FSHD Disease Mechanisms and Models
Silvère M. van der Maarel, Ph.D.
Leiden University Medical Center
An Integrative Approach
Patient
Organizations
FSHD families
Regulatory
Bodies
Industry
Funding
Agencies
Basic &
Medical
Scientists
Modern Research is Teamwork!
Nijmegen
(Padberg/
van Engelen)
Seattle
(Tapscott/
Miller)
Paris
(ButlerBrowne)
Rochester
(Tawil)
Nice
(Sacconi/D
esnuelle)
FSHD AND CHROMATIN DISEASE
Richard Lemmers
Judit Balog
Yvonne Krom
Peter Thijssen
Lucia Clemens-Daxinger
Amanda Mason
Marlinde van den Boogaard
Bianca den Hamer
Kirsten Straasheijm
Patrick van der Vliet
Modern Research is Teamwork!
Geraldi Norton Foundation
& the Eklund Family
George & Jack Shaw
& the Shaw Family Foundation
FSHD and the Fields Center
• Fields Center was established in 2007:
– Strategic Alliance to create a clinical/scientific network
between Rochester-Leiden-Seattle-Nijmegen-Nice
– Expedite Research and Therapy Development
– Non-exclusive
–
–
–
–
–
Protocols freely available
Sharing resources
Standards for Registries
Standards of care, diagnosis
50+ publications
– www.urmc.rochester.edu/fields-center/‎
FSHD at the LUMC
• Long tradition of:
– Genetic research
– Molecular and cellular biology
– DNA diagnosis
– Assistance in diagnosis
FSHD Genetics
23 pairs
For most families, FSHD
is an autosomal
dominant disorder with
incomplete penetrance
transmission
Genetic error
3.2 billion elements
25,000 genes
The Central Dogma of Biology
NUCLEUS
DNA
replication
RNA
transcription
CYTOPLASM
translation
Proteins
How much DNA?
Each cell contains DNA, how much?
6.5 ft of DNA in each nucleus !
Gene regulation: on/off switch
ON
OFF
Breakthrough in 2010
• FSHD is caused by the inappropriate production of
the DUX4 protein in muscle of FSHD individuals
(Lemmers et al., Science 2010)
…“If we were thinking of a collection of the genome’s greatest
hits, this would go on the list,” said Dr.Francis Collins, a human
geneticist and director of the National Institutes of Health.
THE FRONT PAGE
D4Z4, at the heart of FSHD
• Most individuals with FSHD have a
contraction of a repeated DNA
structure on chromosome 4
• This structure is called D4Z4
• Contraction leads to a change in the
3D organization and regulation of
D4Z4
• Some patients have a similar change
in 3D structure and regulation of D4Z4
in the absence of contraction (FSHD2)
• These changes lead to the production
of a protein called DUX4 which should
not be expressed in skeletal muscle.
PAS
11-100
Contraction
(FSHD1)
?
(FSHD2)
PAS
or
1-10
PAS
11-100
AAAAAA
DUX4
Primary disease mechanism in FSHD1
Mutations in SMCHD1 cause FSHD2
• For a long time the existence of
contraction-independent FSHD
was questioned
• We showed that changes in 3D
chromatin structure of D4Z4
seen in FSHD1 patients can
segregate in FSHD2 families
• This led to the identification of
mutations in SMCHD1
underlying 85% of FSHD2
PAS
11-100
Contraction
(FSHD1)
SMCHD1
(FSHD2)
PAS
or
1-10
PAS
11-100
AAAAAA
DUX4
Mutations in SMCHD1 explain 80% of FSHD2
ATPase-ab
ATG
Protein
1
cDNA
2
3
5
4
6
7
8
9
11
10
12
13
14
* M(Rf1196) S+M1(Rf739)
S(Rf744)?
S(Rf833)
S(Rf702) N(Rf391)
* * S(Rf644)
M(Rf1002)
GlyArg
*
M(Rf1247)
* N(Rf1101)
* M2(Rf300)
* AsnSer N(Rf922)
S+D1(Rf1033)
HisAsp
*
CysArg
S1(Rf696)
D(Rf975)
M(Rf866) M(Rf1142)
M(Rf742,
Rf959)
*
D2(Rf393)
GlyGlu LeuPhe
M(Rf1021)
TyrCys
ThrMet
21
23
22
*
N(Rf909)
S(Rf947)
N(Rf947)*
41
24
26
25
27
28
29
30
31
32
33
34
S(Rf1110)
S(Rf725)
M(Rf941)
S4(Rf649, Rf1203)
S3(Rf874, Rf844, Rf580)
S(Rf879)
S(Rf936,Rf878)
*
N(Rf1199)
Hinge-ab
STOP
42
43
*
N(Rf400)
44
45
M(Rf385)
ArgGly
46
+0
+1
+2
+0
-1
-2
48
Variants
Domains
S1-S5 Splice site
Hinge
ATPase
M1-M4 Missence
* Disruption ORF
18
20
19
S+M3(Rf399)
M(Rf917)
LeuPro
*
S(Rf849)
N(Rf1002)
*
N(Rf562)
S(Rf549)
35
36
37
38
S2(Rf268)
M(Rf676)
SerAsn
39
40
PAS
47
D1-D2 Deletion
N Nonsense
17
*
D(Rf753)
S(Rf1043)
3’ Splicesite
16
M4(Rf683)
M(Rf1126)
PheSer
MetIle
*
S5(Rf392, Rf1014) D(Rf1121)
N(Rf629)*
Exons
5’ Splicesite
15
SMCHD1 binds to D4Z4 and represses DUX4
SMCHD1
(FSHD2)
SMCHD1
(FSHD2)
SMCHD1
(FSHD2)
SMCHD1
(FSHD2)
SMCHD1
(FSHD2)
SMCHD1
(FSHD2)
SMCHD1
(FSHD2)
SMCHD1 binds to D4Z4 and represses DUX4
?
(ICF3)
DNMT3B
(ICF1)
SMCHD1
(FSHD2)
ZBTB24
(ICF2)
Chromatin structure:
establishment and maintenance?
Trancription?
Genome-wide
effects?
(Macrosatellite) repeat DNA: silenced chromatin
Repeat length dependent epigenetics?
D4Z4 contraction (FSHD1): impaired silencing
?
(FSHD2)
Clinical Variability
• Large variability in onset, progression and
severity;
• Between families and within families;
• What protects gene-carriers from becoming
affected?;
• Environmental factors?
• Role for SMCHD1?
Landouzy and Dejerine
• Genetic modifiers of D4Z4?
The FSHD2 gene is a modifier for FSHD1
No FSHD
FSHD1 or 2
FSHD1+2
severity
Families with FSHD1 and FSHD2
Sacconi et al., Am J. Hum. Genet. 2013
Consequences of DUX4 in muscle
• DUX4 activates germline and
early stem cell programs in
skeletal muscle;
• DUX4 induces elements that
create an inflammatory
reaction to muscle;
• At the same time, DUX4
suppresses some patways of
our immune system;
• These pathways and
programs lead to muscle
atrophy and cell death.
Geng et al., Dev. Cell 2012
What is next?
• Translational research:
– Increase our understanding of disease
mechanism;
– Translate our findings to models that
allow validation of the mechanism;
– Identify potential targets for therapy;
– Apply disease models for drug
screens;
– Validate hits from drug screens;
– Clinical trials
Yeast models
Muscle cell culture models
Mouse models
Disease Models for FSHD
Any disease model for FSHD should take into account
the bursts of expression pattern of DUX4
DUX4 at the tipping point
Tapscott lab, Seattle
Models for Translational Research
• Cellular and Animal Models:
– Isogenic myoblast clones with or without mutation (coll.
G. Butler-Browne and V. Mouly);
– Mouse models with normal-sized and FSHD-sized D4Z4
arrays;
DUX4-positive nuclei in affected clones only
(De Krom et al., Am J. Path. 2012)
DUX4-positive nuclei in FSHD mouse
(De Krom et al., PLoS Genet, in press)
PAS
11-100
Towards Therapy
• Current knowledge of disease
mechanism already gives leads
to intervention:
– Can we prevent the change in 3D
structure and regulation of D4Z4?
– Can we prevent the production of
DUX4 at RNA or protein level?
– Can we prevent the action of
DUX4?
– Can we treat the downstream
pathways of DUX4?
?
PAS
or
1-10
PAS
11-100
?
AAAAAA
?
DUX4
?
?
Take home messages
• There are at least two genetic forms of FSHD
– The common form FSHD1 (1-10 D4Z4 units)
– The rare form FSHD2 (mostly mutations in SMCHD1)
• Both forms can be genetically confirmed with great
accuracy
• Both forms have an identical disease mechanism
– Expression of DUX4 in skeletal muscle
• Some individuals have FSHD1 and FSHD2
– Individuals have more variable disease severity
• We have uncovered the mechanistic basis of FSHD
How much longer?
• Not possible to predict, but we have the essentials:
– We have a plausible disease mechanism
– We know the target
– We have (animal) models to test the therapeutic
molecules
• The DMD gene was identified in 1987 and only now
there is some hope, but:
– We have learned from the past: translational research
– In the meantime the life expectancy for DMD has
dramatically increased: quality of care