Transcript Congenital myotonic dystrophy

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:
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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
Johnson, et al, 2015
N=150 parents
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 1245.9 (474.9
length (SD)
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
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
2MWT
6MWT
10m walk
Lean mass of the
1.00000
right leg
1.00000
2MWT
0.59239
p=0.0047
0.97743
p<.0001
1.00000
6MWT
0.62179
p=0.0034
10m 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 (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)
 2.2% extremely low
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
7.7
<13
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
2 mo, 1, 3,
7, 11, 16
1150-2200
66.6% male
Pediatric
controls1
9
44.4% male
1 mo-13
NA
DM1 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)
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 DM1 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 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
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
NA
5
BIN1, ex 13
0.36
0.32
0.09
19
MBNL1, ex 5
0.53
0.51
NA
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
dPSICD
M
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
0.38
0.2
0.07
411
0.011
WNT signaling
FERMT2, ex 13
0.37
0.15
0.07
418
0.086
Myogenesis
TNNI2, ex 22
0.15
NA
NA
419
0.022
Ca regulation
1. T-test comparing the PSI value between the 3 youngest CDM samples (2mo-3 yrs) against 3 oldest.
2. 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
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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