Disease Progression and Pathogenesis in Congenital Myotonic Dystrophy

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Disease Progression and Pathogenesis in Congenital Myotonic Dystrophy Nicholas Johnson, MD Assistant Professor of

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 (1 K 23 NS 091511 -01)

Disclosures Funding/Conflicts of Interest: • • • NINDS (1 K 23 NS 091511 -01) Center for Disease Control and Prevention (DD 001108 -02) Muscular Dystrophy Association Myotonic Dystrophy Foundation Ionis Pharmaceuticals Biogen IDEC Ave. Xis Cytokinetics AMO Pharma Utah Neuromuscular Research Fund

Congenital myotonic dystrophy (CDM) • Anticipation can result in unpredictable expansion of CTG repeat

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

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

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

Health Endpoints and Longitudinal Progression in CDM (HELP-CDM) • Children with CDM distributed in

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 1 st

Inclusion/Exclusion criteria (research definition of CDM) • Hospitalized for 72 hours in 1 st 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

Participant demographics • Mean duration of respiratory support at birth: § 25. 9 weeks (1 -156 weeks) • Current respiratory support in CDM group: § 7. 0% Bi. PAP (13. 3 h), 7. 0% supplemental oxygen (9. 6 h) • 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 1245. 9 (474. 9 length (SD)

Motor Assessments: 6 minute walk distance • Children with CDM walked 258. 3 m

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

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

Lean muscle mass: baseline and 12 months

Lean muscle mass: baseline and 12 months

MEASURE Lean mass of the right arm Right grip Right pinch Right jawchuck 1.

MEASURE Lean mass of the right arm Right grip Right pinch Right jawchuck 1. 00000 Lean mass of the right arm 1. 00000 Right grip 0. 91037 p<. 0001 0. 93633 p<. 0001 1. 00000 Right lateral pinch 0. 82621 p<. 0001 0. 86491 p<. 0001 0. 77902 p<. 0001 1. 00000 Right jawchuck 0. 76603 p=0. 0009 MEASURE Lean mass of the right leg 2 MWT 6 MWT 10 m walk Lean mass of the 1. 00000 right leg 1. 00000 2 MWT 0. 59239 p=0. 0047 0. 97743 p<. 0001 1. 00000 6 MWT 0. 62179 p=0. 0034 10 m walk -0. 37817 p=0. 1345 -0. 72268 p<. 0001 -0. 76446 p<. 0001 1. 00000

Age does not affect oral facial weakness • Test-retest reliability of LFM: 0. 99

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

Oral facial weakness: 12 months

Cognitive impairment is variable but severe • Mean IQ at baseline (SD): § 65.

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) § 2. 2% extremely low

Measurement of behavioral phenotype: baseline Instrument N Mean SD Norm Value IQ 36 69.

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 24 18. 1 14. 3 <13 ASSQ Score 24 17. 7 <13

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

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

Pathogenesis of congenital myotonic dystrophy • Does the same spliceopathy affect children with CDM?

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 Cohort Sample size Gender Age (yrs) CTG repeat length CDM cases

Samples and methods Cohort Sample size Gender Age (yrs) CTG repeat length CDM cases 6 2 mo, 1, 3, 7, 11, 16 1150 -2200 66. 6% male Pediatric controls 1 9 44. 4% male 1 mo-13 NA DM 1 cases 16 50% male 29 -57 350 -866 Adult controls 6 33. 3% male 19 -28 NA 1. Pathologically normal muscle samples (courtesy S. Moore) Muscle biopsy (quadriceps or tibialis anterior) RNA extraction 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 Evaluation of top candidates

Local splicing variations (LSVs) using RNA-Seq in CDM and DM 1 muscle Modeling Alternative

Local splicing variations (LSVs) using RNA-Seq in CDM and DM 1 muscle Modeling Alternative Junction Inclusion Quantification (MAJIQ): Vaquero-Garcia et al. , 2016

Weighted Gene Co-expression Network Analysis (WGCNA) <- Count Data PSI value Transcript 1 Transcript

Weighted Gene Co-expression Network Analysis (WGCNA) <- Count Data PSI value Transcript 1 Transcript 2 Transcript 3 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/ <- Find transcripts that have similar PSI values across samples Sample Number <- 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

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

Top shared DM 1 (turquoise) LSVs Gene 1 d. PSICDM d. PSIDM 1 d.

Top shared DM 1 (turquoise) LSVs Gene 1 d. PSICDM d. PSIDM 1 d. PSICo Connectivity rank MAPT, ex 7 0. 79 0. 62 NA 1 CACNA 1 S, ex 27 0. 61 0. 48 0. 04 2 CLASP 1, ex 2 0. 81 0. 70 0. 07 3 LDB 3, ex 6 0. 87 0. 67 0. 03 4 NFIX, ex 7 0. 52 NA 5 BIN 1, ex 13 0. 36 0. 32 0. 09 19 MBNL 1, ex 5 0. 53 0. 51 NA 22 1. MAPT, CACNA 1 S, CLASP 1, NFIX all have multiple events in top 22 candidates

Top developmental (yellow) LSVs • Connectivity ranks starts at 643 • Of top 34

Top developmental (yellow) LSVs • Connectivity ranks starts at 643 • Of top 34 events, titin LSVs represent 29 • MYH 9, ITGAV, ENAH are the rest

Top CDM (pink) LSVs Gene d. PSICD M d. PSIDM 1 d. PSICo Connectivity

Top CDM (pink) LSVs Gene d. PSICD M d. PSIDM 1 d. PSICo Connectivity rank Development difference in CDM (p-value)1 Proposed mechanism STAU 2, ex 4 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 USP 47, ex 22 0. 38 0. 2 0. 07 411 0. 011 WNT signaling FERMT 2, ex 13 0. 37 0. 15 0. 07 418 0. 086 Myogenesis TNNI 2, ex 22 0. 15 NA NA 419 0. 022 Ca regulation 1. T-test comparing the PSI value between the 3 youngest CDM samples (2 mo-3 yrs) against 3 oldest. 2. PALLD, USP 47, TNNI 2 had multiple events in the top 10

Fig 7. Schematic model outlining how reduced expression of palladin affects skeletal muscle differentiation

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. PLo. S ONE 10(4): e 0124762. 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 DM 1, but is also

Summary • Clinical features of CDM are distinct from DM 1, but is also a multisystemic condition • Symptoms in CDM generally improve with age • The underlying pathogenesis of CDM is the same as DM 1 • Regulators of muscle differentiation may be the leading cause of CDM phenotype

Contributors and Support Collaborators • • • • Bob Weiss, Ph. D Craig Campbell,

Contributors and Support Collaborators • • • • Bob Weiss, Ph. D Craig Campbell, MD Donald Mc. Corquodale, MD, Ph. D Russell Butterfield, MD, Ph. D Man Hung, Ph. D Chad Heatwole, MD, MS-CI Steven Moore, MD, Ph. D Missy Dixon Kiera Berggren Heather Hayes Evan Pusillo Deanna Dibella Becky Crockett Caitlin Polanski Brith Otterud Support • National Institute of Neurological Diseases and Stroke (1 K 23 NS 091511 -01) • Muscular Dystrophy Association • Myotonic Dystrophy Foundation • Biogen Idec • Utah Neuromuscular Fund