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
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
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
20.
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.
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
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
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
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