Telomere and q pcr 2013
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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
Qiagen Webinar October 5th, 2011
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TELOMERE STRUCTURE
Calado & Young, Blood 2008
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Chromosome Telomere
Telomere Centrosome
Cell division No telomerase + Telomerase
Telomere erosion
TELOMERES SHORTEN WITH CELL DIVISION
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TELOMERE LENGTH MEASUREMENT
Calado & Young, NEJM 2009
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TELOMERES ARE SHORT IN ACQUIRED APLASTIC ANEMIA
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Nail dystrophy Leukoplakia
DYSKERATOSIS CONGENITA Bone Marrow Failure Associated with Mucocutaneous Triad
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ACQUIRED APLASTIC ANEMIA Telomerase MutaEons in PaEents without Clinical Features of DKC
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
Cohort of 205 pa^ents with acquired aplas^c anemia:
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0
2
4
6
8
10
12
14
16
0 20 40 60 80 100
controls
age, years
telomere length, kb pa^ents
His 412 Tyr
Val 694 Met Ala 202 Thr
Cys 772 Tyr
Val 1090 Met
ACQUIRED APLASTIC ANEMIA Telomeres Are Short in PaEents in with Telomerase MutaEons
Yamaguchi et al., NEJM 2005
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IC
WT:Codon
Co-‐transfec^on Transfec^on of TERT Mutants
-‐ + -‐
Codon
Primary cells
-‐ + -‐ +
codon
FUNCTIONAL ASSAYS OF TELOMERASE ACTIVITY
loss of func^on (hypomorphic)
Yamaguchi et al., NEJM 2005
haploinsufficiency
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HEMATOPOIESIS IN “NORMAL” RELATIVES WITH TERC MUTATIONS
Hematology:
normal peripheral blood counts
mild anemia with macrocytosis mild thrombocytopenia
Hematopoiesis:
severely hypoplas^c ↓CD34 number ↓colony forma^on ↑erythropoie^n, thrombopoie^n
Fogarty et al., Lancet 2003
proband affected sister affected niece unaffected brother
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TELOMERE LENGTH IN PATIENTS WITH ACQUIRED APLASTIC ANEMIA
183 ConsecuEve PaEents Treated with IST
Shorter telomeres
Longer telomeres
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RELAPSE RATE BY TELOMERE QUARTILES
Telomere Length and Relapse
Scheinberg et al. JAMA 2010
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EVOLUTION RATE BY TELOMERE LENGTH
MONOSOMY 7 RATE BY TELOMERE LENGTH
Scheinberg et al. JAMA 2010
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SURVIVAL PROBABILITY BY TELOMERE LENGTH
SURVIVAL PROBABILITY BY TELOMERE LENGTH & ARC
Scheinberg et al. JAMA 2010
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Shorter telomeres % Telomere-‐free ends
INCREASED TELOMERE-‐FREE ENDS IN MARROW CELLS OF AA PATIENTS WITH SHORTER TELOMERES
Long telomeres
Calado et al. Leukemia, in press
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APLASTIC ANEMIA PATIENTS WITH SHORTER TELOMERES AT DIAGNOSIS ALSO DISPLAY MORE ANEUPLOIDY
Long telomeres Short telomeres
% Monosomy 7 cells in BM
Calado et al. Leukemia, in press
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del(1)(p11.2)
t(5;18)
Rob(14;22)
ins(2;10)
t(10;19)
t(X;8)
CHROMOSOMAL INSTABILITY IN APLASTIC ANEMIA PATIENTS WITH SHORTER TELOMERES
Calado et al. Leukemia, in press
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38 y/o liver transplant
26 y/o AA
47 y/o macrocytosis
21 y/o macrocytosis
71 y/o thyroid disease
44 y/o androgen- responsive AA Liver disease IPF
died age 33 yr MDS/AML
26 y/o 18 y/o
4 y/o
died age 65 yr “blood disease” with pallor
wt hTERT
heterozygous K570N not tested
“SHANK’S DISEASE” IN A MENNONITE FAMILY
23 y/o
dead Xin, Calado et al., Blood 2007
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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
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DYSKERATOSIS CONGENITA Predisposes to Cancer
Cumula^ve incidence of cancer Cumula^ve incidence of MDS
Alter et al., Blood 2009
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
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TERT LOCUS AND CANCER Genome-‐Wide AssociaEon Studies (GWAS)
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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.
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Telomere Erosion
↑p53 Senescence Apoptosis
Chromosomal Instability
Marrow Failure
Malignant Transforma^on
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TELOMERE EROSION AND CANCER RISK
Dyskeratosis congenita Tongue SCC, MDS, AML
Aplas4c anemia MDS, AML
Inflammatory bowel disease Colorectal cancer
BarreO’s esophagi4s
Adenocarcinoma
TERT locus and general cancer risk Lung cancer, basal cell
carcinoma, urinary bladder cancer, cervical cancer,
glioma
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TERT MUTATION ASSOCIATES WITH SEVERE LIVER DISEASE
wt TERT heterozygous K570N not tested severe liver disease
Calado et al., PLoS ONE 2009
TERC 341-360 deletion
Barrett esophagus
pancytopenia, cirrhosis,
esophageal cancer
esophageal cancer
COPD
NASH, hypoplastic
marrow “alcoholic cirrhosis”
TERC 28-34 deletion
pulmonary fibrosis, cirrhosis, pancytopenia
aplastic anemia Liver nodular regenerative hyperplasia
MDS
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TERT Muta^ons and Severe Liver Disease
Calado et al., PLoS ONE 2009
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TELOMERASE MUTATIONS AND PULMONARY FIBROSIS
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PULMONARY FIBROSIS IN TERT MUTANT PATIENT
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PULMONARY FIBROSIS IN TERT MUTANT PATIENT
El-‐Chemaly et al, Chest, 2010
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TELOMERES ARE SHORT IN PULMONARY FIBROSIS
Alder et al, PNAS, 2008
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TELOMERE EROSION AND HUMAN DISEASES
Mucocutaneous anomalies Aplas^c
anemia
Pulmonary fibrosis
Liver cirrhosis
EtOH HCV
Virus Chemicals
Smoking
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May/90 Feb/92
Hem
atocrit
16%
48%
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SEX HORMONES INCREASE TELOMERASE ACTIVITY IN CULTURED HUMAN LYMPHOCYTES
(n=10)
900
600
Telomerase Ac^vity
(TPG
units)
Methyltrienolone (synthe^c)
300
0
Nandrolone 6β-‐Hydroxy-‐ Testosterone
β-‐Estradiol
0 0.5 5μM 0 5μM 0 5μM 0 1μM
Androgens
Calado et al, Blood, 2009
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SEX HORMONES MODULATE TERT ACTIVITY
+ Estrogen
Estrogen receptors
TERT gene
Estrogen- estrogen receptors
Estrogen-response element
mRNA
TERT protein
Telomerase activity
OH
O Estradiol
Androgens OH
O
CH3
O
O – C – CH2(CH2)7CH3 O
OH
O
Aromatase
Calado et al, Blood, 2009
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TELOMERE LENGTH MEASUREMENT
Calado & Young, NEJM 2009
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TELOMERE LENGTH MEASUMENT Southern blot
• “Gold standard” • Time-‐consuming (3 to 4 days)
• Limited number of samples
Mol
ecul
ar W
eigh
t
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TELOMERE LENGTH MEASUREMENT Flow-‐FISH
• Telomere length in cell subsets
• Time-‐consuming
• Sensi^ve technique
interphase
metaphase
Courtesy of Peter Lansdorp, UBC
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TELOMERE LENGTH MEASUREMENT qPCR
• High-‐throughput (hundreds to thousands of samples/day
• Reproducible • Telomere length given in a “ra^o”
Cawthon, NAS, 2002
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TELOMERE LENGTH MEASUREMENT qPCR
Cawthon, NAS, 2002
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TELOMERE LENGTH MEASUREMENT qPCR
Cawthon, NAS, 2009
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TELOMERE LENGTH MEASUREMENT qPCR
qPCR for telomere length using the Rotor Gene-Q system
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TELOMERE LENGTH MEASUREMENT qPCR
Telomere length of 300 healthy subjects from 0 to 99 years
Correlation between qPCR and Southern blot
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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
University of São Paulo: Raquel Paiva
Barbara Santana Diego Clé
Flavia Donaires Felipe Silva
Natália Scatena Fernanda Gu^errez
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