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)

Page 24© 2012 Life Length v3.0  All rights reserved. Reproduction of this document or any portion thereof without prior written consent is prohibited.

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

Page 29© 2012 Life Length. All rights reserved. Reproduction of this document or any portion thereof without prior written consent is prohibited.

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