Tackling defective epigenetic assembly in psychopathology:
Towards personalized intervention in Kleefstra syndrome

Next generation genetic studies in rare neurodevelopmental disorders (NDs) have
spectacularly
increased diagnostic yield. The majority of Mendelian causes of intellectual
disability (ID) and autism
spectrum disorders (ASD) can now be identified. For the first time, this opens
doors to detailed
characterization and identification of syndromes based on their genetic
etiology. The aim of our
research group is to design personalized care for genetic NDs.
One of the major issues we experience in dealing with patients affected by ND
syndromes is related to
their psychopathology and behavioural problems, which have a great impact on
quality of life of
patients and their families. A better understanding of the underlying
neurocognitive and biological mechanisms will open doors to investigate new
therapeutic intervention and subsequent improvement of care. To move this field
forward, we have
founded a network of dedicated professionals in the multidisciplinary expert
clinic on rare genetic NDs
(www.radboudumc.nl/ontwikkelingsstoornissen). Our main focus is on patients
with Kleefstra syndrome
(KS). After discovering the genetic cause of KS 10 years ago, we have spent
most of my time unravelling
the pathogenesis of this syndrome and finding ways to improve therapeutic
interventions for those
patients

Based on our historical observations and correspondence from (international)
colleagues and patient
representatives, KS is characterized by (post-)puberty behavioural regression.
Amongst the symptoms
that we have observed in KS patients are extreme apathy (a decline in
motivational behaviours),
catatonia, lack of task focus, sleep disorders (frequent nocturnal awakenings
and daytime sleepiness),
and abnormal posturing of arms and hands. Based on these findings, we
systematically assessed
the psychopathological phenotype in the Dutch cohort (n=24) of KS patients.
Analyses based on
clinical interviews, i.e. ADOS-2 and the mini-PAS-ADD, showed that ASD was
present in all individuals
with KS. We also noted high prevalence of depressive and obsessive compulsive
disorders. Most
importantly, psychotic disorders and severe regression occur in an
exceptionally high
percentage of KS patients. All patients above the age of 18 years in our cohort
study (n=8)
have (had) a psychotic disorder and a severe loss of functioning . The general
population
incidence of psychotic disorders is only 0.8%.
We observed that treatment with relatively high dosages of antipsychotics
contributed
significantly to improvement of daily life functioning. Our experience with
this syndrome forms the basis
for the present project.

We aim to prevent or reverse developmental regression in patients with
Kleefstra syndrome. We hypothesise that the regression we observed is the
result of a psychotic disorder, which is
not properly recognised by clinicians and often not treated according to the
guidelines for psychotic
disorders. Investigating development and sources of the behavioural problems
and regression
requires the development of a strategy to implement longitudinal follow-up of
patients with KS;
this is one of my key-objectives. In our retrospective descriptive study, we
found that KS patients
had better response and fewer side effects on olanzapine, an atypical
antipsychotic acting on multiple
neurotransmitter systems compared to others with predominantly dopaminergic
receptor affinity.

Objective 1: to develop a longitudinal follow-up study of KS patients with
special attention to
behavioural developmental changes

Objective 2: To perform an international study to test the effect of olanzapine
treatment in patients with KS with general regression

Objective 3: to study the in vitro effects of effective drugs like olanzapine
by measuring neural
network activity in patient-derived induced neurons.

We need to develop such robust metrics. However, for patients with
severe intellectual disability (IQ<50), few suitable validated instruments are
available for diagnosing
psychopathology in a detailed and structured manner. Therefore, I will tailor
our test battery to the
needs of KS population based on the explorative findings from our historical
cohort. In addition to a
general examination to assess the level of functioning, this battery will
comprise tests and
questionnaires adapted to the individual capacities of KS patients (table 1,
page 8 application)
Moreover, we will collect baseline measures at t0 to prepare for treatment,
which include a screen for
CYP1A2 and CYP2D6 polymorphisms to determine an optimal drug dosage regime (Box
1, page 9 application). Signs
and symptoms of psychotic disorder - which according to the diagnostic manual
of ID (DM-ID)
characteristically consist of a loss of cognitive functions and (sudden)
significant behavioural changes
in addition to hallucinations and delusions - as well as developmental
regression, will be a particular
focus of our battery, to be assessed by VABS, Mini-PAS-ADD, and PANSS.
We will see the patients annually according to this evaluation scheme to
prospectively collect detailed
longitudinal data on development and behaviour. In total, I expect to evaluate
40 patients at 3 or more
time points. Based on the results from this evaluation, the test battery and
strategy for examining
psychopathology will be further optimized for patients with KS, which will
serve as a model for similar
syndromes with low IQ. Moreover, we will identify additional endpoints to be
used as outcome measures
in future intervention programs. In addition to the annual evaluation,
biobanking of blood, urine, and a
skin biopsy for hIPSC generation are part of the assessment for studying
objective 3

In our open label intervention, our team or the international collaborator
psychiatrist will prescribeantipsychotic treatment, when psychotic disorder is
suspected on the basis of the indicators and
findings by psychiatric examination and observation, as described above. Both
groups will be treated,
when applicable. According to the international guidelines (NICE +
Multidisciplinary Guideline
Schizophrenia from the Netherlands, kinderformularium), the antipsychotic drug
of choice depends on individual factors.
In this study, the treatment of first choice is olanzapine, not only as based
on scientific findings, but
we also found it strikingly more effective in our previously treated patients
compared to other
antipsychotics. In case of contra-indications, prescription of alternative
medication will be discussed for
the specific patient by the expert team. Because of the risk of side effects,
somatic screening will
continue during treatment according to international guidelines.
Adverse drug events will be evaluated by a clinical pharmacologist. A safety
data monitoring team will
evaluate all data once a year.

We will perform an international open label trial according to a wellestablished
design for (ultra)rare diseases using Bayesian statistics and sequential
analyses. The cohort numbers are calculated for a power of .95 (.05
significance) on
the assumption that at least 1/3 of EDC will have PD and treatment will at
least 50% of regression. we will collect data on all KS patients age >=12 years
at t0 and divide them into two groups based on VABS/PANSS/Mini-PAS-ADD
assessments:
I. Late detection Cohort (LDC): KS patients, who score positively on the
life-time
prevalence of psychotic disorder (PD; with or without current suspicion on PD),
with onset of first symptoms >2 years before t0 (already n=8 collected).
II. Early detection Cohort (EDC): those who have been so far without any PD, or
with
suspicion of PD with start of symptoms <=2 years before t0.

Human induced pluripotent stem cells (hIPSCs) and neurons re-programmed from
those,
represent a novel exciting avenue to study the underlying mechanisms of brain
diseases
and for the design and optimization of new therapies (personalized medicine).In
the Stem Cell Facility at our department,
differentiation protocols have been optimized to produce bona fide cortical
excitatory and
dopaminergic neurons from hIPSCs. iNeurons from KS patients
with regression show severe disorganisation of synchronised synaptic bursting
activity.
Here, we will use iNeurons from KS patients to get insight in the mode of
action of drugs
and to predict the efficacy of specific drug interventions.
3a: Mode of action of olanzapine in KS. I am to elucidate how the beneficial
effect of
olanzapine is mediated. In contrast to less effective antipsychotic drugs
(risperidone and
haloperidol), olanzapine affects multiple neurotransmitter systems. Besides
dopaminergic
neurons, which are targeted by all these drugs, olanzapine also acts on
serotonergic and
cholinergic neurons. In addition to the established protocols for
differentiating cortical
excitatory neurons and dopaminergic neurons, we will thus also generate
serotonergic
and cholinergic neurons, using established protocols. Functional analysis will
involve
the use of micro-electrode-arrays (MEAs), which allow repeated (every 2 days),
passive
recording of spontaneously generated action potentials from the same culture.
Such
action potentials become coordinated in space and time during maturation of the
neurons, which we will monitor in 24-well MEAs. We will determine whether
olanzapine
can restore abnormal network activity, and whether its effect is specific for a
particular type of neuron. We speculate that such effect
on MEA cultures will be predictive for its capacity to prevent regression.
3b: Predicting drug efficacy in vitro. It is possible that olanzapine will
proof to be
effective in most, but not in all the KS patients. We will use the conditions
established in
Objective 3a to investigate whether we can predict drug (olanzapine)-efficacy by
comparative analyses with iNeurons from responsive and non-responsive
individuals
(retrospective study). Moreover, I will conduct a prospective study with
iNeurons derived
from patients with a KS-like phenotype caused by other genes that are part of
the *KS
epigenetic module*. Our MEA studies have already established that
haploinsufficiency
of such genes gives rise to highly similar neuronal network defects


.

The study comprises low burden and risks. The burden for all included patients
is In addition to a general examination to assess the level of functioning, the
assessmnet of a battery of tests and questionnaires adapted to the individual
capacities of KS patients . Moreover, we will collect baseline measures at t0
to prepare for treatment, which include a screen for CYP1A2 and CYP2D6
polymorphisms to determine an optimal drug dose regime.
In those individulas where we observe signs of psychotic disorder - which
according to the diagnostic manual of ID (DM-ID) characteristically consist of
a loss of cognitive functions and (sudden) significant behavioral changes in
addition to hallucinations and delusions - as well as developmental regression,
we will implement treatment witth antipsychotics and olanzapine as drug of
first choice, as this is according to current best practice based upon our
historical observation, This will not add extra burden or risks compared to the
already excisting current situation where we advice the same strategy in
clinicla practice. This study aims to evaluate the clinical effect of treatment
with olanzapine.

The particular focus of our battery to screen for psychotic symptoms,, will be
assessed by VABS, Mini-PAS-ADD, and PANSS. We will see the patients annually
according to the evaluation scheme to prospectively collect detailed
longitudinal data. Based on the results from this evaluation, the test battery
and strategy for examining psychopathology will be further optimised for
patients with KS, which will serve as a model for similar ND syndromes. In
addition to the annual evaluation, biobanking of blood (only first occasion)
and urine (all occasions) are part of the assessment.
regarding the tests/questionnaires (listed below) : the majority will be
provided by the caregivers (VABS, MINI-PASSAD, QBCL).
Besides the general examination, only ADOS-2 (wil take 1 hour) , VMI, LTT (both
only take less then 30 min) , (IQ test and CANTAB; depending on their
developmental age which tests), will be requested to be performed by the
patients.

Total list of tests/questionnaires:
-General examination: Includes height, weight, blood pressure, heartbeat
frequency, abdominal circumference, biobank sampling (t0) and video-monitoring
-VABS: The Vineland Adaptive Behavior Scale to assess developmental age and
possible developmental regression
-PANSS: Positive And Negative Syndrome Scale to measure both positive and
negative psychotic symptoms
-Mini PAS-ADDMini Psychiatric Assessment Schedules for Adults with
Developmental Disabilities
to measure a broad range of psychopathology
-ADOS-2: Autism Diagnostic Observation Schedule Standardized psychiatric
observation of Autismspectrum symptoms
-CBCL: Child behavior checklist to asses behavioural difficulties
-Beery-VMI: Test for Visual-Motor Integration
-LLT: Location Learning Test to asses spatial learning and memory
-(IQ test)* Wechsler scale (WAIS-IV/WISC-V)
-(CANTAB) CAmbridge Neuropsychological Test Automated Battery
Computer based assessment of attention, memory and reaction time

Patients with the Kleefstra syndrome have an intellectual disability, so there
is group relatedness.