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

2. 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:

3. 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

4. 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.

5. Adult Onset Myotonic Dystrophy Characteristic appearance: – Myotonia – Muscle weakness and wasting – Low IQ/dementia – Cardiac abnormalities – Hypersomnia/fatigue – Multiple endocrinopathies – Gastrointestinal complaints – Cataracts

6. Disease severity and Repeating numbers Disease severity correlated with (CUG)n repeat size.  Age of Onset, Average age of death, disease symptoms (CTG)n DMPK

7. AUGStop (CUG)5-37 5’-UTR 3’-UTR ORF DMPK RNA DMPK protein AAAA Kinase Coiled coil Trans- mem. mRNA exported and transcribed (CUG)100+ ORF AAAA 3’-UTR mRNA Retained in Nucleus Caused by a CTG expansion in the 3’-UTR of DMPK LRR

8. 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

9. CMV promoterrtTA TRANSGENE (Tet-On System) DMPK 5’UTR7X TRE 5’UTRGFPDMPK 3’UTRDMPK 1 st INTRON GFP-DMPK 3’UTR TRANSGENE Transgenic Mouse Design Dox Induction of Dox leads to a robust disease phenotype similar to the phenotype seen in patients

10. Design 2weeks816 Birth 46 infancychildhoodadolescentadulthood P P 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

11. Generating a Childhood onset DM1 2weeks816 Birth 46 infancychildhoodadolescentadulthood P P P&T CHDM1 Adult DM1 AB CD

12. Skeletal muscle pathology present but not as severe as the adult DM1 ControlDM1 Adult DM1 CHDM1

13. Parent of Origin CHDM1 Pups from 4 WeeksNormal MotherEffected Motherp-value Grip Strength (g force/ g weight)3.3 +/- 0.43.1 +/- 0.70.66 PR Interval (s)0.0317 +/- 0.00380.0325 +/- 0.00720.71 Myotonia (Score 0-3)2.8 +/- 0.43 +/- 00.36 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

14. Molecular Analysis of CHDM1 mice Clcn1 +X7aAverage %StdevP-Value WT (n=4)7.70.8 0.010 313 Het (n=3)21.12.2 Nfix +X7Average %StdevP-Value WT (n=4)24.41.1 0.001 313 Het (n=4)31.81.3 Nrap +X12Average %StdevP-Value WT (n=4)57.13.7 0.013 313 Het (n=4)40.27.1 Input -RT +RT Beads IgG Mbnl1 -RT +RT NTC EGFP-DM5 UTR CHDM1 Adult DM1 EGFP-DM5 UTR A B C MBNL1 +X5Average %StdevP-Value WT (n=4)8.82.9 0.002 313 Het (n=4)20.71.6 WTCHDM1 Smyd1 +39Average %StdevP-Value WT (n=4)64.13.2 0.003 313 Het (n=4)80.34.9 WTCHDM1 Gapdh Cugbp1

15. MBNL1 overexpression, a model for therapeutic testing, corrects myotonia SmyD1 +X39Average %StdevP-Value EGFP (n=2)70.9680.3946 0.028 Mbnl1 (n=2)64.22450.1252 EGFP Mbnl1 CHDM1 EGFP Mbnl1 CHDM1 qPCR of Mbnl1 Relative Mbnl1Stdev EGFP (n=2) 1.000.02 MBNL1 (n=2) 4.643.84 B D Myotonia Score Average ScoreStdev EGFP (n=2) 30 MBNL1 (n=2) 1.50 A Gapdh MBNL1-EGFP Mbnl1 EGFP Leg MBNL1-EGFP Leg C

16. A Reminder 2weeks816 Birth 46 infancychildhoodadolescentadulthood P P P&T CHDM1 Adult DM1 AB CD

17. Early presence of pathological levels of toxic RNA leads to a more severe DM1 phenotype, this is independent of repeat length dCT (Ct EGFP-Gapdh) AverageStdevp-value CHDM1 (n=4)1.410.37 0.148 Adult DM1 (n=5)1.900.50 Remember CHDM1 muscle had a milder pathology than adult muscle

18. 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

19. Acknowledgements Dr. Mani S. Mahadevan Mahua Mandal Dr. Ramesh Yadava Dr. Yun Kim Qing Yu (Jane) Dr. Erin P. Foff Dr. Shagufta Rehman Questions?