Scientific Programme Report,
IASO/ASF Conference Washington DC July 2003
In addition to the presentations with a more scientific content in the
general meeting, there were scientific sessions running concurrently on
two days of the conference. There was a full programme, with nine hours
of scientific presentations in total. The sessions were attended by a
variety of professionals from different disciplines along with several
interested parents. Presentations covered aspects of neurology, neurophysiology,
clinical and molecular genetics, psychology and physiotherapy. There is
no doubt that it was useful for researchers and experts in different fields
to come together to discuss their work on Angelman syndrome. At times
presenters spoke in jargon relative to their particular field that made
it a little difficult for others to follow.
There were several presentations covering the topic of epilepsy and
the EEG. One of these was by Maurizio Elia from Italy, who discussed the
seizure disorders in a group of 32 AS patients he has followed personally,
providing insight into the types of seizures seen (all types!), EEG changes
and medications used (valproate (Depakote) was the most common, others
such as carbamazepine, phenytoin and vigabatrin could make things worse).
This presentation was followed by an overview of epilepsy by Greg Holmes
from New Hampshire, USA. He began by talking generally about the historical
aspects of epilepsy, classification of the epilepsies and what causes
seizure activity. Although many of us with a medical background have covered
this at one stage, it was useful to review this mechanism. Basically,
positively charged sodium ions rush into the cell through special channels
in the cell membrane (ion channels). They alter the electrical charge
of the cell (depolarisation) causing it to fire off an electrical discharge
(action potential) which appears as a spike on the EEG. To combat this
change positively charged potassium ions leave the cell and GABA helps
to get negatively charged chloride ions back in. It seems sensible therefore
that if you want to treat epilepsy you can use drugs which stabilize the
cell membrane (topiramate), drugs which close up the sodium channels (e.g.
lamotrigine or drugs which increase GABA inhibition (valproate, benzodiazepines).
GABA appears to have some role in the seizures in AS, as evidenced by
the fact that seizures are worse in AS with a deletion where GABA receptors
are missing. However, seizures are also seen in the other groups of AS
so UBE3A might have a role, too, perhaps modulated through other proteins
it interacts with such as CaMKII (calcium/calmodulin-dependent protein
kinase II). Dr. Holmes finished by showing some videos of different types
of seizure.
A little later in the programme Bernard Dan from Belgium gave an overview
of the EEG findings in AS. These have been documented right since the
first description of AS. Although not every AS child has a typical AS
EEG, one or more of the EEG features is present in the majority of AS
individuals at some stage (up to 98% of patients in some studies.) The
EEG changes are seen across all groups of AS. There are three typical
patterns: 1) 2-3 cycles per second activity anteriorly, greater than 300µV
2) 4-6 cycles per second activity, more diffuse, > 100µV and
disappearing over the age of 5 years 3) 3-5 cycles per second activity
posteriorly, >200µV and persisting, so this is the activity most
likely to be seen in older patients. It's facilitated by passive eye closure
and although it's difficult to get an AS child to close their eyes, you
can simply hold the eyelids closed gently for a few seconds and this will
bring out the changes (tell your EEG technician this!)
In addition there is another pattern, which Dr Dan referred to as 1B
and this comprises the little notched waves known as delta waves which
are often seen with 1). There are of course other features you might see
on an AS EEG. Some of these are not specific to AS and others represent
seizure activity. Dr. Dan has also done some work on the AS mouse and
has shown that there are similar EEG findings.
A final presentation on epilepsy was by Dr. Gunnar Braathen from Sweden
(who ended up giving three presentations, one on behalf of a colleague
who could not attend). The Swedish group compared 20 AS patients with
abnormal methylation to 20 AS patients with normal methylation. They had
not done UBE3A screening, so some of the second group could have had UBE3A
mutations and the others had a clinical diagnosis only. The group with
methylation abnormalities had higher rates of status epilepticus and were
more likely to have a typical EEG.
There were two presentations which dealt with motor function in AS,
analysing how AS children moved. Dr Braathen from Sweden gave one presentation
on behalf of one of his physiotherapy colleagues. They had studied gross
and fine motor development in a series of 23 AS patients and 10 controls,
using a battery of different tests designed to measure motor function.
One of these was the "sloping plane" test where individuals
had to walk down a slope about a metre wide. The various tests showed
that AS children had more spasticity and more ataxia than the control
group and also frequently used a mechanism called "co-activation",
a situation whereby if you contract one group of muscles to perform a
movement, another, often antagonistic group of muscles contracts as well.
It is thought that this co-activation might be a compensatory measure
to make up for the instability and inco-ordination in AS. Dr Braathen
suggested that if you look at the overall picture, the movements seen
in AS are immature movements rather like a stage of toddler development.
However, you might expect the gait in AS to improve over time, if it was
simple toddler gait, but it doesn't tend to.
Bernard Dan had carried out testing on motor function in AS using an
apparatus that comprised various electrodes attached to the muscles of
the limbs and trunk and to a power pack, which was attached at the waist,
so that there was no trailing wires and the individuals could move freely.
He noted that when asked to carry out movements such as squatting or walking,
AS individuals used different strategies than a normal control group or
individuals with cerebral palsy. The way they moved suggested that they
tended to stiffen themselves up, particularly around the hips to compensate
for instability. Analysis of the movements suggested that the abnormalities
seen were due to involvement both of the corticospinal tracts (messages
running from the brain down the spinal cord) and of the cerebellum at
the back of the brain. Cerebellar involvement was further suggested by
Dr Dan, in studies performed on mouse models. The whole point of doing
studies on motor function is that by analysing movements and muscle tone
you might be able to direct methods of treatment e.g. stretching movements
to combat spasticity, and perhaps methods to lessen co-activation.
Moving on to the genetic presentations, Dr. Karin Buiting from Essen
talked about the experience of her laboratory in analysis of AS patients
with imprinting defects. They receive most of the samples worldwide. In
all, they have analysed samples from 91 patients with imprinting defects.
9 of these had a detectable deletion within the imprinting control centre,
1 had an inversion, (a chromosome rearrangement, which affected the function
of the imprinting centre) and the remaining 81, had abnormal imprinting
with no detectable abnormality of the imprinting centre. There were affected
family members only in the group with imprinting centre deletions, 3 of
the 9 being familial. All the 81 cases without deletions were sporadic
and this has important implications for genetic counselling.
Mosaicism for imprinting defects has also been seen, and accounted for
about a quarter of the group without imprinting centre deletions. Mosaicism
can be detected from looking carefully at the methylation analysis. In
the normal situation one sees both a normal maternal and normal paternal
methylated band. In AS the maternal band is missing. In AS mosaics there
is a maternal band present but this is fainter than normal. Dr. Buiting
and colleagues had devised a technique to measure the percentage of cells,
which were normally methylated and then tried to correlate this with severity
of the clinical features. The correlation was not perfect but there was
a suggestion of a trend showing that the higher the percentage of normally
methylated cells the less severe the clinical features.
On a practical note, Dr. Buiting mentioned that one of the kits currently
in use for methylation analysis could be used with both a single and a
multiplex reaction, and their laboratory had found that it was better
to use the multiplex reaction as the single one could give incorrect results.
Still on the subject of imprinting, Dr. Buiting moved on to discuss
three patients with AS who had been born following assisted conception
using the ICSI (intracytoplasmic sperm infusion) technique. This raised
the question as to whether ICSI might predispose to abnormal imprinting
in the early embryo. Although it's difficult to say whether this is true
based on the small number of cases, imprinting defects in AS are fairly
rare. So three cases born after ICSI does appear to be significant. In
addition, there is also evidence that the incidence of Beckwith-Wiedemann
syndrome, another disorder caused by abnormal imprinting, is increased
after ICSI and that imprinting is altered in animal models following assisted
conception. This subject therefore warrants further study.
The presentation from Dr. Rob Nicholls (USA) first of all discussed
mechanisms leading to chromosome 15q11-13 deletions in AS. There are several
chromosomal regions, 15q11-13 included which are particularly prone to
deletion. The reason for this is that all of these chromosomes contain
multiple copies of short segments of repetitive sequence DNA i.e. segments
of DNA where the genetic code is identical. When a parent comes to have
children they only put one chromosome 15 into each egg or sperm, but before
this the two chromosome 15s pair up and mix up their genetic material
in a process called recombination. Because 15q11-13 contains short repeat
sequences, the two 15s can sometimes pair up unevenly, and when they interchange
their genetic material you can end up with a deletion or a duplication.
This is also why most chromosome 15q11-13 deletions in AS all tend to
be the same size.
Other things that might predispose to deletion formation are if a parent
carries a chromosome rearrangement (inversion or translocation). Deletions
have also been seen in association with a chromosome 15 marker (a small
extra fragment of chromosome 15) although this is perhaps more likely
to predispose to uniparental disomy.
The rest of Rob Nicholls' lecture covered epigenetic modification of
DNA. An epigenetic modification means one where the DNA code is not altered
as in a mutation, but the function of the DNA is altered in some other,
reversible way. Genomic imprinting, the mechanism whereby DNA acquires
a mark or "imprint", telling it which parent it came from is
brought about by epigenetic modification, and Dr Nicholls discussed the
evidence for different types of epigenetic modification. Most people by
now are familiar with the concept that one of the mechanisms causing epigenetic
modification of DNA is methylation. In addition it seems that modification
of histone, the small particles around which the DNA strand is wrapped,
can alter DNA expression. Histone acetylation, for example, also confers
parent of origin specific gene expression on the DNA. In this way we are
gradually beginning to understand more about the process of genomic imprinting
and how it might come about.
Further presentations covering the mechanisms of genomic imprinting
were from Joseph Wagstaff (USA) and Ruth Shemer (Israel). They both described
very elegant laboratory work, which had helped to elucidate control mechanisms
for the imprinting centre. Dr. Wagstaff described identification of several
different control elements (promoters) for the imprinting centre and further
work on histone modification. It seems clear now that, unlike in the mouse,
the imprinting process is going on in humans after the time of fertilization.
Dr. Shemer demonstrated the complex interplay between the Prader Willi
and Angelman parts of the imprinting control region. Whilst the PWS part
has overall control for imprinting of the region the AS centre acts on
the PWS centre and functions as a repressor.
"So why do we want to study all this detail about imprinting?",
someone asked; Principally it's because if we can work out how the imprinting
process works, there is a chance of devising possible therapies which
might perhaps reactivate silenced genes and provide some hope of treatments
for imprinted disorders. We can also learn from studies of other imprinted
conditions although accepting that the processes might be slightly different
in different conditions.
Two further presentations covered analysis of genes within the 15q11-13
region. Dr. Camprubi from Italy described the finding of a novel mutation
(one which hasn't been reported before) in the UBE3A gene.
Dr. Dhar has been working on the ATP10C gene, a further gene with maternal
expression within the 15q11-13 region. Dr. Dhar had become interested
in ATP10C after working on mice with deletions of the Angelman region.
(This is on mouse chromosome 7 which corresponds to human chromosome 15).
They noticed that mice with these deletions were hypopigmented (they were
missing the albinism gene within the region) but were also fatter than
the other mice, and this appears to be due to lack of ATP10C which has
a role in causation of obesity in mice. However, they are not sure whether
this has any relevance for AS in humans. We know from genotype/phenotype
studies described in detail by Dr. Charles Williams that the deletion
group are the ones least likely to get obese with age, and they don't
appear to develop problems with their blood sugar like the mice did. Clearly
more work needs to be done in this area.
Other issues discussed by Dr. Williams were mosaicism (of relevance
to the imprinting group although there have been a couple of deletion
mosaics) and differential diagnosis. He drew attention in particular to
the condition caused by deletion of chromosome 22q13 which is an AS look-alike
condition with very similar appearance and behaviour. Some of these children
have symptoms of mitochondrial disorders. You might not see a 22q13 deletion
on routine cytogenetics and would have to do FISH with the relevant probe
to detect it.
There was some discussion as to why you get characteristic facial features
in patients with UBE3A mutations, UPD and imprinting problems when there
are no actual genes missing. The question is as to whether UBE3A has other
signs apart from affecting the brain. Some thought that the facial features
might actually be related to the effect the brain has on behaviour e.g.
the increase in behaviours such as mouthing, tongue thrusting and smiling
might encourage growth of the jaw and make it more prominent.
Dr. Williams also brought up the topic of autism and AS. Some children
have been reported as having autistic features but in general AS individuals
have good social skills and are "anti-autistic".
Moving on to the last session, the topic of autism arose again. There
were presentations here from two psychologists, Dr. Peters from Baylor
College of Medicine (USA) and Dr. Reason from the UK. They had both been
using the Bayley's scale of infant development (BSID II) and Vineland
scales (based on parental report) to look at behaviour in AS. Dr Peters
has carried out assessments as part of Dr. Beaudet's project on the effect
of folate supplementation in Angelman syndrome. They explained that the
reason for pursuing this type of assessment was that there was very little
objective data about developmental levels in AS in the literature. One
of the main reasons for this is that there are not any validated assessment
tools on which to test children with AS and indeed, the Bayley's scale
includes a lot of language based tests as you move up the scale. AS children
find these, and the fine motor tests more difficult to do and so the Bayley's
probably underestimates developmental level. The level at which the children
were functioning on the Bayley's came out at about 11-13 months in both
studies. Interestingly, UK parents tended to overestimate developmental
level whereas the US parent's assessment of their child's developmental
level agreed more closely with the Bayley's result.
It was argued also that even if the Bayley's didn't give a perfect estimate
of developmental level, it could be used to chart progress in development.
AS children scored better on socialisation skills, as one might expect.
Some had symptoms consistent with autism, and they did less well on testing.
However, overall, the developmental profiles were not those of an autistic
child. Dr. Peters showed an interesting bar chart with the results of
developmental assessments across the different groups of AS. Although
some children with UPD and imprinting mutations scored quite highly overall,
there was a child with UPD who had one of the lowest scores in the group
and a child with a deletion who had one of the highest. This is proof
of the enormous overlap in abilities between the various AS groups.
Dr. Reason found that imitation was not a strength in AS, that they
were good at taking things out of containers but not at putting them in.
Object permanence is a strength in AS. (We all know that if you have hidden
biscuits/cookies somewhere then an AS child will remember and look for
them!) Dr. Reason had also sent out a postal questionnaire to parents
asking about their children's behaviour. The questionnaire had some rather
unusual questions on it such as "Does you child create chaos?" 75%
said yes. It was also clear from the questionnaire that AS individuals
were sociable but stubborn, tended to be clumsy, were often fussy eaters
and had sleep problems.
A study by Dr. Elia from Italy had looked at sleep problems in particular,
by means of a sleep questionnaire. He noted that people with AS, sleep
for shorter periods, moved a lot during sleep and had an increased incidence
of teeth grinding, night terrors and sleep walking. He didn't find an
improvement in sleep with age as other studies have done.
A final presentation by Ginny Paleg, a physiotherapist who has dealt
with several AS children showed how she could help children with AS become
ambulant by means of a gait trainer. This was demonstrated with videos.
She declared a financial interest in the company who produced the equipment,
but nevertheless thought that it was useful in AS, particularly as it
has been proven that global developmental skills improve significantly
once a child becomes ambulant.
There was a lot of information to take home, some of it quite complicated
at times but the good attendance at the scientific meetings demonstrated
that they are a worthwhile part of the conference. More importantly, new
collaborations were forged and people went home with new ideas to pursue.
Many thanks on behalf of the scientific community to the ASF/IASO for
organizing and facilitating the conference.
Jill Clayton-Smith, M.D. Consultant Clinical Geneticist St. Mary's Hospital
Manchester, U.K.
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