Dyskeratosis congenita

Philip J Mason

Departments of Internal Medicine and GeneticsWashington University Medical School

St Louis Missouri

Dyskeratosis congenita (DC)

Different genetic forms - X-linked form and dyskerin- autosomal dominant form and anticipation

Telomerase

Mouse models of X-linked DC- Dyskerin important in cell proliferation- Role of dyskerin in DNA damage response

Variable expression of DC mutations- aplastic anemia- pulmonary fibrosis- leukemia

Dyskeratosis congenita

Skin pigmentation, nail dystrophy, mucosal leukoplakia

Most patients have bone marrow failure, is responsible for death in 85% of cases

Heterogeneous set of other abnormalities include pulmonary disease, short stature, learning difficulties, predisposition to malignancy

Stem cell disorder

Genetically heterogeneous

Dyskeratosis congenita

Malignancy

Epithelial cancers of GI tract

MDS/AML

DCR 039 DCR 070 DCR 073

DCR 101

DCR 108

Patterns of inheritance in DC

11%

31%

4%8%

46%

X-linked dyskerin

Autosomal recessiveNOP10, NHP2, TERT

Autosomal dominantTERT,TERC

Sporadic TINF2

Unknown

Genetics of Dyskeratosis Congenita

X-linked DC locus within Xq28

HpaII- + - + - +

HpaII

(CAG)n

X-inactivation in DC carriers

Exon1 of Humara gene Xq12

Identification of a deletion in an X-linked DC patient

X-linked DC caused by mutations in DKC1 encoding dyskerin

TruB PUA NLS NLS poly-lysine

Dyskerin Human

Rat NAP 57

Cbf5p Yeast

Nop60B/mfl Drosophila

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

A2V

Q31KQ31E F36VL37de

I38TK39EP40RE41KK43ET49M

IVS2 473 C>G

R65TT66AT67IH68QL72Y

S121G

L398PG402EG402RT408IP409LS420Y

M350TM350I

A353V**A353VT357AD359NP384LP384SA386T

1551G>A

1494 insAAG

X-linked dyskeratosis congenitaHoyeraal-Hreidarsson syndromepolymorphisms

2kb deletion

S280R

Mutations in the DKC1 gene

IVS1 +592C>G

URR -142C>G

1461C>T

IVS14 -72G>T

IVS6 +40T>G

K314RL317FL321VR322Q

R158W*

* recurrent

common recurrent de novo mutation**

P10L

R. Rashid et al. Molecular Cell 2006

Clustering of Mutations in Dyskeratosis Congenita

dyskerin

GAR1P

NHP2P

NOP10P

snoRNP

Dyskerin associates with H/ACA small nucleolar RNAs

H ACA

CH3 CH3 CH3CH3 CH3CH3

5.8S

41S

32S21S

12S18SE 28S

18S

3’ ETS18S 5.8S

5’ ETS ITS1 ITS2

47S Pre-rRNA

Box H/ACA snoRNPs

•Pre-rRNA modification

45S

28S

Pre-ribosomesProteinProteinsynthesissynthesis

Mature

ribosomes

•Pre-rRNA processingPre-rRNA processing

Ribosome assemblyRibosome assembly

rDNAPol I transcription

Box H/ACA snoRNPs

The Role of Dyskerin in Ribosome Biogenesis

Dyskerin

Dyskerin

5'

H ACA5’ 3’

rRNA

H/ACA snoRNA

Box H Box ACA

hTERC

mTerc

ANANNA

ACA

ACA

AGAGGA

ACAGGA

ACA

snoRNA

3’

Telomerase RNA

H ACA

5’ Box H/ACA domain

Pseudoknotdomain

Hypervariable

paired region

CR4-CR5 domain

CR7 domain

Template

3’

Telomerase RNA is a H/ACA snoRNA

Pseudoknot domain

Telomerase Mutations in Autosomal Dominant Dyskeratosis Congenita

Polymorphism

small deletion

bp change

large deletion

Pathogenic mutationsCR4-CR5 domain

Box H/ACA domain

Template

TERC

TERT

2001

2005

NOP10 and NHP2 mutated in rare autosomal recessive cases

Walne et al 2007 - NOP10 homozygosity mapping in large consanguineous family - affected members have mutation in conserved aa

NHP2 - 3 mutations found by sequencing Vulliamy et al 2008None found in GAR1

Mutations in TINF2 cause severe DC Savage et al 2008 Found mutation in TINF2 in family withAD DC. Then found them in 4 of 8 AD DC probands with no mutations in DKC1, TERT or TERC

Walne et al 2008 found TINF2 mutations in 33/175 previously uncharacterized cases of DC SevereNearly all de novoTightly clustered - 21 affect residue R282 (14H and 7C)

all within 18aa.

TERTdyskerin

GAR1P

NHP2P

NOP10P

dyskerinGAR1P

NHP2P

NOP10P

others

snoRNP Telomerase RNP

Dyskerin associates with H/ACA snoRNAs and Telomerase RNA (hTERC)

DC due to deficiency of telomerase or rRNA processing?

AD disease due to telomerase RNA

All DC patients have very shorttelomeres

Telomerase activity and TERC levelsdecreased in DC Dyskerin stabilizes TERC

TINF2 mutations cause DC

mTR-/- mice similar to DC Late generation mice have features of DC

Dkc1m mutant mice Hypomorphic mutant causes decreased production of dyskerin due to transcriptional interference - ribosome defects and some

features similar to DC

Other BMF syndromes Diamond Blackfan RPS19 Schwachman Diamond SBDS- nucleolar protein associated with snoRNAs and ribosomal proteins. Cartilage Hair Hypoplasia - mutations in RNA component of RNAse MRP involved in 5.8S rRNA processing

Dyskerin mutants in other species Mutations in yeast and Drosophila cause slow growth and ribosome defects - no association with telomerase

Ribosomes Telomeres

Re

lati

ve

Te

lom

ere

Le

ng

th

(% 4

n C

ell

Lin

e)

Patients with Dyskeratosis Congenita andBone Marrow Failure have Very Short Telomeres

(Flow FISH)

TERC mutations, n=11

0

2

4

6

8

10

12

16

18

0 20 40 60

DYSKERIN mutation, n=3DC no mutation, n=3

Age (years)

14

20

80 100

90%75%50%25%10%

Telomerase

Important in maintaining chromosome ends - solution to end-replication problem

Low levels in most somatic cells - higher in stem cells, cancer cells, germ cells

In somatic cells telomeres get shorter with each cell division

Telomeres are DNA/protein structures at the ends of chromosomes, needed to protect chromosome end from degredation and distinguish them from double stranded breaks. Telomere DNA consists of thousands of repeats of TTAGGG

The end replication problem

5’

5’

3’

3’

leading strand

lagging strand

5’3’

5’ 3’

5’3’

5’ 3’

5’

3’

5’3’

Telomerase action

TTAGGGTTAGGGTTAGGGTTAGGGTTA 3’

AATCCC 5’parental strand

lagging strand

AATCCC 5’ ATCCCAATTTAGGGTTAGGGTTAGGGTTAGGGTTA 3’

AATCCC 5’ ATCCCAATTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTA 3’

AATCCC ATCCCAATCCCAATCCCAATCCCAA 5’

TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTA 3’

DNA POLYMERASE

telomerase reversetranscriptase (TERT)

telomerase RNA(TERC)

Telomere Structure

Telomerase, senescence and cancer

Telomere length

Cell replication

Germ cells (telomerase+)

Stem cells (telomerase+)

Tumor cells (telomerase+)

Somatic cells (telomerase-)

senescence crisis

replicative senescence

p53-

Summary of DC mutations

Gene Inheritance Effects

DKC1 X-linked DC HH

TINF2 AD Severe sporadic

NOP1 AR DC rare

NHP2 AR DC rare

TERC AD DC AA PF MDS

TERT AD DC AA PF MDS AML CLL others

Inheritance of short telomeres - Anticipation

30

52

DCR 082

39

1210

DCR 063

• asymptomatic• hair loss/greying• borderline neutropenia

• diagnosis made at made at younger age in succeeding generations

3 9 1322 17

68

47 48 46 40 2937

DCR 101

• asymptomatic• raised HbF• high MCV

Classical DC incl BMF

72 C->G

• high MCV

• mild anaemia• high MCV• high HbF

110-113 del GACT

Asymptomatic parents of AA patients also carry hTERC mutations

normal

asymptomatic carrier

aplastic anaemia

pancytopeniaPancytopeniaElphic appearance

Thrombocytopenia

9

61

60

20 20

Anticipation by shortening telomeres

Anticipation in family with TERT mutation

Armanios et al 2005

Armanios et al 200773 probands from familial PF registry6 (8%) had TERT (%) or TERC (1) mutations

Telomerase mutations and pulmonary fibrosis

Tsakiri et al 2007

Mapped causative gene to TERT in 2 families with PFScreened another 44 families with PF and 44 patients with IPF.4TERT 1 TERC 1TERT

Primary presentation was PF - some had mild anemia but nofeatures of DC

Telomere length, telomerase mutations and IPF

PF patients have short telomeres that cannot all be accounted for by known mutations

Cronkhite et al 2008

TERT mutations and Leukemia

Calado et al 2008 PNAS8% AML had TERT mutations

decreased telomerase activity6 had A1062T – 3.8X higher frequency than in normalsbut same as CEPH collection!

ASH 2008 not published10% CLL had hypomorphic TERT mutations

Make dyskerin mutations in mice that are copies of human mutations

Mice have very long telomeres - complete absence of telomerase - no abnormalities in early generations - get DC - like effects after 3 or more generations

So effects in early generations should not be due to telomerase defects

Telomerase defects should be seen after in later generations or aftercrossing with short telomere mice

Mouse models of X-linked DC - Rationale

10 11 12 13 14 MPP1

IRES neo

EcoRI HindIIIBamHI BamHI

G G A T G G G G T A T G T G T G A A C C A T G G A

Asp Gly Val STOP

Targeting construct - Del15

15

Telomeres snoRNAsTRAP

Telomerase and snoRNAs in wild type and Del15 ES cells

Proliferative disadvantage of cells expressing the 15 dyskerin protein in aging Dkc1+/15 heterozygous female mice.

Etoposide treatment induces an enhanced ATM-p53/p21 dependent DNA damage response in Dkc115 MEF cells

15 MEFs accumulate more Double strand breaks

Increased in high oxygen

0

1

2

3

4

5

6

7

WT 3% O2 15 3% O2 WT 21% O2 15 21% O2

H

2AX

fo

ci/c

ell

PD 2

PD 6

PD 12

PD 25

Liver Spleen BM

WT 15 WT 15 WT 15

-H2AX

-Actin

p53

15 mice show increased DNA damage response

Spleen

17 M

WT 15 WT 15 WT 15

-H2AX

-Actin

-H2AX

12 M4 M

Increased DNA damage response is related to ageing

N-Acetyl-l-Cysteine (NAC)

NAC is a derivative of the dietary amino acid l-cysteine.

NAC has a high affinity for lung tissue, which it supports through mucolytic and antioxidant action.

NAC also enhances glutathione production and plays a role in heavy metal detoxification

PD 1 3 7 11 15

NAC +

NAC -

NAC can partially rescue the growth disadvantage in female 15/+ MEF cells

100µM

3 weeks 12 weeks

NAC + NAC -

NAC can partially rescue the growth disadvantage in female mice

1mg/ml in drinking water

Summary

Dyskerin is essential for cell proliferation but not for cell survival

Dyskerin mutation causes slow growth due to a telomerase dependent increase in DNA damage at the telomere end - this is independent of telomere length

Hematopoietic stem cells with a dyskerin mutation have a functional defect that increases with age

The growth defect can be partially rescued with antioxidant

Science 30 March 2007

Telomeres shorten in somatic cells with each cell division – leads to replicative senescence

Cancer cells, germ cells and stem cells have telomerase activity.

Activation of telomerase genes is a crucial step in carcinogenesis

Core components are telomerase reverse transcriptase TERT and telomerase RNA TERC which contains a template for the synthesis of TTTAGG repeats

Telomerase has been purified from the ciliate Euplotes aediculatus – consists of a complex of TERT, TERC and protein p43

32 proteins have been found to be associated with human telomerase.

Size measurements show it is bigger than if it contained 1 TERT + 1TERC but smaller than if it contained all proposed proteins.

(Euplotes has millions of chromosome ends and ~300,000 molecules telomerase per cell) QuickTime™ and a

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Which proteins are in telomerase complex?Could be candidates for mutations that affect telomeraseIs telomerase a multimer? May see dominant negative effectsin dominant inheritance

TERTdyskerin

GAR1P

NOP10P

GAR1P

NHP2P

NOP10P

p23Hsp90

Tep1others

snoRNP

Telomerase RNP

Size measurement by sedimentation using a direct primer extension assay for activity

>60% activity in fractions 9 and 10. Thyroglobulin (669kD) in fraction 9.650 to 670 kD

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Glycerol gradients 10-40%

3 step purification scheme achieved 108 –fold enrichment

Step 1 immuno-affinity purification with sheep polyclonal antibody generated against a peptide antigen.HEK lysate incubated with antibody – then antibody-enzyme complex immobilized onto protein G agarose beads. Excess antigenic peptide was added allowing the enzyme to dissociate into solution.

100g cells from fermenter - about 10l cultureLysis buffer - 20mM HEPES-KOH 7.9, 2mM MgCl2, 300mM KCl10% glycerol, 1mM DTT, 1mM EDTA, 1mM PMSFDounced, Triton X-100 added to 0.1%, cleared.

Step 2 Substrate directed affinity purification

Known that dissociation of substrate primer 5’-(TTAGGG)3-3’ from enzyme is very slow Used affinity reagent 5’-Biotin-CTAGACCTGTCATCA(TTAGGG)3-3’ immobilizedon neutravidin beadsTelomerase from 1st step pre-cleared with unmodified neutravidin beads and 5’-CTAGACCTGTCATCA-3’ then treated with (TTAGGG)3 beads to capture Telomerase. Got 90% yield. Assayed by adding dNTPs

Step 3 Elution of active enzyme.

Because binding is to primer is so stable needed to develop rapid elution procedure. Exploited the finding that binding affinity between enzyme primer changes according to the sequence at the 3’end of the primer. GGG-3’ is most stable, TTA-3’ most unstable.

If only TTP and dATP is present the active enzyme should convert primer to end with TTA-3’ and thus be eluted.

Presence of TTP and dATP caused rapid elution. Only active enzyme complex eluted.

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1. Immunoaffinity purified telomerase2. Immunoaffinity purified telomerase after incubation 5h room temp3. Immunoaffinity purified telomerase after preclearing with unmodified neutravidin beads4. Telomerase left in solution after treatment with(TTAGGG)3 beads5. Telomerase eluted in 1h in absence of TTP/dATP6. Telomerase eluted in 10’ in presence of TTP/dATP

Purified telomerase same size as in crude lysate

Yield assessed by quantitative Northern blot.

From 50 fmol HEK-293 cells (100g) 250-300fmol telomerase (5-6 molecules per cell)

Given 30% yield this means 20-50 molecules per HEK-293 cell and purification of 108 fold (100ng from 100g)

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109 cells 50 20 10fmol

Purified telomerase digested with trypsin, peptides captured on cation exchange resin, eluted and analysed by nano liquid chromatography-tandem mass spectrometry.

Compared control elution (with TTAGGG3 - no telomerase activity)with protein eluted by TTP/dATP

Low levels of 6-8 proteins in control elution 5 observed consistentlyTubulin, actin, Y-box, hnRNPA1, hnRNP M

2 new proteins in telomerase sample hTERT and dyskerin. These were the only proteins specifically enriched in telomerase sample.

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Conclude - active telomerase consists of 3 components TERT, RNA and dyskerin.Mutations in these 3 components cause dyskeratosis congenita.

Combined mass of TERT (127kD), TERC (153kD) and dyskerin (57kD) = half the MW (650-670kD)

Therefore 2 molecules of each

But Other proteins may be involved in biogenesis, trafficking, recruitmentto the telomere and degradation.

Only TERT TERC and dyskerin in catalytically active telomerase.

Mutations in these 3 components cause DC - no others so far

No dominant negative mutations in TERT or TERC seen so far