Global  DNA  methyla/on  and  its   role  in    preclamp/c  and  preterm   pregnancies:  Understanding  the   epigene/cs  

Girija  Wagh     Member  of  the  Steering  Commi@ee  Organiza/on   Gestosis     Professor  and  Head  Bhara/  Vidyapeeth  University   Medical  College  ,Pune  India    

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Conrad  Waddington   (1905-­‐1975)  is  oZen   credited  with  coining  the   term  “epigene/cs”  in   1942  as   “the  branch  of  biology   which  studies  the  causal   interac6ons  between   genes  and  their  products,   which  bring  the   phenotype  into  being”  
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HISTORY  :  THE  WISDOM  OF  THE  PAST    
•  Epigene/cs  appears  in  the  literature  as  far   back  as  the  mid  19th  century,  although  the   conceptual  origins  date  back  to  Aristotle   (384-­‐322  BC).   •   He  believed  in  epigenesis:  the  development  of   individual  organic  form  from  the  unformed  

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Contrary  to  the  belief    
•  Having  developed  from  miniscule  fully-­‐formed   bodies.     •  Even  today  the  extent  to  which  we  are   preprogrammed  versus  environmentally   shaped  awaits  universal  consensus.    

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•  The  field  of  epigene/cs  has  emerged  to  bridge   the  gap  between  nature  and  nurture.   •   “  Epigene)cs  has  always  been  all  the  weird   and  wonderful  things  that  can’t  be  explained   by  gene)cs.”  
Denise  Barlow  (Vienna,  Austria)  2006    

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Epigene/cs    
•  The  word  “epigene/c”  literally  means  “in   addi/on  to  changes  in  gene/c  sequence.”     •  The  term  has  evolved  to  include  any  process   that  alters  gene  ac/vity  without  changing  the   DNA  sequence,  and  leads  to  modifica/ons   that  can  be  transmi@ed  to  daughter  cells   (although  experiments  show  that  some  epigene6c   changes  can  be  reversed).    
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Ques/ons  today  ??  
•  Giving  to  understand  the  mul/factorial  e/ology   of  preclampsia  it  is  natural  to  wonder  whether   there  are  any  more  intrinsic  mechanisms  at   intracellular  level  which     •  1  influence  the  pathogenesis    
–  And  help  in  early  detec/on      

•  2  are  influences  as  the  result  of  the  disease  which   can    
–  Affect  the  mother  the  baby  and  the  future   genera/ons    

 

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Intracellular  programming    
•  Many  processes  are  being  elucidated     •  And  there  is  a  lot  of  evidence  today  to  state     •  That  epigene/cs    
–  Cause  as  well  as     –  Result     Due  to  the  pathological  processes  of  preclampsia   and  these  also  an  give  rise  to  las/ng  effect  to  carry   on  for  genera/ons  to  come    
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We  have  had  a  long-­‐standing  deal  with   biology    
Whatever  choices  we  make  during  our  lives  might   ruin  our  short-­‐term  memory  or  make  us  fat  or  hasten   death,  but  they  won't  change  our  genes  —  our  actual   DNA.     Which  meant  that  when  we  had  children    of  our  own,   the  gene/c  slate  would  be  wiped  clean.    
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•  DNA  is  just  a  tape  carrying  informa/on,  and  a   tape  is  no  good  without  a  player.  Epigene/cs   is  about  the  tape  player.”  
        Bryan  Turner  (Birmingham,  UK)  

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•  Bygren  and  other  scien/sts  have  now   amassed  historical  evidence  sugges/ng  that   powerful  environmental  condi/ons  (near   death  from  starva/on,  for  instance)  can   somehow  leave  an  imprint  on  the  gene/c   material  in  eggs  and  sperm.     •  These  gene/c  imprints  can  short-­‐circuit   evolu/on  and  pass  along  new  traits  in  a  single   genera/on.    
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Epigenome    
•  These  pa@erns  of  gene  expression  are   governed  by  the  cellular  material  —  the   epigenome  —  that  sits  on  top  of  the  genome,   just  outside  it  (hence  the  prefix  epi-­‐,  which   means  above).    

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It  is  these  epigene/c  "marks"   that  tell  your  genes  to  switch   on  or  off,  to  speak  loudly  or   whisper.    

It  is  through  epigene/c  marks   that  environmental  factors   like  diet,  stress  and  prenatal   nutri)on  can  make  an  imprint   on  genes  that  is  passed  from   one  genera/on  to  the  next.    

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Epigene/c  processes  
Many  types  of  epigene/c  processes  have  been  iden/fied—they  include    
•  Methyla)on   •  Acetyla)on   •  Phosphoryla)on   •  Ubiquityla)on   •  sumolya)on.  

Other  epigene/c  mechanisms    too  could  exist     These  are    natural  and  essen/al  to  many  organism  func/ons,  but  if  they   occur  improperly,  there  can  be  major  adverse  health  and  behavioral  effects.  

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Out  of  these  …..  

Methyla/on  and  Histone   modifica/on    is  studied   as  it  is  easy  reliable  and   not  easily  influenced  by   storage  and   interpreta/on  related   issues    

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Molecular  Mechanisms  that  Mediate   Epigene/c  Phenomena  
•  DNA  methyla/on  (CpG  dinucleo/des)   •  Histone  modifica/ons  

Nucleosome  

Histone  Modifica/ons  

Adapted from Lund and Lohuizen Genes Dev 2004

Epigene/c  Regula/on  of  Gene  Expression  

Epigene/c  Regula/on  of  Gene  Expression  

Methylated   DNA   Histone  

Histone  Modifica/on  Status  Correlates  with   Transcrip/onal  Ac/vity  

•  Gene  ac/va/on  correlated  with  H3-­‐K9  acetyla)on     •  Gene  silencing  associated  with  H3-­‐K9  methyla)on  

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•  Chroma/n  remodeling  (DNA  methyla/on          and  histone  modifica/on)  regulates  several        biological  processes  affec/ng  embryonic    development  

Li,  E.  (2002).  Chroma/n  modifica/on  and  epigene/c  reprogramming   in  mammalian  development.  Nat  Rev  Genet  3,  662–   673.  
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•  Epigene/c  features  are  implicated  in  the   pathogenesis  of  preeclampsia.     •  Muta/ons  in  STOX1  were  iden/fied  in  some   unique  familial  cases  of  preeclampsia  with   apparent  maternal-­‐only  transmission  of   suscep/bility.  
Van  Dijk  M,  Mulders  J,  Poutsma  A  et  al:  Maternal  segrega6on  of  the  Dutch   preeclampsia   locus  at  10q22  with  a  new  member  of  the  winged  helix  gene  family.  Nat  Genet   2005;  37:  514–519.  
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•  Deficiency  of  the  imprinted  Cdkn1c  gene  in   mice  can  lead  to  hypertension  and  proteinuria   during  pregnancy,  further  implica/ng  the  role   of  imprinted  genes  in  the  development  of   preeclampsia.     •  Epigene/c  altera/ons  of  non-­‐imprinted  genes   have  also  been  suggested  to  be  involved.  
Kanayama  N,  Takahashi  K,  Matsuura  T  et  al:  Deficiency  in  p57Kip2  expression  induces   preeclampsia-­‐like  symptoms  in  mice.  Mol  Hum  Reprod  2002;  8:  1129–1135.  
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•  The  SERPINA3  promoter  was  found  to  be   hypomethylated  in  preeclampsia-­‐associated   placenta  sugges/ng  that  the  epigene/c   altera/on  of  this  gene  may  be  associated  with     reduced  trophoblas/c  invasion  and   implica/ng  this  change  as  a  poten/al   biomarker  for  preeclampsia.  
Chelbi  ST,  Mondon  F,  Jammes  H  et  al:  Expressional  and  epigene/c  altera/ons  of   placental  serine  protease  inhibitors:  SERPINA3  is  a  poten/al  marker  of  preeclampsia.   Hypertension  2007;  49:  76–83.  
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•  Altered  gene-­‐specific  methyla/on  pa@erns  of   serine  protease  inhibitors  (Chelbi  et  al.,  2007),  APC  
(Mu¨ller  et  al.,  2004)  

•  and  RASSF1A  (Wang  et  al.,  2010)  gene  promoters   have  been  reported  in  pre-­‐eclampsia  

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IMPLICATIONS    
•  It  implies  that  certain  factors  in  nurture  and   nature  donot  only  have  influence    in  the   pathogenesis  of  the  disease  process  for  that   par/cular  pregnancy  but  can  have   implica/ons  for  the  future  genera/ons  .   •  We  therefore  need  to  understand  these  in   effects  with  more  details  and  be  able  to   convert  these  into  clinical  prac/ce    
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Preclampsia    
•  Does  seem  to  have  an  associa/on  with  altered   nutri/on  and  is  this  really  so  ???   •  We  always  believed  that  the  baby  is    a   parasite  !!!     •  Also  we  tried  supplemen/ng  Vit  E  ,C    Calcium   with  no  much  conclusive  effect  in  various   trials.    

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•  Maternal  nutri/on  is  an  important  determinant   of  one-­‐carbon  metabolism  that  lies  at  the  heart   of  intrauterine  epigene/c  programming.   •   Exchange  of  nutrients  and  other  vital  molecules   between  the  mother  and  fetus  takes  place  across   the  placenta  and  hence  may  play  direct  role  in   fetal  programming.     •  Pre-­‐eclampsia  (PE)  originates  in  the  placenta   and  altered  maternal  nutri)on  may  influence   epigene)c  paOerns  in  the  placenta,  thereby   affec)ng  birth  outcome.  
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•  Epigene/c  regula/on  of  key  genes  involved  in   adult  disease  is  currently  considered  to  be  the   underlying  mechanism  for  fetal  programming  

Symonds,  M.E.,  Sebert,  S.P.,  Hya@,  M.A.,  and  Budge,  H.  (2009).   Nutri/onal  programming  of  the  metabolic  syndrome.  Nat  Rev   Endocrinol  5,  604–610.  
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It  also  affects  the  angiogeneis    
•  Differen/al  methyla/on  of  genes  related  to   angiogenesis,  blood  vessel  development,        vasculature  development,  and  blood  vessel                         morphogenesis  have  also  been  reported    (Uthus  and  Anderson,  2010).   •   Promoter  hypomethyla/on  of  several  genes   has  been  shown    in  early  onset  pre-­‐eclamp/c   placentas  (Yuen  et  al.,  2010).  
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OUR  EXPERIENCE    
•  We  have  published  our  observa/on  related  to   preclampsia  and  nutri/on  in  rela/onship  to   omega  3  fa@y  acids  and  now    also  DNA   methyla/on  

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•  Our  earlier  studies  in  pre-­‐eclamp/c  women   have  shown  altered  essen/al  polyunsaturated   fa@y  acids  levels,  especially  docosahexaenoic   acid,  which  is  one  of  the  factors  responsible   for  preeclampsia   •  (Mehendale  et  al.,  2008;  Dangat  et  al.,  2010).  

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•  Membrane   phospholipids   are   major   methyl   group  acceptors   •  ⇓⇓   docosahexaenoic   acid   levels   may   result   in   diversion  of  methyl  groups  toward  DNA          ul/mately  resul/ng  in  DNA  methyla/on  as  we     have   recently   described   in   one   carbon    metabolic  pathway  (Kale  et  al.,  2009).  

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•  Further,  several  studies  show  high  levels  of   homocysteine,  another  key  cons/tuent  of   one-­‐carbon  metabolism  in  pre-­‐eclampsia      (Makedos  et  al.,  2007;  Singh  et  al.,  2008;  Guven  et  al.,  2009).  

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•  We  have  further  tested  the  hypothesis  that   global  DNA  methyla/on  pa@erns  in  placenta   vary  between  normotensive  and  pre-­‐ eclamp/c  women  (both  term  and  preterm)   and  may  be  associated  with  maternal  blood   pressure.     •  The  associa/ons  between  global  DNA   methyla/on  pa@erns  and  maternal  and   neonatal  characteris/cs  were  also  studied.  
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STUDY  REVEALED  :  Novel    
(In  normotensive  and  pre-­‐eclamp6c  women  (both  term   and  preterm)  who  were  matched  for  age  and   socioeconomic  status  )   •  ↑    DNA  methyla/on  in  pregnancies  complicated  with   pre-­‐eclampsia  (both  term  and  preterm)   •   ↑  homocysteine  levels  in  pre-­‐eclamp/c  (both  term  and   preterm)  when  compared  with  normotensive  women   •  Posi/ve  associa/on  between  global  DNA  methyla/on          and  SBP  and  DBP  only  in  the    term  pre-­‐eclamp/c      group   •   No  associa/on  of  placental  methyla/on  pa@erns  with   birth  outcome    
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•  The  study  reports  the  associa/on  of  placental   global  methyla/on  pa@erns  with  blood   pressure  only  in  term  preeclamp/c  group   indica/ng  that  differences  may  exist  in  subsets   of  pre-­‐eclamp/c  women  as  suggested  by   others    previously  (Roberts  and  Catov,  2008).  

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•  The  methodology  for  es/ma/on  of  global   methyla/on  levels  used  in  this  study  takes   into  account  methyla/on  of  all  CpGs   irrespec/ve  of  their  posi/on  in  the  genome   (promoter  and  nonpromoter  CpG).     •  This  is  in  concurrence  with  studies  indica/ng   that  CpG  methyla/on  in  intragenic  and   intergenic  regions  are  also  cri/cal  to  gene   expression  (Fazzari  and  Greally,  2004).  
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German  study    
•  Yet  another  study    

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•  Profiled  the  DNA  methyla/on  of  placentas   from  preeclampsia  and  IUGR  pregnancies  and   their  control  counterparts  using  Illumina   GoldenGate  Methyla/on  Cancer  panel  I  array.   •  Although  the  array  mainly  targets  cancer-­‐ related  genes,  the  pseudomalignant        nature  of  the  placenta  makes  it  suitable      for   this  study.  
Chiu  RW,  Chim  SS,  Wong  IH  et  al:  Hypermethyla6on  of  RASSF1A  in  human  and  rhesus  placentas.   Am  J  5/7/12   2007;  170:  941–950.   girijawagh@gmail.com  9422000584   Pathol   42  

•  Among  the  1505  CpG  loci  targeted  by  the   array,  34  loci  were  iden/fied  as   hypomethylated  in  EOPET  but  none  was   differen/ally  methylated  in  LOPET.     •  The  different  epigene/c  profiles  in  EOPET  and   LOPET  placentas  support  the  hypothesis  that   the  two  forms  of  preeclampsia  are  caused  by   different  mechanisms.  
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EOPET    
•   Is  oZen  associated  with  IUGR,  is  a  severe  form   of  preeclampsia  
»  (76%  associated  with  IUGR).  

•   It  is  suggested  to  be  ini/ated  by  abnormal   placenta/on,  caused  by  reduced  perfusion  with   increased  apoptosis  of  trophoblasts.  

Redman  CW,  Sargent  IL:  Latest  advances  in  understanding  preeclampsia.  Science  2005;  308:  1592–1594.   Von  Dadelszen  P,  Magee  LA,  Roberts  JM:  Subclassifica6on  of  preeclampsia.  Hypertens  Pregnancy  2003;  22:  143–148.   Oudejans  CB,  van  Dijk  M,  Oosterkamp  M,  Lachmeijer  A,  Blankenstein  MA:  Gene6cs  of  preeclampsia:  paradigm  shigs.  Hum   Genet  2007;  120:  607–612.  
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LOPET  
•  is  considered  to  be  a  maternal  syndrome,  is  a   mild  form  of  preeclampsia.   •   It  is  usually  associated  with  normal  placental   development  and  a  predisposed  maternal   cons/tu/on,  such  as  hypertension  or  diabetes.  

Redman  CW,  Sargent  IL:  Latest  advances  in  understanding  preeclampsia.  Science  2005;  308:  1592–1594.   Oudejans  CB,  van  Dijk  M,  Oosterkamp  M,  Lachmeijer  A,  Blankenstein  MA:  Gene6cs  of  preeclampsia:  paradigm  shigs.  Hum   Genet  5/7/12  120:  607–612.   2007;   girijawagh@gmail.com  9422000584   45  

•  Epigene/c  change  may  have  a  role  in  EOPET  by   altering  gene    expression  and,  as  a  consequence,   abnormal  placental  development.   •  Epigene/c  changes  may  also  result  from  hypoxic   condi/ons  associated  with  preeclampsia  or  an   altered  trophoblast  composi/on  in  these   placentas.     •  Hypomethyla/on  was  found  in  many  gene   promoter        regions  in  EOPET,  but  there  was  no   difference  in  the  global  DNA  methyla/on  level   compared  with  other  groups  of  placentas  
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•  The  DNA  methyla/on  differences  of  CpGs  in   CAPG,  GLI2,  KRT13  and  TIMP3  were  confirmed   in  an  independent  set  of  26  placentas  with   EOPET  and  gesta/onal  age-­‐matched  control   pregnancies.     •  Among  these  four  genes,  TIMP3  had  the   largest  difference  in  DNA  methyla)on        level  with  an  over  15%  reduc/on  in  EOPET      compared  with  control  placentas.  
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•  TIMP3  gene  expression  can  be  regulated  by   promoter  DNA  methyla/on  in  the  placental   /ssues.  

Feng  H,  Cheung  AN,  Xue  WC  et  al:  Down-­‐regula6on  and  promoter  methyla6on  of   6ssue  inhibitor  of  metalloproteinase  3  in  choriocarcinoma.  Gynecol  Oncol  2004;  94:   375–382.  
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•  TIMP3  is  a  family  member  of  the  matrix   metalloproteinase  inhibitors  
•  Regulates  :   •  cell  growth,  invasion,  migra/on  transforma/on  and   apoptosis.    

•  This  gene  is  highly  expressed  in  placenta  and   suggested  to  be  important  for  implanta/on   and  decidualiza/on  by  regula/ng  trophoblast   invasion.  
Feng  H,  Cheung  AN,  Xue  WC  et  al:  Down-­‐regula6on  and  promoter  methyla6on  of  6ssue  inhibitor  of  metalloproteinase  3  in   choriocarcinoma.  Gynecol  Oncol  2004;  94:  375–382.   Apte  SS,  Maiei  MG,  Olsen  BR:  Cloning  of  the  cDNA  encoding  human  6ssue  inhibitor  of    etalloproteinases-­‐3  (TIMP-­‐3)  and   mapping  of  the  TIMP3  gene  to  chromosome  22.Genomics  1994;  19:  86–90.  
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•  The  hypomethyla/on  of  the  TIMP3  promoter   found  in  this  study  may  increase  TIMP3   expression  and,  in  turn,  reduce  the   invasiveness  of  trophoblast  during  placental   development,  which  leads  to  placental   hypoperfusion  in  EOPET.  

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•  Intriguingly,  hypermethyla/on  of  the  TIMP3   promoter  has  been  reported  in   choriocarcinoma  and  hyda/diform  mole,   condi/ons  that  have  increased    trophoblast   invasiveness,    which  further  supports  the   inverse  rela/onship  between  TIMP3  promoter   methyla/on  and  trophoblast  invasiveness.  

Feng  H,  Cheung  AN,  Xue  WC  et  al:  Down-­‐regula6on  and  promoter  methyla6on  of   6ssue  inhibitor  of  metalloproteinase  3  in  choriocarcinoma.  Gynecol  Oncol  2004;  94:   375–382.   Xue  WC,  Chan  KY,  Feng  HC  et  al:  Promoter  hypermethyla6on  of  mul6ple  genes  in   hyda6diform  mole  and  choriocarcinoma.  J  Mol  Diagn  2004;  6:  326–334  
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•  It  has  also  been  shown  that  TIMP3  could   inhibit  angiogenesis  by  blocking  the  vascular   endothelial  growth  factor  from  binding  its   receptor,  a  well-­‐known  defect  that  is  found  in   the  trophoblast  of  preeclamp/c  pregnancies.  

Qi  JH,  Ebrahem  Q,  Moore  N  et  al:  A  novel  func6on  for  6ssue  inhibitor  of  metalloproteinases-­‐3  (TIMP3):   inhibi6on  of  angiogenesis  by  blockage  of  VEGF  binding  to  VEGF  receptor-­‐2.  Nat  Med  2003;  9:  407–415.   Noris  M,  Perico  N,  Remuzzi  G:  Mechanisms  of  disease:  pre-­‐eclampsia.  Nat  Clin  Pract   Nephrol  2005;  1:  98–114;  quiz  120  
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•  Although  the  cause  of  the  epigene/c   modifica/on  is  unknown,  it  may  be  related  to   the  hypoxic  environment  of  the  cells.   •   TIMP3  expression  was  increased  in  the  first   trimester  trophoblasts  on  hypoxic  treatment.     •  This  implies  that  the  increased  expression  of   TIMP3  under  hypoxic  condi/on,  a  hallmark  in      preeclamp/c  trophoblast,  may  be  mediated  by          the  epigene/c  altera/on  on  its  promoter.  
Shahrzad  S,  Bertrand  K,  Minhas  K,  Coomber  BL:  Induc6on  of  DNA  hypomethyla6on  by  tumor  hypoxia.   Epigene6cs  2007;  2:  119–125.   Gheorghe  CP,  Mohan  S,  Oberg  KC,  Longo  LD:  Gene  expression  paierns  in  the  hypoxic  murine  placenta:  a   role  in  epigenesis?  Reprod  Sci  2007;  14:  223–233.    Koklanaris  N,  Nwachukwu  JC,  Huang  SJ  et  al:  First-­‐trimester  trophoblast  cell  model  gene  response  to   hypoxia.  Am  J  Obstet  Gynecol  2006;  194:  687–693.   5/7/12   girijawagh@gmail.com  9422000584   53  

USEFUL  BIOMARKER  !  
•  The  significant  reduc/on  of  DNA  methyla/on   in  TIMP3  promoter  of  EOPET  placentas  could   be  useful  as  a  biomarker  for  the  disorder.   •  CVS  ?!!  

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•  During  pregnancy,  3  to  6%  of  cell-­‐free  DNA  in  maternal   blood  plasma  is  derived  from  the  placenta.     •  Therefore,  one  can  detect  abnormali/es  in  the  fetal   DNA  directly  from  the  maternal  blood  without  going        through  invasive  methods  such  as  amniocentesis  and                 CVS.    It  has  been  shown  that  there  is  an  over  fivefold  increase   in  circula/ng  fetal  DNA  in  the  maternal  plasma  of   preeclamp/c  pregnancies  compared  with  their  control   counterparts  
Dennis  Lo  YM,  Chiu  RW:  Prenatal  diagnosis:  progress  through  plasma  nucleic  acids.   5/7/12   Nat  Rev  Genet  2007;  8:  71–77.   girijawagh@gmail.com  9422000584  
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Epigene/c  considera/ons  in  preterm   delivery    
•  Spontaneous  preterm  parturi/on  is      syndromic  in  nature,  and  mul/ple    mechanisms  of  disease  are  likely  to  be  involved  

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Many  a@empts  have  been  made    
•  To  study     •  Epigene/c  influences     •  Other  factors  have  been  found  to  be   confounding  !!!  

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•  EPIGENETIC   REGULATION  OF  COX-­‐2   GENE  PROMOTER  IN   PRETERM  LABOR    
Joel  LARMA,  BEGONA  CAMPOS,  LESLIE   MYATT,  University  of  Cincinna6,   Cincinna6,   Ohio,  University  of  Cincinna6,   Obstetrics  and  Gynecology,  Cincinna6,    

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Myometrium  was  studied  at  CS    
•  The  COX  2  gene,  from  the  -­‐1294  promoter   region  to  the  +  275  region  of  the  gene,  did  not   present  a  DNA  methyla/on  pa@ern  in  genomic   DNA  isolated  from  the  myometrium  of   unlabored    and  labored    parturients  at  a   gesta/onal  age  less  than  37  weeks  who   underwent  a  Cesarean  sec/on  

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•  This  study  iden/fied  maternal  and  fetal  DNA   variants  that  predispose  to  preterm  labor  with   intact  membranes  leading  to  preterm  delivery   •  Importantly,TIMP2  consistently  was  found  in          the  different  networks  (maternal  and  fetal)   that  are  associated  with  preterm  labor/  delivery   with  intact  membranes.  

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•  The  observed  associa/on  between  TIMP2  and   spontaneous  preterm  labor  with  intact   membranes  is  novel  and  implicates   extracellular  matrix  metabolism  as  an   important  factor  that  predisposes  to  preterm   parturi/on  

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•  TIMP2  (Tissue  inhibitor  of  metalloproteinase  2)  
–  Modulates  the  ac/on  of    MMP2  
»  Matrix  Metalloproteinase  2      

–  Implicated  in  the  metabolism  of  Collagen  IV  in   uterus  as  well  as  cervix    

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What  do  we  have  then  
•  Environmental  factors  have  intergene/c   influences  which    modify  the  process  of   trophoblas/c  invasion   •  They  affect  cellular  level  mechanisms     •  Harbour  the  poten/al  to  carry  it  not  only  to   the  affected  fetus  but  also  to  the  next   genera/on      

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What  we  need  to  do  ?  
•  •  •  •  Be  more  intense  with  what  we  have     Nutri/onal  assessment     Careful  clinical  assessment     Conscious  interven/ons    

•  Will  go  a  long  way  in  helping  us  alleviate  this   condi/on    
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