Microsoft Word - 57 2016.docx   Vol  4,  No  1  (2016)   ISSN  2167-­‐8677  (online)   DOI  10.5195/d3000.2016.57           http://dentistry3000.pitt.edu     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.     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  Gene+cs,  Istanbul  Medical  Faculty,  Istanbul  University,  Istanbul,  34093,  Turkey   2  Department  of  Medical  Gene2cs,  Koc  University,  School  of  Medicine  (KUSOM),  Istanbul,  34010,  Turkey   3  Department  of  Diagnos/c  and  Biomedical  Sciences  and  Center  for  Craniofacial  Research,  University  of  Texas  Health  Science  Center  at  Houston  School  of  Den:stry,   Houston,  TX,  77054,  USA   4  Pediatric  Research  Center,  University  of  Texas  Health  Science  Center  at  Houston  McGovern  Medical  School,  Houston,  TX,  77030,  USA   Abstract   Tooth   agenesis   is   the  most   common   developmental   abnormality   of   the   human   den''on   characterized  by   the   congenital   absence  of   one  or  more  permanent   teeth.  Oligodon7a   is   the  term  used  to  describe  severe  tooth  agenesis,  where  six  or  more  permanent  teeth  are   missing.  The  WNT  gene  pathway  regulates  mul)ple  developmental  processes  during  cranio-­‐ facial  and  tooth  development,  and  varia2ons  in  WNT  pathway  genes  have  been  reported  in   individuals  with   tooth   agenesis.   In   this   study,  we   inves0gated   the   associa0on  of   37   SNPs   in/nearby   12   WNT   pathway   genes   (WNT3,   WNT3A,   WNT5A,   WNT8A,   WNT9B,   WNT10A,   WNT11,   AXIN1,   AXIN2,   APC,   LRP5,   LRP6)  with   oligodon,a   in   22  mul,plex   families.   Geno-­‐ types  were  generated  using  Taqman  chemistry  in  a  real-­‐!me  polymerase  chain  reac!on  as-­‐ say.    Family-­‐based  associa*on  tests  were  performed  using  FBAT.  Pairwise-­‐haplotype  analy-­‐ sis  was  also  performed.  Bonferroni  correc2on  was  used  to  adjust  for  mul+ple  tes+ng  and  P-­‐ values  ≤  0.001  were   considered   sta0s0cally   significant.  We   found  nominal   associa"on   for   AXIN2   rs7591,   located   in   the   3’   UTR,   with   oligodon:a   (P=0.04).   In   silico   analysis   of   SNP   func%on  predicted  a  binding   site   for  miR-­‐205  with  poten-al   impact  on  AXIN2  expression.   Although  modest,  these  results  con1nue  to  support  a  role  for  AXIN2  in  the  e'ology  of  famil-­‐ ial  tooth  agenesis.       Cita%on:   Dinckan,   et   al.   (2016).   Associa'on   of   AXIN2   gene   polymorphisms  with   nonsyndromic   oligodon'a   in   Turkish   families.   Den$stry   3000.   1:a001  doi:10.5195/d3000.2016.57   Received:  July  19,  2016   Accepted:    August  1,  2016   Published:    October  3,  2016   Copyright:  ©2016  Dinckan,  et  al.  This  is  an  open   access   ar!cle   licensed   under   a   Crea!ve   Com-­‐ mons   A"ribu"on   Work   4.0   United   States   Li-­‐ cense.   Email:  ariadne.m.letra@uth.tmc.edu   Introduction   Tooth  agenesis  is  the  most   common  craniofacial  congenital   malformation  in  humans  [1].  Up  to   20%  of  the  general  population  has   agenesis  of  at  least  one  third  mo-­‐ lar.  Agenesis  of  other  permanent   teeth,  excluding  third  molars,   ranges  from  ~1.6  to  9%,  depend-­‐ ing  on  the  population  studied,  and   in  70-­‐80%  of  these  cases  one  or   two  teeth  are  missing  [2,3].  Tooth   agenesis  can  be  identified  as  hy-­‐ podontia  (up  to  5  teeth  missing,   excluding  third  molars),  or  oligo-­‐ dontia  (lack  of  more  than  6  teeth   missing,  excluding  third  molars)   [1],  in  sporadic  cases  or  segregat-­‐ ing  in  families.  In  most  of  the  fa-­‐ milial  cases,  inheritance  is  auto-­‐ somal  dominant,  however,  auto-­‐ somal  recessive  and  X-­‐linked  in-­‐ heritance  have  also  been  de-­‐ scribed  [4].    Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   2   The  etiology  of   tooth  agenesis  is  com-­‐ plex  and  poorly  under-­‐ stood  [4].  Studies  in   mice  have  allowed  the   identification  of  genes   directly  or  indirectly  in-­‐ volved  in  the  regulation   of  tooth  development,   and  have  been  funda-­‐ mental  to  the  under-­‐ standing  of  the  basic   genetic  principles  of   tooth  development  and   its  defects  [5].  Never-­‐ theless,  very  few  hu-­‐ man  mutations  have   been  described  in  sev-­‐ eral  genes  known  to   arrest  tooth  develop-­‐ ment  in  mice.  This  may   reflect  basic  differences   in  agenesis  mechanisms   because  of  species-­‐ specific  characteristics,   such  as  tooth  type  (only   incisors  and  molars  in   mice)  and  number  of   dentitions  (one  denti-­‐ tion  in  mice  vs.  two   dentitions  -­‐  deciduous   and  permanent  -­‐  in  hu-­‐ mans)  [6].  Mutations  in   PAX9  (Paired  Box  9),   MSX1  (Msh  Homeobox   1),  and  EDA  (Ectodys-­‐ plasin  A),  have  been   shown  to  cause  arrest   of  tooth  development   in  mice  and  humans,   and  these  genes  have   been  extensively  stud-­‐ ied  [1-­‐10].     Table&1.!Details!of!study!families.! Family&No.& Individual&No.& Phenotype& Relationship& No.&of& Missing& Teeth& Type&of&Missing& Teeth& Inheritance&& 1! 111! Oligodontia! Proband! 11! Incisors,!premolars! Complex! ! 112! Unaffected! Mother! 0! !! !! ! 113! Unaffected! Father! 0! !! !! ! 114! Unaffected! Brother! 0! !! !! ! 115! Unaffected! Brother! 0! !! !! ! 116! Oligodontia! Aunt! 10! !! !! ! 117! Oligodontia! Uncle! 7! !! !! ! 118! Oligodontia! Uncle! 7! !! !! 2! 211! Oligodontia! Proband! 28! Incisors,!canines,! premolars,!molars! Complex! ! 212! Unaffected! Mother! 0! !! !! ! 213! Oligodontia! Father! 8! !! !! ! 214! Unaffected! Brother! 0! !! !! ! 215! Hypodontia! Sister! 4! !! !! ! 216! Hypodontia! Brother! 4! !! !! ! 217! Unaffected! Brother! 0! !! !! ! 218! Unaffected! Sister! 0! !! !! ! 219! Hypodontia! Brother! 5! !! !! ! 2110! Oligodontia! Uncle! 7! !! !! ! 2111! Hypodontia! Aunt! 5! !! !! ! 2112! Oligodontia! Cousin! 21! !! !! ! 2113! Oligodontia! Cousin! 23! !! !! ! 2114! Oligodontia! Cousin! 19! !! !! ! 2115! Oligodontia! Cousin! Unk! !! !! 3! 311! Oligodontia! Proband! 20! Incisors,!premolars,! molars! AR! ! 312! Unaffected! Mother! 0! !! !! ! 313! Oligodontia! Father! Unk! !! !! ! 314! Hypodontia! Sister! Unk! !! !! ! 315! Unaffected! Sister! 0! !! !! ! 316! Hypodontia! Brother! Unk! !! !! ! 317! Oligodontia! Sister! 17! !! !! ! 318! Hypodontia! Cousin! Unk! !! !! 4! 411! Oligodontia! Proband! 8! Lower!incisors,! molars! AD! ! 412! Unaffected! Mother! 0! !! !! ! 413! Unaffected! Father! 0! !! !! ! 414! Hypodontia! Brother! 4! !! !! ! 415! Hypodontia! Aunt! Unk! !! !! ! 416! Unaffected! Cousin! 0! !! !! ! 417! Hypodontia! Grandmother! Unk! !! !! Unk,!unknown!missing!tooth!types! Inheritance!patterns!(suspected):!AD,!autosomal!domimant;!AR,!autosomal!recessive.! !  Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   3   The  WNT  gene  pathway   regulates  multiple  developmental   processes  during  craniofacial  and   tooth  development  [11-­‐12].  Previ-­‐ ous  evidence  showing  the  expres-­‐ sion  of  several  Wnt  genes  during   mouse  tooth  de-­‐ velopment   strongly  impli-­‐ cated  this  gene   family  in  the  eti-­‐ ology  of  tooth   agenesis  [11-­‐14].   In  recent  years,   mutations  in   WNT  pathway   genes,  namely   AXIN2  (Axis  Inhi-­‐ bition  Protein  2),   WNT10A  (Wing-­‐ less-­‐Type  MMTV   Integration  Site   Family,  Member   10A),  LRP6  (low-­‐ density  lipopro-­‐ tein  receptor-­‐ related  protein   6),  and  recently   WNT10B  (Wing-­‐ less-­‐Type  MMTV   Integration  Site   Family,  Member   10B),  have  also   been  shown  to   cause  tooth   agenesis  in  hu-­‐ mans  [8,  10,  15-­‐ 18].  Additional   studies  have  also   shown  the  asso-­‐ ciation  of  com-­‐ mon  single  nu-­‐ cleotide  poly-­‐ morphisms  in  a   few  WNT  path-­‐ way  genes  with   the  milder  form   of  tooth  agene-­‐ sis,  hypodontia   [19-­‐22].  Howev-­‐ er,  the  associa-­‐ tion  of  WNT  pathway  gene  poly-­‐ morphisms  in  oligodontia  pheno-­‐ Table&1&(Continued).!Details!of!study!families.! Family&No.& Individual&No.& Phenotype& Relationship& No.&of& Missing& Teeth& Type&of&Missing&Teeth& Inheritance&& 5! 511! Oligodontia! Proband! 13! Incisors,!premolars! AR! ! 512! Unaffected! Mother! 0! !! !! ! 513! Unaffected! Father! 0! !! !! ! 514! Unaffected! Sister! 0! !! !! 6! 611! Oligodontia! Proband! 23! Incisors,!premolars,!molars! Complex! ! 612! Hypodontia! Mother! 2! Upper!lateral!incisors! !! ! 613! Unaffected! Father! 0! !! !! ! 614! Unaffected! Brother! 0! !! !! 7! 711! Oligodontia! Proband! 17! Incisors,!premolars,!molars! AR! ! 712! Unaffected! Mother! 0! !! !! ! 713! Unaffected! Father! 0! !! !! ! 714! Unaffected! Sister! 0! !! !! 8! 811! Oligodontia! Proband! 9! Incisors,!premolars! AD! ! 812! Unaffected! Mother! 0! !! !! ! 813! Hypodontia! Father! 2! Upper!lateral!incisors! !! 9! 911! Oligodontia! Proband! 15! Incisors,!premolars,!molars! Complex! ! 912! Unaffected! Mother! 0! !! !! ! 913! Hypodontia! Father! Unk! !! !! ! 914! Hypodontia! Brother! Unk! !! !! ! 915! Hypodontia! Brother! Unk! !! !! ! 916! Unaffected! Sister! 0! !! !! ! 917! Unaffected! Brother! 0! !! !! ! 918! Unaffected! Sister! 0! !! !! 10! 1011! Oligodontia! Proband! 9! Incisors,!premolars,!molars! X1linked! ! 1012! Unaffected! Mother! 0! !! !! ! 1013! Hypodontia! Father! 3! !! !! ! 1014! Hypodontia! Sister! 4! !! !! ! 1015! Unaffected! Brother! 0! !! !! 11! 1111! Oligodontia! Proband! 8! Incisors,!canines,!premolars! AR! ! 1112! Unaffected! Mother! 0! !! !! ! 1113! Unaffected! Father! 0! !! !! ! 1114! Unaffected! Sister! 0! !! !! 12! 1211! Oligodontia! Proband! 12! Incisors!and!premolars! AD! ! 1212! Oligodontia! Mother! Unk! !! !! ! 1213! Unaffected! Father! 0! !! !! ! 1214! Unaffected! Brother! 0! !! !! ! 1215! Oligodontia! Uncle! 12! !! !! 13! 1311! Oligodontia! Proband! 12! Incisors,!canines,!molars! AR! ! 1312! Unaffected! Mother! 0! !! !! ! 1313! Oligodontia! Father! 7! !! !! ! 1314! Oligodontia! Brother! 8! !! !! Unk,!unknown!missing!tooth!types! Inheritance!patterns!(suspected):!AD,!autosomal!domimant;!AR,!autosomal!recessive.!  Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   4   types  is  still  unclear.  Therefore,  in   this  study,  we  investigated  the  as-­‐ sociation  of  single  nucleotide  pol-­‐ ymorphisms  in  12  WNT  pathway   genes  with  oligodontia  in  multi-­‐ plex  families  from  Turkey.     Material  and   methods   Sample  Popula-­‐ tion   This   study  was  ap-­‐ proved  by  the   Istanbul  Univer-­‐ sity  Institutional   Ethical  Review   Board  and  the   Committee  for   Protection  of   Human  Subjects   at  the  University   of  Texas  Health   Science  Center   at  Houston.  Clin-­‐ ical  and  demo-­‐ graphic  infor-­‐ mation  and  DNA   samples  from   peripheral  blood   were  obtained   from  the  CRANI-­‐ RARE2  Project,   an  European  Un-­‐ ion-­‐funded  col-­‐ loborative  ERA-­‐ net  Project  on   craniofacial  mal-­‐ formations  run   at  the  Istanbul   University,  Is-­‐ tanbul  Medical   Faculty,  Medical   Genetics  De-­‐ partment.  All   registry  partici-­‐ pants  had  signed   an  informed   consent  form  agreeing  to  partici-­‐ pate  in  genetic  studies  and  pro-­‐ Table&1&(Continued).!Details!of!study!families.! Family&No.& Individual&No.& Phenotype& Relationship& No.&of& Missing& Teeth& Type&of&Missing&Teeth& Inheritance&& 14! 1421! Oligodontia! Proband! 12! Incisors!and!premolars! Complex! ! 1422! Hypodontia! Mother! 4! !! !! ! 1423! Unaffected! Father! 0! !! !! ! 1424! Hypodontia! Brother! 4! !! !! ! 1425! Hypodontia! Cousin! 2! Upper!lateral!incisors! !! ! 1426! Unaffected! Uncle! 0! !! !! ! 1427! Hypodontia! Uncle’s!wife! 2! Upper!lateral!incisors! !! 15! 1521! Oligodontia! Proband! 7! Premolars,!molars! Complex! ! 1522! Unaffected! Mother! 0! !! !! ! 1523! Hypodontia! Father! 2! Upper!lateral!incisors! !! ! 1524! Unaffected! Sister! 0! !! !! ! 1525! Hypodontia! Uncle! 2! Upper!lateral!incisors! !! ! 1526! Hypodontia! Uncle! 1! Upper!lateral!incisor! !! ! 1527! Hypodontia! Uncle! 1! Upper!lateral!incisor! !! 16! 1621! Oligodontia! Proband! 10! Incisors,!canines,!premolars,! molars! AD! ! 1622! Unaffected! Mother! 0! !! !! ! 1623! Unaffected! Father! 0! !! !! ! 1624! Unaffected! Brother! 0! !! !! 17! 1721! Oligodontia! Proband! 6! Upper!lateral!incisors,! premolars! AR! ! 1722! Unaffected! Mother! 0! !! !! ! 1723! Hypodontia! Father! 2! Upper!lateral!incisors! !! ! 1724! Unaffected! Brother! 0! !! !! ! 1725! Hypodontia! Sister! 4! Premolars! !! 18! 1821! Oligodontia! Proband! 18! Incisors,!premolars,!molars! AD! ! 1822! Unaffected! Mother! 0! !! !! ! 1823! Unaffected! Father! 0! !! !! ! 1824! Oligodontia! Sister! 28! Incisors,!canines,!premolars,! molars! !! 19! 1921! Oligodontia! Proband! 10! Incisors!and!premolars! Complex! ! 1922! Hypodontia! Mother! 2! Upper!lateral!incisors! !! ! 1923! Unaffected! Father! 0! !! !! 20! 2021! Oligodontia! Proband! 13! !! AD! ! 2022! Unaffected! Mother! 0! !! !! 21! 2121! Oligodontia! Proband! 28! Incisors,!canines,!premolars,! molars! Complex! ! 2122! Unaffected! Mother! 0! !! !! ! 2123! Unaffected! Father! 0! !! !! 22! 2221! Oligodontia! Proband! 16! Incisors,!canines,!premolars,! molars! Complex! ! 2222! Hypodontia! Mother! 2! Upper!lateral!incisors! !! ! 2223! Unaffected! Father! 0! !! !! Unk,!unknown!missing!tooth!types! Inheritance!patterns!(suspected):!AD,!autosomal!domimant;!AR,!autosomal!recessive.! !  Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   5   vided  a  blood  samples  as  a  source   of  genomic  DNA.     Probands  were  selected   from  the  registry  based  on  their   radiographic  records  showing  con-­‐ genital  tooth  agenesis  and  were   invited  to  participate.  Individuals   were  considered  to  have  oligodon-­‐ tia  when  six  or  more  permanent   teeth  were  missing  in  the  oral  cav-­‐ ity,  excluding  third  molars.  Fami-­‐ lies  were  ascertained  through   probands,  and  additional  relatives   were  recruited.  All  probands  and   available  family  members  were   further  examined  clinically  to  con-­‐ firm  the  tooth  agenesis  status  and   exclude  syndromic  cases.  In  a  few   cases,  history  of  tooth  agenesis   was  available  by  self-­‐report  from   family  members,  or  from  state-­‐ ments  by  their  dental  provider.   Our  sample  population  consisted   of  22  multiplex  oligodontia  fami-­‐ lies  (117  total  individuals,  67  af-­‐ fected,  50  unaffected),  in  which   tooth  agenesis  segregated  in  both   autosomal  dominant  and  autoso-­‐ mal  recessive  forms,  and  an  aver-­‐ age  of  2-­‐18  teeth  were  missing  in   each  affected  individual.  Details  of   studied  families  are  presented  in   Table  1.     Selection  of  Candidate  Genes  and   Single  Nucleotide  Polymorphisms   We  selected  37  single  nu-­‐ cleotide  polymorphisms  (SNPs)   in/nearby  APC,  AXIN1,  AXIN2,   LRP5,  LRP6,  WNT3,  WNT3A,   WNT5A,  WNT8A,  WNT9B,   WNT10A,  and  WNT11  genes  for   genotyping  in  our  families.  SNPs   were  selected  based  on  their  loca-­‐ tions  within  the  genes,  on  their   likelihood  to  have  functional  con-­‐ sequences  (i.e.,  located  in  the   promoters,  exons,  or  near  ex-­‐ on/intron  boundaries),  or  if  con-­‐ sidered  tag-­‐SNPs  for  the  linkage   disequilibrium  blocks  surrounding   the  respective  genes  [23].  We   used  information  available  at  the   NCBI  dbSNP   (http://www.ncbi.nlm.gov/SNP/)   and  HapMap  Project   (http://www.hapmap.org)  data-­‐ bases  to  select  polymorphisms.   Details  of  studied  genes  and  pol-­‐ ymorphisms  are  presented  in  Ta-­‐ ble  2.     Genotyping   Genomic  DNA  was  extract-­‐ ed  from  blood  using  established   protocols.  Genotypes  were  gener-­‐ ated  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-­‐ tions,  we  included  a  non-­‐template   control  (water  instead  of  DNA)  as   negative  control  and  a  DNA  sam-­‐ ple  of  known  genotype  as  positive   control  in  each  reaction.   Association  analyses   Family-­‐based  association   tests  were  performed  using  FBAT   software  version  1.06  [25].  We   used  Bonferroni  correction  to  ad-­‐ just  for  multiple  testing  (0.05/37)   and  P-­‐values  ≤  0.001  were  consid-­‐ ered  significant.   In  silico  prediction  of  SNP  function   We  performed  in  silico   analysis  of  SNP  function  to  predict   the  effects  of  the  associated             AXIN2  rs7591  SNPs  function  using   MiRBase  software  [26].       Results   Association  analyses   We  found  evidence  of  al-­‐ tered  allelic  transmission  for             AXIN2  rs7591,  in  the  gene  3’  UTR,   with  oligodontia  (P=0.04).        Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   6   In  silico  prediction  of  SNP  function   In  silico  analysis  of  the  3’   UTR  SNP  in  AXIN2  rs7591  predict-­‐ ed  a  binding  site  for  the  microRNA   miR-­‐205-­‐5p  with  potential  effects   on  gene  expression  (Figure  1).     Discussion   In  this  study,  we  investi-­‐ gated  the  association  of  12  WNT   pathway  genes  (APC,  AXIN1,             AXIN2,  LRP5,  LRP6,  WNT3,   WNT3A,  WNT5A,  WNT8A,  WNT9B,   WNT10A,  and  WNT11)  with  non-­‐ syndromic  oligodontia  in  22  well-­‐ characterized  Turkish  Caucasian   multiplex  families.  Although  mod-­‐ est,  our  results  suggest  a  positive   association  between  AXIN2  and   oligodontia,  and  corroborate  the   results  of  previous  studies  [19-­‐21].   To  our  knowledge,  this  is  the  most   comprehensive  analysis  of  the  as-­‐ sociation  of  WNT/β-­‐catenin  path-­‐ way  genes  with  tooth  agenesis,   particularly  oligodontia.     Over  the  years,  many  sig-­‐ naling  pathways  have  shown  to  be   involved  in  the  organogenesis  and   embryogenesis  of  teeth  [3,  5,  7,   12,  27].  Individuals  with  oligodon-­‐ tia  constitute  approximately  1%  of   all  individuals  with  hypodontia,   and  both  conditions  can  be  found   in  the  same  family,  indicating  vari-­‐ able  expression  of  shared  genetic   factors  [2,  7].  The  importance  of   the  WNT/β-­‐catenin  signaling   pathway  during  tooth  develop-­‐ ment  has  been  reported  by  sever-­‐ al  authors  [11-­‐14].  Many  studies   showed  that  Wnt  pathway  plays  a   critical  role  in  tooth  morphogene-­‐ sis  and  several  Wnt  genes  are  ex-­‐ pressed  in  craniofacial  and  dental   tissues  [11-­‐14].  Wnt  pathway  acti-­‐ vation  has  roles  at  the  lamina-­‐ early  bud  stage  and  also  important   for  molar  cusps  development  [13].   During  tooth  development,  AXIN2   is  expressed  in  the  dental  mesen-­‐ chyme,  the  odontoblasts  and  the   enamel  knot,  and  it  is  needed  as  a   negative  regulator  of  WNT-­‐ signaling  at  specific  stages  [12,   13].   Additional  common  vari-­‐ ants  in  AXIN2  have  also  been  asso-­‐ ciated  with  increased  susceptibil-­‐ ity  to  hypodontia  in  Eastern  Euro-­‐ peans  [19].  However,  the  SNP  as-­‐ sociated  in  the  present  study,   rs7591,  located  in  the  3’  UTR,  has   not  been  previously  reported  in   association  with  tooth  agenesis   and  warrants  additional  confirma-­‐ tory  studies.  Previously,  this  same   SNP  was  reported  in  association   with  oral  clefts  in  families  with  in-­‐ creased  susceptibility  to  colon   cancer  [28,  29].  Interestingly,  in   silico  analyses  predicted  that  this   SNP  harbors  a  binding  site  for  the   miR-­‐205-­‐5p,  with  a  potential  regu-­‐ latory  role  in  gene  expression.  Re-­‐ cent  evidence  has  shown  that  a   number  of  cellular  functions,  in-­‐ cluding  development,  differentia-­‐ tion,  growth,  metabolism,  anabo-­‐ lism,  and  carcinogenesis  can  be   affected  by  miRNA  functions  [30].   Although  the  role  of  miR-­‐205-­‐5p  in   craniofacial  development  is  yet   unknown,  it  has  been  suggested  to   play  a  role  in  cancer  development   and  Parkinson’s  disease  [31].  Fur-­‐ ther,  the  level  of  miR-­‐205-­‐5p  ex-­‐ pression  was  found  to  be  down-­‐ regulated  in  various  cancer  cells,   including  breast,  oral,  prostate   cancer  cells,  and  melanoma  [32].   Additional  studies  on  miR-­‐205-­‐5p   and  its  effect  on  the  regulation  of   AXIN2  might  elucidate  the  role  of   these  molecules  in  tooth  agenesis.   In  addition  to  a  critical  role   in  embryonic  development,  the   WNT/β-­‐catenin  signaling  pathway   is  also  associated  with  tumorigen-­‐ esis  events  [9].  Mutations  in  AXIN2   were  found  segregating  with  auto-­‐ somal  dominant  tooth  agenesis   and  colorectal  cancer  in  a  large   multiplex  family,  suggesting  that  a   same  gene  may  be  involved  in   congenital  anomalies  and  cancer   later  in  life  [15].  AXIN2  mutations   were  also  detected  segregating  in   autosomal  dominant  pattern  with   oligodontia  and  other  findings  in-­‐ cluding  colonic  polyposis,  gastric   polyps,  a  mild  ectodermal  dyspla-­‐ sia  phenotype  with  sparse  hair  and   eyebrows,  and  early  onset  colo-­‐ rectal  and  breast  cancers  [33].  The   AXIN2  gene  encodes  the  axis  inhi-­‐ bition  protein  2  that  regulates  the   stability  of  beta-­‐catenin  and  early   organ  differentiation  and  devel-­‐ opment  and  plays  a  key  role  in   many  basic  cell  functions,  like  cell   homeostasis  [9].  Since  the  report   by  Lammi  et  al.  [15],  numerous   human  genetic  studies  have  fo-­‐ cused  on  identifying  variants  in   AXIN2  in  association  with  tooth   agenesis  or  other  birth  defects   such  as  cleft  lip/palate  [8-­‐10,  19-­‐ 21,  28],  due  to  the  previously  sug-­‐ gested  hypothesis  that  cancer-­‐ related  genes  may  have  a  role  in   tooth  agenesis.  Notwithstanding,   despite  the  positive  associations   reported,  additional  studies  are   needed  to  determine  if  potential   correlations  exist  between  AXIN2,   birth  defects  and  cancer.    Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   7   Recently,  WNT10A  has   been  suggested  as  a  major   candidate  gene  for  tooth   agenesis,  and  oligodontia  in   particular,  and  rare  variants   in  this  gene  have  been  found   in  individuals  with  tooth   agenesis  from  multiple  popu-­‐ lations  [33].  Interestingly,  in   the  present  study,  we  did  not   identify  any  association  be-­‐ tween  common  variants  in   WNT10A  and  oligodontia  in   our  Turkish  families.     In  summary,  although   modest,  our  results  continue   to  support  a  role  for  AXIN2   and  the  WNT/β-­‐catenin  sig-­‐ naling  pathway  in  human   tooth  agenesis.  Discrepancies   between  the  present  and   previous  studies  may  be  due   to  heterogeneity  of  the  con-­‐ dition  across  distinct  popula-­‐ tions,  and/or  the  common   variant-­‐common  disease  ap-­‐ proach  used  in  this  associa-­‐ tion  study,  when  rare  variants   in  relevant  genes  could  be   the  cause  of  the  phenotype.   Future  studies  should  focus   on  the  identification  of  po-­‐ tentially  functional  variants  in   AXIN2  and  additional  WNT   pathway  genes  to  further  es-­‐ tablish  a  biological  role  of  this   pathway  in  tooth  agenesis   phenotypes.   Acknowledgements   We  would  like  to  thank  all   of  the  participating  patients  and   their  families  for  their  support  in   this  research.  This  work  partially   supported  by  Research  Fund  of   Istanbul  University;  Project  num-­‐ ber  48398,  and  from  the  Scientific   and  Technological  Research  Coun-­‐ cil  of  Turkey  (TUBITAK),  grant   number  112S398  [CRANIRARE-­‐2].     References   1.   Genetic  basis  of  tooth   agenesis.  Nieminen  P.  J  Exp  Zool  B   Mol  Dev  Evol.  2009  Jun   15;312B(4):320-­‐42.   PMID:19219933.     2.   A  meta-­‐analysis  of  the   prevalence  of  dental  agenesis  of   permanent  teeth.  Polder  BJ,  Van't   Hof  MA,  Van  der  Linden  FP,  Kui-­‐ jpers-­‐Jagtman  AM.  Community   Table&2.&Details(of&SNPs(genotyped(in(this(study(and(association(results.( SNP& Locus& Gene& Function& Alleles*& MAF& Informative&families**& P>value***& rs861674( Chr.5:(112064475( APC$ Intron( A/T( 0.454( 17( 0.67( rs2431238( Chr.5:(112124369( APC$ Intron( C/T( 0.343( 14( 1( rs454886( Chr.5:(112146117( APC$ Intron( C/T( 0.348( 18( 0.65( rs351771( Chr.5:(112164561( APC$ Synonymous( C/T( 0.009( 1( IIII( rs448475( Chr.5:(112181379( APC$ 3’(UTR( C/G( 0.482( 18( 0.67( 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$ rs7224837( Chr.17:(63528123( AXIN2$ Intron( A/G( 0.491( 23( 0.91( rs11867417( Chr.17:(63537898( AXIN2$ Intron( C/T( 0.201( 13( 0.84( rs3923086( Chr.17:(63549488( AXIN2$ Intron( G/T( 0.438( 18( 0.43( rs2240307( Chr.17:(63554307( AXIN2$ Intron( A/G( 0.179( 7( 0.28( rs740026( Chr.17:(63561681( AXIN2$ Intergenic( A/G( 0.232( 14( 0.86( 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( rs312788( Chr.11:(68122295( LRP5$ Intron( G/T( 0.384( 20( 0.57( 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( rs4477532( Chr.12:(12279361( LRP6$ Intron( A/G( 0.037( 4( IIII( rs7294695( Chr.12:(12323618( LRP6$ Intron( C/G( 0.393( 20( 0.65( rs121908120( Chr.2:(218890289( WNT10A$ Missense( A/T( 1( 0( IIII( rs3806557( Chr.2:(218879152( WNT10A$ Missense( A/G( 0.009( 1( IIII( rs199980023( Chr.2:(218882196( WNT10A$ Missense( C/T( 0.5( 22( 0.81( rs116998555( Chr.2:(218890118( WNT10A$ Missense( C/T( 0.009( 1( IIII( 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( rs3806557( Chr.2:(219743874( WNT10A$ Intron( G/A( 0.221( 12( 0.33( rs1533767( Chr.11:(75905800( WNT11$ Intron( A/G( 0.009( 1( IIII( rs199498( Chr.17:(44865603( WNT3$ Intergenic( C/T( 1( 0( IIII( rs111769( Chr.17:(44871987( WNT3$ Intergenic( C/T( 0.356( 19( 0.31( rs9890413( Chr.17:(44901449( WNT3$ Intergenic( A/G( 0.356( 14( 0.6( 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( rs752107( Chr.1:(228247351( WNT3A$ 3’(UTR( C/T( 0.256( 13( 0.23( rs1745420( Chr.1:(228251732( WNT3A$ Intergenic( C/G( 0.393( 22( 0.15( rs566926( Chr.3:(55520778( WNT5A$ Intron( A/C( 0.348( 18( 0.41( rs2040862( Chr.5:(137419989( WNT8A$ Intron( C/T( 0.446( 23( 0.9( rs2165846( Chr.17:(44941366( WNT9B$ Intron( A/G( 0.5( 18( 0.64( *(Ancestral(allele(listed(first,(NCBI(dbSNP(build(147( **(SNPs(with(less(than(5(informative(families(were(excluded(from(further(analysis( ***FBAT,(P<0.001(denotes(statistical(significance.(PIvalues(<0.05(are(shown(in(italic(font. !  Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   8   Dent  Oral  Epidemiol.  2004   Jun;32(3):217-­‐26.  PMID:   15151692.   3.   Tooth  agenesis:  from  mo-­‐ lecular  genetics  to  molecular  den-­‐ tistry.  Matalova  E,  Fleischmannova   J,  Sharpe  PT,  Tucker  AS.  J  Dent   Res.  2008  Jul;87(7):617-­‐23.  PMID:   18573979.   4.   Anomalies  associated  with   hypodontia  of  the  permanent  lat-­‐ eral  incisor  and  second  premolar.   Symons  AL,  Stritzel  F,  Stamation  J.   J  Clin  Pediatr  Dent.  1993  Win-­‐ ter;17(2):109-­‐11.  PMID:  8466838.   5.   Tooth  morphogenesis  and   cell  differentiation.  Thesleff  I,   Nieminen  P.  Curr  Opin  Cell  Biol.   1996  Dec;8(6):844-­‐50.  PMID:   8939666.   6.   Two  genes  for  missing   teeth.  Thesleff  I.  Nat  Genet.  1996   Aug;13(4):379-­‐80.  PMID:  8696323.   7.   The  genetic  basis  of  inher-­‐ ited  anomalies  of  the  teeth.  Part  1:   clinical  and  molecular  aspects  of   non-­‐syndromic  dental  disorders.   Bailleul-­‐Forestier  I,  Molla  M,  Ver-­‐ loes  A,  Berdal  A.  Eur  J  Med  Genet.   2008  Jul-­‐Aug;51(4):273-­‐91.  PMID:   18499550.   8.   Isolated  oligodontia  asso-­‐ ciated  with  mutations  in   EDARADD,  AXIN2,  MSX1,  and  PAX9   genes.  Bergendal  B,  Klar  J,   Stecksén-­‐Blicks  C,  Norderyd  J,  Dahl   N.  Am  J  Med  Genet  A.  2011   Jul;155A(7):1616-­‐22.  PMID:   21626677.   9.   Exclusion  of  coding  region   mutations  in  MSX1,  PAX9  and  AX-­‐ IN2  in  eight  patients  with  severe   oligodontia  phenotype.  Gerits  A,   Nieminen  P,  DE  Muynck  S,  Carels   C.  Orthod  Craniofac  Res.  2006   Aug;9(3):129-­‐36.  PMID:  16918677.   10.   Mutational  analysis  of  AX-­‐ IN2,  MSX1,  and  PAX9  in  two  Mexi-­‐ can  oligodontia  families.  Mu  YD,   Xu  Z,  Contreras  CI,  McDaniel  JS,   Donly  KJ,  Chen  S.  Genet  Mol  Res.   2013  Oct  10;12(4):4446-­‐58.  PMID:   24222224.   11.   Expression  of  Wnt  signaling   pathway  genes  during  tooth  de-­‐ velopment.  Sarkar  L,  Sharpe  PT.   Mech  Dev.  1999  Jul;85(1-­‐2):197-­‐ 200.  PMID:  10415363.   12.   Expression  patterns  of   WNT/β-­‐CATENIN  signaling  mole-­‐ cules  during  human  tooth  devel-­‐ opment.  Wang  B,  Li  H,  Liu  Y,  Lin  X,   Lin  Y,  Wang  Y,  Hu  X,  Zhang  Y.  J  Mol   Histol.  2014  Oct;45(5):487-­‐96.   PMID:  24647585.     13.   Wnt/beta-­‐catenin  signaling   directs  multiple  stages  of  tooth   morphogenesis.  Liu  F,  Chu  EY,   Watt  B,  Zhang  Y,  Gallant  NM,  Andl   T,  Yang  SH,  Lu  MM,  Piccolo  S,   Schmidt-­‐Ullrich  R,  Taketo  MM,   Morrisey  EE,  Atit  R,  Dlugosz  AA,   Millar  SE.  Dev  Biol.  2008  Jan   1;313(1):210-­‐24.  PMID:  18022614.   14.   Wnt5a  plays  a  crucial  role   in  determining  tooth  size  during   murine  tooth  development.  Cai  J,   Mutoh  N,  Shin  JO,  Tani-­‐Ishii  N,   Ohshima  H,  Cho  SW,  Jung  HS.  Cell   Tissue  Res.  2011  Sep;345(3):367-­‐ 77.  PMID:  21879290.   15.   Mutations  in  AXIN2  cause   familial  tooth  agenesis  and  predis-­‐ pose  to  colorectal  cancer.  Lammi   L,  Arte  S,  Somer  M,  Jarvinen  H,   Lahermo  P,  Thesleff  I,  Pirinen  S,   Nieminen  P.  Am  J  Hum  Genet.   2004  May;74(5):1043-­‐50.  PMID:   15042511.   16.   Mutations  in  WNT10A  are   present  in  more  than  half  of  iso-­‐ lated  hypodontia  cases.  van  den   Boogaard  MJ,  Créton  M,  Bronk-­‐ horst  Y,  van  der  Hout  A,   Hennekam  E,  Lindhout  D,  Cune  M,   Ploos  van  Amstel  HK.  J  Med  Genet.   2012  May;49(5):327-­‐31.  PMID:   22581971.   17.   Loss-­‐of-­‐Function  Mutations   in  the  WNT  Co-­‐receptor  LRP6   Cause  Autosomal-­‐Dominant  Oli-­‐ godontia.  Massink  MP,  Créton  MA,   Spanevello  F,  Fennis  WM,  Cune   MS,  Savelberg  SM,  Nijman  IJ,  Mau-­‐ rice  MM,  van  den  Boogaard  MJ,   van  Haaften  G.  Am  J  Hum  Genet.   2015  Oct  1;97(4):621-­‐6.  PMID:   26387593.   18.   Mutations  in  WNT10B  Are   Identified  in  Individuals  with  Oli-­‐ godontia.  Yu  P,  Yang  W,  Han  D,   Wang  X,  Guo  S,  Li  J,  Li  F,  Zhang  X,   Wong  SW,  Bai  B,  Liu  Y,  Du  J,  Sun   ZS,  Shi  S,  Feng  H,  Cai  T.  Am  J  Hum   Genet.  2016  Jul  7;99(1):195-­‐201.   PMID:  27321946.   19.   Axis  inhibition  protein  2   (AXIN2)  polymorphisms  may  be  a   risk  factor  for  selective  tooth   agenesis.  Mostowska  A,  Biedziak   B,  Jagodzinski  PP.  J  Hum  Genet.   2006;51(3):262-­‐6.  PMID:   16432638.   20.   AXIN2  and  CDH1  polymor-­‐ phisms,  tooth  agenesis,  and  oral   clefts.  Letra  A,  Menezes  R,  Gran-­‐ jeiro  JM,  Vieira  AR.  Birth  Defects   Res  A  Clin  Mol  Teratol.  2009    Associa&on  of  AXIN2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  Turkish  families   Vol  4,  No  1  (2016)        DOI  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   9   Feb;85(2):169-­‐73.  PMID:   18683894.   21.   Axis  inhibition  protein  2   (AXIN2)  polymorphisms  and  tooth   agenesis.  Callahan  N,  Modesto  A,   Meira  R,  Seymen  F,  Patir  A,  Vieira   AR.  Arch  Oral  Biol.  2009   Jan;54(1):45-­‐9.  PMID:  18790474.   22.   Nucleotide  variants  of   genes  encoding  components  of   the  Wnt  signaling  pathway  and  the   risk  of  non-­‐syndromic  tooth  agen-­‐ esis.  Mostowska  A,  Biedziak  B,  Za-­‐ durska  M,  Dunin-­‐Wilczynska  I,  Li-­‐ aneri  M,  Jagodzinski  PP.  Clin   Genet.  2013  Nov;84(5):429-­‐40.   PMID:  23167694.   23.   Selecting  a  maximally  in-­‐ formative  set  of  single-­‐nucleotide   polymorphisms  for  association   analyses  using  linkage  disequilibri-­‐ um.  Carlson  CS,  Eberle  MA,  Rieder   MJ,  Yi  Q,  Kruglyak  L,  Nickerson  DA.   Am  J  Hum  Genet.  2004   Jan;74(1):106-­‐20.  PMID:   14681826.   24.   High-­‐throughput  genotyp-­‐ ing  with  single  nucleotide  poly-­‐ morphisms.  Ranade  K,  Chang  MS,   Ting  CT,  Pei  D,  Hsiao  CF,  Olivier  M,   Pesich  R,  Hebert  J,  Chen  YD,  Dzau   VJ,  Curb  D,  Olshen  R,  Risch  N,  Cox   DR,  Botstein  D.  Genome  Res.  2001   Jul;11(7):1262-­‐8.  PMID:  11435409.   25.   Family-­‐based  tests  of  asso-­‐ ciation  in  the  presence  of  linkage.   Lake  SL,  Blacker  D,  Laird  NM.  Am  J   Hum  Genet.  2000  Dec;67(6):1515-­‐ 25.  PMID:  11058432.   26.    miRBase:  the  microRNA   sequence  database.  Griffiths-­‐Jones   S.  Methods  Mol  Biol.   2006;342:129-­‐38.  PMID:   16957372.   27.   Genetic  basis  for  tooth   malformations:  from  mice  to  men   and  back  again.  Mitsiadis  TA,   Luder  HU.  Clin  Genet.  2011   Oct;80(4):319-­‐29.  PMID:   21819395.   28.   AXIS  inhibition  protein  2,   orofacial  clefts  and  a  family  history   of  cancer.  Menezes  R,  Marazita   ML,  Goldstein  McHenry  T,  Cooper   ME,  Bardi  K,  Brandon  C,  Letra  A,   Martin  RA,  Vieira  AR.  J  Am  Dent   Assoc.  2009  Jan;140(1):80-­‐4.   PMID:19119171.     29.   The  axis  inhibition  protein   2  polymorphisms  and  non-­‐ syndromic  orofacial  clefts  suscep-­‐ tibility  in  a  Chinese  Han  popula-­‐ tion.  Han  Y,  Zhou  L,  Ma  L,  Li  D,  Xu   M,  Yuan  H,  Ma  J,  Zhang  W,  Jiang   H,  Wu  Y,  Wang  L,  Pan  Y.  J  Oral   Pathol  Med.  2014  Aug;43(7):554-­‐ 60.  PMID:  24484320.   30.   MicroRNAs  in  Human  Dis-­‐ eases:  From  Cancer  to  Cardiovas-­‐ cular  Disease.  Ha  TY.  Immune   Netw.  2011  Jun;11(3):135-­‐54.   PMID:  21860607.   31.   A  program  of  microRNAs   controls  osteogenic  lineage  pro-­‐ gression  by  targeting  transcription   factor  Runx2.  Zhang  Y,  Xie  RL,  Cro-­‐ ce  CM,  Stein  JL,  Lian  JB,  van   Wijnen  AJ,  Stein  GS.  Proc  Natl   Acad  Sci  U  S  A.  2011  Jun   14;108(24):9863-­‐8.  PMID:   21628588.   32.   MicroRNA-­‐205  suppresses   the  oral  carcinoma  oncogenic  ac-­‐ tivity  via  down-­‐regulation  of  Axin-­‐ 2  in  KB  human  oral  cancer  cell.   Kim  JS,  Park  SY,  Lee  SA,  Park  MG,   Yu  SK,  Lee  MH,  Park  MR,  Kim  SG,   Oh  JS,  Lee  SY,  Kim  CS,  Kim  HJ,   Chun  HS,  Kim  JS,  Moon  SM,  Kim   DK.  Mol  Cell  Biochem.  2014   Feb;387(1-­‐2):71-­‐9.  PMID:   24166197.   33.   AXIN2-­‐associated  autoso-­‐ mal  dominant  ectodermal  dyspla-­‐ sia  and  neoplastic  syndrome.   Marvin  ML,  Mazzoni  SM,  Herron   CM,  Edwards  S,  Gruber  SB,  Petty   EM.  Am  J  Med  Genet  A.  2011   Apr;155A(4):898-­‐902.  PMID:   21416598.   34.   WNT10A  variants  are  asso-­‐ ciated  with  non-­‐syndromic  tooth   agenesis  in  the  general  popula-­‐ tion.  Song  S,  Zhao  R,  He  H,  Zhang  J,   Feng  H,  Lin  L.  Hum  Genet.  2014   Jan;133(1):117-­‐24.  PMID:   24043634.