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Developmental Synaptopathies Associated with
TSC, PTEN and SHANK3 Mutations
Project Overview
October 7, 2015
Kira A. Dies, ScM, CGC
Clinical Research and Regulatory Affairs Service
Translational Neuroscience Center
Boston Children’s Hospital
Overview of Developmental
Synaptopathies Consortium (DSC)
• 10 site consortium funded by NCATS, NIMH,
NINDS and NICHD
• 5 year grant
• Focusing on three well-established genetic
syndromes that are associated with high
penetrance for ASD/ID caused by TSC1/2,
PTEN and SHANK3 mutations and disruptions in
shared molecular pathways
Developmental Synaptopathies
Consortium (DSC)
What is a Synaptopathy?
• The dysfunction of connectivity in the brain: these
disruptions in synaptic structure and function are major
determinants of the resulting brain disease or disorder.
• Synaptic function and connectivity are implicated in:
- communication
- interpersonal interactions
- functioning and processing
Developmental Synaptopathies Consortium (DSC)
RDCRN
Executive Committee
Scientific Advisory Board
Wendy Chung, MD, PhD (Columbia U)
Anthony Wynshaw-Boris, MD, PhD
(Case)
Helen Tager-Flusberg, PhD (BU)
Catherine Stoodley, PhD (American U)
Michael Aman, PhD (Ohio State U)
MRI Coordinating Center
Simon Warfield, PhD (BCH)
Mustafa Sahin, MD, PhD
Principal Investigator
Administrative Unit
Joseph Buxbaum, PhD (Mt. Sinai), Director
Rajna Filip-Dhima, MS (BCH), Project Manager
Kira Dies, ScM, CGC (BCH), Regulatory Manager
Sahin
Buxbaum
Eng
Hardan
Kolevzon
Krueger
Filip-Dhima
Dies
Bebin
Bernstein
Berry-Kravis
Frazier
Kaufmann
Martinez
Northrup
Powell
Soorya
Thurm
Wu
TSC Alliance
PTEN Foundation
PTEN World
PTEN Life
PMS
Foundation
Seaver
Foundation
Lead Psychologists
Thomas Frazier, PhD (CC)
Deborah Pearson, PhD (UTH)
Audrey Thurm, PhD (NIH)
Training
Hope Northrup, MD (UTH)
Kira Dies, ScM, CGC (BCH)
PTEN Clinical Pilot Study
TSC Longitudinal Study
Project Leader
Darcy Krueger, MD, PhD
(CCHMC)
Co-Investigators
Mustafa Sahin, MD, PhD (BCH)
Martina Bebin, MD (UAB)
Joyce Wu, MD (UCLA)
Hope Northrup, MD (UTH)
PMS Longitudinal Study
Project Leader
Alex Kolevzon, MD
(Mt Sinai)
Co-Investigators
Mustafa Sahin, MD, PhD (BCH)
Audrey Thurm, PhD (NIH)
Elizabeth Berry-Kravis, MD, PhD
/ Latha Soorya, PhD (Rush)
Jon Bernstein, MD (Stanford)
Craig Powell, MD, PhD (UTSW)
PTEN Longitudinal Study
Project Leader
Charis Eng, MD, PhD
(CC)
Co-Investigators
Thomas Frazier, PhD (CC)
Antonio Hardan, MD (Stanford)
Mustafa Sahin, MD, PhD (BCH)
Julian Martinez, MD (UCLA)
DMCC
Project Leader
Antonio Hardan, MD
(Stanford)
Co-Investigators
Mustafa Sahin, MD, PhD (BCH)
Charis Eng, MD, PhD/Thomas Frazier,
PhD (CC)
Julian Martinez, MD (UCLA)
Overall Goals of the DSC
• Long term goal:
• mechanistic analysis of three genetic disorders with high
penetrance of ASD/ID to shed light on molecular pathways
and targets relevant to ASD/ID: Tuberous Sclerosis Complex
(TSC1 and TSC2 genes), PTEN hamartoma syndrome and
Phelan-McDermid syndrome (PMS; SHANK3 gene)
• The short-term goals are:
• (1) to better characterize the neurodevelopmental
phenotype of these three groups of patients
longitudinally
• (2) identify biomarkers that predict risk for disease
severity/progress.
ASD genes to date
Using traditional approaches
Buxbaum, J
The problem in Autism:
500-1000 susceptibility genes
Broad-spectrum
Drugs for
subcategories
Treatment(s)?
One drug
for every gene
TUBEROUS SCLEROSIS COMPLEX
What is Tuberous Sclerosis
Complex (TSC)?
• Multi-system disease
• Causes hamartomas (benign growths) in
brain, eye, skin, kidneys, and heart
• Autosomal dominant (TSC1 and TSC2
genes)
• Usually presents with seizures, cognitive
impairment, autism
• Incidence: 1: 6,000-10,000
Clinical Diagnostic Criteria for
Tuberous Sclerosis Complex
11 Major Features
• Hypomelanotic macules (≥3; at least 5mm diameter)
• Angiofibroma (≥3) or fibrous cephalic plaque
• Ungual fibromas (≥2)
• Shagreen patch
• Multiple retinal hamartomas
• Cortical dysplasias (≥3 )*
• Subependymal nodule (≥2)
• Subependymal giant cell astrocytoma
• Cardiac rhabdomyoma
• Lymphangiomyomatosis (LAM)**
• Angiomyolipomas (≥2)**
6 Minor Features
• “Confetti” skin lesions
• Dental enamel pits (≥3)
• Intraoral fibromas (≥2)
• Retinal achromic patch
• Multiple renal cysts
• Nonrenal hamartomas
Definite TSC: 2 major features or 1 major feature with 2 minor features
Possible TSC: either 1 major feature or >2 minor features
* includes tubers and cerebral white matter radial migration lines
** a combination of the 2 Major clinical features LAM and angiomyolipomas without
Other features does not meet criteria for a Definite Diagnosis
Clinical Features of Tuberous Sclerosis
Dermatologic Findings
Hypomelanotic macules
Facial angiofibromas
Clinical Features of Tuberous Sclerosis
Dermatologic Findings
Ungual fibromas
Shagreen patches
Cephalic plaques
Clinical Features of Tuberous Sclerosis
Central Nervous System Findings
Subependymal nodules, Cortical tubers, SEGAs
Seizures, Intellectual disability/developmental delay
Clinical Features of Tuberous Sclerosis
Renal Findings
Angiomyolipomas, Cysts, RCCs
Clinical Features of Tuberous Sclerosis
Cardiac and Lung Findings
Cardiac rhabdomyoma on
prenatal ultrasound
Lymphangioleiomyomatosis
Frequency of Disease Phenotype
Observed Among TSC Patients
Phenotype
Cortical tuber
Facial angiofibroma
Renal angiomyolipoma
Subependymal nodule
Cardiac rhabdomyomas
Ungual fibroma
Frequencies
~90%
>75%
>80%
~80%
~50%
20-80%
Genetic Aspects of TSC
•
•
•
•
•
Autosomal dominant inheritance
Two-thirds of cases sporadic
One-third of cases familial
Variable expression
Common in the population with
approximately 1:6,000-10,000
individuals affected
Genetic Testing of TSC:
Current Status
• Sanger sequencing identifies a
mutation in 75% to 90% of the
individuals with a definite clinical
diagnosis of TSC1
• In 10% to 25% of patients with TSC,
there is no mutation identified (NMI)
in either TSC1 or TSC22
• Incorporating new DNA-sequencing
technologies into the standard genetic
assessment for patients with TSC can
increase the detection rate of
mutations in TSC3
Current Methods for
TSC Genetic Testing3
Mutation type
Technique used
Point mutations and
small insertions/
deletions
Genomic DNA
sequencing of coding
regions and neighboring
splice sites
Intragenic deletions and
duplications
Analysis by
multiplex ligationdependent probe
amplification (MLPA)
Chromosomal
rearrangements
Oligonucleotide and SNP
array analyses
SNP=single polynucleotide polymorphism.
1.
2.
3.
Crino P. Acta Neuropathol. 2013;125:317-332.
Northrup H, Krueger D. Pediatr Neurol. 2013;49:243-254.
Mayer K et al. Eur J Hum Genet. 2014:22(2): e1-e4.
Rare splice mutations, located deep in introns, or
mutations in promoter regions are not detected by
current DNA-based diagnostics3
NGS Identifies Mosaic Mutations
in Patients With TSC
•
NGS analysis of genomic DNA, including
promoter regions, all exons, and most of
the intronic regions of 46 TSC NMI
patients, revealed that1
– Mutations in TSC1 and TSC2 occur
in over 50% of NMI patients with
TSC
– Mosaic and splice region mutations
were common
Ultra-deep pyrosequencing DNA analysis
of blood samples of 38 NMI patients with
TSC revealed2
– Two TSC2 mutations —each at 5.3%
read frequency in different patients—
consistent with mosaicism
•
1.
2.
Type of mutation identified by NGS in
NMI patients with TSC1 (N=46)
Pathogenic variants
22
Mosaic mutations
11
Splice-site mutation
10
Deletion in TSC2 promoter
1
Heterozygous mutations in
TSC1 and TSC2 missed by
previous analyses
2
Tyburczy ME. Presented at: 2013 International Research Conference on TSC and Related Disorders. June 20-23,2013; Washington DC, USA.
Qin W et al. Hum Genet. 2010;127:573-582.
Timeline of TSC Discoveries
TSC2
von Recklinghausen
cardiac myomata
in newborn
Insulin signaling Rapamycin tx
brain tumors
pathway
Many clinical trials
Linkage to
chromosome 9
mTOR
Genetic
Heterogeneity
mTOR
Bourneville
Rapamycin trial
for kidney and
lung
TSC1
108 years
1862
1879
1997
1987
1993
2008
2003
2001
2006
2014
Simplified TSC pathway
Growth Factors
Energy Level
Akt
AMP
TSC2
AMPK
TSC1
rheb
mTOR
S6K
S6
Protein
synthesis
Cell growth
Rapamycin
• Naturally occurring substance
• Discovered in 1965
• Binds mTOR and inhibits its action, thus
preventing cell division and growth
Major Clinical Trials with mTOR
inhibitors in TSC
• 2007
• 2008
• 2009
• 2010
• 2011
• 2012
• 2013
PTEN HAMARTOMA TUMOR
SYNDROME
PTEN Hamartoma Tumor
Syndrome (PHTS)
• Any patient with germline PTEN
mutation
– Cowden syndrome
– Bannayan-Riley-Ruvalcaba syndrome
(BRRS)
– Proteus-like syndrome
• Areas of greatest clinical concerns
– Increased malignancy risks
– Benign tumors
– Neurodevelopmental issues
Genomic Medicine Institute
Benign Growths in PHTS
• Skin and mucosa
– Trichilemmomas (hair
follicle bumps)
– Keratoses (rough
patches) on extremities
– Papules on tongue,
gums, inside nose
– Lipomas (fatty bumps)
– Fibromas
• Lhermitte-Duclos
(benign tumor of the
cerebellum)
• GI polyps
• Uterine fibroids, other
genitourinary tumors
• Genitourinary
malformations
• Benign breast disease
• Thyroid nodules/goiter/
Hashimoto’s thyroiditis
• Vascular anomalies/
hemangiomas
Genomic Medicine Institute
Key Mucocutaneous Features
Trichilemmoma
Gum papillomas
More keratoses
Palmar pits and
keratoses
Tongue papillomas
Patients provided consent for photographs
Genomic Medicine Institute
Where’s the gene?
• Nelen et al, 1996, Nature Genetics letter: linkage to 10q2223 in 12 CS families, 4 with LDD
• Strict inclusion criteria per International Cowden
Consortium
Pathognomonic criteria
Major criteria
Minor criteria
Mucocutaneous lesions
• Facial trichilemmomas
• Acral keratoses
• Papillomatous papules
Macrocephaly
Breast cancer
Non-medullary thyroid cancer
Adult-onset Lhermitte-Duclos
disease (LDD)
Mental retardation (IQ < 75)
Goiter
GI Hamartomas
Lipomas
Fibrocystic breast disease
Fibromas
GU tumor or malformation
Operational diagnosis given to a person with:
1. Mucocutaneous lesions alone if:
a. > 6 facial papules, > 3 being trichilemmomas, or
b. Cutaneous facial papules + oral papillomas, or
c. Oral papillomas + acral keratoses, or
d. > 6 palmoplantar keratoses
2. 2 major criteria, one being macrocephaly
or LDD
3. 1 major + 3 minor criteria
4. 4 minor criteria
Tumor Suppressor Gene Found
at 10q23
• LOH at 10q23 noted in prostate cancers, glioblastoma
• Li et al, Science 1997: PTEN mutations in somatic
glioblastoma, prostate, breast cancer cell lines
– Protein tyrosine phosphatase domain
– Homologous to chicken tensin
• 2 weeks later: Steck et al, Nature Genetics – MMAC1,
“Mutated in Multiple Advanced Cancers”
– Glioma, prostate, kidney and breast carcinoma cell
lines or tumors
Cancer Risks in PHTS
Tan et al, 2011
Bubien et al, 2013
Nieuwenhuis et al, 2013
Number of patients
368
146
180
Median age (yrs)
39
36
32
Female breast
85%
77%
67%
Thyroid
35%
38%
Women: 25%
Men: 6%
Renal
34%
Elevated in women, N insufficient for
further analysis
Women: 9%
Men: 2%
Endometrial
28%
Elevated, N insufficient for further
analysis
21%
Colorectal
9%
Elevated in men, N insufficient for
further analysis
Women: 17%
Men: 20%
Melanoma
6%
Elevated, N insufficient for further
analysis
Men: 2%
Lifetime cancer risks*
*To age 70 by Tan et al and Bubien et al; to age 60 by Nieuwenhuis et al
Genomic Medicine Institute
Updated Screening
Recommendations
Cancer
General
population
risk
Lifetime Risk
with PHTS
(Average age)
Old Risk Data/
Screening Guidelines
New Screening
Guidelines
Breast
12%
~85% (40s)
25-50%; begin
mammograms at age 30
Starting at age 30:
annual mammogram;
consider MRI for
patients with dense
breasts
Thyroid
1%
35% (30s/40s)
10%; begin annual
ultrasounds at age 18
Annual ultrasound at dx
age
Endometrial
(uterine)
2.6%
28% (40s/50s)
?5-10%; no
recommendations
Starting at age 30:
annual endometrial
biopsy or transvaginal
ultrasound
Renal cell
(kidney)
1.6%
34% (50s)
?elevated; no
recommendations
Starting at age 40: renal
imaging every 2 years
Colon
5%
9% (40s)
??; no recommendations
Starting at age 40:
colonoscopy every 2
years
Melanoma
2%
6% (40s)
??; no recommendations
Annual dermatologic
examination
Tan et al, Clin Cancer Res 2012
Genomic Medicine Institute
Neurodevelopmental Aspects of
PHTS
• Increase in developmental delays in children
– Spectrum from mild to severe
– Some require special education, others excel in school
classes
• Increased risks for autism spectrum disorders
• New research: specific deficits in motor and
executive function
• Recommend thorough developmental evaluation
in children
• Macrocephaly (large head size) very common
Genomic Medicine Institute
White matter hypo-intensities
PTEN ASD
PTEN ASD
Control
PTEN-ASD also had poorly
developed white matter
Slow Processing Speed and
Working Memory Deficits
Also show reductions in Full Scale IQ (p<.001)
White matter abnormalities drive
cognitive deficits
PHELAN-MCDERMID SYNDROME
Rare mutations in autism
Phelan-McDermid Syndrome
global developmental delay
intellectual disability
absent or severely delayed speech
hypotonia
dysmorphic features
The SHANK3 gene
Phelan-McDermid syndrome is caused
by deletions or mutations of the
SHANK3 gene on chromosome 22q
SHANK3 is a master scaffolding
protein which forms a framework for
the connections between brain cells
Previous Phenotyping Research
Studies
Physical and neurological exam
Renal ultrasound
Clinical Genetics Evaluation
Electroencephalography
Medical and Psychiatric History
Laboratory bloodwork
Echocardiography
Height and weight measurement
Electrocardiography
Head circumference
Domain
Measure
Global Cognitive Ability
Mullen Scales for Early Learning or
Stanford Binet-5
Vineland Adaptive Behavior Scales
Mullen and Vineland Subscales
Macarthur Bates Communication Developmental Inventory
Mullen and Vineland Subscales
Developmental Coordination Disorder Questionnaire
Autism Diagnostic Observation Schedule
Pervasive Developmental Disorders Behavior Inventory
Repetitive Behavior Scales-Revised
Nisonger Child Behavior Rating Form
Aberrant Behavior Checklist
Sensory Profile Questionnaire- Short Form
Adaptive Behavior
Language
Motor Functioning
Autism Symptoms
Other Symptoms
Demographic/Genetic Results
Sample Size
32
Male : Female
18:14
Age (years)
1.7- 45.4 (X = 8.8)
Deletion Size (Mb)
.058 (point) – 8.5
Soorya et al., 2013
Rearrangement
N
%
Terminal deletion
21
66
Ring 22
6
19
Unbalanced translocation
2
6
Point mutations
2
6
Interstitial deletion
1
3
ASD and IQ diagnostic
classifications
15.60%
9.40%
Autism
ASD
non-ASD
75%
Nonverbal IQ classification (n=30)
Average (IQ 100-110)
Mild intellectual disability (IQ 50-55 to 70)
Moderate intellectual disability (IQ 35-40 to 50-55)
Severe intellectual disability (IQ 20-25 to 35-40)
Profound intellectual disability (IQ < 20-25
Soorya et al., 2013
N
%
1
3
3
7
16
3.3
10
10
23.3
53.3
*
*Phelan & McDermid, 2012
Kolevzon et al., 2015
Association between deletion
size and phenotypic variables
Phenotypic variable
N
Deletion
size
Number of dysmorphic features
32
.474*
BCa confidence
interval#
Lower
Upper
.145
.738
Number of medical comorbidities
32
.386*
.022
.640
Nonverbal IQ estimate
29
-.332
-.640
.112
Gross motor skills (Vineland)
31
-.402†
-.728
.036
Fine motor skills (Vineland)
31
-.123
-.473
.254
Expressive language skills (Vineland)
32
-.184
-.531
.199
Receptive language skills (Vineland)
32
-.231
-.553
.154
Qualitative abnormalities in reciprocal social interactions (ADI-R)
30
.466*
.073
.723
Qualitative abnormalities in communication (ADI-R)
30
.498*
.091
.740
Restricted, repetitive, and stereotyped patterns of behavior (ADI-R)
30
-.229
-.592
.214
*significant at .05 level (two-tailed test)
significance at .05 level
†approached
Soorya et al., 2013
Refining the neurobehavioral
phenotype
PMS: n=27
ASD/ID: n=38
ASD: n=23
Mieses et al., IMFAR 2014
Diffusion Tensor Imaging
Reduced myelination in areas of the brain associated with language
(superior longitudinal fasciculus; the genu of the corpus callosum;
Broca’s area) and social functioning (medial temporal white matter)
for the PMS group relative to controls.
N = 10
Wang & Lim, unpublished
Reversal of motor deficits in
Shank3-deficient mice (Het)
after treatment with IGF-1
Bozdagi et al, 2013
Effect of IGF-1 on Synaptic Deficits
Bozdagi et al, 2013
A Double-Blind Placebo-Controlled Crossover Trial of IGF1 in Children with Phelan-McDermid Syndrome
Placebo
wash-out
12 weeks
Placebo
12 weeks
4 weeks
IGF-1
IGF-1
Baseline Demographic Characteristics
Subject
Chronological
Age (months)
1
2
3
4
5
6
7
8
9
177.4
103.7
66.3
64.6
109.7
91.8
172.7
71.1
61.8
Kolevzon et al., 2014, Mol Autism
Estimated
Mental Age
Equivalent
(months)
8.5
10.3
11.3
12.3
36
7
30.5
31
9.3
Vineland Adaptive
Behavior Scale
Composite
Standard Score
29
47
52
43
61
50
31
51
45
Effects of IGF-1 on Social Withdrawal
N = 9; t = -2.107; p = 0.040
Kolevzon et al., 2014, Mol Autism
Effects of IGF-1 on Repetitive Behavior
N = 9; t = -2.077; p = 0.042
Kolevzon et al., 2014, Mol Autism
Adverse Events
Kolevzon et al., 2014, Mol Autism
Constipation
Sedation
Decreased appetite
Periobital / facial swelling
Diarrhea
URTI
Sleep Disturbance
Increased appetite
Mood changes
Increased thirst
Increased phlegm
Teeth grinding
Cough
Hand flapping
Increased bowel movements
Increased chewing/biting
Decreased visual acuity
Lethargy/decreased energy
Cooler temp/sweating
Runny nose/congestion
Irritability
Gait changes
Stomach virus
Anxiety
Increased urine frequency
Fever
Increased energy
Gagging
Increased thirst
Conjunctivitis
Erythema / swollen eyes
Vomiting
Rash
Nose swelling
Warmer body temperature
Hair loss
Increased aggression
Hypoglycemia
IGF-1 (N)
4
1
2
1
1
5
7
4
2
1
1
1
1
1
1
1
1
1
1
1
2
1
1
0
2
3
1
1
0
1
1
1
3
1
1
1
1
7
Placebo (N)
3
0
3
0
2
5
2
0
1
0
0
0
2
0
0
0
0
1
0
1
1
2
1
2
0
3
0
0
1
0
0
1
0
0
0
0
0
3
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