Evolu&onary  perspec&ve  suggests  candidate  genes  for  varia&on  in  Turner  Syndrome  phenotype  

Kara  Schaffer  and  Melissa  Wilson  Sayres  School  of  Life  Sciences,  Center  for  Evolu&on  and  Medicine,  The  Biodesign  Ins&tute    Arizona  State  University,  Tempe,  Arizona  

Background  Tremendous   phenotypic   varia&on   exists   across   people   with   Turner  syndrome  (45,X).  This  varia&on  likely  stems  from  differen&al  dosage  of  genes   on   the   X   chromosome.   In   this   study   we   take   an   evolu&onary  approach   to   rank   candidate   genes   that   may   affect   phenotype   across  people  with  Turner  Syndrome.  X-‐inac&va&on  is  the  process  whereby  all  X  chromosomes   in  excess  of  one  are  silenced.  However,  about  15%  of  the  genes  on   the   silenced  X   chromosome  escape   this   inac&va&on  and  are  candidates  for  affec&ng  phenotype  in  people  with  Turner  syndrome.  We   analyze   paRerns   of   DNA   methyla&on   from   46,XX   and   45,X  individuals,   to   inform   about   X-‐inac&va&on   status,   comparing   this  with  studies  about  X-‐inac&va&on   status   from  cell-‐lines,   to   classify   genes  on  the   human   X   chromosome   into   those   that   may   be   more   dosage  sensi&ve.   We   then   analyze   paRerns   of   gene   expression   conserva&on  across   five   &ssues   and   ten   species   by   class   of   X-‐linked   gene,   to   learn  which  may   be  more   evolu&onarily   conserved,   and   thus  more   likely   to  affect  phenotype  when  dosage  is  altered  from  typical  levels.  

Methods  Methyla)on  data  We  used  data  sets  of  methyla&on  levels  of  genes  located  on  the  X-‐chromosome  of  both  normal  46,  XX  females  and  45,X  Turner  syndrome  females,  comparing  the  two  data  sets  to  inform  about  X-‐inac&va&on  status1.  Genes  suscep&ble  to  X-‐inac&va&on  have  the  highest  methyla&on  levels,  while  genes  that  escape  X-‐inac&va&on  will  have  low  methyla&on  levels1.  Measuring  the  difference  between  46,  XX  and  45,X  pa&ents  allows  for  measurement  of  methyla&on  changes  that  occur  solely  with  X-‐inac&va&on1.      Gene  expression  data  AXer  conver&ng  the  MRNA  RefSeq  ID  of  the  genes  present  in  the  methyla&on  data  to  Ensembl  Gene  IDs  4,  we  compared  the  methyla&on  data  to  another  data  set  describing  the  expression  levels  of  genes  in  different  &ssues  (Brain,  Cerebellum,  Heart,  Kidney,  Liver,  and  Tes&s)  and  species(Human,  Chimpanzee,  Bonobo,  Gorilla,  Orangutan,  Macaque,  Mouse,  Opossum)2  .        X-‐inac)va)on  status  We  then  compared  both  of  the  previous  data  sets  to  the  data  of  a  previous  study  that  discovered  the  X-‐inac&va&on  status  of  the  genes  of  the  X-‐chromosomes3.        

Conclusions  and  Further  Analysis    

•  Methyla&on   across   X-‐linked   genes   is   consistently   lower   in   Turner  pa&ents   (45,X)   than   typical   (46,   XX)   females,   regardless   of   X-‐inac&va&on   status.   However,   the   difference   between   46,XX  methyla&on   and   45,X   methyla&on   is   smaller   for   genes   subject   to  inac&va&on  in  46,  X  individuals.  

•  Human  X-‐linked  genes  are  expressed  at  higher  levels  in  the  brain  and  lowest  in  the  testes.  This  trend  is  constant  across  primates.    

•  Genes  subject  to  X-‐inac&va&on  have  higher  levels  of  expression  in  the  brain,   and   lower   levels   of   expression   in   the   testes.   This   trend   is  reversed   in  genes  that  escape   inac&va&on,  which  show  higher   levels  of   expression   in   the   testes,   and   lower   levels     of   expression   in   the  brain.   Further   analysis   will   include   comparing   with   gene   expression  across  &ssues  for  the  autosomes.    

References  1.Sharp,  A.  J.  et  al.  Genome  Res.  21,  1592–1600  (2011).  2.  Brawand,  D.  et  al.  Nature  478,  343–348  (2011).    3.  Carrel,  L.  &  Willard,  H.  F..  Nature  434,  400–404  (2005).  4.  Huang  DW,  Sherman  BT,  Lempicki  RA.  Nature  Protoc.  2009;4(1):44-‐57.  

Acknowledgements  Startup   funds   from   the   School   of   Life   Sciences   and   The   Biodesign  Ins&tute.      

Results    

 

Figure   1.  A   linear   regression   of   the  methyla&on   values   of   genes   of  different   X-‐inac&va&on   status.   The   red   line   represents   the  methyla&on  of  regular  females  (46,XX)  while  the  blue  line  represents  the  methyla&on  of  Turners  females  (45,  X).  

Figure  2a.    The  average    expression  in  RPKM  of    genes  on  the  X  chromosome  for  various  human  &ssues.  

Figure  2b.  The  average  expression  in  RPKM  of  genes  on  the  X  chromosome  for  various  human  &ssues    sorted  by  X-‐inac&va&on  status  

Figure  3a.    The  average    expression  in  RPKM  of    genes  on  the  X  chromosome  for  various  chimpanzee  &ssues.  

Figure  3b.  The  average  expression  in  RPKM  of  genes  on  the  X  chromosome  for  various  chimpanzee  &ssues    sorted  by  X-‐inac&va&on  status  

Figure  4.    The  average    expression  in  RPKM  of    genes  on  the  X  chromosome  for  various  &ssues  in  all  primates  

Female:    P-‐value  =  8.519*10-‐15  Mul&ple  R-‐squared  =  0.197  Turner  :  P-‐value  =  0.2198  Mul&ple  R-‐squared  =  0.00547