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Prof. Inderjeet Dokal
TELOMERES AND TELOMERE BIOLOGY IN AGING
20th September 2013
Prof Inderjeet Dokal
Telomeres cap the ends of all mammalian chromosomes
(TTAGGG)n
Page 4
A telomere measures approximately, 1 / 100,000 of a meter. Life Length’s technology is able to detect differences as small as 1 / 10,000,000 of a meter.
A picture of human telomeres inside living cells. The greater the fluorescent intensity, the longer the telomeres and the healthier the individual.
Telomeres are maintained by the enzyme telomerase
Telomeres and telomerase
Germ line
Telomere binding proteins are also essential for telomere maintenance
DNA damageresponse
telomeredysfunction
absence of telomerase
cancer
no p53
loss of telomere binding proteins
p53
apoptosis
cellularsenescence
stem cell defects
premature aging
Shelterin complex
Mean human telomere length decreases with increasing age
Canela et al, PNAS 2007
disease risk30 70
Telomere length: biomarker of cellular aging
(years)
Severe DC variants
Hoyeraal-Hreidarssonsyndrome (HH)
Revesz syndrome
Classical DC
Cryptic DC variants
Aplastic anaemiaMyelodysplasia
Pulmonary fibrosis
Different faces of dyskeratosis congenita (DC)
Features of premature aging; cancer predispositionCaused by defective telomere maintenance
“The inherited telomereopathies”
Dyskeratosis congenita (DC):Mucocutaneous features of aging
Somatic abnormalities in DC: a disease of premature aging
Abnormality % of patients
Epiphora 30.5Learning difficulties/development/mental retardation 25.4Pulmonary disease 20.3Short stature 19.5Extensive dental caries/loss 16.9Oesophageal stricture 16.9Hair loss/grey hair/sparse eyelashes 16.1Hyperhiderosis 15.3Malignancy 9.8Intrauterine growth retardation 7.6Liver disease/peptic ulceration/enteropathy 7.3Ataxia 6.8Hypogonadism/undescended testes 5.9Microcephaly 5.9Urethral stricture/phimosis 5.1Osteoporosis/aseptic necrosis/scoliosis 5.1Deafness 0.8
Normal bone marrow DC aplastic bone marrow
Haematopoietic stem cell failure in DC
Premature aging of fibroblasts in DC
Normal Dyskeratosis congenita
Chromosomal rearrangements in DC
0
2
4
6
8
10
12
14
16
0 10 20 30 40 50 60 700.00.20.40.60.81.01.21.41.61.82.0
0 10 20 30 40 50 60 70
-10
-8
-6
-4
-2
0
2
4
6
0
2
4
6
8
10
12
14
16
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Age (years)T/
S ra
tio
TRF
(kb)
tel
(kb)
A B C
D E
N DK TR RT TN N DK TR RT TN N DK TR RT TN
Age (years)
TRF
(kb)
T/S
ratio
TELOMERE LENGTH IN DIFFERENT GENETIC DC SUBGROUPS
(tim
e)DKC1 TERCTERTNOP10NHP2TINF2TCAB1CTC1RTEL1
C16orf57
DC disease pathophysiology
Cajal body
TCAB1
Capping complex
STN1
TEN1
CTC1
Genes mutated in dyskeratosis congenita and related bone marrow failure syndromes – the telomereopathies
C16orf57
snRNA processing
Helicases
RTEL1
Complexes involved in telomere maintenance
disease risk30 70
What about “normal”
individuals without mutations in telomere maintenance genes?
TELOMERE SHORTENING AND HUMAN DISEASETELOMERE SHORTENING AND HUMAN DISEASE
1. Hereditary syndromes with short 1. Hereditary syndromes with short telomerestelomeresDyskeratosis congenita (very short telomeres)Dyskeratosis congenita (very short telomeres)Fanconi anaemia + othersFanconi anaemia + others
2. Disease states with short telomeres2. Disease states with short telomeresAtherosclerosis Atherosclerosis Heart failureHeart failureLiver cirrhosisLiver cirrhosis
TELOMERE SHORTENING AND HUMAN DISEASETELOMERE SHORTENING AND HUMAN DISEASE
3. Short telomeres predictive of:3. Short telomeres predictive of:AgeAgeCardiovascular diseaseCardiovascular diseaseDementiaDementia
4.Factors that result in accelerated telomere4.Factors that result in accelerated telomereshorteningshortening
SmokingSmokingAlcoholAlcoholPerceived stressPerceived stressObesityObesity
Telomere shortening correlates with decreased overall survival
Cawthon et al, Lancet 2002
Patients with short telomeres have a mortality rate twice that of those with longer telomeres
Heart disease mortality increased 3X
Infectious disease mortality increased 8X
Van der Harst et al 2007
Short telomeres in Chronic Heart Failure (CHF)
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Short telomeres play a central role in the development of age‐related diseases
Short Telomeres
WEAK
INMUNITY /
ASTHMA /
ALLERGIES
STEM CELLS
DNA
Damage
Senescence &
Apoptosis
CANCER
BLOOD
CELLS
CONNECTIVE TISSUEWRINKLES,
ARTHRITIS,OSTEOPOROSIS
REPR
ODUCT
IVE
ORG
ANSINFERTILITY,
MENOPAUSE &
«ANDROPAUSE»
BETA CELLS
DIABETES
HEART /
ARTERIES
CARDIOVASCULAR
DISEASE,
HYPERTENSION &
ATHEROSCLEROSIS
DNA
Damage
Senescence &
Apoptosis
BRAIN
CNS (CENTRAL NERVOUS SYSTEM)
DISEASES ALZHEIMER’S, PARKINSONS,
DEMENTIA
RETINA
MACULAR
DEGENERATION
DISEASE LEVEL
ORGAN
LEVEL
CELLULAR
Source: Recharge Biomedical Clinic & DR. Ed Park
In this era of personalized medicine, should we use telomere length as a diagnostic tool?
Whole genome sequencing
Disease gene identification
Potential for tailored therapeutic intervention
Still expensive-bioinformatics
Too much information!
Telomere length determination
Identify inherited telomere disorders
Therapeutic intervention-BM transplantation
Identify “healthy”
individuals with acceleratedbiological aging
Potential to enable early intervention-lifestyle modifications to reduce cellular stress
Inexpensive
Common methods to determine telomere lengthTelomere Flow-FISH
Quantitative telomere-PCR
Telome Health
•Intensity of fluorescence signals correlate directly with telomere length
•Ability to process large sample volumes
•Only gives telomere length of a population of cells-
cannot examine length of individual telomeres
•Cannot inform on spatial distribution of cells with the shortest telomeres in a tissue sample
•Does not work well on solid tissues
•Quantity of telomere PCR product correlates directly with telomere length
•Ability to process large sample volumes
•Only gives telomere length of a population of cells-
cannot examine length of individual telomeres
•Cannot inform on spatial distribution of cells with the shortest telomeres in a tissue sample
Quantitative telomere-FISH
•Intensity of signals correlate directly with telomere length
•Only method that could detect the critically shortest telomeres in a cell
•Measures telomere length on metaphase chromosomes or nuclei
•Measures telomere length on clinical tissue samples-valuable spatial information intact
•Automated microscopic imaging techniques enable processing of large sample volumes
ATCCCAATCCC
Telomeric peptide nucleic acid (PNA)-FISH
TTAGGGTTAGGGTTAGGG
(AATCCC)4
PNA probe
TTAGGGTTAGGGTAGGGAATCCC
chromosome
Hybridizationof PNA probeto chromosomes
AATCCC
Rh Rh
GGGATTGGGATTGGGATTCCCTAA
(AATCCC)4Rh
CCCTAAGGGATTGGGATTGGGATTCCCTAACCCTAA
RhRh
•Only a few critically short telomeres needed to push human cells
into senescence
•% shortest telomeres detect more differences between individuals
than average length
•% shortest telomeres show higher dispersion with increasing age
•% shortest telomeres could be a marker for environmentally induced damage
Telomere length measurements: need to determine length of shortest telomeres
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Life Length’s cutting edge technology (TAT)
Most valuable and scalable technology in the market, allowing the determination of telomere length at the cellular level, both
from blood and
tissue samples, with a level of precision not offered by other techniques and multiple sample processing in a short period of time
Telomere length assessment ‐
industry analysis
Scalability
Assessment
value
Flow cytometry
Q ‐
PCR
Southern Blot
STELA
Q ‐
FISH
Multiple
sample
processing
Single
sample
processing
Entire cell
population
analysis
Individual
cell level
analysis
Enhanced
visualization and
improved data
output
Life Length’s technology
High Throughput
Q-FISH (HT Q-FISH)
Telomapping
• Same as Q‐FISH
(determination of
telomere length
on chromosomes
from blood
samples by
hybridization of
specific probes
using fluorescent
microscopy) with
high throughput,
multiple
simultaneous
samples
• Determination of
telomere length
on chromosomes
from tissue
samples by
hybridization of
specific probes
using fluorescent
microscopy
• Average
length and
percentage
of short
telomeres in
individual
cells
• Processes
multiple
samples
• High resolution
to 200 base
pairs
• Individual cells
• Average
length and
percentage
of short
telomeres in
individual
cells within a
tissue
• Visualization
of telomeres
in the
context of
their native
location
within tissue
• Similar to
Q‐FISH• High resolution
to 200 base
pairs
• Individual cells
Description Type of
MeasurementSpeed SensitivityTAT
TAT
TAT is considered a significant improvement over current telomere measurement techniques because of the value associated with
measurement of individual telomeres and percentage of short telomeres compared to the average telomere length
Determination of telomere length in cells within the context of a tissue is also of relevance. An equivalent technology is
not currently available to measure either telomere length or telomerase activity
Inherited factors
mutations in telomere maintenance genescertain inherited cancer syndromesmutations in DNA repair pathways
Environmental factors
carcinogensROS
other stressors?
Accelerated telomere shortening/dysfunction
increased DNA damage
p53 Loss of p53
Stem cell exhaustionPremature aging
Increased cancer incidence
Conclusions
Telomere length measurements as a predictor of increased cellular and disease risk arising from short telomeres
Acknowledgements
Slides
Life Length
Sandy Chang
Tom Vulliamy
Funding
Wellcome Trust
MRC