Oligodontia is defined as the congenital absence of six or more permanent teeth, excluding the third molars. Oligodontia may contribute to masticatory dysfunction, speech alteration, aesthetic problems and malocclusion. Numerous gene mutations have been association with oligodontia. In the present study, we identified a de novo AXIN2 missense mutation (c.314T>G) in a Chinese individual with non-syndromic oligodontia. This mutation results in the substitution of Val at residue 105 for Gly (p.Val105Gly); residue 105 is located in the highly conserved regulator of G protein signaling (RGS) domain of the AXIN2 protein. This is the first report indicating that a mutation in the RGS domain of AXIN2 is responsible for non-syndromic oligodontia. Our study supports the relationship between AXIN2 mutation and non-syndromic oligodontia and extends the mutation spectrum of the AXIN2 gene.
Citation: Liu H, Ding T, Zhan Y, Feng H (2015) A Novel AXIN2 Missense Mutation Is Associated with Non-Syndromic Oligodontia. PLoS ONE 10(9): e0138221. https://doi.org/10.1371/journal.pone.0138221
Editor: Leonard Eisenberg, New York Medical College, UNITED STATES
Received: February 13, 2015; Accepted: August 26, 2015; Published: September 25, 2015
Copyright: © 2015 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All files for the AXIN2 mutation are available from the ClinVar database in NCBI (accession number SCV000223708).
Funding: This work was supported by the National Natural Science Foundation of China (No. 81100725). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Tooth agenesis, the congenital lack of one or more permanent and/or deciduous teeth, is a well-recognized morphologic anomaly in humans. The absence of one to six teeth (excluding the third molars), more than six teeth (excluding the third molars), and the complete absence of teeth have been termed hypodontia, oligodontia, and anodontia, respectively . Tooth agenesis can occur either as isolated findings or as part of a syndrome . Both environmental and genetic factors can cause tooth agenesis, with genetic factors representing by far the most common cause of tooth agenesis .
Numerous genes have been implicated in tooth development and in theory, any of these genes may cause tooth agenesis. To this day, mutations in nine genes (MSX1, PAX9, AXIN2, WNT10A, EDA, EDAR, EDARADD, NEMO and KRT17) have been associated with non-syndromic oligodontia [4–13]. The transcription factor genes MSX1 and PAX9 were the first and second genes to be identified in non-syndromic oligodontia [7,14]. The MSX1 and PAX9 proteins are responsible for the interaction between dental tissues and are essential for the establishment of the odontogenic potential of the mesenchyme . AXIN2 is an intracellular antagonist of Wnt signaling and WNT10A is a member of the Wnt family. Both AXIN2 and WNT10A can cause non-syndromic oligodontia [16–17]. EDA is a well-known gene that causes ectodermal dysplasia; however, several studies also reported that mutations in EDA can cause non-syndromic oligodontia[4,9,18–19]. EDARADD is a downstream signaling mediator of EDA and one study reported that a mutation in this gene led to non-syndromic oligodontia.
In the present study, we investigated six genes (MSX1, PAX9, AXIN2, WNT10A, EDA and EDARADD) in a patient with sporadic non-syndromic oligodontia. Finally, we identified a de novo AXIN2 missense mutation, which was located in a highly conserved region of the encoded protein.
Materials and Methods
Study Individuals and Samples
Subjects of this study were of Chinese descent. The 8-year-old male was recruited from among the patients referred to the Peking University School of Stomatology (the 3rd Dental Center for diagnostic evaluation and treatment of oligodontia). A pedigree was constructed by clinical examination of available family members and through interviews. An experienced dentist determined the status of dentition through oral and panoramic radiographic examinations for the proband and his parents. Peripheral blood samples were taken from the proband and his parents, in addition to 100 unrelated healthy individuals who were not affected with tooth agenesis or other craniofacial abnormalities (control group). Written informed consent was obtained from all the participants and the parents on the behalf of the minors or children participants. The study protocol and subject consent were approved by the Institutional Review Board of Peking University School and Hospital of Stomatology (No.IRBSS2014NNSF01).
Mutation Analysis for Candidate Genes
Genomic DNA was isolated from peripheral blood samples by use of the Biotek DNA Mini-kit (Biotek, Beijing, China), following the manufacturer’s instructions. The MSX1 (NM_002448), PAX9 (NM_006194), AXIN2 (NM_004655), WNT10A (NM_025216), EDA (NM_001399) and EDARADD (NM_080738) genes were selected for genetic analysis, and their exons and exon-intron junctions were amplified by polymerase chain reaction (PCR). Direct DNA sequencing was performed using ABigDye terminator v3.1 (Applied Biosystems, Foster City, USA) and a 3730 DNA sequencer (Applied Biosystems). PCR primers and conditions were consistent with previous studies [6,9,17,20–23].
Conservation Analysis and WebLogo Analysis
Multiple-species amino acid sequence alignment and WebLogo analysis of the AXIN2 protein (NP_004646.3) were carried out separately by ClustalX 2.1 and WebLogo Version 2.8.2 (http://weblogo.berkeley.edu) . AXIN2 sequences from zebrafish to human were obtained from ENSEMBL.
Three-dimensional models of the wild-type regulator of G protein signaling (RGS) domain of the AXIN2 protein and the mutant RGS (p.Val105Gly) domain were designed using the Phyre threading software (www.sbg.bio.ic.ac.uk/phyre/) , based on primary sequence conservation and known protein structures. The models were manipulated using the PyMOL software (PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA).
The proband was an 8-year-old male with a normal appearance. Clinical and radiographic examinations revealed that, in addition to retained deciduous teeth, the patient was missing a total of seven permanent teeth excluding third molars, all of them premolars (Fig 1). The incisors, canines, first molars and second molars were normal. No tooth germ for third molars was detected. Considering his young age, a diagnosis could not yet be made in relation to the lack of third molars. The proband’s facial features, skin, hair and nails appeared normal. Also, he reported normal sweating and lachrymal secretion and denied intolerance to heat, or susceptibility to respiratory tract infections. His parents’ dentition was normal, and they reported no family history of tooth agenesis, ectodermal abnormalities or cancer.
Following mutation analysis of six genes (MSX1, PAX9, AXIN2, WNT10A, EDA and EDARADD), we found a novel missense mutation in AXIN2 in the proband. The nucleotide sequence showed a heterozygous T to G transition at nucleotide 314 (c.314T>G) of the coding sequence in exon 2 of AXIN2 (Fig 2), which resulted in the substitution of Val at residue 105 for Gly. We have submitted the information of this AXIN2 mutation to ClinVar database in NCBI (www.ncbi.nlm.nih.gov/clinvar/; Accession number: SCV000223708). The proband’s parents did not carry the mutation (Fig 2). Also, the mutation was not detected in 100 normal controls (Fig 2). Therefore, this mutation is likely to have occurred de novo.
Val105 is located at the RGS domain of the AXIN2 protein (Fig 3). Evolutionary conservation analysis revealed that the Val105 site is conserved from zebrafish to humans (Fig 3), suggesting that Val105 may play an important role in the function of the RGS domain. To evaluate the conservation of the RGS domain of the AXIN2 protein, we carried out WebLogo analysis (http://weblogo.berkeley.edu) and compared the conservation of all amino acids of the RGS domain from zebrafish to humans based on multiple alignments. We found that the RGS domain was relatively conserved (Fig 3).
(A) The AXIN2 protein is 777 AA long with 4 conserved domains. The 105th AA position is mutated in the proband Val105Gly which resides in the RGS domain. (B) Evolutionary conservation analysis revealed that the Val105 site is conserved from zebrafish to humans. There are 28 out of 33 AA near the Val105 site that are conserved, this is a very high degree of conservation. (C) WebLogo analysis showed that the RGS domain (from zebrafish to humans) was relatively conserved. TNKS_binding: Tankyrase binding N-terminal segment of axin; RGS: Regulator of G protein signaling (RGS) domain; DAX: Domain present in Dishevelled and Axin.
Structural analysis predicted that the 3D structures of the wild-type and mutant RGS domain of AXIN2 are different at residue 105 (Fig 4), which suggests that the p.Val105Gly substitution may affect the structure of the RGS domain of the AXIN2 protein.
Our findings indicate that the novel heterozygous transition found in AXIN2 (c.314T>G) might be responsible for the presentation of non-syndromic oligodontia in the proband. This mutation, which results in a Val105Gly substitution, is localized to the regulator of G protein signaling (RGS) domain found in the Axin protein. RGS domains are protein structural domains that activate GTPases via heterotrimeric G-protein alpha-subunits; RGS proteins have been conserved in evolution . This missense mutation (c.314T>G) may affect the function of the RGS domain of the AXIN2 protein, leading to oligodontia.
In families with severe tooth agenesis and in several mutant mouse lines, the phenotypes are variable in expression, presumably due to the effects of modifying genes and other genetic backgrounds as well as postgenetic factors . In tooth agenesis, this variation in effect of major genetic factors can be reflected as incomplete penetrance. In this study, the proband’s parents displayed normal dentition and reported no family history of tooth agenesis or other ectodermal abnormalities. This may suggest incomplete penetrance of oligodontia in this family. However, the novel mutation was identified only in the proband, and his parents did not carry the mutation (Fig 2). Moreover, the highly conserved character of the mutated nucleotide (Fig 3), as well as the number of analyzed control alleles (100), precludes the possibility that this transition is a rare polymorphism of AXIN2. Hence, we confirm that this missense mutation is a de novo germline mutation. A number of studys have reported SNPs in AXIN2 associated with tooth agenesis [23,28–29], Mu et al.  presented 7 SNPs in the AXIN2, and 3 SNPs were in exons. We did not find any SNPs in AXIN2 in the proband and his parents. It’s seemed that SNPs in AXIN2 is not a risk factor for this family.
Five conserved signaling pathways, including the fibroblast growth factors (Fgf), bone morphogenetic proteins (Bmp), ectodysplasin (Eda), wingless-related (Wnt) and sonic hedgehog (Shh) pathways, play a critical role throughout tooth development . Inactivation of any of these pathways results in early tooth developmental arrest in mice. Mutations in these pathways have also been identified in human patients with tooth agenesis. The MSX1 and PAX9 transcription factors are involved in the Bmp and Fgf pathways during tooth development . The Eda pathway is active during the development of ectodermal organs, including teeth, hairs, feathers, and mammary glands and was discovered by studying human patients affected by anhidrotic/hypohidrotic ectodermal dysplasia. It comprises three main gene products: EDA, a ligand that belongs to the tumor necrosis factor (TNF)-α family, EDAR, a receptor related to the TNFα receptors, and EDARADD, a specific adaptor . Recently, WNT10A, belonging to the Wnt pathway, became a focal candidate gene for tooth agenesis. WNT10A was identified as a causal gene of autosomal recessive ectodermal dysplasia or isolated tooth agenesis [33–35]. Moreover, Pakeeza et al. stated that WNT10A mutations account for one-quarter of population-based isolated oligodontia, and Van den Boogaard et al. reported that mutations in WNT10A are present in more than half of isolated hypodontia cases. In the present study, we did not identify any mutation in WNT10A, but one was identified in AXIN2. AXIN2 is an intracellular antagonist of the Wnt signaling pathway. Thus, this missense mutation (c.314T>G) in AXIN2 may affect tooth development via the Wnt pathway.
To date, four studies reported seven AXIN2 mutations associated with syndromic or non-syndromic tooth agenesis [6,16,21,37]. Among the seven, two mutations caused syndromic tooth agenesis: in 2004, Lammi et al. showed that a nonsense mutation in AXIN2(c.1966C>T, p.Arg656Stop) caused oligodontia and predisposed to cancer. Additionally, in 2009, Marvin et al. described a family with a novel, nonsense AXIN2 mutation (c.1989G>A, p.Tyr663X) segregating in an autosomal dominant pattern with oligodontia and variable other findings, including colonic polyposis, gastric polyps, a mild ectodermal dysplasia phenotype with sparse hair and eyebrows, and early onset colorectal and breast cancers. In the remaining five AXIN2 mutations causing non-syndromic tooth agenesis, four are missense mutations (p.Ala758Thr, p.Ala684Val, p.Thr308Met and p.Met830Ile), and one is a frameshift mutation (p.Gly666GlyfsX42) [6,16,21]. It appears that AXIN2 mutations that lead to a truncated AXIN2 protein are more likely to lead to syndromic oligodontia and predispose to cancer. In this study the missense mutation in AXIN2 (c.314T>G) resulted in the substitution of Val at residue 105 for Gly. Structural analysis predicted that the 3D structures of the wild-type and mutant RGS domain of AXIN2 are different at residue 105 (Fig 4). This results suggests that although the overall structure of the protein is complete, the p.Val105Gly substitution may affect some functional activity of AXIN2 protein. This may lead to the occurrence of clinical symptoms. Of course, functional study of the mutant protein need to be done to confirm the assumption in the future. Because the AXIN2 mutation (c.314T>G) identified in the present study occurred de novo, cancer prevention measures are necessary for the 8-year-old male proband.
Interestingly, in the present study, the proband was missing a total of seven permanent teeth (excluding third molars), all of which were premolars. In previous studies, the positions of missing teeth in patients with AXIN2 mutation were not identical [6,16,21,37]. At present, the phenotype-genotype association in AXIN2-related patients is unclear. However, in all cases, patients with an AXIN2 mutation were missing at least seven teeth. This indicates that any change in the AXIN2 protein can cause severe tooth agenesis.
In conclusion, this study report describes a de novo AXIN2 missense mutation (c.314T>G) in a Chinese individual with non-syndromic oligodontia. This mutation is located at the regulator of G protein signaling (RGS) domain of the AXIN2 protein, which is a highly conserved region. In addition, this study extends the mutation spectrum of the AXIN2 gene in individuals with non-syndromic tooth agenesis. Furthermore, our findings confirm that AXIN2 plays an important role in human tooth development. However, further studies are necessary to clarify the precise role of AXIN2 in tooth development.
Conceived and designed the experiments: HF. Performed the experiments: TD HL YZ. Analyzed the data: TD HL. Contributed reagents/materials/analysis tools: YZ TD HL. Wrote the paper: TD HL HF.
- 1. Chishti MS, Muhammad D, Haider M, Ahmad W. A novel missense mutation in MSX1 underlies autosomal recessive oligodontia with associated dental anomalies in Pakistani families. J Hum Genet. 2006; 51:872–878. pmid:16932841
- 2. Klein OD, Oberoi S, Huysseune A, Hovorakova M, Peterka M, Peterkova R. Developmental disorders of the dentition: An update. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2013; 163:318–332.
- 3. Chhabra N, Goswami M, Chhabra A. Genetic basis of dental agenesis—molecular genetics patterning clinical dentistry. Med Oral Patol Oral Cir Bucal. 2014; 19:e112–e119. pmid:24121910
- 4. Song S, Han D, Qu H, Gong Y, Wu H, Zhang X, et al. EDA gene mutations underlie non-syndromic oligodontia. J Dent Res. 2009; 88:126–131. pmid:19278982
- 5. Wang Y, Wu H, Wu J, Zhao H, Zhang X, Mues G, et al. Identification and functional analysis of two novel PAX9 mutations. Cells Tissues Organs. 2009; 189:80–87. pmid:18701815
- 6. Bergendal B, Klar J, Stecksen-Blicks C, Norderyd J, Dahl N. Isolated oligodontia associated with mutations in EDARADD, AXIN2, MSX1, and PAX9 genes. Am J Med Genet A. 2011; 155A:1616–1622. pmid:21626677
- 7. Stockton DW, Das P, Goldenberg M, D'Souza RN, Patel PI. Mutation of PAX9 is associated with oligodontia. Nat Genet. 2000; 24:18–19. pmid:10615120
- 8. van den Boogaard MJ, Creton M, Bronkhorst Y, van der Hout A, Hennekam E, Lindhout D, et al. Mutations in WNT10A are present in more than half of isolated hypodontia cases. J Med Genet. 2012; 49:327–331. pmid:22581971
- 9. Han D, Gong Y, Wu H, Zhang X, Yan M, Wang X, et al. Novel EDA mutation resulting in X-linked non-syndromic hypodontia and the pattern of EDA-associated isolated tooth agenesis. Eur J Med Genet. 2008; 51:536–546. pmid:18657636
- 10. Arte S, Parmanen S, Pirinen S, Alaluusua S, Nieminen P. Candidate Gene Analysis of Tooth Agenesis Identifies Novel Mutations in Six Genes and Suggests Significant Role for WNT and EDA Signaling and Allele Combinations. PLoS One. 2013; 8:e73705. pmid:23991204
- 11. Pan Y, Wang L, Ma J, Zhang W, Wang M, Zhong W, et al. PAX9 polymorphisms and susceptibility to sporadic tooth agenesis: a case-control study in southeast China. Eur J Oral Sci. 2008; 116:98–103. pmid:18353002
- 12. Silva ER, Reis-Filho CR, Napimoga MH, Alves JB. Polymorphism in the Msx1 gene associated with hypodontia in a Brazilian family. J Oral Sci. 2009; 51:341–345. pmid:19776500
- 13. Ruf S, Klimas D, Honemann M, Jabir S. Genetic background of nonsyndromic oligodontia: a systematic review and meta-analysis. J Orofac Orthop. 2013; 74:295–308. pmid:23828301
- 14. Vastardis H, Karimbux N, Guthua SW, Seidman JG, Seidman CE. A human MSX1 homeodomain missense mutation causes selective tooth agenesis. Nat Genet. 1996; 13:417–421. pmid:8696335
- 15. Peters H, Balling R. Teeth. Where and how to make them. Trends Genet. 1999; 15:59–65. pmid:10098408
- 16. Lammi L, Arte S, Somer M, Jarvinen H, Lahermo P, Thesleff I, et al. Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer. Am J Hum Genet. 2004; 74:1043–1050. pmid:15042511
- 17. Song S, Zhao R, He H, Zhang J, Feng H, Lin L. WNT10A variants are associated with non-syndromic tooth agenesis in the general population. Hum Genet. 2014; 133:117–124. pmid:24043634
- 18. Nikopensius T, Annilo T, Jagomagi T, Gilissen C, Kals M, Krjutskov K, et al. Non-syndromic tooth agenesis associated with a nonsense mutation in ectodysplasin-A (EDA). J Dent Res. 2013; 92:507–511. pmid:23603338
- 19. Yang Y, Luo L, Xu J, Zhu P, Xue W, Wang J, et al. Novel EDA p.Ile260Ser mutation linked to non-syndromic hypodontia. J Dent Res. 2013; 92:500–506. pmid:23625373
- 20. Wong SW, Liu HC, Han D, Chang HG, Zhao HS, Wang YX, et al. A novel non-stop mutation in MSX1 causing autosomal dominant non-syndromic oligodontia. Mutagenesis. 2014; 29:319–323. pmid:24914010
- 21. Wong S, Liu H, Bai B, Chang H, Zhao H, Wang Y, et al. Novel missense mutations in the AXIN2 gene associated with non-syndromic oligodontia. Archives of Oral Biology. 2014; 59:349–353. pmid:24581859
- 22. Cluzeau C, Hadj-Rabia S, Jambou M, Mansour S, Guigue P, Masmoudi S, et al. Only four genes (EDA1, EDAR, EDARADD, and WNT10A) account for 90% of hypohidrotic/anhidrotic ectodermal dysplasia cases. Hum Mutat. 2011; 32:70–72. pmid:20979233
- 23. Mu YD, Xu Z, Contreras CI, McDaniel JS, Donly KJ, Chen S. Mutational analysis of AXIN2, MSX1, and PAX9 in two Mexican oligodontia families. Genet Mol Res. 2013; 12:4446–4458. pmid:24222224
- 24. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004; 14:1188–1190. pmid:15173120
- 25. Kelley LA, Sternberg MJ. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc. 2009; 4:363–371. pmid:19247286
- 26. De Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG. The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol. 2000; 40:235–271. pmid:10836135
- 27. Nieminen P. Genetic basis of tooth agenesis. J Exp Zool B Mol Dev Evol. 2009; 312B:320–342. pmid:19219933
- 28. Callahan N, Modesto A, Meira R, Seymen F, Patir A, Vieira AR Axis inhibition protein 2 (AXIN2) polymorphisms and tooth agenesis. Arch Oral Biol. 2009; 54:45–49. pmid:18790474
- 29. Mostowska A, Biedziak B, Jagodzinski PP. Axis inhibition protein 2 (AXIN2) polymorphisms may be a risk factor for selective tooth agenesis. J Hum Genet. 2006; 51:262–266. pmid:16432638
- 30. Lan Y, Jia S, Jiang R. Molecular patterning of the mammalian dentition. Semin Cell Dev Biol. 2014; 25–26:61–70. pmid:24355560
- 31. Neubuser A, Peters H, Balling R, Martin GR. Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell. 1997; 90:247–255. pmid:9244299
- 32. Sadier A, Viriot L, Pantalacci S, Laudet V. The ectodysplasin pathway: from diseases to adaptations. Trends Genet. 2014; 30:24–31. pmid:24070496
- 33. Nagy N, Wedgeworth E, Hamada T, White JM, Hashimoto T, McGrath JA. Schopf-Schulz-Passarge syndrome resulting from a homozygous nonsense mutation in WNT10A. J Dermatol Sci. 2010; 58:220–222. pmid:20418069
- 34. Petrof G, Fong K, Lai-Cheong JE, Cockayne SE, McGrath JA. Schopf-Schulz-Passarge syndrome resulting from a homozygous nonsense mutation, p.Cys107X, in WNT10A. Australas J Dermatol. 2011; 52:224–226. pmid:21834823
- 35. Nawaz S, Klar J, Wajid M, Aslam M, Tariq M, Schuster J, et al. WNT10A missense mutation associated with a complete odonto-onycho-dermal dysplasia syndrome. Eur J Hum Genet. 2009; 17:1600–1605. pmid:19471313
- 36. Arzoo PS, Klar J, Bergendal B, Norderyd J, Dahl N. WNT10A mutations account for (1/4) of population-based isolated oligodontia and show phenotypic correlations. Am J Med Genet A. 2014; 164A:353–359. pmid:24449199
- 37. Marvin ML, Mazzoni SM, Herron CM, Edwards S, Gruber SB, Petty EM. AXIN2-associated autosomal dominant ectodermal dysplasia and neoplastic syndrome. Am J Med Genet A. 2011; 155A:898–902. pmid:21416598