Transcript Dia 1

Unveiling the Mysteries
Meg Hefner, MS
Genetic Counselor
Advisor, CHARGE Syndrome Foundation
October 26, 2014
Where is CHARGE syndrome (CS)
research being done
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United States
Canada
Europe (UK, Netherlands, France,
Germany, Italy, Spain)
Japan
All over
Many collaborative studies
involving multiple institutions, often
in different countries
Varieties of research
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Human
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Clinical case studies and observations
Clinical series / summaries
Clinical reviews
CHD7
Human research – gathering information
• Observational
• Questionnaires
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Animal models
Molecular research
Webinar caveat
To prepare for this webinar I reviewed scores of
papers, most published in the last three to four
years. I am not providing citations for
everything I reviewed. I will be showing you
information from ~50 papers. There is MUCH
that I am not covering….
If you want to hear more detail about specific
topics, let us know…. Perhaps they can be
future webinars and/or presentations at
conference.
Human case studies: reports of
single or a few cases
 “New” (previously unreported) features
1. Craniosynotosis as an occasional finding
• 8-9 kids - sometimes delaying the diagnosis of CS
• Any suture may be involved
• Complications seen in CS
2. Post-tonsillectomy bleeding with aspiration
and bronchospasm in a 6 year old child
3. Successful cord blood transplantation in a 4
month old child with severe immune
deficiency (doing well at 10 months)
Human case studies: similarities
with other syndromes
1. Chromosome 5q11.2 microdeletion – 5 kids
• Similar features include choanal atresia, heart
defects, external ear anomalies and short
stature
• None had colobomas, semicircular canal
abnormalities or lack of sense of smell
2. Pituitary abnormalities found in CS
• Kallmann syndrome - tested for CHD7
• CHD7 variants – 2 kids
3. One child with Kabuki syndrome and CS
features
• Coloboma and pituitary issues
Human Series: CS features
1. Overview of dental findings – 8 kids
• Lots of problems, no clear patterns
2. Eye findings – 19 kids / 38 eyes
• Asymmetry between the two eyes
• Even a eye with a large coloboma can form
a macula
3. Temporal bone venous malformations
• 10/18 kids has anomalies
• Significant implications for possible surgical
complications
Recent cochlear implant series
• New Zealand (3), Germany (13), Korea (6), Italy (5)
• Common threads from the reports
• All have complex middle and inner ears – BE
CAREFUL!!
• Not children with CS all are eligible for CI due to
absence of auditory nerve
• Variable outcomes – difficult to predict who will have
good outcome
• Speech
• Sound awareness
• No apparent benefit - rare
• The earlier the better to improve potential outcomes
Human series: CHD7 findings
1. Paternal origin of most mutations (12/13)
• Also again confirmed paternal age increase
2. Familial variability : Two sets of parent and
two children with CHD7 mutations
• Family A
• One child with clinical CS
• One child with only coloboma
• Mother with mild vestibular problem and coloboma
• Family B
• Both children with clinical CS
• Father – only one unusual ear
CHD7 - more
1. Kallmann syndrome – CHD7 testing in 209
patients with KS (NOT CS)
• 24/209 (11%) had CHD7 mutations
• Mostly missense mutations
2. Semicircular canal abnormalities – 12
patients
• 6 CHD7 mutations found
• 5 of those clinically had CS
Prenatal features:
Retrospective study (autopsy & CHD7 testing) of 40 cases
(all miscarried or electively terminated due to US findings)
FINDING
 %ear
External
Number
40/40
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100%
Choanal atresia
19/40
47%
Cleft lip
18/40
45%
Heart defect
34/40
85%
SSC abnormal
37/39
95%
Arhinencepahly
35/39
90%
Hydrocephalus
6/40
15%
External genital
abnormality
Urinary system
14/29 male
4/11 female
11/39
48%
28%
28%
Retinal coloboma
16/33
48%
Thymic abnormality 17/39
44%
Esophagus
4/39
10%
Polyhydramnios
9/25
36%
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Severe end of spectrum
• 29 Male 11 Female
• Growth was normal
• CHD7 mutations*
• 17 nonsense
• 19 frameshift
• 2 spice site
• 1 total deletion
• 1 missense
* Nearly all truncating
Legendre , et al. J Med Genet 2012
Review articles: summarize
literature - what is known so far
• Diagnosis and management of CHARGE
syndrome in the NICU - for nurses
• GeneReview – overall diagnosis and
management for physicians
• Many others available
CS Research studies: human
1. Death after neonatal period – 7 cases (11m-22y)
• Respiratory aspiration – 5
• Postoperative complication - 1
• Choked during eating – 1
• Swallowing problems, G-E reflux, aspiration and post-op airway
events due to cranial nerve dysfunction are important contributors
to post-neonatal death in CS
• Multidisciplinary feeding teams and treatment of swallowing and
reflux issues are critical in the management of children with CS
2. Balance 21 children with CS & 31 controls (6-12y)
• 57% of those with CS at medium to high risk of falls
• Increased physical activity with focus on balance and movement
would likely improve balance and balance confidence
Sleep apnea in CS (0-14y)
• Questionnaires completed by 51 parents
• Obstructive sleep apnea diagnosed in 65%
• Treatments reported
• CPAP
• Tonsillectomy and/or adenoidectomy
• Tracheostomy
• All treatments helped
• Even after treatment, still likely some
residual symptoms
Psychomotor testing – 8 children (7-13 y)
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Strengths
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Weaknesses
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Holistic perception
Semantic competencies
Logical reasoning and planning skills
Postural control (static and dynamic)
Visuo-spacial constructive abilities
Sequential processing and selective attention
IQ range 54-92
The extent of the deficits was NOT associated with
severity of sensory deficits. Specific areas of the
brain may be affected in CS
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Other research: there is also a lot out there
on various aspects of behavior in CS
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Information constantly being published on
medical and behavioral aspects of CS
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Professionals (medical, therapeutic and
educational) involved with your children
have an obligation to find out what is
known and use that information
CHD7: Animal models
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Fruit fly (Drosphilia)
Zebra fish
• Embryos are transparent
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Mouse - mammal
Others
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Frog
Chicken (chick embryos)
Llama (can have choanal atresia)
Yeast
CHARGE Syndrome Foundation
supported research
• Early years: CSF supported questionnaire type
research
• Thanks to huge increase in fundraising (THANK
YOU) and surveying membership on priorities,
support of basic science research has become
possible.
• In each of the last 3 years the Foundation has
provided $100,000 per year towards CHARGE
basic science research
Professional Day at Conference
• CSF grantees present their work in progress
at Conferences
• Often unpublished data
• See Professional Day Handouts for 2013
at the CSF Website
• Donna Martin – mouse model PowerPoint
presentation
• Others – brief summaries of their work
• Families are welcome to register and come
to PD
CHD7 is a hot topic
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“A central question in developmental biology is
how chromodomain proteins like CHD7 regulate
important developmental processes, and
whether they directly activate or repress
downstream gene transcription or act more
globally to alter chromatin structure and/or
function. CHD7 is expressed in a wide variety of
tissues during development, suggesting that it
has tissue-specific and developmental stagespecific roles.”
Layman, Hurd and Martin Clin Genet 2011
CDH7 gene and protein
Steps in Gene (DNA) to
Protein Process
DNA is unwound and
transcribed into RNA
RNA is processed into
mRNA– introns removes,
exons spliced together,
Poly A tail added
mRNA leaves the nucleus
Ribosomes move along
the mRNA and translate
the triplet codons to amino
acids to build up the
protein
The protein undergoes
folding and other
modifications to become
functional
Fly CHD7 equivalent is
kismet: fate; fortune
Cover story 2010 –
flies with CHARGE
syndrome
Daniel Marenda, Drexel
University
- CHARGE patients display hypotonia
- Kismet flies have abnormal posture
More fly info
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Kismet knockdown flies have defective
immediate recall memory
• Kismet knockdown flies have defective
motor reflex function.
• Using flies, you can do more to determine
just where the issue lies (nerve or muscle?)
• This behavior is localized to Kismet function
in muscle cells
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Clearly many functional equivalents to
what is seen in children with CS
Chd7 in zebra fish embryos
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Chd7 effects
• Absent retinal nerve cells
• Abnormal organization of
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cranial motor neurons
Defective otoliths (inner ear)
Loss of facial nerves
Irregular vertebrae
Reduced bone mineralization
Patten, et al. PLoSOne 2012
How does Chd7 cause these effects?
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Zebra fish studies in several labs have
shown that Chd7 is acting at two levels
(at least)
Transcription of other genes (turning
them on or not)
Translation from mRNA to protein
(making of the products of the other
genes)
Reversal of the some effects (rescue)
has been shown in zebra fish (two labs)
mRNA rescue !!
Control-MO injected (E) chd7-MO+chd7-mRNA co-injected (F) zebrafish
showed no phenotypic defects at 48 hpf and were comparable to wild type
zebrafish (A) at the same age. Embryos injected with 2 ng/nl chd7-MO (B–
C), or 2 ng/nl (D) chd7-SBMO showed several developmental defects.
Patten et al. PLoS One 2012
How does Chd7 work in zebra fish?
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Chd7 is required for proper organization of neural
crest-derived craniofacial cartilage structures – this
is what they looked at
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In Chd7 knockout, there is elevated expression of
cell-cycle inhibitors -> reduced cell proliferation
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Additionally knocking out a normal repressor gene
(fbxl10) rescues cell proliferation and cartilage
defects – led to complete rescue of CS phenotype
in zebrafish… the enzyme in question might be a
possible therapeutic target in CS
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Balow et al. Dev Biol 2013
First report of Chd7 mutant mice
Chd7 expression in mouse
embryos
Chd7 protein is stained blue so you can see where it
concentrates: Eye, ear, hindbrain, heart
Hurd et al., Mammalian Genome, 2007
Mouse studies
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Chd7 is necessary for
proper craniofacial and
tracheal development
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Use of conditional
knockout mice (Foxg1CKO) to show effect of
eye
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Sperry, et al Developmental
Dynamics, 2014
Other mouse Chd7 findings
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Necessary for development of cerebral
cortex
Regulates neural stem cell and inner ear
development
Causes distinctive abnormalities of
middle and inner ears
• Very similar to human features
• One mouse is whirligig
CHD7 and neurogenesis
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CHD7 contributes to neurogenesis throughout
life in mice and people. Hippocampal
neurogenesis is decreased in adult Chd7 mice
Exercise also contributes to hippocampal
neurogenesis
Chd7 deficient mice were allowed to exercise
(voluntary, on wheels) – neurogenesis
increased, and they got better
• Exercise-induced neurogenesis seems to bypass
Chd7 pathway in mice
“These data suggest that exercise-induced
neurogenesis is probably CHD7independent, indicating an alternative
pathway that can sufficiently drive
neuronal differentiation in the absence
of CHD7….this finding implicates that
exercise might be beneficial for
CHARGE patients, in particular, for the
recovery of the hippocampal-related
learning ability.”
Feng, et al. Cell Stem Cell 2013
Adult neurogenesis – rescue??
Kim and Roberts, Cell Stem Cell 2013
Long story short: CHD7 interacts
with hundreds of other genes
Gene interaction
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Because CHD7 interacts with so many other
genes, it is likely there are other ways to get
to clinical CS – children with all the features
of CS but no CHD7 mutation
• Upstream: There may be genes which
themselves interfere with CHD7 function
• Downstream: there may be genes in the CHD7
function complex that result in similar issues
Applying to people…CHD7 negative
children
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Mouse: looked genome-wide for differences in expression in
Chd7 and wild-type
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Identified 98 genes that were expressed differently in Chd7 &
WT
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Many of the 98 genes are involved in neural crest cell and axon
guidance, including SEMA3A
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Children with CS who are CHD7 negative: Looked for mutations
in one of the human equivalent genes
• Found SEMA3A mutations it in 3/45 patients
• Another way to get to clinical CS
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Schulz, et al Human Genetics 2014
p53 activation
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CHD7 protein binds to p53 promotor,
reducing p53 activity.
• Loss of normal CHD7 (mutation) increases p53
activity
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Increase in p53 activity can also induce
features of CS (without CHD7 mutation)
• Suppression of p53 activity may reduce features
of CS
• Therefore, look at negative regulators of p53 as
possible therapeutic target for people*
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*CSF funding someone looking into this
Putting it all together: knowledge from
people, animals and molecular work
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CHD7 team in the Netherlands collaborated
with 22q team at CHOP (Children’s Hospital of
Philadelphia) to look more closely at the overlap
between the syndromes
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Compared relative frequency of features
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Which gene caused problems in confusing
cases
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Hypothesized about mechanisms
Relative frequency of common clinical features of CHD7 and 22q
Corsten-Jansssen, et al. Mol Syndrome 2013;4:235-245
CS (CHD7) and 22q (TBX1)
Frequency of CS
Frequency of 22q (TBX1)
~ 1/15,000
~1/5,000
1. Out of 802 CHD7+ cases, find those with typical 22q
features
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30/802 (3.7%) …… none had TBX1 mutations
2. Clinical dx of CS but CHD7 negative
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5 cases had 22q deletions
3. Clinical 22q but 22q &TBX1 negative
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5/20 had CHD7 mutations (all truncating)
Hypothesis explaining overlap
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CHD7 and TBX1 likely function in the same
embryonic pathways and/or have common
targets
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Based on information from animal studies,
both TBX1 and CHD7 regulate gene
transcription and might well regulate the
transcription of the same downstream
genes.
Corsten-Jansssen, et al. Mol Syndrome 2013;4:235-245
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Some of those genes cause other
syndromes with overlapping features….
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It is likely that CHD7 (and/or TBX1)
affect the functioning of those genes as
well
Back to Mice to compare CHD7 and
TBX1
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Mice that carry both Chd7 and Tbx1 have
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Severe heart defects
Reduced postnatal viability
Abnormal thymus
Malformed semicircular canals
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research* suggests both genes act in the
same developmental pathway
*supported by the CHARGE Syndrome Foundation
Syndromes with clinical overlap with CHARGE and 22q –
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Corsten-Jansssen, et al. Mol Syndrome 2013;4:235-245
2012 & 2013 CSF Grants
Chd7 and…. (mostly mouse studies)
• Craniofacial development
• Regulation of chromatin structure, histone
modification and DNA transcription
• Heart structure & conotruncal defects
• Sensory deficits & brain patterning
• Auditory nerves and hair cells - interaction
between CHD7 and SOX2
• Cerebral cortex – and relationship to genes
linked to autism and intelligence
2014 CSF Grants
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Examine brain MRIs of people with CS to
see if there are any patterns /brain
anomalies
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The role of CHD7 in nerve function
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CHD7 and gene regulation
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Identifying negative regulators of CHD7 as
potential therapeutic targets for CHARGE
syndrome (p53)
CSCDP: CHARGE Syndrome
Clinical Database Project
• Supported by the CSF (Blue button on
homepage)
• Supported by Saint Louis University
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Bigger numbers for clinical findings
Identify occasional features
Registry for additional studies
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Provide baseline info for other studies
I NEED YOU!!