CHD7 AND CHARGE SYNDROME RESEARCH

Unveiling the Mysteries

Meg Hefner, MS

Genetic Counselor

Advisor, CHARGE Syndrome Foundation

October 26, 2014

Where is CHARGE syndrome (CS) research being done

• 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

• Human• Clinical case studies and observations• Clinical series / summaries• Clinical reviews• CHD7• Human research – gathering information

• Observational• Questionnaires

• Animal models• Molecular research

Webinar caveatTo 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) features1. Craniosynotosis as an occasional finding

• 8-9 kids - sometimes delaying the diagnosis of CS• Any suture may be involved

• Complications seen in CS2. 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)

% • 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

FINDING Number %External ear 40/40 100%

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

14/29 male4/11 female

48%28%

Urinary system 11/39 28%

Retinal coloboma 16/33 48%

Thymic abnormality 17/39 44%

Esophagus 4/39 10%

Polyhydramnios 9/25 36%

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: human1. 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)

• Strengths• Holistic perception• Semantic competencies• Logical reasoning and planning skills

• Weaknesses• 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

• Other research: there is also a lot out there on various aspects of behavior in CS

• Information constantly being published on medical and behavioral aspects of CS

• 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

• Fruit fly (Drosphilia)• Zebra fish

• Embryos are transparent

• Mouse - mammal• Others

• 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• “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 stage-specific 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

• 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

• Clearly many functional equivalents to what is seen in children with CS

Chd7 in zebra fish embryos

• Chd7 effects • Absent retinal nerve cells• Abnormal organization of

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?• 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?• Chd7 is required for proper organization of neural crest-

derived craniofacial cartilage structures – this is what they looked at

• In Chd7 knockout, there is elevated expression of cell-cycle inhibitors -> reduced cell proliferation

• 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

• Balow et al. Dev Biol 2013

First report of Chd7 mutant mice

Chd7 expression in mouse embryos

Hurd et al., Mammalian Genome, 2007

Chd7 protein is stained blue so you can see where it concentrates: Eye, ear, hindbrain, heart

Mouse studies• Chd7 is necessary for

proper craniofacial and tracheal development

• Use of conditional knockout mice (Foxg1-CKO) to show effect of eye

• Sperry, et al Developmental Dynamics, 2014

Other mouse Chd7 findings• 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

• 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 CHD7-independent, 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

• 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• Mouse: looked genome-wide for differences in expression in

Chd7 and wild-type

• Identified 98 genes that were expressed differently in Chd7 & WT

• Many of the 98 genes are involved in neural crest cell and axon guidance, including SEMA3A

• 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

• Schulz, et al Human Genetics 2014

p53 activation• CHD7 protein binds to p53 promotor, reducing

p53 activity. • Loss of normal CHD7 (mutation) increases p53

activity

• 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*

• *CSF funding someone looking into this

Putting it all together: knowledge from people, animals and molecular work

• 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

• Compared relative frequency of features

• Which gene caused problems in confusing cases

• Hypothesized about mechanisms

Click icon to add picture

Corsten-Jansssen, et al. Mol Syndrome 2013;4:235-245

Relative frequency of common clinical features of CHD7 and 22q

CS (CHD7) and 22q (TBX1)

Frequency of CS ~ 1/15,000

Frequency of 22q (TBX1) ~1/5,000

1. Out of 802 CHD7+ cases, find those with typical 22q features

• 30/802 (3.7%) …… none had TBX1 mutations

2. Clinical dx of CS but CHD7 negative • 5 cases had 22q deletions

3. Clinical 22q but 22q &TBX1 negative• 5/20 had CHD7 mutations (all truncating)

Hypothesis explaining overlap

• CHD7 and TBX1 likely function in the same embryonic pathways and/or have common targets

• 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

• Some of those genes cause other syndromes with overlapping features….

• It is likely that CHD7 (and/or TBX1) affect the functioning of those genes as well

Back to Mice to compare CHD7 and TBX1• Mice that carry both Chd7 and Tbx1 have

• Severe heart defects• Reduced postnatal viability• Abnormal thymus• Malformed semicircular canals

•This research* suggests both genes act in the same developmental pathway

*supported by the CHARGE Syndrome Foundation

Corsten-Jansssen, et al. Mol Syndrome 2013;4:235-245

Syndromes with clinical overlap with CHARGE and 22q –

2012 & 2013 CSF GrantsChd7 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

• Examine brain MRIs of people with CS to see if there are any patterns /brain anomalies

• The role of CHD7 in nerve function

• CHD7 and gene regulation

• 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

• Bigger numbers for clinical findings• Identify occasional features• Registry for additional studies

• Provide baseline info for other studies

I NEED YOU!!