Archives of Oral Biology 61 (2016) 144–148
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A novel initiation codon mutation of PAX9 in a family with oligodontia Jia Lianga,b,1, Chuanqi Qina,1, Haitang Yuea , Hong Hec,**, Zhuan Biana,* a The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China b Department Two of Endodontics, Hospital and School of Stomatology, Wuhan University, Wuhan, China c Department One of Orthodontics, Hospital and School of Stomatology, Wuhan University, Wuhan, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 February 2015 Received in revised form 21 October 2015 Accepted 25 October 2015
Objective: Recent studies have attributed non-syndromic tooth agenesis to mutations in several genes, including MSX1, PAX9, AXIN2, WNT10A and EDA. In this study, mutation of PAX9gene was investigated in a four-generation Chinese family with oligodontia. Design: Genomic DNA was isolated from the blood samples of all the available family members. Candidate genes MSX1 and PAX9 were ampliﬁed using polymerase chain reaction and then directly sequenced. Results: A novel initiation codon mutation was identiﬁed; it consisted of a heterozygous c.2T > G mutation in the PAX9 gene which changed the ATG initiation codon to AGG. Restriction-enzyme analysis was performed to verify this mutation, which was segregated amongst the members with the oligodontia phenotype. Conclusions: Our results demonstrate a new initiation codon mutation in the PAX9 gene. This mutation probably caused the oligodontia in the investigated Chinese family through haplo-insufﬁciency. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: Oligodontia PAX9 Initiation codon
1. Introduction Tooth agenesis is a common developmental anomaly in humans, which occurs in 3–10% of the population (De, Oster, Marks, Martens, & Huysseune, 2009). Three terms are used to describe tooth agenesis according to the number of missing teeth: hypodontia is deﬁned as absence of one to six permanent teeth, excluding third molars; oligodontia refers to the absence of more than six permanent teeth, excluding third molars; and anodontia denotes the absence of all teeth (Schalk-van der Weide, Beemer, Faber, & Bosman, 1994). Agenesis can also be classiﬁed into nonsyndromic and syndromic tooth agenesis according to the accompanying symptoms (Vastardis, 2000). Any disturbance in tooth development which involves a series of inductive interactions between epithelium and underlying mesenchyme may result in tooth agenesis or other dental defects (Thesleff, 2006). Mutations in MSX1, PAX9, AXIN2, WNT10A and EDA have been proven to cause non-syndromic tooth agenesis (Vastardis, Karimbux, Guthua, Seidman, & Seidman, 1996; Stockton, Das,
* Corresponding author at: School & Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, China. ** Corresponding author at: School & Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, China. E-mail addresses: [email protected]
(H. He), [email protected]
(Z. Bian). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.archoralbio.2015.10.022 0003-9969/ ã 2015 Elsevier Ltd. All rights reserved.
Goldenberg, D’Souza, & Patel, 2000; Lammi et al., 2004; van den Boogaard, Creton, Bronkhorst, van der Hout, Hennekam, & Lindhout, 2012; Tao et al., 2006) PAX9 is localised in chromosome 14(14q12–q13) and is a member of the PAX gene family, which encodes a group of transcription factors that are critical for foetal growth and organogenesis. PAX proteins are deﬁned by the presence of a DNA-binding domain (the paired-domain), which makes sequence-speciﬁc contact with DNA (Chi & Epstein, 2002). Pax9 is widely expressed in neural crestderived mesenchyme, which is involved in craniofacial and tooth development (Peters, Neubuser, Kratochwil, & Balling, 1998). Homozygous Pax9-deﬁcient mice die shortly after birth, lack pharyngeal pouch derivatives and exhibit numerous craniofacial and limb anomalies. All mutant mice fail to form teeth beyond the bud stage and have a cleft secondary palate. Heterozygous Pax9 mutant mice do not exhibit any obvious abnormalities, indicating that Pax9 is haploid sufﬁcient (Peters et al., 1998). Since the ﬁrst discovery of PAX9 mutation (Stockton et al., 2000), a number of autosomal dominant mutations ranging from in-frame to out-offrame mutations have been associated with this disorder. Most of these mutations cluster in and around the paired-domain, indicating that this region might be a mutation hotspot of PAX9. In the present study, we describe a four-generation family in which tooth agenesis is inherited in an autosomal dominant manner. Mutational screening for candidate genes was performed to associate genotype with phenotype.
J. Liang et al. / Archives of Oral Biology 61 (2016) 144–148
2. Materials and methods 2.1. Subjects
sequencing of exons and exon–intron boundaries of two genes, namely, MSX1 (GenBank accession number M97676) and PAX9 (GenBank accession number AJ238381) as described previously (Klein, Nieminen, Lammi, Niebuhr, & Kreiborg, 2005).
The study included subjects from a family of Chinese descent who were referred to the orthodontic department at the School of Stomatology, Wuhan University. Pedigree construction was conducted by clinical examination and interview of the available family members. Panoramic radiographs and photographs were obtained to verify tooth agenesis. Blood samples were collected from the available family members and 100 unrelated healthy controls. The study was conducted with the informed consent of the family members and approval of the Institutional Review Board.
Genomic DNA of all the available family members was ampliﬁed with speciﬁc primers PAX9initialF 50 -CCAGGTGGGGAGCTAGCCTG-30 and PAX9initialR 50 -AGTCAATAGAGAATGTGAGCGCCT-30 . The ampliﬁed fragments were then subjected to restriction digestion with BsrDI (NEB) following the manufacturer’s instructions. Digestion products were analyzed by agarose gel electrophoresis.
2.2. Mutation detection
Genomic DNA was extracted from the peripheral blood samples of all the available members and controls following the standard salt extraction procedure. Pathogenic mutations were screened using polymerase chain reaction (PCR) ampliﬁcation and
3.1. Pedigree and phenotype analyses
2.3. Restriction analysis
Pedigree analysis indicated that oligodontia in this family was segregated in an autosomal dominant pattern (Fig. 1a). Oligodontia
Fig. 1. (a) Pedigree of the four-generation Chinese family with oligodontia. (b) Panoramic radiograph of the proband. The white star indicates missing permanent tooth. (c) Clinical photograph of the proband; upper jaw and lower jaw. (d) Tooth phenotypes of the family members with oligodontia. $denotes congenitally missing tooth and ~denotes cone-shaped tooth.
J. Liang et al. / Archives of Oral Biology 61 (2016) 144–148
could be traced back to four generations in this family. Nine family members are involved in this study, and seven of them could be diagnosed with oligodontia based on panoramic radiographs and photographs (Fig. 1b and c). The proband, a 12-year-old boy, together with his relatives, lacked most of their permanent molars. Most individuals also lacked maxillary second premolars and incisors. Moreover, some individuals lacked at least one of their canines, indicating that the pattern of missing teeth involved all classes of teeth. Some patients were reported to have cone-shaped teeth (Fig. 1d). IV4 and IV10 were reported to have normal primary dentition, the status of which was not available for other individuals. Given that several extractions were performed to accommodate prostheses, we could not ascertain the exact number or types of missing II1 and II3 teeth. III2 and II2 were not available for clinical examination; their phenotypes were obtained based on the interview of available family members. The members had no other ectodermal abnormalities in the sweat glands, hair or nails, and their clinical ﬁndings did not suggest the presence of any syndrome or systemic disorder. 3.2. Mutation analysis A heterozygous c.2T > G mutation was detected in exon 1 of PAX9 in all available affected individuals (Fig. 2a). This c.2T > G transversion changed the ATG initiation codon to AGG. In addition, sequencing of MSX1 and other exons of PAX9 did not show mutation in any of the affected individuals. The mutation was not detected in any of the unaffected relatives and the 100 controls (data not shown). The single base change of a T to a G destroys a BsrDI restriction site. Thus, we performed restriction-enzyme analysis. The DNA of all available family members was ampliﬁed with the
Fig. 2. (a) Chromatogram showing the c.2T > G mutation, which changes the initiation codon from ATG to AGG. WT indicates wild type. (b) Restriction-enzyme analysis of PCR-ampliﬁed DNA fragments around the initiation codon of PAX9. The mutation destroyed the cleavage site for BsrDI. The PCR fragments (517 bp) were digested with BsrDI. The mutant allele was not cleaved at this site (longer fragment), whereas the WT was cleaved normally (shorter fragment).
speciﬁc primers. The PCR products were digested with BsrDI and analyzed using 3% agarose gel electrophoresis. The presence of the mutation in the affected family member was conﬁrmed (Fig. 2b). 4. Discussion Several genes, including MSX1, PAX9, AXIN2, WNT10AandEDA, have been involved in non-syndromic tooth agenesis. MSX1 mutations cause second premolar agenesis, whereas PAX9 mutations cause molar agenesis (Kim, Simmer, Lin, & Hu, 2006). AXIN2-associated tooth agenesis is often accompanied with predisposing to colorectal cancer (Lammi et al., 2004). In addition, EDA mutations are more likely to cause anterior teeth agenesis (Han et al., 2008), whereas WNT10A aberrations usually cause autosomal recessive or isolated tooth agenesis (van den Boogaard et al., 2012). In this four-generation oligodontia family, the pattern of missing teeth involved all classes of teeth. Therefore, we selected MSX1 and PAX9 prior to the other candidate genes. To date, about thirty PAX9 mutations have been found (Stockton et al., 2000; van den Boogaard et al., 2012; Klein et al., 2005; Wang et al., 2009a; Wang, Chan, Makovey, Simmer, & Hu, 2012; Das et al., 2003; Mitsui et al., 2014; Lammi et al., 2003; Liang, Song, Li, & Bian, 2012; Jumlongras et al., 2004; Zhao et al., 2007; Mostowska, Kobielak, Biedziak, & Trzeciak, 2003; Bergendal, Klar, StecksenBlicks, Norderyd, & Dahl, 2011; Kapadia, Frazier-Bowers, Ogawa, & D’Souza, 2006; Tallon-Walton et al., 2007; Nieminen et al., 2001; Hansen, Kreiborg, Jarlov, Niebuhr, & Eiberg, 2007; Zhu, Yang, Zhang, Ge, & Zheng, 2012; Mostowska, Zadurska, Rakowska, Lianeri, & Jagodzinski, 2013a; Suda, Ogawa, Kojima, Saito, & Moriyama, 2011; Mostowska, Biedziak, & Trzeciak, 2006; FrazierBowers et al., 2002; Mostowska et al., 2013b; Das et al., 2002) (Table 1). And we differentiated all these mutations into two subsets (Zhong et al., 2009). The ﬁrst subset, in-frame mutations, includes missense mutations and in-frame insertions or deletions. The second subset, truncating mutations, includes nonsense mutations, out-of-frame insertions or deletions, initiation codon mutations and deletion of the entire gene. The phenotypes caused by truncating mutations are more severe than those caused by inframe mutations (Fig. 3): ﬁrst, the average tooth missing number in truncating mutations subset is a little larger than that in in-frame mutations subset; second, primary tooth agenesis only appeared in truncating mutations subset. Combined with other functional studies of PAX9 (Liang et al., 2012; Suda et al., 2011; Wang et al., 2009b), we suggest that truncating mutations have a more serious effect on PAX9 protein than in-frame mutations and PAX9 mutations cause tooth agenesis as a dose-sensitive manner. In the present study, we report a second initiation codon mutation of PAX9, c.2T > G, which is associated with nonsyndromic oligodontia. The mutation is present in all affected family members, whereas all unaffected family members and 100 controls are negative for this mutation. The ﬁrst initiation codon mutation of PAX9, c.1A > G (Klein et al., 2005), was also associated with non-syndromic oligodontia in the family of Chinese decent. Some similarities of the phenotypes were found in the two families. Tooth agenesis was severe and affected all types of teeth, particularly the permanent molars, which correlated well with reports on PAX9 mutation families. Pegshaped teeth were found in patients of both families. By contrast, the primary teeth in this family were not affected. This difference may be attributed to phenotypic heterogeneity and to the availability of only two patients for primary agenesis evaluation. In this study, the mutation was found to change a methionine codon at position 1 to an arginine. We hypothesised that such change might lead to a low level of initiation of mRNA translation from the mutated allele. The scanning mechanism for the translation initiation proposes that the ribosome normally scans
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Table 1 Two subsets of PAX9 mutations. Type of mutation
Paired domain affected
Total missing number
Number of patients References
c.16G > A c.43T > A c.62T > C c.73–75 del ATC c.76C > T c.80T > C c.83G > C c.86T > C c.128G > A; c.129C > A c.139C > T c.146C > T c.151G > A c.152G > C c.238A > G c.259A > T c.271A > G c.428A > G
p.G6R p.F15I p.L21P p.I25del p.R26W p.L27P p.R28P p.I29T p.S43K
Yes Yes Yes Yes Yes Yes Yes Yes Yes
3 14 91 6 45 19 13 20 13
1 2 9 1 4 2 1 2 2
Wang et al. (2009a) Wang et al. (2012) Das et al. (2003) Mitsui et al. (2014) Lammi et al. (2003) Liang et al. (2012) Jumlongras et al. (2004) Liang et al. (2012) Wang et al. (2009a)
p.R47W p.S49L p.G51S p.G51A p.T80A p.I87F p.K91E p.Y143C
Yes Yes Yes Yes Yes Yes Yes No
16 10 9 7 8 23 44 11
1 1 1 1 1 2 6 1
Zhao et al. (2007) Mitsui et al. (2014) Mostowska et al. (2003) Bergendal et al. (2011) Bergendal et al. (2011) Kapadia et al. (2006) Das et al. (2003) Bergendal et al. (2011)
Out-of-frame mutation Nonsense c.175C > T Nonsense c.340A > T Nonsense c.433C > T Nonsense c.480C > G Frameshift c.59del C Frameshift c.175_183delGATACAAins288bp Frameshift c.218_219insG
p.R59X p.K114X p.Q145X p.Y160X p.P20fsX65 p.A59fsX119 p.G73fsX243
Yes Yes No No Yes Yes Yes
35a 32a 38a 20 31a 19 122
4 3 5 1 2 2 12
Frameshift Frameshift Frameshift Frameshift Frameshift Initial Initial Entire gene del
p.R77fsX4 p.G108fsX209 p.S119fsX199 p.I207fsX5 p.V265fsX25 p.0? p.0? –
Yes Yes Yes No No Yes Yes Yes
13 44 9 70 78 30a 57 39a
1 4 1 5 9 2 5 2
Tallon-Walton et al. (2007) Nieminen et al. (2001) Hansen et al. (2007) Zhu et al. (2012) Mostowska et al. (2013a) Das et al. (2003) Stockton et al. (2000) Zhu et al. (2012) Bergendal et al. (2011) Suda et al. (2011) Mostowska et al. (2013b) Mostowska et al. (2006) Frazier-Bowers et al. (2002) Klein et al. (2005) This study Das et al. (2002)
In-frame mutation Missense Missense Missense Del Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense
c.230_242del13bp c.321_322insG c.353_354insTGCC c.619_621delATCin-s24bp c.792_793insC c.1A > G c.2T > G –
*All mutations are annotated using HGVS (Human Genome Variation Society) system. a Primary tooth affected.
Fig. 3. Summary of teeth missing number caused by PAX9 mutations. The dashed line divided the mutations into two subsets (Left: In-frame mutations; Right: Out-of-frame mutations). The bars indicate the average missing number with error bars representing maximum and minimum missing number. The grey bars indicate primary tooth affected.
in a 50 –30 direction, searching for the ﬁrst AUG that initiates translation. In addition, the sequences surrounding AUG are also important for efﬁcient initiation to proceed. The sequence near the ﬁrst initiation codon in the PAX9 (GGAGCAaugG) matches well with the known recognition sequence with a purine at position 3 and G at position +4 (Kozak, 1991, 1999). Therefore, the ﬁrst mutated AUG may not be recognised by most of the ribosomes. The next in-frame unmutated AUG is located at codon 188; however, the surrounding sequence (GTGGCCaugC) does not completely
match with the known recognition sequence. Even if PAX9 could be translated to be similar with the other in-frame or out-frame AUG, the paired domain of PAX9 could be affected. In conclusion, we suggest that the heterozygous c.2T > G mutation in PAX9 causes severe or complete inhibition of PAX9 translation at one allele and acts by inactivating one protein copy, leading to haplo-insufﬁciency. And this mutation caused oligodontia in the Chinese family.
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Conﬂict of interest The authors declare that they have no conﬂict of interest. Funding National Natural Science Foundation of China (30930099, 81120108010). Ethical approval Institutional Review Board of hospital and School of Stomatology, Wuhan University. The relevant Judgement’s reference number 54. Acknowledgements We are extremely grateful to all the family members for their enthusiastic participation in this study. The assistance of doctors in the Departments of Clinical Laboratory and Radiology is much appreciated. This study was supported by grants from the National Natural Science Foundation of China (81470727, 81120108010) and China Scholarship Council. References Bergendal, B., Klar, J., Stecksen-Blicks, C., Norderyd, J., & Dahl, N. (2011). Isolated oligodontia associated with mutations in EDARADD, AXIN2, MSX1, and PAX9 genes. American Journal of Medical Genetics Part A, 155A(7), 1616–1622. Chi, N., & Epstein, J. A. (2002). Getting your Pax straight: Pax proteins in development and disease. Trends in Genetics, 18(1), 41–47. Das, P., Stockton, D. W., Bauer, C., Shaffer, L. G., D’Souza, R. N., Wright, T., et al. (2002). Haploinsufﬁciency of PAX9 is associated with autosomal dominant hypodontia. Human Genetics, 110(4), 371–376. Das, P., Hai, M., Elcock, C., Leal, S. M., Brown, D. T., Brook, A. H., et al. (2003). Novel missense mutations and a 288-bp exonic insertion in PAX9 in families with autosomal dominant hypodontia. American Journal of Medical Genetics Part A, 118A(1), 35–42. De, C., Oster, P. J., Marks, L. A., Martens, L. C., & Huysseune, A. (2009). Dental agenesis: genetic and clinical perspectives. Journal of Oral Pathology and Medicine, 38(1), 1–17. van den Boogaard, M. J., Creton, M., Bronkhorst, Y., van der Hout, A., Hennekam, E., Lindhout, D., et al. (2012). Mutations in WNT10A are present in more than half of isolated hypodontia cases. Journal of Medical Genetics, 49(5), 327–331. Frazier-Bowers, S. A., Guo, D. C., Cavender, A., Xue, L., Evans, B., King, T., et al. (2002). A novel mutation in human PAX9 causes molar oligodontia. Journal of Dental Research, 81(2), 129–133. Han, D., Gong, Y., Wu, H., Zhang, X., Yan, M., Wang, X., et al. (2008). Novel EDA mutation resulting in X-linked non-syndromic hypodontia and the pattern of EDA-associated isolated tooth agenesis. European Journal of Medical Genetics, 51 (6), 536–546. Hansen, L., Kreiborg, S., Jarlov, H., Niebuhr, E., & Eiberg, H. (2007). A novel nonsense mutation in PAX9 is associated with marked variability in number of missing teeth. European Journal of Oral Sciences, 115(4), 330–333. Jumlongras, D., Lin, J. Y., Chapra, A., Seidman, C. E., Seidman, J. G., Maas, R. L., et al. (2004). A novel missense mutation in the paired domain of PAX9 causes nonsyndromic oligodontia. Human Genetics, 114(3), 242–249. Kapadia, H., Frazier-Bowers, S., Ogawa, T., & D’Souza, R. N. (2006). Molecular characterization of a novel PAX9 missense mutation causing posterior tooth agenesis. European Journal of Human Genetics, 14(4), 403–409. Kim, J. W., Simmer, J. P., Lin, B. P., & Hu, J. C. (2006). Novel MSX1 frameshift causes autosomal-dominant oligodontia. Journal of Dental Research, 85(3), 267–271. Klein, M. L., Nieminen, P., Lammi, L., Niebuhr, E., & Kreiborg, S. (2005). Novel mutation of the initiation codon of PAX9 causes oligodontia. Journal of Dental Research, 84(1), 43–47. Kozak, M. (1991). An analysis of vertebrate mRNA sequences: intimations of translational control. Journal of Cell Biology, 115(4), 887–903.
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