Novel genetic markers of surgical candidacy in refractory epilepsy: Whole Exome Sequencing in historical cohorts of patients

Epilepsy surgery is the only treatment that can fully cure patients from their
seizures without the need for lifelong medication. During surgery, the
epileptogenic zone is removed or disconnected with sparing of surrounding
normal functional tissue. The chance of postoperative seizure-freedom is
maximized when the epileptogenic lesion is completely removed/disconnected.
Whereas in the past only patients with visible structural MRI- lesions
(considered causative for the seizures) were considered surgical candidates, we
now increasingly evaluate people with refractory epilepsy and normal imaging.
Currently 60-70% of these MRI-negative (but presumed lesional/structural)
patients are rejected for surgery, often after extensive and invasive
source-localizing diagnostics. Operated MRI-negative patients have a lower
chance of reaching seizure freedom. Furthermore, the absence of a
histopathological abnormality (~8% of all operated patients) is a major
predictor of poor outcome. When evaluating MRI-negative patients with
refractory focal seizures for surgical candidacy, the epilepsy is either caused
by an *invisible* microstructural lesion or malformation of cortical
development -probably implying a high chance of postoperative seizure-freedom-,
by a genetic syndrome without an identifiable lesion -probably reflecting a low
chance of surgical success -, or by a combination of the two (e.g. tuberous
sclerosis). The crucial differentiation between people with operable and
non-operable epilepsy (i.e. between a presumed lesional and non-lesional cause
of seizures) requires new and reliable biomarkers. Pathogenic variants in novel
and known epilepsy genes are such biomarkers that are currently not routinely
implemented in the presurgical evaluation. This project will will extend the
scope of genetic biomarkers in a more experimental phase to find novel
biomarkers of focal epilepsy that will be functionally validated and rapidly
transferred to the clinical presurgical evaluation phase.

Primary Objective:
Our overall aim is to improve selection of patients for epilepsy surgery and
predict surgical candidacy with use of genetic test results as a biomarker. The
specific objective is:

Cohort
1. perform trio-WES in MRI-negative patients who were shown to be poor surgical
candidates (cohort I) and patients who proved to be good surgical candidates
(cohort II) to discover new genetic variants that can serve as new biomarkers
to predict surgical candidacy in patients with refractory focal epilepsy.

Hypothesis
We hypothesize that by discovery of new pathogenic variants and functional
validation, we will expand the diagnostic utility and validity of genetic
screening. We hypothesize that routine testing for germline epilepsy gene
mutations in MRI-negative
patients with refractory epilepsy can improve the differentiation between good
surgical candidates (i.e. those with a focal structural cause of seizures) and
those who
are less eligible for epilepsy surgery (particularly those with non-lesional
genetic epilepsy
syndromes), ultimately improving the rate of seizure-freedom after surgery and
preventing unnecessary invasive source localization techniques. In addition, we
hypothesize that the detection of somatic mutations in resected tissue can
improve post-surgery diagnosis, outcome prediction and postsurgical AED
regimens. Furthermore, we assume that detection of somatic mosaicism gene
mutations in saliva of operated patients with use of ultra-deep targeted
sequencing could improve the diagnostic yield of genetic testing during the
presurgical evaluation phase. This could ultimately improve the presurgical
differentiation between good ** presumed lesional focal epilepsy patients
** and poor surgical candidates.

Overall approach
In this study we will extend the scope of genetic biomarkers in a more
experimental phase to find novel biomarkers of focal epilepsy that will be
functionally validated and rapidly transferred to the clinical presurgical
evaluation phase. In a parallel study, we start with the clinical presurgical
evaluation phase in which we apply routine genetic testing in MRI-negative
patients and in resected tissue, demonstrate proof-of-concept, and evaluate the
effect of detecting genetic biomarkers on surgical decision-making, lesion
characterization, and outcome prediction. For this parallel study, a separate
application for ethical approval will be submitted.

General relevance
Seizure control is not reached in ~30% of all patients 12 months after epilepsy
surgery. Those with normal MRI scans have an even higher chance of poor
surgical outcome up to ~50%, because the preoperative differentiation between
patients with either focal lesional (structural) epilepsy, or with focal
genetic non-lesional epilepsy has proven to be difficult. It is of crucial
importance to improve the selection of suitable surgical candidates, and to
reduce the risk of unnecessary (deemed unsuccessful) invasive diagnostics and
resective surgery. We hypothesize that routine testing for the recently
discovered genetic causes of epilepsy in MRI-negative patients can improve the
recognition of poor surgical candidates and prevent further presurgical and
invasive diagnostics. Vice versa, demonstration of a genetic abnormality that
is likely to underlie a structural cause (such as FCD or mMCD) may improve the
recognition of patients who are good candidates for surgery, despite normal
MRI. Most importantly, in patients with refractory focal non-lesional epilepsy,
in whom semiology and ictal EEG findings are not concordant with other
functional imaging techniques to localize the epileptogenic zone (such as MEG,
PET, and SPECT), the demonstration of a genetic underlying cause of the
epilepsy syndrome should prevent invasive and expensive (further) presurgical
diagnostics, such as grid or SEEG monitoring. Currently, genetic studies have
presented the prelude for personalized medicine by demonstrating the potential
relevance of genetic mutations for focal epilepsy, yet these fundamental
discoveries have not been translated to improved care and clinical utility. Our
proposal will accelerate the implementation of genetic discoveries into the
epilepsy surgery program, by immediately implementing genetic testing in
MRI-negative patents that are enrolled for surgery. By implementing genetic
screening in clinical practice, and assessing prospectively and retrospectively
the impact on presurgical evaluation, we aim to provide the evidence needed for
long term application. The combination of basic and applied studies from at
least two different institutions, in collaboration with at least two renowned
foreign centers, will provide the clinical evidence needed to implement
molecular diagnostics in epilepsy surgery.

We will perform trio-(both parents and affected child) WES in a retrospective
cohort of 50 patients (and their parents) who were shown to be poor surgical
candidates, defined as: a) MRI-negative, stereo-EEG (SEEG) no focus, rejected
for surgery, or b) MRI-negative, resected, pathology-negative and poor seizure
outcome. Furthermore, we will screen 50 MRI-negative and pathology-negative
surgical patients (in particular FCD) that reached seizure freedom. We will
functionally validate the mutations in genes previously unknown to be related
to FE in this study. Patients will be selected from the epilepsy surgery
databases of the three participating centers (UMC Utrecht, Netherlands; UCL
Great Ormond Street Institute of Child Health & Great Ormond Street Hospital
for Children, London, UK).

Sequencing results will be analysed and interpreted by a collaborative team of
bioinformaticians. We will select all de novo mutations that are predicted to
be damaging and are absent in available control databases. Surgical outcome is
collected from the databases and electronic medical patient files. Genetic
findings will be compared between both groups, and related with seizure
outcome. Furthermore, we will share results with our international partners
through several genetic consortia that are aimed at identifying epilepsy genes.
By sharing our results we will increase the likelihood of finding similar
patients with mutations in the same gene, which is necessary evidence for
causality.

The current proposal aims to describe the implications of genetic testing in
patients with refractory epilepsy who received a clinical assessment for
epilepsy surgery, and to confirm the as well as elucidate the association
between different subtypes of gene mutations and surgical candidacy, and to
discover new genetic pathogenic variants. The burden of patients in both cohort
studies is minimal and consists of drawing one tube of blood for the purpose of
the study.
All in all, the overall burden of participation in this study is low, while the
associated risk is negligible (vene puncture for DNA blood draw). There is no
clear benefit associated with this study, however knowledge obtained from this
study will contribute to the decision making and outcome prediction in future
patients with focal refractory epilepsy who are evaluated for epilepsy surgery.
The inclusion of minors in the current study is required and justified;
meaningful results can only be acquired through this group-related design.