Slide 1

Modeling Childhood-onset Myotonic Dystrophy Jordan Gladman Ph.D.PRPR 9/24/2012Myotonic Dystrophy Type 1 Autosomal dominant 1 in 8000 - most common adult muscular dystrophy Variable age of onset and phenotypeCongenitalChildhoodAdult

Progressive skeletal muscle lossCardiac defectsSmooth muscle dysfunctionOther multisystem effects including cataracts, insulin resistance, mental retardation

Common features:Congenital Myotonic DystrophyChildren often born from mothers with DM1Respiratory and swallowing difficultiesmoderate to severe intellectual disabilities cardiomyopathy Often need extensive neonatal careSurvivors may strengthen somewhat, but ultimately develop a progressive myopathy similar to the more common forms of the diseaseMyotonia absent in neonates

Childhood-Onset Myotonic DystrophyMilder than congenital myotonic dystrophy but still more severe than Adult DM1No parent of origin effectUnlike 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 DystrophyCharacteristic appearance:MyotoniaMuscle weakness and wastingLow IQ/dementiaCardiac abnormalitiesHypersomnia/fatigueMultiple endocrinopathiesGastrointestinal complaintsCataracts

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

(CTG)nDMPK

AUGStop(CUG)5-375-UTR3-UTRORFDMPK RNADMPK proteinAAAAKinase Coiled coilTrans- mem.mRNA exported and transcribed

(CUG)100+ORFAAAA3-UTRmRNA Retained in Nucleus

Caused by a CTG expansion in the 3-UTR of DMPK

LRRMouse Models of DM1The perfect mouse model, especially to study therapeutics, does not existMultiple labs working on addressing this issueMost 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 DM1CMV promoterrtTArtTA TRANSGENE(Tet-On System)DMPK 5UTR7X TRE 5UTRGFPDMPK 3UTRDMPK 1st INTRONGFP-DMPK 3UTRTRANSGENETransgenic Mouse DesignDoxDoxInduction of Dox leads to a robust disease phenotype similar to the phenotype seen in patientsDesign2weeks816Birth46infancychildhoodadolescentadulthoodPPP&TP&TCHDM1Adult DM1Start 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 rearingGenerating a Childhood onset DM12weeks816Birth46infancychildhoodadolescentadulthoodPPP&TP&TCHDM1Adult DM1ABCDSkeletal muscle pathology present but not as severe as the adult DM1ControlDM1Adult DM1CHDM1

Parent of OriginCHDM1 Pups from4 WeeksNormal MotherEffected Motherp-valueGrip Strength (g force/ g weight)3.3 +/- 0.43.1 +/- 0.70.66PR Interval (s)0.0317 +/- 0.00380.0325 +/- 0.00720.71Myotonia (Score 0-3)2.8 +/- 0.43 +/- 00.36Both sick DM1 sexes produce pups that are equally sick- Like childhood DM1 in humans there does not seem to be a parent of origin effectMolecular Analysis of CHDM1 mice Clcn1 +X7aAverage %StdevP-ValueWT (n=4)7.70.80.010313 Het (n=3)21.12.2 Nfix +X7Average %StdevP-ValueWT (n=4)24.41.10.001313 Het (n=4)31.81.3 Nrap +X12Average % StdevP-ValueWT (n=4) 57.13.70.013313 Het (n=4)40.27.1

Input-RT +RTBeadsIgGMbnl1-RT +RTNTCEGFP-DM5 UTR

CHDM1Adult DM1EGFP-DM5 UTRABC MBNL1 +X5 Average % StdevP-ValueWT (n=4) 8.82.90.002313 Het (n=4) 20.71.6WTCHDM1

Smyd1 +39Average % StdevP-ValueWT (n=4) 64.13.20.003313 Het (n=4) 80.34.9

WTCHDM1GapdhCugbp1MBNL1 overexpression, a model for therapeutic testing, corrects myotonia

SmyD1 +X39Average % StdevP-ValueEGFP (n=2) 70.9680.39460.028Mbnl1 (n=2)64.22450.1252EGFP Mbnl1CHDM1 EGFP Mbnl1CHDM1 qPCR of Mbnl1Relative Mbnl1StdevEGFP (n=2)1.000.02MBNL1 (n=2)4.643.84BDMyotonia ScoreAverage ScoreStdevEGFP (n=2)30MBNL1 (n=2)1.50A

GapdhMBNL1-EGFP

Mbnl1EGFP LegMBNL1-EGFP LegCA Reminder2weeks816Birth46infancychildhoodadolescentadulthoodPPP&TP&TCHDM1Adult DM1ABCDEarly presence of pathological levels of toxic RNA leads to a more severe DM1 phenotype, this is independent of repeat lengthdCT (Ct EGFP-Gapdh)AverageStdevp-valueCHDM1 (n=4)1.410.370.148Adult DM1 (n=5)1.900.50Remember CHDM1 muscle had a milder pathology than adult muscleFuture DirectionsComplete this work by evaluation more miceMove into the DM200 mouse modeExamine neurological phenotype in these miceConclusionsExpressing the toxic RNA during development leads to a CHDM1 mouse modelThis model is more severe than its adult counterpartIt can be used to test therapeuticsAge of onset, independent of repeat length, has an effect on disease phenotypeAcknowledgements

Dr. Mani S. Mahadevan

Mahua Mandal

Dr. Ramesh YadavaDr. Yun KimQing Yu (Jane)

Dr. Erin P. FoffDr. Shagufta Rehman

Questions?