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Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
Telomere and q pcr 2013
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Telomere and q pcr 2013

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  1. Qiagen  Webinar   October  5th,  2011   Telomere  shortening,  human  disease,  and  cancer   Rodrigo  T  Calado   Associate  Professor,  Department  of  Medicine   University  of  São  Paulo  at  Ribeirão  Preto  Medical  School   Ribeirão  Preto,  SP,  Brasil  
  2. TELOMERE  STRUCTURE   Calado  &  Young,  Blood  2008  
  3. TELOMERES  SHORTEN  WITH  CELL  DIVISION   Chromosome   Telomere   No  telomerase   Telomere  erosion   Telomere   Centrosome   Cell  division   +  Telomerase  
  4. TELOMERE  LENGTH  MEASUREMENT   Calado  &  Young,  NEJM  2009  
  5. TELOMERES  ARE  SHORT  IN  ACQUIRED  APLASTIC  ANEMIA  
  6. DYSKERATOSIS  CONGENITA   Bone  Marrow  Failure  Associated  with  Mucocutaneous  Triad   Nail  dystrophy   Leukoplakia  
  7. ACQUIRED  APLASTIC  ANEMIA   Telomerase  MutaEons  in  PaEents  without  Clinical  Features  of  DKC   Cohort  of  205  pa^ents  with  acquired  aplas^c  anemia:   Muta4on   Pa4ent   Diagnosis   Family  History   Ala  202  Thr   41  M  Hispanic   SAA   -­‐-­‐   Ala  202  Thr   75  F  Hispanic   MAA   brother  MDS   His  412  Tyr   56  M  White   MAA   mother  MDS   His  412  Tyr   31  M  Hispanic   SAA   -­‐-­‐   Val  694  Met   34  M  White   MAA   sister  MDS/AML   Tyr  772  Cys   53  M  White   MAA   sister  “anemia”   Val  1090  Met   64  F  Hispanic   SAA   -­‐-­‐   Yamaguchi  et  al.,  NEJM  2005  
  8. ACQUIRED  APLASTIC  ANEMIA   Telomeres  Are  Short  in  PaEents  in  with  Telomerase  MutaEons   telomere  length,  kb   16   14   controls   pa^ents   12   10   8   6   4   His  412  Tyr   2   0   Val  694  Met   0   20   Ala  202  Thr   Val  1090  Met   Cys  772  Tyr   40   60   80   100   age,  years   Yamaguchi  et  al.,  NEJM  2005  
  9. FUNCTIONAL  ASSAYS  OF  TELOMERASE  ACTIVITY   Primary  cells   Transfec^on  of  TERT  Mutants   Co-­‐transfec^on   loss  of  func^on   (hypomorphic)   haploinsufficiency   IC   -­‐  +  -­‐     -­‐    +   -­‐    +   codon   Codon   WT:Codon   Yamaguchi  et  al.,  NEJM  2005  
  10. HEMATOPOIESIS  IN  “NORMAL”  RELATIVES  WITH  TERC  MUTATIONS   Hematology:   Hematopoiesis:        normal  peripheral  blood  counts        mild  anemia  with  macrocytosis        mild  thrombocytopenia        severely  hypoplas^c        ↓CD34  number        ↓colony  forma^on        ↑erythropoie^n,  thrombopoie^n   proband   affected  sister   affected  niece   unaffected  brother   Fogarty  et  al.,  Lancet  2003  
  11. TELOMERE  LENGTH  IN  PATIENTS  WITH   ACQUIRED  APLASTIC  ANEMIA   183  ConsecuEve  PaEents  Treated  with  IST   Longer  telomeres   Shorter  telomeres  
  12. Telomere Length and Relapse RELAPSE RATE BY TELOMERE QUARTILES Scheinberg  et  al.  JAMA  2010  
  13. EVOLUTION RATE BY TELOMERE LENGTH MONOSOMY 7 RATE BY TELOMERE LENGTH Scheinberg  et  al.  JAMA  2010  
  14. SURVIVAL PROBABILITY BY TELOMERE LENGTH SURVIVAL PROBABILITY BY TELOMERE LENGTH & ARC Scheinberg  et  al.  JAMA  2010  
  15. INCREASED  TELOMERE-­‐FREE  ENDS  IN  MARROW  CELLS   OF  AA  PATIENTS  WITH  SHORTER  TELOMERES   Shorter  telomeres   %  Telomere-­‐free  ends   Long  telomeres   Calado  et  al.  Leukemia,  in  press  
  16. APLASTIC  ANEMIA  PATIENTS  WITH  SHORTER  TELOMERES  AT   DIAGNOSIS  ALSO  DISPLAY  MORE  ANEUPLOIDY   %  Monosomy  7  cells  in  BM   Short  telomeres   Long  telomeres   Calado  et  al.  Leukemia,  in  press  
  17. CHROMOSOMAL  INSTABILITY  IN  APLASTIC  ANEMIA  PATIENTS    WITH  SHORTER  TELOMERES   t(10;19)   Rob(14;22)   del(1)(p11.2)   t(5;18)   ins(2;10)   t(X;8)   Calado  et  al.  Leukemia,  in  press  
  18. “SHANK’S  DISEASE”  IN  A  MENNONITE  FAMILY   died age 65 yr “blood disease” with pallor 71 y/o thyroid disease 38 y/o liver transplant died age 33 yr MDS/AML 47 y/o 44 y/o macrocytosis androgenresponsive AA Liver disease IPF 26 y/o 18 y/o 23 y/o 21 y/o macrocytosis 26 y/o AA wt hTERT heterozygous K570N not tested dead 4 y/o Xin,  Calado  et  al.,  Blood  2007  
  19. TERT  MUTATIONS  IN  ACUTE  MYELOID  LEUKEMIA   Brazil  Cohort   N=133  consecuEve  AML  pa^ents  (200  matched  blood  donors;  p  =  0.0001)   11  TERT  muta^ons:    7  heterozygous,  1  homozygous  for  A1062T          1  heterozygous  H412Y          1  heterozygous  R522K          1  heterozygous  P65A   TERT-­‐  AML:    2  inv(16),  1  complex,  t(5;11)(q35;q13)  +  del(10)(p15),  3  t(15,17)   MD  Anderson  Cohort   N=  89  selected  AML  by  cytogene^cs  (528  healthy  controls;  P=0.028)   4  TERT  muta^ons:      2  heterozygous  for  A1062T          1  homozygous  for  411E  dele^on          1  heterozygous  for  V299M   Muta^ons  associated  with  trisomy  8  and  inv(16)   TERT  muta^ons  are  cons^tu^onal  and  dominant  loss-­‐of-­‐func^on.   Calado  et  al.,  PNAS  2009  
  20. DYSKERATOSIS  CONGENITA   Predisposes  to  Cancer   Cumula^ve  incidence  of  cancer   Cumula^ve  incidence  of  MDS   Types  of  cancers  and  observed/expected  (O/E)  ra^o   Cancer      O/E    95%  C.I.   All  sites      11*    4-­‐23   Tongue      1154*    232-­‐3372   AML      196*    22-­‐707   Cervical  SCC    43*    0.6-­‐236   *P<0.05   Alter  et  al.,  Blood  2009  
  21. TERT  LOCUS  AND  CANCER   Genome-­‐Wide  AssociaEon  Studies  (GWAS)  
  22. CANCER  INCIDENCE  AND  CANCER  MORTALITY  AND  TELOMERE  LENGTH   General  PopulaEon  >  40  years,  The  Bruneck  Study,  Italy   Willeit,  P.  et  al.  JAMA  2010;304:69-­‐75.  
  23. Telomere  Erosion   Chromosomal   Instability   ↑p53   Senescence   Apoptosis   Marrow  Failure   Malignant  Transforma^on  
  24. TELOMERE  EROSION  AND  CANCER  RISK   TERT  locus  and   general  cancer  risk   Lung  cancer,  basal  cell   carcinoma,  urinary  bladder   cancer,  cervical  cancer,   glioma   Aplas4c  anemia   MDS,  AML   Inflammatory   bowel  disease   Colorectal  cancer   BarreO’s   esophagi4s   Dyskeratosis   congenita   Tongue  SCC,   MDS,  AML   Adenocarcinoma  
  25. TERT  MUTATION  ASSOCIATES  WITH  SEVERE  LIVER  DISEASE     wt TERT heterozygous K570N not tested severe liver disease TERC 28-34 deletion TERC 341-360 deletion Barrett esophagus pancytopenia, cirrhosis, esophageal cancer pulmonary fibrosis, cirrhosis, pancytopenia esophageal cancer MDS COPD aplastic anemia “alcoholic cirrhosis” NASH, hypoplastic marrow Liver nodular regenerative hyperplasia Calado  et  al.,  PLoS  ONE  2009  
  26. TERT  Muta^ons  and  Severe  Liver  Disease   Calado  et  al.,  PLoS  ONE  2009  
  27. TELOMERASE  MUTATIONS  AND  PULMONARY  FIBROSIS  
  28. PULMONARY FIBROSIS IN TERT MUTANT PATIENT
  29. PULMONARY FIBROSIS IN TERT MUTANT PATIENT El-­‐Chemaly  et  al,  Chest,  2010  
  30. TELOMERES ARE SHORT IN PULMONARY FIBROSIS Alder  et  al,  PNAS,  2008  
  31. TELOMERE  EROSION  AND  HUMAN  DISEASES   Smoking   EtOH   HCV   Virus   Chemicals   Liver   cirrhosis Pulmonary   fibrosis Mucocutaneous   Aplas^c   anomalies anemia
  32. Hematocrit   48%   16%   May/90   Feb/92  
  33. SEX  HORMONES  INCREASE  TELOMERASE  ACTIVITY  IN   CULTURED  HUMAN  LYMPHOCYTES   900   Telomerase  Ac^vity   (TPG  units)   (n=10)   600   300   0   0      0.5    5μM                0    5μM                    0    5μM                    0    1μM     Methyltrienolone   Nandrolone   6β-­‐Hydroxy-­‐   β-­‐Estradiol   (synthe^c)   Testosterone   Androgens   Calado    et  al,  Blood,  2009  
  34. SEX  HORMONES  MODULATE  TERT  ACTIVITY   OH Androgens O O – C – CH2(CH2)7CH3 CH3 OH Estradiol O O O OH Aromatase   Estrogen + O Estrogen receptors Estrogenestrogen receptors TERT gene Estrogen-response element mRNA TERT protein Telomerase activity Calado    et  al,  Blood,  2009  
  35. TELOMERE  LENGTH  MEASUREMENT   Calado  &  Young,  NEJM  2009  
  36. TELOMERE  LENGTH  MEASUMENT   Southern  blot   •   “Gold  standard”   •   Time-­‐consuming  (3  to  4  days)   Molecular Weight •   Limited  number  of  samples  
  37. TELOMERE  LENGTH  MEASUREMENT   Flow-­‐FISH   •   Telomere  length  in  cell  subsets       •   Time-­‐consuming   •   Sensi^ve  technique   metaphase   interphase   Courtesy  of  Peter  Lansdorp,  UBC  
  38. TELOMERE  LENGTH  MEASUREMENT   qPCR   •   High-­‐throughput  (hundreds  to  thousands  of  samples/day   •   Reproducible   •   Telomere  length  given  in  a  “ra^o”   Cawthon,  NAS,  2002  
  39. TELOMERE  LENGTH  MEASUREMENT   qPCR   Cawthon,  NAS,  2002  
  40. TELOMERE  LENGTH  MEASUREMENT   qPCR   Cawthon,  NAS,  2009  
  41. TELOMERE  LENGTH  MEASUREMENT   qPCR   qPCR for telomere length using the Rotor Gene-Q system
  42. TELOMERE  LENGTH  MEASUREMENT   qPCR   Telomere length of 300 healthy subjects from 0 to 99 years Correlation between qPCR and Southern blot
  43. ACKNOWLEDGEMENTS   NHLBI:   Neal  Young   Elaine  Sloand   Jeffrey  Abrams   Jen  Brudno   James  Cooper   Jake  Decker   Solomon  Graf   Sachiko  Kajigaya   Keyvan  Keyvanfar   Daniela  Malide   Paulomi  Mehta   Nate  Peterson   Joshua  Regal   Phillip  Scheinberg   NCI:   Stephen  Chanock   David  Kleiner   Hesed  Padilla-­‐Nash   Tom  Ried   Sharon  Savage   University  of  BriEsh  Columbia:   Peter  Lansdorp   Mark  Hills   Irma  Vulto   M.D.  Anderson  Cancer  Center:   Elihu  Estey   University  of  Arizona:   Thomas  Boyer   University  of  São  Paulo:   Raquel  Paiva   Barbara  Santana   Diego  Clé   Flavia  Donaires   Felipe  Silva   Natália  Scatena   Fernanda  Gu^errez  

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