Modeling Childhood-onset Myotonic Dystrophy
Jordan Gladman Ph.D.PRPR 9/24/2012
Myotonic Dystrophy Type 1
Autosomal dominant 1 in 8000 - most common adult muscular dystrophy Variable age of onset and phenotype
Congenital Childhood Adult
Progressive skeletal muscle loss Cardiac defects Smooth muscle dysfunction Other multisystem effects including cataracts, insulin resistance,
mental retardation
Common features:
Congenital Myotonic Dystrophy
• Children often born from mothers with DM1• Respiratory and swallowing difficulties• moderate to severe intellectual disabilities • cardiomyopathy • Often need extensive neonatal care• Survivors may strengthen somewhat, but
ultimately develop a progressive myopathy similar to the more common forms of the disease
• Myotonia absent in neonates
Childhood-Onset Myotonic Dystrophy
• Milder than congenital myotonic dystrophy but still more severe than Adult DM1
• No parent of origin effect• Unlike congenital myotonic dystrophy individuals with
childhood-onset myotonic dystrophy do not have in utero abnormalities, delayed early motor development, and if present only have mild hypotonia or respiratory problems
• Childhood-onset DM1 patients usually have myotonia and frequently have mental handicaps such as a decrease in mean IQ and a range of psychosocial difficulties
• As patients age they tend to then also develop features seen in adult onset DM1.
Adult Onset Myotonic Dystrophy
• Characteristic appearance:– Myotonia– Muscle weakness and
wasting– Low IQ/dementia– Cardiac abnormalities– Hypersomnia/fatigue– Multiple endocrinopathies– Gastrointestinal complaints– Cataracts
Disease severity and Repeating numbers
• Disease severity correlated with (CUG)n repeat size.Age of Onset, Average age of death, disease symptoms
(CTG)nDMPK
AUG Stop(CUG)5-37
5’-UTR3’-UTR
ORFDMPK RNA
DMPK protein
AAAA
Kinase Coiled coil
Trans- mem.
mRNA exported and transcribed
(CUG)100+
ORFAAAA
3’-UTR
mRNA Retained in Nucleus
Caused by a CTG expansion in the 3’-UTR of DMPK
LRR
Mouse Models of DM1
• The perfect mouse model, especially to study therapeutics, does not exist
• Multiple labs working on addressing this issue• Most efforts are aimed at adult DM1
Goal: Develop a childhood onset mouse model of DM1 that allows therapeutic testing and can be used to better understand DM1
CMV promoter rtTArtTA TRANSGENE(Tet-On System)
DMPK 5’UTR 7X TRE 5’UTR GFP DMPK 3’UTR DMPK 1st INTRONGFP-DMPK 3’UTRTRANSGENE
Transgenic Mouse Design
Dox
Dox
Induction of Dox leads to a robust disease phenotype similar to the phenotype seen in patients
Design
2weeks 8 16
Birth
4 6
infancy childhood adolescent adulthood
P P P&T
P&T
CHDM1
Adult DM1
Start induction of the toxic RNA before birth, as early as conception and monitor disease phenotype
- Start with the DM5 mice as we have them well characterized- Mate a DM5 +/- who is induced for 1 months with a DM5 -/-
- Keep dox present during mating, pregnancy and rearing
Generating a Childhood onset DM1
CHDM1 4wks CHDM1 6 wks Adult DM1 8wks
Control 4.46465216529632 5.29838897378426 4.10421366425186
DM1 3.23903015950851 4.46600719720004 3.95333483745084
0.5
1.5
2.5
3.5
4.5
5.5
Forelimb Grip Strength
Gri
p S
tre
ng
th (
g f
orc
e p
ulle
d/g
bo
dy
w
eig
ht)
***
***n.s.
CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks
Control 0 0 0 0
DM1 1.3 2.88888888888888
2.88888888888888
2.25
0.5
1.5
2.5
Myotonia Score
My
oto
nia
Sc
ore
**
*** *** ***
CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks
Control 0.023125 0.024423076923077
0.0232692307692308
0.0290625
DM1 0.022 0.0319444444444444
0.035 0.0346428571428572
0.0050
0.0150
0.0250
0.0350
ECG PR Interval
PR
In
terv
al (
s)
**** ***
n.s.
2weeks 8 16
Birth
4 6infancy childhood adolescent adulthood
P P P&T
P&T
CHDM1
Adult DM1
A B
C D
Skeletal muscle pathology present but not as severe as the adult DM1
Control DM1
Adul
t DM
1CH
DM
1
Parent of Origin CHDM1 Pups from
4 Weeks Normal Mother Effected Mother p-value
Grip Strength (g force/ g weight) 3.3 +/- 0.4 3.1 +/- 0.7 0.66
PR Interval (s) 0.0317 +/- 0.0038 0.0325 +/- 0.0072 0.71
Myotonia (Score 0-3) 2.8 +/- 0.4 3 +/- 0 0.36
Both sick DM1 sexes produce pups that are equally sick- Like childhood DM1 in humans there does not seem to be a parent of origin effect
Molecular Analysis of CHDM1 mice
Clcn1 +X7a Average % Stdev P-ValueWT (n=4) 7.7 0.8
0.010313 Het (n=3) 21.1 2.2
Nfix +X7 Average % Stdev P-ValueWT (n=4) 24.4 1.1
0.001313 Het (n=4) 31.8 1.3
Nrap +X12 Average % Stdev P-ValueWT (n=4) 57.1 3.7
0.013313 Het (n=4) 40.2 7.1
Input-RT +RT Beads IgG
Mbnl1-RT +RT NTC
EGFP-DM5 UTRCHDM1
Adult DM1 EGFP-DM5 UTR
A
B
C
MBNL1 +X5 Average % Stdev P-ValueWT (n=4) 8.8 2.9
0.002313 Het (n=4) 20.7 1.6
WT CHDM1
Smyd1 +39 Average % Stdev P-ValueWT (n=4) 64.1 3.2
0.003313 Het (n=4) 80.3 4.9
WT CHDM1
WT CHDM1
0
0.5
1
1.5
Relative CUGBP1 Protein
Re
lativ
e A
mo
un
t (C
ug
p1
/Ga
pd
h)
**
Gapdh
Cugbp1
MBNL1 overexpression, a model for therapeutic testing, corrects myotonia
SmyD1 +X39 Average % Stdev P-ValueEGFP (n=2) 70.968 0.3946
0.028Mbnl1 (n=2) 64.2245 0.1252
EGFP Mbnl1CHDM1
EGFP Mbnl1CHDM1
qPCR of Mbnl1Relative Mbnl1 Stdev
EGFP (n=2) 1.00 0.02MBNL1 (n=2) 4.64 3.84
B
D
Myotonia ScoreAverage
Score StdevEGFP (n=2) 3 0
MBNL1 (n=2) 1.5 0
A
Gapdh
MBNL1-EGFP
Mbnl1
EG
FP
Leg
MB
NL1
-EG
FP
Le
g
C
A Reminder
CHDM1 4wks CHDM1 6 wks Adult DM1 8wks
Control 4.46465216529632 5.29838897378426 4.10421366425186
DM1 3.23903015950851 4.46600719720004 3.95333483745084
0.5
1.5
2.5
3.5
4.5
5.5
Forelimb Grip Strength
Gri
p S
tre
ng
th (
g f
orc
e p
ulle
d/g
bo
dy
w
eig
ht)
***
***n.s.
CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks
Control 0 0 0 0
DM1 1.3 2.88888888888888
2.88888888888888
2.25
0.5
1.5
2.5
Myotonia Score
My
oto
nia
Sc
ore
**
*** *** ***
CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks
Control 0.023125 0.024423076923077
0.0232692307692308
0.0290625
DM1 0.022 0.0319444444444444
0.035 0.0346428571428572
0.0050
0.0150
0.0250
0.0350
ECG PR Interval
PR
In
terv
al (
s)
**** ***
n.s.
2weeks 8 16
Birth
4 6infancy childhood adolescent adulthood
P P P&T
P&T
CHDM1
Adult DM1
A B
C D
Early presence of pathological levels of toxic RNA leads to a more severe DM1 phenotype, this is independent of repeat length
-22.50
-7.50
7.50
Relative change in forelimb
Per
cent
Cha
nge
*
10.00
30.00
50.00
Relative change in PR interval
Per
cent
Cha
nge
**
0.5
1.5
2.5
Change in Myotonia Score
Myo
toni
a S
core
*
dCT (Ct EGFP-Gapdh) Average Stdev p-valueCHDM1 (n=4) 1.41 0.37 0.148Adult DM1 (n=5) 1.90 0.50
Remember CHDM1 muscle had a milder pathology than adult muscle
Future Directions• Complete this work by evaluation more mice• Move into the DM200 mouse mode• Examine neurological phenotype in these mice
Conclusions• Expressing the toxic RNA during development leads
to a CHDM1 mouse model• This model is more severe than its adult counterpart• It can be used to test therapeutics• Age of onset, independent of repeat length, has an
effect on disease phenotype
Acknowledgements
Dr. Mani S. Mahadevan
Mahua Mandal
Dr. Ramesh YadavaDr. Yun KimQing Yu (Jane)
Dr. Erin P. FoffDr. Shagufta Rehman
Questions?