Association of AXIN2 Gene Polymorphisms with Nonsyndromic Oligodontia in Turkish Families Nuriye Dinckan1,3, Zehra Oya Uyguner1, Hulya Kayserili2, Ariadne Letra3,4

Vol 4, No 1 (2016) ISSN 2167-8677 (online) DOI 10.5195/d3000.2016.57

Association of AXIN2 gene polymorphisms with nonsyndromic oligodontia in Turkish families Nuriye Dinckan1,3, Zehra Oya Uyguner1, Hulya Kayserili2, Ariadne Letra3,4

1Department of Medical Genecs, Istanbul Medical Faculty, Istanbul University, Istanbul, 34093, Turkey 2 Department of Medical Genecs, Koc University, School of Medicine (KUSOM), Istanbul, 34010, Turkey 3 Department of Diagnosc and Biomedical Sciences and Center for Craniofacial Research, University of Texas Health Science Center at Houston School of Denstry, Houston, TX, 77054, USA 4 Pediatric Research Center, University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX, 77030, USA

Abstract

Citaon: Dinckan, et al. (2016). Associaon of Tooth agenesis is the most common developmental abnormality of the human denon AXIN2 gene polymorphisms with nonsyndromic characterized by the congenital absence of one or more permanent teeth. Oligodona is oligodona in Turkish families. Denstry 3000. the term used to describe severe tooth agenesis, where six or more permanent teeth are 1:a001 doi:10.5195/d3000.2016.57 missing. The WNT gene pathway regulates mulple developmental processes during cranio- Received: July 19, 2016 facial and tooth development, and variaons in WNT pathway genes have been reported in Accepted: August 1, 2016 individuals with tooth agenesis. In this study, we invesgated the associaon of 37 SNPs Published: October 3, 2016 in/nearby 12 WNT pathway genes (WNT3, WNT3A, WNT5A, WNT8A, WNT9B, WNT10A, Copyright: ©2016 Dinckan, et al. This is an open access arcle licensed under a Creave Com- WNT11, AXIN1, AXIN2, APC, LRP5, LRP6) with oligodona in 22 mulplex families. Geno- mons Aribuon Work 4.0 United States Li- types were generated using Taqman chemistry in a real-me polymerase chain reacon as- cense. say. Family-based associaon tests were performed using FBAT. Pairwise-haplotype analy- Email: [email protected] sis was also performed. Bonferroni correcon was used to adjust for mulple tesng and P- values ≤ 0.001 were considered stascally signiﬁcant. We found nominal associaon for AXIN2 rs7591, located in the 3’ UTR, with oligodona (P=0.04). In silico analysis of SNP funcon predicted a binding site for miR-205 with potenal impact on AXIN2 expression. Although modest, these results connue to support a role for AXIN2 in the eology of familial tooth agenesis.

Introduction ranges from ~1.6 to 9%, depend- [1], in sporadic cases or segregating on the population studied, and ing in families. In most of the fa- Tooth agenesis is the most in 70-80% of these cases one or milial cases, inheritance is auto- common craniofacial congenital two teeth are missing [2,3]. Tooth somal dominant, however, auto- malformation in humans [1]. Up to agenesis can be identified as hy- somal recessive and X-linked in- 20% of the general population has podontia (up to 5 teeth missing, heritance have also been de- agenesis of at least one third mo- excluding third molars), or oligo- scribed [4]. lar. Agenesis of other permanent dontia (lack of more than 6 teeth teeth, excluding third molars, missing, excluding third molars)

New articles in this journal are licensed under a Creative Commons Attribution 4.0 United States License. This journal is published by the University Library System, University of Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponored by the University of Pittsburgh Press. http://dentistry3000.pitt.edu Associaon of AXIN2 gene polymorphisms with nonsyndromic oligodona in Turkish families Vol 4, No 1 (2016) DOI 10.5195/d3000.2016.57 Table&1.!Details!of!study!families.! The etiology of Family&No.& Individual&No.& Phenotype& Relationship& No.&of& Type&of&Missing& Inheritance&& tooth agenesis is com- Missing& Teeth& Teeth& plex and poorly under- 1! 111! Oligodontia! Proband! 11! Incisors,!premolars! Complex! stood [4]. Studies in 112! Unaffected! Mother! 0! !! !! mice have allowed the ! 113! Unaffected! Father! 0! !! !! identification of genes ! 114! Unaffected! Brother! 0! !! !! directly or indirectly in- ! 115! Unaffected! Brother! 0! !! !! volved in the regulation ! 116! Oligodontia! Aunt! 10! !! !! of tooth development, ! 117! Oligodontia! Uncle! 7! !! !! and have been funda- ! 118! Oligodontia! Uncle! 7! !! !! mental to the under- ! Incisors,!canines,! 2! 211! Oligodontia! Proband! 28! premolars,!molars! Complex! standing of the basic

! 212! Unaffected! Mother! 0! !! !! genetic principles of

! 213! Oligodontia! Father! 8! !! !! tooth development and

! 214! Unaffected! Brother! 0! !! !! its defects [5]. Never-

! 215! Hypodontia! Sister! 4! !! !! theless, very few hu- ! 216! Hypodontia! Brother! 4! !! !! man mutations have

! 217! Unaffected! Brother! 0! !! !! been described in sev- ! 218! Unaffected! Sister! 0! !! !! eral genes known to

! 219! Hypodontia! Brother! 5! !! !! arrest tooth develop- ! 2110! Oligodontia! Uncle! 7! !! !! ment in mice. This may ! 2111! Hypodontia! Aunt! 5! !! !! reflect basic differences ! 2112! Oligodontia! Cousin! 21! !! !! in agenesis mechanisms ! 2113! Oligodontia! Cousin! 23! !! !! because of species- 2114! Oligodontia! Cousin! 19! !! !! specific characteristics, ! 2115! Oligodontia! Cousin! Unk! !! !! such as tooth type (only ! Incisors,!premolars,! 3! 311! Oligodontia! Proband! 20! molars! AR! incisors and molars in

312! Unaffected! Mother! 0! !! !! mice) and number of ! 313! Oligodontia! Father! Unk! !! !! dentitions (one denti- ! 314! Hypodontia! Sister! Unk! !! !! tion in mice vs. two ! 315! Unaffected! Sister! 0! !! !! dentitions - deciduous ! 316! Hypodontia! Brother! Unk! !! !! and permanent - in hu- ! 317! Oligodontia! Sister! 17! !! !! mans) [6]. Mutations in ! 318! Hypodontia! Cousin! Unk! !! !! PAX9 (Paired Box 9), ! Lower!incisors,! 4! 411! Oligodontia! Proband! 8! molars! AD! MSX1 (Msh Homeobox

412! Unaffected! Mother! 0! !! !! 1), and EDA (Ectodys- ! 413! Unaffected! Father! 0! !! !! plasin A), have been ! 414! Hypodontia! Brother! 4! !! !! shown to cause arrest ! 415! Hypodontia! Aunt! Unk! !! !! of tooth development ! 416! Unaffected! Cousin! 0! !! !! in mice and humans, ! 417! Hypodontia! Grandmother! Unk! !! !! and these genes have Unk,!unknown!missing!tooth!types! ! been extensively stud- Inheritance!patterns!(suspected):!AD,!autosomal!domimant;!AR,!autosomal!recessive.! ! ied [1-10].

http://dentistry3000.pitt.edu 2 Associaon of AXIN2 gene polymorphisms with nonsyndromic oligodona in Turkish families Vol 4, No 1 (2016) DOI 10.5195/d3000.2016.57

Table&1&(Continued).!Details!of!study!families.! mouse tooth de- Family&No.& Individual&No.& Phenotype& Relationship& No.&of& Type&of&Missing&Teeth& Inheritance&& Missing& velopment Teeth& strongly impli- 5! 511! Oligodontia! Proband! 13! Incisors,!premolars! AR! cated this gene 512! Unaffected! Mother! 0! !! !! ! family in the eti- 513! Unaffected! Father! 0! !! !! ! ology of tooth 514! Unaffected! Sister! 0! !! !! ! agenesis [11-14]. 6! 611! Oligodontia! Proband! 23! Incisors,!premolars,!molars! Complex! In recent years, 612! Hypodontia! Mother! 2! Upper!lateral!incisors! !! ! mutations in 613! Unaffected! Father! 0! !! !! ! WNT pathway 614! Unaffected! Brother! 0! !! !! ! genes, namely 7! 711! Oligodontia! Proband! 17! Incisors,!premolars,!molars! AR! AXIN2 (Axis Inhi- 712! Unaffected! Mother! 0! !! !! ! bition Protein 2), 713! Unaffected! Father! 0! !! !! ! WNT10A (Wing- 714! Unaffected! Sister! 0! !! !! ! less-Type MMTV 8! 811! Oligodontia! Proband! 9! Incisors,!premolars! AD! Integration Site 812! Unaffected! Mother! 0! !! !! ! Family, Member 813! Hypodontia! Father! 2! Upper!lateral!incisors! !! ! 10A), LRP6 (low- 9! 911! Oligodontia! Proband! 15! Incisors,!premolars,!molars! Complex! density lipopro- ! 912! Unaffected! Mother! 0! !! !! tein receptor- ! 913! Hypodontia! Father! Unk! !! !! related protein ! 914! Hypodontia! Brother! Unk! !! !! 6), and recently ! 915! Hypodontia! Brother! Unk! !! !! WNT10B (Wing- ! 916! Unaffected! Sister! 0! !! !! less-Type MMTV ! 917! Unaffected! Brother! 0! !! !! Integration Site ! 918! Unaffected! Sister! 0! !! !! Family, Member 10! 1011! Oligodontia! Proband! 9! Incisors,!premolars,!molars! X1linked! ! 1012! Unaffected! Mother! 0! !! !! 10B), have also been shown to ! 1013! Hypodontia! Father! 3! !! !! ! 1014! Hypodontia! Sister! 4! !! !! cause tooth

! 1015! Unaffected! Brother! 0! !! !! agenesis in hu-

11! 1111! Oligodontia! Proband! 8! Incisors,!canines,!premolars! AR! mans [8, 10, 15-

! 1112! Unaffected! Mother! 0! !! !! 18]. Additional

! 1113! Unaffected! Father! 0! !! !! studies have also

! 1114! Unaffected! Sister! 0! !! !! shown the asso-

12! 1211! Oligodontia! Proband! 12! Incisors!and!premolars! AD! ciation of com-

! 1212! Oligodontia! Mother! Unk! !! !! mon single nu- ! 1213! Unaffected! Father! 0! !! !! cleotide poly-

! 1214! Unaffected! Brother! 0! !! !! morphisms in a ! 1215! Oligodontia! Uncle! 12! !! !! few WNT path-

13! 1311! Oligodontia! Proband! 12! Incisors,!canines,!molars! AR! way genes with 1312! Unaffected! Mother! 0! !! !! the milder form ! 1313! Oligodontia! Father! 7! !! !! of tooth agene- ! 1314! Oligodontia! Brother! 8! !! !! sis, hypodontia Unk,!unknown!missing!tooth!types! ! Inheritance!patterns!(suspected):!AD,!autosomal!domimant;!AR,!autosomal!recessive.! [19-22]. Howev- The WNT gene pathway tooth development [11-12]. Previ- er, the associa- regulates multiple developmental ous evidence showing the expres- tion of WNT pathway gene poly- processes during craniofacial and sion of several Wnt genes during morphisms in oligodontia pheno-

http://dentistry3000.pitt.edu 3 Associaon of AXIN2 gene polymorphisms with nonsyndromic oligodona in Turkish families Vol 4, No 1 (2016) DOI 10.5195/d3000.2016.57 Table&1&(Continued).!Details!of!study!families.! Family&No.& Individual&No.& Phenotype& Relationship& No.&of& Type&of&Missing&Teeth& Inheritance&& Missing& Teeth& Material and 14! 1421! Oligodontia! Proband! 12! Incisors!and!premolars! Complex! methods ! 1422! Hypodontia! Mother! 4! !! !! ! 1423! Unaffected! Father! 0! !! !! Sample Popula- ! 1424! Hypodontia! Brother! 4! !! !! tion ! 1425! Hypodontia! Cousin! 2! Upper!lateral!incisors! !!

! 1426! Unaffected! Uncle! 0! !! !! This ! 1427! Hypodontia! Uncle’s!wife! 2! Upper!lateral!incisors! !! study was ap-

15! 1521! Oligodontia! Proband! 7! Premolars,!molars! Complex! proved by the ! 1522! Unaffected! Mother! 0! !! !! Istanbul Univer-

! 1523! Hypodontia! Father! 2! Upper!lateral!incisors! !! sity Institutional ! 1524! Unaffected! Sister! 0! !! !! Ethical Review

! 1525! Hypodontia! Uncle! 2! Upper!lateral!incisors! !! Board and the ! 1526! Hypodontia! Uncle! 1! Upper!lateral!incisor! !! Committee for

! 1527! Hypodontia! Uncle! 1! Upper!lateral!incisor! !! Protection of Incisors,!canines,!premolars,! 16! 1621! Oligodontia! Proband! 10! molars! AD! Human Subjects ! 1622! Unaffected! Mother! 0! !! !! at the University

! 1623! Unaffected! Father! 0! !! !! of Texas Health

! 1624! Unaffected! Brother! 0! !! !! Science Center Upper!lateral!incisors,! at Houston. Clin- 17! 1721! Oligodontia! Proband! 6! premolars! AR! ical and demo- ! 1722! Unaffected! Mother! 0! !! !! ! 1723! Hypodontia! Father! 2! Upper!lateral!incisors! !! graphic information and DNA ! 1724! Unaffected! Brother! 0! !! !! ! 1725! Hypodontia! Sister! 4! Premolars! !! samples from peripheral blood 18! 1821! Oligodontia! Proband! 18! Incisors,!premolars,!molars! AD!

! 1822! Unaffected! Mother! 0! !! !! were obtained

! 1823! Unaffected! Father! 0! !! !! from the CRANI- Incisors,!canines,!premolars,! RARE2 Project, ! 1824! Oligodontia! Sister! 28! molars! !! an European Un- 19! 1921! Oligodontia! Proband! 10! Incisors!and!premolars! Complex! ion-funded col- ! 1922! Hypodontia! Mother! 2! Upper!lateral!incisors! !! loborative ERA- ! 1923! Unaffected! Father! 0! !! !! net Project on 20! 2021! Oligodontia! Proband! 13! !! AD! craniofacial mal- 2022! Unaffected! Mother! 0! !! !! ! Incisors,!canines,!premolars,! formations run 21! 2121! Oligodontia! Proband! 28! molars! Complex! at the Istanbul 2122! Unaffected! Mother! 0! !! !! ! University, Is- 2123! Unaffected! Father! 0! !! !! ! Incisors,!canines,!premolars,! tanbul Medical 22! 2221! Oligodontia! Proband! 16! molars! Complex! Faculty, Medical 2222! Hypodontia! Mother! 2! Upper!lateral!incisors! !! ! Genetics De- 2223! Unaffected! Father! 0! !! !! Unk,!unknown!missing!tooth!types! ! partment. All Inheritance!patterns!(suspected):!AD,!autosomal!domimant;!AR,!autosomal!recessive.! registry partici- ! pants had signed an informed types is still unclear. Therefore, in ymorphisms in 12 WNT pathway consent form agreeing to partici- this study, we investigated the as- genes with oligodontia in multi- pate in genetic studies and pro- sociation of single nucleotide pol- plex families from Turkey.

http://dentistry3000.pitt.edu 4 Associaon of AXIN2 gene polymorphisms with nonsyndromic oligodona in Turkish families Vol 4, No 1 (2016) DOI 10.5195/d3000.2016.57 Genotyping

Genomic DNA was extract- ed from blood using established protocols. Genotypes were generated using Taqman chemistry [24]. Reactions were carried out in 5-μL volumes in a ViiA7 Sequence De- tection System (Applied Biosys- tems, Foster City, CA). Assays and reagents were supplied by Applied Biosystems. The results were ana- lyzed using EDS software (Applied Biosystems). In order to ensure quality control of genotyping reac- vided a blood samples as a source studied families are presented in tions, we included a non-template of genomic DNA. Table 1. control (water instead of DNA) as Probands were selected Selection of Candidate Genes and negative control and a DNA sam- from the registry based on their Single Nucleotide Polymorphisms ple of known genotype as positive radiographic records showing con- control in each reaction. We selected 37 single nu- genital tooth agenesis and were Association analyses invited to participate. Individuals cleotide polymorphisms (SNPs) were considered to have oligodon- in/nearby APC, AXIN1, AXIN2, Family-based association tia when six or more permanent LRP5, LRP6, WNT3, WNT3A, tests were performed using FBAT teeth were missing in the oral cav- WNT5A, WNT8A, WNT9B, software version 1.06 [25]. We ity, excluding third molars. Fami- WNT10A, and WNT11 genes for used Bonferroni correction to ad- lies were ascertained through genotyping in our families. SNPs just for multiple testing (0.05/37) probands, and additional relatives were selected based on their loca- and P-values ≤ 0.001 were consid- were recruited. All probands and tions within the genes, on their ered significant. available family members were likelihood to have functional con- further examined clinically to con- sequences (i.e., located in the In silico prediction of SNP function firm the tooth agenesis status and promoters, exons, or near ex- We performed in silico exclude syndromic cases. In a few on/intron boundaries), or if con- analysis of SNP function to predict cases, history of tooth agenesis sidered tag-SNPs for the linkage the effects of the associated was available by self-report from disequilibrium blocks surrounding AXIN2 rs7591 SNPs function using family members, or from state- the respective genes [23]. We MiRBase software [26]. ments by their dental provider. used information available at the NCBI dbSNP Our sample population consisted Results of 22 multiplex oligodontia fami- (http://www.ncbi.nlm.gov/SNP/) lies (117 total individuals, 67 af- and HapMap Project Association analyses fected, 50 unaffected), in which (http://www.hapmap.org) data- We found evidence of al- tooth agenesis segregated in both bases to select polymorphisms. tered allelic transmission for autosomal dominant and autoso- Details of studied genes and pol- AXIN2 rs7591, in the gene 3’ UTR, mal recessive forms, and an aver- ymorphisms are presented in Ta- with oligodontia (P=0.04). age of 2-18 teeth were missing in ble 2. each affected individual. Details of

http://dentistry3000.pitt.edu 5 Associaon of AXIN2 gene polymorphisms with nonsyndromic oligodona in Turkish families Vol 4, No 1 (2016) DOI 10.5195/d3000.2016.57 In silico prediction of SNP function tissues [11-14]. Wnt pathway acti- Additional studies on miR-205-5p vation has roles at the lamina- and its effect on the regulation of In silico analysis of the 3’ early bud stage and also important AXIN2 might elucidate the role of UTR SNP in AXIN2 rs7591 predict- for molar cusps development [13]. these molecules in tooth agenesis. ed a binding site for the microRNA During tooth development, AXIN2 miR-205-5p with potential effects is expressed in the dental mesen- In addition to a critical role on gene expression (Figure 1). chyme, the odontoblasts and the in embryonic development, the WNT/β-catenin signaling pathway Discussion enamel knot, and it is needed as a negative regulator of WNT- is also associated with tumorigen- In this study, we investi- signaling at specific stages [12, esis events [9]. Mutations in AXIN2 gated the association of 12 WNT 13]. were found segregating with auto- pathway genes (APC, AXIN1, somal dominant tooth agenesis AXIN2, LRP5, LRP6, WNT3, Additional common vari- and colorectal cancer in a large WNT3A, WNT5A, WNT8A, WNT9B, ants in AXIN2 have also been asso- multiplex family, suggesting that a WNT10A, and WNT11) with non- ciated with increased susceptibil- same gene may be involved in syndromic oligodontia in 22 well- ity to hypodontia in Eastern Euro- congenital anomalies and cancer characterized Turkish Caucasian peans [19]. However, the SNP as- later in life [15]. AXIN2 mutations multiplex families. Although mod- sociated in the present study, were also detected segregating in est, our results suggest a positive rs7591, located in the 3’ UTR, has autosomal dominant pattern with association between AXIN2 and not been previously reported in oligodontia and other findings in- oligodontia, and corroborate the association with tooth agenesis cluding colonic polyposis, gastric results of previous studies [19-21]. and warrants additional confirma- polyps, a mild ectodermal dyspla- To our knowledge, this is the most tory studies. Previously, this same sia phenotype with sparse hair and comprehensive analysis of the as- SNP was reported in association eyebrows, and early onset colo- sociation of WNT/β-catenin path- with oral clefts in families with in- rectal and breast cancers [33]. The way genes with tooth agenesis, creased susceptibility to colon AXIN2 gene encodes the axis inhi- particularly oligodontia. cancer [28, 29]. Interestingly, in bition protein 2 that regulates the silico analyses predicted that this stability of beta-catenin and early Over the years, many sig- SNP harbors a binding site for the organ differentiation and devel- naling pathways have shown to be miR-205-5p, with a potential regu- opment and plays a key role in involved in the organogenesis and latory role in gene expression. Re- many basic cell functions, like cell embryogenesis of teeth [3, 5, 7, cent evidence has shown that a homeostasis [9]. Since the report 12, 27]. Individuals with oligodon- number of cellular functions, in- by Lammi et al. [15], numerous tia constitute approximately 1% of cluding development, differentia- human genetic studies have fo- all individuals with hypodontia, tion, growth, metabolism, anabo- cused on identifying variants in and both conditions can be found lism, and carcinogenesis can be AXIN2 in association with tooth in the same family, indicating vari- affected by miRNA functions [30]. agenesis or other birth defects able expression of shared genetic Although the role of miR-205-5p in such as cleft lip/palate [8-10, 19- factors [2, 7]. The importance of craniofacial development is yet 21, 28], due to the previously sug- the WNT/β-catenin signaling unknown, it has been suggested to gested hypothesis that cancer- pathway during tooth develop- play a role in cancer development related genes may have a role in ment has been reported by sever- and Parkinson’s disease [31]. Fur- tooth agenesis. Notwithstanding, al authors [11-14]. Many studies ther, the level of miR-205-5p ex- despite the positive associations showed that Wnt pathway plays a pression was found to be down- reported, additional studies are critical role in tooth morphogene- regulated in various cancer cells, needed to determine if potential sis and several Wnt genes are ex- including breast, oral, prostate correlations exist between AXIN2, pressed in craniofacial and dental cancer cells, and melanoma [32]. birth defects and cancer.

http://dentistry3000.pitt.edu 6 Associaon of AXIN2 gene polymorphisms with nonsyndromic oligodona in Turkish families Vol 4, No 1 (2016) DOI 10.5195/d3000.2016.57 Recently, WNT10A has been suggested as a major Table&2.&Details(of&SNPs(genotyped(in(this(study(and(association(results.( SNP& Locus& Gene& Function& Alleles*& MAF& Informative&families**& P>value***& candidate gene for tooth rs861674( Chr.5:(112064475( APC$ Intron( A/T( 0.454( 17( 0.67( agenesis, and oligodontia in rs2431238( Chr.5:(112124369( APC$ Intron( C/T( 0.343( 14( 1( particular, and rare variants rs454886( Chr.5:(112146117( APC$ Intron( C/T( 0.348( 18( 0.65( in this gene have been found rs351771( Chr.5:(112164561( APC$ Synonymous( C/T( 0.009( 1( IIII( in individuals with tooth rs448475( Chr.5:(112181379( APC$ 3’(UTR( C/G( 0.482( 18( 0.67( agenesis from multiple popu- rs2301522( Chr.16:(359953( AXIN1$ Intron( A/G( 0.4( 16( 0.5( rs7591( Chr.17:(63525082( AXIN2$ 3’(UTR( A/T( 0.143( 11( 0.04$ lations [33]. Interestingly, in rs7224837( Chr.17:(63528123( AXIN2$ Intron( A/G( 0.491( 23( 0.91( the present study, we did not rs11867417( Chr.17:(63537898( AXIN2$ Intron( C/T( 0.201( 13( 0.84( identify any association be- rs3923086( Chr.17:(63549488( AXIN2$ Intron( G/T( 0.438( 18( 0.43( tween common variants in rs2240307( Chr.17:(63554307( AXIN2$ Intron( A/G( 0.179( 7( 0.28( WNT10A and oligodontia in rs740026( Chr.17:(63561681( AXIN2$ Intergenic( A/G( 0.232( 14( 0.86( our Turkish families. rs634008( Chr.11:(68094741( LRP5$ Intron( C/T( 0.402( 20( 0.46( rs667126( Chr.11:(68177728( LRP5$ Intron( C/T( 0.267( 16( 0.29( In summary, although rs312788( Chr.11:(68122295( LRP5$ Intron( G/T( 0.384( 20( 0.57( modest, our results continue rs312014( Chr.11:(68084962( LRP5$ Intron( C/G( 0.438( 17( 0.77( rs10743980( Chr.12:(12412795( LRP6$ Intron( C/T( 0.438( 17( 0.45( to support a role for AXIN2 rs4477532( Chr.12:(12279361( LRP6$ Intron( A/G( 0.037( 4( IIII( and the WNT/β-catenin sig- rs7294695( Chr.12:(12323618( LRP6$ Intron( C/G( 0.393( 20( 0.65( naling pathway in human rs121908120( Chr.2:(218890289( WNT10A$ Missense( A/T( 1( 0( IIII( tooth agenesis. Discrepancies rs3806557( Chr.2:(218879152( WNT10A$ Missense( A/G( 0.009( 1( IIII( between the present and rs199980023( Chr.2:(218882196( WNT10A$ Missense( C/T( 0.5( 22( 0.81( previous studies may be due rs116998555( Chr.2:(218890118( WNT10A$ Missense( C/T( 0.009( 1( IIII( to heterogeneity of the con- rs4574113( Chr.2:(219762662( WNT10A$ Intron( A/T( 0.194( 11( 1( rs10177996( Chr.2:(219746561( WNT10A$ Intron( C/T( 0.223( 12( 0.41( dition across distinct popula- rs3806557( Chr.2:(219743874( WNT10A$ Intron( G/A( 0.221( 12( 0.33( tions, and/or the common rs1533767( Chr.11:(75905800( WNT11$ Intron( A/G( 0.009( 1( IIII( variant-common disease ap- rs199498( Chr.17:(44865603( WNT3$ Intergenic( C/T( 1( 0( IIII( proach used in this associa- rs111769( Chr.17:(44871987( WNT3$ Intergenic( C/T( 0.356( 19( 0.31( tion study, when rare variants rs9890413( Chr.17:(44901449( WNT3$ Intergenic( A/G( 0.356( 14( 0.6( in relevant genes could be rs708111( Chr.1:(228191365( WNT3A$ Intergenic( C/T( 0.143( 13( 0.5( rs3094912( Chr.1:(228209815( WNT3A$ Intron( A/T( 0.38( 18( 0.24( the cause of the phenotype. rs752107( Chr.1:(228247351( WNT3A$ 3’(UTR( C/T( 0.256( 13( 0.23( Future studies should focus rs1745420( Chr.1:(228251732( WNT3A$ Intergenic( C/G( 0.393( 22( 0.15( on the identification of po- rs566926( Chr.3:(55520778( WNT5A$ Intron( A/C( 0.348( 18( 0.41( tentially functional variants in rs2040862( Chr.5:(137419989( WNT8A$ Intron( C/T( 0.446( 23( 0.9( AXIN2 and additional WNT rs2165846( Chr.17:(44941366( WNT9B$ Intron( A/G( 0.5( 18( 0.64( *(Ancestral(allele(listed(first,(NCBI(dbSNP(build(147( pathway genes to further es- **(SNPs(with(less(than(5(informative(families(were(excluded(from(further(analysis( tablish a biological role of this ***FBAT,(P<0.001(denotes(statistical(significance.(PIvalues(<0.05(are(shown(in(italic(font. pathway in tooth agenesis ! phenotypes. ber 48398, and from the Scientific Mol Dev Evol. 2009 Jun Acknowledgements and Technological Research Coun- 15;312B(4):320-42. We would like to thank all cil of Turkey (TUBITAK), grant PMID:19219933. number 112S398 [CRANIRARE-2]. of the participating patients and 2. A meta-analysis of the their families for their support in References prevalence of dental agenesis of this research. This work partially permanent teeth. Polder BJ, Van't supported by Research Fund of 1. Genetic basis of tooth Hof MA, Van der Linden FP, Kui- Istanbul University; Project num- agenesis. Nieminen P. J Exp Zool B jpers-Jagtman AM. Community

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26. miRBase: the microRNA 32. MicroRNA-205 suppresses sequence database. Griffiths-Jones the oral carcinoma oncogenic ac- S. Methods Mol Biol. tivity via down-regulation of Axin- 2 in KB human oral cancer cell.

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