modeling childhood-onset myotonic dystrophy

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Modeling Childhood- onset Myotonic Dystrophy Jordan Gladman Ph.D. PRPR 9/24/2012

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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. Common features:. - PowerPoint PPT Presentation

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Page 1: Modeling Childhood-onset Myotonic Dystrophy

Modeling Childhood-onset Myotonic Dystrophy

Jordan Gladman Ph.D.PRPR 9/24/2012

Page 2: Modeling Childhood-onset Myotonic Dystrophy

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:

Page 3: Modeling Childhood-onset Myotonic Dystrophy

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

Page 4: Modeling Childhood-onset Myotonic Dystrophy

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.

Page 5: Modeling Childhood-onset Myotonic Dystrophy

Adult Onset Myotonic Dystrophy

• Characteristic appearance:– Myotonia– Muscle weakness and

wasting– Low IQ/dementia– Cardiac abnormalities– Hypersomnia/fatigue– Multiple endocrinopathies– Gastrointestinal complaints– Cataracts

Page 6: Modeling Childhood-onset Myotonic Dystrophy

Disease severity and Repeating numbers

• Disease severity correlated with (CUG)n repeat size.Age of Onset, Average age of death, disease symptoms

(CTG)nDMPK

Page 7: Modeling Childhood-onset Myotonic Dystrophy

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

Page 8: Modeling Childhood-onset Myotonic Dystrophy

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

Page 9: Modeling Childhood-onset Myotonic Dystrophy

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

Page 10: Modeling Childhood-onset Myotonic Dystrophy

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

Page 11: Modeling Childhood-onset Myotonic Dystrophy

Generating a Childhood onset DM1

CHDM1 4wks CHDM1 6 wks Adult DM1 8wks0.0

1.0

2.0

3.0

4.0

5.0

6.0

Forelimb Grip Strength

Grip

Str

engt

h (g

forc

e pu

lled/

g bo

dy

wei

ght)

***

***n.s.

CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks0.0

1.0

2.0

3.0

Myotonia Score

Myo

toni

a S

core

**

*** *** ***

CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks0.0000

0.0100

0.0200

0.0300

0.0400

ECG PR Interval

PR

Inte

rval

(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

Page 12: Modeling Childhood-onset Myotonic Dystrophy

Skeletal muscle pathology present but not as severe as the adult DM1

Control DM1

Adul

t DM

1CH

DM1

Page 13: Modeling Childhood-onset Myotonic Dystrophy

Parent of Origin  CHDM1 Pups from  

4 Weeks Normal Mother Effected Mother p-valueGrip 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

Page 14: Modeling Childhood-onset Myotonic Dystrophy

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 0.001

313 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 0.002

313 Het (n=4) 20.7 1.6

WT CHDM1

  Smyd1 +39 Average % Stdev P-ValueWT (n=4) 64.1 3.2 0.003

313 Het (n=4) 80.3 4.9

WT CHDM1

WT CHDM10

0.5

1

1.5

Relative CUGBP1 Protein

Rel

ativ

e A

mou

nt

(Cug

p1/G

apdh

)

**

Gapdh

Cugbp1

Page 15: Modeling Childhood-onset Myotonic Dystrophy

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 L

eg

MB

NL1

-EG

FP

Leg

C

Page 16: Modeling Childhood-onset Myotonic Dystrophy

A Reminder

CHDM1 4wks CHDM1 6 wks Adult DM1 8wks0.0

1.0

2.0

3.0

4.0

5.0

6.0

Forelimb Grip Strength

Grip

Str

engt

h (g

forc

e pu

lled/

g bo

dy

wei

ght)

***

***n.s.

CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks0.0

1.0

2.0

3.0

Myotonia Score

Myo

toni

a S

core

**

*** *** ***

CHDM1 2wks CHDM1 4wks CHDM1 6wks Adult DM1 8wks0.0000

0.0100

0.0200

0.0300

0.0400

ECG PR Interval

PR

Inte

rval

(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

Page 17: Modeling Childhood-onset Myotonic Dystrophy

Early presence of pathological levels of toxic RNA leads to a more severe DM1 phenotype, this is independent of repeat length

-30.00

-15.00

0.00

15.00Relative change in forelimb

Per

cent

Cha

nge

*

0.00

20.00

40.00

60.00

Relative change in PR interval

Per

cent

Cha

nge

**

0.0

1.0

2.0

3.0

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

Page 18: Modeling Childhood-onset Myotonic Dystrophy

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

Page 19: Modeling Childhood-onset Myotonic Dystrophy

Acknowledgements

Dr. Mani S. Mahadevan

Mahua Mandal

Dr. Ramesh YadavaDr. Yun KimQing Yu (Jane)

Dr. Erin P. FoffDr. Shagufta Rehman

Questions?