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Proof: Sunita Jagani
Dr. Percy
Neuroscience
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corticotopin releaseing hormone- CRH MR- mental retardation DS- Down’s Syndrome
please refer to the graph for the slides with graphs because the transcript doesn’t make sense unless you look at
the picture
RETT syndrome
I. Understanding RETT syndrome and role of MECP2 [S1]
a. It will make some sense later on in the course even though it’s early in the course for clinical
b. RETT syndrome
i. Disorder that burst out in the scene 25 years ago that was recognized in the late1950s, early 1960s
c. In the subsequent slides, will show basic aspects of the disorder
i. Movie of a child that was first seen in 1983
ii. Clinical background
iii. Gene associated with the disorder that is known
II. Outline [S2]
a. Clinical background
b. Molecular implications
i. Gene associated with the disorder is known, will be discussed briefly.
c. Phenotype-Genotype relation
i. Very broad
d. Functional effect of mutations
e. Animal models that now exist
f. Therapy- both fundamental and ongoing therapies
g. Dr. Percy’s clinic meets Thursday and Fridays at the Civitan Center- anyone is welcome
III. A Progressive Syndrome of Autism, Dementia, Ataxia in RETT’s syndrome[S3]
a. First described in English speaking literature1983 and at that time it was unknown in the western world
b. Described by Andrias Ratt (?) a developmental pediatrician
c. From that point and forward that it made a remarkable progress
IV. RETT syndrome is caused by mutations in X-linked MECP2 [S4]
a. Mutations were discovered in the gene MECP2 sixteen years later
b. Mutation was discovered in the laboratory of Baylor College of Medicine (Sic Em Bears!!)
c. More than 250 gene mutations have been identified since then
V. Video [S5]
a. Girl demonstrates classical clinical features
i. Hand stereotopis
1. begin between ages 1 and 2
2. hand clapping only while awake
3. breathes fast and holds her breath
4. has strabismus that is intermittent and has gone away,
ii. Retropulsion (first step is backward)
1. Dyspraxia- poor motor planning, poor conducting acts
2. First step is backwards, broad based, and on the toes
3. Wandering is typical- they don’t go from point A to point B
4. this girl doesn’t walk and never has walked
iii. Has anxiety (which is a principal characteristic) when you surprise them.
iv. Unusual ankle position that is not related to aspestocity instead it is dystonia- difference is that it can be
overcome by AFO
VI. RETT Syndrome an unfinished story [S6]
a. Effects females predominately, with some exceptions
b. Worldwide
c. Incidence in 1/10,000 female births
d. Occurs in all ethnic groups
e. Still there is a lot to learn about how it works and how it functions in the brain
f. Protein is a controller gene and works within the nucleus to effect other genes
VII. RETT cases by state [S7]
a. In the US, International RETT syndrome foundation collects voluntary basis in the states and territory that have
this disorder- not all encompassing of all the people that have the syndrome.
b. The numbers are around 4400 based last year,
i. Can see general representation in the states,
ii. Does mildly reflect the population density in the states
Neuroscience 2:00-3:00
Scribe: Sally Hamissou
Monday, January 5, 2009
Proof: Sunita Jagani
Dr. Percy
Neuroscience
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c. Ubiquitous and is challenging to physicians, therapists, allied health professions, Optometry, and Dental
VIII. RETT syndrome: characterized by [S8]
a. It is a Neurodevelopmental disorder, as opposed to a neurodegenerative disorder (Parkinson’s, Alzheimer’s)
which is a relentless downhill course
b. Something is wrong in the programming that causing the nervous system to mature in an improper fashion and
it sets the tone for the disorder.
c. But it is not progressive and the women can live a long time,
d. Profound cognitive impairment
i. Difficult to asses because they don’t speak or use their hands- so typical cognitive testing is out
e. Communication dysfunction
i. Don’t speak in general or have few words
ii. Give excellent eye contact- they go through a period where they appear autistic
iii. Hand stereotypic
iv. Pervasive Failure to grow
1. End up small, short, and low in weight
2. Also have acceleration of rate of head growth
IX. RETT syndrome: consensus criteria [S9]
a. Not going to go through these- don’t have to remember these but you can look at it
b. Diagnosis is made in a strict way- unless a girl goes through a period of regression and loss of previous skills
X. RETT Syndrome: Temporal profile [S10]
a. Has a specific temporal profile
b. Girls appear normal at birth
i. Normal development at 16 months of age- this is a broad range of development and sometimes are not
picked up
ii. After period of development, there is arrest and regression in the skills that are retained purposeful fine
motor use, feeding themselves, and walk.
c. 80 % learn to walk and 1/4 lose ability to walk around the period of regression
d. Overall 50 % can walk throughout life
e. After period of regression and apparent loss of communication and interaction, there is a period of rebound in
social sense that can develop a communication system (as simple as hold up a glass of juice and make a
choice or use pictures- some girls can use computers that are rigged in one way or another)
XI. RETT Syndrome: what do we know? [S11]
a. What do we know?
b. A genetic disorder
i. Not inherited
ii. Sporadic- based on new mutations that occur at the germ cell level, at the sperm or the egg- more likely to
occur at the sperm (75-80% new mutation in the sperm).
iii. Only 1/10 occur in families
iv. In genetic counseling, has a positive outlook that because is unlikely it won’t reoccur but with the presence
of mutation in the gene, we can guide you for further children.
1. In other normally developed daughters can be tested when they are at child baring age.
c. Diagnosis on clinical criterion
d. Variable clinical expression
i. Varies in girls that don’t do anything at all to those that can carry on a normal conversation with you- but is
a broad range of clinical involvement
ii. Some girls will die prematurely and we will be shown data that shows longevity.
e. Neuropathology is consistent
XII. Brain morphology [S12]
a. Bain looks normal and is formed in a normal way
b. But brain is small- in autopsy of someone that has dies 60 to 80 percent of the anticipated weight for a person
that is that age
c. Frontal and temporal lobe is smaller in volume than in typical individuals
d. Deep grey matter- ganglions (which we will learn about)
e. Some areas in the brain are pigmented pestantita nigra (sp?)- missing melanin completely or markedly
diminished
f. Small neurons and neurons are grow close together because the dendrites don’t develop and the synaptic
development isn’t how it should be
g. No evidence of recognizable diseases no suggestion of cell loss or progress neuropathology process
XIII. Overview Title [S13]
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a. Data from Baylor College of Medicine
b. Open circle= patient with RETT syndrome
c. At age 20- would be a line parallel to x axis- no progressive loss to brain wave but the brain is just small (when
we measure circumference)
d. Brain waves are below what you would expect in a normal population
XIV.
Spine Dysgenesis in Mental Retardation[S14]
a. The problem is in the spine and dendrites, very small structures
b. Normal dendrite looks like (on slide)- stubby projections are the areas of connections between cells, how
messages are exchanged back and forth
c. In DS, FragX, MR have odd looking spines that are too long; the short stubby spines are more effected in
communication
d. In DS and MR there are large areas of dendrites that are devoid of spines
e. A graduate student at UAB- spines are odd shaped, short stubby mature spines that you would expect to see
f. Failure to develop normal spines and failure for cells to communicate properly
XV. Other neurodevelopmental disorder [S15]
a. Can see similar changes in DS, autism, and Angelman & fragile x syndrome
b. Aside from Autism, these disorders have a specific genetic basis that is different from RETT syndrome
c. Something in the common pathway that relates to the functional abnormalities of these individuals.
XVI.
RETT Syndrome and MECP2 [S16]
a. What is the relationship between RETT and MECP2?
b. RETT syndrome is a clinical diagnosis- only 95 % of girls that meet criteria for RETT syndrome actually have the
mutation in the gene; 5 % don’t have a mutation
c. May have disorder that is like RETT syndrome but is based on another genetic abnormality
d. Mutation is not synonymous with people with this disorder- can see RETT syndrome with our without the
mutation and you can see RETT syndrome with other features
XVII. MECP2 and RETT Syndrome [S17]
a. 90-05 who meet clinical criteria for RETT syndrome will have a genetic mutation
b. 250 mutations in the gene- 8 (4 missense and 4 nonsense) makes up 2/3 of total
c. Deletions near the end of the gene, and large deletions (15-18%) make up; the remaining 10% are mutations
held in a few individuals
d. RETT Syndrome is largely sporadic with a paternal origin
e. Familial RETT (under 1%) is due to large deletions- the whole exons of the genes are missing
XVIII. Continuum of MECPS associate phenotypes chart [S18]
a. Phenotypes extend well beyond classic RETT syndrome
b. On the female side of the chart
i. One can have mutations and still seem normal
ii. Can have learning disabilities
iii. Or they can have behavioral phenotypes that appear autistic, obsessive compulsive disorder, aggressive
hyperactive disorder
c. Or you can have various forms of RETT in the classical form or the invariant forms and both are based on the
position and the type of the mutation
d. X chromosome Activation (unique to women)
i. Half of female cells express genes one from X chromosome and the other from the other X chromosome
ii. in general it is random, like a coin toss but is not always 50/50 but once you get beyond that it gets
skewed- so if 70-90 % are expression the normal gene- you may not have RETT syndrome but rather
another phenotypeiii. The normal individuals and the learning disability individuals are parents of girls that had siblings that
have RETT- they are transmitting female without expressing the disorder themselves
e. More complicated on the male side- Somatic mosaism or Klinefelter syndrome (47XXY)
i. Kleinefelter- a normal X balancing an abnormal X in half of your cells like in females
ii. Boys who have the same mutation as girls do that don’t have the extra X chromosome end up with severe
encephalopathy at birth- they are floppy, have difficulty breathing, and will ultimately stop breathing and die
iii. On the other side you can have duplications on the area of the X chromosome at the far end of the long
arm (xq28); MACP2 and other genes are duplicated as well
1. These boys don’t have RETT syndrome but they have similar features: they don’t communicate with
people well. On the other hand, they are not small and in some cases they are quite large in stature
2. In some cases they have recurrent infections due to one of the other duplicated genes and are upper
respiratory infections, particularly sinusitis
XIX.
Medical issues [S19]
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a. Medical issues in disorder are broad and we wont cover all of them but we will discuss longevity and growth in
terms of genotype/ phenotype seizures
b. Principal target is the brain that affects all systems
c. GI dysfunction
i. They don’t grow well, they don’t eat well, they have problems in the GI tract from the top to the bottom, they
don’t chew food well, swallowing dynamics are abnormal, high frequency of acid reflux, don’t empty GI tract
well, constipation, and increased frequency in gall bladder diseases
ii. We have to know from top to bottom why they are in pain because they may not complain of pain
d. Scoliosis
i. Curvature of the spine- over 80 % of the girls will develop scoliosis and 10-20% will require surgery
because of the extreme degree especially seen in girls that don’t walk
e. It is not he curvature that bothers us but rotation of the trunk that causes compression of the lungs- can
compromise pulmonary functions that can cause infections that can be difficult to manage
f. Ostopenia- bones that are under mineralized, increased chance for bones to fracture. 25-30% will have
fractures as they approach maturity as opposed to 4-5% of the general population
g. Breathing abnormalities only while they are awake- particularly in age 5-16
i. Sleep is disrupted and they often sleep in short bursts
1. They can go to sleep but have difficulty staying asleep
h. Cardiac conduction abnormality which can cause Prolonged Cutey (?) Syndrome
i. Pathologically have an immaturity in the conduction system- specialized cardiac muscles conduct impulses
in the heart, this going back to the immaturity of the developing nervous system
ii. Sexual maturation occurs on time and the onset of menstruation is at about the same time as the general
female population
1. They are quite capable of having children- this is a genetic disorder because of the vertical
transmission (woman in Sweden that was assaulted and had a baby that developed RETT
Syndrome showed evidence that this is a genetic disorder)
2. When these children become mature, someone has to watch out for them throughout life because
they can’t protect themselves
XX. Longevity in RETT syndrome [S20]
a. Study from Texas (15 years ago0): Survival to age 10 was the same in most populations; at age 35 70 % of the
population was living
b. In a recent data in Australia showed information roughly in the same range 78% survival at age 25
c. [S21] Graph data was collected from 2000 women from USA and Canada, created a captain meyer survival
curve based on birth, don’t try to discern the individual curves based on the periods of birth then is in decades
after each line is smaller the earlier group may have had more survivals. It is possible in the later group that
they had picked up more severely involved girls look at the 50 % survival period is 50 years of age- survival
compared to 75-80 years in the general US population
i. Life span it is shorter than normal and is significant- if you have RETT syndrome and survive to age 50,
your parents are around the age of 80 presents health and social problems.
XXI.
Head circumference graph [S22]
a. Growth is an overwhelming problem because they don’t feed well and they have a higher than normal
cordinal(?) requirement
b. Gene leads to poor overall growth
c. Referring to graph: this is a pattern of head circumference growth from 0-18 years of age, data from 500 girls
d. Orange color line = normal; heavy orange line = 50 percentile; red girls in bluish line is the 50 th percentile
e. By 2 or so months of age the 50th percentile lines begin to diverge and by the time they reach age 2, they are
well out below the second percentile. By age 18, a substantial number are below the normal range
f. Original description of RETT was they had microcephaly- below normal head circumference but there are
some that are well in the normal range so it is not an overriding conclusion
g. By in large they all go through this pattern of deceleration and is one of the key criteria that is made in the
diagnosis
XXII. Graph decrease in weight (height follows the same pattern) [S23]
a. At around age 1, they begin to fall way from 50th percentile line so by the time they reach 18 more than 50% are
well below the percentile of normal population
b. Weight is a problem but on one sense it isn’t a bad thing if they have to be carried around and lifted by their
parent. On the other hand, some girls can achieve a very robust size.
XXIII. Molecular implications [S24]
a. This is an interesting protein and we are still learning about it
XXIV. MECP2 [S25]
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a. Family of proteins that bind to methylated cytosine in the promoter region or in front of the promoter region so
the protein acting in the nucleus
b. Protein has nuclear localization signal and helps it get into the nucleus
c. It appears to be initially involved in transcriptional silencing or regulation
d. This gene was known in cancer literature because they were looking at potential regulators of cell growth and
MECP2 and its family members popped up
e. Protein is ubiquitous in brain
f. There are a number of targets but the whole list of targets is not known
g. Function in the maintenance and maturation of developing neurons
XXV.
MECP2 distribution in human brain development [S26]
a. In a developmental study of the protein process in the developing brain, 10 week gestation to 10 years
b. Can see some staining at the peripheral hemisphere- principally the early staining is at the brain stem (GI
functions and breathing center which are critical sites of action for the protein)
c. In the hemisphere, in the periphery the early appearance is in the guiding peripheral inaudible 32:00
d. As time goes by in gestation after the second trimester begin to see regions of the hemisphere being populated
more intensely, pattern seen here at 10 years of age
e. It is an important protein in the function of the brain
f. Does occur in other organs but is not clear if they are responsible directly for their function besides the
conduction system of the heart
XXVI. MECP2 complex role in the nucleus [S27]
a. Ignore the top part
b. Initial idea that MECP2 is involved in the regulation of transcription- and that it could silence genes and was
based on literature from cancer research group
c. MECPs combine with the two elements and prevent transcription of whatever gene that was involved
d. In a recent paper from last year, using Chip technology- MECP2 down regulates the gene and with association
of CREB1 up regulates a significant number of genes (up regulates about 5X as many genes as it down
regulates)
e. It is unclear about the specific relationship with any other given gene
XXVII. Cartoon of down regulation [S28]
a. MECP2 binds to methylated CpG and in ordinary circumstances, it should connect with the two other elements
in the HDCA and lead to the chromatin becoming compacted
b. When this can’t happen, the chromatin is un-compacted and the gene is silenced and not transcribed
c. The mechanism of up regulation (other than involving CREB1) of gene remains to be seen
XXVIII. Does mutation predict outcome [S30]
a. Relationship between the clinical features and the gene mutation
b. Three point mutations in the C terminal shortening of the gene are associated with lower severity, lower being
better in the rubric.
c. Slower progression and in many cases preservation of conversational speech
d. There is a clear relationship between specific mutation and the clinical features
e. It is modified to some extent by X chromosome activation
f. Girls with typical RETT have missense or nonsense mutations
g. Those with variant RETT forms have nonsense mutations
h. Those who don’t have RETT have a missense mutation or have a C terminal truncation (frameshift)
XXIX. Medical issues Chart [S31]
a. Don’t remember this
b. Certain mutations have higher frequency of seizures than others and some have higher frequency of scoliosis
than in others. Those requiring scoliosis surgery numbers are too small to say a whole lot about.
c. We can make some predications when we see a girl in the clinic
XXX. Medical issue Chart [S32]
a. Constipation
i. Certain mutations are worse than others G reflux, and fractures
ii. R133 C in milder in many of these things and inaudible 36:30 seen in constipation
XXXI.
Mouse models [S33]
a. Knock out mouse
i. Gene is removed or portions of the gene is removed and can be done selectively in given elements in the
nervous system
ii. We can learn exactly how the gene works in the forebrain versus the hindbrain versus the cerebellum
iii. Can be done in a conditional way that allows the dissection of how exactly the gene or protein is working
b. Knock in mouse
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Dr. Percy
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i. Gene mutation seen in RETT syndrome is recreated in a mouse or a mutation that is made up and is
knocked out of a particular nucleotide
XXXII. Knock out Mutant [S34]
a. Question is this disorder reversible?
b. Using an estrogen receptor controlling the MACP2 promoter, created an animal with the normal gene in there
and after the animals were allowed to develop the disorder or before they developed the disorder were given the
extra analog Tamoxifen (done in groups who worked with cancer)
c. Tamoxifen reverses phenotypes in animals and the animals went from being affected to being virtually normal
d. So this confirms the principle that the disorder can be reversible
XXXIII. Knock in mutant [S35]
a. Clinically looks like the female situations and it’s where the four limbs come together
b. Appear disheveled, don’t have bright eyed appearance of the controlled litter mates, and have hump in the back
that is not necessarily scoliosis but is in the same ballpark
c. Like in the Zoghbi lab, they had problems with memory, conditioned fear, anxious, long term depression and
long term potentiation.
d. [S36] Reduction in post-synaptic densities, indicating a problem in the connection between cells
e. [S37] Anxiety and fear are related to elevated corticosterone levels in the blood and is related to an elevation of
the CRH.
i. MACP2 is a regulator of CRH released in the hypothalamus.
ii. CRH occurs in the hypothalamus and in other areas of the brain, including the amygdala and other areas of
the deep brain matter
iii. Corticosterone analog in the mouse of cortisole in humans
iv. [S38] Amygdala has down stream connections into the brainstem that relays GI dysfunctions, breathing
abnormality, and cardiac dysfunctions.
v. Amygdala also has upstream features into the cortex where it helps regulate motor function and
association cortex function.
vi. This explains the overriding neurological features that the girls have.
XXXIV. But over expression is detrimental [S39] As seen in Zygohbi lab
a. If you over express the gene in mice, they become very sick
b. This is an EEG from a mouse, this is a wildtype that is nice and quiet
c. MECP2 over expression mutant at 20 weeks
d. MECP2 over expression at 50 weeks
i. EEG becomes very chaotic, and at that time the mouse enters statsiclimatic (inaudible) and die.
ii. It is clear that dosing of the gene is a very critical amount (one dose) that is required for normal cell function
and that has significant implications for therapy, particularly gene based gene therapy when you are trying
to re-express the normal gene.
XXXV. Therapy [S40-41]
a. Gene therapy and the potential problem of over expression. Another problem is some cells have a normal
genes that is already at work
i. How do you make a gene go into a cell where it is abnormal and how do you how do you suppress the
abnormal gene that is already there?
ii. One can repair the protein in the post transcriptional way or at the RNA way level and some experiment are
ongoing and trying to do this.
b. You can attempt to reintroduce the protein and this has been achieved in animals and has increased survival
50% in male mice
c. You can modify the mutant protein itself
d. [S42] Said all of the same things and will be on our handout that we will have so he said he won’t go into
detail
i. There are many avenues of approach that are ongoing now in the laboratory
XXXVI. Challenges [S43]
a. A problem at the clinical level is we don’t have a good outcome measure because it is tough to measure
cognitive function and behavior.
b. They are pressing the RETT Center Association to fund a mechanism to develop outcome measures.
i. A natural history study from across the county that has enrolled 700 girls and we are following them over
time and following the natural course of the disorder.
c. EEG would be a useful outcome measure
d. Heterogeneity of population makes picking clinical trial population difficult and even using cells in the lab is a
problem, maybe the mice will help us?
XXXVII. What about gene therapy? [S44]
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a. In order for gene therapy to work you have to know the single gene disorder, you have to know what the protein
does, and you have to know what adding a normal protein will do
b. You have to know if the affected tissues can be accessed and it is hard to do a brain biopsy and get that by
human studies board
XXXVIII.
Gene therapy cartoon [S45]
a. Is you character dominant or recessive? In this case it’s a dominant gene
XXXIX. Post transcriptional repair [S46]
a. The most novel therapy known in microbiology literature.
b. If you use Aminoglycosides (Gentamycin), you can cause a mutation that can be read through. If you have
normal protein being made but there is a “stop” here and you get short piece of protein, when you add the
aminoglycoside, you get the full length protein because it jumped over where the missing nucleotide was.
c. Problem is that aminoglycosides are toxic and can make you deaf and ruin your kidneys if you take them for a
long period of time.
d. [S47] PTC 124, agent capable of entering the brain (key for RETT syndrome). Currently in clinical trials for
Cystic Fibrosis and Duchenne Muscular Dystrophy and has shown some efficacy. But unlike RETT syndrome,
these disorders are involved in sites outside of the brain.
i. They are waiting now for a proper animal model to test the compound.
XL. Therapies for RETT [S48]
a. Aggressive with nutritional guidance
b. [S49]Toe walking
i. Have to be able to deal with toe walking like what was seen in the video.
c. Ankle- foot dystonia [S50]
i. AFO can deal with the dystonia. If the AFO doesn’t work, you can use botox, botulinum injection into the
muscles to alleviate the problem
d. [S51] Physical therapy is important- girls generally like it
e. [S52] Hippothearpy they tend to like
f. [S53-54] Skiing is a good thing to do and they can ski. This is a fancy rig for someone that doesn’t stand or
walk
g. [S53] Companion dog interact well in very well in a social way with animals and humans
XLI.
Conclusion [S55]
a. Interesting and quite unique disorder and is relatively young
b. Learned a lot about the clinical features
c. Are currently actively engaged in developing appropriate interventions at the clinical and expanding activities in
ongoing therapies.
d. [S56] Large group of people working on this at Baylor College of Medicine and at the Davis Center Greenwood
Genetic Center funding through NIH.
e. [S57] Rolling with
i. Same girl without her toes tethered and has instructor behind her
[End 48:40]