Metabolomics in Dravet syndrome

To function optimally, the brain requires a large energy supply. In healthy
individuals, the required energy is mostly derived from glucose metabolism, in
the form of ATP1.Glucose metabolism is altered in epileptic brains. During
seizures, the oxygen and energy consumption of the brain enhances due to the
increase in demand. Post-ictal elevated lactate levels are common and are
likely due to the mismatch in glycolysis and use of pyruvate1,2. Interictal
FDG-PET imaging has shown glucose hypometabolism in epileptic foci without
corresponding brain atrophy, indicating a permanently altered metabolic state
in these brain regions3,4. The interictal reduced availability of ATP may
contribute to seizure generation, since the post-ictal stabilization of ion
gradients and membrane potentials, sufficient neuronal signal transduction and
neuronal damage repair demands ample amounts of energy1Moreover, components of
the second step of glucose metabolism, the tricarboxylic acid cycle (TCA
cycle), function as building blocks for amino acids and proteins. Chronic
depletion of these vital elements prevents neuronal repair as well as
replenishing of neurotransmitters and may in part contribute to comorbidities
of chronic epilepsy, such as cognitive regression. The role of impaired glucose
metabolism in epileptogenesis has been affirmed in clinical practice,
illustrated by the therapeutic success of the ketogenic diet in refractory
epilepsy, which induces a metabolism shift to fatty acid metabolism5,6

The role of energy metabolism in epilepsy is however poorly understood and
possibly differs per type of epilepsy. One of the epilepsy syndromes for which
an altered metabolism has been suggested in earlier studies is Dravet syndrome.
Dravet syndrome (DS) is a severe epileptic and developmental encephalopathy,
presenting in the first year of life with prolonged generalized or unilateral
tonic-clonic or clonic seizures, that progress to often therapy-resistant
seizures over the course of the disease7). After the second year of life,
cognitive and motor development slows. In more than 80% of patients, DS is
caused by a mutation in the SCN1A-gene, which codes for the alpha subunit of a
voltage-gated sodium channel, predominantly expressed in GABAergic inhibitory
interneurons, resulting in increased neuronal excitability8 . A longitudinal
follow-up study compared FDG-PET scanning over time in three patients with an
SCN1A-mutation to age- and gender matched controls and found normal cerebral
glucose metabolism up until the first year of life but a gradual bilateral
cortical glucose hypometabolism by the age of four, concurring with the
clinical manifestation of cognitive regression. Interestingly, the three SCN1A
patients showed similar patterns, suggesting a disease-specific process9. In a
small study of fibroblasts in four Dravet patients a severe reduction in
efficacy of the third step of glucose metabolism, the electron transport chain,
compared to a control group was revealed. These defects were not seen in
patients with a PCDH19 mutation with a similar phenotype10. A zebrafish model
of Dravet syndrome demonstrated a decrease in the rate of glycolysis and of
oxygen consumption, compared to wildtype, that was effectively resolved with
the ketogenic diet11. A recent metabolomics analysis that was performed in
Dravet mouse model revealed altered concentrations of several intermediates of
glycolysis and the TCA cycle, indicating a shift in metabolism. Furthermore, an
increased GABA-to-glutamate ratio was detected, a possible compensatory
mechanism for increased excitability12.

Untargeted high-resolution metabolomics enables the profiling and analysis of
thousands of metabolites. Over recent years, it has been applied in various
fields of neuroscience to investigate biomarkers for disease and to identify
metabolic pathways in pathophysiology. Research into biomarkers for mild
cognitive impairment (MCI) en Alzheimer*s disease has shown promising results
by identifying several candidate metabolites as prognostic factors13. For
amyotrophic lateral sclerosis, metabolomics have been applied to elucidate the
pathophysiological mechanisms13. In epilepsy, studies have applied metabolomics
for various purposes. A comparative study between patients with different types
of epilepsy and healthy controls could identify changes in the metabolic
profile of patients with epilepsy in general, but could not distinguish types
of epilepsy14. Comparing metabolic profiles of responders to non-responders to
anti-seizure medication (ASM) did result in distinct characteristics15. However
this difference was not observed in a different study in patients with newly
diagnosed epilepsy16.
The heterogeneity of patients with epilepsy complicated the interpretation of
results. Besides patient demographics such as age, gender and comorbidities,
there is a large variety in seizure severity and use of different anti-seizure
medication, that have an enormous influence on the metabolome.

Primary Objectives:
1. To assess the metabolic profile of patients with Dravet syndrome and
identify metabolic variations that contribute to the pathophysiology of
seizures and developmental delay

Secondary Objectives:
2. To assess the association between the metabolomics variations and response
to treatment.
3. 3. To assess the effect of anti-seizure medication on the metabolome.

To achieve these objectives, we have formulated the following research
questions.

For the primary objective 1:
a. What is the metabolic profile of patients with Dravet syndrome?
b. What are the differences in metabolic profile between Dravet patients,
patients with other types of refractory epilepsy and patients without epilepsy?
c. Is there an association between specific metabolic variations and the
phenotype in Dravet patients?

For secondary objective 2:
a. Is there an association between specific metabolic variations and the
response to specific treatments, including the ketogenic diet?

For secondary objective 3:
a. What are the effects of commonly used anti-seizure medication, such as
valproate, on the metabolome?

To assess the metabolomic profile in Dravet syndrome and to evaluate the effect
of other factors on the metabolome, we will perform a case-control study in a
cohort of patients with Dravet syndrome. As control groups, we will include
patients with refractory epilepsies and a control group based on remnant
samples available at the metabolic diagnostics section of the Genetic
department of the UMC Utrecht.

With respect to potential benefits for participants: the aim of this study is
to assess the metabolome of Dravet syndrome and to describe metabolic
variations that can be linked to the severity of the phenotype and to responses
to treatment. On a group level, this will increase our understanding of the
pathophysiology of Dravet syndrome and may lead to improved therapeutic choices
that may improve long-term outcome. On a broader level, elucidating the effect
of anti-seizure medication and the ketogenic diet on the metabolome will
improve our understanding of the method of action of these drugs and enable us
to identify likely responders. The benefit for individual participants is
possible improvement of treatment strategy based on metabolic profile.

With respect to group-relatedness: it is only possible to extend the knowledge
of a genomic disorder (such as DS) by studying individuals with these specific
disorders. As a large proportion of children and adults with genomic disorders
has intellectual disability, the study needs to include participants who lack
mental capacity to provide informed consent. The study cannot be carried out
solely in patients who have capacity to provide informed consent. Therefore,
the required criteria group-relatedness has been fulfilled for these patients.

With respect to burden, patients will be asked to undergo venepuncture on one
occasion. This might give some discomfort or anxiety, in the worst case a
vasovagal syncope might occur. We will ensure to minimize this risk by using
personnel trained to work with mentally impaired subjects. No severe adverse
events are to be expected from this procedure.