Disease Progression and Pathogenesis in Congenital Myotonic Dystrophy Nicholas Johnson, MD Assistant Professor of Neurology, Pediatrics, Pathology University of Utah

Disclosures Funding/Conflicts of Interest: NINDS (1K23NS091511-01) Center for Disease Control and Prevention (DD001108-02) Muscular Dystrophy Association Myotonic Dystrophy Foundation Ionis Pharmaceuticals Biogen IDEC AveXis Cytokinetics AMO Pharma Utah Neuromuscular Research Fund

Congenital myotonic dystrophy (CDM) Anticipation can result in unpredictable expansion of CTG repeat length Onset in 1st month of life with: Hypotonia Respiratory failure Feeding difficulties Clubfoot 30% mortality in 1st yr of life if ventilated >3 months

Phases of myotonic dystrophy in childhood N=150 parents Johnson, et al, 2015

Health Endpoints and Longitudinal Progression in CDM (HELP-CDM) Children with CDM distributed in 3 age groups 0-2 years 3-5 years 6-13 years Control subjects distributed in the 3 age groups 2 day visit at baseline, 12 months, 24 months Enrolled at U of Utah, University of Western Ontario

Inclusion/Exclusion criteria (research definition of CDM) Hospitalized for 72 hours in 1st month Required breathing assistance, or Feeding assistance, or Clubfeet, or Hypotonia, and CTG repeat length >200 No other significant medical illness

Participant demographics Mean duration of respiratory support at birth: 25.9 weeks (1-156 weeks) Current respiratory support in CDM group: 7.0% BiPAP (13.3h), 7.0% supplemental oxygen (9.6h) Five children with CDM had ECG abnormalities: Left anterior fascicular block (1), prolonged QT (2), and first degree AV block (2) Demographic CDM Control N 41 29 Age (years) (SD) 6.8 (3.3) 9.1 (3.1) Female (%) 49 59 Race 98% Caucasian, 2% Asian 100% Caucasian Ethnicity 12% Hispanic, 88% non-hispanic 7% Hispanic, 93% non-hispanic Mean CTG Repeat length (SD) 1245.9 (474.9

Motor Assessments: 6 minute walk distance Children with CDM walked 258.3 m (SD 176), while healthy controls walked 568.3 m (SD 73.2) (p<0.001) Test-retest reliability: 0.96 (ICC) Johnson et al., 2016

Grip assessments: baseline and 12 months Johnson et al., 2016

Lean muscle mass: baseline and 12 months

Lean mass of the right arm Lean mass of the right leg MEASURE Lean mass of the right arm Right grip Right pinch Right jawchuck 1.00000   0.91037 p<.0001 Right lateral pinch 0.82621 p<.0001 0.93633 p<.0001 0.76603 p=0.0009 0.86491 p<.0001 0.77902 p<.0001 Lean mass of the right leg 2MWT 6MWT 10m walk Lean mass of the right leg 0.59239 p=0.0047 0.62179 p=0.0034 0.97743 p<.0001 -0.37817 p=0.1345 -0.72268 p<.0001 -0.76446 p<.0001

Age does not affect oral facial weakness Test-retest reliability of LFM: 0.99 (ICC) Test-retest reliability of IOPI: 0.92 (ICC) Johnson et al., 2016

Oral facial weakness: 12 months

Cognitive impairment is variable but severe Mean IQ at baseline (SD): 65.6 (14.9) 2.2% extremely low Mean IQ at 12 months (SD): 61.7 (13.7)

Measurement of behavioral phenotype: baseline Instrument N Mean SD Norm Value IQ 36 69.4 17 >80 Social Communication Score 24 12.9 6.1 <15 Repetitive Behavior Score 18.1 14.3 <13 ASSQ Score 17.7 7.7

Daytime sleepiness and quality of life: baseline Johnson et al., 2016

Pathogenesis of congenital myotonic dystrophy Does the same spliceopathy affect children with CDM? Are the same targets affected, given the distinct phenotype?

Samples and methods Muscle biopsy (quadriceps or tibialis anterior) RNA extraction Cohort Sample size Gender Age (yrs) CTG repeat length CDM cases 6 66.6% male 2 mo, 1, 3, 7, 11, 16 1150-2200 Pediatric controls1 9 44.4% male 1 mo-13 NA DM1 cases 16 50% male 29-57 350-866 Adult controls 33.3% male 19-28 Paired-end 125 RNA-Seq (at least 25 million paired reads) MAJIQ analysis for PSI Keep candidates if PSI>0.15; reads >50/sample= 1974 events WGCNA analysis for top candidates 1. Pathologically normal muscle samples (courtesy S. Moore) Evaluation of top candidates

Local splicing variations (LSVs) using RNA-Seq in CDM and DM1 muscle Modeling Alternative Junction Inclusion Quantification (MAJIQ): Vaquero-Garcia et al., 2016

Weighted Gene Co-expression Network Analysis (WGCNA) WGCNA developed by Steve Horvath, Professor of Human Genetics & Biostatics at UCLA. His group develops biostatistics for studying complex phenotypes http://www.genetics.ucla.edu/labs/horvath/ <- Count Data Sample Number PSI value Transcript 1 Transcript 2 Transcript 3 <- Find transcripts that have similar PSI values across samples <- Create a correlation matrix that can be used as input for co-expression network aij =│cor(xi,xj)│β Adjacency of two transcripts =│ absolute correlation of two transcript PSI profiles across multiple samples│using soft thresholding power to assess connection strength <- Analyze the distances between nodes (PSI) in co-expression network to look for groups (modules)

Many turquoise events are previously described Nakamori, et al., 2013

Top shared DM1 (turquoise) LSVs Gene1 dPSICDM dPSIDM1 dPSICo Connectivity rank MAPT, ex 7 0.79 0.62 NA 1 CACNA1S, ex 27 0.61 0.48 0.04 2 CLASP1, ex 2 0.81 0.70 0.07 3 LDB3, ex 6 0.87 0.67 0.03 4 NFIX, ex7 0.57 0.52 5 BIN1, ex 13 0.36 0.32 0.09 19 MBNL1, ex 5 0.53 0.51 22 1. MAPT, CACNA1S, CLASP1, NFIX all have multiple events in top 22 candidates

Top developmental (yellow) LSVs Connectivity ranks starts at 643 Of top 34 events, titin LSVs represent 29 MYH9, ITGAV, ENAH are the rest

Top CDM (pink) LSVs Gene dPSICDM dPSIDM1 dPSICo Connectivity rank Development difference in CDM (p-value)1 Proposed mechanism STAU2, ex4 0.26 0.23 0.1 349 0.007 RNA binding protein PALLD, ex 212 0.38 0.2 0.06 352 0.08 Myoblast differentiation USP47, ex 22 0.07 411 0.011 WNT signaling FERMT2, ex 13 0.37 0.15 418 0.086 Myogenesis TNNI2, ex 22 NA 419 0.022 Ca regulation T-test comparing the PSI value between the 3 youngest CDM samples (2mo-3 yrs) against 3 oldest. PALLD, USP47, TNNI2 had multiple events in the top 10

Fig 7. Schematic model outlining how reduced expression of palladin affects skeletal muscle differentiation processes. Nguyen NUN, Wang HV (2015) Dual Roles of Palladin Protein in In Vitro Myogenesis: Inhibition of Early Induction but Promotion of Myotube Maturation. PLoS ONE 10(4): e0124762. doi:10.1371/journal.pone.0124762 http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0124762

Summary Clinical features of CDM are distinct from DM1, but is also a multisystemic condition Symptoms in CDM generally improve with age The underlying pathogenesis of CDM is the same as DM1 Regulators of muscle differentiation may be the leading cause of CDM phenotype

Contributors and Support Collaborators Bob Weiss, PhD Craig Campbell, MD Donald McCorquodale, MD, PhD Russell Butterfield, MD, PhD Man Hung, PhD Chad Heatwole, MD,MS-CI Steven Moore, MD, PhD Missy Dixon Kiera Berggren Heather Hayes Evan Pusillo Deanna Dibella Becky Crockett Caitlin Polanski Brith Otterud Support National Institute of Neurological Diseases and Stroke (1K23NS091511-01) Muscular Dystrophy Association Myotonic Dystrophy Foundation Biogen Idec Utah Neuromuscular Fund