“interrogating rettsyndrome: developing ideas for research ... · that matters” angus clarke,...
TRANSCRIPT
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“Interrogating Rett Syndrome:
developing ideas for research
that matters”
Angus Clarke,
Clinical Genetics, Prifysgol Caerdydd,
Cymru
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Research into Rett Syndrome
Four Factors:
• Unique clinical entity
• Biology is fascinating, intriguing
• The patients (and their families) are waiting
• An effective, rational treatment may just be
a possibility
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Research THAT MATTERS
The research you would do if your daughter
had Rett syndrome:
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Research THAT MATTERS:
• Natural History
• Genotype-Phenotype research (and
other loci)
• Outcome Measures for Possible Trials
• Molecular Mechanisms
• Potential therapies
• Experiences and Ethics
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Career of a Diagnosis
• Recognition of a clinical entity
• Narrow diagnostic criteria =>
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Career of a Diagnosis
• Recognition of a clinical entity
• Narrow diagnostic criteria =>
• Progress in recognising (+/- understanding)
the underlying pathology
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Career of a Diagnosis
• Recognition of a clinical entity
• Narrow diagnostic criteria =>
• Progress in recognising (? understanding)
the underlying pathology
• Recognition of wider range of phenotypes
associated with the same pathology
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Career of a Diagnosis
• Recognition of a clinical entity
• Narrow diagnostic criteria =>
• Progress in recognising (? understanding)
the underlying pathology
• Recognition of wider range of phenotypes
associated with the same pathology
• Appropriate clinical indications for further
investigation
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Career of Rett Syndrome
• Recognition of “cerebral atrophy with
hyperammonaemia” by Andreas Rett 1966
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Career of Rett Syndrome
• Recognition of “cerebral atrophy with
hyperammonaemia” by Andreas Rett 1966
• English language publication 1983 by
Hagberg et al
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Career of Rett Syndrome
• Recognition of “cerebral atrophy with
hyperammonaemia” by Andreas Rett 1966
• English language publication 1983 by
Hagberg et al
• Diagnostic criteria formalised ..…….
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Career of Rett Syndrome
• Recognition of “cerebral atrophy with
hyperammonaemia” by Andreas Rett 1966
• English language publication 1983 by
Hagberg et al
• Diagnostic criteria formalised ..…
and then progressively revised
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Career of Rett Syndrome
• Recognition of “cerebral atrophy with
hyperammonaemia” by Andreas Rett 1966
• English language publication 1983 by
Hagberg et al
• Diagnostic criteria formalised ..…
and then progressively revised ….
• Diagnostic landscape
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Initial Diagnostic Criteria
• Normal development to 6 months
• Developmental stagnation
• Regression - social contact, hand use
• Hand stereotypies
• Recovery of social contact
• Persisting profound cognitive impairment
• Gait and truncal ataxia
• (Only girls)
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Other associated features
• Muscle tone, including spasticity in legs
• Ventilatory rhythm
• Vasomotor disturbances including cool,
atrophic feet
• Seizures
• Scoliosis
• Impaired growth
– including 4th metatarsal
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Rett syndrome is primarily a
CLINICAL diagnosis
with a highly characteristic timecourse
and evolution, although:
(i) recognition of clinical features before 6
months in (otherwise) classic cases,
(ii) molecular studies have shown us that
there are variant forms of Rett syndrome
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Variant / Atypical forms
– Forme fruste (late stagnation, no regression)
– Preserved speech (Zapella)
– Congenital onset (no regression)
– Early onset of seizures (Hagberg)
– Angelman-like
– Male cases (some with 47,XXY)
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How was RTT not “spotted” until
1966 ?• 6-12 months: “She’ll catch up”
• Acute regression
– degenerative cerebral disease
– undiagnosed encephalopathy
– childhood psychosis or autism
• Dystonic, ataxic or diplegic cerebral palsy; microcephaly and epilepsy
Recognition of the temporal pattern
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”Early concerns”
Systemic disease
Non-progressive intellectual impairment
? Dysmorphism – dx by genetic test or opinion
Regression – severe autism
- diagnosis by biochemical methodsLeigh’s disease
- diagnosis by MRIBrain tumour
Neurological problem – seizures
- microcephaly
- cerebral palsy
Socio-emotional problem
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original diagnostic landscape
Microcephaly
Cerebral palsy
Severe
Epilepsies cognitive
impairment
Psychosis and
autism Neurodegenerative
disorders
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original diagnostic landscape
Cerebral palsy
Epilepsies
Neurodegenerative disordersAutism
Microcephaly
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then ...........
Cerebral palsy
Epilepsies
Neurodegenerative disordersAutism
Microcephaly
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then Rett syndrome ....
Cerebral palsy
Epilepsies
Neurodegenerative disordersAutism
Microcephaly
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then Rett syndrome ... arrives
Cerebral palsy
Epilepsies
Neurodegenerative disordersAutism
Microcephaly
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revised diagnostic landscape ...
Cerebral palsy
Epilepsies
Neurodegenerative disordersAutism
Microcephaly
Rett Syndrome
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the later diagnostic landscape ...
post-encephalitic
severe
cerebral palsy autism
microcephaly, MATURE
delay, spasticity RETT West’s and
SYNDROME severe
syndromic epilepsies
disorders Angelman
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the diagnostic landscape - 4
”MECP2 disease” (UBE3A)
Angelman
forme – mild – CLASSIC – severe – early - congenital
fruste RTT RTT RTT fits onset(no regression) (with regression) (no regression)
infantile spasms
(CDKL5)
(TSC1/2)
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Cerebral atrophy
“with hyperammonaemia” ?
• Initial observations not supported
• BUT many cases found to have some
metabolic anomalies, although
– Although often variable and inconsistent
– Typically an increase in blood lactic acid or
ammonia or in 24 hour orotic acid excretion
• One very unusual family:
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Search for genetic basis
• X-linked dominant, male-lethal disorder
would account for unusual pattern of
occurrence
– Sporadic
– Occurring only in females
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Search for genetic basis
• X-linked dominant, male-lethal disorder
would account for unusual pattern of
occurrence
– Sporadic
– Only in females
• Gender bias in (high) mutation rates
=> a better explanation
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Search for genetic basis
• X-linked dominant, male-lethal disorder
would account for unusual pattern of
occurrence
– Sporadic
– Only in females
• Gender bias in (high) mutation rates
=> a better explanation
• Wide clinical variability from X inactivation
– Apparent in familial cases and MZ twins
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Search for genetic basis
• Cytogenetic “clues” … t(Xp;A) x 2
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Search for genetic basis
• Cytogenetic “clues” … t(Xp;A) x 2
• Uncertain significance of common
ancestry in Swedish genealogy cases
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Search for genetic basis
• Cytogenetic “clues” … t(Xp;A) x 2
• Uncertain significance of common
ancestry in Swedish genealogy cases
• Linkage analysis difficult
– Few families
– Familial cases perhaps atypical
(criteria too relaxed ??)
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Search for genetic basis
• Cytogenetic “clues” … t(Xp;A) x 2
• Uncertain significance of common ancestry
in Swedish genealogy cases
• Linkage analysis difficult
– Few families
– Familial cases perhaps atypical
(criteria relaxed ??)
• Xq28 a likely region
– Amir et al 1999 => MECP2 gene
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MeCP2 Protein: Adrian Bird 1992
• Already implicated in repression of
transcription via methylated CpG groups
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MeCP2 Protein: Adrian Bird 1992
• Already implicated in repression of
transcription via methylated CpG groups
• Rapid progress in the molecular biology
• Steady progress in diagnostic utility
• Slower progress in understanding the
pathogenesis
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Rett syndrome is caused by
mutations in MECP2
•Methyl-CpG-binding protein 2
•Global transcription repressor
•Locus at Xq28
Methyl Binding
Domain
AT hooks
Transcription
Repression Domain
Amir et al 1999
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Correlation between the
mutation and the disorder
• truncating vs missense mutations
• early truncating vs late truncating
mutations
• some common mutations associated
statistically with greater or lesser severity
– R133C and C-terminal deletions milder
– R270X more severe
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Large deletions in MECP2
Exon 4.2 - 4.3 (n=1)
Exon 1 & 2 (n=3)
Exon 3 & 4.1 (n=3)
Exon 3 – 4.3 (n=5)
Exon 4.1 - 4.3 (n=1)
Exon 4.3 – IRAK1 (n=1)
Exon 3 & 4 (n=3)
Exon 4.2 (n=1)
Exon 4.2 - 4.4 (n=1)
Exon 4.3 (n=1)
Exon 4 (n=1)
MECP2
Exon 1 Exon 2 Exon 3 Exon 4
~2Mb ~5kb ~60kb <1kb 1.6kb ~124kb
IRAK1 L1CAMSYBL1
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MeCP2 has 2 isoforms
Exon 1 Exon 2 Exon 3 Exon 4
5’UTR MBD TRD 3’UTR (8.5kb)
ATG
ATG
•Previously exon 1 was thought to be non coding
•Now known that exon 1 – containing isoform is
10x more abundant in the brain
Mnatzakanian et al 2004, Kriaucionis and Bird 2004
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Mild Rett syndrome
• Walk
• Swim
• Ride a bike
• Talk
• Use hands – self-feed, write
• Better growth
• Greater survival
• But significant learning disability
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How do the mutations cause
the disease ?
• MeCP2 deficiency => ~2 fold up-regulation
of many genes (Ballestar et al 2005)
• Rett syndrome is a disease of “chromatin
configuration” that “should” have global
consequences
– hard to understand how mutation leads to the
very specific disease phenotype
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Some of the MeCP2 target genes
• UBE3A/GABRB3 (Samaco et al 2005, Makedonski et al 2005)
related to Angelman phenotype
• BDNF (Chen et al 2003, Martinowich et al 2003)
neuronal plasticity, eating behaviour
• DLX5 (Horike et al 2005)
silent chromatin loop, GABA synthesis
• FMR1 (Harikrishnan et al 2005)
• Hairy2a (Stancheva et al 2003)
• Glucocorticoid response elements
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Diagnostic Applications of
MECP2 testing
• Classical Rett Syndrome
>85% mutations
• ‘Atypical’ Rett syndrome
50% mutations
• Early seizure variant
<10% mutations, nil (so far) with infantile spasms
• Is the mutation pathogenic ?
– de novo ? synonymous ? conserved ?
– present in healthy male ?
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Family Consequences of
Mutation Testing for RTT
• Confirmation of diagnosis
– Reproductive confidence in face of mosaicism
– But still an emotional kick
• “Disconfirmation” of diagnosis
– An anomalous category
– A different emotional kick
• “Disconfirmation of normality” when MECP2mutation found in absence of RTT
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Diagnostic Test => 2 x 2 Table
expected! New and
anomalous
Clinical diagnosis:
NOT
typical of RTT
! New and
anomalousexpected
Clinical diagnosis:
typical or ‘atypical’
case of RTT
Test Negative:
mutation
NOT found
Test Positive:
mutation
found
Mutation
Test
Clinical
assessment
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No (MECP2) mutation ? …
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No MECP2 mutation ? …
• Look harder in MECP2 (promoter, 3’UTR, miRNAbinding sites, …)
• In the mouse, duplications of MECP2 in males associated with some features of RTT
• In boys, duplications of Xq28 associated with delay (and ? Some features of RTT)– Duplications including MECP2, filamin A, …
• In girls, CDKL5 gene disrupted in girls with infantile spasms and ? RTT or autism– Mutations in 3/20 girls with early seizure variant of RTT, including infantile spasms (15%)
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Sin3a
MeCP2
HDAC1HDAC2
MeCP2
HDAC2
HDAC1
Sin3a
P:P: P:
P:
- +
P:
P:
Presence of CDKL5 results in phosphorylation of
MeCP2, releasing it from the methylated CpGs –
CDKL5 mutations amplify effects of MeCP2
Inactive state Active state
CDKL5
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Pathogenesis
What could plausibly count as an
“explanation” of RTT pathogenesis ?
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RTT Pathogenesis
• Descriptive explanations
Pattern recognition
= natural history
• Mechanistic / Linear explanations
A => B => C => D; upstream and target loci
= science
• Systemic explanations
Complex web of interactions; neuronal plasticity
……. = ? despair or reality ?
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Conclusion: The Jigsaw of
Neurodevelopmental Disease
• Clinical delineation of RTT led to MECP2
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Conclusion: The Jigsaw of
Neurodevelopmental Disease
• Clinical delineation of RTT led to MECP2
• Mutations in MECP2 can be associated
with classic or atypical RTT, non-RTT, or
normal female phenotypes
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Conclusion: The Jigsaw of
Neurodevelopmental Disease
• Clinical delineation of RTT led to MECP2
• Mutations in MECP2 can be associated
with classic or atypical RTT, non-RTT, or
normal female phenotypes
• Mutations at other loci (CDKL5, FOXG1, …)
result in related disorders
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Conclusion: The Jigsaw of
Neurodevelopmental Disease
• Clinical delineation of RTT led to MECP2
• Mutations in MECP2 can be associated with
classic or atypical RTT, non-RTT, or normal
female phenotypes
• Mutations at other (interacting?) loci result
in related disorders
(BUT do remember that families can suffer
distress from - or during - our attempts to
understand the disease)
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Back to research areas:
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Natural History
• Ageing – pattern of problems
– Value of surveillance
– Need to monitor osteoporosis ?
– Neurophysiological changes (spasticity and late motor deterioration)
– (Very) late deterioration ?
• Response to conventional treatments:
– Seizures, ‘Rett episodes’
– Scoliosis
– Agitation and mood
– Environmental triggers for episodes, behaviours
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Genotype-Phenotype research
and other loci
• We do not need a single, ‘global’ Rettsyndrome database
• Joint (even global) collaborative analysis of anonymised data is possible without a global database – but needs coordination
• The fact of X chromosome inactivation makes a focus on genotype-phenotype analysis futile for predictive prognosis of the individual case
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InterRett (and EuroRett?)
• Can coordinate analyses of pooled, anonymised data from national registers
• Need to support, strengthen and coordinate the national databases and registers:
– Data quality: checking, updating, …
– Compatibility of IT systems and data fields
– Links with Family Support Groups
– Identify potential participants in therapeutic trials within a country
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Genotype-Phenotype research:
modifiers and other loci
• Phenotypic subgroups are emerging
CDKL5, FOXG1, …
• Remember modifying polymorphisms as with
BDNF: ? effect of other loci, eg HIPK2, PARP1.
• SNPs and CNVs – as modifiers within mutation
categories –
BUT may need parent-of-origin information
• CNVs may identify new loci
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Progression and Outcomes for
Clinical Trials
• Seizure severity and activity scale
– Include “Rett episodes” and “challenging
behaviours”
• Scoliosis
• Agitation, mood
• Nutrition
• Cognition
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Neurophysiological outcome
measures
• Transcranial magnetic stimulation (with
surface EMG on biceps, triceps, ….)
• Sensory evoked potentials
– SSER, ABR, VER
• Rett episodes and autonomic events
– System of carer scoring severity
– Monitoring respiratory patterns
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Molecular Mechanisms
• Neuronal iPS cells … as best material for
ChIP on chip: where does MeCP2 bind ?
• Detailed studies of MeCP2 interaction with
other DNA sequences and proteins
• miRNA and the 3’UTR
• Synapse biology + proteome …
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Potential therapies
• Read-through of stop codons, PTC124 …
• TAT-MeCP2 fusion protein …
• High-throughput screening of small
molecules
• ‘Transmitters’ etc given orally or IM
• WHAT IF a treatment was really very
nearly ‘complete’
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Experiences, Ethics and Engagement
• Family experiences => a needs assessment
• Ways of helping RTT support groups from becoming too optimistic about progress
• Preparation of family support groups to give consent for clinical trials
– equipoise
– concept of the RCT
• Imagine the experiences of patients undergoing treatment. How do we prepare for this ?
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Ideas to Forget ??
• More and bigger genotype-phenotype
studies of each mutation
• Mapping disease features to specific areas
of the brain ???
(is there such a 1:1 correlation ?)
• Osteoporosis mechanisms - unless this
really is a major clinical problem …
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Acknowledgements
• Patients and their families
• Drs Hayley Archer and Julie Evans
• The Health Foundation
• Other colleagues in Cardiff– including Julian Sampson, David Millar, Peter Thompson, Martin Horan, David Cooper, Lazarus Lazarou, Rachel Butler, Sharon Whatley, Daniela Pilz
• Colleagues elsewhere– including Alison Kerr, Franco Laccone, Peter Huppke, Moritz Meins, Kirsten Ravn, David Ravine, Helen Leonard, John Christodoulou, Jozef Gecz, John Osborne, John Tolmie, … and many more …