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Telomere and q pcr 2013
- 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
- 6. TELOMERES ARE SHORT IN ACQUIRED APLASTIC ANEMIA
- 7. DYSKERATOSIS CONGENITA Bone Marrow Failure Associated with Mucocutaneous Triad Nail dystrophy Leukoplakia
- 9. 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
- 10. 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
- 11. 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
- 12. 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
- 13. TELOMERE LENGTH IN PATIENTS WITH ACQUIRED APLASTIC ANEMIA 183 ConsecuEve PaEents Treated with IST Longer telomeres Shorter telomeres
- 14. Telomere Length and Relapse RELAPSE RATE BY TELOMERE QUARTILES Scheinberg et al. JAMA 2010
- 15. EVOLUTION RATE BY TELOMERE LENGTH MONOSOMY 7 RATE BY TELOMERE LENGTH Scheinberg et al. JAMA 2010
- 16. SURVIVAL PROBABILITY BY TELOMERE LENGTH SURVIVAL PROBABILITY BY TELOMERE LENGTH & ARC Scheinberg et al. JAMA 2010
- 17. 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
- 18. 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
- 19. 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
- 21. “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
- 22. 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
- 23. 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
- 24. TERT LOCUS AND CANCER Genome-‐Wide AssociaEon Studies (GWAS)
- 25. 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.
- 26. Telomere Erosion Chromosomal Instability ↑p53 Senescence Apoptosis Marrow Failure Malignant Transforma^on
- 27. 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
- 28. 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
- 29. TERT Muta^ons and Severe Liver Disease Calado et al., PLoS ONE 2009
- 30. TELOMERASE MUTATIONS AND PULMONARY FIBROSIS
- 31. PULMONARY FIBROSIS IN TERT MUTANT PATIENT
- 32. PULMONARY FIBROSIS IN TERT MUTANT PATIENT El-‐Chemaly et al, Chest, 2010
- 33. TELOMERES ARE SHORT IN PULMONARY FIBROSIS Alder et al, PNAS, 2008
- 34. TELOMERE EROSION AND HUMAN DISEASES Smoking EtOH HCV Virus Chemicals Liver cirrhosis Pulmonary fibrosis Mucocutaneous Aplas^c anomalies anemia
- 35. Hematocrit 48% 16% May/90 Feb/92
- 36. 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
- 37. 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
- 38. TELOMERE LENGTH MEASUREMENT Calado & Young, NEJM 2009
- 39. TELOMERE LENGTH MEASUMENT Southern blot • “Gold standard” • Time-‐consuming (3 to 4 days) Molecular Weight • Limited number of samples
- 40. TELOMERE LENGTH MEASUREMENT Flow-‐FISH • Telomere length in cell subsets • Time-‐consuming • Sensi^ve technique metaphase interphase Courtesy of Peter Lansdorp, UBC
- 41. TELOMERE LENGTH MEASUREMENT qPCR • High-‐throughput (hundreds to thousands of samples/day • Reproducible • Telomere length given in a “ra^o” Cawthon, NAS, 2002
- 42. TELOMERE LENGTH MEASUREMENT qPCR Cawthon, NAS, 2002
- 43. TELOMERE LENGTH MEASUREMENT qPCR Cawthon, NAS, 2009
- 44. TELOMERE LENGTH MEASUREMENT qPCR qPCR for telomere length using the Rotor Gene-Q system
- 45. TELOMERE LENGTH MEASUREMENT qPCR Telomere length of 300 healthy subjects from 0 to 99 years Correlation between qPCR and Southern blot
- 46. 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