Fragile X-associated Tremor/Ataxia

Syndrome (FXTAS)
What our children teach us
IV Congreso Internacional del Sndrome X Frgil
Associaci Catalana SXF
October 10, 2015
Paul J Hagerman MD. PhD
Professor, Department of Biochemistry
and Molecular Medicine,
Investigator, UC Davis MIND Institute
University of California, Davis, School of
Medicine

Adult-onset problems among

premutation carriers of the fragile X gene
Es necesario
explicar claramente
su problema

Mothers of children with FXS:

Child with fragile X

syndrome

Mothers of children with FXS:

problems with their own (carrier) fathers
Frequent falls/ balance problems
Difficulty writing, eating
Memory loss
Numbness/tingling in hands legs
Loss of bladder/bowel control

Child with fragile X

syndrome

Fragile X-associated tremor/ataxia

syndrome (FXTAS)
Case DR: Premutation carrier with 89 CGG repeats, identified through
two grandchildren with fragile X syndrome.
First identified case of a carrier grandfather with progressive
neurological dysfunction

Core features
Tremor Onset in right hand at
age 54, left hand within two
years; writing illegible at 58 yr;
retired early as an electrician at
58 yr.
Ataxia Progressive difficulty
with balance and gait; frequent
falls

Core clinical features of FXTAS

Non-resting
tremor

Gait ataxia

Associated forms of clinical involvement

- Peripheral neuropathy

- Cognitive decline
- Autonomic dysfunction
- Anxiety, mood instability
- Parkinsonism

Core clinical features of FXTAS

Non-resting
tremor

Gait ataxia

Associated forms of clinical involvement

- Peripheral neuropathy

- Cognitive decline
- Autonomic dysfunction
- Anxiety, mood instability
- Parkinsonism

Prior diagnoses for individuals

with FXTAS
Sixty-two patients FXTAS and family history of fragile X
syndrome (55 men, 7 women; age of onset, 60.2 7.1 years)
56 sought medical help 98 prior diagnoses
Miscellaneous
16%
Cerebrovascular
10%
Dementia
13%

Ataxia
17%

Parkinsonism
24%

Tremor
20%

Hall et al. (2005) Neurol 65:299

Penetrance of FXTAS in families with

known fragile X syndrome
Approximately 40% of male premutation carriers (>50 yr)
have combined tremor and ataxia
The percent with clinical features increases with age
- males: 17% (50s)
75% (80s)
- far lower in females Jacquemont et al. (2004)
Screening studies find the premutation in 2 5% of
unexplained ataxia cases over 50 yr
Jacquemont et al. (2006)

Women with FXTAS have less severe

involvement than men
FXTAS is less frequent in women: 16% vs >50%
(Jacquemont et al 2004; Coffey et al 2008; Rodriquez-Revenga et al
2009)

Less severe white matter disease; less severe brain

atrophy; less frequent MCP sign
(Adams et al 2007)

Less frequent (rare) dementia

(Seritan et al 2010; Schneider et al 2014)

When dementia does occur in women it is associated

with Alzheimer changes in brain pathology
(Tassone et al 2012)

 Medical History 146 women carriers

Coffey et al., 2008 AJMG

**Fishers exact test for 22 contingency table analysis p<0.05

FXTAS: Imaging correlates

Brunberg et al. (2002) AJNR 23:1757-1766
MRI T2-weighted image
Male 75y

MCP sign ~60% of cases

Increased signal (T2/FLAIR) in cerebral white matter
and middle cerebellar peduncles (MCP)
Moderate/severe cortical atrophy/ ventricular enlargement
correlates with CGG repeat length

What is causing FXTAS?

The fragile X gene is turned off in the

full mutation range
Typical
(CGG)

< 45

Full mutation
(CGG)

> 200

mRNA

FMRP
Clinical Typical

Fragile X syndrome

The fragile X gene is too active in the

premutation range
Typical
(CGG)

< 45

Premutation
(CGG)

55 - 200

Full mutation
(CGG)

> 200

mRNA

FMRP
Clinical Typical

Fragile X syndrome
Primary Ovarian Insufficiency (POI)
FXTAS
Neurodevelopmental problems

The RNA itself may be the cause of the premutation-specific

disorders concept of RNA toxicity

FXTAS is an inclusion disorder

- Ubiquitin-positive
- Distinct from nucleoli in neuronal nuclei
- Never observed in oligodendroglia
Greco
nucleolus

Astrocytes

inclusion

Ubiquitin immunostaining of cortical neurons and astrocytes

Inclusions can be formed in cultured

neural cells
Elimination of all of the FMR1 gene except the CGG repeat
region still gives rise to cellular abnormalities of FXTAS
FMR1 promoter

CGG

Viral promoter

CGG

FMRP coding

Reporter (GFP) coding

SK neural cells
88 CGG repeats
DAPI

crystallin

- Inclusions can be induced

in cell culture
- Induction requires expanded
CGG repeat as RNA
- Expanded CGG repeat as DNA
does not cause inclusion formation

Garcia-Arocena et al., 2005

merged

FXTAS is an inclusion disorder

Also in multiple cell culture systems
DAPI

Greco et al. (2002, 2006)

Willemsen et al., 2003

Jin et al., 2003

crystallin

merged

SK cells 88 CGG Garcia-Arocena et al., 2005

Pure inclusion preparations has led

to identification of numerous proteins
of interest to pathogenic mechanism

Iwahashi et al. (2006)

Broad distribution of intranuclear

inclusions in FXTAS
in brain, exclusively in nuclei
of neurons and astrocytesl
Also present in numerous peripheral tissues
anterior and posterior pituitary
thyroid
dorsal root ganglia
paraspinal sympathetic ganglia
subepicardial autonomic ganglia of the heart
ganglion cells of adrenal medulla
myenteric ganglia of the stomach/intestine
ovarian stromal cells
testicular (Leydig) cells
Greco et al., 2002 Brain; Willemsen et al., 2003 Hum Mol Genet; Greco et al., 2006 Brain
Greco et al., 2007 J Urology; Brouwer et al., 2008 Psychoneuroendocrinology
Godken et al., 2009 Neuropathology; Hunsaeker et al., 2011

The concept of RNA toxicity

Myotonic dystrophy model: The expanded repeat binds to one or more
proteins, thus removing them from their normal function.

Myotonic dystrophy
DMPK 3UTR

Increased binding of specific

proteins (e.g., MBNL1) to the
expanded CUG repeat
reduces free protein levels
AAAAAAAAAAA

Loss of proteins prevents

their normal function

The concept of RNA toxicity

Sequestration model applied to FXTAS: The expanded CGG repeat
binds to one or more proteins, thus removing them from their normal
function.

Myotonic dystrophy

DMPK 3UTR

FXTAS

Increased binding of specific

proteins (e.g., MBNL1) to the
expanded CUG repeat
reduces free protein levels
AAAAAAAAAAA

FMR1 5UTR

DGCR8/DROSHA
Loss of proteins prevents
their normal function

Pur
hnRNP A2/B1
CUGBP
Etc.

Expanded CGG repeat inhibits DGCR8/

DROSHA acOvity
C
G
CGGCGGCGGCGGCGGCGGCGGCGG G

FMR1 5UTR
CGGCGGCGGCGGCGGCGGCGGCGG

DGCR8

DROSHA
DGCR8

DROSHA
DGCR8

DROSHA

Sellier C et al. (2013) Cell Rep 3:869-80.

Expanded CGG repeat inhibits DGCR8/

DROSHA acOvity
miRNA sequence
FMR1 5UTR
DGCR8

CGGCGGCGGCGGCGGCGGCGGCGG

Transcription

C
G
CGGCGGCGGCGGCGGCGGCGGCGG G

DNA

DROSHA

pri-miR

DGCR8
DROSHA

pre-miR

DGCR8

DROSHA
DGCR8

mature
miRNA

DROSHA

decreased
miRNA levels

Sellier C et al. (2013) Cell Rep 3:869-80.

Expanded CGG repeat inhibits DGCR8/

DROSHA acOvity
miRNA sequence
DGCR8

CGGCGGCGGCGGCGGCGGCGGCGG

Transcription

C
G
CGGCGGCGGCGGCGGCGGCGGCGG G

DNA

DROSHA

pri-miR
pre-miR

DGCR8
DROSHA

DGCR8

DROSHA
DGCR8

mature
miRNA

Observed decreases in
miRNA levels
1
0.75

CTL

0.50
0.25
0

FXTAS
miR-26a1

qRT-PCR

DROSHA
1
0.75
0.50
0.25
0

Sellier C et al. (2013) Cell Rep 3:869-80.

miR-190

Possible mechanism(s) of FXTAS

FMR1
pathogenesis
transcription

Post-transcriptional
mechanisms
Protein
sequestration

Inclusion
Formation
Evidence of cellular
pathology

A Repeat-associated non-AUG (RAN)

translaOon model for pathogenesis
Todd et al. (2013) Neuron 78: 440455

RAN products (poly glycine) are

detected in mouse and human
inclusions.
Such products are capable of
eliciting neurodegenerative
phenotypes in Drosophila
FMRpolyG
Hoechst

Ubiquitin
Merge

Hukema et al. (2015) Human Molecular

GeneOcs 24:49484957

Possible mechanism(s) of FXTAS

FMR1
pathogenesis
transcription

Post-transcriptional
mechanisms
Protein
sequestration

RAN translation

RAN
products

FMRP

Todd 2013

Inclusion
Formation

Inclusion
Formation

Evidence of cellular
pathology

Evidence of cellular
pathology

FXTAS inclusions point to a role of DNA

damage repair in FXTAS
DNA damage and repair:
- Evidence of DNA damage/repair response in the
FXTAS inclusions. Presence of the
phosphorylated variant of the H2A histone variant
(H2AX). Hoem et al. (2011)
Intranuclear inclusion

Working model: Unrepaired DNA damage

at/near the CGG repeat leads to inclusion
formation and neuronal cell damage.

A co-transcriptional (R-loop) model

Newly made RNA reinvades the transcribing DNA to form an R-loop,
which is known to lead to DNA damage if not quickly eliminated.
Skourti-Stathaki K and Proud foot NJ
(2015) Genes Devel 28:13841396

We observe R-loop formation near the start of transcription of the FMR1

gene, and this observation may provide an explanation for other
features of the pathogenesis of FXTAS. Loomis E et al., (2014)

Pathogenesis of FXTAS
DNA damage response (DDR) model
C Iwahashi

nucleolus
inclusion

inclusion

Pathogenesis of FXTAS (DDR model)

Inclusion

Pathogenesis of FXTAS (DDR model)

Inclusion

Pathogenesis of FXTAS (DDR model)

Inclusion

Pathogenesis of FXTAS (DDR model)

Inclusion

The DNA Damage Repair (DDR) model

The DNA Damage Repair (DDR) model

predicts elevated p53 levels

Neuronal
cell death

Evidence of mitochondrial involvement

in human FXTAS fibroblasts
Several patients with FXTAS were initially diagnosed with mitochondrial
myopathy, prompting us to look at mitochondrial function
Ca2+

ROS

Progressive
mitochondrial
dysfuncOon

Increased oxidative damage

Ross-Inta et al., 2010

Decreased mito MnSOD

what our children teach us

FXTAS may reflect a broader (much earlier onset) disease
process among children with the premutation
~8-13% with childhood seizures
~10-15% with ASD
~5% with intellectual impairment
~50% with ADHD

Important for two reasons:

- likely reflects common underlying
mechanism(s) so common target for
therapy
- should allow treatment at far earlier
ages, before onset of clinical
symptoms

Reduced dendritic complexity in neonatal

premutation mice
Expanded CGG (premutation) neurons show reduced
dendrite length and dendritic complexity -

6,13, and 20
DIV (days in vitro)

Chen et al., (2010)

Chen et al., (2010) Hum Mol Genet 19:196-208

Neurons from neonatal premutation mice

show reduced cell survival
Perhaps reflecting
increased susceptibility
to:

To environmental toxins
To seizures
To oxidative stress
To second genetic hits

Chen et al 2009 HMG

Mitochondrial dysfunction begins

early in development
Reduced mitochondrial
motility in mouse
premutation
hippocampal neurons
in culture (4DIV)
Decreased
mitochondrial density
in proximal
neurite of premutation
hippocampal neurons
compared to wild type
Kaplan et al 2012

Cultured mouse premutation neurons

have increased spike/burst activity
Consistent with
increase seizures
(8-13%) in
children with the
premutation

Chen et al 2009

Reversal of spike frequency and burst

activity with allopregnanolone

Expectations for research on FXTAS

Some neurodegenerative diseases may be reversible
Huntington disease mouse
created with abnormal gene that can be turned off
Control

18 wk

34 wk

gene
on

Brain pathology: inclusions,

loss of brain mass

Expectations for research on FXTAS

Some neurodegenerative diseases may be reversible
Huntington disease mouse
created with abnormal gene that can be turned off
Control

18 wk

34 wk

gene
on

Control

18 wk

Brain pathology: inclusions,

loss of brain mass

34 wk

gene
off

Reversal of brain pathology,

recovery of normal behavior

ParOal reversibility of FXTAS inclusions in

the premutaOon mouse
Hukema et al, 2015.

Mice engineered to have premutation CGG repeat (~100 CGG)

Gene can be turned on / off with an antibiotic (doxycycline)
Weeks CGG-repeat gene on (green)
Start (3 wks)

12 weeks gene off (yellow)

CGG-repeat reporter gene

On
Off
Mouse brain

Stopping expression aaer eight weeks in

FXTAS mouse:
substan'al reversal of inclusion size and number

100 Inclusion number

90
80
70
dox
60
wash-out
50
*
40
30
20
10
0
8 wks

12 weeks gene off

2.00
1.80
1.60
1.40
area (um)

percentage

8 weeks gene on

Inclusion size
dox
wash-out

But no reversal seen if the

gene was turned off after 12
weeks

1.20
1.00

**

0.80

Need early diagnosis

and treatment

0.60
0.40
0.20
0.00
8 wks

Current and Recent Collaborators

UC Davis School of Medicine
Biochem Molec Med
Flora Tassone
Chris Iwahashi
Anna Ludwig
Chris Raske
Gry Hoem
Erick Loomis
Kasia Koscielska
Sean Roenspie
Khaled Amiri (UAE University)
Glenda Espinal
Jun Yin
Jun Yi Wang
Biosta9s9cs
Danh Nguyen
Kiyoung Kim
Molecular Biosciences
Isaac Pessah
Yucui Chen
Cecilia Giulivi
Zhongwei Cao
Susan Hulsizer
Neurosurgery
Robert Berman
Ryan Hunsaker
Dolores Garcia-Arocena

MIND Ins9tute
Randi Hagerman
Louise Gane
Jennifer Cogswell
Patrick Adams
Michele Ono
Susan Rivera
Iin Wirnarni
Len Abbeduto

Radiology

James Brunberg

Rehab Med

UCHSC (Denver)
Neurology
Maureen Leehey

Medicine
James Grigsby

RUSH Med Ctr (Chicago)

Pediatrics
Elizabeth Berry-Kravis

Neurology
Christopher Goetz
Deborah Hall

Erasmus MC (Rotterdam)
Clinical Gene9cs

Veronica MarOnez-Cerdeno Edwin Mientjes

Neurology
Rob Willemsen
Lin Zhang
IGBMC (Illkirch))
Michael Rogawski
Nicolas Charlet
John Olichney
Chantal Sellier

Psychiatry

David Hessl
Andreea Seritan
Susan Rivera
Noelle LEtoile
Andrea Schneider
Steve Noctor
Chris Cunningham

Stem Cell Program

Jan Nolta
Jeanie Liu

Pacific Biosciences
Jackie Yen
John Major
David Rank
John Eid
Paul Peluso
Luke Hickey
Thang Pham

LaTrobe University (Melbourne)

Psychology

Danuta Loesch

10/06/15