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

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

Lead Psychologists Thomas Frazier, PhD (CC) Deborah Pearson, PhD (UTH) Audrey Thurm, PhD (NIH)

Training Hope Northrup, MD (UTH) Kira Dies, ScM, CGC (BCH)

MRI Coordinating Center Simon Warfield, PhD (BCH)

TSC Longitudinal Study PMS Longitudinal Study

Project Leader Alex Kolevzon, MD (Mt Sinai)

PTEN Longitudinal Study PTEN Clinical Pilot Study

Project Leader Antonio Hardan, MD (Stanford)

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)

Project Leader Charis Eng, MD, PhD (CC)

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)

Co-Investigators Mustafa Sahin, MD, PhD (BCH) Charis Eng, MD, PhD/Thomas Frazier, PhD (CC) Julian Martinez, MD (UCLA)

Co-Investigators

Thomas Frazier, PhD (CC) Antonio Hardan, MD (Stanford) Mustafa Sahin, MD, PhD (BCH) Julian Martinez, MD (UCLA)

DMCC

Executive Committee Sahin Bebin Buxbaum Bernstein Eng Berry-Kravis Hardan Frazier Kolevzon Kaufmann Krueger Martinez Filip-Dhima Northrup Dies Powell Soorya Thurm Wu

TSC Alliance PTEN Foundation

PTEN World PTEN Life

PMS

Foundation

Seaver Foundation

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)

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

Treatment(s)?

Broad-spectrum Drugs for subcategories

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

Facial angiofibromas

Hypomelanotic macules

Clinical Features of Tuberous Sclerosis Dermatologic Findings

Ungual fibromas

Shagreen patches

Cephalic plaques

Clinical Features of Tuberous Sclerosis

Seizures, Intellectual disability/developmental delay

Central Nervous System Findings Subependymal nodules, Cortical tubers, SEGAs

Clinical Features of Tuberous Sclerosis

Angiomyolipomas, Cysts, RCCs

Renal Findings

Clinical Features of Tuberous Sclerosis

Cardiac rhabdomyoma on prenatal ultrasound

Cardiac and Lung Findings

Lymphangioleiomyomatosis

Frequency of Disease Phenotype Observed Among TSC Patients

Phenotype Frequencies Cortical tuber Facial angiofibroma Renal angiomyolipoma Subependymal nodule Cardiac rhabdomyomas Ungual fibroma

~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 ligation-dependent probe amplification (MLPA)

Chromosomal rearrangements

Oligonucleotide and SNP array analyses

1. Crino P. Acta Neuropathol. 2013;125:317-332. 2. Northrup H, Krueger D. Pediatr Neurol. 2013;49:243-254. 3. 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

SNP=single polynucleotide polymorphism.

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

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

1. Tyburczy ME. Presented at: 2013 International Research Conference on TSC and Related Disorders. June 20-23,2013; Washington DC, USA. 2. Qin W et al. Hum Genet. 2010;127:573-582.

Timeline of TSC Discoveries

108 years

von Recklinghausen cardiac myomata

in newborn

1862 1879

1993

1997

2001 2006 2008 1987

2014

Bourneville

Linkage to chromosome 9

Genetic Heterogeneity

TSC2

TSC1

Insulin signaling pathway

Rapamycin tx brain tumors

Many clinical trials

Rapamycin trial for kidney and

lung

2003

mTOR mTORmTOR

AMP

AMPK

Energy Level

Protein synthesis

Cell growth

rheb

mTOR S6K S6

TSC2

TSC1

Growth Factors

Akt

Simplified TSC pathway

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

Genomic Medicine Institute

Trichilemmoma

Palmar pits and keratoses

Gum papillomas

Tongue papillomas

More keratoses

Patients provided consent for photographs

Where’s the gene? • Nelen et al, 1996, Nature Genetics letter: linkage to 10q22-

23 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

Lifetime cancer risks*

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%

Genomic Medicine Institute

*To age 70 by Tan et al and Bubien et al; to age 60 by Nieuwenhuis et al

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

Genomic Medicine Institute Tan et al, Clin Cancer Res 2012

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 Control PTEN ASD

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

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 Adaptive Behavior Vineland Adaptive Behavior Scales Language Mullen and Vineland Subscales

Macarthur Bates Communication Developmental Inventory Motor Functioning Mullen and Vineland Subscales

Developmental Coordination Disorder Questionnaire Autism Symptoms Autism Diagnostic Observation Schedule

Pervasive Developmental Disorders Behavior Inventory Repetitive Behavior Scales-Revised

Other Symptoms Nisonger Child Behavior Rating Form Aberrant Behavior Checklist Sensory Profile Questionnaire- Short Form

Previous Phenotyping Research Studies

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

Rearrangement N %

Terminal deletion 21 66

Ring 22 6 19

Unbalanced translocation 2 6

Point mutations 2 6

Interstitial deletion 1 3

Soorya et al., 2013

N % Nonverbal IQ classification (n=30)

Average (IQ 100-110) 1 3.3 Mild intellectual disability (IQ 50-55 to 70) 3 10 Moderate intellectual disability (IQ 35-40 to 50-55) 3 10 Severe intellectual disability (IQ 20-25 to 35-40) 7 23.3 Profound intellectual disability (IQ < 20-25 16 53.3

75%

9.40%

15.60%

Autism

ASD

non-ASD

ASD and IQ diagnostic classifications

Soorya et al., 2013

*Phelan & McDermid, 2012

*

Kolevzon et al., 2015

Association between deletion size and phenotypic variables

Phenotypic variable N Deletion size

BCa confidence interval# Lower Upper

Number of dysmorphic features 32 .474* .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) †approached significance at .05 level

Soorya et al., 2013

Refining the neurobehavioral phenotype

Mieses et al., IMFAR 2014

PMS: n=27 ASD/ID: n=38 ASD: n=23

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.

Diffusion Tensor Imaging

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 IGF-1 in Children with Phelan-McDermid Syndrome

Placebo Placebo

IGF-1 IGF-1

12 weeks 12 weeks

wash-out

4 weeks

Subject Chronological Age (months)

Estimated Mental Age Equivalent (months)

Vineland Adaptive Behavior Scale

Composite Standard Score

1 177.4 8.5 29 2 103.7 10.3 47 3 66.3 11.3 52 4 64.6 12.3 43 5 109.7 36 61 6 91.8 7 50 7 172.7 30.5 31 8 71.1 31 51 9 61.8 9.3 45

Baseline Demographic Characteristics

Kolevzon et al., 2014, Mol Autism

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 IGF-1 (N) Placebo (N) Constipation 4 3 Sedation 1 0 Decreased appetite 2 3 Periobital / facial swelling 1 0 Diarrhea 1 2 URTI 5 5 Sleep Disturbance 7 2 Increased appetite 4 0 Mood changes 2 1 Increased thirst 1 0 Increased phlegm 1 0 Teeth grinding 1 0 Cough 1 2 Hand flapping 1 0 Increased bowel movements 1 0 Increased chewing/biting 1 0 Decreased visual acuity 1 0 Lethargy/decreased energy 1 1 Cooler temp/sweating 1 0 Runny nose/congestion 1 1 Irritability 2 1 Gait changes 1 2 Stomach virus 1 1 Anxiety 0 2 Increased urine frequency 2 0 Fever 3 3 Increased energy 1 0 Gagging 1 0 Increased thirst 0 1 Conjunctivitis 1 0 Erythema / swollen eyes 1 0 Vomiting 1 1 Rash 3 0 Nose swelling 1 0 Warmer body temperature 1 0 Hair loss 1 0 Increased aggression 1 0 Hypoglycemia 7 3

Kolevzon et al., 2014, Mol Autism

THE DEVELOPMENTAL SYNAPTOPATHIES CONSORTIUM (U54NS092090) IS PART OF NCATS RARE DISEASE CLINICAL RESEARCH NETWORK (RDCRN), AN INITIATIVE OF THE OFFICE OF RARE DISEASE RESEARCH (ORDR). THIS CONSORTIUM IS FUNDED THROUGH COLLABORATION BETWEEN NCATS, AND THE NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE (NINDS) OF THE NATIONAL INSTITUTES OF HEALTH. THE CONTENT OF THIS PUBLICATION IS SOLELY THE RESPONSIBILITY OF THE AUTHORS AND DOES NOT NECESSARILY REPRESENT THE OFFICIAL VIEWS OF THE NATIONAL INSTITUTES OF HEALTH

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