SCN1A-related seizure disorders: prediction of clinical course based on advanced genotyping

Dravet syndrome (DS) is a severe neurological disorder of childhood, of which
the first symptoms usually occur within the first year of life. Its frequency
is about 1 in 20,000 - 40,000 children. The disorder usually starts with
generalized or unilateral clonic seizures associated with fever, illness or
vaccination. At a later stage, other seizure types occur as well and patients
may have prolonged status epilepticus. Psychomotor development is initially
normal, but slows down in the second year of life. In addition, other
neurological symptoms may appear, like pyramidal tract signs. Many patients
with DS also have behavioural problems. Outcome is poor, with intellectual
disability in most patients and ongoing seizures. Intellectual impairment
varies from severe in 50% of the patients, to moderate and mild intellectual
disability each accounting for 25% of cases. Rare patients have normal
intellect. Gait studies have shown that in many patients with DS walking
deteriorates and patients may gradually develop a crouch pattern.

In about 75% of children with DS, a mutation is found in the SCN1A gene, which
is encoding for the *-subunit of a neuronal sodium channel, Nav1.1. In 95% of
the cases, it represents a de novo mutation in the affected child.
Certain anti-epileptic drugs might worsen seizures in patients with Dravet
syndrome. A positive SCN1A test therefore influences treatment choice and may
thereby have a beneficial effect on clinical course and cognitive outcome,
especially in the very young. Therefore early genetic testing may be
beneficial, and might be considered in each infant under age 12 months with a
febrile seizure or a seizure after vaccination, or even as part of neonatal
screening programmes.

However, SCN1A-mutations are also identified in patients with febrile seizures
and in patients with the milder epilepsy syndrome GEFS+ (Genetic Epilepsy
Febrile Seizures Plus). Moreover, in patients with DS there is a wide
phenotypic variability, with on the one end of the phenotypic spectrum patients
with ongoing seizures who are institutionalized and severely disabled, and on
the other end patients who live independent lives. One of the prerequisites for
early genetic testing would be that the clinical course of a patient could be
accurately predicted according to the genotype. Genetic testing by mutation
analysis of SCN1A only, would not allow a reliable prognosis.

Mutations causing truncation of the SCN1A protein and missense mutations
affecting the voltage and/or ion-pore regions of the protein are more frequent
in DS than in GEFS+. However, no positive correlation has been found between a
specific mutation and a specific phenotype, and variable phenotypes have been
associated with the same mutation. Mosaicism for the SCN1A mutation and the
percentage of mosaicism may contribute to the variable clinical expression. In
addition, variants in other genes, like the SCN9A-gene and CACNA1A-gene, may
modulate the phenotype in SCN1A-related DS. Since DS is caused by decreased
levels of Nav1.1, variants in the SCN1A promoter region and in 5*- and 3*
untranslated exons may affect phenotype as well, by modulating SCN1A-
expression.

The objective of the study is to investigate if it is possible to predict the
clinical outcome of a patient with SCN1A related epilepsy based on the findings
of advanced genotyping.

We aim to answer the following questions:

1. Can clinical outcome in SCN1A-related febrile seizures / epilepsy be
predicted based on *advanced genotyping*?
a. Is the degree of somatic mosaicism for the SCN1A mutation associated with
disease severity?
b. Are DNA-variants in the promoter region and/or the 5*- and 3*untranslated
exons of SCN1A, and other variations than the pathogenic mutation in the coding
regions of SCN1A associated with disease severity?
c. Are mutations in modifier genes associated with disease severity?

2. Does an early genetic diagnosis have a beneficial effect on clinical course
and outcome in patients with SCN1A-related febrile seizure(s)/ epilepsy?

a. Approach of possible study participants
The parents / caretakers / legal representatives (from here called: *parents*)
of potential participants will be contacted by the physician who had requested
analysis of the SCN1A-gene, or by the neurologist, or, if the medical genetics
department has been visited before, by the clinical geneticist.

b. Informed consent procedure
Patients and their parents who fulfil the criteria for inclusion will receive
oral and written information about the study.
The written information includes three informed consent forms: one for
participation of the patient with SCN1A-related febrile seizures / epilepsy,
and one for each of the parents.
For patients with Dravet syndrome with intellectual disability, both parents
will be asked to give written consent. For participation of normally
intelligent children aged 12-18 years, both the parents and the child will be
asked for written informed consent.

Patients and their parents will be asked for permission:
* to obtain data from medical records of the patient
* to undergo a telephone interview
* to respond to questionnaires on mobility, quality of life and behavior
* to make videos of the patient for gait analysis
* to use their DNA stored at the UMC Utrecht laboratory for genome diagnostics
* to have the laboratory test performed for the detection of: somatic
mosaicism; variants in the promoter region and /or 5*- and 3* untranslated
region; additional variants in the coding region of SCN1A; and, variants in
modifier genes.

c. Medical data collection
When informed consent has been obtained the following medical data will be
collected:
- date of birth
- gender
- gestational age, birth weight, head circumference at birth, Apgar scores,
perinatal complications
- age at clinical and at molecular diagnosis
- calendar year of clinical and molecular diagnosis
- age at onset per seizure type
- frequency and type of seizures
- fever sensitivity
- anticonvulsive treatment that has been / is being used
- response to treatment
- the results of neurological examination
- the results of EEG and MRI
- use of walking aids and procedures that the child underwent to improve
walking ability
- comorbidity
- psychomotor development; including early developmental milestones
- school performance
- results of neuropsychological examination, including instruments that have
been used and date of assessment
- behavioral abnormalities, autism, attention deficit (hyperactivity) disorder,
aggression
- family history
- age of the parents at the time of conception

In case these data are not available in the patient*s records, additional
information will be collected during a telephone interview with patient or
parents.

d. Questionnaires
To evaluate clinical outcomes, we will interview parents by phone about
mobility, quality of life, and behavioral problems using short standardized
questionnaires. Parents receive the questionnaires in advance by mail.


e. Laboratory methods

Mosaicism
To establish whether the patient is mosaic for the pathogenic SCN1A mutation,
and if so, at what grade, high coverage massive parallel sequencing of the DNA
will used. After initial validation of the procedure using mixtures of DNA, the
ratio of reads carrying the mutation relative to those carrying the wildtype
can be used to establish the degree of mosaicism.

Regulatory variants
Variants in the promoter region, the 5*UTRs and the 3*UTRs of the gene SCN1A
may affect expression of the gene. Additional non-pathogenic variants in the
gene may affect the protein function or splicing. Presence of such variants
will be established by massive parallel sequencing. Follow-up of identified
variants will depend on the type of variant. In addition to bioinformatics
prediction, cell culture experiments may be used to get better idea of the
effect of the variant on SCN1A expression.

Modifier genes
To identify variants in modifier genes, again massive parallel sequencing will
be used. Establishing a list of possible modifier genes and filtering
strategies for possible modifier variants will be part of this project.

The only potential risk of participating in this study is related to a venous
puncture in parents from whom no DNA is available yet. These risks include
hematomas and local infection on the site of puncture, and for some patients a
vasovagal response on the venous puncture.