paired domain of PAX9, is associated with tooth agenesis. The mutation may have its effect in one of several ways: (i) loss of function due to the absence of the DNA-binding domain; (ii) haploinsufficiency secondary to mu-

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We thank the family for their cooperation; C. Egger for PAX9 genomic sequence; and D. Nguyen and P. Mai in the Kleberg Genotyping Center. This work was supported in part by a grant from the Kleberg Foundation, Basic Research Grant No. 1FY98-0119 from the March of Dimes Birth Defects Foundation (P.I.P.), and NIH grants EY00375 (D.W.S.), DE11663 and DE13368 (R.D.S.). David W. Stockton1,2*, Parimal Das3*, Monica Goldenberg4, Rena N. D’Souza4 & Pragna I. Patel1,3

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1Departments

of Molecular and Human Genetics, 2Medicine and 3Neurology, Baylor College of Medicine, and 4Department of Orthodontics, University of Texas Houston Health Science Center Dental Branch, Houston, Texas, USA. Correspondence should be addressed to R.D.S. (e-mail: [email protected]) or P.I.P. (e-mail: [email protected]). 1.

2. 3. 4.

5. 6.

7. 8. 9.

*These authors contributed equally to this work.

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Fig. 2 Molecular analysis of human PAX9. a, Two-point linkage analysis results for the microsatellite markers flanking PAX9. b, DNA sequence electropherogram from individual III:11 with the normal and mutant PAX9 sequence and predicted protein sequences. Note the guanine insertion (arrow) and the resulting frameshift. c, A representative SSCP autoradiogram showing co-segregation of the abnormal conformer band with the phenotype. d, Schematic representation of PAX9 structure with the exons indicated (boxes), and normal and putative mutated proteins. Note the paired domain (PD), octapeptide domain (OD) and the frameshifted amino acid sequence (hashed region) in the mutated protein. The guanine insertion at nt 219 extends a series of 5 guanines to 6, causing a frameshift and premature termination of the protein. The mutation alters the amino acid sequence starting at residue 73, which is in a highly conserved region 70 aa into the paired domain, and between its amino-terminal and carboxy-terminal DNA-binding domains. A premature stop is created 243 codons after the insertion, shortening the protein by 25 aa.

tant mRNA or protein instability; (iii) gain of function due to creation of a novel protein; or (iv) dominant-negative effect by interference with the product of the normal PAX9 allele. In vitro expression and functional characterization of the mutant gene and creation of a transgenic mouse with the mutation may advance our understanding. Acknowledgements

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Gorlin, R.J., Cohen, M.M. & Levin, L.S. Syndromes of the Head and Neck (Oxford University Press, New York, 1990). Jorgenson, R.J. J. Am. Dent. Assoc. 101, 283–286 (1980). Graber, L.W. J. Am. Dent. Assoc. 96, 266–275 (1978). Vastardis, H., Karimbux, N., Guthua, S.W., Seidman, J.G. & Seidman, C.E. Nature Genet. 13, 417–421 (1996). Nieminen, P., Arte, S., Pirinen, S., Peltonen, L. & Thesleff, I. Hum. Genet. 96, 305–308 (1995). Vastardis, H. Genetic approaches to understanding tooth development: a human MSX1 homeodomain missense mutation causes selective tooth agenesis. Thesis, Harvard Univ. (1996). Stapleton, P., Weith, A., Urbánek, P., Kozmik, Z. & Busslinger, M. Nature Genet. 3, 292–298 (1993). Semenza, G.L. PAX Proteins 169–198 (Oxford University Press, New York, 1998). Peters, H., Neubüser, A., Kratochwil, K. & Balling, R. Genes Dev. 12, 2735–2747 (1998).

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