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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
TIFF (Uncompressed) decompressorare needed to see this picture.
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
QuickTime™ and aTIFF (Uncompressed) decompressor
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QuickTime™ and aTIFF (Uncompressed) decompressor
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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)
QuickTime™ and aTIFF (Uncompressed) decompressor
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QuickTime™ and aTIFF (Uncompressed) decompressor
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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.
QuickTime™ and aTIFF (Uncompressed) decompressor
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QuickTime™ and aTIFF (Uncompressed) decompressor
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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