Silvère M. van der Maarel, Ph.D. Leiden University Medical Center

FSHD Disease Mechanisms and Models

An Integrative Approach

Patient Organizations

Regulatory Bodies

Funding Agencies

Basic & Medical

Scientists

Industry

FSHD families

Modern Research is Teamwork!

Seattle (Tapscott/

Miller)

Rochester (Tawil)

Nice (Sacconi/Desnuelle)

Nijmegen (Padberg/

van Engelen) Paris

(Butler-Browne)

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

• 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 and the Fields Center

• Long tradition of: – Genetic research – Molecular and cellular biology

– DNA diagnosis – Assistance in diagnosis

FSHD at the LUMC

FSHD Genetics

For most families, FSHD is an autosomal dominant disorder with incomplete penetrance

23 pairs 25,000 genes

transmission

Genetic error

3.2 billion elements

The Central Dogma of Biology

DNA RNA Proteins

replication

transcription translation

NUCLEUS CYTOPLASM

How much DNA?

6.5 ft of DNA in each nucleus !

Each cell contains DNA, how much?

Gene regulation: on/off switch

ON

OFF

• FSHD is caused by the inappropriate production of the DUX4 protein in muscle of FSHD individuals (Lemmers et al., Science 2010)

Breakthrough in 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

• 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.

D4Z4, at the heart of FSHD

AAAAAA

DUX4

11-100

1-10

11-100

Contraction

(FSHD1)

?

(FSHD2)

or

PAS

PAS

PAS

Primary disease mechanism in FSHD1

• 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

Mutations in SMCHD1 cause FSHD2

AAAAAA

DUX4

11-100

1-10

11-100

Contraction

(FSHD1)

SMCHD1

(FSHD2)

or

PAS

PAS

PAS

Mutations in SMCHD1 explain 80% of FSHD2

M(Rf1196) GlyArg

M1-M4 Missence

+0

+1

+2

S1-S5 Splice site

D1-D2 Deletion

Exons

5’ Splicesite Variants Domains

Hinge

ATPase +0

-2

-1

3’ Splicesite

2 1 5 7 6 8 9 10 3

ATG ATPase-ab

Protein

cDNA 4

S+D1(Rf1033)

D2(Rf393)

S+M1(Rf739)

M2(Rf300) CysArg

13 20 17 18

S1(Rf696)

15 19 14

S2(Rf268)

43 45 41 42 47 48 44

PAS Hinge-ab STOP

36 37 32 39 34 40

S5(Rf392, Rf1014)

31 33

M4(Rf683) PheSer

26 29 23 27 24 25

S3(Rf874, Rf844, Rf580) S4(Rf649, Rf1203)

30 35 22 21

11 16

46

38

12

28

* * *

* *

Disruption ORF *

S+M3(Rf399)

N Nonsense

S(Rf833) S(Rf702)

N(Rf909)

S(Rf879)

S(Rf1043)

S(Rf947)

S(Rf549)

S(Rf849)

S(Rf1110)

S(Rf744)?

D(Rf1121)

N(Rf400)

N(Rf947)

N(Rf629)

N(Rf1101) N(Rf922)

S(Rf936,Rf878)

N(Rf1002)

M(Rf742, Rf959) TyrCys

M(Rf1021) ThrMet

M(Rf1126) MetIle

M(Rf385) ArgGly

M(Rf941)

* * * M(Rf676)

SerAsn

* *

*

N(Rf562) * D(Rf975) M(Rf866) GlyGlu

M(Rf1002) AsnSer M(Rf1247)

HisAsp

N(Rf391) * S(Rf644)

M(Rf917) LeuPro

S(Rf725)

N(Rf1199) * D(Rf753)

M(Rf1142) LeuPhe

*

*

*

*

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

Repeat length dependent epigenetics? D4Z4 contraction (FSHD1): impaired silencing

(Macrosatellite) repeat DNA: silenced chromatin

SMCHD1 (FSHD2) ZBTB24

(ICF2)

Chromatin structure: establishment and maintenance?

Genome-wide effects?

Trancription?

DNMT3B (ICF1)

? (FSHD2) ?

(ICF3)

Clinical Variability

• Large variability in onset, progression and

severity;

• Between families and within families;

• What protects gene-carriers from becoming

affected?;

• Environmental factors?

• Genetic modifiers of D4Z4?

• Role for SMCHD1?

Lan

do

uzy

an

d D

ejer

ine

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

• 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.

Consequences of DUX4 in muscle

Geng et al., Dev. Cell 2012

• 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

What is next?

Mouse models

Muscle cell culture models

Yeast models

Disease Models for FSHD

Any disease model for FSHD should take into account the bursts of expression pattern of DUX4

Tapscott lab, Seattle

DUX4 at the tipping point

• 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;

Models for Translational Research

DUX4-positive nuclei in FSHD mouse (De Krom et al., PLoS Genet, in press)

DUX4-positive nuclei in affected clones only (De Krom et al., Am J. Path. 2012)

• 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?

Towards Therapy

AAAAAA

DUX4

11-100

1-10

11-100

or

PAS

PAS

PAS

?

?

?

?

?

• 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

Take home messages

• 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

How much longer?