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Mutations in AXIN2 Cause Familial Tooth Agenesis and Predispose to Colorectal Cancer

2004; Elsevier BV; Volume: 74; Issue: 5 Linguagem: Inglês

10.1086/386293

1537-6605

Laura Lammi, Sirpa Arte, Mirja Somer, Heikki Järvinen, Päivi Lahermo, Irma Thesleff, Sinikka Pirinen, Pekka Nieminen,

Connective tissue disorders research

Wnt signaling regulates embryonic pattern formation and morphogenesis of most organs. Aberrations of regulation of Wnt signaling may lead to cancer. Here, we have used positional cloning to identify the causative mutation in a Finnish family in which severe permanent tooth agenesis (oligodontia) and colorectal neoplasia segregate with dominant inheritance. Eleven members of the family lacked at least eight permanent teeth, two of whom developed only three permanent teeth. Colorectal cancer or precancerous lesions of variable types were found in eight of the patients with oligodontia. We show that oligodontia and predisposition to cancer are caused by a nonsense mutation, Arg656Stop, in the Wnt-signaling regulator AXIN2. In addition, we identified a de novo frameshift mutation 1994-1995insG in AXIN2 in an unrelated young patient with severe tooth agenesis. Both mutations are expected to activate Wnt signaling. The results provide the first evidence of the importance of Wnt signaling for the development of dentition in humans and suggest that an intricate control of Wnt-signal activity is necessary for normal tooth development, since both inhibition and stimulation of Wnt signaling may lead to tooth agenesis. Our findings introduce a new gene for hereditary colorectal cancer and suggest that tooth agenesis may be an indicator of cancer susceptibility. Wnt signaling regulates embryonic pattern formation and morphogenesis of most organs. Aberrations of regulation of Wnt signaling may lead to cancer. Here, we have used positional cloning to identify the causative mutation in a Finnish family in which severe permanent tooth agenesis (oligodontia) and colorectal neoplasia segregate with dominant inheritance. Eleven members of the family lacked at least eight permanent teeth, two of whom developed only three permanent teeth. Colorectal cancer or precancerous lesions of variable types were found in eight of the patients with oligodontia. We show that oligodontia and predisposition to cancer are caused by a nonsense mutation, Arg656Stop, in the Wnt-signaling regulator AXIN2. In addition, we identified a de novo frameshift mutation 1994-1995insG in AXIN2 in an unrelated young patient with severe tooth agenesis. Both mutations are expected to activate Wnt signaling. The results provide the first evidence of the importance of Wnt signaling for the development of dentition in humans and suggest that an intricate control of Wnt-signal activity is necessary for normal tooth development, since both inhibition and stimulation of Wnt signaling may lead to tooth agenesis. Our findings introduce a new gene for hereditary colorectal cancer and suggest that tooth agenesis may be an indicator of cancer susceptibility. Morphogenesis and tissue regeneration are based on tight control of cell homeostasis. Wnt signaling has been implicated in regulation of diverse developmental events, as well as in aberrations of cell homeostasis that may lead to cancer (Huelsken and Birchmeier Huelsken and Birchmeier, 2001Huelsken J Birchmeier W New aspects of Wnt signaling pathways in higher vertebrates.Curr Opin Genet Dev. 2001; 11: 547-553Crossref PubMed Scopus (475) Google Scholar; Alonso and Fuchs Alonso and Fuchs, 2003Alonso L Fuchs E Stem cells in the skin: waste not, Wnt not.Genes Dev. 2003; 17: 1189-1200Crossref PubMed Scopus (273) Google Scholar; Lustig and Behrens Lustig and Behrens, 2003Lustig B Behrens J The Wnt signaling pathway and its role in tumor development.J Cancer Res Clin Oncol. 2003; 129: 199-221Crossref PubMed Scopus (445) Google Scholar; Giles et al. Giles et al., 2003Giles RH van Es JH Clevers H Caught up in a Wnt storm: Wnt signaling in cancer.Biochim Biophys Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1284) Google Scholar). Central to the mediation of Wnt signals is the regulation of β-catenin level and localization. When cells receive a Wnt signal, β-catenin is stabilized and binds transcription factors of the TCF family regulating expression of Wnt target genes. In the absence of Wnt signal, β-catenin is subjected to phosphorylation and subsequent degradation by action of a multiprotein complex (Seidensticker and Behrens Seidensticker and Behrens, 2000Seidensticker MJ Behrens J Biochemical interactions in the Wnt pathway.Biochim Biophys Acta. 2000; 1495: 168-182Crossref PubMed Scopus (229) Google Scholar). The complex is organized by APC (product of the adenomatous polyposis coli gene) and Axin1 or its homolog Axin2, which serve as scaffolding components for the complex. Axin1 and Axin2 (also called “conductin” or “axil”) share a similar domain structure with binding sites for β-catenin, GSK3β, APC, and disheveled (Behrens et al. Behrens et al., 1998Behrens J Jerchow BA Wurtele M Grimm J Asbrand C Wirtz R Kuhl M Wedlich D Birchmeier W Functional interaction of an Axin homolog, conductin, with β-catenin, APC, and GSK3β.Science. 1998; 280: 596-599Crossref PubMed Scopus (1061) Google Scholar; Yamamoto et al. Yamamoto et al., 1998Yamamoto H Kishida S Uochi T Ikeda S Koyama S Asashima M Kikuchi A Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3β and β-catenin and inhibits axis formation of Xenopus embryos.Mol Cell Biol. 1998; 18: 2867-2875Crossref PubMed Scopus (170) Google Scholar; Mai et al. Mai et al., 1999Mai M Qian C Yokomizo A Smith DI Liu W Cloning of the human homolog of conductin (AXIN2), a gene mapping to chromosome 17q23–q24.Genomics. 1999; 55: 341-344Crossref PubMed Scopus (57) Google Scholar; Seidensticker and Behrens Seidensticker and Behrens, 2000Seidensticker MJ Behrens J Biochemical interactions in the Wnt pathway.Biochim Biophys Acta. 2000; 1495: 168-182Crossref PubMed Scopus (229) Google Scholar). Axin1 is expressed uniformly during development, whereas Axin2 is expressed in a tissue- and stage-specific manner; for example, in somites, branchial arches, and limb buds (Jho et al. Jho et al., 2002Jho EH Zhang T Domon C Joo CK Freund JN Costantini F Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.Mol Cell Biol. 2002; 22: 1172-1183Crossref PubMed Scopus (1224) Google Scholar; Aulehla et al. Aulehla et al., 2003Aulehla A Wehrle C Brand-Saberi B Kemler R Gossler A Kanzler B Herrmann BG Wnt3a plays a major role in the segmentation clock controlling somitogenesis.Dev Cell. 2003; 4: 395-406Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Axin2 is induced by Wnt signaling, suggesting that Axin2 expression serves as a negative-feedback regulator of Wnt signaling (Jho et al. Jho et al., 2002Jho EH Zhang T Domon C Joo CK Freund JN Costantini F Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.Mol Cell Biol. 2002; 22: 1172-1183Crossref PubMed Scopus (1224) Google Scholar; Leung et al. Leung et al., 2002Leung JY Kolligs FT Wu R Zhai Y Kuick R Hanash S Cho KR Fearon ER Activation of AXIN2 expression by β-catenin-T cell factor: a feedback repressor pathway regulating Wnt signaling.J Biol Chem. 2002; 277: 21657-21665Crossref PubMed Scopus (290) Google Scholar). Mutations that facilitate the escape of β-catenin from the action of the degradation complex may lead to cancer due to increased transcription of Wnt target genes. Somatic mutations in β-catenin and molecules of the degradation complex have been found in cancer tissues, including skin, gastrointestinal, ovarian, and hepatocellular tumors (Huelsken and Birchmeier Huelsken and Birchmeier, 2001Huelsken J Birchmeier W New aspects of Wnt signaling pathways in higher vertebrates.Curr Opin Genet Dev. 2001; 11: 547-553Crossref PubMed Scopus (475) Google Scholar; Giles et al. Giles et al., 2003Giles RH van Es JH Clevers H Caught up in a Wnt storm: Wnt signaling in cancer.Biochim Biophys Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1284) Google Scholar; Lustig and Behrens Lustig and Behrens, 2003Lustig B Behrens J The Wnt signaling pathway and its role in tumor development.J Cancer Res Clin Oncol. 2003; 129: 199-221Crossref PubMed Scopus (445) Google Scholar). Germline loss-of-function mutations in APC cause familial adenomatous polyposis (FAP [MIM 175100]), comprising ∼10% of hereditary colorectal cancer (Groden et al. Groden et al., 1991Groden J Thliveris A Samowitz W Carlson M Gelbert L Albertsen H Joslyn G Stevens J Spirio L Robertson M Identification and characterization of the familial adenomatous polyposis coli gene.Cell. 1991; 66: 589-600Abstract Full Text PDF PubMed Scopus (2313) Google Scholar; Kinzler et al. Kinzler et al., 1991Kinzler KW Nilbert MC Su LK Vogelstein B Bryan TM Levy DB Smith KJ Preisinger AC Hedge P McKechnie D Identification of FAP locus genes from chromosome 5q21.Science. 1991; 253: 661-665Crossref PubMed Scopus (1930) Google Scholar). It is interesting that familial colorectal polyposis is often accompanied by extracolonic neoplasia, especially cysts, osteomas, and odontomas (Gardner Gardner, 1962Gardner EJ Follow-up study of a family group exhibiting dominant inheritance for a syndrome including intestinal polyps, osteomas, fibromas and epidermal cysts.Am J Hum Genet. 1962; 14: 376-390PubMed Google Scholar). Agenesis of one or more permanent teeth (hypodontia [MIM 106600]) is the most common congenital malformation in humans. More than 20% of the population lacks one or more third molars (wisdom teeth) congenitally, and >5% lack other permanent teeth, most commonly some of the second premolars or upper lateral incisors (Vastardis Vastardis, 2000Vastardis H The genetics of human tooth agenesis: new discoveries for understanding dental anomalies.Am J Orthod Dentofacial Orthop. 2000; 117: 650-656PubMed Scopus (274) Google Scholar; Arte Arte, 2001Arte S (2001) Phenotypic and genotypic features of familial hypodontia. PhD thesis, Institute of Dentistry, University of Helsinki, Helsinki; http://ethesis.helsinki.fi/julkaisut/laa/hamma/vk/arte/ (accessed March 16, 2004)Google Scholar). Genetic factors causing these common types of hypodontia have remained unknown. The genetic background for more-severe tooth agenesis, oligodontia (MIM 604625), is better understood. Oligodontia, defined as congenital lack of six or more permanent teeth, third molars excluded, is relatively rare and is most often associated with some multiorgan syndrome. It has become obvious that both hypodontia and oligodontia are genetically very heterogeneous. It is plausible that mutations or common variants in genes in which loss-of-function mutations cause severe tooth agenesis may significantly contribute also to the background of the common hypodontia. For nonsyndromic oligodontia, dominant mutations in transcription factors MSX1 and PAX9 have been described (Vastardis et al. Vastardis et al., 1996Vastardis H Karimbux N Guthua SW Seidman JG Seidman CE A human MSX1 homeodomain missense mutation causes selective tooth agenesis.Nat Genet. 1996; 13: 417-421Crossref PubMed Scopus (516) Google Scholar; Stockton et al. Stockton et al., 2000Stockton DW Das P Goldenberg M D’Souza RN Patel PI Mutation of PAX9 is associated with oligodontia.Nat Genet. 2000; 24: 18-19Crossref PubMed Scopus (378) Google Scholar; Nieminen et al. Nieminen et al., 2001Nieminen P Arte S Tanner D Paulin L Alaluusua S Thesleff I Pirinen S Identification of a nonsense mutation in the PAX9 gene in molar oligodontia.Eur J Hum Genet. 2001; 9: 743-746Crossref PubMed Scopus (131) Google Scholar; Lammi et al. Lammi et al., 2003Lammi L Halonen K Pirinen S Thesleff I Arte S Nieminen P A missense mutation in PAX9 in a family with distinct phenotype of oligodontia.Eur J Hum Genet. 2003; 11: 866-871Crossref PubMed Scopus (87) Google Scholar). In homozygous Msx1- and Pax9-null mutant mice, tooth development is arrested at an early stage, whereas heterozygotes have normal dentition (Satokata and Maas Satokata and Maas, 1994Satokata I Maas R Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development.Nat Genet. 1994; 6: 348-356Crossref PubMed Scopus (1018) Google Scholar; Peters et al. Peters et al., 1998Peters H Neubuser A Kratochwil K Balling R Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities.Genes Dev. 1998; 12: 2735-2747Crossref PubMed Scopus (622) Google Scholar). There is experimental evidence indicating that Msx1 and Pax9 are required for the mediation of BMP and FGF signaling, respectively. Experiments in mice have also shown that Shh and Wnt signals are necessary for normal tooth development. It is thought that integrated networks of signaling pathways are the key regulators of tooth morphogenesis (Jernvall and Thesleff Jernvall and Thesleff, 2000Jernvall J Thesleff I Reiterative signaling and patterning during mammalian tooth morphogenesis.Mech Dev. 2000; 92: 19-29Crossref PubMed Scopus (767) Google Scholar; Thesleff Thesleff, 2003Thesleff I Epithelial-mesenchymal signalling regulating tooth morphogenesis.J Cell Sci. 2003; 116: 1647-1648Crossref PubMed Scopus (496) Google Scholar). Here, we describe a Finnish four-generation family in which oligodontia segregated as an autosomal dominant trait (fig. 1A). The proband and her father were orthodontic patients at the Institute of Dentistry, University of Helsinki. The diagnosis of oligodontia was based on clinical examination, panoramic radiographs, and interviews. The study was approved by the Ethics Committee of the Institute of Dentistry, and informed consent was obtained from all participating individuals. Altogether, 11 family members lacked at least eight permanent teeth, and, in 2 of them, only three permanent teeth had developed (figs. 1B, 1C, and 2). The oligodontia phenotype appeared completely penetrant. The majority of the affected family members lacked most permanent molars, premolars, lower incisors, and upper lateral incisors. Three also lacked all canines. Most often present were upper central incisors (always present), canines, first premolars, and first molars. One affected individual (IV:1) also lacked four deciduous incisors. Other individuals reported no defects in primary dentition, but dental records were not always available. The considerable variation in the number and type of missing teeth is typical for familial oligodontia (Vastardis et al. Vastardis et al., 1996Vastardis H Karimbux N Guthua SW Seidman JG Seidman CE A human MSX1 homeodomain missense mutation causes selective tooth agenesis.Nat Genet. 1996; 13: 417-421Crossref PubMed Scopus (516) Google Scholar; Stockton et al. Stockton et al., 2000Stockton DW Das P Goldenberg M D’Souza RN Patel PI Mutation of PAX9 is associated with oligodontia.Nat Genet. 2000; 24: 18-19Crossref PubMed Scopus (378) Google Scholar; Lammi et al. Lammi et al., 2003Lammi L Halonen K Pirinen S Thesleff I Arte S Nieminen P A missense mutation in PAX9 in a family with distinct phenotype of oligodontia.Eur J Hum Genet. 2003; 11: 866-871Crossref PubMed Scopus (87) Google Scholar; Nieminen et al. Nieminen et al., 2003Nieminen P Kotilainen J Aalto Y Knuutila S Pirinen S Thesleff I MSX1 gene is deleted in Wolf-Hirschhorn syndrome patients with oligodontia.J Dent Res. 2003; 82: 1013-1017Crossref PubMed Scopus (52) Google Scholar). However, the tooth phenotypes in this family were more severe than and different from oligodontia in families with mutations reported elsewhere. MSX1 mutations are associated especially with agenesis of the second premolars and third molars and PAX9 mutations with molar tooth agenesis (Vastardis et al. Vastardis et al., 1996Vastardis H Karimbux N Guthua SW Seidman JG Seidman CE A human MSX1 homeodomain missense mutation causes selective tooth agenesis.Nat Genet. 1996; 13: 417-421Crossref PubMed Scopus (516) Google Scholar; Stockton et al. Stockton et al., 2000Stockton DW Das P Goldenberg M D’Souza RN Patel PI Mutation of PAX9 is associated with oligodontia.Nat Genet. 2000; 24: 18-19Crossref PubMed Scopus (378) Google Scholar; Nieminen et al. Nieminen et al., 2001Nieminen P Arte S Tanner D Paulin L Alaluusua S Thesleff I Pirinen S Identification of a nonsense mutation in the PAX9 gene in molar oligodontia.Eur J Hum Genet. 2001; 9: 743-746Crossref PubMed Scopus (131) Google Scholar, Nieminen et al., 2003Nieminen P Kotilainen J Aalto Y Knuutila S Pirinen S Thesleff I MSX1 gene is deleted in Wolf-Hirschhorn syndrome patients with oligodontia.J Dent Res. 2003; 82: 1013-1017Crossref PubMed Scopus (52) Google Scholar; Lammi et al. Lammi et al., 2003Lammi L Halonen K Pirinen S Thesleff I Arte S Nieminen P A missense mutation in PAX9 in a family with distinct phenotype of oligodontia.Eur J Hum Genet. 2003; 11: 866-871Crossref PubMed Scopus (87) Google Scholar). The phenotype also differed from hypohidrotic ectodermal dysplasia (HED [MIM 305100]), in which both deciduous and permanent teeth are severely affected and the teeth that develop are often conical in shape. Furthermore, the other ectodermal symptoms in nails, hair, or skin typical for HED were not found in our patients. The patient records initially revealed two patients with oligodontia from the oldest generation (individuals II:2 and II:7) who had a history of colorectal neoplasia (fig. 1A; table 1). During this study, 10 other family members (7 subjects with oligodontia and 3 healthy individuals) were studied through use of colonoscopy or sigmoidoscopy. Colorectal neoplasia was found in six family members with oligodontia (individuals II:4, II:8, III:2, III:4, III:5, and III:7). No signs of neoplasia were found in family members with normal dentition. In the family, risk for colorectal neoplasia thus appeared to be associated with oligodontia. The colorectal findings were variable, since polyposis was found in a few patients, whereas it was not otherwise evident, even in the patients with severe neoplasia (table 1).Table 1Colorectal Findings in Family MembersPatientAgeaAge at the time of examination. (years)Colorectal FindingsII:1bPatient not affected with oligodontia.60No abnormal findings in colonoscopyII:254A widely metastasized adenocarcinoma originating from the hepatic flexure of the colonII:462Two hyperplastic polyps and two tubular adenomas with mild dysplasia (cecum and ascending colon)II:75768 adenomatous polyps of different sizes in colon, with severe dysplastic changesII:858A tubular adenoma in the rectum and the hepatic flexure of colon showing mild dysplasiaIII:235A 5–6-cm-wide sessile polypous tumor of the ascending colon, histopathology showing a tubulovillous adenoma with severe dysplasia but no invasive carcinomaIII:3bPatient not affected with oligodontia.29No abnormal findings in colonoscopyIII:4312 6-mm polyps removed at colonoscopy (cecum and upper rectum); histology showed hyperplastic polyp mixed with tubular adenoma, mild dysplasiaIII:527A sessile hyperplastic polyp of 10 mm in diameter removed at colonoscopy from the ascending colonIII:6bPatient not affected with oligodontia.36No abnormal findings in sigmoidoscopyIII:73510–20 hyperplastic polyps in the ascending colon; a large sessile hyperplastic polyp in cecum; 2 small adenomas in the sigmoid colonIII:826No abnormal findings in colonoscopya Age at the time of examination.b Patient not affected with oligodontia. Open table in a new tab Seventeen members of the family were available for molecular analysis. DNA was isolated from venous blood samples with the Puregene DNA purification system (Gentra). Sequencing of the coding regions of the oligodontia candidate genes PAX9 (Nieminen et al. Nieminen et al., 2001Nieminen P Arte S Tanner D Paulin L Alaluusua S Thesleff I Pirinen S Identification of a nonsense mutation in the PAX9 gene in molar oligodontia.Eur J Hum Genet. 2001; 9: 743-746Crossref PubMed Scopus (131) Google Scholar) and MSX1 of the proband and his father did not reveal mutations. For a genomewide search with linkage analysis, tooth agenesis was modeled as an autosomal dominant trait with 95% penetrance and a gene frequency of 0.0001. Linkage analysis excluded loci for PAX9, MSX1, LEF1, and PITX2. Analysis of chromosomes of the patients with colorectal neoplasia excluded also the APC locus. In pairwise analysis with Mlink (Lathrop et al. Lathrop et al., 1984Lathrop GM Lalouel JM Julier C Ott J Strategies for multilocus linkage analysis in humans.Proc Natl Acad Sci USA. 1984; 81: 3443-3446Crossref PubMed Scopus (2173) Google Scholar), highest pairwise LOD scores were obtained in chromosome 17 for two neighboring markers, 3.07 for D17S944 and 3.23 for D17S949. No recombinants were found for those markers and the disease. Genotyping more markers defined a maximal recombination-free region of ∼15 cM between markers D17S948 and D17S1352, according to Généthon and Marshfield genetic maps (University of California–Santa Cruz Web site). Multipoint analysis with Simwalk (Sobel and Lange Sobel and Lange, 1996Sobel E Lange K Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics.Am J Hum Genet. 1996; 58: 1323-1337PubMed Google Scholar) gave a maximum LOD score of 3.55 between markers D17S1792 and D17S1351 (fig. 3), providing evidence for linkage of oligodontia to a locus in that region. The region includes ⩾80 known or predicted genes. None of these had been previously implicated in tooth agenesis in humans or mice. AXIN2 (MIM 604425) was selected as a candidate gene because it is involved in Wnt signaling and because somatic mutations of AXIN2 have been found in colorectal tumors with defective mismatch repair (Behrens et al. Behrens et al., 1998Behrens J Jerchow BA Wurtele M Grimm J Asbrand C Wirtz R Kuhl M Wedlich D Birchmeier W Functional interaction of an Axin homolog, conductin, with β-catenin, APC, and GSK3β.Science. 1998; 280: 596-599Crossref PubMed Scopus (1061) Google Scholar; Liu et al. Liu et al., 2000Liu W Dong X Mai M Seelan RS Taniguchi K Krishnadath KK Halling KC Cunningham JM Boardman LA Qian C Christensen E Schmidt SS Roche PC Smith DI Thibodeau SN Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating β-catenin/TCF signalling.Nat Genet. 2000; 26: 146-147Crossref PubMed Scopus (419) Google Scholar). Direct sequencing of AXIN2-coding regions and flanking intronic sequences (fig. 4; table A [online only]; Dental Genetics Group Web site) revealed a 1966C→T transition in exon 7, leading to a change of arginine 656 to a stop codon and premature termination of translation (fig. 4A and 4C). Further sequencing revealed the presence of the same nonsense mutation in all family members who had oligodontia but not in the healthy family members or in >100 unrelated control individuals.Table APCR Primers and Conditions for Sequencing of AXIN2TargetSize of Product (bp)Forward PrimerReverse PrimerPCR ConditionsaHot start, annealing temperature in °C, and number of cycles.Exon 1 5′727TGGGTTTTTGGAAGGTTGTGGAACAGGTAAGCACCGTCTTGHot +59 ×32Exon 1 3′684CATCTCCGGATTCCCCTCTTCCACCCATCCACCATACTTHot +59 ×32Exon 2465GCTGCCTCTGGAATACTCTCTGTAAGTGCTCAGGTGGCATCCHot +59 ×32Exon 3327AGCACCGATGGTATCTGGAGCCACCACCCATTTCTTTTCTTHot +59 ×32Exon 4549GATGGTTGACAACAGTCTTTGAAGCTAACGCACCCCATGCACHot +62 ×32Exon 5673CTTCTGCTTCCTGGGTCACTCTGCCGCCCTCTTAGAAACTHot +59 ×32Exon 6330AGGAGTCCCGGAGATTTAACCAACAGCCATTCCCACAATACCHot +59 ×32Exon 7398TTCCAGTTCTTCTAACCCAGTTTCTTGAGACCCAGGCAGAAAGAGHot +59 ×32Exon 8320AATTGCTCTGGGGACAACAGGGACATGGATGGCAACATCTHot +59 ×32Exon 9300GCACGTGTGTGTTTGCTTTAGTCTGGCTCTTGGTTCTGAGCHot +59 ×32Exon 10684TCAACAATGTGGAAAATGCAGAGAAACCATGAACGCACTCCHot +59 ×32a Hot start, annealing temperature in °C, and number of cycles. Open table in a new tab While screening the AXIN2 gene for mutations in other patients with oligodontia, we identified a heterozygous 1-bp insertion (fig. 4B), after nucleotide 1994 in exon 7, in a 13-year-old boy with a very similar tooth phenotype as that in the family described above (fig. 2). The mutation was not found in his healthy parents, indicating that it is a de novo germline mutation in one of the parents. The mutation expands one of the several mononucleotide repeats in exon 7 of AXIN2 and recodes the amino acids starting at Asn666, incorporating a stop codon 40 codons later. This mutation was identical to one of the somatic frameshift mutations described for colorectal cancer tissue (Liu et al. Liu et al., 2000Liu W Dong X Mai M Seelan RS Taniguchi K Krishnadath KK Halling KC Cunningham JM Boardman LA Qian C Christensen E Schmidt SS Roche PC Smith DI Thibodeau SN Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating β-catenin/TCF signalling.Nat Genet. 2000; 26: 146-147Crossref PubMed Scopus (419) Google Scholar). The finding of this de novo mutation suggests the vulnerability of the mononucleotide repeats in exon 7 of AXIN2 for frameshift mutations also in absence of defective mismatch repair (Liu et al. Liu et al., 2000Liu W Dong X Mai M Seelan RS Taniguchi K Krishnadath KK Halling KC Cunningham JM Boardman LA Qian C Christensen E Schmidt SS Roche PC Smith DI Thibodeau SN Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating β-catenin/TCF signalling.Nat Genet. 2000; 26: 146-147Crossref PubMed Scopus (419) Google Scholar). The young age of the patient prevents the confirmation of cancer predisposition. Both mutations introduce a premature stop codon to exon 7 of the AXIN2 gene. According to current knowledge, in-frame stop codons introduced by mutations may lead to degradation of mRNA via the nonsense-mediated mRNA decay mechanism (Wilusz et al. Wilusz et al., 2001Wilusz CJ Wang W Peltz SW Curbing the nonsense: the activation and regulation of mRNA surveillance.Genes Dev. 2001; 15: 2781-2785PubMed Google Scholar; Cartegni et al. Cartegni et al., 2002Cartegni L Chew SL Krainer AR Listening to silence and understanding nonsense: exonic mutations that affect splicing.Nat Rev Genet. 2002; 3: 285-298Crossref PubMed Scopus (1645) Google Scholar). The mRNAs transcribed from the mutated copies of AXIN2 fulfill the requirement that the premature stop codon be located ⩾50 bp upstream of the last exon-exon junction, suggesting that the mutated AXIN2 mRNAs are guided to the degradation pathway. A protein product translated from the mutated mRNA would be truncated and lacking the C-terminal domain (fig. 4C and 4D), which, in Axin1, mediates oligomerization and interactions with protein phosphatase 2A, disheveled, and LRP5/6 (Seidensticker and Behrens Seidensticker and Behrens, 2000Seidensticker MJ Behrens J Biochemical interactions in the Wnt pathway.Biochim Biophys Acta. 2000; 1495: 168-182Crossref PubMed Scopus (229) Google Scholar; Mao et al. Mao et al., 2001Mao J Wang J Liu B Pan W Farr GH Flynn C Yuan H Takada S Kimelman D Li L Wu D Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Since the truncations would cause the predicted oligomerization domain to be lost and, in the case of the frameshift mutation, the length of recoded amino acids would be short, a dominant negative effect would be improbable. Stop-codon–creating exon 7 frameshift mutants of AXIN2 are associated with accumulation of β-catenin in the nucleus of cancer cells and, in transfection experiments, activate a TCF-promoted reporter gene (Liu et al. Liu et al., 2000Liu W Dong X Mai M Seelan RS Taniguchi K Krishnadath KK Halling KC Cunningham JM Boardman LA Qian C Christensen E Schmidt SS Roche PC Smith DI Thibodeau SN Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating β-catenin/TCF signalling.Nat Genet. 2000; 26: 146-147Crossref PubMed Scopus (419) Google Scholar). C-terminal deletions in mouse Axin1 have been shown to abolish its ability to inhibit Wnt reporter-gene activity (Hsu et al. Hsu et al., 1999Hsu W Zeng L Costantini F Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain.J Biol Chem. 1999; 274: 3439-3445Crossref PubMed Scopus (210) Google Scholar). These data indicate that Arg656Stop and 1994-1995insG lead to decreased AXIN2 function and most probably represent loss-of-function mutations that cause activation of Wnt signaling. It has been shown that, in mice, Axin2 exhibits tissue- and stage-specific expression patterns during development (Jho et al. Jho et al., 2002Jho EH Zhang T Domon C Joo CK Freund JN Costantini F Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.Mol Cell Biol. 2002; 22: 1172-1183Crossref PubMed Scopus (1224) Google Scholar; Aulehla et al. Aulehla et al., 2003Aulehla A Wehrle C Brand-Saberi B Kemler R Gossler A Kanzler B Herrmann BG Wnt3a plays a major role in the segmentation clock controlling somitogenesis.Dev Cell. 2003; 4: 395-406Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). It is expressed in the primitive streak and later in the dorsal neural tube and developing somites, as well as in the branchial arches and the limb buds of mouse embryos (Jho et al. Jho et al., 2002Jho EH Zhang T Domon C Joo CK Freund JN Costantini F Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.Mol Cell Biol. 2002; 22: 1172-1183Crossref PubMed Scopus (1224) Google Scholar). In the presomitic mesoderm, it is expressed in an oscillating pattern, and an orchestrating role for Axin2 and Wnt3a was suggested in the establishment of the segmental pattern during somitogenesis (Aulehla et al. Aulehla et al., 2003Aulehla A Wehrle C Brand-Saberi B Kemler R Gossler A Kanzler B Herrmann BG Wnt3a plays a major role in the segmentation clock controlling somitogenesis.Dev Cell. 2003; 4: